Trogocytosis-inducing bispecific molecules and methods of use

Abstract

Provided are soluble bispecific molecules. In some instances, the soluble bispecific molecules comprise a first moiety that specifically binds a cell surface molecule on a donor cell, and a second moiety that specifically binds a cell surface molecule on an acceptor cell. The soluble bispecific molecule comprises the first moiety stably associated with the second moiety and induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis. Compositions comprising the bispecific molecules and methods of using the bispecific molecules (e.g., for therapeutic and / or research purposes) are also provided.

Description

[0001] T ROGOCYTOSIS-lNDUCING BlSPECIFIC MOLECULES AND METHODS OF USE

[0002] CROSS-REFERENCE

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 563,082, filed March 8, 2024, which application is incorporated herein by reference in its entirety.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with Government support under contracts GM058867 and GM143843 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0006] INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

[0007] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN- 2166WO_SEQ_LIST_3-7-25” created on March 7, 2025 and having a size of 16,736 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0008] INTRODUCTION

[0009] Cell surface proteins are key to defining cell identity in addition to driving many physiological and pathological functions. This central importance of cell surface proteins has spurred the development of technologies to manipulate the cell surface proteome. In particular, multiple methods for depleting cell surface proteins through targeted degradation have been reported over the past three years. These proof-of-concept strategies address therapeutic challenges rooted in the pathogenic presence of a cell surface protein, yet multiple pathologies result from the absence of functional cell surface proteins1Despite this fact, raising levels of an otherwise non-expressed cell surface protein without genetic intervention remains challenging.

[0010] Trogocytosis, the contact-dependent transfer of plasma membrane components (proteins, lipids, and glycolipids) between cells, is a natural process that has been observed during therapeutic intervention,5 7normal physiology,8 0and parasitic infection11 12. This process is typically rapid (minutes timescale), requires synapse formation between donor and acceptor cells, and results in the transfer of intact proteins to the acceptor cell. Through trogocytosis, it is possible for acceptor cells to express proteins they are otherwise incapable of expressing due to genetic or biochemical deficiencies. While mechanistic details underpinning trogocytic protein transfer have yet to be fully elucidated, multiple studies have demonstrated that transferred proteins maintain topological presentation and function on the receiver cell surface. These studies include immune cell recognition of the transferred protein, evasion of complement lysis through acquired CD46 / CD55,13and maintained signaling function through the CD28 / TCR axis14. SUMMARY

[0011] Provided are soluble bispecific molecules. In some instances, the soluble bispecific molecules comprise a first moiety that specifically binds a cell surface molecule on a donor cell, and a second moiety that specifically binds a cell surface molecule on an acceptor cell. The soluble bispecific molecule comprises the first moiety stably associated with the second moiety and induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis. Compositions comprising the bispecific molecules and methods of using the bispecific molecules (e.g., for therapeutic and / or research purposes) are also provided.

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 : TrogoTACs that engage CD22 induce cell surface protein transfer. A, Concept for TrogoTACs where CD22L-conjugated antibodies induce synapse formation between different cell types and cause transfer of cell surface proteins. B, Preparation of the lra-CD22L trogoTAC through a two-step functionalization-click reaction scheme. C, lra-CD22L binding to CD30-expressing Karpas-299 cells measured by flow cytometry. D, lra-CD22L binding to CD22- expressing Daudi-GFP cells measured by flow cytometry. E, Increased CD30 level on Daudi- GFP after treatment with 5 nM lra-CD22L for 2 h at 37 °C as measured by flow cytometry. F, Visualization of punctate CD30 localization on Daudi-GFP cells by imaging flow cytometry. G, Dose response curve for CD30 transfer from Karpas-299 to Daudi-GFP with lra-CD22L and Ira. H, CD30 transfer to CD22-KO Daudi, WT Daudi, and Daudi electroporated with non-targeting guide RNA. CD30 transfer from Karpas-299 to Daudi cells treated with ligand-blocking anti-CD22, transfer in the presence of a microporous membrane (0.4 pm) physically segregating Karpas-299 from Daudi cells, and transfer based on treatment with co-culture supernatant in place of Karpas- 299.

[0014] FIG. 2: CD22-binding ligand structure and LAR modulate protein transfer efficiency. A, Overall reaction scheme and azide-linked glycans employed for TrogoTAC construction. B, CD30 transfer from Karpas-299 cells to Daudi-GFP cells following treatment with 5 nM TrogoTAC at 37 °C for 2 hours with varied ligands, R. C, CD30 transfer as in B, following treatment with lra-CD22L of various ligand-antibody ratios (LAR, n). BPC = biphenylcarboxyl.

[0015] FIG. 3: TrogoTACs direct protein transfer to primary human B cells. A, Scheme for B cell isolation and CellTrace Violet labeling. B, Flow cytometry measurements of CD30 transfer to primary B cells from Karpas-299 upon 5 nM lra-CD22L or Ira treatment of a 2-hour 1 :1 B cell / Karpas-299 co-culture at 37 °C (left) and selected images of double-positive Karpas-299 / B cell heteroconjugates from imaging flow cytometry (right). C, CD30 transfer levels for 4 donors at 2 and 24-hour timepoints. D, Confocal microscopy images of Karpas-299 / B cell co-cultures after treatment with 5 nM lra-CD22L or Ira (white arrows highlight CD30-positive staining on B cells). E, CD30 transfer to different immune cell subsets following Karpas-299 / PBMC 1 :1 co-culture treatment with 5 nM lra-CD22L or Ira (left) or the Fc-silenced LALA-PG-lra-CD22L or LALAPG- Ira (right). G, Scheme for trogocytic bystander protein transfer and quantification of transfer to B cells for various antigens expressed at higher levels on Karpas-299 than B cells (left) and protein transfer to Karpas-299 for those antigens with higher expression on B cells than Karpas-299 (right) following treatment with lra-CD22L. H, Time course for CD30 transfer to B cells from 15 minutes to 4 hours. I, CD30 transfer to B cells following incubation with ligand-blocking anti-CD22 (10 pg / mL, 30 minutes at 37 °C, MAB19682, R&D Systems).

[0016] FIG. 4: CD22L-based TrogoTACs targeting receptors beyond CD30 elicit cell surface protein transfer. A, IL2Ra (CD25) transfer from SUDHL-1 cells to B cells following CD25-targeting Daclizumab-CD22L treatment. B, PD1 transfer from jurkat-PD1 cells to B cells following PD1 -targeting Pembrolizumab-CD22L treatment. C, EGFR transfer from MDA-MB-468 cells to B cells following EGFR-targeting Cetuximab-CD22L treatment. D, HER2 transfer from SK-BR-3 cells to B cells following HER2-targeting Trastuzumab-CD22L treatment. All experiments carried out with 5 nM TrogoTAC treatment at 37 °C for 2 h with a B cell :cell line ratio of 1 :1 using B cells from different anonymous healthy donors (n = 3). Transfer levels measured by MFI fold-change on B cells by flow cytometry using an antibody recognizing an epitope orthogonal to the TrogoTAC antibody. E, CD30 transfer to primary B cells from Karpas-299 upon 5 nM lra-CD22L or 0.5 nM Ira-Pas33 nanobody-antibody fusion treatment of a 10:1 PBMC / Karpas-299 co-culture at 37 °C for 2 hours. F, Scheme for transfer-sort-kill workflow demonstrating T cell stimulation and killing following MHC I transfer from CD30-mCherry expressing MDA-MB-231 cells. G, Key for proteins transferred and treatments in F. H, MHC I (HLA-A2) bystander transfer from HLA-A2+ MDA-MB231 -CD30-mCherry cells to naturally HLA- A2- HeLa cells expressing CD22. I, IFNy positivity in CD8+ T cells stimulated with HeLa-CD22 cells treated with Ira or Ira-Pas33 in the presence of MDA-MB231 -CD30-mCherry prior to FACS- based separation and treatment with 5 nM tebentafusp and 30 pM YLEPGPVTA (1 :1 CD8T:Hel_a-CD22). J, HeLa-CD22 lysis following treatment analogous to I, but with 5:1 CD8T:Hel_a-CD22.

[0017] FIG. 5: (A) Schematic illustration of trogocytosis using a proof-of-concept full-protein TrogoTAC according to embodiments of the present disclosure. In this example, the TrogoTAC is an Ofatumumab-IL2mutein fusion protein. Ofatumumab binds CD20 on B cells while the IL2mutein binds CD25 on regulatory T cells (Tregs). (B) Flow cytometry data demonstrating transfer of CD25 from Tregs to B cells. (C) Quantification of CD25 transfer from Tregs to B cells by the Ofatumumab-IL2mutein TrogoTAC.

[0018] FIG. 6: (A) Flow cytometry data demonstrating transfer of CD5 from Tregs to B cells (bystander transfer). (B) Quantification of CD5 transfer from Tregs to B cells by the Ofatumumab- IL2mutein TrogoTAC.

[0019] FIG. 7: Amino acid sequences of the heavy and light chains of the Ofatumumab- IL2mutein fusion protein (SEQ ID NOs:1 and 2, respectively). A signal peptide in the heavy and light chains is underlined. FIG. 8: Amino acid sequences of the heavy and light chains of the Iratumumab-Pas33 fusion protein (SEQ ID NOs: 3 and 4, respectively).

[0020] DETAILED DESCRIPTION

[0021] Before the bispecific molecules, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the bispecific molecules, compositions and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the bispecific molecules, compositions and methods will be limited only by the appended claims.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the bispecific molecules, compositions and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the bispecific molecules, compositions and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the bispecific molecules, compositions and methods.

[0023] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the bispecific molecules, compositions and methods belong. Although any bispecific molecules, compositions and methods similar or equivalent to those described herein can also be used in the practice or testing of the bispecific molecules, compositions and methods, representative illustrative bispecific molecules, compositions and methods are now described.

[0025] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present bispecific molecules, compositions and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0026] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0027] It is appreciated that certain features of the bispecific molecules, compositions and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the bispecific molecules, compositions and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present bispecific molecules, compositions and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0028] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0029] SOLUBLE BISPECIFIC MOLECULES

[0030] Aspects of the present disclosure include soluble bispecific molecules. In certain embodiments, the soluble bispecific molecules comprise a first moiety that specifically binds a cell surface molecule on a donor cell, and a second moiety that specifically binds a cell surface molecule on an acceptor cell. Such soluble bispecific molecules comprise the first moiety stably associated with the second moiety, and induce transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis. By “soluble” is meant the bispecific molecule is not associated with a cell, e.g., neither the first moiety nor the second moiety is associated with a cell membrane.

[0031] The soluble bispecific molecules (sometimes referred to herein as “Troqocvtosis- TArqetinq Chimeras” or “TrogoTACs”) are based in part on the inventors’ surprising findings that an exogenous molecule directly acting on the cell surface can increase protein levels on an acceptor cell in a targeted manner without using exogenous genetic material or the target protein. Redirecting the process of trogocytosis enables the modulation of protein levels on the cell surface in an additive rather than degradative manner. Demonstrated herein is that the soluble bispecific molecules of the present disclosure can serve as biochemical tools for manipulating cell surface protein levels in a manner that is complementary to currently available methods for cell surface protein depletion. Details regarding embodiments of the soluble bispecific molecules will now be described.

[0032] In some instances, the first and second moieties may be independently selected from a polypeptide, an antibody, a ligand, a small molecule, and an aptamer. The terms “polypeptide”, “peptide”, or “protein” are used interchangeably herein to designate a linear series of amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The amino acids may include the 20 “standard” genetically encodable amino acids, amino acid analogs, or a combination thereof. The term “amino acid” includes, but is not limited to, naturally-occurring amino acids and their stereoisomers. “Stereoisomers” of amino acids refer to mirror image isomers of the amino acids, such as L-amino acids or D-amino acids. For example, a stereoisomer of a naturally-occurring amino acid refers to the mirror image isomer of the naturally-occurring amino acid (i.e., the D-amino acid). A moiety of a bispecific molecule of the present disclosure may comprise only of L-amino acids, may comprise only of D-amino acids, or may comprise a mixture of L-amino acids and D-amino acids. Moieties comprised of D-amino acids (e.g., all-D amino acids) may be advantageous compared to their all-L counterparts in terms of PK, reduced immunogenicity, and / or the like. See Mandal et al. (2012) PNAS 109:14779-14784. Moreover, any proteinaceous moiety of a bispecific molecule of the present disclosure may comprise one or more beta amino acids, one or more gamma amino acids, and / or one or more of any other amino acid types that suitably replace alpha-amino acids. See, e.g., Sang et al. Acc. Chem. Res. 53, 10, 2425-2442.

[0033] By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., IgG 1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the target molecule (e.g., a cell surface molecule of a target cell), including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv); and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In certain embodiments, the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH). According to some embodiments, the antibody is a VHH (sometimes referred to as a “nanobody”). The antibody may be detectably labeled, e.g., with an in vivo imaging agent, a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like. The first and / or second moiety of a bispecific molecule of the present disclosure may be independently selected from known antibodies including, by way of example, iratumumab (anti- CD30; CAS number: 640735-09-7), daclizumab (anti-CD25; CAS number: 152923-56-3), pembrolizumab (anti-PD-1 ; CAS number: 1374853-91 -4), cetuximab (anti-EGFR; CAS number: 205923-56-4), and trastuzumab (anti-HER2; CAS number: 180288-69-1 ), pas33 (anti-CD22; described, e.g., in McComb, Scott, et al. "Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22." Molecular Therapy Oncology 32.1 (2024)).

[0034] In certain embodiments, when a bispecific molecule of the present disclosure comprises an antibody as the first and / or second moiety and the antibody comprises an Fc region, one or more amino acid modifications may be introduced into the Fc region, thereby generating an Fc region variant. The Fc region variant may comprise a murine Fc region sequence (e.g.: lgG1 , lgG2a or lgG2b) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions. The Fc region variant may comprise a human Fc region sequence (e.g., a human lgG1 , lgG2, lgG3 or lgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions (e.g., an lgG4 isotype including the S228P mutation).

[0035] According to some embodiments, the Fc region is mutated to increase its affinity to FcRn at pH 6.0 and consequently extend the antibody half-life. Antibodies with enhanced affinity to FcRn include those with substitution of one or more of Fc region residues 252, 253, 254, 256, 428, 434, including the so called YTE mutation with substitution M252Y / S254T / T256E (Dall’ Acqua et al, J Immunol. 169:5171 -5180 (2002)) or LS mutation M428UN434S (Zalevsky et al, Nat Biotechnol. 28(2): 157-159 (2010)).

[0036] In certain embodiments, an antibody variant may possess some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement activation and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes and microglia express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991 ). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is described in U.S. Patent No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat’IAcad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat’l Acad. Sci. USA 82:1499- 1502 (1985); 5,821 ,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351 - 1361 (1987)). Antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235, 238, 265, 269, 270, 297, 327 and 329 (U.S. Patent No. 6,737,056). Certain antibody variants with improved or diminished binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591 -6604 (2001 )). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (US Patent No. 7,332,581 ) or the so-called “DANG” FC mutant with substitution of residues 265 to alanine and 297 to Glycine. Alternatively, antibodies with reduced effector function include those with substitution of one or more of Fc region residues 234, 235 and 329, so-called “LALA-PG” Fc mutant with substitution of residues 234 and 235 to alanine and 329 to glycine (Lo, M. et al., Journal of Biochemistry, 292, 3900- 3908). Other known mutations at position 234, 235 and 321 , the so called TM mutant containing mutations L234F / L235E / P331 S in the CH2 domain, can be used (Oganesyan et al. Acta Cryst. D64, 700-704. (2008)). Antibodies from the human lgG4 isotype include mutations S228P / L235E to stabilize the hinge and to reduce FgR binding (Schlothauer et al, PEDS, 29 (10):457-466).

[0037] Other Fc variants include those with substitutions at one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311 , 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc region residue 434 (US Patent No. 7,371 ,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821.

[0038] According to certain embodiments, the first or second moiety is a ligand. As used herein, a “ligand” is a substance that forms a complex with a biomolecule in nature to serve a biological purpose. The ligand may be a substance selected from a circulating factor, a secreted factor, a cytokine, a growth factor, a hormone, a peptide, a polypeptide, a small molecule, and a nucleic acid, that forms a complex with the target molecule, e.g., a cell surface molecule on the surface of the donor or acceptor cell. In some embodiments, when the first or second moiety is a ligand, the ligand is modified in such a way that complex formation with the cell surface molecule occurs, but the normal biological result of such complex formation does not occur.

[0039] In certain embodiments, the first moiety or second moiety binds to a cell surface receptor present on the donor or acceptor cell, respectively. Cell surface receptors of interest include, but are not limited to, stem cell receptors, immune cell receptors, growth factor receptors, cytokine receptors, hormone receptors, receptor tyrosine kinases, immune receptors such as CD22, CD28, CD80, ICOS, CTLA4, PD1 , PD-L1 , BTLA, HVEM, CD27, 4-1 BB, 4-1 BBL, 0X40, OX40L, DR3, GITR, CD30, SLAM, CD2, 2B4, TIM1 , TIM2, TIM3, TIGIT, CD226, CD160, LAG3, LAIR1 , B7-1 , B7-H1 , and B7-H3, a type I cytokine receptor such as lnterleukin-1 receptor, lnterleukin-2 receptor, lnterleukin-3 receptor, lnterleukin-4 receptor, lnterleukin-5 receptor, lnterleukin-6 receptor, lnterleukin-7 receptor, lnterleukin-9 receptor, Interleukin-1 1 receptor, Interleukin-12 receptor, Interleukin-13 receptor, Interleukin-15 receptor, Interleukin-18 receptor, Interleukin-21 receptor, Interleukin-23 receptor, Interleukin-27 receptor, Erythropoietin receptor, GM-CSF receptor, G-CSF receptor, Growth hormone receptor, Prolactin receptor, Leptin receptor, Oncostatin M receptor, Leukemia inhibitory factor, a type II cytokine receptor such as interferon- alpha / beta receptor, interferon-gamma receptor, Interferon type III receptor, Interleukin-10 receptor, Interleukin-20 receptor, Interleukin-22 receptor, Interleukin-28 receptor, a receptor in the tumor necrosis factor receptor superfamily such as Tumor necrosis factor receptor 2 (1 B), Tumor necrosis factor receptor 1 , Lymphotoxin beta receptor, 0X40, CD40, Fas receptor, Decoy receptor 3, CD27, CD30, 4-1 BB, Decoy receptor 2, Decoy receptor 1 , Death receptor 5, Death receptor 4, RANK, Osteoprotegerin, TWEAK receptor, TACI, BAFF receptor, Herpesvirus entry mediator, Nerve growth factor receptor, B-cell maturation antigen, Glucocorticoid-induced TNFR- related, TROY, Death receptor 6, Death receptor 3, Ectodysplasin A2 receptor, a chemokine receptor such as CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 , CX3CR1. XCR1 , ACKR1 , ACKR2, ACKR3 , ACKR4, CCRL2, a receptor in the epidermal growth factor receptor (EGFR) family, a receptor in the fibroblast growth factor receptor (FGFR) family, a receptor in the vascular endothelial growth factor receptor (VEGFR) family, a receptor in the rearranged during transfection (RET) receptor family, a receptor in the Eph receptor family, a receptor that can induce cell differentiation (e.g., a Notch receptor), a cell adhesion molecule (CAM), an adhesion receptor such as integrin receptor, cadherin, selectin, and a receptor in the discoidin domain receptor (DDR) family, transforming growth factor beta receptor 1 , and transforming growth factor beta receptor 2. In some embodiments, such a receptor is an immune cell receptor selected from a T cell receptor, a B cell receptor, a natural killer (NK) cell receptor, a macrophage receptor, a monocyte receptor, a neutrophil receptor, a dendritic cell receptor, a mast cell receptor, a basophil receptor, and an eosinophil receptor.

[0040] In some embodiments, the first moiety and / or the second moiety is a small molecule. By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain aspects, the small molecule is not made of repeating molecular units such as are present in a polymer.

[0041] In certain embodiments, the first moiety and / or the second moiety is an aptamer. By “aptamer” is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the target cell surface molecule. Aptamers exhibit certain desirable properties, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C). Aptamers that find use in the bispecific molecules of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1 ):39-45; Sun et al. (2014) Mol. Ther. Nucleic Acids 3: e182; and Zhang et al. (201 1 ) Curr. Med. Chem. 18(27):4185-4194. In some embodiments, the first moiety and / or the second moiety specifically binds its respective cell surface molecule. As used herein, a first molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances, e.g., in a sample. In certain embodiments, the moiety “specifically binds” the cell surface molecule if it binds to or associates with the cell surface molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1 / M) of, for example, greater than or equal to about 104M1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 102M to 1013M, or less). In certain aspects, specific binding means the moiety binds to the cell surface molecule with a KD of less than or equal to about 105M, less than or equal to about 106M, less than or equal to about 107M, less than or equal to about 108M, or less than or equal to about 109M, 101° M, 1011M, or 1012M or less. The binding affinity of the moiety for the cell surface molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.

[0042] In some instances, the donor and acceptor cells are independently selected from an immune cell, a tumor cell, a stem cell, or a cell of a tissue. Non-limiting examples of immune cells include T cells, B cells, natural killer (NK) cells, macrophages, monocytes, neutrophils, dendritic cells, mast cells, basophils, and eosinophils. In certain embodiments, the immune cell is a T cell selected from a naive T cell (TN), a cytotoxic T cell (TCTL), a memory T cell (TMEM), a T memory stem cell (TSCM), a central memory T cell (TCM), an effector memory T cell (TEM), a tissue resident memory T cell (TRM), an effector T cell (TEFF), a regulatory T cell (TREGS), a helper T cell (TH, TH1 , TH2, TH17), a CD4+ T cell, a CD8+ T cell, a virus-specific T cell, an alpha beta T cell (Tap), or a gamma delta T cell (Tv5). In some instances, the immune cell is a B cell. According to certain embodiments, the immune cell is a regulatory T cell (Treg).

[0043] By “tumor cell” is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and / or development in an immunocompromised nonhuman animal model, and / or any appropriate indicator of cellular transformation. “Tumor cell” may be used interchangeably herein with “cancer cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like. In certain embodiments, the tumor cell is a carcinoma cell, a lymphoma cell, a blastoma cell, or a sarcoma cell.

[0044] In some embodiments, when the first or second moiety binds a cell surface molecule on a tumor cell, the tumor cell may be a cell of a tumor selected from renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone cancer and connective tissue sarcomas such as but not limited to bone sarcoma, myeloma bone disease, multiple myeloma, cholesteatoma-induced bone osteosarcoma, Paget's disease of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft- tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangio sarcoma, neurilemmoma, rhabdomyosarcoma, and synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, and primary brain lymphoma; breast cancer including but not limited to adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease) and inflammatory breast cancer; adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers such as but limited to Cushing's disease, prolactinsecreting tumor, acromegaly, and diabetes insipius; eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers such as squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer such as squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers such as but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers such as but not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers such as but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; cervical carcinoma; esophageal cancers such as but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers such as but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; colorectal cancer, KRAS mutated colorectal cancer; colon carcinoma; rectal cancers; liver cancers such as but not limited to hepatocellular carcinoma and hepatoblastoma, gallbladder cancers such as adenocarcinoma; cholangiocarcinomas such as but not limited to papillary, nodular, and diffuse; lung cancers such as KRAS-mutated non-small cell lung cancer, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; lung carcinoma; testicular cancers such as but not limited to germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, androgen-independent prostate cancer, androgendependent prostate cancer, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acrallentiginous melanoma; kidney cancers such as but not limited to renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and / or uterer); renal carcinoma; Wilms' tumor; and bladder cancers such as but not limited to transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. In some embodiments, the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.

[0045] In some instances, the donor cell is a tumor cell and the first moiety specifically binds a tumor antigen. In certain embodiments, the acceptor cell is a tumor cell and the second moiety specifically binds a tumor antigen. Non-limiting examples of tumor antigens to which the first or second moiety may specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , deltalike canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1 ), glycoprotein non-metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1 ), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), trophoblast cell-surface antigen (TROP-2), and Wilms' tumor 1 (WT1 ).

[0046] Exemplary non-limiting examples of bispecific molecules of the present disclosure will now be described.

[0047] In some embodiments, the acceptor cell is a B cell (e.g., the second moiety may specifically bind CD22 or CD20) and the donor cell is a Treg, e.g., the first moiety (e.g., an IL2 mutein (e.g., comprising an N88D mutation) that preferentially binds Tregs over effector T cells) may specifically bind CD25. Such bispecific molecules find use, e.g., in transferring CD25 from Tregs to B cells. Boosting CD25 levels on B cells is useful when increased regulation of activation of the B cells is desirable, e.g., for preventing or reducing autoimmunity in a subject in need thereof. Increased CD25 expression on B cells can better control B cell receptor (BCR) signaling, preventing excessive activation of B cells. Such a regulatory mechanism is useful for maintaining immune tolerance and preventing / reducing autoimmune responses. A non-limiting example of a soluble bispecific molecule useful in the transfer of CD25 from Tregs to B cells is the Ofatumumab-IL2mutein TrogoTAC described herein, e.g., in Example 2 and FIGs. 5-7.

[0048] According to some embodiments, the acceptor cell is a B cell (e.g., the second moiety may specifically bind CD22 or CD20) and the donor cell is a tumor cell comprising tumor antigens (e.g., HER2, EGFR, or the like), optionally complexed with MHC molecules. Such a bispecific molecule enables targeted transfer of tumor antigens or MHC-tumor antigen complexes from the tumor cell to the B cells - thereby transforming the B cells into antigen-presenting cells (APCs) and amplifying the immune response against the tumor. Moreover, transfer of tumor antigens such as HER2, EGFR, or the like to B cells finds use in boosting activity of the B cells and promoting an enhanced immune response. According to some embodiments, the acceptor cell is an MHC negative cell (e.g., an MHC-negative B cell, where the second moiety may specifically bind CD22 or CD20) and the donor cell is an MHC-positive cell. Such a bispecific molecule enables targeted transfer of MHC class I molecules from the donor to the acceptor cell - thereby transforming the acceptor cell into an MHC class I positive cell. For example, in the context of MHC-negative cancers that have escaped immune surveillance (e.g., an MHC-negative B cell lymphoma), the transfer of MHC class I molecules to an MHC-negative cancer cell can enable therapeutic modalities that employ the redirection of cytotoxic T cells to target and kill the cancer cell.

[0049] In certain embodiments, the acceptor cell is a B cell (e.g., the second moiety may specifically bind CD22 or CD20) and the donor cell is a second type of immune cell. For example, the bispecific molecule may find use in transferring immune checkpoint molecules (e.g., PD-1 ) from donor immune cells (e.g., T cells) to acceptor B cells. In the context of autoimmunity, transfer of immune checkpoint molecules to B cells can reduce overactive B cell activity, which in turn can reduce inflammation and autoantibody production by B cells. In the context of tumors and chronic infection, T cells are often exhausted and express immune checkpoint molecules (e.g., PD-1 ). By transferring immune checkpoint molecules from T cells to B cells, an anti-tumor or antiinfection T cell response may be enhanced by removing the immune checkpoint molecule- mediated inhibition of T cell signaling.

[0050] According to some embodiments, the acceptor cells are present in a transplanted tissue or organ (e.g., liver), and the bispecific molecule is designed to transfer immunosuppressive cell surface molecules (e.g., CD39, CD73, GITR, or the like) from Tregs to the cells of the transplanted tissue or organ, thereby shielding the transplanted tissue or organ from attack by the host immune system. Additionally, the MHC on the transplanted tissue or organ is different from the MHC of the host, and Tregs displaying the MHC molecules from the transplanted tissue or organ to the host immune system can tolerize the host to the new MHC molecules derived from the transplanted tissue or organ. Thus, one- or two-way transfer of surface proteins between transplanted tissue or organ and Tregs can promote tolerization. When the transplanted tissue or organ is a transplanted liver, an exemplary cell surface molecule which may be bound by the first or second moiety is asialoglycoprotein receptor (ASGPR).

[0051] According to some embodiments, the first moiety is stably associated with the first moiety via conjugation. The term “conjugation” or “conjugated” generally refers to a chemical linkage, either covalent or non-covalent, usually covalent, that proximally associates one molecule of interest with a second molecule of interest. In certain embodiments, the first moiety is conjugated to the second moiety via a linker. If present, the linker molecule(s) may be of sufficient length to permit the first moiety and second moiety to allow some flexible movement between the first moiety and second moiety. Linker molecules may be, e.g., about 6-50 atoms long. Linker molecules may also be, e.g., aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.

[0052] Where the linkers are peptides, the linkers can be of any suitable length, such as from 1 amino acid (e.g., Gly) to 20 or more amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1 , 2, 3, 4, 5, 6, or 7 amino acids in length. Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers may be used where relatively unstructured amino acids are of interest, and may serve as a neutral tether between components. The ordinarily skilled artisan will recognize that design of conjugates can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer a less flexible structure.

[0053] According to some embodiments, the first moiety is conjugated to the second moiety via a non-cleavable linker. Non-cleavable linkers of interest include, but are not limited to, thioether linkers. An example of a thioether linker that may be employed includes a succinimidyl 4-(N- maleimidomethyl)cyclohexane-1 -carboxylate (SMCC) linker.

[0054] Numerous strategies are available for linking the first moiety and second moiety directly, or indirectly via a linker. For example, the first moiety may be derivatized by covalently attaching a linker to the first moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the second moiety. Also by way of example, the second moiety may be derivatized by covalently attaching a linker to the second moiety, where the linker has a functional group capable of reacting with a “chemical handle” on the first moiety. The functional group on the linker may vary and may be selected based on compatibility with the chemical handle on the first moiety or second moiety. According to one embodiment, the chemical handle is provided by incorporation of an unnatural amino acid having the chemical handle into the first moiety or second moiety. Unnatural amino acids which find use for preparing the conjugates of the present disclosure include those having a functional group selected from an azide, alkyne, alkene, aminooxy, hydrazine, aldehyde (e.g., formylglycine, e.g., SMARTag™ technology from Catalent Pharma Solutions), nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, and boronic acid functional group. Unnatural amino acids which may be incorporated into a first moiety or second moiety of a conjugate of the present disclosure, which unnatural amino acid may be selected to provide a functional group of interest, are known and described in, e.g., Maza et al. (2015) Bioconjug. Chem. 26(9): 1884-9; Patterson et al. (2014) ACS Chem. Biol. 9:592-605; Adumeau et al. (2016) Mol. Imaging Biol. (2):153-65; and elsewhere. An unnatural amino acid may be incorporated into a first moiety or second moiety via chemical synthesis or recombinant approaches, e.g., using a suitable orthogonal amino acyl tRNA synthetase-tRNA pair for incorporation of the unnatural amino acid during translation of a first moiety or second moiety in a host cell.

[0055] The functional group of an unnatural amino acid present in the first moiety or second moiety may be an azide, alkyne, alkene, amino-oxy, hydrazine, aldehyde, asaldehyde, nitrone, nitrile oxide, cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, diazo, tetrazine, tetrazole, quadrocyclane, iodobenzene, or other suitable functional group, and the functional group on the linker is selected to react with the functional group of the unnatural amino acid (or vice versa). As just one example, an azide-bearing unnatural amino acid (e.g., 5-azido-L- norvaline, or the like) may be incorporated into the first moiety or second moiety and the linker portion of a linker-agent moiety may include an alkyne functional group, such that the first moiety or second moiety and linker-agent moiety are covalently conjugated via azide-alkyne cycloaddition. Conjugation may be carried out using, e.g., a copper-catalyzed azide-alkyne cycloaddition reaction.

[0056] In certain embodiments, the chemical handle on the first moiety or second moiety does not involve an unnatural amino acid. A first moiety or second moiety containing no unnatural amino acids may be conjugated by utilizing, e.g., nucleophilic functional groups of the first moiety or second moiety (such as the N-terminal amine or the primary amine of lysine, or any other nucleophilic amino acid residue) as a nucleophile in a substitution reaction with a moiety bearing a reactive leaving group or other electrophilic group. An example would be to prepare a first moiety-linker moiety bearing an N-hydroxysuccinimidyl (NHS) ester and allow it to react with the second moiety under aqueous conditions at elevated pH (~10) or in polar organic solvents such as DMSO with an added non-nucleophilic base, such as N,N-diisopropylethylamine.

[0057] It will be appreciated that the particular approach for attaching a linker, first moiety and / or second moiety to each other may vary depending upon the particular linker, first moiety and / or second moiety and functional groups selected and employed for conjugating the various components to each other.

[0058] In certain embodiments, the first moiety is a polypeptide (or multimer, e.g., a multimeric antibody), the second moiety is a polypeptide (or multimer, e.g., a multimeric antibody), and the soluble bispecific molecule comprises or consists of a fusion protein comprising the first moiety fused to the second moiety, either directly or via one or more linkers and / or additional protein domains. According to some embodiments, a rigid linker is employed. Rigid linkers of interest include those that form alpha helices. Examples of such rigid linkers include those having the following formula: A(EAAAK)nA (SEQ ID NO:12); (n = 2-5). The amino acid sequence of a nonlimiting example of a rigid linker that finds use in operably linking the first and second moieties is provided in the sequence table herein. Alternatively or additionally, in some instances, a flexible linker is employed. By “flexible” is meant the linker is of sufficient length and flexibility to freely permit association of the first and second moieties to their respective targets under the desired conditions, e.g., in vivo. Flexible linkers are often rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. In certain embodiments, a Gly-Ser (GS) linker is employed, non-limiting examples of which are those comprising one or more GGGGS (SEQ ID NO:14) units, i.e., (GGGGS)n(SEQ ID NO:13) where n is an integer of 1 or greater, e.g., 1 to 10. In some embodiments, a flexible linker comprising Gly and Ser, and one or more threonine (Thr) residues is employed. According to some embodiments, a flexible linker comprising Gly or Ser residues, and one or more Thr residues is employed. The amino acid sequence of a non-limiting example of such linkers that find use in operably linking the first and second moieties are provided in the sequence table herein. Flexible linkers include glycine polymers (G)n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Detailed guidance regarding the design of flexible linkers is provided in, e.g., Chen et al. (2013) Adv Drug Deliv Rev. 65(10):1357-1369, van Rosmalen et al. (2017) Biochemistry 56(50):6565-6574, and elsewhere.

[0059] Two non-limiting proof-of-concept soluble bispecific molecules comprising a fusion protein according to the present disclosure include Ofa-IL2mutein (Ofatumumab in which the heavy chain is fused to IL2mutein) and CD25lra-Pas33 (Iratumumab in which the heavy chain is fused to the CD22-binding nanobody, Pas33). The amino acid sequences of these bispecific fusion proteins are provided in Table 1. In these examples, Ofa-IL2mutein comprises an Ofatumumab heavy chain comprising LALA-PG mutations (bold and underlined) fused to IL2mutein (IL2 N88D mutein) via a flexible linker (italicized and underlined). Ira-Pas33 comprises an Iratumumab heavy chain comprising LALA-PG mutations (bold and underlined) fused to Pas33 via a rigid linker (italicized and underlined). Table 1 - Fusion protein amino acid sequences

[0060]

[0061] When a bispecific molecule of the present disclosure comprises a moiety for binding to CD25 (e.g., on regulatory T cells) and the moiety is an IL2mutein, the IL2mutein may comprise the amino acid sequence of the IL2mutein set forth in Table 1. Similarly, when a bispecific molecule of the present disclosure comprises a moiety for binding to CD22 (e.g., on B cells) and the moiety is Pas33, the Pas33 may comprise the Pas33 amino acid sequence set forth in Table 1. Alternatively, the IL2mutein or Pas33 moiety may comprise a variant amino acid sequence relative to the sequence set forth in Table 1. In some instances, a variant amino acid sequence may comprise 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91 % or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater amino acid sequence identity to the sequence set forth in Table 1.

[0062] Methods of Producing Bispecific Molecules

[0063] Using the information provided herein, the bispecific molecules of the present disclosure may be prepared using standard techniques well known to those of skill in the art. For example, a nucleic acid sequence(s) encoding the amino acid sequence of a bispecific molecule of the present disclosure can be used to express the bispecific molecules. The polypeptide sequences provided herein (see, e.g., FIG. 7) can be used to determine appropriate nucleic acid sequences encoding the bispecific molecules and the nucleic acids sequences then used to express one or more bispecific molecules. The nucleic acid sequence(s) can be optimized to reflect particular codon “preferences” for various expression systems according to standard methods well known to those of skill in the art. Using the sequence information provided, the nucleic acids may be synthesized according to a number of standard methods known to those of skill in the art.

[0064] Once a nucleic acid(s) encoding a subject bispecific molecule is synthesized, it can be amplified and / or cloned according to standard methods. Molecular cloning techniques to achieve these ends are known in the art. A wide variety of cloning and in vitro amplification methods suitable for the construction of recombinant nucleic acids are known to persons of skill in the art and are the subjects of numerous textbooks and laboratory manuals.

[0065] Expression of natural or synthetic nucleic acids encoding the bispecific molecules of the present disclosure can be achieved by operably linking a nucleic acid encoding the bispecific molecule to a promoter (which is either constitutive or inducible), and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the bispecific molecule. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0066] To obtain high levels of expression of a cloned nucleic acid it is common to construct expression plasmids which typically contain a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription / translation terminator, each in functional orientation to each other and to the protein-encoding sequence. Examples of regulatory regions suitable for this purpose in E. coli are the promoter and operator region of the E. coli tryptophan biosynthetic pathway, the leftward promoter of phage lambda (PL), and the L-arabinose (araBAD) operon. The inclusion of selection markers in DNA vectors transformed in E. coli is also useful. Examples of such markers include genes specifying resistance to ampicillin, tetracycline, or chloramphenicol. Expression systems for expressing bispecific molecules are available using, for example, E. coli, Bacillus sp. and Salmonella. E. coli systems may also be used.

[0067] The bispecific molecule gene(s) may also be subcloned into an expression vector that allows for the addition of a tag (e.g., FLAG, his (e.g., hexahistidine), and the like) at the C-terminal end or the N-terminal end of the bispecific molecule to facilitate purification. Methods of transfecting and expressing genes in mammalian cells are known in the art. Transducing cells with nucleic acids can involve, for example, incubating lipidic microparticles containing nucleic acids with cells or incubating viral vectors containing nucleic acids with cells within the host range of the vector. The culture of cells used in the present disclosure, including cell lines and cultured cells from tissue (e.g., tumor) or blood samples is well known in the art.

[0068] Once the nucleic acid encoding a subject bispecific molecule is isolated and cloned, one can express the nucleic acid in a variety of recombinantly engineered cells known to those of skill in the art. Examples of such cells include bacteria, yeast, filamentous fungi, insect (e.g., those employing baculoviral vectors), and mammalian cells.

[0069] Isolation and purification of a subject bispecific molecule can be accomplished according to methods known in the art. For example, a protein can be isolated from a lysate of cells genetically modified to express the protein constitutively and / or upon induction, or from a synthetic reaction mixture, by immunoaffinity purification (or precipitation using Protein L or A), washing to remove non-specifically bound material, and eluting the specifically bound bispecific molecule. The isolated bispecific molecule can be further purified by dialysis and other methods normally employed in protein purification methods. In one embodiment, the bispecific molecule may be isolated using metal chelate chromatography methods. Bispecific molecules of the present disclosure may contain modifications to facilitate isolation, as discussed elsewhere herein.

[0070] The bispecific molecules may be prepared in substantially pure or isolated form (e.g., free from other polypeptides). The protein can be present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components). Purified bispecific molecules may be provided such that the bispecific molecule is present in a composition that is substantially free of other expressed proteins, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed proteins. The bispecific molecules produced by prokaryotic cells may require exposure to chaotropic agents for proper folding. During purification from E. coli, for example, the expressed protein can be optionally denatured and then renatured. This can be accomplished, e.g., by solubilizing the bacterially produced bispecific molecules in a chaotropic agent such as guanidine HCI. The bispecific molecule is then renatured, either by slow dialysis or by gel filtration. Alternatively, nucleic acid encoding the bispecific molecule may be operably linked to a secretion signal sequence such as pelB so that the bispecific molecules are secreted into the periplasm in correctly-folded form.

[0071] The present disclosure also provides cells that produce the bispecific molecules of the present disclosure, where suitable cells include eukaryotic cells (e.g., mammalian cells) and prokaryotic cells, e.g., bacterial cells. When bacterial cells are employed, in some embodiments, endotoxin is removed from the bispecific molecule subsequent to expression, and / or the bacterial cells are genetically modified such that they do not produce endotoxin. The present disclosure provides a recombinant host cell (also referred to herein as a “genetically modified host cell”) that is genetically modified with one or more nucleic acids comprising a nucleotide sequence encoding a bispecific molecule of the present disclosure.

[0072] NUCLEIC ACIDS, EXPRESSION VECTORS AND CELLS

[0073] In view of the section above regarding methods of producing the bispecific molecules of the present disclosure, it will be appreciated that the present disclosure also provides nucleic acids, expression vectors and cells.

[0074] According to some embodiments, provided is a nucleic acid encoding a bispecific molecule of the present disclosure. Because of the knowledge of the codons corresponding to the various amino acids, availability of an amino acid sequence of a bispecific molecule or moiety thereof of interest provides a description of all the polynucleotides capable of encoding the bispecific molecule or moiety thereof of interest. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the bispecific molecules disclosed herein. Thus, having identified a particular amino acid sequence, those of ordinary skill in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the bispecific molecule of interest. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based upon the possible codon choices, and all such variations are to be considered specifically disclosed for any bispecific molecule or moiety thereof.

[0075] Also provided are expression vectors comprising any of the nucleic acids of the present disclosure. Expression of natural or synthetic nucleic acids encoding the bispecific molecules of the present disclosure can be achieved by operably linking a nucleic acid encoding the bispecific molecule to a promoter (which is either constitutive or inducible) and incorporating the construct into an expression vector to generate a recombinant expression vector. The vectors can be suitable for replication and integration in prokaryotes, eukaryotes, or both. Typical cloning vectors contain functionally appropriately oriented transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the nucleic acid encoding the bispecific molecule. The vectors optionally contain generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in both eukaryotes and prokaryotes, e.g., as found in shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

[0076] Cells that comprise any of the nucleic acids and / or expression vectors of the present disclosure are also provided. Also provided are methods of making a bispecific molecule of the present disclosure, the methods including culturing a cell of the present disclosure under conditions suitable for the cell to express the bispecific molecule, where the bispecific molecule is produced. The conditions for culturing the cell such that the bispecific molecule is expressed may vary. Such conditions may include culturing the cell in a suitable container (e.g., a cell culture plate or well thereof), in suitable medium (e.g., cell culture medium, such as DMEM, RPMI, MEM, IMDM, DMEM / F-12, or the like) at a suitable temperature (e.g., 32°C - 42°C, such as 37°C) and pH (e.g., pH 7.0 - 7.7, such as pH 7.4) in an environment having a suitable percentage of CO2, e.g., 3% to 10%, such as 5%).

[0077] COMPOSITIONS

[0078] Aspects of the present disclosure further include compositions. According to some embodiments, a composition of the present disclosure comprises a bispecific molecule of the present disclosure. For example, the bispecific molecule may be any of the bispecific molecules described in the Soluble Bispecific Molecules section hereinabove or in the Experimental section below, which descriptions are incorporated but not reiterated herein for purposes of brevity.

[0079] In certain aspects, a composition of the present disclosure includes the bispecific molecule present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCI, MgCh, KCI, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, a protease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0080] Aspects of the present disclosure further include pharmaceutical compositions. In some embodiments, a pharmaceutical composition of the present disclosure includes a bispecific molecule of the present disclosure, and a pharmaceutically acceptable carrier. The bispecific molecule can be incorporated into a variety of formulations for therapeutic administration. More particularly, the bispecific molecules can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, injections, inhalants and aerosols.

[0081] Formulations of the bispecific molecules for administration to a subject (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0082] In pharmaceutical dosage forms, the bispecific molecules can be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and carriers / excipients are merely examples and are in no way limiting.

[0083] For oral preparations, the bispecific molecules can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[0084] The bispecific molecules can be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration. In certain aspects, the bispecific molecules are formulated for injection by dissolving, suspending or emulsifying the bispecific molecules in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[0085] Pharmaceutical compositions that include the bispecific molecules may be prepared by mixing the bispecific molecules having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and / or tonicity agents. Acceptable carriers, excipients and / or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and / or nonionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG).

[0086] The pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration.

[0087] An aqueous formulation of the bispecific molecules may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0088] A tonicity agent may be included to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term "isotonic" denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0089] A surfactant may also be added to the formulation to reduce aggregation and / or minimize the formation of particulates in the formulation and / or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Example concentrations of surfactant may range from about 0.001% to about 1% w / v. A lyoprotectant may also be added in order to protect the bispecific molecules against destabilizing conditions during a lyophilization process. For example, known lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included, e.g., in an amount of about 10 mM to 500 nM.

[0090] In some embodiments, the pharmaceutical composition includes the bispecific molecule, and one or more of the above-identified components (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% weight / volume (w / v).

[0091] METHODS OF USE

[0092] Aspects of the present disclosure further include methods of using the bispecific molecules of the present disclosure. For example, in certain embodiments, provided are methods of transferring a cell surface molecule from a donor cell to an acceptor cell. Such methods comprise contacting the donor and acceptor cells with any of the soluble bispecific molecules of the present disclosure under conditions in which the soluble bispecific molecule induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis. Such methods may be performed in vitro, in vivo, or ex vivo.

[0093] In some instances, the methods are performed ex vivo anti are directed to transferring a cell surface molecule from a donor cell to an acceptor cell in a subject in need thereof. Such methods comprise administering to the subject the soluble bispecific molecule in an amount effective to transfer the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

[0094] The methods of the present disclosure find use in treating a variety of conditions. In certain embodiments, the condition is a cell proliferative disorder. By “cell proliferative disorder” is meant a disorder wherein unwanted cell proliferation of one or more subset(s) of cells in a multicellular organism occurs, resulting in harm, for example, pain or decreased life expectancy to the organism. Cell proliferative disorders include, but are not limited to, cancer, pre-cancer, benign tumors, blood vessel proliferative disorders (e.g., arthritis, restenosis, and the like), fibrotic disorders (e.g., hepatic cirrhosis, atherosclerosis, and the like), psoriasis, epidermic and dermoid cysts, lipomas, adenomas, capillary and cutaneous hemangiomas, lymphangiomas, nevi lesions, teratomas, nephromas, myofibromatosis, osteoplastic tumors, dysplastic masses, mesangial cell proliferative disorders, and the like.

[0095] In some embodiments, the condition is cancer. The subject methods may be employed for the treatment of a large variety of cancers. “Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of cancers that may be treated using the subject methods include any of the cancers described above in the section above entitled Soluble Bispecific Molecules, which for purposes of brevity is not repeated herein.

[0096] In certain embodiments, the condition is cancer, and the cancer comprises a solid tumor. According to some embodiments, the solid tumor is a carcinoma or a sarcoma. When the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. According to some embodiments, when the cancer comprises a solid tumor, the solid tumor is immune-infiltrated. In certain embodiments, the condition is cancer, and the cancer is a myeloma, a leukemia, a lymphoma, or mixed type.

[0097] The methods of the present disclosure may be used to treat a variety of other conditions, non-limiting examples of which include autoimmune disorders, infection (e.g., viral or bacterial infection), etc. In some embodiments, the subject is a tissue or organ transplant recipient.

[0098] The bispecific molecules of the present disclosure may be administered via a route of administration selected from oral (e.g., in tablet form, capsule form, liquid form, or the like), parenteral (e.g., by intravenous, intra-arterial, subcutaneous, intramuscular, epidural injection), topical, intra-nasal, intra-tumoral administration, or intraperitoneal (IP) administration.

[0099] The bispecific molecules of the present disclosure may be administered (e.g., in a pharmaceutical composition) in a therapeutically effective amount. By “therapeutically effective amount” is meant a dosage sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a cancer, infection, tissue or organ transplant, etc., as compared to a control. With respect to cancer, in some embodiments, the therapeutically effective amount is sufficient to slow the growth of a tumor, reduce the size of a tumor, and / or the like. An effective amount can be administered in one or more administrations.

[0100] Provided are methods of treating a condition in a subject in need thereof. By treatment is meant at least an amelioration of one or more symptoms associated with the condition of the individual, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the individual no longer suffers from the condition, or at least the symptoms that characterize the condition. KITS

[0101] Aspects of the present disclosure further include kits. In certain embodiments, the kits find use in practicing the methods of the present disclosure, e.g., methods of treating a condition in a subject in need thereof.

[0102] Accordingly, in certain embodiments, a kit of the present disclosure comprises any of the bispecific molecules of the present disclosure (e.g., present in a pharmaceutical composition), and instructions for administering the bispecific molecule to an individual in need thereof. As will be appreciated, the kits of the present disclosure may include any of the bispecific molecules having any of the features (e.g., first moieties, second moieties, etc.) described above in the section relating to the Soluble Bispecific Molecules of the present disclosure, which are not reiterated herein for purposes of brevity.

[0103] The kits of the present disclosure may include a quantity of the bispecific molecule, present in unit dosages, e.g., ampoules, or a multi-dosage format. As such, in certain embodiments, the kits may include one or more (e.g., two or more) unit dosages (e.g., ampoules) of a bispecific molecule of the present disclosure. The term “unit dosage”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the bispecific molecule calculated in an amount sufficient to produce the desired effect. The amount of the unit dosage depends on various factors, such as the bispecific molecule employed, the effect to be achieved, and the pharmacodynamics associated with the bispecific molecule, in the individual. In yet other embodiments, the kits may include a single multi dosage amount of the bispecific molecule.

[0104] The instructions (e.g., instructions for use (I FU)) included in the kits may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet) are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.

[0105] For purposes of completeness, the present disclosure is further defined in the following numbered clauses.

[0106] 1 . A soluble bispecific molecule comprising: a first moiety that specifically binds a cell surface molecule on a donor cell; and a second moiety that specifically binds a cell surface molecule on an acceptor cell, wherein the soluble bispecific molecule comprises the first moiety stably associated with the second moiety, and wherein the soluble bispecific molecule induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

[0107] 2. The soluble bispecific molecule of clause 1 , wherein the first and second moieties are independently selected from an antibody, a ligand, a small molecule, and an aptamer.

[0108] 3. The soluble bispecific molecule of clause 1 or 2, wherein the donor and acceptor cells are independently selected from an immune cell, a tumor cell, a stem cell, or a cell of a tissue.

[0109] 4. The soluble bispecific molecule of clause 3, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, or an eosinophil.

[0110] 5. The soluble bispecific molecule of clause 4, wherein the immune cell is a B cell.

[0111] 6. The soluble bispecific molecule of clause 4, wherein the immune cell is a regulatory T cell (Treg).

[0112] 7. The soluble bispecific molecule of any one of clauses 1-4, wherein the donor cell is a tumor cell and the first moiety specifically binds a tumor antigen.

[0113] 8. The soluble bispecific molecule of any one of clauses 1-4, wherein the acceptor cell is a tumor cell and the second moiety specifically binds a tumor antigen.

[0114] 9. The soluble bispecific molecule of clause 7 or 8, wherein the tumor antigen is 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), glycoprotein non- metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1 ), mucin 16 (MUC16), sodiumdependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p- CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), trophoblast cell-surface antigen (TROP-2), and Wilms' tumor 1 (WT1 ). 10. The soluble bispecific molecule of clause 3, wherein the acceptor cell is a B cell.

[0115] 11 . The soluble bispecific molecule of clause 10, wherein the second moiety specifically binds CD22 or CD20.

[0116] 12. The soluble bispecific molecule of clause 10 or 11 , wherein the donor cell is a Treg.

[0117] 13. The soluble bispecific molecule of any one of clauses 10-12, wherein the first moiety specifically binds CD25.

[0118] 14. The soluble bispecific molecule of clause 10 or 11 , wherein the donor cell is a tumor cell.

[0119] 15. The soluble bispecific molecule of clause 14, wherein the first moiety specifically binds a tumor antigen, optionally wherein the tumor antigen is as defined in clause 9.

[0120] 16. The soluble bispecific molecule of clause 15, wherein the tumor antigen is complexed with a major histocompatibility complex (MHC) molecule.

[0121] 17. The soluble bispecific molecule of clause 10 or 11 , wherein the first moiety specifically binds an immune checkpoint molecule.

[0122] 18. The soluble bispecific molecule of clause 17, wherein the immune checkpoint molecule is cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death-1 (PD-1 ), programmed cell death ligand-1 (PD-L1 ), lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), indoleamine (2,3)-dioxygenase (IDO), T cell immunoreceptor with Ig and ITIM domains (TIGIT), or V-domain Ig suppressor of T cell activation (VISTA).

[0123] 19. The soluble bispecific molecule of clause 3, wherein the acceptor cell is a cell of a tissue.

[0124] 20. The soluble bispecific molecule of clause 19, wherein the tissue is of a transplanted organ.

[0125] 21 . The soluble bispecific molecule of clause 19 or 20, wherein the transplanted organ is a transplanted liver.

[0126] 22. The soluble bispecific molecule of clause 21 , wherein the second moiety specifically binds asialoglycoprotein receptor (ASGPR).

[0127] 23. The soluble bispecific molecule of any one of clauses 19-22, wherein the donor cell is a Treg.

[0128] 24. The soluble bispecific molecule of clause 23, wherein the cell surface molecule on the donor cell is immunosuppressive.

[0129] 25. The soluble bispecific molecule of clause 24, wherein the cell surface molecule on the donor cell is CD39, CD73, or GITR.

[0130] 26. The soluble bispecific molecule of clause 3, wherein the acceptor cell is a Treg. 27. The soluble bispecific molecule of clause 26, wherein the cell surface molecule on the donor cell is an MHC molecule.

[0131] 28. The soluble bispecific molecule of clause 27, wherein the donor cell is a cell of transplanted tissue.

[0132] 29. The soluble bispecific molecule of clause 28, wherein the donor cell is a cell of a transplanted liver.

[0133] 30. The soluble bispecific molecule of any one of clauses 1-29, wherein the first moiety is stably associated with the second moiety via conjugation.

[0134] 31 . The soluble bispecific molecule of any one of clauses 1-29, wherein the first moiety is a polypeptide, the second moiety is a polypeptide, and the soluble bispecific molecule is a fusion protein.

[0135] 32. A nucleic acid encoding the soluble bispecific molecule of clause 31 .

[0136] 33. An expression vector comprising the nucleic acid of clause 32.

[0137] 34. A cell comprising the nucleic acid of clause 32 or the expression vector of clause 33.

[0138] 35. A composition comprising the soluble bispecific molecule of any one of clauses 1 -31 .

[0139] 36. The composition of clause 35 formulated for administration to a subject.

[0140] 37. The composition of clause 36 formulated for parenteral administration to a subject.

[0141] 38. The composition of clause 37 formulated for intravenous, intra-arterial, subcutaneous, intramuscular, epidural, topical, intra-nasal, intra-tumoral, or intraperitoneal (IP) administration.

[0142] 39. A method of transferring a cell surface molecule from a donor cell to an acceptor cell, the method comprising: contacting the donor and acceptor cells with the soluble bispecific molecule of any one of clauses 1 -31 under conditions in which the soluble bispecific molecule induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

[0143] 40. The method of clause 39, wherein the method is performed in vitro.

[0144] 41 . The method of clause 39, wherein the method is performed in vivo.

[0145] 42. A method of transferring a cell surface molecule from a donor cell to an acceptor cell in a subject in need thereof, the method comprising administering to the subject the soluble bispecific molecule of any one of clauses 1-31 in an amount effective to transfer the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

[0146] 43. The method of clause 42, wherein the subject has cancer.

[0147] 44. The method of clause 42, wherein the subject is a transplant recipient.

[0148] 45. The method of clause 42, wherein the subject has an autoimmune disorder.

[0149] 46. The method of clause 42, wherein the subject is in need of reduced T cell exhaustion. The following examples are offered by way of illustration and not by way of limitation.

[0150] EXPERIMENTAL

[0151] Cell surface proteins are key to defining cell identity in addition to driving many physiological and pathological functions. This central importance of cell surface proteins has spurred the development of technologies to manipulate the cell surface proteome. In particular, multiple methods for depleting cell surface proteins through targeted degradation have been reported over the past three years. These strategies address therapeutic challenges rooted in the pathogenic presence of a cell surface protein, yet multiple pathologies result from the absence of functional cell surface proteins. Despite this fact, raising levels of an otherwise non-expressed cell surface protein without genetic intervention remains challenging.

[0152] The present studies sought to address this challenge by exploiting a natural process for cell-to-cell transfer of membrane proteins known as trogocytosis. The Examples herein disclose the development of soluble bispecific molecules (sometimes referred to herein as Trogocytosis- TArgeting Chimeras (or “TrogoTACs”) capable of inducing protein transfer between distinct cells. These proof-of-concept TrogoTACs include those that bind CD22 (Siglec-2) through a synthetic small molecule ligand chemically linked to an antibody which recognizes a cell surface molecule expressed on the target cell. These CD22-targeted TrogoTACs induce cell surface protein transfer to human B cells from the target cell by redirecting trogocytosis in a targeted fashion. Further experiments show that the process depends on cell-cell contact, expression of CD22, and requires high-affinity CD22 binding. TrogoTACs induce targeted protein transfer in model cell lines, purified primary B cells, and in healthy donor PBMCs. The scope of this platform is demonstrated through targeting therapeutically relevant proteins programmed death receptor 1 (PD-1 ), interleukin 2 receptor subunit alpha (CD25), epidermal growth factor receptor (EGFR), and receptor tyrosine-protein kinase erbB-2 (HER2). These antibody-small molecule chimeras can remodel the cell surface proteome in a non-genetic manner, with potential applications in protein delivery, ex-vivo cell surface editing, and in studying mechanisms of trogocytic protein transfer.

[0153] Example 1 - Development of a proof-of-concept trogocytic bispecific conjugate

[0154] As a prototypical TrogoTAC target, CD30 was selected based on its high expression in several established T cell lymphoma lines and the availability of the anti-CD30 clinical candidate Iratumumab (Ira). Preparation of the corresponding CD22-targeted bispecific chimera (Ira- CD22L) was carried out through a two-step procedure involving i) surface-exposed lysine labeling with NHS-PEG4-alkyne followed by ii) Cu-catalyzed azide / alkyne click reaction with azide- functionalized CD22L (Figure 1 B). MALDI-TOF MS analysis of lra-CD22L was used to confirm formation of the desired conjugate (CD22L:lra = 13:1 ) and flow cytometry based binding measurements with Daudi (CD22+CD30 ", EC50 = 14 nM) and Karpas-299 (CD22 CD30+, EC50 = 1.0 nM) cells were used to determine the apparent affinities of lra-CD22L for CD22 and CD30 respectively (Figure 1 C-D). These cell surface binding affinities are consistent with previous measurements of CD22L binding to CD2217and Iratumumab binding to Karpas-299 cells. To test the ability of lra-CD22L to induce trogocytic cell surface protein transfer, Daudi stably expressing cytosolic GFP (Daudi-GFP) and wild-type Karpas-299 cells were co-incubated in the presence of 5 nM lra-CD22L. Following a 2-hour incubation, the coculture was analyzed by flow cytometry, using the GFP marker to distinguish Daudi and Karpas-299 cells and Brentuximab-AF647, which binds a CD30 epitope distinct from Iratumumab, to detect CD30 levels on the two populations. This experiment revealed a significant increase in CD30 levels on Daudi-GFP in the presence of 5 nM lra-CD22L when compared to either untreated cells or cells treated with an equal concentration of unconjugated Iratumumab, as visualized by the right-shifting of the Daudi-GFP population only in the presence of lra-CD22L (Figure 1 E). To probe the morphology of transferred CD30 on the Daudi-GFP population, an identical set of treatment conditions was analyzed by imaging flow cytometry (IFC). This hybrid sorting / imaging platform revealed that transferred CD30 exists in puncta on the surface of recipient Daudi-GFP cells (Figure 1 F), consistent with previous imaging studies of trogocytosis18 20. The concentration dependence of lra-CD22L induced trogocytosis was determined, as measured by the fold-change in CD30 levels present on Daudi-GFP, revealing that lra-CD22L is a highly potent inducer of protein transfer (Figure 1 G, EC5O = 200 pM). Interestingly, this potency exceeds the apparent binding affinities of lra-CD22L to Karpas-299 and Daudi-GFP (Figure 1 C-D), indicating that a cooperative effect, likely facilitated through increased avidity, takes place when the bispecific chimera engages both cell types.

[0155] Having demonstrated the ability of lra-CD22L to induce surface protein transfer between CD22 and CD30 expressing Daudi and Karpas-299 cells, probed next was the mechanism of this process. Sought was to confirm the involvement of CD22, the putative target of synthetic CD22L, and to determine whether this process was indeed taking place in a contact-dependent manner. A Daudi cell line with CD22 knocked out was generated alongside the non-targeting sgRNA control line. While the non-targeting sgRNA control line exhibited similar CD30 transfer levels to the wild-type cells, CD30 transfer was completely ablated in the CD22KO line (Figure 1 H). CD30 transfer was also blocked by incubation with an anti-CD22 antibody (clone 219903) recognizing the ligand-binding site (Figure 1 H). Taking these results together strongly support the necessity of CD22 engagement by lra-CD22L to facilitate trogocytosis. Contact dependency was first examined using a transwell incubation experiment, where Daudi-GFP and Karpas-299 cells are physically separated by a microporous (0.4 pm) membrane, but otherwise treated in a manner identical to the standard co-incubation (Figure 1 H). In this setup, exosomes, microvesicles, and shed proteins can pass freely between the two cell populations, but cell-cell contact is prohibited21 22(Figure 1 H). To further corroborate the contact dependency of lra-CD22L mediated cell surface protein transfer, Karpas-299 and Daudi-GFP were co-incubated for the treatment time (2 hours) before removing the supernatant and exposing naive Daudi-GFP to the coculture supernatant for 2 hours. Under these conditions as well, no transfer of CD30 to Daudi-GFP was observed (Figure 1 H).

[0156] In order to determine the parameters necessary to redirect trogocytosis for protein transfer to B cells, multiple CD22-targeted TrogoTACs were prepared and used as treatments. Iratumumab conjugates were prepared from alternative CD22-binding glycans including a variant of CD22L lacking the N-acetyl group (BPC-Sia-Lac), the natural ligand for CD22 (Sia-LacNAc), and a control glycan not recognized by CD22 (LacNAc) (Figure 3A). Consistent with the need for high-affinity sialoside to overcome cis-binding effects23, the synthetic sialosides BPC-Sia- LacNAc (CD22L) and BPC-Sia-Lac were the only ligands to efficiently facilitate CD30 transfer. Measuring Daudi cell surface staining with these conjugates revealed a trend that paralleled the transfer results, wherein only the synthetic BPC-containing ligands showed significant staining. Examined next was the effect of ligand-antibody ratio (LAR) on trogocytosis efficiency by generating lra-CD22L from various equivalents of NHS-PEG4-alkyne (5-60 equiv) followed by Cu-catalyzed click to afford the final sialoside-linked antibody (LAR = 2.7-18).

[0157] Following initial studies on the transfer of CD30 to the immortalized Daudi B cell lymphoma line, the efficiency of this process in primary peripheral B cells was assessed. B cells were isolated from healthy donor-derived peripheral blood mononuclear cells (PBMCs), labelled with CellTrace Violet, and treated with lra-CD22L or unconjugated Ira in the presence of Karpas- 299 cells at a cell ratio of 1 :1 (Figure 3A-B). Upon treatment, primary B cells (CTV+ cells) significantly increase in CD30 levels (Figure 3B). Moreover, a double-positive (CTV+ / CD30-high) population is present in the lra-CD22L treatment condition and significantly enriched relative to Ira or no treatment conditions. This population was further analyzed by imaging flow cytometry, revealing multiple B cell / Karpas-299 heteroconjugates, providing direct evidence of lra-CD22L inducing cell-cell contact. Four donors were analyzed following a 2-hour treatment with lra-CD22L or unconjugated Ira (Figure 3C). While live cell antibody-based detection demonstrates the surface localization of transferred CD30, subsequent internalization and lysosomal degradation pathways are possible routes to limiting its surface lifetime. To probe this, these four donors were examined at a 24-hour timepoint, which revealed that significant CD30 increase remained on the cell surface of B cells, yet levels were diminished from the 2-hour timepoint in many cases (Figure 3C). Finally, mixed populations of Karpas-299 and B cells were analyzed by confocal microscopy following the standard 2-hour treatment with lra-CD22L or Ira. Ira-CD22L treatment gives rise to punctate CD30 staining on the surface of B cells (Figure 3D) in a manner consistent with results observed with Daudi-GFP cells (Figure 1 F). The timescale of trogocytosis varied between donors, but generally followed a rapid transfer on the minutes to early hours timescale (Figure 3H). CD22 dependence of the transfer event was probed using the anti-CD22 ligand-blocking antibody (MAB19682, R&D Systems) and showed a clear reduction in transfer following CD22 blockade (> 90% reduction, Figure 3I). It is well known that Fc-gamma receptor (FcYR)-mediated trogocytosis can facilitate protein transfer between antibody-opsonized target cells and FcyR-expressing NK cells, monocytes, and macrophages.524Based on this, assessed was whether lra-CD22L, which is derived from human lgG1 , would engage these receptors and transfer CD30 from Karpas-299 cells to NK cells and monocytes found in PBMCs. Indeed, lra-CD22L treatment of Karpas-299 co-cultured with PBMCs (donor-derived NK, T, and B cells, monocytes) led to significant transfer of CD30 to monocytes, wherein both unconjugated Iratumumab and lra-CD22L caused increased CD30 levels after a 2-hour treatment at 37 °C (Figure 3E). While CD22L conjugation dampens the transfer to NK cells and monocytes when compared to unconjugated Iratumumab (Figure 3E), it was set out to obtain a molecule capable of B cell-selective protein transfer in the context of competing Fc receptor-mediated pathways. To this end, LALAPG-lra-CD22L was generated from the parent Iratumumab containing Fc-silencing mutations L234A / L235A / P329G (‘LALAPG’)25in a manner otherwise identical to the construction of IgG 1 -based lra-CD22L. When compared to lra-CD22L, LALAPG-lra-CD22L reduces monocyte-directed CD30 transfer (Figure 3E) while maintaining the desired B cell directed protein transfer. Taken together, these results demonstrate that LALAPG-lra-CD22L delivers cell surface CD30 from the donor cell line Karpas- 299 to B cells selectively in the presence of other peripheral blood immune cells.

[0158] Beyond proteins directly engaged in trogocytic synapse formation (TCR / MHC, antibody target / FcR), multiple studies have demonstrated additional bystander proteins undergo trogocytic transfer.6’726If TrogoTACs transfer proteins beyond their direct targets, this has implications for applications of the technology and the underlying mechanism of the process. To determine the extent to which bystander proteins are transferred by lra-CD22L treatment, a panel of cell surface proteins was monitored, focusing on targets with higher expression on Karpas-299 cells compared to primary B cells (Figure 3G left). While CD30 exhibits the highest level of transfer, significant transfer takes place with additional proteins including HLA-A3, CD26, and CD4 (Figure 3G). The presence of HLA-A3 on HLA-A3-negative donor B cells is particularly striking as this demonstrates the use of directed protein transfer to introduce proteins that are otherwise inaccessible due to genetic constraints of the acceptor B cell. These findings also allude to the possibility of divorcing the targeting element (CD30 in this case) from the protein transfer objective. FcR-mediated trogocytosis is thought to occur in both unidirectional and bidirectional fashions, where the identity of the FcR-expressing cell determines the directionality of transfer.27To probe the directionality of lra-CD22L-mediated transfer, the transfer of CD22 to Karpas-299 was monitored along with additional B cell markers which could participate in bystander transfer. It was determined that CD22 and other B cell markers transfer to Karpas-299, yet with dramatically lower efficiency than CD30 transfer to B cells.

[0159] While 1s,-generation TrogoTACs can be rapidly assembled through chemical conjugation, a fully protein-based 2nd-generation TrogoTAC platform would obviate the challenges inherent to antibody conjugate heterogeneity. To this end, Ira-Pas33 (Iratumumab fused to the CD22-binding nanobody, Pas33) was designed and found to successfully directCD30 transfer from Karpas-299 cells to primary B cells with significantly improved efficiency over the 1st-generation TrogoTAC lra-CD22L (10X improvement, Figure 4E). Ira-Pas33 was then utilized in a transfer-sort-kill experiment (Figure 4F-G) to probe the functional capacity of adoptively transferred proteins following TrogoTAC treatment. MDA-MB231 -CD30-mCherry cells naturally express high levels of the HLA-A2 allele of MHC I, whereas CD22-expressing HeLa are negative for HLA-A2. This set of cell lines provided an ideal proof-of-concept system to examine the function of MHC I molecules following transfer to a cell type that is otherwise incapable of expressing the HLA-A2 allele. Using Ira-Pas33 (5 nM), HLA-A2 can be transferred to Hel_a-CD22 cells through a bystander transfer process (Figure 4H). Following cell sorting to remove excess MDA-MB-231 (CTV+) cells, HLA-A2 function was probed by treatment with CD8+ T cells, the T cell engager tebentafusp (5 nM) and the MHC I peptide YLEPGPVTA (30 pM) that tebentafusp recognizes. Ira-Pas33 treatment led to significantly higher IFNy release (Figure 4I) and HeLa-CD22 lysis (Figure 4J) when compared to treatment with Ira alone. Taken together, these findings support the use of TrogoTACs to redirect T cell cytotoxicity through targeted protein transfer. Such a strategy holds promise for rewiring T cell responses in the context of heterogeneous target expression in tumor microenvironments and in engaging immunosuppressive tumor-supporting cells.

[0160] Having demonstrated the selective transfer of the model antigen, CD30, examined next were additional cell surface protein targets expressed on additional cell types. Daclizumab- CD22L was generated from the FDA-approved IL2Ra (CD25)-targeting antibody Daclizumab and used to treat the large cell lymphoma line SU-DHL-1 co-cultured with primary B cells (1 :1 cell ratio, 5 nM Daclizumab-CD22L at 37 °C for 2 h). Flow cytometry measurements of CD25 transfer revealed significant transfer to B cells upon Daclizumab-CD22L treatment, but not with unconjugated Daclizumab. The acute T cell leukemia line Jurkat with stable over-expression of PD1 was treated with Pembrolizumab-CD22L (5 nM, 37 °C, 2 h) in a 1 :1 coculture with primary B cells. This treatment resulted in significant PD1 transfer, as measured by an orthogonal antibody to detect transferred PD1 (clone MIH4) (Figure 4B). Next, cell surface protein transfer from donor cells of epithelial origin was examined using EGFR- and HER2-targeting TrogoTACs. The EGFR-targeting antibody Cetuximab was used to generate the corresponding CD22L conjugate (Ctx-CD22L) for treatment of the EGFR-expressing breast cancer cell line MDA-MB- 468. Upon treatment of MDA-MB-468 cells with primary B cells (1 :1 ) and Ctx-CD22L (5 nM, 37 °C, 2 h), EGFR transfer to B cells can be detected by flow cytometry using an orthogonal EGFR antibody (clone 199.12) (Figure 4D). In a similar manner, the anti-HER2 antibody Trastuzumab was used to generate the TrogoTAC Tras-CD22L for targeting the HER2-expressing breast cancer line SK-BR-3. Treatment of SK-BR-3 cells with primary B cells (1 :1 ) and Tras-CD22L (5 nM, 37 °C, 2 h) induced HER2 transfer to B cells as measured by flow cytometry using an orthogonal HER2 antibody (clone 24D4) (Figure 4D). Taking these results together demonstrates that cells of epithelial or immune origin can participate as donors in cell surface protein transfer induced by appropriately designed TrogoTACs. Successful transfer of the signaling molecules EGFR and HER2 further opens the door to modulating signaling function in the receiver cell.

[0161] In summary, the TrogoTAC platform makes possible the targeted transfer of cell surface proteins between cells. Through this process membrane proteins remain on the cell surface with the extracellular face maintaining natural orientation. This strategy was applied to targeting protein transfer to the surface of B cells in a selective fashion, sparing other circulating immune cells present in blood. Targeted protein transfer offers a non-genetic mechanism to introduce proteins to the surface of cells that would otherwise not be expressed or even genetically encoded. The modular nature of TrogoTAC construction through chemical conjugation will enable rapid generation of molecules capable of targeted protein transfer in a wide variety of biological contexts. This approach enables manipulation of the cell surface proteome of target cells of interest in research settings as well as for therapeutic interventions.

[0162] Methods for Example 1

[0163] General Synthetic Chemistry Procedures

[0164] All reactions were performed in standard, dry glassware fitted with rubber septa under an inert atmosphere of nitrogen unless otherwise described. Reagents were purchased in reagent grade from commercial suppliers and used as received, unless otherwise described. Anhydrous solvents were prepared by passing the solvent through an activated alumina column. CD22 ligands BPC-Sia-LacNAc-N3,17BPC-Sia-Lac-N3,28Sia-LacNAc-N3,29and LacNAc-N330were prepared according to previously described syntheses.

[0165] TrogoTAC construction by antibody conjugation

[0166] Standard procedure for antibody conjugation

[0167] Antibody solution (50 pg, 50 pL at 1 mg / mL in PBS) was combined with 40 equiv. of NHS- PEG4-alkyne (10 mM stock solution in DMSO), and the reaction was incubated overnight at room temperature. The resulting mixture was purified using a PBS-equilibrated 0.5 ml_ 40 KDa Zeba size-exclusion column and taken forward with Cu-catalyzed click elaboration without further purification. To the alkyne-labeled antibody conjugate, 40 equiv. of CD22L-N3was added from a 10 mM stock in ddH2O, followed by Cu(l)«BTTP mixture (7.5 vol% to obtain a final concentration of 250 pM Cu). The Cu-catalyzed azide / alkyne click reaction was allowed to proceed at room temperature for 1 hour before purification by PBS-equilibrated 0.5 ml_ 40 KDa Zeba sizeexclusion column. The resulting antibody conjugate concentration was quantified by Pierce 660 protein quantification assay, diluted to 100 pg / mL in PBS, and stored at 4 °C until further use.

[0168] Preparation of Cu(l)-BTTP stock solution To a 10 mM stock solution of Cu(ll)SO4 in dd H2O, an equal volume of BTTP stock solution (40 mM in DMSO) is added. To the resulting solution, a freshly-made sodium ascorbate solution (100 mM in ddH2O) is added in a volume equal to the starting Cu(l l)SO4, resulting in a 1 :1 :1 ratio by volume and a 1 :4:10 molar ratio of the three components.

[0169] Cell Culture

[0170] All cell lines were purchased from the American Type Culture Collection (ATCC) and were cultured in RPMI supplemented with 10% heat-inactivated fetal bovine serum (HI FBS), 100U / ml_ Penicillin, and 100 pg / mL Streptomycin in T-75 flasks (Fisher Scientific) at 37 °C and 5% CO2, unless otherwise noted. The Daudi-GFP cell line was purchased from FenicsBio and the base media was supplemented with 1 .0 pg / mL puromycin. The Karpas-299 cell line was purchased from Sigma Aldrich. The Hel_a-CD22 cell line was a generous gift from Dr. Michael Bassik (Stanford University). The Jurkat-PD1 cell line was purchased from Invivogen and the base media was supplemented with 10 pg / mL blasticidin, 10 pg / mL hygromycin, 100 pg / mL zeocin, and 250 pg / mL geneticin. The Daudi-CD22-KO and NT-sgRNA control cell lines were generated as described below.

[0171] CD22 Gene Knockout Pool

[0172] CRISPR-Cas9 mediated CD22 knockout cell pool in Daudi cells was generated using Synthego Gene Knockout Kit v2 (Synthego Corporation). Cells were electroporated with Streptococcus pyogenes Cas9 (Sp-Cas9) precomplexed to sgRNAs (8:1 sgRNA:Sp-Cas9 ratio) targeting CD22 in the SF buffer using the Lonza 4D Nucleofector X on CM-138 setting following Synthego’s instructions. Cells were expanded for one week before CD22-positive cells were removed by FACS following staining by anti-Hu CD22-AF647 (HIB22, 2 pg / mL). NT-sgRNA control cells were generated by electroporating Sp-Cas9 complexed with a non-targeting sgRNA provided by Synthego in a manner otherwise identical to the targeting sgRNA procedure. sgRNA guides: CD22 5'- CCCAGGCGUAGAGGGUUUCA-3' (SEQ ID NO:5); 5'-

[0173] GCUUUCCAGGUCACCAUCUA-3' (SEQ ID NO:6); 5'- AAGACUCUAUGAAAGCACAA-3' (SEQ ID NO:7).

[0174] Cell-surface protein transfer assay via flow cytometry

[0175] For protein transfer experiments with suspension cells, cells were incubated at a total density of 1 x 106cells / mL (1 :1 , 0.5 x 106cells / mL of each cell type) in RPMI (10% HI FBS) for 2 hours with either unconjugated antibody, TrogoTAC conjugate, or no treatment. For adherent cell lines, cells were lifted with TrypLE Express for a minimal duration directly prior to treatment and were otherwise treated identically to suspension cells. Cells were then pelleted and incubated with FACS buffer (0.5% BSA, 2 mM EDTA in PBS) containing fluorophore-conjugated primary antibodies not blocked by the T rogoTAC antibody for 30 minutes at 4 °C. Cells were then washed 3 times with FACS buffer and incubated with either Sytox Blue or Sytox Green according to the manufacturer’s specifications for 15 minutes at 4 °C. Cells were then analyzed by flow cytometry using a MACSQuant Analyzer 10 (Miltenyi Biotec), and FlowJo software was used to gate on single cells, viable cells, and GFP+ / or CTV+ / populations for analysis.

[0176] Cell-surface TrogoTAC binding measurements via flow cytometry

[0177] Cells were incubated at a density of 1 x 106cells / mL in FACS buffer (0.5% BSA, 2 mM EDTA in PBS) for 30 minutes with lra-CD22L. Cells were then pelleted and washed with FACS buffer 3 times before resuspending in Gt anti-Hu-AF647 (1 .5 pg / mL). Cells were then pelleted and incubated with FACS buffer (0.5% BSA, 2 mM EDTA in PBS) containing fluorophore- conjugated primary antibodies for 30 minutes at 4 °C. Cells were then washed 3 times with FACS buffer and incubated with either Sytox Blue or Sytox Green according to the manufacturer’s specifications for 15 minutes at 4 °C. Cells were then analyzed by flow cytometry using a MACSQuant Analyzer 10 (Miltenyi Biotec), and FlowJo software was used to gate on single cells, viable cells, and GFP+ / or CTV+ / populations for analysis.

[0178] Primary immune cell isolation

[0179] Leukoreduction system chambers were obtained from healthy anonymous blood bank donors. PBMCs were isolated using Ficoll-Paque (GE Healthcare Life Sciences) density gradient separation. Following PBMC isolation, cells were left to rest in RPMI media (10% HI FBS + 100U / mL Penicillin, and 100 pg / mL streptomycin) for 12 hours. For experiments using bulk PBMCs, cells were used directly at this stage. B cells were isolated at this point using the EasySep Human B Cell Isolation Kit (Stemcell Technologies) according to the manufacturer’s protocol, washed once with PBS, and labelled with CellTrace Violet (CTV) (Invitrogen) following the manufacturer’s protocol.

[0180] Confocal microscopy

[0181] Cells were treated as described for the flow cytometry-based protein transfer assay through the antibody staining step. Following antibody staining, cells were plated on human fibronectin-coated slides and let rest for 90 minutes at 4 °C before fixing (4% PFA, 10 minutes, room temperature). Confocal images were acquired using Leica TCS SP8 confocal microscopy with a Fluotar VISIR 25X objective, 0.95-NA water objective using a 405 nm diode, white light laser at 653 nm.

[0182] Matrix-assisted laser desorption / ionization-mass spectrometry

[0183] A solution of the antibody conjugate (0.2 - 1 .0 mg / mL) was buffer exchanged into ddH2O using a 40K Zeba size-exclusion column to remove excess salts. Samples were prepared by mixing 2 pL of sinapinic acid (SPA) matrix (10 mg / mL in 0.1 % trifluoroacetic acid and 50% acetonitrile) and 2 pL of the antibody sample. The mixture was vortexed, and 1 pL was loaded onto a MALDI stainless steel plated pre-coated with 1 pL dried blank SPA matrix. The sample was dried at room temperature for 15 min, and the MALDI-MS was acquired by AB SCIEX TOF / TOF and a 5800 CovalX High Mass Detector with a mass range of 10,000 - 250,000 Da and a fixed laser intensity of 5,900. MALDI data represent an average of at least 3 desorption / ionization scans per sample.

[0184] Example 2 - Development of a proof-of-concept trogocvtic bispecific fusion protein

[0185] Upon establishing that bispecific conjugates are able to induce targeted protein transfer between cells as demonstrated in Example 1 , the present example describes the development and validation of a proof-of-concept fully-proteinaceous trogocytic bispecific molecule. In this particular example, a TrogoTAC consisting of an Ofatumumab-IL2mutein fusion protein was developed.

[0186] FIG. 5A is a schematic illustration of trogocytosis using the proof-of-concept full-protein TrogoTAC consisting of an Ofatumumab-IL2mutein fusion protein. Ofatumumab binds CD20 on B cells while the IL2mutein binds CD25 on regulatory T cells (Tregs). Flow cytometry data demonstrating transfer of CD25 from Tregs to B cells induced by the Ofatumumab-IL2mutein fusion protein is shown in FIG. 5B. FIG. 5C is a bar graph in which CD25 transfer from Tregs to B cells induced by the Ofatumumab-IL2mutein TrogoTAC is quantified.

[0187] FIG. 6A provides flow cytometry data demonstrating transfer of CD5 from Tregs to B cells (bystander transfer). A bar graph showing quantification of CD5 transfer from Tregs to B cells by the Ofatumumab-IL2mutein TrogoTAC is provided in FIG. 6B.

[0188] The amino acid sequences of the heavy and light chains of the Ofatumumab-IL2mutein fusion protein are provided in FIG. 7. The Ofatumumab sequence comprises LALA-PG mutations to prevent Fc-mediated effector functions. The IL2 mutein comprises an amino acid substitution (N88D) for selective binding to CD25 (IL2 alpha) which is highly expressed in Tregs, thus ensuring Treg selectivity.

[0189] References

[0190] (1 ) Dalemans, W.; Barbry, P.; Champigny, G.; Jallat, S.; Jallat, S.; Dott, K.; Dreyer, D.; Crystal, R. G.; Pavirani, A.; Lecocq, J.-P.; Lazdunski, M. Altered Chloride Ion Channel Kinetics Associated with the AF508 Cystic Fibrosis Mutation. Nature 1991 , 354 (6354), 526-528. https: / / doi.Org / 10.1038 / 354526a0.

[0191] (2) Hill, A. ; DeZern, A. E.; Kinoshita, T.; Brodsky, R. A. Paroxysmal Nocturnal Haemoglobinuria. Nat. Rev. Dis. Primer20V7, 3 (1 ), 1-14. https: / / doi.org / 10.1038 / nrdp.2017.28.

[0192] (3) Soli, A. H.; Kahn, C. R. ; Neville, D. M.; Roth, J. Insulin Receptor Deficiency in Genetic and Acquired Obesity. J. Clin. Invest. 1975, 56 (4), 769-780.

[0193] (4) Hanna, S.; Etzioni, A. MHC Class I and II Deficiencies. J. Allergy Clin. Immunol. 2014, 134 (2), 269-275. https: / / doi.Org / 10.1016 / j.jaci.2014.06.001 . (5) Taylor, R. P.; Lindorfer, M. A. Fcy-Receptor-Mediated Trogocytosis Impacts mAb-Based Therapies: Historical Precedence and Recent Developments. Blood 2015, 125 (5), 762- 766. https: / / doi.org / 10.1182 / blood-2014-10-569244.

[0194] (6) Rossi, E. A.; Goldenberg, D. M.; Michel, R.; Rossi, D. L; Wallace, D. J.; Chang, C.-H. Trogocytosis of Multiple B-Cell Surface Markers by CD22 Targeting with Epratuzumab. Blood 2013, 122 (17), 3020-3029. https: / / doi.org / 10.1182 / blood-2012-12-473744.

[0195] (7) Hamieh, M.; Dobrin, A.; Cabriolu, A.; van der Stegen, S. J. C.; Giavridis, T.; Mansilla-Soto, J.; Eyquem, J.; Zhao, Z.; Whitlock, B. M.; Miele, M. M.; Li, Z.; Cunanan, K. M.; Huse, M.; Hendrickson, R. C.; Wang, X.; Riviere, I.; Sadelain, M. CAR T Cell Trogocytosis and Cooperative Killing Regulate Tumour Antigen Escape. Nature 2019, 568 (7750), 112-116. https: / / doi.Org / 10.1038 / S41586-019-1054-1 .

[0196] (8) Bettadapur, A.; Miller, H. W.; Ralston, K. S. Biting Off What Can Be Chewed: Trogocytosis in Health, Infection, and Disease. Infect. Immun. 2020, 88 (7), 10.1128 / iai.00930-19. https : / / doi . org / 10.1128 / iai .00930- 19.

[0197] (9) Schriek, P.; Ching, A. C.; Moily, N. S.; Moffat, J.; Beattie, L.; Steiner, T. M.; Hosking, L. M.;

[0198] Thurman, J. M.; Holers, V. M.; Ishido, S.; Lahoud, M. H.; Caminschi, I.; Heath, W. R.; Mintern, J. D.; Villadangos, J. A. Marginal Zone B Cells Acquire Dendritic Cell Functions by Trogocytosis. Science 2022, 375 (6581), eabf7470. https: / / d0i.0rg / l 0.1126 / science.abf7470.

[0199] (10) Zenke, S.; Sica, M. P.; Steinberg, F.; Braun, J.; Zink, A.; Gavrilov, A.; Hilger, A.; Arra, A.; Brunner-Weinzierl, M.; Eiling, R.; Beyersdorf, N.; Lammermann, T.; Smulski, C. R.; Rohr, J. C. Differential Trafficking of Ligands Trogocytosed via CD28 versus CTLA4 Promotes Collective Cellular Control of Co-Stimulation. Nat. Common. 2022, 13 (1), 6459. https: / / doi.Org / 10.1038 / S41467-022-34156-1 .

[0200] (11) Ralston, K. S. Chew on This: Amoebic Trogocytosis and Host Cell Killing by Entamoeba

[0201] Histolytica. Trends Parasitol. 2015, 31 (9), 442-452. https: / / doi.Org / 10.1016 / j.pt.2O15.05.003.

[0202] (12) Brown, T. OBSERVATIONS BY IMMUNOFLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY ON THE CYTOPATHOGENICITY OF NAEGLERIA FOWLERI IN MOUSE EMBRYO-CELL CULTURES. J. Med. Microbiol. 1979, 12 (3), 363-371. https: / / doi.Org / 10.1099 / 00222615-12-3-363.

[0203] (13) Miller, H. W.; Tam, T. S. Y.; Ralston, K. S. Entamoeba Histolytica Develops Resistance to

[0204] Complement Deposition and Lysis after Acquisition of Human Complement-Regulatory Proteins through Trogocytosis. mBio 2022, 13 (2), e03163-21. https: / / doi.Org / 10.1128 / mbio.03163-21 .

[0205] (14) Osborne, D. G.; Wetzel, S. A. Trogocytosis Results in Sustained Intracellular Signaling in

[0206] CD4+ T Cells. J. Immunol. 2012, 189 (10), 4728-4739. https: / / d0i.0rg / l 0.4049 / jimmunol.1201507. (15) Campana, D.; Janossy, G.; Bofill, M.; Trejdosiewicz, L. K.; Ma, D.; Hoffbrand, A. V.; Mason, D. Y.; Lebacq, A. M.; Forster, H. K. Human B Cell Development. I. Phenotypic Differences of B Lymphocytes in the Bone Marrow and Peripheral Lymphoid Tissue. J. Immunol. 1985, 134 (3), 1524-1530. https: / / doi.Org / 10.4049 / jimmunol.134.3.1524.

[0207] (16) Kaltgrad, E.; O’Reilly, M. K.; Liao, L.; Han, S.; Paulson, J. C.; Finn, M. G. On-Virus Construction of Polyvalent Glycan Ligands for Cell-Surface Receptors. J. Am. Chem. Soc. 2008, 130 (14), 4578-4579. https: / / doi.org / 10.1021 / ja077801 n.

[0208] (17) Peng, W.; Paulson, J. C. CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells. J. Am. Chem. Soc. 2017, 139 (36), 12450-12458. https: / / doi.Org / 10.1021 / jacs.7b03208.

[0209] (18) Miller, H. W.; Suleiman, R. L.; Ralston, K. S. Trogocytosis by Entamoeba Histolytica

[0210] Mediates Acquisition and Display of Human Cell Membrane Proteins and Evasion of Lysis by Human Serum. mBio 2019, 10 (2), 10.1128 / mbio.00068-19. https: / / d0i.0rg / l 0.1128 / mbio.00068-19.

[0211] (19) Pham, T.; Mero, P.; Booth, J. W. Dynamics of Macrophage Trogocytosis of Rituximab-

[0212] Coated B Cells. PLOS ONE 2011 , 6 (1 ), e14498. https: / / doi.org / 10.1371 / journal.pone.0014498.

[0213] (20) Matlung, H. L.; Babes, L.; Zhao, X. W.; van Houdt, M.; Treffers, L. W.; van Rees, D. J.; Franke, K.; Schornagel, K.; Verkuijlen, P.; Janssen, H.; Halonen, P.; Lieftink, C.; Beijersbergen, R. L.; Leusen, J. H. W.; Boelens, J. J.; Kuhnle, I.; van der Werff Ten Bosch, J.; Seeger, K.; Rutella, S.; Pagliara, D.; Matozaki, T.; Suzuki, E.; Menke-van der Houven van Oordt, C. W.; van Bruggen, R.; Roos, D.; van Lier, R. A. W.; Kuijpers, T. W.; Kubes, P.; van den Berg, T. K. Neutrophils Kill Antibody-Opsonized Cancer Cells by Trogoptosis. Cell Rep. 2018, 23 (13), 3946-3959.e6. https: / / doi.Org / 10.1016 / j.celrep.2018.05.082.

[0214] (21) Game, D. S.; Rogersl, N. J.; Lechler, R. I. Acquisition of HLA-DR and Costimulatory Molecules by T Cells from Allogeneic Antigen Presenting Cells. Am. J. Transplant. 2005, 5 (7), 1614-1625. https: / / doi.Org / 10.1111 / j.1600-6143.2005.00916.x.

[0215] (22) Bella, D.; Antonietta, M. Overview and Update on Extracellular Vesicles: Considerations on Exosomes and Their Application in Modern Medicine. Biology 2022, 11 (6), 804. https: / / doi.Org / 10.3390 / biology11060804.

[0216] (23) Collins, B. E.; Blixt, O.; Han, S.; Duong, B.; Li, H.; Nathan, J. K.; Bovin, N.; Paulson, J. C. High-Affinity Ligand Probes of CD22 Overcome the Threshold Set by Cis Ligands to Allow for Binding, Endocytosis, and Killing of B Cells. J. Immunol. 2006, 177 (5), 2994-3003. https: / / doi.Org / 10.4049 / jimmunol.177.5.2994.

[0217] (24) Lindorfer, M. A.; Taylor, R. P. FcyR-Mediated Trogocytosis 2.0: Revisiting History Gives

[0218] Rise to a Unifying Hypothesis. Antibodies 2022, 11 (3), 45. https: / / d0i.0rg / l 0.3390 / antibl 1030045. (25) Schlothauer, T.; Herter, S.; Koller, C. F.; Grau-Richards, S.; Steinhart, V.; Spick, C.; Kubbies, M.; Klein, C.; Umana, P.; Mossner, E. Novel Human lgG1 and lgG4 Fc-Engineered Antibodies with Completely Abolished Immune Effector Functions. Protein Eng. Des. Sei. 2016, 29 (10), 457-466. https: / / doi.org / 10.1093 / protein / gzw040.

[0219] (26) Daubeuf, S.; Aucher, A.; Bordier, C.; Salles, A.; Serre, L; Gaibelet, G.; Faye, J.-C.; Favre, G.; Joly, E.; Hudrisier, D. Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis. PLOS ONE 2010, 5 (1 ), e8716. https: / / doi.org / 10.1371 / journal.pone.0008716.

[0220] (27) Daubeuf, S.; Lindorfer, M. A.; Taylor, R. P.; Joly, E.; Hudrisier, D. The Direction of Plasma Membrane Exchange between Lymphocytes and Accessory Cells by Trogocytosis Is Influenced by the Nature of the Accessory Cell. J. Immunol. 2010, 184 (4), 1897-1908. https: / / doi.Org / 10.4049 / jimmunol.0901570.

[0221] (28) Rillahan, C. D.; Macauley, M. S.; Schwartz, E. ; He, Y.; McBride, R.; Arlian, B. M.; Rangarajan, J.; Fokin, V. V.; Paulson, J. C. Disubstituted Sialic Acid Ligands Targeting Siglecs CD33 and CD22 Associated with Myeloid Leukaemias and B Cell Lymphomas. Chem. Sci. 2014, 5 (6), 2398-2406. https: / / doi.org / 10.1039 / C4SC00451 E.

[0222] (29) Vasiliu, D.; Razi, N.; Zhang, Y.; Jacobsen, N.; Allin, K.; Liu, X. ; Hoffmann, J.; Bohorov, O.; Blixt, O. Large-Scale Chemoenzymatic Synthesis of Blood Group and Tumor-Associated Poly-N-Acetyllactosamine Antigens. Carbohydr. Res. 2006, 341 (10), 1447-1457. https: / / doi.Org / 10.1016 / j.carres.2006.03.043.

[0223] (30) Gao, Y. ; Eguchi, A.; Kakehi, K.; Lee, Y. C. Synthesis and Molecular Recognition of Carbohydrate-Centered Multivalent Glycoclusters by a Plant Lectin RCA120. Bioorg. Med. Chem. 2005, 13 (22), 6151-6157. https: / / doi.Org / 10.1016 / j.bmc.2005.06.036.

[0224] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

WHAT IS CLAIMED IS:1 . A soluble bispecific molecule comprising: a first moiety that specifically binds a cell surface molecule on a donor cell; and a second moiety that specifically binds a cell surface molecule on an acceptor cell, wherein the soluble bispecific molecule comprises the first moiety stably associated with the second moiety, and wherein the soluble bispecific molecule induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

2. The soluble bispecific molecule of claim 1 , wherein the first and second moieties are independently selected from an antibody, a ligand, a small molecule, and an aptamer.

3. The soluble bispecific molecule of claim 1 or 2, wherein the donor and acceptor cells are independently selected from an immune cell, a tumor cell, a stem cell, or a cell of a tissue.

4. The soluble bispecific molecule of claim 3, wherein the immune cell is a T cell, a B cell, a natural killer (NK) cell, a macrophage, a monocyte, a neutrophil, a dendritic cell, a mast cell, a basophil, or an eosinophil.

5. The soluble bispecific molecule of claim 4, wherein the immune cell is a B cell.

6. The soluble bispecific molecule of claim 4, wherein the immune cell is a regulatory T cell (Treg).

7. The soluble bispecific molecule of any one of claims 1 -4, wherein the donor cell is a tumor cell and the first moiety specifically binds a tumor antigen.

8. The soluble bispecific molecule of any one of claims 1 -4, wherein the acceptor cell is a tumor cell and the second moiety specifically binds a tumor antigen.

9. The soluble bispecific molecule of claim 7 or 8, wherein the tumor antigen is 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3(FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), glycoprotein non- metastatic B (GPNMB), guanylate cyclase 20 (GUCY2C), human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1 ), mucin 16 (MUC16), sodiumdependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p- CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), trophoblast cell-surface antigen (TROP-2), and Wilms' tumor 1 (WT1 ).

10. The soluble bispecific molecule of claim 3, wherein the acceptor cell is a B cell.11 . The soluble bispecific molecule of claim 10, wherein the second moiety specifically binds CD22 or CD20.

12. The soluble bispecific molecule of claim 10 or 11 , wherein the donor cell is a Treg.

13. The soluble bispecific molecule of any one of claims 10-12, wherein the first moiety specifically binds CD25.

14. The soluble bispecific molecule of claim 10 or 11 , wherein the donor cell is a tumor cell.

15. The soluble bispecific molecule of claim 14, wherein the first moiety specifically binds a tumor antigen, optionally wherein the tumor antigen is as defined in claim 9.

16. The soluble bispecific molecule of claim 15, wherein the tumor antigen is complexed with a major histocompatibility complex (MHO) molecule.

17. The soluble bispecific molecule of claim 10 or 11 , wherein the first moiety specifically binds an immune checkpoint molecule.

18. The soluble bispecific molecule of claim 17, wherein the immune checkpoint molecule is cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death-1 (PD-1 ), programmed cell death ligand-1 (PD-L1 ), lymphocyte activation gene-3 (LAG-3), T-cell immunoglobulin domain and mucin domain 3 (TIM-3), indoleamine (2,3)-dioxygenase (IDO), T cell immunoreceptor with Ig and ITIM domains (TIGIT), or V-domain Ig suppressor of T cell activation (VISTA).

19. The soluble bispecific molecule of claim 3, wherein the acceptor cell is a cell of a tissue.

20. The soluble bispecific molecule of claim 19, wherein the tissue is of a transplanted organ.21 . The soluble bispecific molecule of claim 19 or 20, wherein the transplanted organ is a transplanted liver.

22. The soluble bispecific molecule of claim 21 , wherein the second moiety specifically binds asialoglycoprotein receptor (ASGPR).

23. The soluble bispecific molecule of any one of claims 19-22, wherein the donor cell is a Treg.

24. The soluble bispecific molecule of claim 23, wherein the cell surface molecule on the donor cell is immunosuppressive.

25. The soluble bispecific molecule of claim 24, wherein the cell surface molecule on the donor cell is CD39, CD73, or GITR.

26. The soluble bispecific molecule of claim 3, wherein the acceptor cell is a Treg.

27. The soluble bispecific molecule of claim 26, wherein the cell surface molecule on the donor cell is an MHC molecule.

28. The soluble bispecific molecule of claim 27, wherein the donor cell is a cell of transplanted tissue.

29. The soluble bispecific molecule of claim 28, wherein the donor cell is a cell of a transplanted liver.

30. The soluble bispecific molecule of any one of claims 1 -29, wherein the first moiety is stably associated with the second moiety via conjugation.31 . The soluble bispecific molecule of any one of claims 1 -29, wherein the first moiety is a polypeptide, the second moiety is a polypeptide, and the soluble bispecific molecule is a fusion protein.

32. A nucleic acid encoding the soluble bispecific molecule of claim 31 .

33. An expression vector comprising the nucleic acid of claim 32.

34. A cell comprising the nucleic acid of claim 32 or the expression vector of claim 33.

35. A composition comprising the soluble bispecific molecule of any one of claims 1 -31 .

36. The composition of claim 35 formulated for administration to a subject.

37. The composition of claim 36 formulated for parenteral administration to a subject.

38. The composition of claim 37 formulated for intravenous, intra-arterial, subcutaneous, intramuscular, epidural, topical, intra-nasal, intra-tumoral, or intraperitoneal (IP) administration.

39. A method of transferring a cell surface molecule from a donor cell to an acceptor cell, the method comprising: contacting the donor and acceptor cells with the soluble bispecific molecule of any one of claims 1-31 under conditions in which the soluble bispecific molecule induces transfer of the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

40. The method of claim 39, wherein the method is performed in vitro.41 . The method of claim 39, wherein the method is performed in vivo.

42. A method of transferring a cell surface molecule from a donor cell to an acceptor cell in a subject in need thereof, the method comprising administering to the subject the soluble bispecific molecule of any one of claims 1-31 in an amount effective to transfer the cell surface molecule on the donor cell to the surface of the acceptor cell via trogocytosis.

43. The method of claim 42, wherein the subject has cancer.

44. The method of claim 42, wherein the subject is a transplant recipient.

45. The method of claim 42, wherein the subject has an autoimmune disorder.

46. The method of claim 42, wherein the subject is in need of reduced T cell exhaustion.

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