Anti-whitlow linker binding domains and uses thereof

Abstract

Binding domains that bind the Whitlow linker and subsequences thereof are described. The binding domains can be used as research, diagnostic, and therapeutic tools in combination with Whitlow-containing molecules, such as single chain variable fragments (scFv) and chimeric antigen receptors (CAR).

Description

ANTI-WHITLOW LINKER BINDING DOMAINS AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 656,004 filed June 4, 2024, which is incorporated herein by reference in its entirety as if fully set forth herein.REFERENCE TO SEQUENCE LISTING

[0002] 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 3FS3962.xml. The file is 244,065 bytes, was created on June 4, 2025, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0003] The current disclosure describes binding domains that bind the Whitlow linker and subsequences thereof. The binding domains can be used as research, diagnostic, or therapeutic tools with Whitlow linker-containing molecules, such as single chain variable fragments (scFv) and chimeric antigen receptors (CAR).BACKGROUND OF THE DISCLOSURE

[0004] In recent years, advances in medical technology have led to the emerging use of immunotherapies to treat different types of illnesses and diseases, including various forms of cancer. Significant progress, for example, has been made in genetically engineering cells of the immune system to target and kill unwanted cell types (e.g., cancer cells). Many of these immune cells are T cells that have been genetically engineered to express a recombinant receptor, such as a chimeric antigen receptor (CAR). CAR are proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill unwanted cell types. The subcomponents include at least an extracellular component and an intracellular component expressed as a single protein or assembling into a functional unit. The extracellular component includes a binding domain that specifically binds a marker (e.g., an antigen) that is preferentially present on the surface of unwanted cells. Oftentimes, the binding domain is a single chain variable fragment (scFv) including a variable heavy chain attached to a variable light chain using a linker, such as a Whitlow linker. When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell.

[0005] Identification of CAR-T cells is a key step in research and diagnostic applications to establish factors associated with success and failure of CAR-T cell immunotherapy. Despite this, most CAR-binding antibodies are used primarily for flow cytometry, because of the difficulty in generating reagents that function in formalin-fixed paraffin-embedded (FFPE) tissues. Anti-CARand / or Whitlow-linker antibodies that are suitable for use in multiplex immunohistochemistry (mIHC) imaging of FFPE archival or frozen tissue sections are needed.SUMMARY OF THE DISCLOSURE

[0006] Because recombinant proteins, such as chimeric antigen receptors (CAR) are largely made of proteins or fragments thereof that are found in the body, it can be difficult to differentiate cells genetically modified to express CAR from the native proteins when detection is performed in vivo or in situ. The current disclosure, however, provides binding domains that bind the artificial Whitlow linker and subsequences thereof. The Whitlow linker is a peptide linker defined by the amino acid sequence GSTSGSGKPGSGEGSTKG (SEQ ID NO: 1) and is incorporated in many recombinant proteins. In the cellular immunotherapy field, this linker is often present in single chain variable fragments (scFvs) that are used to endow antigen-targeting specificity in many CAR constructs and T cell engagers (TCEs). If a molecule (e.g., CAR) includes the Whitlow linker, the binding domains provided herein can be useful in detecting, modulating the activity of, or eliminating said molecules. For example, the disclosed anti-Whitlow linker binding domains can be used as antibodies for detection or therapy, as multi-specific antibodies for detection or therapy, or as antibody conjugates to bring payloads to the Whitlow linker-containing molecule. The disclosed binding domains are suitable for use in multiplex immunohistochemistry (mIHC) imaging of formalin-fixed paraffin-embedded (FFPE) archival or frozen tissue sections, as well as in other methods such as flow cytometry or immunohistochemistry.

[0007] While the Background and Summary of this disclosure focus on scFv, CAR, and immunotherapy, the disclosure is not so limited. The binding domains disclosed herein can be used to interact with any Whitlow-linker containing molecule and / or any cell type expressing a Whitlow-linker containing molecule.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0008] Some of the drawings submitted herewith 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.

[0009] FIG. 1. Anti CAR-T mouse monoclonal antibody (mAb) screening workflow. Bead-bound antigens (B1-B5) and CAR-T cells were stained together with hybridoma supernatants. The different targets in the screen are indicated in the boxes at the top of the figure. Bead-bound peptides and proteins (B1-B5) were presented in both native and fixed (formalin-fixed, heated, and ethanol treated; F / H / E) forms. The flowchart illustrates the workflow for selection of hybridoma clones for further analysis.

[0010] FIG. 2. Format of formalin-fixed paraffin-embedded (FFPE) cell pellet admixtures used for secondary screening by immunohistochemistry (IHC). Schematic diagrams of IHC slides used for screening hybridoma supernatants. Three FFPE cell pellets containing the indicated proportions of CAR-T cells were cut and placed on a slide for each type of CAR-T cell. The number of positive hybridoma pools obtained in this secondary screen of 86 pools is shown below for each type of CAR-T cell.

[0011] FIG. 3. Example images of secondary IHC screening of hybridoma pool supernatants. An example image is shown for positive staining (brown stain, Ab membrane stain) for FMC63- Whitlow CAR-T cells from the 1 B4 hybridoma pool. No significant staining was seen with FMC63- G4S CAR-T cells or human PBMC control. Hematoxylin (blue) is shown as a counter stain for all cells.

[0012] FIG. 4. Summary of secondary IHC screening results. Columns from left to right: clone identifier, supernatant dilution factor, concentration of antibody in supernatant, volume of supernatant, volume of diluent, results (visual inspection in microscope). Anti-P2A antibody (Novus Biologicals, clone 3H4) was used as a positive control as well as a universal negative control Ab (Enzo, ADI-950-231-0025) for each staining batch.

[0013] FIG. 5. Summary of characteristics of heavy and light chain protein sequences in antiWhitlow clones. Each of the 5 mAb clones utilize unique variable heavy chain (VH) sequences paired with one of a limited number of shared variable light chain (VL) sequences.

[0014] FIGs. 6A-6G. Detection of Whitlow-containing CAR T cells by IHC in FFPE tissue. (6A, 6B) Jurkat T cells were transduced with an FMC63-Whitlow CAR containing an EGFR transduction maker, sorted by flow cytometry, and then admixed with parental CAR(-) Jurkat T cells at the displayed dilution factors. The cell admixtures were stained with (anti-tEGFR) by flow cytometry, and the rest were fixed in formalin and embedded for IHC staining. (6A) The relative percentage of CAR positive cells as detected by anti-EGFRt by flow cytometry and by 1 B4A1 IHC are shown. Error bars indicate error in duplicate measurements. (6B) The correlation of percent CAR positive cells detected by each method is shown. (6C-6G) Fluorescent imaging of multiplex IHC staining of FFPE tissues. Anti-CD3 highlights all T cells and 1 B4A1 anti-Whitlow labels Whitlow linker expressing cells. All images are counterstained with DAPI to visualize all nucleated cells. Scale bars are as displayed. (6C) Fluorescent images of IHC staining of an FDA Standard Tissue Array (Novus) containing cores from 30 different normal human tissues (6D) FFPE tissue sample obtained from a patient 28 days after infusion with the Whitlow linker-containing CD19 directed CAR T cell product, JCAR014. (6E-6G) Fluorescent images of IHC staining for detection of three different commercial CAR engineered T cells in archival patient biopsies. (6E) Lymphnode biopsy from a patient with large B cell lymphoma treated with lisocabtagene maraleucel (71 days post-infusion). (6F) Lymph node biopsy from patient with large B cell lymphoma treated with axicabtagene ciloleucel (6 days post-infusion). (6G) Bone marrow biopsy from a patient with multiple myeloma treated with idecabtagene vicleucel (30 days post-infusion) are shown.

[0015] FIG. 7. Maximal staining index (SI) achieved with optimized doses of anti-Whitlow monoclonal antibody (mAb). Staining of a mix of FMC63-Whitlow CAR-T cells and untransduced T cells using serial dilutions of labeled mAbs. Staining index was calculated with the formula: Stain Index = (Median of Positive - Median of Negative) I (Standard Deviation of Negative x 2). Staining index was measured for the detection by flow cytometry with fresh CAR-T cells using the fluorophore Corlite 647 (Proteintech).

[0016] FIGs. 8A, 8B. Flow cytometry staining of fresh FMC63-Whitlow and FMC63-G4S CAR-T cells. Histograms of gated live, single lymphocytes from human healthy donor peripheral blood mononuclear cells (T cells, light grey) are overlayed with histograms for (8A) FMC63-Whitlow CAR-T cells (dark grey) and (8B) FMC63-G4S CAR-T cells (dark grey) stained with purified mAbs labeled with Corlite 647 (Proteintech). Antibody used in each histogram is indicated. Staining with anti-human epidermal growth factor receptor (EGFR) mAb against the co-expressed transduction marker, truncated EGFR (EGFRt) was used as a positive control for transduced cells. A second positive control was demonstrated by staining with a known Whitlow linker-specific antibody (clone E3U7Q). Mean florescent intensity values for the five different clones staining FMC63- Whitlow CAR-T cells are shown in parenthesis.

[0017] FIGs. 9A, 9B. Screening for stimulatory effects using CAR-expressing Jurkat reporter cell lines. A nur77-neon green Jurkat reporter cell line was transduced with the CD19-targeting CAR incorporating a Whitlow linker-containing FMC63 scFv. EGFRt was also contained in the transduction vector to serve as a marker of successful transduction. EGFRt positive and negative Jurkat cells were mixed 1:1 and then cultured with 1C3C3, 1 B4A1 , 1 B3b, 1 H5b, 1G2G2 or lgG1 and lgG2a isotype control antibodies, either coated on the culture plates (solid phase) or added into the culture solution (soluble). (9A) Dose-response curves are shown for solid phase mAb and soluble mAb (indicated in legend). The highly stimulating clone, 1C3C3, is shown in comparison to lgG2a. Clones with low / no stimulation are shown in separate response graphs. (9B) Comparison of neon green expression for each antibody at peak activity for solid phase is shown compared to positive controls (PMA / ionomycin or CD19 antigen expressing K562 cells) and negative controls (PBS or K562 parental cells). Lanes marked “+” indicate flow cytometry analyses gated on EGFRt-positive (CAR-transduced) Jurkat-nur77 reporter cells, and those marked were gated on EGFRt-negative (untransduced) Jurkat-nur77 reporter cells.

[0018] FIGs. 10A-10C. Targeted stimulation of Whitlow peptide containing primary CAR-T cells. Primary healthy donor derived T cells transduced with a Whitlow-peptide containing CD19- targeting CAR, and control T cells were evaluated for activation (CD69) and proliferative (Ki67) phenotype, live cell accumulation, and cytokine production in response to coculture with 25 ug / ml of soluble 1 C3C3, 1 B4A1 or isotype over 7 days. (10A) 1 B4A1 and 1 C3C3 elicit a specific gradient upregulation of Ki67 and Ki67 / CD69 coexpression in CAR T cells relative to isotype control, indicating that these antibodies induce an activated and proliferative phenotype in T cells transduced with a Whitlow linker containing CAR. This effect was not observed in control T cells. (10B) Consistent with this phenotypic modulation, 1 B4A1 and 1C3C3 coculture induces proliferation and accumulation of live Whitlow-containing CAR-T cells. Furthermore, an increase in fold expansion of these CAR T cells can be achieved with serial antibody dosing, shown here for the 1C3C3 antibody. (10C) 1 B4A1 and 1C3C3 induce effector cytokine secretion by Whitlow containing CAR-T cells to different levels, and this effect can be enhanced by serial antibody dosing.

[0019] FIG. 11. Summary of five mAbs with differing activities identified.

[0020] FIG. 12. Monoclonal anti-Whitlow linker antibodies can bind engineered cells expressing the Whitlow peptide within an scFv other than FMC63.. Flow cytometry histogram overlays of 1 H7-Whitlow or I H7-G4S CAR T cells and untransduced T cell controls all stained with optimized concentrations of each of five anti-Whitlow antibody clones labeled with a rapid high-affinity fluorescent labeling system (FlexAble, Proteintech) and titrated to determine their optimal staining concentration. An anti-CD19 transduction control antibody staining is shown for comparison.

[0021] FIG. 13. Equilibrium dissociation constant (KD) for anti-Whitlow recombinant antibodies. To analyze the interactions between the binding domains of each monoclonal antibody and the Whitlow linker peptide, chimeric antibodies were generated in which the murine variable domains were fused to human lgG1 constant regions and then tested using a high-throughput surface plasmon resonance (SPR) assay. The binding kinetics demonstrate the range of KDvalues for binding to recombinant FMC63- Whitlow scFv (0.1-3.4 pM) and the Whitlow peptide alone (0.11- 0.96 pM). Except for 1 H5b, the antibodies tested exhibited a two to four-fold improvement in affinity for the Whitlow peptide compared to FMC63-Whitlow scFv, while one antibody (1G2G2) showed no reactivity under the conditions tested. As expected, there was no observable binding to the G4S peptide. The affinity ranking of the recombinant antibodies toward the Whitlow peptide alone or contained within the FMC63 recombinant protein is 1 H5b > 1 B4A1 > 1 B3b = 1 C3C3. SD = standard deviation; nM = nanomolar; NB = no observable binding; NA = not applicable.

[0022] FIGs. 14A-14E. 3D-modeling of anti-Whitlow mAb fragment variables (Fv) and theirinteraction with the Whitlow peptide. (14A) Molecular models were generated for the Fv of each clone and are displayed with indicators of surface charge distribution (blue (+) = positive charge and red (-)= negative charge). (14B) Top interface predicted template modeling (iPTM) scores were generated to quantify the confidence in the predicted binding interface between each Fv and the Whitlow Peptide. An iPTM of >0.8 indicates high confidence that the predicted interface is structurally correct. (14C) Representative model of the 1 B4A1 Fv and its interaction with the Whitlow peptide. (14D) Predicted net charge of the solvent accessible surface area for each antibody Fv. (14E) Molecular model of simultaneous binding of CD19 antigen and 1 B4A1 antiWhitlow mAb Fab region showing non-competitive binding to FMC63 scFv.

[0023] FIGs. 15A-15F. Flow cytometry-based detection of CAR T cell products in clinical samples using anti-Whitlow antibodies. (15A, 15B) Admixtures of healthy donor FMC63- Whitlow CAR engineered T cells serially diluted with unmodified peripheral blood mononuclear cells (PBMCs) evaluated by flow cytometry using the 1 B4A1 anti-Whitlow antibody or anti-FMC63 idiotype. (15A) Pearson correlation with simple linear regression and 95% confidence intervals for detection of CAR-T cells by the 1 B4A1 anti-Whitlow antibody vs the anti-FMC63 idiotype antibody. R2=0.9980, p<0.0001. (15B) Bland Altman plot representing the difference in the % of CAR T cells detected by 1 B4A1 anti-Whitlow antibody vs the anti-FMC63 idiotype (Y axis) and plotted against the average of the two measurements (X axis). Bias and SD of bias are as displayed. The 95% limits of agreement are shown as two dotted lines. (15C-15F) PBMCs obtained from patients treated with distinct commercial CAR T cell products were stained for detection of CAR engineered cells using the 1B4A1 anti-Whitlow antibody and either anti-idiotype antibody or recombinant CAR- target protein. Shown are graphs representing the measured CAR expressing T cells / ul for prior to lymphodepletion chemotherapy (PreLD) and at multiple time points after infusion of (15C) lisocabtagene maraleucel (15D) axicabtagene ciloleucel (15E) idecabtagene vicleucel, or (15F) tisagenlecleucel. CAR+ CD3+ T cells / ul were calculated using the following formula: absolute lymphocyte count x 1000 x (CD37100) x (CAR+in CD3+gate / 100). Inserts represent zebra plots with outliers of live CD3+events at individual peak expansion time points.

[0024] FIG. 16. The average background staining of 1 B4A1 is comparable to FMC63 antiidiotype. The average nonspecific background positive events detected by flow cytometry in unmodified healthy donor PBMCs is 0.07% (SD=0.018) for 1 B4A1 anti-Whitlow mAb, and 0.058% (SD=0.011) for FMC63 anti-idiotype antibodies. No statistically significant differences were found between the two reagents (paired t test, p=0.5626). Data represents triplicate samples. Assuming this background represents the limit of blank, the limit of CAR-T cell detection per sample is estimated to be 0.1 % (limit of detection = Mean of blank + 3 SD of blank).

[0025] FIG. 17. Detection of Whitlow-containing CAR-T by immunohistochemistry (IHC) in formalin-fixed, paraffin embedded tissue. 1B4A1, 1G2G2, 1C3C3, and 1B3b show strong membrane staining of FMC63-Whitlow CAR-T cell pellets (top panels) but not on FMC63-G4S CAR-T cell pellets (bottom panel) In these brightfield images, anti-whitlow antibody staining is visualized as brown stain, with blue hematoxylin counter stain for all nucleated cells. Cell pellets were prepared with 50% healthy donor CAR-T cells and 50% unmodified healthy donor PBMCs. Scale bars (100 pm) are shown for each image.

[0026] FIG. 18. Summary of multiplexed IHC performed on archival biopsies. To confirm the ability of 1 B4A1 to detect diverse CAR T cell products, archival tumor biopsies from patients (n=10) treated with commercial Whitlow-containing CAR T cells were stained. As expected, 1 B4A1 staining was negative in all pre-treatment samples tested (n=3). Notably, the total number of T cells, CAR-T cells, and the proportion of T cells expressing CARs varied substantially across the cohort. Nevertheless, strong and specific staining of CAR-expressing cells was observed-not only in lymph node tissue but also in bone marrow particle preparations and gastrointestinal biopsies.

[0027] FIG. 19. Highly multiplexed spatial immunophenotyping of CAR T cells in situ using antiWhitlow mAbs. Click-chemistry-based custom site-specific methods targeting the antibody hinge region were employed to barcode the 1 B4A1 anti-Whitlow mAb. The barcoded antibody was then successfully incorporated into a custom staining panel for highly multiplexed spatial immunophenotyping. Here, FFPE biopsies from a patient with B cell lymphoma obtained after infusion with the Whitlow-linker CAR T cell product, lisocabtagene maraleucel, was stained with a panel of DNA-barcoded antibodies. Visual rendition was then achieved by sequential iterations of hybridization of corresponding fluorescent oligonucleotides individual antibodies, image capture and then stripping of the fluorescent oligonucleotides using the PhenoCycler Fusion instrument. This process was repeated on all antibodies in the panel to achieve highly multiplexed spatial immunophenotyping. CAR T cells can be distinguished from unmodified T cells and immunophenotyped in their spatial contexture.

[0028] FIG. 20. Oligonucleotide-barcoded anti-Whitlow antibody for CAR T cell identification and characterization using sequencing-based proteomics. The 1 B4A1 antibody was conjugated to a custom oligonucleotide barcode to enable identification of Whitlow-linker-expressing cells via sequencing. Cells were stained with the barcoded antibody, followed by secondary staining with an APC-conjugated anti-mouse antibody. Histogram plots show APC fluorescence intensity. The dashed line represents the fluorescence signal from a no-primary antibody control, while the solid line with grey fill indicates cells stained with the oligonucleotide-conjugated 1 B4A1 primaryantibody. The barcoded antibody retained specific reactivity against Whitlow-expressing cells, demonstrating selective binding to FMC63-Whitlow CAR T cells and no detectable binding to untransduced T cells. This barcoded antibody has been incorporated into custom antibody panels for the identification and characterization of CAR T cells in sequencing-based multiomic platforms (e.g., cellular indexing of transcriptomes and epitopes (CITE)-seq).

[0029] FIGs. 21 A, 21 B. Anti-Whitlow mAbs can be leveraged for CAR T cell manufacturing procedures Enrichment of CAR expressing T cells using 1 B4A1. (21 A) FMC63-Whitlow CAR expressing T cells were admixed with untransduced T cells at different ratios (left panel), stained with APC-labeled 1 B4A1 and sorted using anti-APC magnetic beads-based column purification or flow-based fluorescent activated cell sorting (FACS). Cells were expanded for 48 hours and then tested for expression of the tEGFR transduction marker by flow cytometry. The middle panel represents histograms of tEGFR fluorescence intensity of cell sorted by magnetic beads (middle panel) or FACS (right panel) and untransduced cells were measured for a negative control. (21 B) Purity of CAR T cell product achieved by enrichment with 1 B4A1 using magnetic beads or FACS.

[0030] FIG. 22. Additional screening for activation of CAR T cells expressing an scFv other than FMC63 using the Jurkat nur77-GFP reporter cell line. Jurkat nur77-GFP cells were transduced to express the CD33 targeting 1 H7 construct containing either a G4S or Whitlow linker and then cultured with a PMA-lonomycin, a CD33 expressing cell line (ML1) the 1C3C3 antibody in plate bound format or controls as displayed.

[0031] FIG. 23. CAR T cells can remain responsive to target-expressing cells despite the presence of anti-Whitlow antibodies. A Jurkat reporter cell line expressing Nur77-driven GFP was transduced with an FMC63-Whitlow CAR construct and co-cultured for 4 hours with CD19- expressing K562 (K562-CD19) target cells in the presence of 0, 25, or 50 pg / mL of anti-Whitlow monoclonal antibodies. GFP expression, measured by flow cytometry, served as a readout for T cell activation. These data suggest that CAR-expressing T cells may still be activated by targetexpressing cells even in the presence of bivalent anti-Whitlow antibodies.

[0032] FIGs. 24A-24D. Engineered bispecific molecules enable redirection of the cytotoxic activity of CAR T cells. Redirecting molecules engineered with the variable domains of anti-Whitlow antibodies can be designed to target antigens distinct from the cognate target of a CAR. As one example, the bispecific molecule 1 B4A1-CD33 was constructed by fusing the VH and VL domains of the anti-Whitlow antibody 1 B4A1 into a scFv, which was then linked to the CD33-targeting 1 H7 scFv to generate a bispecific engager. These bispecific engagers were tested using Whitlowcontaining FMC63 CAR T cells, which naturally recognize CD19, and human cell lines expressing varying levels of wild-type (WT) CD33 (24A). (24B) FMC63- Whitlow CAR T cells were co-culturedwith fluorescently-labeled CD33+target cells (ML1 , HL60, MOLM14) at an effector-to-target (E:T) ratio of 1 :2 in the presence of varying concentrations of the bispecific engager for 24 hours. (24C) A CD33-knockout (KO) ML1 cell line was included as a control for non-specific killing. Cytotoxicity was measured by flow cytometry based on the proportion of viable (Annexin V", 7-AAD-) CD3“ GFP+target cells, normalized to a no-treatment control. Dose-dependent killing of CD33+target cells was observed across all three cell lines, while no increased killing was seen in the CD33 KO ML1 control. (24D) Additionally, no reduction in CD19-specific killing of the K562-CD19 cell line was observed, indicating that the engager does not significantly impair the ability of CAR T cells to eliminate their cognate target.

[0033] FIGs. 25, 25B. Blocking of antigen binding by CAR T cells using anti-Whitlow antibodies. CD19-targeted FMC63-Whitlow CAR T cells were pre-incubated with unlabeled anti-Whitlow antibodies at the indicated concentrations and then stained with biotinylated recombinant CD19, followed by allophycocyanin (APC)-conjugated streptavidin to assess CD19 binding in the presence of blocking antibodies. (25A) Anti-Whitlow antibodies in their native bivalent format showed varying degrees of dose-dependent inhibition of CAR binding to its cognate antigen, as indicated by reduced CD19 fluorescence intensity compared to a no-blocking antibody control. (25B) Representative histograms illustrate the reduction in CD19 binding: the dashed line represents fluorescence from cells stained without anti-Whitlow antibodies, and the solid line with dark grey fill shows fluorescence from cells pretreated with 20 pg / mL of anti-Whitlow antibody.

[0034] FIG. 26. Variable heavy (VH) and variable light (VL) chain amino acid sequences of antibody clones: 1B3b, 1H5b, 1G2G2, 1B4A1, and 1C3C3; and FMC63.

[0035] FIG. 27. Variable heavy (VH) and variable light (VL) chain nucleic acid sequences encoding antibody clones: 1 B3b, 1H5b, 1G2G2, 1 B4A1 , and 1C3C3.

[0036] FIG. 28. Protein sequences of antibody Fab regions (heavy (HC) and light (LC) chains) for antibody clones: 1B3b, 1H5b, 1G2G2, 1B4A1, and 1C3C3.

[0037] FIG. 29. DNA sequences of antibody Fab regions (HC and LC) for antibody clones: 1 B3b, 1 H5b, 1G2G2, 1B4A1, and 1C3C3.

[0038] FIG. 30. Exemplary heavy chain and light chain signal peptides.DETAILED DESCRIPTION

[0039] In recent years, advances in medical technology have led to the emerging use of immunotherapies to treat different types of illnesses and diseases, including various forms of cancer. Significant progress, for example, has been made in genetically engineering cells of the immune system to target and kill unwanted cell types (e.g., cancer cells). Many of these immune cells are T cells that have been genetically engineered to express a recombinant receptor, suchas a chimeric antigen receptor (CAR). CARs are proteins including several distinct subcomponents that allow the genetically modified T cells to recognize and kill unwanted cell types. CARs can be engineered into a variety of structures with different subcomponents. Conventionally, CAR subcomponents include at least an extracellular component and an intracellular component expressed as a single protein or assembling into a functional unit. The extracellular component includes a binding domain that specifically binds a marker (e.g., an antigen) that is preferentially present on the surface of unwanted cells. Oftentimes, the binding domain is an scFv including a variable heavy chain conjugated to a variable light chain using a linker, such as a Whitlow linker. When the binding domain binds such markers, the intracellular component signals the T cell to destroy the bound cell.

[0040] Identification of CAR-T cells is a key step in research and diagnostic applications to establish factors associated with success and failure of CAR-T cell immunotherapy. Despite this, most CAR-binding antibodies are used primarily for flow cytometry, because of the difficulty in generating reagents that function in formalin-fixed paraffin-embedded (FFPE) tissues. Anti-CAR antibodies that are suitable for use in multiplex immunohistochemistry (mIHC) imaging of FFPE archival or frozen tissue sections are needed.

[0041] Furthermore, because recombinant proteins, such as CAR T cells are largely made of proteins or fragments thereof that are found in the body, it can be difficult to differentiate cells genetically modified to express the recombinant protein from the native proteins when detection is performed in vivo or in situ.

[0042] Finally, although CAR T cells are approved for treatment of select malignancies, there remain challenges in monitoring CAR T cell activity, persistence, and trafficking after administration. Overall, there is a need for tools to modulate the activity of CAR T cells in vivio and to detect CAR within fixed biopsy tissues, fresh biopsy tissues, and in vivo for research, diagnostic, and therapeutic purposes.

[0043] While the Background and Summary of this disclosure focus scFv, CAR, and immunotherapy, the disclosure is not so limited. The binding domains disclosed herein can be used to interact with any Whitlow-linker containing molecule and / or any cell type expressing a Whitlow-linker containing molecule.

[0044] The current disclosure provides binding domains that bind the artificial Whitlow linker and subsequences thereof. The Whitlow linker is a peptide linker defined by the amino acid sequence of SEQ ID NO: 1 and is incorporated in many recombinant proteins. In the cellular immunotherapy field, the linker is often present in single chain variable fragments (scFvs) that are used to endow antigen-targeting specificity in many CAR constructs and T cell engagers (TCEs). If a molecule(e.g., CAR or bispecific antibody) includes the Whitlow linker, the binding domains provided herein can be useful in detection of said molecules. Research, diagnostic, and / or therapeutic uses of these binding domains are provided herein. For example, the disclosed anti-Whitlow linker binding domains can be used as antibodies for detection, as multi-specific antibodies for detection or therapy, as antibody conjugates to bring payloads to the Whitlow linker-containing molecule, or as part of a recombinant receptor (e.g., CAR).

[0045] In particular embodiments, a binding domain that binds the Whitlow linker (e.g., an antiWhitlow linker antibody) includes binding domains from the following antibody clones: 1 B3b, 1 H5b, 1G2G2, 1 B4A1 , or 1C3C3. These five antibody clones demonstrated superior staining index values compared to a commercial anti-Whitlow linker antibody (Cell Signaling T echnologies clone E3U7Q).

[0046] In particular embodiments, a binding domain that binds the Whitlow linker includes a heavy chain sequence and associated light chain sequence set forth in FIG. 28. In particular embodiments, a binding domain that binds the Whitlow linker is encoded by a heavy chain sequence and associated light chain sequence set forth in FIG. 29.

[0047] In particular embodiments, a humanized binding domain from 1 B3b includes a variable heavy chain including the sequence of SEQ ID NO: 218 and a variable light chain including the sequence of SEQ ID NO: 225. In particular embodiments, a humanized binding domain from 1B3b includes a variable heavy chain including the sequence of SEQ ID NO: 219 and a variable light chain including the sequence of SEQ ID NO: 226. In particular embodiments, a humanized binding domain from 1 B3b includes a variable heavy chain including the sequence of SEQ ID NO: 220 and a variable light chain including the sequence of SEQ ID NO: 227. In particular embodiments, a humanized binding domain from 1B3b includes a variable heavy chain including the sequence of SEQ ID NO: 220 and a variable light chain including the sequence of SEQ ID NO: 225. In particular embodiments, a humanized binding domain from 1 B3b includes a variable heavy chain including the sequence of SEQ ID NO: 221 and a variable light chain including the sequence of SEQ ID NO: 225. In particular embodiments, a humanized binding domain from 1B3b includes a variable heavy chain including the sequence of SEQ ID NO: 222 and a variable light chain including the sequence of SEQ ID NO: 225. In particular embodiments, a humanized binding domain from 1 B3b includes a variable heavy chain including the sequence of SEQ ID NO: 223 and a variable light chain including the sequence of SEQ ID NO: 225. In particular embodiments, a humanized binding domain from 1B3b includes a variable heavy chain including the sequence of SEQ ID NO: 224 and a variable light chain including the sequence of SEQ ID NO: 225.

[0048] In particular embodiments, a binding domain from 1 B3b includes a variable heavy chain including the sequence of SEQ I D NO: 2 and a variable light chain including the sequence of SEQ ID NO: 3. In particular embodiments, a binding domain from 1 B3b includes a variable heavy chain encoded by the sequence of SEQ ID NO: 12 and a variable light chain encoded the sequence of SEQ ID NO: 13.

[0049] In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 228 and a variable light chain including the sequence of SEQ ID NO: 235. In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 229 and a variable light chain including the sequence of SEQ ID NO: 235. In particular embodiments, a humanized binding domain from 1H5b includes a variable heavy chain including the sequence of SEQ ID NO: 230 and a variable light chain including the sequence of SEQ ID NO: 235. In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 231 and a variable light chain including the sequence of SEQ ID NO: 235. In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 232 and a variable light chain including the sequence of SEQ ID NO: 236. In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 233 and a variable light chain including the sequence of SEQ ID NO: 235. In particular embodiments, a humanized binding domain from 1H5b includes a variable heavy chain including the sequence of SEQ ID NO: 234 and a variable light chain including the sequence of SEQ ID NO: 237. In particular embodiments, a humanized binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ ID NO: 234 and a variable light chain including the sequence of SEQ ID NO: 235.

[0050] In particular embodiments, a binding domain from 1 H5b includes a variable heavy chain including the sequence of SEQ I D NO: 4 and a variable light chain including the sequence of SEQ ID NO: 5. In particular embodiments, a binding domain from 1 H5b includes a variable heavy chain encoded the sequence of SEQ ID NO: 14 and a variable light chain encoded the sequence of SEQ ID NO: 15.

[0051] In particular embodiments, a binding domain from 1G2G2 includes a variable heavy chain including the sequence of SEQ I D NO: 6 and a variable light chain including the sequence of SEQ ID NO: 5. In particular embodiments, a binding domain from 1G2G2 includes a variable heavy chain encoded the sequence of SEQ ID NO: 16 and a variable light chain encoded the sequence of SEQ ID NO: 15.

[0052] In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 238 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 239 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 240 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 241 and a variable light chain including the sequence of SEQ ID NO: 246. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 241 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 242 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 243 and a variable light chain including the sequence of SEQ ID NO: 245. In particular embodiments, a humanized binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ ID NO: 244 and a variable light chain including the sequence of SEQ ID NO: 245.

[0053] In particular embodiments, a binding domain from 1 B4A1 includes a variable heavy chain including the sequence of SEQ I D NO: 8 and a variable light chain including the sequence of SEQ ID NO: 3. In particular embodiments, a binding domain from 1 B4A1 includes a variable heavy chain encoded the sequence of SEQ I D NO: 18 and a variable light chain encoded the sequence of SEQ ID NO: 13.

[0054] In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 247 and a variable light chain including the sequence of SEQ ID NO: 254. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 248 and a variable light chain including the sequence of SEQ ID NO: 254. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 249 and a variable light chain including the sequence of SEQ ID NO: 255. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 250 and a variable light chain including the sequence of SEQ ID NO: 256. In particular embodiments, a humanized binding domain from 1C3C3 includesa variable heavy chain including the sequence of SEQ ID NO: 250 and a variable light chain including the sequence of SEQ ID NO: 254. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 251 and a variable light chain including the sequence of SEQ ID NO: 254. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 252 and a variable light chain including the sequence of SEQ ID NO: 254. In particular embodiments, a humanized binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 253 and a variable light chain including the sequence of SEQ ID NO: 254.

[0055] In particular embodiments, a binding domain from 1C3C3 includes a variable heavy chain including the sequence of SEQ ID NO: 10 and a variable light chain including the sequence of SEQ ID NO: 3. In particular embodiments, a binding domain from 1C3C3 includes a variable heavy chain encoded the sequence of SEQ ID NO: 20 and a variable light chain encoded the sequence of SEQ ID NO: 13.

[0056] In particular embodiments, the variable heavy chain and variable light chain are connected by a linker to form an scFv. The linker can include any linker known in the art or described herein. In particular embodiments, the linker includes the sequence as set forth in SEQ ID NO: 126. Referring to the binding domains provided herein, the following CDR sets are provided. A CDR set refers to 3 heavy chain CDRs and 3 light chain CDRs that together result in binding to the Whitlow linker.

[0057] Table 1. CDR sequences for humanized 1B3b, 1 H5b, 1 B4A1 , and 1C3C3.

[0058] Table 2. CDR sequences for 1B3b, 1H5b, 1G2G2, 1B4A1, and 1C3C3.

[0059] CDR predictions were generated using ABodyBuilder2 within the SAbPrep program opig.stats.ox.ac.uk / webapps / newsabdab / sabpred / ).

[0060] The present disclosure provides these binding domain CDR sets, variable light chain sequences, variable heavy chain sequences, cells expressing the binding domains, nucleic acid sequences encoding the binding domains, manufacturing methods, and methods of use, among other aspects of the disclosure.

[0061] The binding domains disclosed herein can be engineered into numerous formats for research, detection, and / or treatment purposes. For example, binding domains can be used in any format or method known for antibodies in general. Example uses include for selection or enrichment, imaging, spatial mapping, sorting, quantification, in vivo stimulation, in vivo blocking, or immunotherapy.

[0062] In particular embodiments, binding domains derived from 1 B3b, 1H5b, 1G2G2, 1 B4A1 , or 1C3C3 can be used in flow cytometry, immunohistochemistry, or modulation (e.g., activation, inhibition, or redirection) of Whitlow linker-containing molecules (also referred to herein as Whitlow-containing molecules).

[0063] In particular embodiments, binding domains derived from 1 B3b, 1H5b, 1G2G2, 1 B4A1 , or 1C3C3 can be used in flow cytometry.

[0064] In particular embodiments, binding domains derived from 1B3b, 1G2G2, 1 B4A1 , or 1C3C3 can be used in immunohistochemistry assays.

[0065] In particular embodiments, binding domains derived from 1 B3b, 1H5b, 1G2G2, 1 B4A1 , or 1C3C3 can be used to modulate the activity of Whitlow linker-containing molecules (e.g., CAR). In particular embodiments, binding domains derived from 1C3C3 can be used to strongly stimulate Whitlow linker-containing molecules (e.g., CAR). In particular embodiments, stimulation of Whitlow-expressing CAR T cells includes cytokine production such as production of interferon-gamma (IFN-y) and / or tumor necrosis factor-alpha (TNF-a). In particular embodiments, stimulation of Whitlow-expressing CAR T cells includes activation of CAR T cells. In particular embodiments, activation includes increase in CD69 expression. In particular embodiments, stimulation of Whitlow-expressing CAR T cells includes proliferation. In particular embodiments, proliferation includes an increase in Ki67 expression.

[0066] In particular embodiments, binding domains derived from 1 B3b, 1H5b, 1G2G2, or 1 B4A1 have lower stimulation or no stimulation of Whitlow linker-containing molecules (e.g., CAR), particularly compared to 1C3C3. In particular embodiments, select assays or therapies require or prefer an antibody having low stimulation or no stimulation of Whitlow linker-containing molecules.

[0067] A Whitlow-containing molecule refers to any molecule that includes the Whitlow linker or a subsequence thereof, wherein the subsequence is bound by a binding domain disclosed herein. In particular embodiments, the subsequence of the Whitlow linker includes at least 10 consecutive amino acids of the sequence of SEQ ID NO: 1. In particular embodiments, the subsequence of the Whitlow linker includes at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 consecutive amino acids of the sequence of SEQ ID NO: 1. In particular embodiments, an anti-Whitlow binding domain binds a Whitlow linker-containing molecule. In particular embodiments, an anti-Whitlow binding domain binds a cell expressing a Whitlow linkercontaining molecule. Examples of Whitlow linker-containing molecules include antibodies (e.g., scFv) and recombinant receptors (e.g., CAR). In particular embodiments, a Whitlow linkercontaining molecule can include a Whitlow linker as a linker or as a separate domain such as a binding domain.

[0068] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Antibodies; (ii) Antibody Variants; (iii) Multi-Domain Binding Molecules; (iv) Expression of Recombinant Proteins; (v) Antibody Conjugates; (vi) Recombinant Receptors; (vii) Cells; (viii) Cell Sample Collection and Enrichment; (ix) Genetic Engineering Techniques; (x) Nanoparticles; (xi) Compositions or Formulations for Administration; (xii) Ex Vivo Methods of Use; (xiii) In Vivo Methods of Use; (xiv) Kits; (xv) Exemplary Embodiments; (xvi) Experimental Examples; and (xvii) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0069] (i) Antibodies. Binding domain disclosed herein can be in the form of antibodies or fragments thereof. Conventional 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 amino-terminal 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 ofrelatively 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 are aligned 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.

[0070] The assignment of amino acids to each domain can be in accordance with 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 et al., 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)), 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)), 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 et al., J Mol Biol. 406(2) :228-256 (2011), “A new clustering of antibody CDR loop conformations”), or other numbering schemes.

[0071] 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 ABodyBuilder2 and Paratome can also be used to determine CDR sequences.

[0072] The carboxy-terminal portion of each chain of a naturally occurring antibody 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); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B-cell receptors); and B-cell activation.

[0073] Within full-length light and heavy chains, the variable and constant regions are joined bya “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)).

[0074] The constant region of an antibody may be of any suitable immunoglobulin subtype or subclass. In particular embodiments, a constant region includes a light chain constant region and a heavy chain constant region.

[0075] Human light chains are classified as kappa (IgK) and lambda (IgA) light chains. In particular embodiments, a human IgK Fc region includes the sequence: TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 113). In particular embodiments, a human IgA Fc region includes the sequence: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 114).

[0076] In particular embodiments, the light chain includes an IgK Fc region.

[0077] 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. IgG causes opsonization and cellular cytotoxicity and crosses the placenta, IgA functions on the mucosal surface, IgM is most effective in complement fixation, and IgE mediates degranulation of mast cells and basophils. Resting B cells, which are immunocompetent but not yet activated, express IgM and IgD. Once activated and committed to secrete antibodies, these B cells can express any of the five isotypes. The heavy chain isotypes of IgG, IgA, IgM, IgD and IgE are respectively designated the y, a, p, 5, and £ chains.

[0078] In particular embodiments, a human lgG1 Fc region includes the sequence: THTCPPCPAPEFFGGPSVFFFPPKPKDTFMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVETVFHQDWENGKEYKCKVSNKAFPVPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115).

[0079] In particular embodiments, a human lgG2 Fc region includes the amino acid sequence: PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 116). In particular embodiments, the lgG2 Fc region includes an lgG2a Fc region.

[0080] In particular embodiments, a human lgG3 Fc region includes the amino acid sequence: PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPR EEQFNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQ QGNIFSCSVMHEALHNRFTQKSLSLSPGK (SEQ ID NO: 117).

[0081] In particular embodiments, a human lgG4 Fc region includes the amino acid sequence: PAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQ EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 118).

[0082] The human IgD constant region typically includes the amino acid sequence: APTKAPDVFPIISGCRHPKDNSPVVLACLITGYHPTSVTVTWYMGTQSQPQRTFPEIQRRDSYY MTSSQLSTPLQQWRQGEYKCVVQHTASKSKKEIFRWPESPKAQASSVPTAQPQAEGSLAKAT TAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFT CFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTL NHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMWLEDQRE VNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSY VTDHGPMK (SEQ ID NO: 119).

[0083] The human IgE constant region typically includes the amino acid sequence: ASTQSPSVFPLTRCCKNIPSNATSVTLGCLATGYFPEPVMVTWDTGSLNGTTMTLPATTLTLSG HYATISLLTVSGAWAKQMFTCRVAHTPSSTDWVDNKTFSVCSRDFTPPTVKILQSSCDGGGHF PPTIQLLCLVSGYTPGTINITWLEDGQVMDVDLSTASTTQEGELASTQSELTLSQKHWLSDRTY TCQVTYQGHTFEDSTKKCADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTW SRASGKPVNHSTRKEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTS GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTK GSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK (SEQ ID NO: 120).

[0084] IgA and IgM constant regions are described elsewhere herein.

[0085] 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 thatcontact the variable region of an antibody.

[0086] In general, an epitope denotes the binding site on a viral peptide, bacterial peptide, cancer protein, or other antigen bound by a corresponding variable region of an antibody. In particular embodiments, particular embodiments, the epitope includes the binding site on the Whitlow linker bound by a corresponding variable region of the binding domains disclosed herein. The variable region either binds to a linear epitope, (e.g., an epitope including a stretch of 5 to 12 consecutive amino acids), or the variable region binds to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target. Three-dimensional epitopes recognized by a variable region, e.g., by the epitope recognition site or paratope of an antibody or antibody fragment, can be thought of as three-dimensional surface features of an epitope molecule. These features fit precisely (in)to the corresponding binding site of the variable region and thereby binding between the variable region and its target protein (more generally, antigen) is facilitated. In particular embodiments, an epitope can be considered to have two levels: (i) the “covered patch” which can be thought of as the shadow an antibody variable region would cast on the antigen to which it binds; and (ii) the individual participating side chains and backbone residues that facilitate binding. Binding is then due to the aggregate of ionic interactions, hydrogen bonds, and hydrophobic interactions.

[0087] Epitopes of the currently disclosed binding domain (that is, epitopes to which the antibodies bind) can be found on a Whitlow linker (e.g., Whitlow linker of an scFv as part of a chimeric antigen receptor).

[0088] In particular embodiments, Kd can be characterized using BIAcore. For example, in particular embodiments, Kd can be measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25°C with immobilized antigen CM5 chips at 10 response units (RU).

[0089] In particular embodiments, the term “antibody” refers to naturally occurring antibodies (having two full-length heavy chains and two full-length light chains as described above), and variants, derivatives, and fragments thereof, examples of which are described below. In particular embodiments, antibodies include monoclonal antibodies (mAbs), human or humanized antibodies, multi-specific antibodies, bi-specific antibodies, tri-specific antibodies, tetra-specific antibodies, penta-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 the antibodies disclosed herein.

[0090] 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.

[0091] 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-encoding sequences.

[0092] 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 VL or VH sequences 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 V , the subgroup is subgroup kappa I as in Kabat et al. (supra). In particular embodiments, for the VH, the subgroup is subgroup III as in Kabat et al. (supra).

[0093] A chimeric antibody is a molecule made up of domains from different species. In a chimeric antibody, at least a portion of the constant region of an antibody of a first species is replaced with at least a portion of the constant region from a second species. For example, a chimeric antibody can include a murine variable chain region and a human constant chain region.

[0094] In certain examples, a chimeric antibody is an antibody having some or all CDRs and variable region framework sequences entirely or substantially from a non-human “donor” antibody; and constant regions, if present, entirely or substantially from human antibody sequences. Similarly, a chimeric heavy chain has at least one, two and usually all three CDRs and variable heavy chain framework sequence entirely or substantially from a donor antibody heavy chain; and heavy chain constant region, if present, substantially from human heavy chainconstant region sequences. Similarly, a chimeric light chain has at least one, two and usually all three CDRs and light chain variable region framework sequence entirely or substantially from a donor antibody light chain; and a light chain constant region, if present, substantially from human light chain constant region sequences. Other than nanobodies and diabodies, a chimeric antibody typically includes a non-human donor heavy chain variable region and a non-human donor light chain variable region. A CDR in a chimeric antibody is substantially from or substantially identical to a corresponding CDR in a non-human antibody with at least 60%, 85%, 90%, 95% or 100% of corresponding residues are identical between the respective CDRs. In particular embodiments, a CDR in a chimeric antibody is substantially from or substantially identical to a corresponding CDR in a non-human antibody when there are no more than 3 conservative amino acid substitutions in each CDR. In certain examples, the variable region framework sequences of an antibody chain are substantially from a non-human donor variable region framework sequence when at least 70%, 80%, 85%, 90%, 95% or 100% of corresponding residues are identical to the non-human variable framework sequence. The constant region of an antibody chain are substantially from a human constant region sequence when at least 70%, 80%, 85%, 90%, 95% or 100% of corresponding residues are identical to the human constant region sequence.

[0095] The binding domains described herein can be readily humanized using standard methods or methods described herein. For example, the binding domain can be humanized using recombinant methods to reduce its immunogenicity a human. A humanized antibody is an engineered antibody in which the CDRs from a non-human donor antibody are grafted into human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881 ,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. In particular embodiments, a humanized antibody includes humanized variable chain regions and human constant regions.

[0096] Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a non-human donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a variable heavy chain framework sequence and heavy chain constant region, if present, substantially from human variable heavy chain framework and human heavy chain constant region sequences. Similarly, a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain,and a variable light chain framework sequence and light chain constant region, if present, substantially from human variable light chain framework and human light chain constant region sequences. Other than nanobodies and diabodies, a humanized antibody typically includes a humanized heavy chain and a humanized light chain. A CDR in a humanized or human antibody is substantially from or substantially identical to a corresponding CDR in a non-human antibody with at least 60%, 85%, 90%, 95% or 100% of corresponding residues are identical between the respective CDRs. In particular embodiments, a CDR in a humanized antibody or human antibody is substantially from or substantially identical to a corresponding CDR in a non-human antibody when there are no more than 3 conservative amino acid substitutions in each CDR. The variable region framework sequences of an antibody chain or the constant region of an antibody are substantially from a human variable region framework sequence or human constant region respectively when at least 70%, 80%, 85%, 90%, 95% or 100% of corresponding residues are identical to reference human sequences.

[0097] Chimeric and 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”); Kim, et al., PLoS One 6(5):e19867, 2011 (describing production and characterization of chimeric monoclonal antibodies); Dall'Acqua et al., Methods 36:43- 60,2005 (describing “FR shuffling”); and Osbourn et al., 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.

[0098] In humanized antibodies, certain amino acids from the human variable region framework residues can be selected for substitution based on their possible influence on CDR conformation and / or binding to antigen. Investigation of such possible influences is by modeling, examination of the characteristics of the amino acids at particular locations, or empirical observation of the effects of substitution or mutagenesis of particular amino acids. 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).

[0099] The choice of constant region can depend, in part, whether antibody-dependent cell- mediated cytotoxicity, antibody dependent cellular phagocytosis and / or complement dependent cytotoxicity are desired. For example, human isotopes lgG1 and lgG3 have strong complementdependent cytotoxicity, human isotype lgG2 has weak complement-dependent cytotoxicity and human lgG4 lacks complement-dependent cytotoxicity. Human lgG1 and lgG3 also induce stronger cell mediated effector functions than human lgG2 and lgG4.

[0100] Although chimeric and humanized antibodies often incorporate all six CDRs from a nonhuman antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5) CDRs from a non-human antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091 , 1999; Tamura et al, Journal of Immunology, 164:1432-1441 , 2000).

[0101] (ii) Antibody Variants. Binding domains disclosed herein can be utilized to prepare various forms of relevant antibodies. 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.

[0102] In particular embodiments, an antibody fragment is used. An “antibody fragment” denotes a portion of a 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.

[0103] A single chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins connected with a short linker peptide. Fv fragments include the VL and VH domains of a single arm of an antibody but lack the constant regions. Although the two domains of the Fv fragment, V 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.

[0104] Linker sequences that are used to connect the VL and VH of an scFv 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. Example Gly-Ser linker sequences includes 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: 121), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 122), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 123), and (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 124). In particular embodiments, the linker is (Gly4Ser)4(SEQ ID NO: 125), (Gly4Ser)3(SEQ ID NO: 126), (Gly4Ser)2(SEQ ID NO: 127), (Gly4Ser)i (SEQ ID NO: 128), (Gly3Ser)2(SEQ ID NO: 129), (Gly3Ser)i (SEQ ID NO: 130), (Gly2Ser)2(SEQ ID NO: 131) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 132), GGSGGGSGSG (SEQ ID NO: 133), or GGSGGGSG (SEQ ID NO: 134). Linker sequences of scFv are also commonly the Whitlow linker (GSTSGSGKPGSGEGSTKG; SEQ ID NO: 1).

[0105] 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 et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0106] A Fab fragment is a monovalent antibody fragment including VL, VH, CL and CH1 domains. A F(ab')2fragment is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region. For discussion of Fab and F(ab')2fragments 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; WQ1993 / 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 , 2011)) 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. In particular embodiments, the binding domains disclosed herein are expressed as a Fab.

[0107] 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 an Fc region sequence (e.g., a lgG1 , 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.

[0108] In particular embodiments, variants (including Fc variants) have been modified from a reference sequence to produce an administration benefit. Exemplary administration benefits can include reduced susceptibility to proteolysis, reduced susceptibility to oxidation, altered binding affinities, reduced immunogenicity; increased immunogenicity, and / or extended half-life. While the disclosure below describes these modifications in terms of their application to antibodies, when applicable to another particular anti-Whitlow binding domain format (e.g., bispecific antibodies), the modifications can also be applied to these other formats.

[0109] 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 C1q 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.

[0110] 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 (EU 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.

[0111] 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 fucoseis 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 / 085119; W02003 / 084570; US2003 / 0115614; US2003 / 0157108; US2004 / 0093621 ; US2004 / 0110704; US2004 / 0132140; US2004 / 0110282; 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).

[0112] 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, 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.

[0113] 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 anN-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.

[0114] 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 site-specific PEGylation of acetyl-phenylalanine residues can be performed.

[0115] 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 al., 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 et al., J. Biol. Chem. 252: 3582, 1977), and increase protein solubility as described, for example, in PCT Publication No. WO 92 / 16221).

[0116] 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.

[0117] 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 et al., 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 a bispecific antibody as described herein.

[0118] In particular embodiments, one or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be missing or derivatized in a proportion or all of the molecules. Substitutions can be made in the constant regions to reduce or increase effector function such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., US Patent No. 5,624,821 ; Tso et al., US Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). For additional information regarding Fc mutations that create administration benefits, see Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Frontiers in Immunology (2019) Vol. 10, Article 1296.

[0119] (iii) Multi-Domain Binding Molecules. Binding domains disclosed herein can be expressed on multi-domain binding molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes an anti- Whitlow binding domain 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 the Whitlow linker. In particular embodiments, all of the binding domains of a multi-domain binding molecule bind the Whitlow linker.

[0120] Multi-domain binding molecules include bispecific antibodies which bind at least two epitopes wherein at least one of the epitopes is located on the Whitlow linker. Multi-domainbinding molecules include trispecific antibodies which binds at least 3 epitopes, wherein at least one of the epitopes is located on the Whitlow linker, and so on.

[0121] Bispecific antibodies can be prepared utilizing antibody fragments (for example, F(ab')2 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.

[0122] 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.

[0123] Two antibodies or fragments thereof can be linked through a linker to form a bispecific antibody. In particular embodiments, the two antibodies or fragments thereof can bind the same epitope or different epitopes. 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.

[0124] Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). Example Gly-Ser linkers are described elsewhere herein. In particular embodiments, the multi-domain binding molecule does not include a Whitlow linker.

[0125] 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.

[0126] 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 linkeris 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).

[0127] In particular embodiments, binding domains disclosed herein can be used to create bi-, tri, (or more) specific immune cell engaging molecules. Immune cell engaging molecules have at least one binding domain that binds a receptor on an immune cell and modulates the activity of the immune cell. Examples of multi-domain immune cell engaging molecules include those which bind both an immune cell (e.g., T-cell or NK-cells) epitope and Whitlow linker, with the goal of bringing immune cells to Whitlow linker-containing molecules or cells expressing these target molecules, for example, in order to destroy them. See, for example, US 2008 / 0145362. Such molecules are referred to herein as immune-activating multi-specifics or l-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, B-cells, T-cells, natural killer (NK) cells, and macrophages which are discussed in more detail herein.

[0128] Herein, a target molecule refers to any molecule including a Whitlow linker to which the binding domains disclosed herein can bind. The target molecule can include the Whitlow linker itself or any molecule or cell expressing a Whitlow linker as part of a recombinant protein, such as an scFv or CAR.

[0129] In particular embodiments, l-AMS disclosed herein can bind 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 (CD137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3.

[0130] In particular embodiments, the CD3 binding domain (e.g., scFv) is derived from the OKT3 antibody (the same as the one utilized in blinatumomab), otelixizumab, teplizumab, visilizumab, 20G6-F3, 4B4-D7, 4E7-C9, 18F5-H10, or TR66. The OKT3 antibody is described in detail in U.S. Patent No. 5,929,212.

[0131] In particular embodiments, the OKT3 binding domain includes a light chain variable region ofQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFR GSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINR (SEQ ID NO: 135) and a heavy chain variable region of QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYN QKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVTSS (SEQ ID NO: 136).

[0132] In particular embodiments, the binding domain includes a light chain variable region including a CDRL1 sequence including SASSSVSYMN (SEQ ID NO: 137), a CDRL2 sequence including DTSKLAS (SEQ ID NO: 138), a CDRL3 sequence including QQWSSNPFTF (SEQ ID NO: 139), a CDRH1 sequence including RYTMH (SEQ ID NO: 140), a CDRH2 sequence including YINPSRGYTNYNQKFKD (SEQ ID NO: 141), and a CDRH3 sequence including YYDDHYCL (SEQ ID NO: 142). In particular embodiments, the binding domain is human or humanized. For more information regarding binding domains that bind CD3, see U.S. Pat. No. 8785604, PCT / US 17 / 42264, and / or WQ02051871.

[0133] In particular embodiments, a binding domain is “derived from” a reference antibody when the binding domain includes the CDRs of the reference antibody, according to a known numbering scheme (e.g., Kabat, Chothia, Martin, or others).

[0134] CD28 binds to B7-1 (CD80) and B7-2 (CD86) and is the most potent of the known costimulatory molecules (June et al., Immunol. Today 15:321 , 1994; Linsley et al., Ann. Rev. Immunol. 11 :191 , 1993). In particular embodiments, the CD28 binding domain is derived from TGN1412, CD80, CD86 or the 9D7 antibody. Additional antibodies that bind CD28 include 9.3, KOLT-2, 15E8, 248.23.2, and EX5.3D10.

[0135] In particular embodiments, the binding domain that binds CD28 is derived from TGN-1412 and / or theralizumab. In particular embodiments, the binding domain includes a light chain variable region of DIQMTQSPSSLSASVGDRVTITCKTNENIYSNLAWYQQKDGKSPQLLIYAATHLVEGVPSRFSG SGSGTQYSLTISSLQPEDFGNYYCQHFWGTPXTFGGGTKLEI KR, wherein X=C, A, or N. (SEQ ID NO: 143) and a heavy chain variable region of VQLQQSGAELKKPGASVKVSCKASGYTFTEYIIHWIKLRSGQGLEWIGWFYPGSNDIQYNAQF KGKATLTADKSSSTVYMELTGLTPEDSAVYFCARRDDFSGYDALPYWGQGTLVTVSA (SEQ ID NO: 144). In particular embodiments, the binding domain includes a light chain variable region including a CDRL1 sequence including HASQNIYVWLN (SEQ ID NO: 145), a CDRL2 sequence including KASNLHT (SEQ ID NO: 146), a CDRL3 sequence including QQGQTYPYT (SEQ ID NO: 147), a CDRH1 sequence including SYYIH (SEQ ID NO: 148), a CDRH2 sequence including CIYPGNVNTNYNEKFKD (SEQ ID NO: 149), and a CDRH3 sequence including SHYGLDWNFDV (SEQ ID NO: 150). In particular embodiments, the binding domain is human or humanized. For more information regarding binding domains that bind CD28, see U.S. Pat. No. US8785604 and / or WQ02051871.

[0136] Activated T-cells express 4-1 BB (CD137). In particular embodiments, the 4-1BB binding domain includes a light chain variable region including a CDRL1 sequence including RASQSVS (SEQ ID NO: 151), a CDRL2 sequence including ASNRAT (SEQ ID NO: 152), and a CDRL3 sequence including QRSNWPPALT (SEQ ID NO: 153) and a heavy chain variable region including a CDRH1 sequence including YYWS (SEQ ID NO: 154), a CDRH2 sequence including INH, and a CDRH3 sequence including YGPGNYDWYFDL (SEQ ID NO: 155).

[0137] 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.

[0138] 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.

[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 1D11 , 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 certain examples, multi-domain binding molecules can be used to increase the avidity of and / or re-target a cell extracellularly expressing a Whitlow-containing molecule linked to a binding domain that binds a first antigen. For example, to increase avidity, a multi-domain binding molecule would include an anti-Whitlow binding domain disclosed herein linked to one or more binding domains that bind the same first antigen. To re-target a cell, a multi-domain binding molecule would include an anti-Whitlow binding domain disclosed herein linked to one or more binding domains that bind an antigen that is different from the first antigen. To increase avidity and re-target a cell, a multi-domain binding molecule would include an anti-Whitlow binding domain disclosed herein linked to one or more binding domains that bind the same first antigen and one or more binding domains that bind an antigen that is different from the first antigen.

[0141] Examples cancer antigens include A33; BAGE; Bcl-2; p-catenin; CA125; CA19-9; CD5; CD19; CD20; CD21 ; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1 ; cyclin B1 ; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2 / neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1 ; ras; RORI; survivin; tenascin; TSTA tyrosinase; VEGF; and WT1.

[0142] Without limiting the foregoing, the particular following cancers can be treated by targeting the associated provided antigens: leukemia / lymphoma (CD19, CD20, CD22, ROR1 , CD33); multiple myeloma (B-cell maturation antigen (BCMA)); prostate cancer (PSMA, WT1 , Prostate Stem Cell antigen (PSCA), SV40 T); breast cancer (HER2, ERBB2); stem cell cancer (CD133); ovarian cancer (L1-CAM, extracellular domain of MUC16 (MUC-CD), folate binding protein (folate receptor), Lewis Y); renal cell carcinoma (carboxy-anhydrase-IX (CAIX); melanoma (GD2); and pancreatic cancer (mesothelin, CEA, CD24).

[0143] Viral antigen markers include peptides expressed by CMV, cold viruses, Epstein-Barr, flu viruses, hepatitis A, B, and C viruses, herpes simplex, HIV, influenza, Japanese encephalitis, measles, polio, rabies, respiratory syncytial, rubella, smallpox, varicella zoster or West Nile virus.

[0144] As further particular examples, cytomegaloviral antigens include envelope glycoprotein B and CMV pp65; Epstein-Barr antigens include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3 and HCV NS4; herpes simplex viral antigens include immediate early proteins and glycoprotein D; HIV antigens include gene products of the gag, pol, and env genes such as HIV gp32, HIV gp41 , HIV gp120, HIV gp160, HIV P17 / 24, HIV P24, HIV P55 GAG, HIV P66 POL, HIV TAT, HIV GP36, the Nef protein and reverse transcriptase; influenza antigens include hemagglutinin and neuraminidase; Japanese encephalitis viral antigens include proteins E, M-E, M-E-NS1, NS1 , NS1-NS2A and 80% E; measles antigens include the measles virus fusion protein; rabies antigens include rabies glycoprotein and rabies nucleoprotein; respiratory syncytial viral antigens include the RSV fusion protein and the M2 protein; rotaviral antigens include VP7sc; rubella antigens include proteins E1 and E2; and varicella zoster viral antigens include gpl and gpll. See Fundamental Virology, Second Edition, eds. Fields, B. N. and Knipe, D. M. (Raven Press, New York, 1991) for additional examples of viral antigens.

[0145] As particular examples of bacterial antigen markers, anthrax antigens include anthrax protective antigen; gram-negative bacilli antigens include lipopolysaccharides; haemophilus influenza antigens include capsular polysaccharides; diptheria antigens include diptheria toxin; Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein and antigen 85A; pertussis toxin antigens include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase; pneumococcal antigens include pneumolysin and pneumococcal capsular polysaccharides; rickettsiae antigens include rompA; streptococcal antigens include M proteins; and tetanus antigens include tetanus toxin.

[0146] As examples of fungal antigens, coccidiodes antigens include spherule antigens;cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heat shock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; plasmodium falciparum antigens include merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte / gamete surface antigens, protozoal and other parasitic antigens including the blood-stage antigen pf 155 / RESA; schistosomae antigens include glutathione-S-transferase and paramyosin; tinea fungal antigens include trichophytin; toxoplasma antigens include SAG-1 and p30; and trypanosoma cruzi antigens include the 75-77 kDa antigen and the 56 kDa antigen.

[0147] Examples of autoimmune antigens include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor. Examples of allergic antigens include pollen antigens such as Japanese cedar pollen antigens, ragweed pollen antigens, rye grass pollen antigens, animal derived antigens (such as dust mite antigens and feline antigens), histocompatibility antigens, and penicillin and other therapeutic drugs.

[0148] Without limiting the foregoing examples, antigens can also include CEACAM6, c-Met, EGFR, ErbB2, ErbB3, ErbB4, EphA2, IGF1 R, GHRHR, GHR, FLT1 , KDR, FLT4, CD44v6, CA125, CEA, BTLA, TGFBR2, TGFBR1, IL6R, gp130, TNFR1, TNFR2, PD1 , PD-L1 , PD-L2, HVEM, mesothelin, PSMA, RANK, ROR1 , TNFRSF4, TWEAK-R, HLA, tumor or pathogen derived peptides bound to HLA (such as from hTERT, tyrosinase, or WT-1), LT R, LIFR|3, LRP5, MUC1 , OSMRp, TCRa, TCR[3, B7H4, TLR7, TLR9, PTCH1 , PTCH1 , Robol , a-fetoprotein (AFP) and Frizzled.

[0149] Binding domains for exemplary antigens described herein are available commercially and in the publicly available art.

[0150] Binding domains of l-AMS and other engineered formats described herein may be joined through a linker. As indicated previously, a linker is an amino acid sequence which can provide flexibility and room for conformational movement between the binding domains of a l-AM. Any appropriate linker may be used.

[0151] Other forms of bispecific binding molecules include the single chain “Janusins” described in Traunecker et al. (Embo Journal, 10, 3655-3659, 1991).

[0152] Bispecific binding molecules with extended half-lives are described in, for example, US Patent No. 8,921,528 and US Patent Publication No. 2014 / 0308285.

[0153] Because albumin has an extended serum half-life, it can be of use in improving the pharmacokinetics of administered anti-Whitlow linker antibodies. In particular embodiments, antiWhitlow linker antibodies can be linked to albumin. In other particular embodiments, anti-Whitlowlinker antibodies can be linked to albumin-binding domains (ABDs). ABDs include, for example, albumin-binding peptides, antibodies, antibody fragments, and designed ankyrin repeat proteins (DARPins).

[0154] 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 other directly or through intervening linker segments such that the function of each included domain is retained.

[0155] Certain examples include fusion protein with two or three copies of an antibody or binding domain disclosed herein, each linked with the Gly-Ser linker (Gly4Ser)4 (SEQ ID NO: 125).

[0156] 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 are highly conserved protein sequences that can include different types of sequence motifs such as leucine zipper, helix loop-helix, ankyrin and PAS (Feuerstein et al, Proc. Natl. Acad. Sci. USA, 91 :10655-10659, 1994). Multimerization domains present in proteins can bind to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer, and / or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type (US Patent No. 10030065).

[0157] Dimerization domains can include protein sequence motifs such as coiled coils, acid patches, zinc fingers, calcium hands, a CH1-CL pair, an "interface" with an engineered "knob" and / or "protruberance" (US 5821333), leucine zippers (US 5932448), SH2 and SH3 (Vidal et al., Biochemistry, 43:7336- 44, 2004), PTB (Zhou et al., Nature, 378:584- 592, 1995), WW (Sudol Prog Biochys MoL Bio, 65:113-132, 1996), PDZ (Kim et al., Nature, 378: 85-88, 1995; Komau et al., Science, 269:1737-1740, 1995) and WD40 (Hu et al., J Biol Chem., 273:33489- 33494, 1998). Additional examples of molecules that contain dimerization domains / motifs are receptor dimer pairs such as the interleukin-8 receptor (IL-8R), integrin heterodimers such as LFA-I and GPU Ib / ll la, dimeric ligand polypeptides such as nerve growth factor (NGF), neurotrophin-3 (NT- 3), interleukin-8 (IL-8), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrophic factor (BDNF) (Arakawa et al., J Biol. Chem., 269:27833-27839, 1994; Radziejewski et al., Biochem, 32: 1350, 1993) and variants of some of these domains with modified affinities (PCT Publication No. WO 2012 / 001647).

[0158] In particular embodiments, the sequence corresponding to a dimerization motif / domain includes the leucine zipper domain of Jun (US5932448;RIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMN (SEQ ID NO: 156)), the dimerization domain of Fos (US 5932448; LTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAA (SEQ ID NO: 157)), a consensus sequence for a WW motif (PCT Publication No. WO 1997 / 037223), the dimerization domain of the SH2B adapter protein from GenBank Accession no. AAF73912.1 (Nishi et al., Mol Cell Biol, 25: 2607-2621 , 2005;WREFCESHARAAALDFARRFRLYLASHPQYAGPGAEAAFSRRFAELFLQHFEAEVARAS (SEQ ID NO: 158)), the SH3 domain of IB1 from GenBank Accession no. AAD22543.1 (Kristensen et al., EMBO J., 25: 785-797, 2006;THRAIFRFVPRHEDELELEVDDPLLVELQAEDYWYEAYNMRTGARGVFPAYYAIE (SEQ ID. NO: 159)), 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;LGEVHRFHVTVAPGTKLESGPATLHLCNDVLVLARDIPPAVTGQWKLSDLRRYGAVPSGFIFEG GTRCGYWAGVFFLSSAEGEQISFLFDCIVRGISPTKG (SEQ ID NO: 160)), the PDZ-like domain of SATB1 from UniProt Accession No. Q01826 (Galande et al., Mol Cell Biol. Aug; 21 : 5591- 5604, 2001;DCKEEHAEFVLVRKDMLFNQLIEMALLSLGYSHSSAAQAKGLIQVGKWNPVPLSYVTDAPDAT VADMLQDVYHVVTLKIQLHSCPKLEDLPPEQWSHTTVRNALKDLLKDMNQSS (SEQ ID NO: 161)), the WD40 repeats of APAF from UniProt Accession No. 014727 (Jorgensen et al., 2009. PLOS One. 4(12):e8463;CAPWPMVEKLIKQCLKENPQERPTSAQVFDILNSAELVCLTRRILLPKNVIVECMVATHHNSRN ASIWLGCGHTDRGQLSFLDLNTEGYTSEEVADSRILCLALVHLPVEKESWIVSGTQSGTLLVINT EDGKKRHTLEKMTDSVTCLYCNSFSKQSKQKNFLLVGTADGKLAIFEDKTVKLKGAAPLKILNIG NVSTPLMCLSESTNSTERNVMWGGCGSQLFSYAAFSDSNIITVVVDTALYIAKQNSPWEVWD KKTEKLCGLIDCVHFLREVMVKETKIFSFSNDFTIQKLIETRTNKESKHKMSYSGRVKTLCLQKN TALWIGTGGGHILLLDLSTRRLIRVIYNFCNSVRVMMTAQLGSLKNVMLVLGYNRKNTEGTQKQ KEIQSCLTVWDINLPHEVQNLEKHIEVRKELAEKMRRTSVE (SEQ ID NO: 162)), the PAS motif of the dioxin receptor from UniProt Accession No. I6L9E7 (Pongratz et al., Mol Cell Biol, 18:4079- 4088, 1998;DQELKHLILEAADGFLFIVSCETGRVVYVSDSVTPVLNQQQSEWFGSTLYDQVHPDDVDKLRE QLSTSENALTGR (SEQ ID NO: 163)) and the EF hand motif of parvalbumin from UniProt Accession No. P20472 (Jamalian et al., Int J Proteomics, 2014: 153712, 2014;LSAKETKMLMAAGDKDGDGKIGVDEFSTLVAES (SEQ ID NO: 164)).

[0159] In particular embodiments, the dimerization domain can be a dimerization and docking domain (DDD) on one antibody and an anchoring domain (AD) on another antibody to facilitate a stably tethered structure. In particular embodiments, the DDD (DDD1 and DDD2) are derived from the regulatory subunits of a cAMP-dependent protein kinase (PKA), and the AD (AD1 and AD2) are derived from a specific region found in various A-kinase anchoring proteins (AKAPs) that mediates association with the R subunits of PKA. In particular embodiments, DDD1 includes the amino acid sequence:SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 165). In particular embodiments, DDD2 includes the amino acid sequence:CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 166). In particular embodiments, AD1 includes the amino acid sequence: QIEYI.AKQIVDNAIQQA (SEQ ID NO: 167). In particular embodiments, AD2 includes the amino acid sequence: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO: 168). However, one skilled in the art will realize that other DDDs and ADs are known and can be used such as: the 4-helix bundle type DDD domains may be obtained from p53, DCoH (pterin 4 alpha carbinolamine dehydratase / dimerization cofactor of hepatocyte nuclear factor 1 alpha (TCF1)) and HNF-1 (hepatocyte nuclear factor 1). Other AD sequences of potential use may be found in Patent Publication No. US2003 / 0232420A1.

[0160] The X-type four-helix bundle dimerization motif that is a structural characteristic of the DDD (Newlon, et al. EMBO J. 2001 ; 20: 1651-1662; Newlon, et al. Nature Struct Biol. 1999; 3: 222-227) is found in other classes of proteins, such as the S100 proteins (for example, S100B and calcyclin), and the hepatocyte nuclear factor (HNF) family of transcriptional factors (for example, HNF-1 a and HNF-1 P). Over 300 proteins that are involved in either signal transduction or transcriptional activation also contain a module of 65-70 amino acids termed the sterile a motif (SAM) domain, which has a variation of the X-type four-helix bundle present on its dimerization interface. For S100B, this X-type four-helix bundle enables the binding of each dimer to two p53 peptides derived from the c-terminal regulatory domain (residues 367-388) with micromolar affinity (Rustandi, et al. Biochemistry. 1998; 37: 1951-1960). Similarly, the N-terminal dimerization domain of HNF-1a (HNF-p1) was shown to associate with a dimer of DCoH (dimerization cofactor for HNF-1) via a dimer of HNF-p1 (Rose, et al. Nature Struct Biol. 2000; 7: 744-748). In alternative embodiments, these naturally occurring systems can also be used to provide stable multimeric structures with multiple functions or binding specificities. Other binding events such as those between an enzyme and its substrate / inhibitor, for example, cutinase and phosphonates (Hodneland, et al. Proc Natl Acd Sci USA. 2002; 99: 5048-5052), may also be utilized to generatethe two associating components (the “docking” step), which are subsequently stabilized covalently (the “lock” step).

[0161] In particular embodiments, dimerization of antibodies can be induced by a chemical inducer. This method of dimerization requires one antibody to contain a chemical inducer of dimerization binding domain 1 (CBD1) and the second antibody to contain 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). If the CID is rapamycin, CBD1 and CBD2 can be the rapamycin binding domain of FK-binding protein 12 (FKBP12) and the FKBP12-Rapamycin Binding (FRB) domain of mTOR. In particular embodiments, FKBP12 includes the sequence:MGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNPFKFMLGKQEVIRGWEEG VAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 169).

[0162] In particular embodiments, FRB includes the sequence:MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLES (SEQ ID NO: 170). If the CID is FK506 / cyclosporin fusion protein or a derivative thereof, CBD1 and CBD2 can be the FK506 (Tacrolimus) binding domain of FK-binding protein 12 (FKBP12) and the cyclosporin binding domain of cylcophilin A. If the CID is estrone / biotin fusion protein or a derivative thereof, CBD1 and CBD2 can be an oestrogen-binding domain (EBD) and a streptavidin binding domain. If the CID is dexamethasone / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be a glucocorticoid-binding domain (GBD) and a dihydrofolate reductase (DHFR) binding domain. If the CID is O6-benzylguanine derivative / methotrexate fusion molecule or a derivative thereof, CBD1 and CBD2 can be an O6-alkylguanine-DNA alkyltransferase (AGT) binding domain and a dihydrofolate reductase (DHFR) binding domain. If the CID is RSL1 or a derivative thereof, CBD1 and CBD2 can be a retinoic acid receptor domain and an ecodysone receptor domain. If the CID is AP1903 or a derivative thereof, CBD1 and CBD2 can be the FK506 binding protein (FKBP12) binding domains including a F36V mutation. Use of the CID binding domains can also be used to alter the affinity to the CID. For instance, altering amino acids at positions 2095, 2098, and 2101 of FRB can alter binding to Rapamycin: KTW has high, KHF intermediate and PLW is low (Bayle et al, Chemistry & Biology 13, 99-107, January 2006).

[0163] In particular embodiments, antibodies can multimerize using a transmembrane polypeptide derived from a FCERI chain. In particular embodiments, an antibody can include a part of a FCERI alpha chain and another antibody can include a part of an FCERI beta chain or variant thereof such that said FCERI chains spontaneously dimerize together to form adimeric antibody. In particular embodiments, antibodies can include a part of a FCERI alpha chain and a part of a FCERI gamma chain or variant thereof such that said FCERI chains spontaneously trimerize together to form a trimeric antibody, and in another embodiment the multi-chain antibody can include a part of FCERI alpha chain, a part of FCERI beta chain and a part of FCERI gamma chain or variants thereof such that said FCERI chains spontaneously tetramerize together to form a tetrameric antibody.

[0164] In particular embodiments, additional methods of causing dimerization can be utilized. Additional modifications to generate a dimerization domain in antibody could include: replacing the C-terminus domain with murine counterparts; generating a second interchain disulfide bond in the C-terminus domain by introducing a second cysteine residue into both antibodies; swapping interacting residues in each of the antibodies in the C-terminus domains (“knob-in-hole”); and fusing the variable domains of the antibodies directly to CD3£ (CD3 fusion) (Schmitt et al., Hum. Gene Ther. 2009. 20:1240-1248).

[0165] Particular embodiments can utilize multimerization domains, such as C4b multimerization domains or ferritin multimerization domains. Full-length native C4b includes seven a-chains linked together by a multimerization (i.e., heptamerization) domain at the C-terminus of the a-chains. Blom et al., (2004) Mol Immunol 40: 1333-1346. Ferritin is an iron storage protein found in almost all living organisms, and has been extensively studied and engineered for a number of biochemical / biomedical purposes (US 20090233377; Meldrum, et al. Science 257, 522-523 (1992); U.S. 20110038025; Yamashita, Biochim Biophys Acta 1800, 846-857 (2010), including as a multimerizing vaccine platform for displaying peptide epitopes (US 20060251679 (2006); Li, et al. Industrial Biotechnol 2, 143-147 (2006)).

[0166] Mutlimerization with encapsulin and lumazine synthase can also be performed. Both can be linked to antibodies to create self-assembling 60mer particles (Jardine et al., 2013, Science 340, 711-716 and Kanekiyo et al., 2015, Cell 162, 1090-1100).

[0167] 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 , US20180118816A1 , US20190185570A1 , and US20180265596A1 , and PCT Publication Nos. WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761, and WO 2019 / 165340.

[0168] 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 orhexamers. 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).

[0169] 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. I gA1 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.

[0170] 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 "tail-piece" (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 of the 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.

[0171] The human lgA1 constant region typically includes the amino acid sequence: ASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDL YTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPR LSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVL PGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLAR GFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMV GHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 171). Referring to this SEQ ID NO: 171 , the human CA1 domain extends from amino acid 6 to amino acid 98; the humanlgA1 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.

[0172] The human lgA2 constant region typically includes the amino acid sequence ASPTSPKVFPLSLDSTPQDGNVVVACLVQGFFPQEPLSVTWSESGQNVTARNFPPSQDASGD LYTTSSQLTLPATQCPDGKSVTCHVKHYTNPSQDVTVPCPVPPPPPCCHPRLSLHRPALEDLL LGSEANLTCTLTGLRDASGATFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAQPWNHG ETFTCTAAHPELKTPLTANITKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRW LQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQK TIDRLAGKPTHVNVSVVMAEVDGTCY (SEQ ID NO: 172). Referring to this SEQ ID NO: 172, 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.

[0173] As indicated, two IgA binding units can form a complex with two additional polypeptide chains, the J chain (e.g., SEQ ID NO: 173, 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 ITLISSEGYVSSKYAGRANLTNFPENGTFWNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVS QGPGLLNDTKVYTVDLGRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRL DIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFH CALGPEVANVAKFLCRQSSGENCDWVNTLGKRAPAFEGRILLNPQDKDGSFSVVITGLRKED AGRYLCGAHSDGQLQEGSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIK YWCLWEGAQNGRCPLLVDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTN GDTLWRTTVEIKIIEGEPNLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALP SQDEGPSKAFVNCDENSRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAA GSRDVSLAKADAAPDEKVLDSGFREIENKAIQDPRLFAEEKAVADTRDQADGSRASVDSGSSE EQGGSSRALVSTLVPLGLVLAVGAVAVGVARARHRKNVDRVSIRSYRTDISMSDFENSREFGA NDNMGASSITQETSLGGKEEFVATTESTTETKEPKKAKRSSKEEAEMAYKDFLLQSSTVAAEA QDGPQEA (SEQ ID NO: 174). An exemplary mature secretory component includes KSPIFGPEEVNSVEGNSVSITCYYPPTSVNRHTRKYWCRQGARGGCITLISSEGYVSSKYAGR ANLTNFPENGTFVVNIAQLSQDDSGRYKCGLGINSRGLSFDVSLEVSQGPGLLNDTKVYTVDL GRTVTINCPFKTENAQKRKSLYKQIGLYPVLVIDSSGYVNPNYTGRIRLDIQGTGQLLFSVVINQLRLSDAGQYLCQAGDDSNSNKKNADLQVLKPEPELVYEDLRGSVTFHCALGPEVANVAKFLCR QSSGENCDVVVNTLGKRAPAFEGRILLNPQDKDGSFSWITGLRKEDAGRYLCGAHSDGQLQE GSPIQAWQLFVNEESTIPRSPTVVKGVAGGSVAVLCPYNRKESKSIKYWCLWEGAQNGRCPLL VDSEGWVKAQYEGRLSLLEEPGNGTFTVILNQLTSRDAGFYWCLTNGDTLWRTTVEIKIIEGEP NLKVPGNVTAVLGETLKVPCHFPCKFSSYEKYWCKWNNTGCQALPSQDEGPSKAFVNCDEN SRLVSLTLNLVTRADEGWYWCGVKQGHFYGETAAVYVAVEERKAAGSRDVSLAKADAAPDEK VLDSGFREIENKAIQDPR (SEQ ID NO: 175). 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., et 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.

[0174] 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: 171 or SEQ ID NO: 172), or a variant, derivative, or analog thereof.

[0175] In particular embodiments, the IgA heavy chain constant regions can include amino acids 125 to 353 of SEQ ID NO: 171 or amino acids 113 to 340 of SEQ ID NO: 172. 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: 171 or amino acids 102 to 340 of SEQ ID NO: 172. 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.

[0176] Each of the strategies discussed above can be used to create IgA antibody-based dimers.

[0177] Particular embodiments include IgM immunoglobulin constant region domains that allow the binding portion of molecules provided herein to readily multimerize into pentamers or hexamers.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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), anantibody 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.

[0183] 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 Cp3 domain situated N- terminal to the IgM Cp4 and IgM tp domains.

[0184] 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 Cp2, Cp3, and Cp4-tp domains as follows: VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAE AKESGPTTYKVTSTLTIKESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSF ASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWN SGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADV FVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNR VTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 176).

[0185] 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: 177). An exemplary IgM constant region of this type includes the variant human I gG 1 hinge region fused to a multimerizing fragment of the human IgM constant region including the Cp3, Cp4, and tp domains, and includes the amino acid sequence:VEPKSSDKTHTCPPCPAPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNG EAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHR PDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPG RYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY (SEQ ID NO: 178).

[0186] 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: 179 or SEQ ID NO: 180 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.

[0187] The human IgM heavy chain constant region typically includes the amino acid sequence GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 179; identical to, e.g., GenBank Accession Nos. pir||S37768, CAA47708.1 , and CAA47714.1). Referring to this SEQ ID NO: 179, the human Cp1 region ranges from amino acid 5 to amino acid 102; the human C 2 region ranges from amino acid 114 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.

[0188] In particular embodiments, an IgM heavy chain constant region includes the sequence: GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP R KSKLI CQATG FSP RQ I Q VSWLR EG KQVGSGVTTDQ VQAEA KESG PTTYKVTSTLTI KES D WL GQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDS VTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTI SRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSA PMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLV MSDTAGTCY (SEQ ID NO: 180; (UniProt ID P01871)— allele IGHM*04). This sequence differs from SEQ ID NO: 179 by one amino acid at position 191.

[0189] 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: 179 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.

[0190] 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: 179. These positions correspond to the Kabat numbering system as follows: S401 of SEQ ID NO: 179 corresponds to S524 of Kabat; E402 of SEQ ID NO: 179 corresponds to E525 of Kabat; E403 of SEQ ID NO: 179 corresponds to E526 of Kabat; R344 of SEQ ID NO: 179 corresponds to R467 of Kabat; and E345 of SEQ ID NO: 179 corresponds to E468 of Kabat.

[0191] In particular embodiments, “corresponds to” means the designated position of SEQ ID NO: 179 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.

[0192] In particular embodiments, P311 of SEQ ID NO: 179 can be substituted, e.g., with alanine (P311A), serine (P311S), or glycine (P311G) and / or P313 of SEQ ID NO: 179 can be substituted, e.g., with alanine (P313A), serine (P313S), or glycine (P313G). P311 and P313 of SEQ ID NO: 179 can be substituted with alanine (P311A) and serine (P313S), respectively as shown in the following sequence: (mutations in bold underline) GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP R KSKLI CQATG FSP RQ I Q VSWLR EG KQVGSGVTTDQ VQAEA KESG PTTYKVTSTLTI KES D WL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLASSLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 217).

[0193] In certain aspects, S401 of SEQ ID NO: 179 can be substituted with any amino acid. In certain aspects, S401 of SEQ ID NO: 179 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEA KESG PTTYKVTSTLTI KESDWLSQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVAEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 181).

[0194] In certain aspects, E402 of SEQ ID NO: 179 can be substituted with any amino acid. In certain aspects, E402 of SEQ ID NO: 179 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP R KSKLI CQATG FSP RQ I Q VSWLR EG KQVGSGVTTDQ VQAEA KESG PTTYKVTSTLTI KES D WL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSAEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 182).

[0195] In certain aspects, E403 of SEQ ID NO: 179 can be substituted with any amino acid. In certain aspects, E403 of SEQ ID NO: 179 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP RKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEA KESG PTTYKVTSTLTI KESDWL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSV TISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEAEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 183).

[0196] In certain aspects, R344 of SEQ ID NO: 179 can be substituted with any amino acid. In certain aspects, R344 of SEQ ID NO: 179 can be substituted with alanine (A) as follows (alanine substitution indicated by bold underline):GSASAPTLFPLVSCENSPSDTSSVAVGCLAQDFLPDSITFSWKYKNNSDISSTRGFPSVLRGGK YAATSQVLLPSKDVMQGTDEHVVCKVQHPNGNKEKNVPLPVIAELPPKVSVFVPPRDGFFGNP R KSKLI CQATG FSP RQ I Q VSWLR EG KQVGSGVTTDQ VQAEA KESG PTTYKVTSTLTI KES D WL SQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTIS RPKGVALHRPDVYLLPPAREQLNLAESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAP MPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVM SDTAGTCY (SEQ ID NO: 184).

[0197] In certain aspects, E345 of SEQ ID NO: 179 can be substituted with any amino acid. In certain aspects, E345 of SEQ ID NO: 179 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: 185).

[0198] 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 YTAWPLVYGGETKMVETALTPDACYPD (SEQ ID NO: 186). The signal peptide extends from amino acid 1 to amino acid 22 of SEQ ID NO: 186 and the mature human J-chain extends from amino acid 23 to amino acid 159 of SEQ ID NO: 186.

[0199] The mature human J-chain includes the amino acid sequenceQEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 173).

[0200] 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: 173. 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.

[0201] 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 S51of SEQ ID NO: 173.

[0202] By “an amino acid corresponding to” a position of SEQ ID NO: 173 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: 173 is conserved in the J-chain amino acid sequences of at least 43 other species. The position corresponding to T103 in SEQ ID NO: 173 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: 173 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.

[0203] In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 173 can be substituted with any amino acid. In certain aspects, the amino acid corresponding to Y102 of SEQ ID NO: 173 can be substituted with alanine (alanine substitution indicated by bold underline): QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCATYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 187), with serine (serine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCSTYDRNKCYTAVVPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 188),Or with arginine (arginine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCRTYDRNKCYTA VPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 189).

[0204] In certain aspects, the amino acid corresponding to T103 of SEQ ID NO: 173 can be substituted with any amino acid. In a particular aspect, the amino acid corresponding to T103 of SEQ ID NO: 173 can be substituted with alanine as follows (alanine substitution indicated by bold underline):QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDL CKKCDPTEVELDNQIVTATQSNICDEDSATETCYAYDRNKCYTAVVPLVYGGETKMVETALTP DACYPD (SEQ ID NO: 190).

[0205] 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: 173, provided that S51 is not substituted with threonine (T), or wherein the J-chain includes amino acid substitutions at the amino acidpositions corresponding to both amino acids N49 and S51 of SEQ ID NO: 173.

[0206] The amino acids corresponding to N49 and S51 of SEQ ID NO: 173 along with the amino acid corresponding to 150 of SEQ ID NO: 173 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: 173 can be substituted with any amino acid. In certain aspects, the asparagine at the position corresponding to N49 of SEQ ID NO: 173 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: 173 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 CKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAWPLVYGGETKMVETALTPD ACYPD (SEQ ID NO: 191).

[0207] In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 173 can be substituted with any amino acid except threonine. In certain aspects, the serine at the position corresponding to S51 of SEQ ID NO: 173 can be substituted with alanine (A) or glycine (G). In a particular aspect the position corresponding to S51 of SEQ ID NO: 173 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: 192).

[0208] 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: 121) wherein n is 1-5.

[0209] 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: 173 between cysteine residues 92 and 101 of SEQ ID NO: 173. In a further aspect, the antigen-bindingdomain can be introduced into the human J-chain of SEQ ID NO: 173 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: 173 within 10 amino acid residues from the C- terminus, or within 10 amino acids from the N-terminus.

[0210] In particular embodiments, the single-domain antibody is introduced into the native human J-chain sequence of SEQ ID NO: 173 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: 173 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.

[0211] 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.

[0212] 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 undulatus, Anas platyrhynchos, Gallus gallus, Meleagris gallopavo, Falco peregrinus, Zonotrichia albicollis, and Pteropus alecto.

[0213] (iv) Expression of Recombinant Proteins. Proteins (e.g., antibodies, antibody binding fragments, multidomain binding molecules, CAR) described herein can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expressioncontrol sequence operably linked to the coding sequences of the encoded protein, 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 nucleotide sequences, and the collection and purification of expressed proteins.

[0214] In particular embodiments, mammalian cells are used as a host for expressing nucleotide segments encoding proteins. 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. In particular embodiments, 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 necessary processing information 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).

[0215] Once expressed, proteins 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)).

[0216] In particular embodiments, recombinant proteins 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- translational ly 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 beneeded due to the purity of manufacture according to the methods described herein.

[0217] In particular embodiments, binding domains (e.g., antibodies) are expressed by a cell. In particular embodiments, the binding domain (e.g., antibody) can be secreted or expressed on the surface of a cell (e.g., recombinant receptor such as a CAR).

[0218] (v) Antibody Conjugates. Antibody conjugates include binding domains disclosed herein linked to another molecule or payload, other than an additional binding domain. Examples of payloads include a drug, a toxin, a detectable label, a radioisotope, or a particle. Examples of antibody conjugates include antibody-drug conjugates (ADCs), antibody immunotoxins, antibody- detectable label conjugates, antibody radioisotope conjugates, and antibody-particle conjugates.

[0219] Antibody-drug conjugates allow for the targeted delivery of a drug moiety to a molecule or cell expressing the Whitlow linker, in particular embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387). In particular embodiments, the drug can be a cytotoxic drug or cell stimulatory factors.

[0220] In particular embodiments, the antibody-drug conjugates include an antibody conjugated, i.e. , covalently attached, to the drug moiety. In particular embodiments, the anti-Whitlow binding domain 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) 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 antibody-drug conjugate selectively delivers an effective dose of a drug to cells (e.g., CAR- expressing cells) whereby greater selectivity, i.e., a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0221] To prepare antibody-drug conjugates, linker-drug conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 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 thiolconcentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the 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 ADC 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 ADC can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage.

[0222] Cell stimulatory factors can include molecules that promote cell growth, differentiation, and / or efficacy. Drug moieties that impart cell growth, differentiation, and / or efficacy effects can include growth factors, cytokines, chemokines, antigens, fusion proteins, receptors, and any other agent designed to activate cells, promote growth, and / or drive differentiation. “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhi biting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; pl ate let- growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-l and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and - gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-I, IL- 1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-I I, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines. In particular embodiments, cytokines includes interleukin (I L)- 2, IL-7, IL-15 and / or IL-21.

[0223] Cell stimulatory factors can include cell activating epitopes such as CD3, CD27, CD2, CD4, CD5, CD7, CD8, CD28, CD30, CD40, CD56, CD83, CD90, CD95, 4-1 BB (CD 137), B7-H3, CTLA-4, Frizzled-1 (FZD1), FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, HVEM, ICOS, IL-1 R, LAT, LFA-1 , LIGHT, MHCI, MHCII, NKG2D, 0X40, ROR2 and RTK.

[0224] Cell stimulatory factors can include growth factors such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).

[0225] Drug moieties that impart their cytotoxic and cytostatic effects can do so 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.

[0226] The drug may be obtained from essentially any source; it may be synthetic or a natural product isolated from a selected source, e.g., a plant, bacterial, insect, mammalian or fungal source. The drug may also be a synthetically modified natural product or an analogue of a natural product.

[0227] In particular embodiments, an anti-Whitlow binding domain can be formed as an antibody immunotoxin. Antibody immunotoxins include an anti-Whitlow antibody 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.

[0228] 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 the 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.

[0229] Antibody-detectable label conjugates include an anti-Whitlow binding domain linked to a detectable label. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In particular embodiments, detectable labels can include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymatic labels, affinity tags, and / or oligonucleotides. In particular embodiments, a radiolabel can also be used as a detectable label.

[0230] Fluorescent labels can be particularly useful in cell staining, identification, imaging, 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.

[0231] Chemiluminescent labels can include lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

[0232] Spectral colorimetric labels can include colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

[0233] Enzymatic labels can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes can include malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase,asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI- phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

[0234] Affinity tags can include, for example, His tag (HHHHHH (SEQ ID NO: 193)), Flag tag (DYKDDDD (SEQ ID NO: 194), Xpress tag (DLYDDDDK (SEQ ID NO: 195)), Avi tag (GLNDIFEAQKIEWHE (SEQ ID NO: 196)), Calmodulin binding peptide (CBP) tag (KRRWKKNFIAVSAANRFKKISSSGAL (SEQ ID NO: 197)), Polyglutamate tag (EEEEEE (SEQ ID NO: 198)), HA tag (YPYDVPDYA (SEQ ID NO: 199)), Myc tag (EQKLISEEDL (SEQ ID NO: 200)), Strep tag (WRHPQFGG (SEQ ID NO: 201)), STREP® tag II (WSHPQFEK (SEQ ID NO: 202); IBA Institut fur Bioanalytik, Germany; see, e.g., US 7,981,632), Softag 1 (SLAELLNAGLGGS (SEQ ID NO: 203)), Softag 3 (TQDPSRVG (SEQ ID NO: 204)), and V5 tag (GKPIPNPLLGLDST (SEQ ID NO: 205)).

[0235] Oligonucleotides are polynucleotides having a small number of nucleotides. Oligonucleotides can be associated, directly or indirectly, to an anti-Whitlow binding domain and sequencing the oligonucleotides can be used to detect the presence of the anti-Whitlow binding domains associated with oligonucleotides. Adding oligonucleotides can be done based on any of the compositions or methods disclosed in patent publication WO 2014047561 A1. Examples of oligonucleotides includes barcodes and unique molecular identifiers (UMI). The term “barcode” as used herein refers to a short sequence of nucleotides (for example, DNA or RNA) that is used as an identifier for an associated molecule, such as a target molecule and / or target nucleic acid, or as an identifier of the source of an associated molecule, such as a cell-of-origin. In particular embodiments, an oligonucleotide can be identified using cellular indexing of transcriptomes and epitopes sequencing (CITE-Seq) or CODEX.

[0236] A barcode may also refer to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment. A nucleic-acid based barcode is a short sequence of nucleotides (for example, DNA, RNA, or combinations thereof) that is used as an identifier for an associated molecule, such as a target molecule and / or target nucleic acid. A nucleic acid barcode can have a length of at least, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nucleotides, and can be in single- or double-stranded form. Target molecule and / or target nucleic acid can be associated with multiple nucleic acid barcodes to provide information about all of these features (and more). Methods of generating nucleic acidbarcodes are disclosed, for example, in International Patent Application Publication No. WQ / 2014 / 047561.

[0237] The term “unique molecular identifiers” (U I) as used herein refers to a sequencing linkeror a subtype of nucleic acid barcode used in a method that uses molecular tags to detect and quantify unique amplified products. A UMI is used to distinguish effects through a single clone from multiple clones. The term “clone” as used herein may refer to a single mRNA or target nucleic acid to be sequenced. The UMI may also be used to determine the number of transcripts that gave rise to an amplified product, or in the case of target barcodes as described herein, the number of binding events. In preferred embodiments, the amplification is by PCR or multiple displacement amplification (MDA).

[0238] Antibody-radioisotope conjugates include an anti-Whitlow binding domain (e.g., antiWhitlow antibody) linked to a radioisotope 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 (RIT).

[0239] Examples of radioactive isotopes that can be conjugated to an anti-Whitlow binding domain (e.g., anti-Whitlow antibody) of the present disclosure include iodine-131 yttrium-90, arsenic-72, arsenic-74, iodine-131 , indium-1 11 , and lutetium-177, as well as alpha-emitting radionuclides such as astatine-21 1 , actinium-225, 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.

[0240] 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.

[0241] Additional examples of useful radioisotopes include228Ac,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,174ml_u,176mLu,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, "Y,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 includes131l, "Y, and / or211At. In particular embodiments, a radioisotope is selected that includes a half-life (ti / 2) that enables high-yieldradiolabeling and drug delivery. In particular embodiments, a radioisotope is selected that includes a half-life (ti / 2) of 7.2 hours. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0242] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and / or colored beads, and can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of capture proteins, such as the anti-Whitlow binding domains disclosed herein, to their surface. Examples of suitable materials include: ceramics, glass, polymers, and magnetic materials. Suitable polymers include polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co - glycolic acid)), polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials. See, e.g., "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. In particular embodiments, a particle includes magnetic beads, agarose beads, sepharose beads, polymer microspheres, silica particles, or quantum dots. In particular embodiments, microparticles can be made of one or more materials. In particular embodiments, microparticles are paramagnetic microparticles. Particular embodiments utilize carboxy-modified polystyrene latex (CML) flow cytometry beads and / or magnetic MagPlex® (Luminex, Austin, TX) flow cytometry beads. In particular embodiments, particles can carry a payload.

[0243] In particular embodiments, an antibody as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the constant region can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags, and transglutaminase recognition sequences. Antibody fragments can also be modified for sitespecific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8).

[0244] (vi) Recombinant Receptors. Anti-Whitlow binding domains disclosed herein can be utilized within recombinant receptors such as chimeric antigen receptors (CAR). In particular embodiments, the anti-Whitlow linker can act as a bridge for a universal binder recombinant receptor cell system. In this approach a recombinant receptor (e.g., CAR) is generated that includes an anti-Whitlow binding domain or a Whitlow linker in the extracellular portion of the recombinant receptor. Immune cells that express the anti-Whitlow or Whitlow recombinantreceptor would not be reactive against a target in vivo. Recombinant receptor-expressing cells could be modulated by infusion of a recombinant molecule that includes the Whitlow linker or antiWhitlow binding domain, respectively, conjugated to an antigen-targeting moiety (e.g., an scFv against a tumor antigen or a ligand for a receptor on a tumor cell). This would allow multiple specificities to be targeted by one cellular immunotherapy product.

[0245] Alternatively, an anti-Whitlow recombinant receptor could be used to elicit cytotoxic effects on Whitlow linker-containing molecules. For example, to mitigate toxicity of a CAR product that has been administered to a subject, anti-Whitlow recombinant receptor-expressing immune cells could be administered.

[0246] CAR, for example, include several distinct subcomponents that allow genetically modified cells (e.g., regulatory T cells) to recognize and kill cells expressing an antigen (e.g., Whitlow linker). The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds an antigen that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such antigen, 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.

[0247] Examples of antigens that can be bound by a recombinant receptor are described elsewhere herein, and include cancer antigens, viral antigens, bacterial antigens, parasitic antigens, degenerative cell antigens, autoantigens, and / or antigens expressed by a cell that is not the cell expressing the Whitlow-containing molecule

[0248] Particular embodiments of binding domains include an anti-Whitlow antibody and / or the CDRs thereof as disclosed herein.

[0249] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, transduction markers, and tags.

[0250] 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.

[0251] 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 aminoacids. The structure of a transmembrane domain can include an a helix, a £ 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; CD45, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0252] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane 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).

[0253] Intracellular effector domains activate the expressing cell when the binding domain binds antigen (Whitlow linker). The term “effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0254] 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, CD3E, CD3 , CD27, CD28, CD40, BAFFR, DAP10, ICOS, LAG3, NKG2D, NOTCH 1, 0X40, ROR2, SLAMF1 , TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory 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 w CD19, CD4, CD8a, 6, CD49f, ITGAD,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.

[0255] 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, CD40, BAFFR, CD66d, CD79a, CD79b, and common FcRy (FCER1G), FcyRlla, FcR[3 (Fee Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0256] A co-stimulatory domain is a domain whose activation can be required for an efficientlymphocyte 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.

[0257] Transduction 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 Wang et 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). Methods to genetically modify cells to express recombinant receptors are well-known in the art.

[0258] Recombinant receptors can additionally include tags, such as the tags described as affinity tags elsewhere herein.

[0259] (vii) Cells. The present disclosure describes cells genetically modified to express a recombinant protein disclosed herein. Genetically modified cells can include immune cells, stem cells, epithelial cells, muscle cells, neural cells, and connective tissue cells, among others.

[0260] Immune cells can include T-cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC). In particular embodiments, genetically modified cells include T-cells.

[0261] Several different subsets of T-cells have been discovered, each with a distinct function. For example, a majority of T-cells have a T-cell receptor (TCR) existing as a complex of several proteins. The actual T-cell receptor is composed of two separate peptide chains, which are produced from the independent T-cell receptor alpha and beta (TCRa and TCRP) genes and are called a- and p-TCR chains.

[0262] y8 T-cells represent a small subset of T-cells that possess a distinct T-cell receptor (TCR) on their surface. In y8 T-cells, the TCR is made up of one y-chain and one 8-chain. This group of T-cells is much less common (2% of total T-cells) than the op T-cells.

[0263] CD3 is expressed on all mature T cells. Activated T-cells express 4-1 BB (CD137), CD69, and CD25. CD5 and transferrin receptor are also expressed on T-cells.

[0264] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ T-cells because they express the CD4 protein on their surface. Helper T-cells become activatedwhen they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of antigen presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response.

[0265] Cytotoxic T-cells destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T-cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body.

[0266] "Central memory" T-cells (or "TCM") as used herein refers to an antigen experienced CTL that expresses CD62L or CCR7 and CD45RO on the surface thereof and does not express or has decreased expression of CD45RA as compared to naive cells. In particular embodiments, central memory cells are positive for expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have decreased expression of CD45RA as compared to naive cells.

[0267] "Effector memory" T-cell (or "TEM") as used herein refers to an antigen experienced T- cell that does not express or has decreased expression of CD62L on the surface thereof as compared to central memory cells and does not express or has decreased expression of CD45RA as compared to a naive cell. In particular embodiments, effector memory cells are negative for expression of CD62L and CCR7, compared to naive cells or central memory cells, and have variable expression of CD28 and CD45RA. Effector T-cells are positive for granzyme B and perforin as compared to memory or naive T-cells.

[0268] "Naive" T-cells as used herein refers to a non-antigen experienced T cell that expresses CD62L and CD45RA and does not express CD45RO as compared to central or effector memory cells. In particular embodiments, naive CD8+ T lymphocytes are characterized by the expression of phenotypic markers of naive T-cells including CD62L, CCR7, CD28, CD127, and CD45RA.

[0269] Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. NK cells express CD8, CD16 and CD56 but do not express CD3.

[0270] NK cells include NK-T cells. NK-T cells are a specialized population of T cells that express a semi invariant T cell receptor (TCR ab) and surface antigens typically associated with natural killer cells. NK-T cells contribute to antibacterial and antiviral immune responses and promote tumor-related immunosurveillance or immunosuppression. Like natural killer cells, NK-T cells can also induce perforin-, Fas-, and TNF-related cytotoxicity. Activated NK-T cells are capable of producing IFN-y and IL-4. In particular embodiments, NK-T cells are CD3+ / CD56+.

[0271] Macrophages (and their precursors, monocytes) reside in every tissue of the body (incertain instances as microglia, Kupffer cells and osteoclasts) where they engulf apoptotic cells, pathogens and other non-self-components. Monocytes / macrophages express CD11b, F4 / 80; CD68; CD11c; IL-4Ra; and / or CD163.

[0272] Immature dendritic cells (i.e., pre-activation) engulf antigens and other non-self- components in the periphery and subsequently, in activated form, migrate to T-cell areas of lymphoid tissues where they provide antigen presentation to T cells. Dendritic cells express CD1a, CD1b, CD1c, CD1d, CD21 , CD35, CD39, CD40, CD86, CD101 , CD148, CD209, and DEC-205.

[0273] Stem cells can include hematopoietic stem cells or induced pluripotent stem cells. Hematopoietic Stem / Progenitor Cells or HSPC refer to a combination of hematopoietic stem cells and hematopoietic progenitor cells.

[0274] Hematopoietic stem cells refer to undifferentiated hematopoietic cells that are capable of self-renewal either in vivo, essentially unlimited propagation in vitro, and capable of differentiation to all other hematopoietic cell types.

[0275] A hematopoietic progenitor cell is a cell derived from hematopoietic stem cells or fetal tissue that is capable of further differentiation into mature cell types. In certain embodiments, hematopoietic progenitor cells are CD24|0Lin_CD117+hematopoietic progenitor cells. HPC can differentiate into (i) myeloid progenitor cells which ultimately give rise to monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, or dendritic cells; or (ii) lymphoid progenitor cells which ultimately give rise to T-cells, B-cells, and NK-cells.

[0276] HSPC can be positive for a specific marker expressed in increased levels on HSPC relative to other types of hematopoietic cells. For example, such markers include CD34, CD43, CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof. Also, the HSPC can be negative for an expressed marker relative to other types of hematopoietic cells. For example, such markers include Lin, CD38, or a combination thereof. Preferably, the HSPC are CD34+ cells.

[0277] An induced pluripotent stem cell or iPSC refers to a pluripotent cell induced by artificially dedifferentiating (reprogramming) the adult cells that have already been differentiated. The term "adult cell" as used herein refers to a cell derived from an adult that is born and alive, as opposed to an embryonic cell. As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. iPSC can be reprogrammed from adult stem cells using any method known in the art.

[0278] A statement that a cell or population of cells is "positive" for or expressing a particularmarker refers to the detectable presence on or in the cell of the particular marker. When referring to a surface marker, the term can refer to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype- matched control under otherwise identical conditions and / or at a level substantially similar to that for cell known to be positive for the marker, and / or at a level substantially higher than that for a cell known to be negative for the marker.

[0279] A statement that a cell or population of cells is "negative" for a particular marker or lacks expression of a marker refers to the absence of substantial detectable presence on or in the cell of a particular marker. When referring to a surface marker, the term can refer to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0280] Cells to be genetically modified according to the teachings of the current disclosure can be patient-derived cells (autologous) or allogeneic when appropriate, and can also be in vivo or ex vivo. In particular embodiments, the immune cell is a CD4+ T cell or CD8+ T cell.

[0281] (viii) Cell Sample Collection and Cell Enrichment. Methods of sample collection and enrichment are known by those skilled in the art. In some embodiments, cells are derived from cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, or pig. In particular embodiments, cells are derived from humans, for example a patient to be treated.

[0282] In some embodiments, T cells are derived or isolated from samples such as whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. In particular embodiments, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in particular embodiments, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, HSC, HPC, HSPC, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cellsand platelets and further processing is necessary. In particular embodiments, T cells are derived from PBMCs.

[0283] In some embodiments, blood cells collected from a subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In particular embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. 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 re-suspended in a variety of biocompatible buffers after washing, such as, Ca++ / Mg++ free PBS.

[0284] 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.

[0285] In particular embodiments, a sample can be enriched for T cells by using density-based cell separation methods and related methods. For example, white blood cells can be separated from other cell types in the peripheral blood by lysing red blood cells and centrifuging the sample through a Percoll or Ficoll gradient.

[0286] In particular embodiments, a bulk T cell population can be used that has not been enriched for a particular T cell type. In particular embodiments, a selected T cell type can be enriched for and / or isolated based on cell-marker based positive and / or negative selection. In positive selection, cells having bound cellular markers are retained for further use. In negative selection, cells not bound by a capture agent, such as an antibody to a cellular marker are retained for further use. In some examples, both fractions can be retained for a further use. In particular embodiments, CD4+ and / or CD8+ T cells are enriched from PBMCs.

[0287] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type refers to decreasing thenumber or percentage of such cells but need not result in a complete removal of all such cells.

[0288] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.

[0289] In some embodiments, an antibody or binding domain for a cellular marker is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ); see also US 4,452,773; US 4,795,698; US 5,200,084; and EP 452342.

[0290] In some embodiments, affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). MACS systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0291] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5):355 — 376). In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined cell subsets at high purity.

[0292] Cell-markers for different T cell subpopulations are described above. In particular embodiments, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CCR7, CD45RO, CD8, CD27, CD28, CD62L, CD127, CD4, and / or CD45RA T cells, are isolated by positive or negative selection techniques.

[0293] CD3+, CD28+ T cells can be positively selected for and expanded using anti-CD3 / anti- CD28 conjugated magnetic beads (e.g., DYNABEADS® (Life Technologies AS, Norway) M-450 CD3 / CD28 T Cell Expander).

[0294] In particular embodiments, a CD8+ or CD4+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD8+ and CD4+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0295] In some embodiments, enrichment for central memory T (TCM) cells is carried out. In particular embodiments, memory T cells are present in both CD62L subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L, CD8 and / or CD62L+CD8+ fractions, such as by using anti-CD8 and anti-CD62L antibodies.

[0296] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD45RO, CD27, CD62L, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CCR7, CD45RO, and / or CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained, optionally following one or more further positive or negative selection steps.

[0297] Other cell types can be enriched based on known marker profiles and techniques. For example, CD34+ HSC, HSP, and HSPC can be enriched using anti-CD34 antibodies directly or indirectly conjugated to magnetic particles in connection with a magnetic cell separator, for example, the CliniMACS® Cell Separation System (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0298] (ix) Genetic Engineering Techniques. Desired genes, genetic constructs, and sequences encoding the binding domains and other proteins 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 includingthe gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, in vivo nanoparticle-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. Then. 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.

[0299] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence). The gene can encode a binding domain disclosed herein (e.g., anti-Whitlow binding domain). This definition includes various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not substantially affect the function of the encoded genetic construct. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the open reading frame(s) within the genetic construct. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non- full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.

[0300] Gene sequences encoding genetic constructs are provided herein and can also be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence. In embodiments, the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.

[0301] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for the synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biologicalsystem. A "gene sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.

[0302] Polynucleotide gene sequences encoding more than one portion of an expressed genetic constructs can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0303] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids, cosmids, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0304] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and nondividing cells. 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).

[0305] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al, Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. In particular embodiments, cells are genetically engineered to express genetic constructs using a lentivirus or lentiviral vector.

[0306] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0307] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequencesnecessary for the packaging and integration of the viral genome, i.e. , (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Cunn. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991 , Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75.2Q7-282).

[0308] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles including transgenes are described in, e.g., US 8,119,772; Walchli, et al., 2011 , PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Then 14:1155; Frecha, et al., 2010, Mol. Then 18:1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0309] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas 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 related thereto; 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,WO20 14 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351, WO2015 / 089354, WO20 15 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 and WO2015 / 089486, W02016205711 , WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0310] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells. A zinc finger is a domain of 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A well-known example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; US 6,607,882; US 6,746,838; US 6,794, 136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US 7,013,219; US 7,030,215; US 7,220,719; US 7,241 ,573; US 7,241 ,574; US 7,585,849; US 7,595,376; US 6,903,185; US 6,479,626; US 2003 / 0232410 and US 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, etal. Nature biotechnology 25, 778-785 (2007); Bibikova, etal. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1169-1175 (2002); Wolfe, etal. 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, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0311] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431 ; US 8,440,432; US 8,450,471 ; US 8,586,363; and US 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762;Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, etal. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0312] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0313] 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.

[0314] 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 lucifugusy 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.

[0315] (x) Nanoparticles. Particular embodiments disclosed herein utilize nanoparticles to carry or deliver components. One example of nanoparticles includes 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 is an outer layer surrounding a porous particle.

[0316] Liposomes can be neutral (cholesterol) or bipolar and include phospholipids, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM) and other types of bipolar lipids including dioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain length in the range of 14-22, and saturated or with one or moredouble 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.

[0317] 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).

[0318] Particular embodiments can utilize lipid nanoparticles (LNPs) as described in US2018 / 0303925. Particles as described in Pardi et al., Nature Communications (2018) 9:3361 can also be used. Particular embodiments can include mRNA and poly(C) RNA (Sigma) encapsulated in LNPs using a self-assembly process. In particular embodiments, an aqueous solution of mRNA at pH = 4.0 can be rapidly mixed with a solution of lipids dissolved in ethanol, as described in Maier, et al., Mol. Ther. 21 ,1570-1578 (2013). The LNPs can include an ionizable cationic lipid including phosphatidylcholine / cholesterol / PEG-lipid (50:10:38.5:1.5 mol / mol) with RNA encapsulated at an RNA to total lipid ratio of 0.05 (wt / wt). Particular embodiments can utilize LNPs with a diameter of 80 nm as measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd., Malvern, UK) instrument.

[0319] In particular embodiments, particles can include features that enhance the delivery and / or expression of a nucleic acid. For example, in particular embodiments, the particle includes a carrier molecule that condenses and protects a nucleic acid from enzymatic degradation. Suchcarriers are positively charged (e.g., poly([3-amino ester)). Additional examples of positively charged polymers include 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.

[0320] Examples of positively charged lipids 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 3|3- [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.

[0321] Examples of negatively charged polymers include alginic acids; carboxylic acid polysaccharides; carboxymethyl cellulose; carboxymethyl cellulose-cysteine; carrageenan (e.g., Gelcarin® 209, Gelcarin® 379); 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); sodium carboxymethyl cellulose (CMC); sulfated polysaccharides (heparins, agaropectins); pectin, gelatin and hyalouronic acid.

[0322] Neutrally charged polymers 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.

[0323] 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).

[0324] In particular embodiments, polymers 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.

[0325] In particular embodiments, the branches are zwitterionic or negatively-charged polymeric branches. For star polymers, the branch precursors can be converted to zwitterionic or negatively- charged polymers via hydrolysis, ultraviolet irradiation, or heat. The polymers also may be obtained by any polymerization method effective for polymerization of unsaturated monomers, including atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer polymerization (RAFT), photo-polymerization, ring-opening polymerization (ROP), condensation, Michael addition, branch generation / propagation reaction, or other reactions.

[0326] 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.

[0327] Without limiting the foregoing, 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 nanocarriers include silica. The exceptionally high surface area of mesoporous silica (exceeding 1,000 m2 / g) enables nucleic acid loading at levels exceeding conventional DNA carriers such as liposomes.

[0328] Particles can be formed in a variety of different shapes, including spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like.

[0329] (xi) Compositions and Formulations for Administration. Any of the binding domains described herein (e.g., antibodies, multi-domain binding molecules, antibody conjugates) in any exemplary format, can be formulated alone or in combination into compositions for administration to subjects. Additionally, nucleic acids encoding the antibodies can also be formulated into compositions for administration (e.g., nucleic acids encapsulated within nanoparticles (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., anti-Whitlow antibodies, multi-domain binding molecules, antibody conjugates) and / or nucleic acids encoding binding domains are collectively referred to herein as “active ingredients”. Certain examples may include formulations. Formulations include cells genetically modified to express a therapeutic molecule within apharmaceutically acceptable carrier.

[0330] Salts and / or pro-drugs of the active ingredients can also be used.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of an active ingredient or by hydrolysis of a biologically labile group.

[0335] 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.

[0336] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0337] 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.

[0338] An exemplary chelating agent is EDTA (ethylene-diamine-tetra-acetic acid).

[0339] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0340] 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.

[0341] 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 th ioglycol ate, 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.

[0342] The compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. The compositions disclosed herein can further be formulated for bone marrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, sublingual, and / or subcutaneous administration and more particularly by intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, intravesicular, and / or subcutaneous injection.

[0343] For injection, compositions 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.

[0344] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid compositions such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g., lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose,and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0345] 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. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredient and a suitable powder base such as lactose or starch.

[0346] Compositions and / or formulations can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0347] Additionally, compositions and / or formulations can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release one or more active ingredients following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0348] Depot compositions and / or formulations can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0349] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solventsinclude water, ethanol, dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0350] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0351] Excipients that partition into the external phase boundary of nanoparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0352] Additional processing of the disclosed sustained release depot compositions can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0353] In particular embodiments, 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.

[0354] In certain examples, cells are genetically modified to express a therapeutic molecule, for example, 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. In particular embodiments, the cells are T cells genetically modified to express a recombinant receptor.

[0355] In particular embodiments, genetically modified cells can be harvested from a culture medium and washed and concentrated into a carrier in a therapeutically-effective amount. 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.

[0356] In particular embodiments, carriers can be supplemented with human serum albumin (HSA) or other human serum components or fetal bovine serum. In particular embodiments, a carrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol.

[0357] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0358] Where necessary or beneficial, formulations can include a local anesthetic such as lidocaine to ease pain at a site of injection.

[0359] 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 1011.

[0360] In formulations disclosed herein, cells 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 / ml or 108cells / ml.

[0361] As indicated, formulations can include at least one genetically modified cell type (e.g., modified T cells, NK cells, or stem cells). Formulations can include different types of genetically- modified cells (e.g., T cells, NK cells, and / or stem cells in combination).

[0362] Different types of genetically-modified cells or cell subsets (e.g., modified T cells, NK cells, and / or stem cells) can be provided in different ratios e.g., a 1 :1 :1 ratio, 2:1 :1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5:1 :1 ratio, 1 :5:1 ratio, 1 :1:5 ratio, 10:1:1 ratio, 1 :10:1 ratio, 1 :1 :10 ratio, 2:2:1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1:5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1:10:10 ratio, 10:1 :10 ratio, etc. These ratios can also apply to numbers of cells expressing the same or different recombinant receptor components. If only two of the cell types are combined or only 2 combinations of expressed recombinant receptor components are included within a formulation, the ratio can include any 2-number combination that can be created from the 3 number combinations provided above. In embodiments, the combined cell populations are tested for efficacy and / or cellproliferation in vitro, in vivo and / or ex vivo, and the ratio of cells that provides for efficacy and / or proliferation of cells is selected. Particular embodiments include a 1:1 ratio of CD4 T cells and CD8 T cells.

[0363] Any composition and / or formulations 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 and / 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.

[0364] (xii) Ex Vivo Methods of Use. Methods disclosed herein include using binding domains that bind the Whitlow linker for research and / or ex vivo diagnostic uses. Binding domains can be used to select and / or identify engineered cells or molecules that present a Whitlow linker. Methods include imaging and detection of the Whitlow linker or molecules containing the Whitlow linker ex vivo or in vitro. For detection and imaging purposes, an effective amount is applied to a specimen, for example, a tissue sample.

[0365] An “effective amount” is the amount of a composition necessary to result in a desired physiological change in the specimen. For example, an effective amount can provide a detectable signal enabling the visualization or measurement of the Whitlow linker-containing molecules. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistical ly-significant effect in an in vitro assay.

[0366] “Specimen” refers to a component which contains cells and is extracted from a living body. Examples include cells, tissue, organs, or bodily fluids taken from a subject. Subjects include humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish).

[0367] Binding domains described herein bind the Whitlow linker. Binding domains described herein bind the Whitlow linker, wherein the Whitlow linker is part of a target molecule. For example, many chimeric antigen receptors (CAR) include a Whitlow linker within the scFv portion of the CAR binding domain. As such, binding domains disclosed herein are useful for imaging and detection of target molecules, such as CAR. Binding domains can be incorporated into antibodies, multi-domain binding molecules, or antibody conjugates.

[0368] Compositions disclosed herein can be used for ex vivo detection or isolation of Whitlow linker-containing molecules (e.g., CAR). In particular embodiments, detection is for research and / or diagnostic uses. In particular embodiments, methods of detection include administering aneffective amount of a binding domain (e.g., anti-Whitlow linker antibody) disclosed herein. The antibody can be, directly or indirectly, associated with or linked to a detectable label, and the composition can be suitable for detection of a Whitlow linker-containing molecule (e.g., CAR).

[0369] For isolation of Whitlow linker-containing molecules, immunoprecipitation or affinity chromatography can be used. In immunoprecipitation, the anti-Whitlow antibody can be conjugated to an object in order to isolate the specific antigen from a mixture. In affinity chromatography, separation is conducted in a column, relying on an interaction between a binding site on a protein and a ligand bound to a resin. In particular embodiments, the anti-Whitlow binding domains disclosed herein can be used to enrich for Whitlow linker-containing molecules (e.g., CAR) during the manufacturing process. For example, the antibodies can be conjugated to a particle, magnetic bead, or solid support, wherein the antibody binds the target antigen and the particle, magnetic bead, or solid support allows for the separation of the antibody-bound antigen from the mixture. In particular embodiments, a solid support can be made of glass, cellulose, agarose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. Separation or isolation can be performed in the manufacturing process to enrich for Whitlow linker-containing molecules or anti-Whitlow binding domains. Alternatively, separation or isolation can be performed on a sample such as a biological sample. In particular embodiments, a method of separating a Whitlow linker-containing molecule from a sample includes contacting the sample with an anti-Whitlow binding domain disclosed herein that is directly or indirectly associated with a solid support; and isolating the solid support from the sample. In particular embodiments, a method of separating an anti-Whitlow binding domain from a sample includes contacting the sample with a Whitlow linker-containing molecule disclosed herein that is directly or indirectly associated with a solid support; and isolating the solid support from the sample. In particular embodiments, the sample is a biological sample. In particular embodiments, the sample is a harvest medium. A harvest medium refers to a solution containing a molecule of interest (e.g., manufactured molecule of interest). In particular embodiments, a harvest medium is a manufactured solution before it has undergone purification. In particular embodiments, a harvest medium is a crude extract from a biological sample.

[0370] For detection applications, the binding domain of the presently disclosed subject matter can be labeled with a detectable label. The detectable label can be any label that is capable of producing, either directly or indirectly, a detectable signal. For example, detectable labels are described elsewhere herein and include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymatic labels, and affinity tags. Radiolabels can also be used as a detectable label. In particular embodiments, the binding domain is conjugated to the detectablelabel and methods for detection include contacting the binding domain with the sample and detecting the detectable label. In other embodiments, a method of detecting a target molecule includes providing the binding domain; contacting the binding domain with the sample containing the target molecule; contacting the binding domain with a binding molecule detection agent, wherein the binding molecule detection agent includes a binding domain that binds the binding molecule and a detectable label; and detecting the detectable label. A binding molecule detection agent can include a secondary antibody conjugated to a fluorophore, for example.

[0371] Detection and imaging of the antibody is tunable, such that imaging can be performed in under 1 , 2, 4, 6, 12, or 18, 24, 36, or 48 hours, or any amount below, above, or between this amount. It has been demonstrated that PEGs / larger molecules increase serum half-life by 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times compared to a smaller molecule. This allows for imaging at different time points.

[0372] In particular embodiments, a composition as disclosed herein can be used for ex vivo imaging and detection. In particular embodiments, ex vivo imaging methods include detecting a Whitlow linker-containing molecule by (a) contacting a biological sample with a composition; and (b) detecting the composition, thereby detecting the Whitlow linker-containing molecule. In particular embodiments, the composition includes an anti-Whitlow binding domain, a multi-domain binding molecule, or an antibody conjugate. In particular embodiments, the multi-domain binding molecule includes an anti-Whitlow binding domain and a binding domain that binds a molecule including a detectable label. In particular embodiments, the antibody conjugate includes an anti- Whitlow binding domain and a detectable label. In particular embodiments, ex vivo imaging methods include detecting a Whitlow linker-containing molecule by (a) contacting a biological sample with an antibody conjugate; and (b) detecting the antibody conjugate, thereby detecting the Whitlow linker-containing molecule.

[0373] Methods may be used to monitor progression of a Whitlow-linker-containing molecule (e.g., CAR), for example, using biopsy samples at different times or imaging the subject at different times. In such aspects, instead of comparing the expression of the target molecule (e.g., Whitlow linker-containing molecule) against a control sample from, e.g., a different tissue source or subject known not to have a Whitlow linker- containing molecule, the expression of the target molecule is compared against a biological sample obtained from the same tissue or the same subject at an earlier time point, for example, from days, weeks or months earlier. In particular embodiments, methods may be used to monitor the progression of a Whitlow linker-containing molecule by detecting the existence or quantity of the target molecule in different tissues of thesame subject.

[0374] Any suitable biological sample may be used; the nature of the disease or condition may determine the nature of the sample which is to be used in the methods. The sample may be, for example, a sample from a tissue biopsy, tumor tissue biopsy, bone marrow biopsy, or circulating cells in, e.g., blood. Alternatively, e.g., where, for example, the methods are being used to diagnose or monitor a gastrointestinal tumor, tumor cells may be isolated from feces (stool) samples. Other sources of biological sample may include plasma, serum, cerebrospinal fluid, urine, interstitial fluid, ascites fluid or the like.

[0375] For example, solid tumor samples may be collected in complete tissue culture medium with, for example, antibiotics. Cells may be manually teased from the tumor specimen or, where necessary, are enzymatically disaggregated by incubation with collagenase / DNAse and suspended in appropriate media containing, for example, human or animal sera.

[0376] In other aspects, biopsy samples may be isolated and frozen or fixed in fixatives such as formalin. The samples may then be tested for expression levels of Whitlow linker-containing molecules or cells. Formalin-fixed paraffin-embedded (FFPE) tissue, for example, is prepared by collecting tissue, fixing the tissue in a neutral-buffered formalin (e.g., 10% neutral-buffered formalin) for a time period necessary to fix the sample according to the tissue size (e.g., 6-48 hours), dehydrate the tissue by transferring the sample to increasing concentrations of ethanol, clearing the ethanol, then infiltrating with molten paraffin wax at a temperature and time necessary to permeate the tissue and provide a supportive matrix (e.g., 60°C for 2 hours), and slicing the tissue into thin sections and embedded on a slide. Frozen or fixed (e.g., FFPE) tissues can be analyzed using binding domains disclosed herein using methods disclosed herein. Exemplary methods include applying antibody-conjugates including a binding domain disclosed herein conjugated to a detectable label (e.g., fluorescent or chemiluminescent detectable label).

[0377] The binding domains of the presently disclosed subject matter can be employed in various ex vivo assay methods, such as detection of an affinity tag, flow cytometry, immunohistochemistry, fluorescence imaging, oligonucleotide-conjugated identification, ELISA, electron microscopy, latex agglutination, lateral flow immunoassays, immunoblotting, and Dip Stick Immuno testing, competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays (see e.g., Zola, 1987; Harlow & Lane, 1988), and as affinity purification agents. Some of these procedures are described in more detail in the following paragraphs.

[0378] If an affinity tag has been used, a protein or compound that binds the affinity tag can be used to detect the affinity tag. Representative affinity tags are described elsewhere herein. Inparticular embodiments, a protein or compound that binds the affinity tag can be conjugated to a detectable label. In particular embodiments, a protein or compound that binds the affinity tag is conjugated to an enzymatic label. In particular embodiments, a protein or compound that binds the affinity tag is conjugated to an enzymatic label and is detected by the production of a colorimetric or luminescent product that is measurable using a spectrophotometer or luminometer, respectively.

[0379] Flow cytometry is a technique used to detect and measure the physical and chemical characteristics of a population of cells or particles. The term “cytometry” technically refers to the counting or enumeration of cells, particularly blood cells. The term “cytometry” is used generically in this disclosure to refer to the enumeration of any of a number of analytes, particularly particle analytes, described in more detail below. “Flow cytometry,” a technical method in which target analytes (e.g., cells or particles) are identified and / or enumerated as they move past a detector or sets of detectors. In many cases, the detector includes a laser which detects a fluorescently- tagged antibody bound to the analyte of interest.

[0380] Immunohistochemistry (IHC) utilizes a binding domain disclosed herein to detect Whitlow linker-containing molecules. The antibodies can identify Whitlow linker-containing molecules in frozen or FFPE tissues. IHC detects target molecules through antigen-antibody complexes in a pathological specimen using enzyme-linked antigens or antibodies. The presence of the target molecule can then be detected via an enzyme immunoassay.

[0381] A multitude of benefits are realized with IHC versus traditional immunofluorescence. For example, unlike immunofluorescence, IHC can be used with commonly used formalin-fixed paraffin-embedded tissue specimens. Specimens, including histological tissue sections and / or other biological preparations such as tissue culture cells, are commonly used in diagnostic pathology and can be easily screened via IHC. Further, IHC staining is permanent and preserves cell morphology. A comparison of the cell morphology and antigen proliferation on two different slides can be useful in monitoring the progression, location, and / or proliferation of a Whitlow-linker molecule.

[0382] Once an antibody detectable label conjugate has attached, either directly or indirectly, to the specimen (or biological sample), a substrate, specific for the enzyme, is added to the specimen. When the substrate is added, the enzyme label converts the substrate causing a color change that can be seen with light microscopy. The presence of a color change indicates the presence of the target molecule and allows an observer to determine, assess, and / or diagnose.

[0383] Non-invasive imaging methods can also include detection of a fluorescent label (i.e., fluorescence imaging). Examples of fluorescent labels are described elsewhere herein.Fluorescence imaging can be performed ex vivo or in vivo. For in vivo detection of a fluorescent label, an image is created using emission and absorbance spectra that are appropriate for the particular label used. The image can be visualized, for example, by diffuse optical spectroscopy. Additional methods and imaging systems are described in U.S. Pat. Nos. 5,865,754; 6,083,486; and 6,246,901 , elsewhere herein, among other places.

[0384] Oligonucleotide-conjugated identification uses binding domains conjugated to an oligonucleotide for identification of a target molecule. For example, in methods such as cellular indexing of transcriptomes and epitopes (CITE)-Seq or CODEX, antibodies conjugated to an oligonucleotide can be used for spatial mapping of target molecule. The oligonucleotide can include a barcode, unique molecular identifier (UMI), and / or any additional sequence to aid in library preparation and amplification.

[0385] (xiii) In Vivo Methods of Use. Methods disclosed herein include using binding domains that bind the Whitlow linker for in vivo imaging (e.g., diagnostic) and / or therapeutic uses. Binding domains can be used to select and / or identify engineered cells or molecules that present a Whitlow linker. Furthermore, binding domains can be administered to treat subjects in need thereof.

[0386] Methods disclosed herein include treating or imaging subjects (humans, veterinary animals (dogs, cats, reptiles, birds, etc.) livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice, fish, etc.) with formulations and / or compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0387] An "effective amount," in the context of in vivo methods of use, is the amount of a formulation necessary to result in a desired physiological change in the subject. For example, an effective amount can provide a detectable signal enabling the visualization or measurement of the Whitlow linker-containing molecules, a cytotoxic effect to Whitlow linker-expressing cells, anticancer effects, or an immunogenic effect. Therapeutically effective amounts disclosed herein can cause a statistically significant effect in an animal model relevant to the assessment of a therapeutic treatment’s success.

[0388] In vivo methods disclosed here use compositions disclosed here in (e.g., binding domains) for in vivo imaging. In particular embodiments, a composition of the presently disclosed subject matter includes a label that can be detected in vivo. The term “in vivo” as used herein to describe imaging or detection methods, refers to generally non-invasive methods such as fluorescence, scintigraphic methods, magnetic resonance imaging, autoradiographic detection, orradioimmunoguided systems, each described briefly herein below. The term “non-invasive methods” includes methods employing administration of a contrast agent to facilitate in vivo imaging. In vivo imaging can be useful in determining the location and quantity of Whitlow linkercontaining molecules (e.g., CAR) within a body.

[0389] In particular embodiments, a method for detecting target molecules (i.e. , Whitlow linkercontaining molecules) in a subject includes (a) administering, to the subject, a composition disclosed herein; and (b) detecting the composition, thereby detecting the target molecules. In particular embodiments, a method for detecting target molecules in a subject includes (a) administering, to the subject, a composition including the anti-Whitlow binding domain (i.e., binding domain that binds Whitlow linker or subsequence thereof); and (b) detecting the antiWhitlow binding domain, thereby detecting the target molecules. In particular embodiments, the composition includes an anti-Whitlow binding domain, a multi-domain binding molecule, or an antibody conjugate. In particular embodiments, the multi-domain binding molecule includes a binding domain disclosed herein and a binding domain that eventually binds a molecule with a detectable label. In particular embodiments, the antibody conjugate includes a binding domain disclosed herein linked to a detectable label.

[0390] In particular embodiments, the detectable label can be conjugated or otherwise associated with an binding domain disclosed herein or the detectable label can associate with the binding domain during the methods. Following administration of the labeled composition to a subject, and after a time sufficient for binding, the biodistribution of the composition can be visualized. The term “time sufficient for binding” refers to a temporal duration that permits binding of the labeled agent to a target molecule.

[0391] Examples of in vivo imaging include scintigraphic imaging, magnetic resonance imaging (MRI), autoradiographic detection, and radioimmunographic system. These procedures are described in more detail in the following paragraphs.

[0392] Scintigraphic imaging methods include SPECT (Single Photon Emission Computed Tomography). PET (Positron Emission Tomography), gamma camera imaging, and rectilinear scanning. A gamma camera and a rectilinear scanner each represent instruments that detect radioactivity in a single plane. Most SPECT systems are based on the use of one or more gamma cameras that are rotated about the subject of analysis, and thus integrate radioactivity in more than one dimension. PET systems include an array of detectors in a ring that also detect radioactivity in multiple dimensions.

[0393] Imaging instruments suitable for practicing the detection and / or imaging methods of the presently disclosed subject matter, and instruction for using the same, are readily available fromcommercial sources. For example, a SPECT scanner can be used with a computed tomography (CT) scanner, with coregistration of images. As in PET / CT, this allows location of tumors or tissues which may be seen on SPECT scintigraphy but are difficult to precisely locate with regard to other anatomical structures. Both PET and SPECT systems are offered by ADAC Laboratories of Milpitas, Calif., United States of America, and Siemens of Hoffman Estates, III., United States of America. Related devices for scintigraphic imaging can also be used, such as a radio-imaging device that includes a plurality of sensors with collimating structures having a common source focus.

[0394] When scintigraphic imaging is employed, the detectable label can include a radiolabel as described elsewhere herein. When the labeling moiety is a radionuclide, stabilizers to prevent or minimize radiolytic damage, such as ascorbic acid, gentisic acid, or other appropriate antioxidants, can be administered to the subject being scintigraphically imaged.

[0395] Autoradiographic detection uses a radioisotope (also referred to herein as radiolabel) as a detectable label and conventional autoradiography or a phosphorimager using methods known to one of skill in the art. In particular embodiments, an autoradiographic method employs photostimulable luminescence imaging plates (Fuji Medical Systems of Stamford, Conn., United States of America). Briefly, photostimulable luminescence is the quantity of light emitted from irradiated phosphorous plates following stimulation with a laser during scanning. The luminescent response of the plates is linearly proportional to the activity.

[0396] Another application of the antibodies disclosed herein is in the radioimmunoguided surgery (RIGS) system. This technique involves the intravenous administration of a radiolabeled antibody prior to surgery. After allowing for tumor uptake and blood clearance of radioactivity, the patient is taken to the operating room where surgical exploration is affected with the aid of a handheld gamma activity probe, e.g., Neoprobe®1000 (Neoprobe Corporation, Dublin, Ohio). This helps the surgeon identify the tumor metastases and improve the complications of excision. The RIGS system could be used in a new context, wherein the RIGS system is used to determine the infiltration of CAR (Whitlow linker-containing molecule) into a tumor.

[0397] MRI-based techniques create images based on the relative relaxation rates of water protons in unique chemical environments. As used herein, the term “magnetic resonance imaging” refers to magnetic source techniques including conventional magnetic resonance imaging, magnetization transfer imaging (MTI), proton magnetic resonance spectroscopy (MRS), diffusion- weighted imaging (DWI) and functional MR imaging.

[0398] Those skilled in the art of diagnostic labeling recognize that metal ions can be bound by chelating moieties, which in turn can be conjugated to a therapeutic agent in accordance with themethods of the presently disclosed subject matter. For example, gadolinium ions are chelated by diethylenetriaminepentaacetic acid (DTPA). Lanthanide ions are chelated by tetraazacyclododocane compounds. See U.S. Pat. Nos. 5,738,837 and 5,707,605. Alternatively, a contrast agent can be carried in a liposome.

[0399] Images derived used a magnetic source can be acquired using, for example, a superconducting quantum interference device magnetometer (SQUID, available with instruction from Quantum Design of San Diego, Calif., United States of America; see also U.S. Pat. 5,738,837).

[0400] In addition to imaging and detection, therapeutic methods are described herein. In particular embodiments, binding domains can be used to treat a subject in need thereof. In particular embodiments, a method of treating a subject in need thereof includes administering binding domains to the subject. Treating subjects includes delivering therapeutically effective amounts of the compositions and / or formulations disclosed herein (including antibodies, multidomain binding molecules, antibody conjugates, or genetically engineered cells). Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0401] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of a condition or displays only early signs or symptoms of a condition such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the condition further. Thus, a prophylactic treatment functions as a preventative treatment against a condition. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of a condition.

[0402] 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 diminishing or eliminating those signs or symptoms of the condition or effects of the condition. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the condition and / or reduce, control or eliminate side effects of the condition.

[0403] 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.

[0404] In particular embodiments, therapeutically effective amounts provide anti-cancer or antiinfection effects. Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases, prevented or reduced metastases, a decrease in tumor volume, inhibited tumor growth, an increase in life expectancy, prolonged subject life, induced chemo- orradiosensitivity in cancer cells, inhibited cancer cell proliferation, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0405] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre-malignant or malignant.

[0406] In particular embodiments, an infection includes a bacterial infection, viral infection, or parasitic infection. Anti-infection effects include a reducing or preventing a virus from infecting a cell, decreasing the number of infected cells, decreasing the volume of infected tissue, increasing lifespan, increasing life expectancy, and / or reducing or eliminating infection-associated symptoms. In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against an infection.

[0407] 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. Such information can be used to more accurately determine useful doses in subjects of interest. 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 condition, stage of condition, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0408] Effective amounts of formulations and / or compositions can range from 0.1 to 5 pg / kg or from 0.5 to 1 pg / kg. In other examples, a dose can include 1 pg / 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 or more.

[0409] Therapeutically effective amounts 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 1011.

[0410] 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, every4 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, the treatment protocol may be dictated by a clinical trial protocol or an FDA- approved treatment protocol. Therapeutically effective amounts can be administered for any duration (e.g., for one dose, for a year, for 3 years, for 5 years, for 10 years, for 15 years, for 20 years, or for life).

[0411] The pharmaceutical compositions and / or formulations described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual administration and more particularly by intravenous, intradermal, intraarterial, intraparenteral, intranasal, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral, subcutaneous, and / or sublingual injection.

[0412] In particular embodiments, a therapeutic method includes administering a composition disclosed herein. In particular embodiments, a therapeutic method includes administering a formulation disclosed herein. In particular embodiments, a therapeutic method includes administering binding domains. In particular embodiments, binding domains are used to deliver payloads, stimulate, block, and / or alter the function of Whitlow-containing molecules. In particular embodiments, binding domains are used to deliver payloads, stimulate, block, and / or alter the function of a CAR. In particular embodiments, compositions and / or formulations disclosed herein modulate Whitlow linker-containing molecules. Methods of modulating Whitlow linker-containing molecules are provided herein. The term “modulating” refers to exerting a modifying or controlling influence on a cell. In particular embodiments, modulating includes increasing activity of a cell expressing a Whitlow linker-containing molecule, decreasing activity of a cell expressing a Whitlow linker-containing molecule, increasing the avidity of a cell expressing a Whitlow linkercontaining molecule and / or retargeting the activity of a cell expressing a Whitlow linker-containing molecule.

[0413] As an example of increasing avidity of a cell expressing a Whitlow linker-containing molecule, a cell could extracellularly express a Whitlow-containing molecule linked to a receptor that binds a first antigen (e.g., a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell thatis not the cell expressing the Whitlow-containing molecule). To increase the avidity of the cell, a recombinant protein that includes a Whitlow linker binding domain disclosed herein and a receptor that binds the same antigen increases avidity.

[0414] In particular embodiments, a therapeutic method includes administering an antibody conjugate. In particular embodiments, the antibody conjugate is used to deliver payloads to a target molecule. If the target molecule is a CAR, for example, the payload might include growth factors to provide a growth signal to the CAR-expressing cell or the microenvironment. In particular embodiments, the growth factor includes a cytokine, an innate immune system stimulating molecule (e.g., TLR ligand), or a microbe-derived molecule. “Cytokine” as used herein is meant to refer to proteins released by one cell population that act on another cell as intercellular mediators. In particular embodiments, the cytokine includes interleukins (ILs) such as IL-1 , IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12; IL-15, IL-18, IL-21 , or IL-23; growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGFa and TGF[3; insulin-like growth factor-l and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and - gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte- macrophage-CSF (GM-CSF); granulocyte-CSF (G-CSF); a tumor necrosis factor such as TNF-alpha or TNF-beta; high affinity variants, engineered hybrid molecules, and other polypeptide factors. In particular embodiments, a recombinant protein including an anti-Whitlow binding domain conjugated to a cytokine, such as IL-15 could support T cell survival. Similarly, cytokines, such as IL-2, IL-21 , IL- 7 and others could support T cell survival. Other cytokines (e.g., IL-18, IL-12, IL-23) and engineered variants conjugated to an anti-Whitlow binding domain could support CAR-T cell function in the tumor microenvironment. In particular embodiments, the payload can inhibit signals, such as inhibit inflammation.

[0415] In particular embodiments, the payload can include a drug. In particular embodiments, the drug elicits a cytotoxic effect to kill the cell expressing the target molecule. In particular embodiments, an anti-Whitlow binding domain complexed to a cytotoxic moiety could be used to eliminate CAR-T cells that harbor the Whitlow linker. In particular embodiments, the antibodyconjugate delivers a drug to the environment surrounding the target molecule.

[0416] In particular embodiments, the antibody conjugate delivers a detectable label to the target molecule in order to image or detect the target molecule within the body. Antibody conjugates can deliver payloads to the target molecule, wherein the payloads provide a function such as increasing the half-life, decreasing the half-life, altering the location or circulation of the target molecule.

[0417] In particular embodiments, a therapeutic method includes administering a binding domain disclosed herein. In particular embodiments, the binding domain can bind a Whitlow linkercontaining molecule and modulate its activity. For example, the binding domain can increase, decrease, or inhibit the activity of the Whitlow linker-containing molecule. In particular embodiments, the binding domain is a stimulatory antibody. In particular embodiments, the binding domain is an inhibitory antibody. In particular embodiments, the binding domains disclosed herein can be cloned into an antibody backbone having cytolytic properties. Antibody dependent cellular cytotoxicity, is an immune mechanism through which effector cells can recognize and kill antibody-coated target cells. In particular embodiments, the binding domains cloned into antibody backbones having cytolytic properties can be used for cell depletion.

[0418] Unlike CAR T cell use in hematological malignancies, the efficacy of CAR T cells in solid tumors can be limited by the tumor microenvironment, T cell exhaustion, and the T cells’ persistence and potency. In particular embodiments, the binding domains disclosed herein can bind the target molecule (e.g., an scFv of a CAR) and either stimulate activity or block activity. For example, a stimulatory binding domain can cause specific or targeted activation of a CAR- expressing cell and induce proliferation whereas a non-stimulatory binding domain can bind and prevent stimulation through the CAR. In particular embodiments, a stimulatory binding domain promotes cell expansion of cells expressing a molecule containing the Whitlow linker. In particular embodiments, cell expansion occurs during manufacturing. In particular embodiments, cell expansion occurs in vivo. This could be used to increase in vivo CAR-T cell counts, which correlate with disease clearance, or for patients with low burden disease, or to eliminate residual disease after CAR-T cell treatment. In particular embodiments, binding domains with different stimulatory levels produce cell products with different qualities. In particular embodiments, a blocking anti-Whitlow binding domain might be used in vivo to mitigate or prevent toxicity of CAR- T cells, by allowing titrated control of cognate antigen-induced CAR-T cell activation.

[0419] In some cases, tumor cells have developed mechanisms to evade CAR-T cell recognition and elimination, such as tumor antigen escape, e.g., the expression level of tumor antigen may be reduced to a level where CAR-T cells cannot participate in and mediate cytotoxic activity. Insome cases, the tumor cells can evade killing by expressing an alternative form of the target antigen that lacks the binding epitope of the CAR. In other cases, tumor cells may evade killing by switching to genetically related but phenotypically different diseases (so-called lineage switching). Given the high cost of cellular therapies such as CAR T cell therapy - typically over $400,000 USD and sometimes over $1 million USD per patient - it is not practical to develop and administer an entirely new CAR T cell therapy program with every mutation of the malignancy. As such, the binding domains disclosed herein can be useful in altering the function of CAR that already exist in a subject from a prior administration. Specifically, a bispecific antibody including an anti-Whitlow linker binding domain and a second binding domain, can bind the Whitlow linker of a CAR. If the second binding domain binds a second antigen, the CAR can now bind the second antigen and elicit cytotoxic effects on cells expressing the second antigen (e.g., cancer antigen).

[0420] In particular embodiments, the binding domains disclosed herein can modulate the function of a target molecule. In particular embodiments modulating includes retargeting. In particular embodiments, a therapeutic method includes administering a multi-domain binding molecule to retarget a pre-existing Whitlow linker-containing molecule. For example, a binding domain can be engineered into a bispecific antibody. In particular embodiments, the bispecific antibody includes a first binding domain that binds the Whitlow linker and a second binding domain that binds a second antigen. In particular embodiments, the second antigen includes a cancer antigen, viral antigen, bacterial antigen, or parasitic antigen. In particular embodiments, the first binding domain binds the Whitlow linker-containing molecule and the second binding domain is exposed and acts as a target binding domain. This method can retarget the use of, for example, a CAR that was administered to bind a first antigen into binding a second antigen. In particular embodiments, a target binding domain binds a target antigen. In particular embodiments, the target antigen includes a cancer antigen, viral antigen, bacterial antigen, a parasitic antigen, a degenerative cell antigen, or an autoantigen.

[0421] As an example of retargeting a cell expressing a Whitlow linker-containing molecule, a cell could extracellularly express a Whitlow-containing molecule linked to a receptor that binds a first antigen (e.g., a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule). To retarget the cell, a recombinant protein that includes a Whitlow linker binding domain disclosed herein and a receptor that binds a different antigen retargets the cell to an additional antigen type.

[0422] Modulating activity can also include affecting the activity of the Whitlow linker-containing molecule itself rather than or in addition to modulating the activity of the cell that expresses it.

[0423] Because the second binding domain can be changed to bind almost any antigen, therapeutic treatments can treat many conditions including cancer, disease, or infection. Example of cancers include bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, gall bladder cancer, gastrointestinal cancer, glioma, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, neuroblastoma, oral cancer, oropharyngeal cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, sarcoma, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, or vaginal cancer. Examples of diseases include diseases where depletion of a select cell population is desired. An example disease includes autoimmune disorders such as systemic lupus, rheumatoid arthritis, thyroid diseases, celiac disease, vitiligo, vasculitis, Addison disease, dermatomyositis, inflammatory bowel disease, multiple sclerosis, myasthenia gravis, pernicious anemia, psoriasis, reactive arthritis, Sjogren syndrome, or type I diabetes. For autoimmune disorders, an autoantigen can be a target antigen. An example disease includes degenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington’s disease, Lewy body disease, Parkinson’s disease, spinal muscular atrophy. For degenerative diseases, the target antigen can be a degenerative cell antigen. Examples of infection include bacterial infection (e.g., Clostridioides difficile, bacterial pneumonia, Otitis media, meningitis, tuberculosis), viral infection (e.g., influenza, coronavirus, human immunodeficiency virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papillomavirus (HPV)), or parasitic infection (e.g., giardiasis, trichomoniasis, pinworm, malaria, hookworm, leishmaniasis, toxoplasmosis, schistosomiasis).

[0424] In particular embodiments, a CAR T cell targeting a first antigen and harboring a Whitlow linker can be redirected to target a second antigen and / or a third antigen by administering a multispecific antibody with an anti-Whitlow binding domain and a binding domain targeting the second antigen and / or third antigen. In particular embodiments, a CD19 CAR T cell harboring a Whitlow linker can be redirected to target CD22 and / or CD20 on B cell malignancies by administering a multispecific antibody with an anti-Whitlow binding domain and an anti-CD22 and / or anti-CD20 binding domain. In particular embodiments, a CD33 CAR T cell harboring a Whitlow linker can be redirected to target CLL1 and / or CD123 on myeloid malignancies by administering a multispecific antibody with an anti-Whitlow binding domain and an anti-CLL1 and / or anti-CD123 binding domain.

[0425] Furthermore, in particular embodiments, , an anti-Whitlow binding domain can serve as a bridge for a universal binder system. In particular embodiments, a recombinant receptor (e.g., CAR) is generated with a binding domain containing an anti-Whitlow binding domain (e.g., scFv). Cells can be genetically modified to express the recombinant receptor and the recombinantreceptor would not be reactive against a target in vivo. In particular embodiments, the recombinant receptor-expressing cells could be activated by infusion of a recombinant molecule that includes the Whitlow linker conjugated to a second binding domain (e.g., an scFv against a tumor antigen or a ligand for a receptor on a tumor cell). In other embodiments, the cell is further genetically modified to express the recombinant molecule. This would allow multiple specificities to be targeted by one universal binder product. Alternatively, in particular embodiments, a universal binder system includes a recombinant receptor (e.g., CAR) expressed by a cell, wherein the recombinant receptor includes a Whitlow linker binding domain. In particular embodiments, the recombinant receptor can be targeted to a second binding domain by administering a multidomain binding molecule (or recombinant molecule) including an anti-Whitlow-binding domain and a second binding domain. In particular embodiments, the recombinant receptor can be targeted to a second binding domain by genetically modifying the cell to express a multi-domain binding molecule (or recombinant molecule) including an anti-Whitlow-binding domain and a second binding domain.

[0426] (xiv) Kits. Also provided herein are kits including one or more containers including one or more of the binding domains described herein. Kits may be formed with components to practice, for example, the methods described herein. In particular embodiments, the kit includes an antiWhitlow binding domain, an anti-Whitlow antibody, a multi-domain binding molecule, an antibody conjugate, a multimerized antibody, or sequences encoding an anti-Whitlow antibody, a multidomain binding molecule, or an antibody conjugate as described herein. 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 materials useful in sample processing, washing, or conducting any other step of the methods described herein. Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, in which notice reflects approval by the agency of manufacture, use, or sale for human administration.

[0427] 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.

[0428] 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 the present 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.

[0429] (xv) Exemplary Embodiments.1. A binding domain that binds a Whitlow-containing molecule, wherein the binding domain includes 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 , CDRL2, and CDRL3; wherein: the CDRH1 includes the sequence of SEQ ID NO: 46, the CDRH2 includes the sequence of SEQ I D NO: 100, and the CDRH3 includes the sequence of SEQ I D NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 52, the CDRH2 includes the sequence of SEQ I D NO: 101 , and the CDRH3 includes the sequence of SEQ I D NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 102, the CDRH2 includes the sequence of SEQ I D NO: 103, and the CDRH3 includes the sequence of SEQ I D NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 56, the CDRL2 includes the sequence GAS, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 209, the CDRH2 includes the sequence of SEQ I D NO: 105, and the CDRH3 includes the sequence of SEQ I D NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; the CDRH1 includes the sequence of SEQ ID NO: 60, the CDRH2 includes the sequence of SEQ ID NO: 106, and the CDRH3 includes the sequence of SEQ ID NO: 107, and the CDRL1 includes the sequence of SEQ ID NO: 63, the CDRL2 includes the sequence of SEQ ID NO: 64, and the CDRL3 includes the sequence of SEQ ID NO: 65 according to Contact;the CDRH1 includes the sequence of SEQ ID NO: 57, the CDRH2 includes the sequence of SEQ I D NO: 105, and the CDRH3 includes the sequence of SEQ I D NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; the CDRH1 includes the sequence of SEQ ID NO: 46, the CDRH2 includes the sequence of SEQ ID NO: 47, and the CDRH3 includes the sequence of SEQ ID NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 52, the CDRH2 includes the sequence of SEQ ID NO: 53, and the CDRH3 includes the sequence of SEQ ID NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 102, the CDRH2 includes the sequence of SEQ ID NO: 54, and the CDRH3 includes the sequence of SEQ ID NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 56, the CDRL2 includes the sequence GAS, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 209, the CDRH2 includes the sequence of SEQ ID NO: 58, and the CDRH3 includes the sequence of SEQ ID NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; the CDRH1 includes the sequence of SEQ ID NO: 60, the CDRH2 includes the sequence of SEQ ID NO: 61 , and the CDRH3 includes the sequence of SEQ ID NO: 62, and the CDRL1 includes the sequence of SEQ ID NO: 63, the CDRL2 includes the sequence of SEQ ID NO: 64, and the CDRL3 includes the sequence of SEQ ID NO: 65 according to Contact; the CDRH1 includes the sequence of SEQ ID NO: 66, the CDRH2 includes the sequence of SEQ ID NO: 67, and the CDRH3 includes the sequence of SEQ ID NO: 68, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 70, and the CDRL3 includes the sequence of SEQ ID NO: 71according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 210, the CDRH2 includes the sequence of SEQ ID NO: 73, and the CDRH3 includes the sequence of SEQ ID NO: 68, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 70, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 211 , the CDRH2 includes the sequence of SEQ ID NO: 75, and the CDRH3 includes the sequence of SEQ ID NO: 212, and the CDRL1 includes the sequence of SEQ ID NO: 77, the CDRL2 includes the sequence KVS, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 213, the CDRH2 includes the sequence of SEQ ID NO: 79, and the CDRH3 includes the sequence of SEQ ID NO: 212, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 80, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to North; the CDRH1 includes the sequence of SEQ ID NO: 81 , the CDRH2 includes the sequence of SEQ ID NO: 214, and the CDRH3 includes the sequence of SEQ ID NO: 215, and the CDRL1 includes the sequence of SEQ ID NO: 84, the CDRL2 includes the sequence of SEQ ID NO: 85, and the CDRL3 includes the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 includes the sequence of SEQ ID NO: 46, the CDRH2 includes the sequence of SEQ I D NO: 108, and the CDRH3 includes the sequence of SEQ I D NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 52, the CDRH2 includes the sequence of SEQ I D NO: 109, and the CDRH3 includes the sequence of SEQ I D NO: 48, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 50, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 102, the CDRH2 includes the sequence of SEQ ID NO: 110, and the CDRH3 includes the sequence of SEQ ID NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 56, the CDRL2 includes thesequence GAS, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 209, the CDRH2 includes the sequence of SEQ I D NO: 111 , and the CDRH3 includes the sequence of SEQ I D NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; the CDRH1 includes the sequence of SEQ ID NO: 60, the CDRH2 includes the sequence of SEQ ID NO: 112, and the CDRH3 includes the sequence of SEQ ID NO: 62, and the CDRL1 includes the sequence of SEQ ID NO: 63, the CDRL2 includes the sequence of SEQ ID NO: 216, and the CDRL3 includes the sequence of SEQ ID NO: 65 according to Contact; the CDRH1 includes the sequence of SEQ ID NO: 52, the CDRH2 includes the sequence of SEQ ID NO: 54, and the CDRH3 includes the sequence of SEQ ID NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 56, the CDRL2 includes the sequence GAS, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 57, the CDRH2 includes the sequence of SEQ ID NO: 58, and the CDRH3 includes the sequence of SEQ ID NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; the CDRH1 includes the sequence of SEQ ID NO: 72, the CDRH2 includes the sequence of SEQ ID NO: 73, and the CDRH3 includes the sequence of SEQ ID NO: 68, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 70, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 74, the CDRH2 includes the sequence of SEQ ID NO: 75, and the CDRH3 includes the sequence of SEQ ID NO: 76, and the CDRL1 includes the sequence of SEQ ID NO: 77, the CDRL2 includes the sequence KVS, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 78, the CDRH2 includes the sequence of SEQ ID NO: 79, and the CDRH3 includes the sequence of SEQ ID NO: 76,and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 80, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to North; the CDRH1 includes the sequence of SEQ ID NO: 81 , the CDRH2 includes the sequence of SEQ ID NO: 82, and the CDRH3 includes the sequence of SEQ ID NO: 83, and the CDRL1 includes the sequence of SEQ ID NO: 84, the CDRL2 includes the sequence of SEQ ID NO: 85, and the CDRL3 includes the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 includes the sequence of SEQ ID NO: 87, the CDRH2 includes the sequence of SEQ ID NO: 88, and the CDRH3 includes the sequence of SEQ ID NO: 89, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 70, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to Kabat; the CDRH1 includes the sequence of SEQ ID NO: 90, the CDRH2 includes the sequence of SEQ ID NO: 91 , and the CDRH3 includes the sequence of SEQ ID NO: 89, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 70, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 includes the sequence of SEQ ID NO: 92, the CDRH2 includes the sequence of SEQ ID NO: 93, and the CDRH3 includes the sequence of SEQ ID NO: 94, and the CDRL1 includes the sequence of SEQ ID NO: 77, the CDRL2 includes the sequence KVS, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 includes the sequence of SEQ ID NO: 95, the CDRH2 includes the sequence of SEQ ID NO: 96, and the CDRH3 includes the sequence of SEQ ID NO: 94, and the CDRL1 includes the sequence of SEQ ID NO: 69, the CDRL2 includes the sequence of SEQ ID NO: 80, and the CDRL3 includes the sequence of SEQ ID NO: 71 according to North; the CDRH1 includes the sequence of SEQ ID NO: 97, the CDRH2 includes the sequence of SEQ ID NO: 98, and the CDRH3 includes the sequence of SEQ ID NO: 99, and the CDRL1 includes the sequence of SEQ ID NO: 84, the CDRL2 includes the sequence of SEQ ID NO: 85, and the CDRL3 includes the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 includes the sequence of SEQ ID NO: 57, the CDRH2 includes thesequence of SEQ I D NO: 111 , and the CDRH3 includes the sequence of SEQ I D NO: 55, and the CDRL1 includes the sequence of SEQ ID NO: 49, the CDRL2 includes the sequence of SEQ ID NO: 59, and the CDRL3 includes the sequence of SEQ ID NO: 51 according to North; or the CDRH1 includes the sequence of SEQ ID NO: 60, the CDRH2 includes the sequence of SEQ ID NO: 112, and the CDRH3 includes the sequence of SEQ ID NO: 62, and the CDRL1 includes the sequence of SEQ ID NO: 63, the CDRL2 includes the sequence of SEQ ID NO: 64, and the CDRL3 includes the sequence of SEQ ID NO: 65 according to Contact. The binding domain of embodiment 1 , wherein the binding domain is humanized or murine. The binding domain of embodiments 1 or 2, wherein the binding domain is humanized. The binding domain of any of embodiments 1-3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 238 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1 -4, wherein the variable heavy chain includes the sequence of SEQ ID NO: 238 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1 -3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 239 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 6, wherein the variable heavy chain includes the sequence of SEQ ID NO: 239 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1 -3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 240 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 8, wherein the variable heavy chain includes the sequence of SEQ ID NO: 240 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1 -3, wherein the variable heavy chain includesa sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 241 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 246. The binding domain of any of embodiments 1-3 or 10, wherein the variable heavy chain includes the sequence of SEQ ID NO: 241 and the variable light chain includes the sequence of SEQ ID NO: 246. The binding domain of any of embodiments 1-3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 241 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 12, wherein the variable heavy chain includes the sequence of SEQ ID NO: 241 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 242 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 14, wherein the variable heavy chain includes the sequence of SEQ ID NO: 242 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 243 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 16, wherein the variable heavy chain includes the sequence of SEQ ID NO: 243 and the variable light chain includes the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1 -3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 244 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245. The binding domain of any of embodiments 1-3 or 18, wherein the variable heavy chain includes the sequence of SEQ ID NO: 244 and the variable light chain includes the sequence of SEQ ID NO: 245.The binding domain of any of embodiments 1 -3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 218 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225. The binding domain of any of embodiments 1-3 or 20, wherein the variable heavy chain includes the sequence of SEQ ID NO: 218 and the variable light chain includes the sequence of SEQ ID NO: 225. The binding domain of any of embodiments 1-3, wherein the variable heavy chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 219 and the variable light chain includes a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 226. The binding domain of any of embodiments 1-3 or 22, wherein the variable heavy chain includes the sequence...

Claims

CLAIMSWhat is claimed is:

1. A binding domain that binds a Whitlow-containing molecule, wherein the binding domain comprises 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 , CDRL2, and CDRL3; wherein: the CDRH1 comprises the sequence of SEQ ID NO: 46, the CDRH2 comprises the sequence of SEQ ID NO: 100, and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 52, the CDRH2 comprises the sequence of SEQ ID NO: 101 , and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 102, the CDRH2 comprises the sequence of SEQ ID NO: 103, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 56, the CDRL2 comprises the sequence GAS, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 209, the CDRH2 comprises the sequence of SEQ ID NO: 105, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 60, the CDRH2 comprises the sequence of SEQ ID NO: 106, and the CDRH3 comprises the sequence of SEQ ID NO: 107, and the CDRL1 comprises the sequence of SEQ ID NO: 63, the CDRL2 comprises the sequence of SEQ ID NO: 64, and the CDRL3 comprises the sequence of SEQ ID NO: 65 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 57, the CDRH2 comprises the sequence of SEQ ID NO: 105, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprisesthe sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 46, the CDRH2 comprises the sequence of SEQ ID NO: 47, and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 52, the CDRH2 comprises the sequence of SEQ ID NO: 53, and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 102, the CDRH2 comprises the sequence of SEQ ID NO: 54, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 56, the CDRL2 comprises the sequence GAS, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 209, the CDRH2 comprises the sequence of SEQ ID NO: 58, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 60, the CDRH2 comprises the sequence of SEQ ID NO: 61 , and the CDRH3 comprises the sequence of SEQ ID NO: 62, and the CDRL1 comprises the sequence of SEQ ID NO: 63, the CDRL2 comprises the sequence of SEQ ID NO: 64, and the CDRL3 comprises the sequence of SEQ ID NO: 65 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 66, the CDRH2 comprises the sequence of SEQ ID NO: 67, and the CDRH3 comprises the sequence of SEQ ID NO: 68, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 70, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 210, the CDRH2 comprises the sequence of SEQ ID NO: 73, and the CDRH3 comprises the sequence of SEQ ID NO:68, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 70, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 211 , the CDRH2 comprises the sequence of SEQ ID NO: 75, and the CDRH3 comprises the sequence of SEQ ID NO: 212, and the CDRL1 comprises the sequence of SEQ ID NO: 77, the CDRL2 comprises the sequence KVS, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 213, the CDRH2 comprises the sequence of SEQ ID NO: 79, and the CDRH3 comprises the sequence of SEQ ID NO: 212, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 80, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 81 , the CDRH2 comprises the sequence of SEQ ID NO: 214, and the CDRH3 comprises the sequence of SEQ ID NO: 215, and the CDRL1 comprises the sequence of SEQ ID NO: 84, the CDRL2 comprises the sequence of SEQ ID NO: 85, and the CDRL3 comprises the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 46, the CDRH2 comprises the sequence of SEQ ID NO: 108, and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 52, the CDRH2 comprises the sequence of SEQ ID NO: 109, and the CDRH3 comprises the sequence of SEQ ID NO: 48, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 50, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 102, the CDRH2 comprises the sequence of SEQ ID NO: 110, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 56, the CDRL2 comprises the sequence GAS, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 209, the CDRH2 comprisesthe sequence of SEQ ID NO: 111 , and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 60, the CDRH2 comprises the sequence of SEQ ID NO: 112, and the CDRH3 comprises the sequence of SEQ ID NO: 62, and the CDRL1 comprises the sequence of SEQ ID NO: 63, the CDRL2 comprises the sequence of SEQ ID NO: 216, and the CDRL3 comprises the sequence of SEQ ID NO: 65 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 52, the CDRH2 comprises the sequence of SEQ ID NO: 54, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 56, the CDRL2 comprises the sequence GAS, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 57, the CDRH2 comprises the sequence of SEQ ID NO: 58, and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO: 51 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 72, the CDRH2 comprises the sequence of SEQ ID NO: 73, and the CDRH3 comprises the sequence of SEQ ID NO: 68, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 70, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 74, the CDRH2 comprises the sequence of SEQ ID NO: 75, and the CDRH3 comprises the sequence of SEQ ID NO: 76, and the CDRL1 comprises the sequence of SEQ ID NO: 77, the CDRL2 comprises the sequence KVS, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 78, the CDRH2 comprises the sequence of SEQ ID NO: 79, and the CDRH3 comprises the sequence of SEQ ID NO: 76, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 80, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to North;the CDRH1 comprises the sequence of SEQ ID NO: 81 , the CDRH2 comprises the sequence of SEQ ID NO: 82, and the CDRH3 comprises the sequence of SEQ ID NO: 83, and the CDRL1 comprises the sequence of SEQ ID NO: 84, the CDRL2 comprises the sequence of SEQ ID NO: 85, and the CDRL3 comprises the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 87, the CDRH2 comprises the sequence of SEQ ID NO: 88, and the CDRH3 comprises the sequence of SEQ ID NO: 89, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 70, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to Kabat; the CDRH1 comprises the sequence of SEQ ID NO: 90, the CDRH2 comprises the sequence of SEQ ID NO: 91 , and the CDRH3 comprises the sequence of SEQ ID NO: 89, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 70, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to Chothia; the CDRH1 comprises the sequence of SEQ ID NO: 92, the CDRH2 comprises the sequence of SEQ ID NO: 93, and the CDRH3 comprises the sequence of SEQ ID NO: 94, and the CDRL1 comprises the sequence of SEQ ID NO: 77, the CDRL2 comprises the sequence KVS, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to IMGT; the CDRH1 comprises the sequence of SEQ ID NO: 95, the CDRH2 comprises the sequence of SEQ ID NO: 96, and the CDRH3 comprises the sequence of SEQ ID NO: 94, and the CDRL1 comprises the sequence of SEQ ID NO: 69, the CDRL2 comprises the sequence of SEQ ID NO: 80, and the CDRL3 comprises the sequence of SEQ ID NO: 71 according to North; the CDRH1 comprises the sequence of SEQ ID NO: 97, the CDRH2 comprises the sequence of SEQ ID NO: 98, and the CDRH3 comprises the sequence of SEQ ID NO: 99, and the CDRL1 comprises the sequence of SEQ ID NO: 84, the CDRL2 comprises the sequence of SEQ ID NO: 85, and the CDRL3 comprises the sequence of SEQ ID NO: 86 according to Contact; the CDRH1 comprises the sequence of SEQ ID NO: 57, the CDRH2 comprises the sequence of SEQ ID NO: 111 , and the CDRH3 comprises the sequence of SEQ ID NO: 55, and the CDRL1 comprises the sequence of SEQ ID NO: 49, the CDRL2 comprises the sequence of SEQ ID NO: 59, and the CDRL3 comprises the sequence of SEQ ID NO:51 according to North; or the CDRH1 comprises the sequence of SEQ ID NO: 60, the CDRH2 comprises the sequence of SEQ ID NO: 112, and the CDRH3 comprises the sequence of SEQ ID NO: 62, and the CDRL1 comprises the sequence of SEQ ID NO: 63, the CDRL2 comprises the sequence of SEQ ID NO: 64, and the CDRL3 comprises the sequence of SEQ ID NO: 65 according to Contact.

2. The binding domain of claim 1 , wherein the binding domain is humanized or murine.

3. The binding domain of claim 1 , wherein the binding domain is humanized.

4. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 238 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

5. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 238 and the variable light chain comprises the sequence of SEQ ID NO: 245.

6. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 239 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

7. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 239 and the variable light chain comprises the sequence of SEQ ID NO: 245.

8. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 240 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

9. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 240 and the variable light chain comprises the sequence of SEQ ID NO: 245.

10. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 241 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 246.

11. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequenceof SEQ ID NO: 241 and the variable light chain comprises the sequence of SEQ ID NO: 246.

12. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 241 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

13. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 241 and the variable light chain comprises the sequence of SEQ ID NO: 245.

14. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 242 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

15. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 242 and the variable light chain comprises the sequence of SEQ ID NO: 245.

16. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 243 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

17. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 243 and the variable light chain comprises the sequence of SEQ ID NO: 245.

18. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 244 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 245.

19. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 244 and the variable light chain comprises the sequence of SEQ ID NO: 245.

20. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 218 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225.

21. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 218 and the variable light chain comprises the sequence of SEQ ID NO:225.

22. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 219 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 226.

23. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 219 and the variable light chain comprises the sequence of SEQ ID NO:226.

24. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 220 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 227.

25. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 220 and the variable light chain comprises the sequence of SEQ ID NO:227.

26. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 220 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225.

27. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 220 and the variable light chain comprises the sequence of SEQ ID NO: 225.

28. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 221 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225.

29. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 221 and the variable light chain comprises the sequence of SEQ ID NO: 225.

30. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 222 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequenceof SEQ ID NO: 225.

31. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 222 and the variable light chain comprises the sequence of SEQ ID NO: 225.

32. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 223 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225.

33. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 223 and the variable light chain comprises the sequence of SEQ ID NO: 225.

34. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 224 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 225.

35. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 224 and the variable light chain comprises the sequence of SEQ ID NO: 225.

36. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 228 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

37. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 228 and the variable light chain comprises the sequence of SEQ ID NO: 235.

38. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 229 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

39. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 229 and the variable light chain comprises the sequence of SEQ ID NO: 235.

40. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 230 and the variablelight chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

41. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 230 and the variable light chain comprises the sequence of SEQ ID NO: 235.

42. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 231 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

43. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 231 and the variable light chain comprises the sequence of SEQ ID NO:235.

44. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 232 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 236.

45. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 232 and the variable light chain comprises the sequence of SEQ ID NO:236.

46. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 233 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

47. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 233 and the variable light chain comprises the sequence of SEQ ID NO: 235.

48. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 234 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 237.

49. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 234 and the variable light chain comprises the sequence of SEQ ID NO:237.

50. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequencehaving at least 95% sequence identity to the sequence of SEQ I D NO: 234 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 235.

51. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 234 and the variable light chain comprises the sequence of SEQ ID NO: 235.

52. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 247 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

53. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 247 and the variable light chain comprises the sequence of SEQ ID NO: 254.

54. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 248 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

55. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 248 and the variable light chain comprises the sequence of SEQ ID NO:254.

56. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 249 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 255.

57. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 249 and the variable light chain comprises the sequence of SEQ ID NO:255.

58. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 250 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 256.

59. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 250 and the variable light chain comprises the sequence of SEQ ID NO:256.

60. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 250 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

61. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 250 and the variable light chain comprises the sequence of SEQ ID NO: 254.

62. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 251 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

63. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 251 and the variable light chain comprises the sequence of SEQ ID NO: 254.

64. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 252 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

65. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 252 and the variable light chain comprises the sequence of SEQ ID NO: 254.

66. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ I D NO: 253 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 254.

67. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 253 and the variable light chain comprises the sequence of SEQ ID NO: 254.

68. The binding domain of claim 1 , wherein the binding domain is murine.

69. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 8 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3.

70. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequenceof SEQ ID NO: 8 and the variable light chain comprises the sequence of SEQ ID NO: 3.

71. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 2 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3.

72. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 2 and the variable light chain comprises the sequence of SEQ ID NO: 3.

73. The binding domain of claim 1 , wherein the variable heavy chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 12 and the variable light chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 13.

74. The binding domain of claim 1, wherein the variable heavy chain is encoded by the sequence of SEQ ID NO: 12 and the variable light chain is encoded by the sequence of SEQ ID NO: 13.

75. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 4 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5.

76. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 4 and the variable light chain comprises the sequence of SEQ ID NO: 5.

77. The binding domain of claim 1 , wherein the variable heavy chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 14 and the variable light chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 15.

78. The binding domain of claim 1, wherein the variable heavy chain is encoded by the sequence of SEQ ID NO: 14 and the variable light chain is encoded by the sequence of SEQ ID NO: 15.

79. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 6 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 5.

80. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 6 and the variable light chain comprises the sequence of SEQ ID NO: 5.

81. The binding domain of claim 1 , wherein the variable heavy chain is encoded by asequence having at least 95% sequence identity to the sequence of SEQ ID NO: 16 and the variable light chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 15.

82. The binding domain of claim 1, wherein the variable heavy chain is encoded by the sequence of SEQ ID NO: 16 and the variable light chain is encoded by the sequence of SEQ ID NO: 15.

83. The binding domain of claim 1, wherein the variable heavy chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 18 and the variable light chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 13.

84. The binding domain of claim 1, wherein the variable heavy chain is encoded by the sequence of SEQ ID NO: 18 and the variable light chain is encoded by the sequence of SEQ ID NO: 13.

85. The binding domain of claim 1 , wherein the variable heavy chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 10 and the variable light chain comprises a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 3.

86. The binding domain of claim 1 , wherein the variable heavy chain comprises the sequence of SEQ ID NO: 10 and the variable light chain comprises the sequence of SEQ ID NO: 3.

87. The binding domain of claim 1 , wherein the variable heavy chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 20 and the variable light chain is encoded by a sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 13.

88. The binding domain of claim 1, wherein the variable heavy chain is encoded by the sequence of SEQ ID NO: 20 and the variable light chain is encoded by the sequence of SEQ ID NO: 13.

89. The binding domain of claim 1 , comprising a heavy chain constant region and a light chain constant region.

90. The binding domain of claim 89, wherein the heavy chain constant region comprises the heavy chain constant region of an IgG antibody, an IgA antibody, an IgM antibody, an IgD antibody, or an IgE antibody.

91. The binding domain of claim 89, wherein the heavy chain constant region comprises the heavy chain constant region of an IgG antibody.

92. The binding domain of claim 91 , wherein the IgG antibody comprises an lgG1 antibody,an lgG2 antibody, an lgG3 antibody, or an lgG4 antibody.

93. The binding domain of claim 91 , wherein the IgG antibody comprises an lgG1 antibody.

94. The binding domain of claim 91 , wherein the IgG antibody comprises an lgG2 antibody.

95. The binding domain of claim 94, wherein the lgG2 antibody comprises an lgG2a antibody.

96. The binding domain of claim 89, wherein the light chain constant region comprises an IgK light chain constant region or an IgA light chain constant region.

97. The binding domain of claim 89, wherein the light chain constant region comprises an IgK light chain constant region.

98. The binding domain of claim 1 , wherein the binding domain is an scFv or a Fab.

99. The binding domain of claim 1 , wherein the light chain variable region further comprises a signal peptide.

100. The binding domain of claim 99, wherein the signal peptide comprises the sequence of SEQ ID NO: 44 or SEQ ID NO: 45.

101. The binding domain of claim 1 , wherein the heavy chain variable region further comprises a signal peptide.

102. The binding domain of claim 101 , wherein the signal peptide comprises the sequence of SEQ ID NO: 42 or SEQ ID NO: 43.

103. A multi-domain binding molecule comprising at least two binding domains wherein at least one binding domain comprises the binding domain of claim 1.

104. The multi-domain binding molecule of claim 103, wherein the at least two binding domains comprise at least two copies of the binding domain of claim 1.

105. The multi-domain binding molecule of claim 104, wherein the at least two copies are joined by a protein linker.

106. The multi-domain binding molecule of claim 105, wherein the protein linker is a Gly-Ser linker.

107. The multi-domain binding molecule of claim 106, wherein the Gly-Ser linker is (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.

108. The multi-domain binding molecule of claim 103, comprising 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the binding domain of claim 1 .

109. The multi-domain binding molecule of claim 103, wherein the multi-domain binding molecule is a dimer, trimer, tetramer, pentamer, hexamer, or heptamer.

110. The multi-domain binding molecule of claim 104, wherein the at least two copies are linked to an Fc region of an antibody.

111. The multi-domain binding molecule of claim 110, wherein the Fc region is an IgA Fc region or an IgM Fc region.

112. The multi-domain binding molecule of claim 111 , wherein the Fc region comprises a multimerizing fragment of the IgA Fc region or a multimerizing fragment of the IgM Fc region.

113. The multi-domain binding molecule of claim 103, wherein the multi-domain binding molecule comprises a binding domain that binds a target antigen.

114. The multi-domain binding molecule of claim 113, wherein the target antigen comprises a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

115. The multi-domain binding molecule of claim 103, wherein the multi-domain binding molecule comprises an immune cell engaging molecule.

116. The multi-domain binding molecule of claim 115, wherein the immune cell engaging molecule modulates activity of a B cell, T cell, natural killer (NK) cell, or macrophage.

117. The multi-domain binding molecule of claim 116, 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.

118. The multi-domain binding molecule of claim 115, wherein a binding domain of the immune cell engaging molecule binds CD3, CD28, CD8, NKG2D, CDS, 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.

119. A conjugate comprising the binding domain of claim 1 , linked to a drug, a toxin, a detectable label, a radioisotope, or a particle.

120. The conjugate of claim 119, wherein the drug comprises a cell stimulatory factor.

121. The conjugate of claim 120, wherein the cell stimulatory factor comprises a growth factor, a cytokine, or a chemokine.

122. The conjugate of claim 119, wherein the drug comprises a cytotoxic drug.

123. The conjugate of claim 122, wherein the cytotoxic 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.

124. The conjugate of claim 119, wherein the toxin comprises a plant toxin or bacterial toxin.

125. The conjugate of claim 124, wherein the plant toxin comprises ricin, abrin, mistletoe lectin, modeccin, pokeweed antiviral protein, saporin, Bryodin 1 , bouganin, or gelonin.

126. The conjugate of claim 124, wherein the bacterial toxin comprises diphtheria toxin or Pseudomonas exotoxin.

127. The conjugate of claim 119, wherein the detectable label comprises a fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, an affinity tag, an oligonucleotide, or a radioisotope.

128. The conjugate of claim 127, wherein the fluorescent label comprises blue fluorescent protein, cyan fluorescent protein, green fluorescent protein, luciferase, orange fluorescent protein, red fluorescent protein, far red fluorescent protein, or yellow fluorescent protein.

129. The conjugate of claim 127, wherein the chemiluminescent label comprises lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, or oxalate ester.

130. The conjugate of claim 127, wherein the spectral colorimetric label comprises colloidal gold.

131. The conjugate of claim 127, wherein the enzymatic label comprises malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, betagalactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, or acetylcholinesterase.

132. The conjugate of claim 127, wherein the affinity tag comprises a tag with the sequence of SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, or SEQ ID NO: 205.

133. The conjugate of claim 127, wherein the oligonucleotide comprises a barcode or aunique molecular identifier (UMI).

134. The conjugate of claim 127, wherein the radioisotope comprises228Ac,111Ag, 124Am,74As,211At,209At,194Au,128Ba,7Be,206Bi,245Bk,246Bk,76Br,11C,14C,47Ca,254Cf, 242Cm,51Cr,S7Cu,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,1S9Lu,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.

135. A recombinant receptor that, when expressed by a cell, comprises an extracellular component comprising the binding domain of claim 1.

136. The recombinant receptor of claim 135, wherein the binding domain comprises an scFv.

137. The recombinant receptor of claim 135, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR).

138. The recombinant receptor of claim 137, wherein the CAR comprises an intracellular component linked to the extracellular component by a transmembrane domain.

139. The recombinant receptor of claim 138, wherein the intracellular component comprises an effector domain comprising: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3^, CD27, CD28, CD40, BAFFR, DAP10, ICOS, LAG3, NKG2D, NOTCH 1 , 0X40, ROR2, SI.AMF1 , TCRa, TCR , TRIM, Wnt, Zap70, or a combination thereof.

140. The recombinant receptor of claim 138, 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.

141. The recombinant receptor of claim 137, wherein the CAR further comprises a spacer region.

142. A nucleic acid encoding the binding domain of claim 1 , the multi-domain binding molecule of claim 103, the conjugate of claim 119, and / or the recombinant receptor of claim 135.

143. The nucleic acid of claim 142, wherein the nucleic acid comprises DNA or RNA.

144. The nucleic acid of claim 143, wherein the DNA comprises plasmid DNA.

145. The nucleic acid of claim 143, wherein the RNA comprises synthetic RNA.

146. The nucleic acid of claim 145, wherein the synthetic RNA comprises in vitro transcribed RNA.

147. The nucleic acid of claim 142, wherein the nucleic acid encodes a recombinant receptor and a recombinant molecule.

148. The nucleic acid of claim 147, wherein the recombinant receptor comprises an extracellular binding domain comprising the binding domain of claim 1 ; and the recombinant molecule comprises a Whitlow-containing molecule and a target binding domain.

149. The nucleic acid of claim 147, wherein the recombinant receptor comprises an extracellular binding domain comprising a Whitlow-containing molecule; and the recombinant molecule comprises a multi-domain binding molecule.

150. The nucleic acid of claim 149, wherein the multi-domain binding molecule comprises the binding domain of claim 1 and a target binding domain.

151. A vector comprising the nucleic acid of claim 142.

152. The vector of claim 151, wherein the vector is a viral vector.

153. The vector of claim 152, wherein the viral vector is a lentiviral vector.

154. A nanoparticle comprising the nucleic acid of claim 142.

155. A cell genetically modified to express the binding domain of claim 1, the multidomain binding molecule of claim 103, the conjugate of claim 119, and / or the recombinant receptor of claim 135.

156. The cell of claim 155, wherein the cell is genetically modified to express a recombinant receptor and a recombinant molecule.

157. The cell of claim 156, wherein the cell is genetically modified to express multiple copies of the recombinant receptor or multiple copies of a Whitlow-containing molecule.

158. The cell of claim 156, wherein the cell is genetically modified to express multiple copies of the recombinant molecule or multiple copies of a Whitlow-containing molecule.

159. The cell of claim 156, wherein the recombinant receptor comprises an extracellular component comprising the binding domain of claim 1 ; and the recombinant molecule comprises a Whitlow-containing molecule and a target binding domain.

160. The cell of claim 159, wherein the target binding domain binds a target antigen selected from a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that isnot the cell expressing the Whitlow-containing molecule.

161. The cell of claim 156, wherein the recombinant receptor comprises an extracellular binding domain comprising a Whitlow-containing molecule; and the recombinant molecule comprises a multi-domain binding molecule.

162. The cell of claim 161 , wherein the multi-domain binding molecule comprises the binding domain of claim 1 and a target binding domain.

163. The cell of claim 162, wherein the target binding domain binds a target antigen selected from a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

164. The cell of claim 156, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

165. The cell of claim 155, wherein the cell is a stem cell.

166. The cell of claim 165, wherein the stem cell is a hematopoietic stem cell or an induced pluripotent stem cell.

167. The cell of claim 155, wherein the cell is an immune cell.

168. The cell of claim 167, wherein the immune cell is a T cell, B cell, natural killer cell, or macrophage.

169. The cell of claim 155, wherein the cell is an epithelial cell, a muscle cell, a neural cell, or a connective tissue cell.

170. A composition comprising the binding domain of claim 1 , the multi-domain binding molecule of claim 103, the conjugate of claim 119, the nucleic acid of claim 142, the vector of claim 151, and / or the nanoparticle of claim 154; and a pharmaceutically acceptable carrier.

171. A formulation comprising the cell of claim 155 and a pharmaceutically acceptable carrier.

172. A system comprising: a cell expressing a recombinant receptor, wherein the recombinant receptor comprises an extracellular component comprising the binding domain of claim 1 ; and a recombinant molecule comprising a Whitlow-containing molecule and a target binding domain.

173. The system of claim 172, wherein the cell expresses multiple copies of the binding domain of claim 1.

174. The system of claim 172, wherein the recombinant receptor is a chimeric antigenreceptor (CAR).

175. The system of claim 172, wherein the binding domain of claim 1 is an scFv.

176. The system of claim 172, wherein the binding domain of claim 1 binds the Whitlowcontaining molecule in vivo.

177. The system of claim 172, wherein the target binding domain binds a target antigen.

178. The system of claim 177, wherein the target antigen comprises a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

179. A system comprising: a cell expressing a recombinant receptor, wherein the recombinant receptor comprises an extracellular component comprising a Whitlow-containing molecule; and a recombinant molecule comprising the binding domain of claim 1 and a target binding domain.

180. The system of claim 179, wherein the cell expresses multiple copies of the Whitlow-containing molecule.

181. The system of claim 179, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

182. The system of claim 179, wherein the recombinant molecule is a multi-domain binding molecule.

183. The system of claim 179, wherein the target binding domain binds a target antigen.

184. The system of claim 183, wherein the target antigen comprises a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

185. The system of claim 179, wherein the binding domain of claim 1 binds the Whitlowcontaining molecule in vivo.

186. A kit comprising the binding domain of claim 1 , the multi-domain binding molecule of claim 103, the conjugate of claim 119, the recombinant receptor of claim 135, the nucleic acid of claim 142, the vector of claim 151 , the nanoparticle of claim 154, the cell of claim 155, a nucleic acid encoding the components of the systems of claims 172 or 179, and / or the system of claims 172 or 179.

187. A method of separating a Whitlow -containing molecule from a sample comprising: contacting the sample with the binding domain of claim 1 that is directly or indirectlyassociated with a solid support under conditions suitable for binding; and isolating the solid support from the sample, thereby separating the Whitlowcontaining molecule from the sample.

188. The method of claim 187, wherein the solid support comprises magnetic beads, agarose beads, sepharose beads, polymer microspheres, silica particles, or quantum dots.

189. The method of claim 187, wherein the solid support is made of glass, cellulose, agarose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

190. The method of claim 187, wherein the Whitlow-containing molecule comprises an antibody.

191. The method of claim 190, wherein the antibody comprises an scFv.

192. The method of claim 187, wherein the Whitlow-containing molecule comprises a recombinant receptor.

193. The method of claim 192, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR).

194. The method of claim 187, wherein the sample is a biological sample.

195. The method of claim 187, wherein the sample comprises a harvested medium.

196. The method of claim 187, wherein separating the Whitlow-containing molecule comprises enriching for the Whitlow-containing molecule.

197. The method of claim 187, wherein the method comprises immunoprecipitation or affinity chromatography.

198. The method of claim 187, further comprising eluting the Whitlow-containing molecule from the solid support.

199. A method of detecting a Whitlow-containing molecule comprising: administering, to a subject or a biological sample derived from the subject, a binding domain of claim 1 that is directly or indirectly associated with a detectable label; and detecting the detectable label, thereby detecting the Whitlow-containing molecule.

200. The method of claim 199, wherein the method is performed in vivo or ex vivo.

201. The method of claim 199, wherein the subject is a mammal.

202. The method of claim 199, wherein the subject is a mouse, nonhuman primate, or human.

203. The method of claim 199, wherein the Whitlow-containing molecule is expressedby a cell of the subject.

204. The method of claim 203, wherein the cell expresses multiple copies of the Whitlow-containing molecule.

205. The method of claim 199, wherein the Whitlow-containing molecule comprises a recombinant receptor.

206. The method of claim 205, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR).

207. The method of claim 199, wherein the method is ex vivo.

208. The method of claim 199, wherein the administering comprises pipetting.

209. The method of claim 199, wherein the biological sample derived from the subject comprises tissue, blood, bone marrow, or stool.

210. The method of claim 209, wherein the tissue comprises tumor tissue.

211. The method of claim 209, wherein the tissue comprises formalin-fixed paraffin- embedded (FFPE) tissue.

212. The method of claim 209, wherein the tissue was frozen.

213. The method of claim 199, wherein the detectable label comprises fluorescent label, a chemiluminescent label, a spectral colorimetric label, an enzymatic label, an affinity tag, or an oligonucleotide.

214. The method of claim 213, wherein the oligonucleotide comprises a barcode or a unique molecular identifier (UM I).

215. The method of claim 199, wherein the detectable label comprises a radiolabel.

216. The method of claim 199, wherein the detecting utilizes flow cytometry, immunohistochemistry, fluorescence imaging, enzyme-linked immunosorbent assay (ELISA), electron microscopy, latex agglutination, lateral flow immunoassay, immunoblotting, Dip Stick Immuno testing, a competitive binding assay, sandwich assay, an immunoprecipitation assay, or oligonucleotide-conjugated identification.

217. The method of claim 216, wherein the oligonucleotide-conjugated identification comprises cellular indexing of transcriptomes and epitopes sequence (CITE-Seq) or CODEX.

218. The method of claim 216, wherein the detecting utilizes flow cytometry.

219. The method of claim 216, wherein the detecting utilizes immunohistochemistry.

220. The method of claim 199, wherein the detecting comprises optical spectroscopy.

221. The method of claim 199, wherein the method is in vivo.

222. The method of claim 199, wherein the administering is through intravenous,intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

223. The method of claim 199, wherein the detecting comprises fluorescence, scintigraphic imaging, magnetic resonance imaging, autoradiographic detection, or a radioimmunoguided system.

224. The method of claim 223, wherein the scintigraphic imaging comprises Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT).

225. The method of claim 199, further comprising repeating the method.

226. The method of claim 225, wherein the repeating is performed at different time points to monitor a condition of the subject.

227. A method of treating a subject in need thereof comprising: administering, to the subject, a therapeutically-effective amount of a cell genetically modified to express a Whitlow-containing molecule; and administering, to the subject, a therapeutically effective amount of the composition of claim 170, thereby treating the subject in need thereof.

228. The method of claim 227, wherein the Whitlow-containing molecule comprises a Whitlow linker.

229. The method of claim 227, wherein the cell is genetically modified to express multiple copies of the Whitlow-containing molecule.

230. The method of claim 227, wherein the Whitlow-containing molecule comprises a recombinant receptor.

231. The method of claim 230, wherein the recombinant receptor comprises an extracellular domain comprising a first binding domain and a Whitlow-containing molecule.

232. The method of claim 231 , wherein the first binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

233. The method of claim 227, wherein the composition comprises a stimulating antibody.

234. The method of claim 227, wherein the composition comprises an antibody conjugate.

235. The method of claim 234, wherein the antibody conjugate comprises the binding domain of claim 1 and a payload.

236. The method of claim 235, wherein the payload comprises a drug.

237. The method of claim 236, wherein the drug comprises a cell stimulatory factor.

238. The method of claim 237, wherein the cell stimulatory factor comprises a growth factor, a cytokine, or a chemokine.

239. The method of claim 227, wherein the composition comprises a multi-domain binding molecule.

240. The method of claim 239, wherein the multi-domain binding molecule comprises binding domain of claim 1 and a second binding domain.

241. The method of claim 240, wherein the second binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

242. The method of claim 227, wherein the subject has cancer, an infection, a degenerative condition, or an autoimmune disorder.

243. The method of claim 227, wherein the subject has cancer.

244. The method of claim 227, wherein the subject has an infection.

245. The method of claim 227, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

246. A method of treating a subject in need thereof comprising: administering, to the subject, a therapeutically-effective amount of a cell genetically modified to express a binding domain of claim 1 ; and administering, to the subject, a therapeutically effective amount of a Whitlow-containing molecule, thereby treating the subject in need thereof.

247. The method of claim 246, wherein the Whitlow-containing molecule comprises a target binding domain.

248. The method of claim 247, wherein the target binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

249. The method of claim 246, wherein the cell is genetically modified to express multiple copies of the binding domain of claim 1 .

250. The method of claim 246, wherein the binding domain of claim 1 is a recombinant receptor.

251. The method of claim 250, wherein the recombinant receptor is a chimeric antigen receptor (CAR).

252. The method of claim 250, wherein the recombinant receptor comprises an extracellular domain comprising the binding domain of claim 1.

253. The method of claim 246, wherein the subject has cancer, an infection, a degenerative condition, or an autoimmune disorder.

254. The method of claim 246, wherein the subject has cancer.

255. The method of claim 246, wherein the subject has an infection.

256. The method of claim 246, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

257. A method of treating a subject in need thereof comprising: administering, to the subject, a therapeutically-effective amount of a cell genetically modified to express a recombinant molecule and a Whitlow-containing molecule; or administering, to the subject, a therapeutically effective amount of a nanoparticle or nanoparticles comprising a nucleic acid encoding the recombinant molecule and the Whitlow-containing molecule, wherein the recombinant molecule or Whitlow-containing molecule is a recombinant receptor, thereby treating the subject in need thereof.

258. The method of claim 257, wherein the recombinant molecule is the recombinant receptor.

259. The method of claim 258, wherein the recombinant receptor comprises an extracellular component comprising a binding domain of claim 1.

260. The method of claim 258, wherein the Whitlow-containing molecule is linked to a target binding domain.

261. The method of claim 260, wherein the target binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cellexpressing the Whitlow-containing molecule.

262. The method of claim 257, wherein the Whitlow-containing molecule is the recombinant receptor.

263. The method of claim 262, wherein the recombinant receptor comprises an extracellular component comprising a Whitlow-containing molecule.

264. The method of claim 262, wherein the recombinant receptor comprises an extracellular component comprising a first binding domain and a Whitlow-containing molecule.

265. The method of claim 257, wherein the recombinant molecule comprises a multidomain binding molecule.

266. The method of claim 265, wherein the multi-domain binding molecule comprises a binding domain of claim 1 and a target binding domain.

267. The method of claim 266, wherein the target binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

268. The method of claim 257, wherein the subject has cancer, an infection, a degenerative condition, or an autoimmune disorder.

269. The method of claim 257, wherein the administering is through intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual administering.

270. A method of modulating a cell genetically modified to express a Whitlow-containing molecule comprising delivering a therapeutically effective amount of the composition of claim 170 and / or formulation of claim 171 , thereby modulating cell activity.

271. The method of claim 270, wherein the modulating is in vivo or ex vivo.

272. The method of claim 270, wherein the modulating is in vivo.

273. The method of claim 270, wherein the cell is genetically modified to express multiple copies of the Whitlow-containing molecule.

274. The method of claim 270, wherein the Whitlow-containing molecule is a recombinant receptor.

275. The method of claim 274, wherein the recombinant receptor comprises an extracellular component comprising a Whitlow-containing molecule.

276. The method of claim 274, wherein the recombinant receptor comprises anextracellular component comprising an scFv.

277. The method of claim 276, wherein the scFv comprises a first binding domain having a Whitlow-containing molecule.

278. The method of claim 277, wherein the first binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

279. The method of claim 270, wherein the modulating comprises stimulating activity of the Whitlow-containing molecule.

280. The method of claim 279, wherein the composition comprises a stimulating antibody.

281. The method of claim 279, wherein the composition comprises an antibody conjugate.

282. The method of claim 281 , wherein the antibody conjugate comprises a binding domain of claim 1 and a payload.

283. The method of claim 282, wherein the payload comprises a drug .

284. The method of claim 283, wherein the drug comprises a cell stimulatory factor.

285. The method of claim 284, wherein the cell stimulatory factor comprises a growth factor, a cytokine, or a chemokine.

286. The method of claim 279, wherein the stimulating activity results in cell expansion.

287. The method of claim 279, wherein the stimulating activity comprises activating cytotoxicity of the cell genetically modified to express the Whitlow-containing molecule.

288. The method of claim 270, wherein the modulating comprises inhibiting or reducing activity of the Whitlow-containing molecule.

289. The method of claim 288, wherein the composition comprises an inhibitory antibody.

290. The method of claim 288, wherein the composition comprises an antibody with a cytolytic antibody backbone.

291. The method of claim 288, wherein the composition comprises an antibody conjugate.

292. The method of claim 291 , wherein the antibody conjugate comprises a binding domain of claim 1 and a payload.

293. The method of claim 292, wherein the payload comprises a drug, a toxin, or a radioisotope.

294. The method of claim 293, wherein the drug comprises a cytotoxic drug.

295. The method of claim 288, wherein the inhibiting activity mitigates or prevents toxicity.

296. The method of claim 288, wherein the formulation comprises a recombinant receptor-expressing cell.

297. The method of claim 296, wherein the recombinant receptor-expressing cell elicits cytotoxic effects on the Whitlow-containing molecule.

298. The method of claim 270, wherein the modulating comprises retargeting the Whitlow-containing molecule.

299. The method of claim 298, wherein the retargeting comprises administering a multidomain binding molecule comprising a binding domain of claim 1 and a second binding domain.

300. The method of claim 299, wherein the second binding domain binds a cancer antigen, a viral antigen, a bacterial antigen, a parasitic antigen, a degenerative cell antigen, an autoantigen, and / or an antigen expressed by a cell that is not the cell expressing the Whitlow-containing molecule.

301. The method of claim 299, wherein the second binding domain binds CD22 and / or CD20 and the Whitlow-containing molecule comprises a first binding domain that binds CD19; the second binding domain binds CD22 and / or CD19 and the Whitlow-containing molecule comprises a first binding domain that binds CD20; or the second binding domain binds CD20 and / or CD19 and the Whitlow-containing molecule comprises a first binding domain that binds CD22.

302. The method of claim 299, wherein the second binding domain binds CLL1 and / or CD123 and the Whitlow-containing molecule comprises a first binding domain that binds CD33; the second binding domain binds CD33 and / or CD123 and the Whitlow-containing molecule comprises a first binding domain that binds CLL1 ; or the second binding domain binds CD33 and / or CLL1 and the Whitlow-containing molecule comprises a first binding domain that binds CD123.

303. The method of claim 270, wherein the delivering is intravenous, intradermal, intraarterial, intranodal, intravesicular, intrathecal, intraperitoneal, intraparenteral, intranasal, intralesional, intramuscular, oral, intrapulmonary, subcutaneous, or sublingual.

304. The method of claim 270, wherein the method is ex vivo.

305. The method of claim 270, wherein the modulating comprises stimulating the Whitlow-containing molecule ex vivo.

306. The method of claim 270, wherein the delivering comprises pipetting.

307. A method of manufacturing the binding domain of claim 1 , the multi-domain binding molecule of claim 103, the conjugate of claim 119, the recombinant receptor of claim 135, the nucleic acid of claim 142, the vector of claim 151 , the nanoparticle of claim 154, the cell of claim 155, the composition of claim 170, the formulation of claim 171 , and / or the system of claims 172 or 179.

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