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WO2025235604A1 - Fusion protein for use as immune cell engager - Google Patents

Fusion protein for use as immune cell engager

Info

Publication number
WO2025235604A1
WO2025235604A1 PCT/US2025/028136 US2025028136W WO2025235604A1 WO 2025235604 A1 WO2025235604 A1 WO 2025235604A1 US 2025028136 W US2025028136 W US 2025028136W WO 2025235604 A1 WO2025235604 A1 WO 2025235604A1
Authority
WO
WIPO (PCT)
Prior art keywords
cel
immune
engager
cels
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/028136
Other languages
French (fr)
Inventor
Seungjin Shin
Weiliang TANG
Mollie M. MCDONNELL
Yong-Ha Youn
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Umoja Biopharma Inc
Original Assignee
Umoja Biopharma Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Umoja Biopharma Inc filed Critical Umoja Biopharma Inc
Publication of WO2025235604A1 publication Critical patent/WO2025235604A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • T cels may be geneticaly engineered for use as therapeutic agents.
  • CAR chimeric antigen receptor
  • Improved methods and compositions for enhancing T cels are needed.
  • Genetic engineering of T cels may require delivery of polynucleotides into the T cels selected for engineering, a procedure termed transduction.
  • an immune cel engager is used to enhance activation of immune cels.
  • the immune cel engager may be designed to display molecules that enhance transduction.
  • An antibody or antibody fragment against a component of a T cel receptor may be incorporated into the immune cel engager to target the engager to T cels. Additionaly, engagement of CD3 by an immune cel engager comprising a binding domain targeting CD3 may cause the T cels to activate via a primary activation signal. Incorporation of one or more ligands for a co-receptor into the immune cel engager, such as CD28, a molecule expressed by T cels, may cause the T cels to activate via a secondary activation signal. Activation of a T cel, via the primary and optionaly secondary activation signals, may make them more susceptible to transduction. Transduction of T cels may be achieved using various viral and non-viral delivery vehicles.
  • a ligand for CD28 may include a B7 protein.
  • Ligands for CD28 may include, for example, CD80 and CD86.
  • an immune cel engager that includes an adhesion molecule, such as a binding domain that binds CD2 and/or activates CD2 signaling in a target cel (e.g., a T cel or other immune cel) may be used to activate cels (e.g., T cels or other immune cels), and may, for example, be used as a reagent alone or in combination with a polynucleotide delivery vehicle.
  • an immune cel engager described herein is able to bind and activate T cels independent of a virus particle.
  • transduction of target cels may be enhanced by co-administering the immune cel engager with viral particles.
  • the immune cel engager may be enhanced by fusing an adhesion molecule to a costimulatory molecule, an immune cel binding domain, or both.
  • an immune cel engager as disclosed herein e.g. comprising adhesion molecule domain(s), costimulatory molecule domain(s), and optionaly immune cel binding domain(s) was able to bind and activate target cels indicating that each domain was not only appropriately positioned to enable binding its cognate ligand, but maintained its functional characteristics (e.g.
  • the present disclosure provides an engager, comprising: an cel binding domain that specificaly binds a receptor on a target cel comprising a target immune cel or a target tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target cel or on an immune cel, wherein concurent binding of the cel binding domain and the adhesion molecule to the target cel forms a supramolecular adhesion complex on the target cel.
  • the target cel may be an immune cel.
  • An example immune cel is a T cel.
  • the target cel may be a B cel.
  • the target cel may be a cancer cel.
  • the target cel may be a tumor cel.
  • the target cel may be a malignant immune cel.
  • the target cel may be a malignant B cel.
  • the present disclosure provides an immune cel engager for activating a target immune cel, comprising: an cel binding domain that specificaly binds a receptor on a target immune cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cel forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel.
  • the engager further comprises a multimerization domain.
  • the engager further comprises a dimerization domain.
  • the engager is a homodimer.
  • the target immune cel is a T cel.
  • the cel binding domain is a binding domain that specificaly binds CD3.
  • the cel binding domain is a binding domain that comprises complementarity determining regions of an anti- CD3 antibody.
  • Atorney Docket No.061479-511001WO the adhesion molecule is a binding domain that specificaly binds CD2.
  • the adhesion molecule is CD58, CD48, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112, or a functional fragment thereof.
  • engager further comprises an immune cel costimulatory molecule that provide a costimulatory signal to the immune cel.
  • the costimulatory molecule is a binding domain that specificaly binds CD28.
  • the costimulatory molecule is CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD70, CD30L, LIGHT, LTalpha, MICA, or MICB, or a functional fragment thereof.
  • the present disclosure provides an immune cel engager comprising a fusion molecule, the fusion molecule comprising an antigen-binding domain of an anti-CD3 antibody, optionaly a single- chain variable fragment (CD3scFv); a CD58 extracellular domain; a CD80 extracellular domain or a CD86 extracellular domain, wherein the fusion molecule forms an artificial supramolecular adhesion complex with T cels.
  • the fusion molecule further comprises a dimerization domain and the complex is a homodimer.
  • the dimerization domain is a crystalization fragment (Fc) of an immunoglobulin.
  • the domains of the fusion molecule are fused via polypeptide linkers to form a single polypeptide chain.
  • the domains are linked in N- to C-terminal order as CD58-CD3scFv-CD80/CD86-Fc.
  • the domains are linked in N- to C-terminal order as CD3scFv-CD58-CD80/CD86-Fc.
  • the engager is a monomer.
  • the domains of the fusion molecule are fused via polypeptide linkers to form a single polypeptide chain.
  • the domains are linked in N- to C-terminal order as CD58-CD3scFv-CD80/CD86. In some embodiments, the domains are linked in N- to C-terminal order as CD3scFv-CD58- CD80/CD86. [0017] In some embodiments, the engager is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% to a sequence in Table 1.
  • the engager is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% to a sequence in Table 2.
  • the fusion molecule forms an artificial supramolecular adhesion complex with T cels and specificaly activates T cels by engaging, adhering to, and/or costimulating T cels via the CD3scFv, CD58, and CD80/CD86 domains, respectively.
  • the engager is isolated. In some embodiments, the engager is soluble.
  • the engager lacks a transmembrane or a lipid associating domain. In some embodiments, the engager is not associated with a viral particle.
  • cel engagers comprising: a cel binding domain that specificaly binds an antigen on a target cel; and an immune cel adhesion molecule Atorney Docket No.061479-511001WO that specificaly binds a reciprocal adhesion molecule of an immune cel, wherein binding of the immune cel adhesion molecule to the immune cel activates the immune cel, and wherein the cel engager is soluble.
  • the target cel is a tumor cel.
  • the antigen comprises CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, BCMA, ACTN4, BAGE-1, BCR-ABL, beta-catenin, CA 125, CA 15-3, CA 195, CA 242, CA 50, CAM43, Casp-8, CDC27, CDK4, CDKN2A, CEA, COA-1, DEK-CAN fusion protein, EBNA1, EF2, an Epstein Bar virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, HSP70-2, KIAA0205, MART2, MUM-1, MUM-2, MUM-3, neo-PAP, myosin class I proteins, OS-9, PML-RAR ⁇ fusion protein, PTPRK, K-RAS, N-RAS, TPI, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GNT-V, HERV-K- MEL, LAGE-1,
  • the target cel is the immune cel or another immune cel.
  • the antigen comprises CD3.
  • the reciprocal adhesion molecule comprises CD2.
  • the immune cel adhesion molecule comprises a CD58 protein.
  • Some embodiments include an immune cel costimulatory molecule.
  • the immune cel costimulatory molecule binds CD28.
  • the immune cel costimulatory molecule comprises a CD80 or CD86 protein.
  • the disease comprises cancer.
  • the disease comprises an autoimmune disease.
  • the engager is associated with a reagent comprising a substrate.
  • the substrate is a solid support.
  • the substrate is a bead.
  • the substrate is a resin.
  • the substrate is a chromatography resin.
  • engager is atached to a solid support. In some embodiments, the engager is atached to an expamer.
  • Atorney Docket No.061479-511001WO The present disclosure provides an in vivo method of generating engineered immune cels in a patient, comprising: a. administering to the patient an immune cel engager, or a vector encoding said immune cel engager, wherein the immune cel engager forms an artificial supramolecular adhesion complex with target immune cels and specificaly activates target immune cels by engaging, adhering to, and costimulating target immune cels, and c.
  • a gene therapy vector capable of transducing the target immune cels, wherein the gene therapy vector transduces target immune cels thereby generating engineered immune cels, and wherein transduction of the immune cels and/or generation of engineered immune cels is increased compared to administering only the gene therapy vector.
  • the present disclosure provides a method of treating or preventing cancer, comprising administering an immune cel engager and a gene therapy vector that transduces target immune cels to generate, in vivo, engineered immune cels capable of kiling cancer cels, wherein the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector.
  • the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal
  • the present disclosure provides a method of making engineered immune cels, comprising contacting target immune cels with an immune cel engager and with a gene therapy vector that generates, ex vivo, engineered immune cels, wherein the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector.
  • the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target
  • the present disclosure provides a method of activating immune cels, comprising: a. selecting an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cel, and optionaly an immune cel costimulatory molecule; Atorney Docket No.061479-511001WO b. preparing an immune cel engager comprising functional fragments of the immune cel binding domain, immune cel adhesion molecule, and optionaly the immune cel costimulatory molecule; c. contacting target immune cels with the immune cel engager.
  • the contacting comprises administering the immune cel engager to a patient, thereby contacting the immune cel engager with target immune cels in vivo and activating the immune cels.
  • the method further comprises administering a gene therapy vector specific to the target immune cels, wherein co-administration of the immune cel engager with the vector increases transduction of target immune cels by the vector in the patient.
  • the target immune cels are T cels.
  • FIG.1 includes diagrams ilustrating examples of immune cel engagers that include a soluble multi-domain fusion 1 (sMDF1) monomer, a sMDF1 dimer, and a soluble multi-domain fusion 2 (sMDF2) dimer.
  • FIG.2A is a schematic that shows an illustrateative immune cel engager comprising a CD58 extracellular region and anti-CD3 scFv fused to the N-terminus of a CD80 protein via a linker The construct is termed “498.”
  • the schematic may include an N terminus on the left side, and a C terminus on the right side.
  • FIG.2B is a schematic that shows an illustrateative immune cel engager comprising a CD58 extracellular region fused to an N-terminus of a CD80 protein via a linker. The construct is termed “455.” anti-CD3 scFv is expressed as a separate polypeptide in the producer cels. The schematic may include an N terminus on the left side, and a C terminus on the right side.
  • FIG.3A includes diagrams ilustrating example immune cel engagers.
  • FIG.3B includes diagrams ilustrating example immune cel engagers.
  • FIG.4 is a diagram summarizing an example production, purification, and analysis workflow for generation of some immune cels engagers (e.g.
  • FIG.5 includes diagrams ilustrating example immune cel engagers that include soluble multi-domain fusion 1 (sMDF1) and soluble multi-domain fusion 2 (sMDF2).
  • TM_CT transmembrane domain
  • FIG.6 includes an image of an SDS-PAGE gel that shows sMDF1 and sMDF2 produced in 293T cels have structural and post-translational modifications (e.g., glycosylation). Isolated sMDF proteins were treated with the indicated enzymes and run on a non-reducing SDS page gel.
  • FIG.7 summarizes example parameters for isolating sMDF1 on a His-Tag column or sMDF1 IgG Fc or sMDF2 IgG Fc on a Protein A Sepharose column. Example buffers and flow rates for each type of column are listed.
  • FIG.8 includes SDS-PAGE gel images showing successful isolation of example immune cel engagers that included sMDF variants: sMDF1 (HisTag), sMDF1 IgG Fc and sMDF2 IgG Fc.
  • the sMDF1 (HisTag) gel indicates the volume loaded, purity and calculated concentration.
  • FIG.9 shows a western blot of purified sMDF1 HisTag (monomer) and Fc (Dimer) run on the SDS-PAGE.
  • sMDF was detected with an anti-CD58 antibody.
  • dimer MDF1 showed expected 200 kD size (2X 905 aa), while monomer MDF1 showed expected 75 kD (674 aa).
  • the dimer was reduced to monomer form (100 kD, 905 aa).
  • FIG.10 shows a graph that quantifies T cel activation after purified sMDFs were incubated with NFAT reporter cels. The sMDFs and cels were incubated for 2 hours and then NFAT activation was measured. sMDF was added at the listed concentrations.
  • FIG.11 shows a capilary electrophoresis (Jess) analysis of different amounts of purified sMDFs and lentivirus particles containing the MDF. A standard curve of sMDF concentration was generated and was used to quantify the amount of virus expressing the MDF (VPL 128 and VPL 390).
  • FIG.12 shows percentages of CD25+ PBMCs incubated with the indicated amount of sMDF after 3 days of culture.
  • CD4+ and CD8+ T cels were evaluated separately for their activation after treatment with the immune cel engagers (sMDF1 and sMDF2).
  • FIG.13 shows percentages of CD25+ PBMCs incubated with the indicated amount of sMDF after 7 days of culture.
  • CD4+ and CD8+ T cels were evaluated separately for their activation after treatment with sMDF.
  • FIG.14 shows percentages of CD8+ PBMCs incubated with the indicated amount of sMDF after 7 days of culture.
  • FIG.15 shows percentages of sMDF bound to CD3+ PMBCs.
  • PBMCs were incubated with sMDF for 1.5 hours and washed.
  • the presence of sMDFs on the surface of the PBMCS was assessed by FACS using Fc antibody Atorney Docket No.061479-511001WO DETAILED DESCRIPTION
  • the present disclosure relates generaly to an immune cel engager for use in the binding and activation of target cels, such as immune cels, or specificaly T cels.
  • the disclosure provides, an immune cel engager for binding and activation of target cels, comprising an adhesion molecule linked to a costimulatory molecule, an immune cel binding domain, or both.
  • the immune cel engager of the present disclosure combines an adhesion molecule with a costimulatory molecule or an immune cel binding domain, or both.
  • the term “transduction” is used in its broadest sense to mean delivery of an agent to a cel, such as a therapeutic agent.
  • the agent may be a smal molecule, polynucleotide, or polypeptide.
  • a combination of agents may be delivered, such as several polynucleotides or a protein-nucleic acid complex (e.g., a gene-editing nuclease in complex with guide nucleic acid).
  • an immune cel engager may be engineered to incorporate any of the adhesion molecules or costimulatory molecules; extracellular fragments thereof; or functional fragments thereof discussed in this application. Extracellular portions of these molecules may be identified in databases such as UniProt, which is available at www.uniprot.org, or may be predicted using methods, such as a method implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk.
  • functional fragments of each are identified in scientific literature or they may be identified using laboratory methods. For example, one may identify the fragments of a protein likely to form wel-folded domains. Fragments may be tested in binding assays against a cognate molecule or used in pul-down assays compared to the ful molecule.
  • Functional assays such as expression of a fluorescence reporter under the control of a promoter activated by T-cel signaling (e.g., the NKkB promoter) when a T cel is contacted with a cel, an immune cel engager or particle expressing a putative functional fragment.
  • the sequence of the adhesion molecule, costimulatory molecule, or immune cel binding domain may be varied to identify and use variants that retain function. For example, conservative mutations may be made to a molecule, or a molecule may be randomly mutated with the function of the variant confirmed experimentaly. [0051] In each case, a molecule may be incorporated as a ful-length form, without its native transmembrane domain. Alternatively, the extracellular portion of the molecule may be replaced with a heterologous protein. Each immune cel engager may be a monomer, bound to another engager to form a dimer or bound to an additional antigen binding domain.
  • an adhesion molecule, costimulatory molecule, and immune cel binding domain may be linked in any order with only the most N-terminal or C-terminal of the molecules.
  • the immune cel engager comprises or is associated with another membrane-associated molecule.
  • display is used, in a broad sense, to mean positioned on the surface of the immune cel engager such that the molecule Atorney Docket No.061479-511001WO may contact cognate molecules on the target cel.
  • the immune cel engager may be loaded onto a solid support either by association with a component of the particle (e.g., a capsid protein) or by the addition of an additional molecule with afinity for the solid support (e.g., as an immune cel engager comprising a capsid protein).
  • Immune Cel Engagers [0052] Disclosed herein, in some embodiments, are immune cel engagers. As used herein, an “engager” refers to a binding molecule or a complex of binding molecules capable a binding to a target cel or target cels.
  • the target cel may be an immune cel, in which case the engager may be termed an “immune cel engager.”
  • the engager may be a T-cel engager.
  • the target cel may be an tumor cel, in which case the engager may be termed a “tumor cel engager.”
  • the immune cel engager may include a fusion molecule.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, or a cel binding molecule.
  • the fusion molecule may include an adhesion molecule.
  • the fusion molecule may include a costimulatory molecule.
  • the fusion molecule may include an immune cel binding domain.
  • the fusion molecule may include a tumor cel binding domain.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, and an immune cel binding domain.
  • the fusion molecule may include an adhesion molecule, a costimulatory molecule, and a tumor cel binding domain.
  • the fusion molecule may include an adhesion molecule and an immune cel binding domain.
  • the fusion molecule may include an adhesion molecule and a tumor immune cel binding domain.
  • the fusion molecule may include a costimulatory molecule and an immune cel binding domain.
  • the fusion molecule may include a costimulatory molecule and a tumor cel binding domain.
  • the fusion molecule may be or include a fusion protein.
  • the immune cel engager may be isolated.
  • the immune cel engager may be soluble.
  • the immune cel engager may be or include a soluble fusion protein.
  • the immune cel engager (or the multispecific immune cel engager) may be used in a method described herein.
  • the disclosure provides an immune cel engager comprising a combination of an adhesion molecule, a costimulatory molecule, and an immune cel binding domain (e.g., a TCR-binding molecule), thereof each component linked directly or indirectly to the other components.
  • the immune cel engager comprises adhesion molecule, a costimulatory molecule, and an immune cel binding domain (e.g., a TCR-binding molecule).
  • the immune cel engager comprises adhesion molecule and a costimulatory molecule, but not a TCR-binding molecule.
  • the immune cel engager comprises an adhesion molecule and an immune cel binding domain (e.g., a TCR-binding molecule), but not a costimulatory molecule.
  • the immune cel engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cel binding domains (e.g., TCR-binding molecules).
  • TCR-binding molecules e.g., TCR-binding molecules
  • the disclosure provides a tumor cel engager comprising a combination of an adhesion molecule, a costimulatory molecule, and a tumor cel binding domain, thereof each component linked directly or indirectly to the other components.
  • the tumor cel engager comprises adhesion molecule, a costimulatory molecule, and a tumor cel binding domain.
  • the immune cel engager comprises an adhesion molecule and a tumor cel binding domain, but not a costimulatory molecule.
  • the tumor cel engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cel binding domains (e.g., TCR-binding molecules).
  • An immune cel engager may be soluble.
  • An immune cel engager may exclude a transmembrane domain. As used herein, the term “soluble” refers to any molecule that does not contain a transmembrane domain.
  • the engager is a soluble macromolecule, substantialy free of lipids. In some embodiments, the soluble engager is not part of a viral particle.
  • the term “macromolecular complex” refers to a stable assembly of two or more fusion proteins.
  • the immune cel engager may be a monomer.
  • the immune cel engager may comprise an Fc domain.
  • the immune cel engager may comprise an Fc domain on the C terminus.
  • the immune cel engager may comprise an Fc domain on the N terminus.
  • the Fc domain of the immune cel engager may facilitate dimer formation. In some embodiments, the dimer is a homodimer.
  • the term “transmembrane domain” refers to the portion of a protein that spans the phospholipid bilayer of a biological membrane, such as the plasma membrane of a cel.
  • the immune cel engager may be comprised of proteins without their transmembrane domain.
  • the immune cel engager may be comprised of proteins without their transmembrane domain.
  • the transmembrane domain is replaced with a heterologous protein.
  • the term “fusion molecule” refers to any molecule having multiple components linked together, directly or indirectly, covalently or non-covalently.
  • the fusion molecule may be soluble.
  • the fusion molecule may be made up of more than one protein.
  • the immune cel engager may be made up of one or more fusion molecules. When those proteins are linked together into a single molecule by peptide bonds, the fusion molecule is termed a “fusion protein.” [0059]
  • the fusion molecule may be made using various linkers, including chemical (covalent) bonds (e.g., by click chemistry) or by peptide bounds.
  • the linker between each component of the fusion protein may be a single peptide bound (e.g., a direct C- to N- peptide bound in a polypeptide chain) or via a polypeptide linker.
  • Ilustrative polypeptide linkers may include, but are not limited to, the glycine-serine linkers, such as GGSGGS (SEQ ID NO.304), GSSGSS (SEQ ID NO.305), or others. Atorney Docket No.061479-511001WO [0060]
  • the fusion molecule is or includes a fusion protein.
  • the fusion protein may comprise an adhesion protein, a polypeptide linker, and a costimulatory portion.
  • the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an immune cel binding domain.
  • the adhesion molecule is N-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is N-terminal to the immune cel binding domain. In some embodiments, the adhesion molecule is C-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the immune cel binding domain. [0062] In some embodiments of the fusion protein, the immune cel binding domain is N-terminal to the costimulatory molecule. In some embodiments, the immune cel binding domain is N-terminal to the adhesion molecule. In some embodiments, the immune cel binding domain is C-terminal to the costimulatory molecule.
  • the immune cel binding domain is C-terminal to the adhesion molecule.
  • the costimulatory molecule is N-terminal to the immune cel binding domain. In some embodiments, the costimulatory molecule is N-terminal to the adhesion molecule. In some embodiments, the costimulatory molecule is C-terminal to the immune cel binding domain. In some embodiments, the costimulatory molecule is C-terminal to the adhesion molecule.
  • Some embodiments of the fusion protein include a linker. Some embodiments include multiple linkers. In some embodiments, a linker directly connects the costimulatory molecule with the adhesion molecule.
  • a linker directly connects the costimulatory molecule with the immune cel binding domain. In some embodiments, a linker directly connects the adhesion molecule with the immune cel binding domain. [0065] In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed (via a polypeptide bond or a polypeptide linker sequence) with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the costimulatory molecule is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed with an end of the immune cel binding domain.
  • a C terminal end of the costimulatory molecule is juxtaposed with an end of the immune cel binding domain.
  • an N terminal end of the immune cel binding domain is juxtaposed with an end of the adhesion molecule.
  • a C terminal end of the immune cel binding domain is juxtaposed with an end of the adhesion molecule.
  • an N terminal end of the immune cel binding domain is juxtaposed with an end of the costimulatory molecule.
  • a C terminal end of the immune cel binding domain is juxtaposed with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule.
  • a C terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule.
  • an N terminal end of the adhesion molecule is juxtaposed with an end of the immune cel binding domain.
  • a C terminal end of the adhesion molecule is juxtaposed with an end of the immune cel binding domain.
  • the fusion protein comprises a Fc fusion.
  • Fc- based fusion proteins are composed of an immunoglobin Fc domain that is directly linked to another peptide.
  • the fused partner can be any other proteinaceous molecule of interest, such as a ligand that activates upon interaction with a cel-surface receptor, a peptide antigen (Ag) against a chalenging pathogen or a ‘bait’ protein to identify binding partners assembled in a protein microarray.
  • the transmembrane domain is replaced with another protein of interest. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc fusion. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgA. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgE. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgM.
  • the transmembrane domain is replaced with a Fc domain from IgG1. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG2. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG3. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG4. In some embodiments, the Fc domain alows the fusion protein to form a monomer. In some embodiments, the Fc domain alows the fusion protein to form a dimer. In some embodiments, the Fc domain alows the fusion protein to form a multimer.
  • the Fc domain alows the fusion protein to form an oligomer.
  • the transmembrane domain is replaced with a protein tag.
  • the transmembrane domain is replaced with a streptavidin tag.
  • Protein tags can be used to afinity purify proteins of interest or eliminate specific proteins from a solution (negative selection), to help solubilize proteins through maintaining proper protein folding and keep them from aggregating, to alter chromatographic properties of the protein to afford different resolution across a particular separation technique, to add an additional epitope to the protein to alow its purification, to add a fluorescent tag, for enzymatic modification (biotinylation) or chemical modification.
  • the transmembrane domain is replaced with an additional binding domain.
  • the additional binding domain can be an antibody or scFv. Incorporation of the antibody or scFv into the fusion protein may alow for targeting the fusion protein to specific cel types.
  • the transmembrane domain is replaced with a cytokine. Cytokines are smal proteins that are crucial in controling the growth and activity of immune system cels and bloods cels.
  • the cytokine is an interleukin.
  • the cytokine is an interferon.
  • the cytokine is a tumor necrosis factor. In some embodiments, the cytokine is a growth factor. Linking the fusion protein (or immune cel engager) to a cytokine may alow the cytokine to bind to its cognate receptor upon binding of the fusion protein to a cel. The addition of the cytokine to the fusion protein may alow for the fusion protein to further activate immune cels in vivo and in vitro. [0072] In some embodiments of the fusion protein, the transmembrane domain is replaced with a protein that binds viral particles. In some embodiments of the fusion protein, the transmembrane domain is replaced with heparin.
  • Heparin is a naturaly occuring glycosaminoglycan that can be used an anticoagulant. Heparin has a natural affinity for viral particles and has been used in chromatography to purify viruses. Incorporating a heparin molecule into the fusion protein may alow for the fusion protein to coat the outside of the viral particle with the fusion protein.
  • the viral particle generated in this protocol can be used in any method or composition described herein.
  • the fusion protein is atached to a substrate. In some embodiments of the fusion protein, the fusion protein is atached to a solid surface. In some embodiments of the fusion protein, the fusion protein is atached to a nanoparticle.
  • the fusion protein is atached to a lipid nanoparticle. In some embodiments, the fusion protein is atached to a bead.
  • a bead can be a particle comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, celulose, nylon, silicone, silica gel, controled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose, celulose, or a combination thereof.
  • PDMS polydimethylsiloxane
  • the bead is a streptavidin bead. In some embodiments, the bead is an agarose bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the bead is a hydrogel bead. In some embodiments, the bead is an antibody conjugated bead. In some embodiments, the bead is a protein A conjugated bead. In some embodiments, the bead is a protein G conjugated bead. In some embodiments, the bead is a protein NG conjugated bead. In some embodiments, the bead is a protein L conjugated bead. In some embodiments, the bead is an oligodT conjugated bear.
  • the bead is a silica bead. In some embodiments, the bead is a silica like bead. In some embodiments, the bead is an anti-biotin microbead. In some embodiments, the bead is an anti-fluorochrome microbead. Atorney Docket No.061479-511001WO [0074] In some embodiments, the fusion protein is atached to a resin. In some embodiments, the fusion protein is atached to a chromatography resin. In some embodiments, the resin is a protein G resin. In some embodiments, the resin is a protein A resin. In some embodiments, the resin is a protein L resin.
  • the resin is glutathione resin. In some embodiments, the resin is a GST fusion protein resin. In some embodiments, the resin is a nickel charge resin. In some embodiments, the resin is a streptavidin resin. In some embodiments, the resin is an anti- DYKDDDDK (Flag Tag) (SEQ ID NO.306) resin. In some embodiments, the resin is a NI-IDS resin. In some embodiments, the resin is an Iodoacetyl resin. In some embodiments, the resin is an anti-HIS affinity resin. [0075] In some embodiments, the fusion protein atached to a resin is used in a column.
  • the fusion protein atached to a resin is used in a column for protein purification.
  • the fusion protein is atached to a polymer.
  • Expamers are proteinaceous multimers that enable precise regulation of T cel stimulation duration and provide promise of control over T cel profiles in future products.
  • the expamer with a fusion protein atached may be used for immune cel activation.
  • the fusion protein is atached to a hydrogel.
  • the fusion protein is atached via covalent bond.
  • the fusion protein is atached via a non-covalent bond.
  • the term “substrate” refers, in a broad sense, to a material which provides the surface on which something is deposited.
  • the fusion protein may be associated with the substrate by covalent or non-covalent interactions.
  • the substrate may be a solid support (e.g., a microtiter plate), a bead, a resin, a column or any other commonly used substrate in protein biology, cel biology, or virology.
  • the fusion protein may comprise, in any order, a cluster of differentiation 80 (CD80), a CD80 extracellular domain, or a functional fragment of CD80; a cluster of diferentiation 58 (CD58), a CD58 extracellular domain; or a functional fragment of CD58; an immune cel binding domain (e.g., a TCR-binding molecule); and polypeptide linkers.
  • the fusion protein may comprise, in any order, a cluster of differentiation 58 (CD58), a CD58 extracellular domain; or a functional fragment of CD58; a tumor cel binding domain; and polypeptide linkers.
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular domain; or a functional fragment of CD58.
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular domain, or a functional fragment of CD80.
  • the fusion protein may comprise, in N- to C-terminal order, an immune cel binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD80, a CD80 extracellular domain, or a Atorney Docket No.061479-511001WO functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracellular domain; or a functional fragment of CD58.
  • an immune cel binding domain e.g., a TCR-binding protein
  • the fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and an immune cel binding domain (e.g., a TCR-binding protein).
  • the fusion protein may comprise, in N- to C-terminal order, an immune cel binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracellular domain, or a functional fragment of CD80.
  • an immune cel binding domain e.g., a TCR-binding protein
  • the fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracellular domain; or a functional fragment of CD58; a polypeptide linker; CD80, a CD80 extracellular domain, or a functional fragment of CD80; a polypeptide linker; and an immune cel binding domain (e.g., a TCR-binding protein).
  • An illustrated fusion protein comprises a CD58 extracellular domain and anti-cluster of diferentiation 3 (CD3) scFv fused to the N-terminus of a CD80 via a linker; this construct is termed a tri-fusion polypeptide and/or termed “498.”
  • An illustrated fusion protein comprises a CD58 extracellular domain fused to the N- terminus of CD80 via a linker; this construct is termed a bi-fusion polypeptide and/or termed “455.”
  • an anti-CD3 scFv is expressed as a separate polypeptide in the producer cels.
  • the polypeptide linker may be optional.
  • fusion proteins may be made by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art.
  • the fusion molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). In some embodiments, the disclosure provides polynucleotides encoding such fusion proteins.
  • the polynucleotide may be an isolated polynucleotide, or it may be part of a vector (e.g., a plasmid) or it may be introduced into and propagated in a host cel.
  • a vector e.g., a plasmid
  • FIGs.1-3B Some example immune cel engagers are shown in FIGs.1-3B. An immune cel engager may include any aspect in any of these figures. Some aspects of such figures may be optional, such as a tag or linker.
  • Polypeptide sequences of illustrate dual CD58+CD80 fusion proteins are provided in Table 1. In each case, an optional signal peptide is shown in parentheses.
  • the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 1.
  • the fusion protein may comprise a polypeptide Atorney Docket No.061479-511001WO having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 1.
  • an optional signal peptide is shown in parentheses.
  • the signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Diagrams of some example fusions are provided in FIG.2. Table 1 Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO [0090] Polypeptide sequences of illustrate triple CD58+CD80+anti-CD3scFv fusion proteins are provided in Table 2. In each case, the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 2.
  • the fusion protein may comprise a polypeptide less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 2.
  • an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues.
  • the present disclosure provides an engager, comprising: a cel binding domain that specificaly binds a receptor on a target cel comprising a target immune cel or a target tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target cel or on an immune cel.
  • Concurent binding of the cel binding domain and the adhesion molecule may form a supramolecular adhesion complex.
  • the supramolecular adhesion complex may be formed on the target cel.
  • the supramolecular adhesion complex may be formed on an immune cel.
  • the target cel may be an immune cel.
  • An example immune cel is a T cel.
  • An example immune cel is a natural kiler (NK) cel.
  • the target cel may be a B cel.
  • the target cel may be a cancer cel.
  • the target cel may be a tumor cel.
  • some embodiments relate to or include an engager, comprising: a cel binding domain that specificaly binds a receptor on a target Atorney Docket No.061479-511001WO tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on an immune cel.
  • the target cel may be a malignant immune cel.
  • the target cel may be a malignant B cel.
  • the engager may include a cel binding domain that specificaly binds an antigen on a target cel.
  • the engager may include an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of an immune cel. Binding of the cel binding domain to the immune cel may activate the immune cel. Binding of the cel binding domain to the antigen on the target cel and binding of the immune cel adhesion molecule to the immune cel may activate the immune cel.
  • the engager may be soluble.
  • cel engagers comprising: a cel binding domain that specificaly binds an antigen on a target cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of an immune cel, wherein binding of the immune cel adhesion molecule to the immune cel activates the immune cel, and wherein the cel engager is soluble.
  • the target cel is a tumor cel.
  • the antigen comprises cluster of differentiation 19 (CD19), cluster of differentiation 20 (CD20), cluster of diferentiation 22 (CD22), cluster of differentiation 27(CD27), cluster of differentiation 30 (CD30), cluster of diferentiation 70 (CD70), cluster of diferentiation 123 (CD123), cluster of differentiation (CD133), B-cel maturation antigen (BCMA), alpha-actinin-4 (ACTN4), B melanoma antigen 1 (BAGE-1), breakpoint cluster region (BCR) – Abelson murine leukemia (ABL) fusion protein (BCR-ABL), beta-catenin, cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3, also known as CA 27.29), cancer antigen 195 (CA195), cancer antigen 242 (CA 242), cancer antigen 50 (CA 50), tumor associated antigen defined by CT43 monoclonal antibody (CAM43), Caspase-8 (Casp-8), cel division cycle protein 27 (CDC27), cyclin-dependent
  • the target cel is the immune cel or another immune cel. In some embodiments, the target cel is the immune cel. In some embodiments, the target cel is another immune cel. In some embodiments, the antigen comprises cluster of differentiation 3 (CD3). In some embodiments, the reciprocal adhesion molecule comprises cluster of differentiation 2 (CD2). In some embodiments, the immune cel adhesion molecule comprises a cluster of diferentiation 58 (CD58) protein. Some embodiments include an immune cel costimulatory molecule. In some embodiments, the immune cel costimulatory molecule binds cluster of diferentiation 28 (CD28).
  • CD3 cluster of differentiation 3
  • the reciprocal adhesion molecule comprises cluster of differentiation 2 (CD2).
  • the immune cel adhesion molecule comprises a cluster of diferentiation 58 (CD58) protein.
  • Some embodiments include an immune cel costimulatory molecule. In some embodiments, the immune cel costimulatory molecule binds cluster of diferentiation 28 (CD28).
  • the immune cel costimulatory molecule comprises a cluster of diferentiation 80 (CD80) or a cluster of differentiation 86 (CD86) protein.
  • Adhesion Molecules [0093] Disclosed herein, in some embodiments, are adhesion molecules.
  • the adhesion molecule(s) may be included as part of a fusion protein.
  • the adhesion molecule may be included as part of an immune cel engager.
  • adhesion molecule refers, in a broad sense, to a molecular component of a SMAC or other immune synapse, other than an immune cel binding domain (e.g., TCR-binding agent) or a costimulatory molecule, which contributes to adhesion of a particle to target cels.
  • Adhesion molecules from natural sources may be molecules expressed, natively, on antigen- presenting cels and adapted for use here on particles. Both naturaly occuring adhesion molecules, and their variants, and artificial adhesion molecules, such as antibodies, or fragments thereof, are contemplated.
  • adhesion molecule as the term is used herein, specificaly binds a conjugate molecule with affinity adequate to cause increased adhesion between the immune cel engager and the target cel compared to the adhesion of a reference immune cel engager lacking the adhesion molecule to the same or similar target cel.
  • adhesion molecule includes but is not limited to CD58, a CD58 extracellular domain, and functional fragments of CD58.
  • the term “functional fragment” is used herein to a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide.
  • a functional fragment of CD58 is a fragment of CD58 that specificaly binds cluster of differentiation 2 (CD2).
  • the adhesion molecule may be a protein, termed herein an “adhesion protein.”
  • the adhesion molecule is or includes a binding domain that specificaly binds CD2.
  • the adhesion molecule is or includes CD58, cluster of diferentiation 48 (CD48), intercellular adhesion molecule 1 (ICAM-1), intercellular adhesion molecule 2 (ICAM-2), intercellular adhesion molecule 3 (ICAM-3), intercellular adhesion molecule 4 (ICAM-4), intercellular adhesion molecule 5 (ICAM-5), junctional adhesion molecule A (JAM- A), cluster of differentiation 155 (CD155) or cluster of differentiation 112 (CD112), or a functional fragment thereof.
  • the costimulatory and/or adhesion molecule comprises an amino acid sequence 100% identical to a sequence in Table 3A or Table 3B. In some embodiments, the costimulatory and/or adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 3A or Table 3B.
  • the costimulatory and/or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 3A or Table 3B.
  • Polypeptide sequences of illustrated adhesion molecules are provided in Table 3A, with the “start” and “end” positions of the extracellular portion of each.
  • the adhesion molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 3A, or functional fragments thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 (or any range thereof) amino acid portion that retains binding affinity to its cognate molecule, when measured using afinity assays such as biolayer interferometry or other assays that may be known in the art.
  • Some embodiments include an immune cel adhesion molecule.
  • Some examples of immune cel adhesion molecule that include a CD58 protein fragment are provided in Table 3C.
  • a CD58 protein may bind CD2.
  • a CD58 protein that binds CD2 may be or include a CD58 protein fragment.
  • an adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence in Table 3C.
  • an adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 3C.
  • the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 309.
  • the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 310. In some embodiments, the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311.
  • the adhesion molecule is CD58.
  • CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3).
  • LFA-3 lymphocyte function-associated antigen 3
  • CD58 binds to CD2 (LFA-2) on T cels.
  • the extracellular portion of CD58 is residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYN LTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCP MEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSILTTCIPSSGHSRHR (SEQ ID NO: 10) [0100]
  • the polypeptide sequence of CD58 shares at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 248: FSQQIYGVVYGNVTFH
  • the extracellular portion of CD58 has a ligand-binding domain and a second extracellular domain.
  • the ligand-binding domain may be used as the functional fragment of CD58—e.g., without the second extracellular domain.
  • the adhesion molecule (or immune cel engager) comprises the polypeptide sequence of SEQ ID NO: 1 or 10, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule (or immune cel engager) comprises a sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 1 or 10.
  • the adhesion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide).
  • the adhesion molecule may be encoded by the polynucleotide sequence of CD58, SEQ ID NO: 11, or by a subsequence encoding the extracellular portion or a functional fragment.
  • SEQ ID NO: 11 (5’ to 3’): ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCAC TGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTT CCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGC AGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACT GTGTCAGGTAGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAA TCGCCAAATATTACTGATACCATGAAGTTCTTTATGTGCTTGAGTCTCTCTTCCATC
  • the polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 11, or a suitable subsequence, which may be used to express the adhesion molecule.
  • SEQ ID NO: 11 or a suitable subsequence
  • adhesion molecules useful in the practice of an immune cel engager may include any molecule that specificaly binds CD2, LFA-1, or DNAM-1.
  • the adhesion molecule may be a molecule that comprises an antibody, or antigen-binding domain thereof, specific to CD2, Atorney Docket No.061479-511001WO LFA-1, or DNAM-1.
  • the adhesion molecule is a VHH, an scFv, DARPin or any other antigen binding proteins.
  • the adhesion molecule binds to CD2.
  • CD2 is also known as T11, LFA-2, and the erythrocyte rosete receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cels. CD2 is a natural ligand for CD58.
  • engagement of CD2 by CD58 provides a costimulatory signal that may enhance activation and effector functions.
  • the immune cel engager comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof.
  • the immune cel engager comprises an antibody, single domain antibody, antibody fragment, and/or nanobody specific for CD2.
  • the adhesion molecule may comprise any polypeptide sequence in Table 1, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 1, to an extracellular portion thereof, or to a functional fragment thereof.
  • Costimulatory Molecules Disclosed herein, in some embodiments, are costimulatory molecules.
  • the costimulatory molecule may be included as part of a fusion molecule.
  • the costimulatory molecule may be included as part of an immune cel engager.
  • the fusion molecule comprising the immune cel engager may include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule.
  • the fusion molecule may incorporate a costimulatory molecule as a separate molecule, or the fusion molecule may lack any costimulatory molecule.
  • the costimulatory molecule may be a protein, termed herein a “costimulatory protein.”
  • costimulatory protein refers to a molecule capable of providing a costimulatory signal to target cels.
  • the binding of the T cel receptor by an antigen can provide the primary stimulatory signal to the cel.
  • So-caled costimulatory signals are provided by accessory molecules.
  • An example costimulatory signal is the signal provided by binding of cluster of differentiation 28 (CD28) on T cels by a ligand.
  • Some examples of ligands of CD28 include CD80 and cluster of differentiation 86 (CD86).
  • Ilustrative costimulatory molecules include, but are not limited to, CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD27, CD30L, LIGHT, LTalpha, MICA, MICB and anti-CD28.
  • CD80, CD86, CD40L also known as CD154
  • GITRL OX40L
  • 41BBL OX40L
  • ICOSL CD27
  • CD30L LIGHT
  • LIGHT LTalpha
  • MICA MICB
  • anti-CD28 anti-CD28.
  • engager Atorney Docket No.061479-511001WO includes an immune cel costimulatory molecule that provide a costimulatory signal to the immune cel.
  • the costimulatory molecule is or includes a binding domain that specificaly binds CD28.
  • the costimulatory molecule is or includes cluster of diferentiation 80 (CD80), cluster of diferentiation 86 (CD86), CD40 ligand (CD40L also known as CD154), glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 4 (TNFSF4 also known as OX40L), tumor necrosis factor superfamily member 9 (TNFSF9 also known as 41BBL), inducible T cel costimulatory ligand (ICOSL), cluster of diferentiation 70 (CD70), CD30 ligand (CD30L), tumor necrosis factor superfamily member 14 (TNFSF14 also known as LIGHT), lymphotoxin-alpha (LTalpha), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB), or a functional fragment thereof.
  • CD80 cluster of diferent
  • the costimulatory molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any sequence in Table 4, or functional fragments thereof.
  • the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any sequence in Table 4, or a functional fragment thereof.
  • Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays known in the art.
  • the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28.
  • the extracellular portion of CD80 includes residues 35-230 of SEQ ID NO: 12, which includes an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), either or both of which may be included to form the costimulatory molecule.
  • the costimulatory molecule is or includes CD86.
  • CD86 binds to CD28.
  • the extracellular portion of CD86 includes residues 33-225 of SEQ ID NO: 13, which includes an Ig-like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), either or both of which may be included to form the costimulatory molecule.
  • one or both of the domains may be used as the functional fragment of CD86.
  • CD80 or CD86 may be used.
  • homologs of CD80 or CD86 from other species may be identified and tested for use in transducing human, or non-human, target cels. It is expected that at least some non-human homologs wil retain costimulatory molecule function when used with human target cels.
  • the costimulatory molecule (or immune cel engager) comprises the polypeptide sequence of one or more of SEQ ID NO: 12-13 and 25-28, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to one or more of SEQ ID NO: 12-13 and 25-28.
  • the costimulatory molecule comprises a CD80 protein.
  • the CD80 protein may be or include the polypeptide sequence of SEQ ID NO: 250, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 250.
  • the costimulatory molecule (or immune cel engager) comprises a polypeptide sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to one or more of SEQ ID NO: 12-13 and 25- 28.
  • the costimulatory molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).
  • the costimulatory molecule may be encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding the extracellular domain or a functional fragment.
  • costimulatory molecules useful in the practice of the present disclosure may include any molecule that specificaly binds CD28.
  • the costimulatory molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD28.
  • CD28 is a receptor expressed on T cels that provide costimulatory signal. T cel co- stimulation through CD28, resulting in, for example, the production of various interleukins (in Atorney Docket No.061479-511001WO particular IL-6).
  • the costimulatory molecule is an antibody, or fragment thereof, that specificaly binds to CD28.
  • 15E8 is a mouse monoclonal antibody to human CD28. Its complementarity determining regions (CDRs) are as folows: [0126] CDRH1: GFSLTSY (SEQ ID NO: 36) [0127] CDRH2: WAGGS (SEQ ID NO.37) [0128] CDRH3: DKRAPGKLYYGYPDY (SEQ ID NO.38) [0129] CDRL1: RASESVEYYVTSLMQ (SEQ ID NO.39) [0130] CDRL2: AASNYES (SEQ ID NO.40) [0131] CDRL3: QQTRKVPST (SEQ ID NO.41) [0132] TGN1412 (also known as CD28-SuperMAB) is a humanized monoclonal antibody that not only binds to, but also is a strong agonist for,
  • CDRHl GYTFSY (SEQ ID NO.42)
  • CDRH2 YPGNVN (SEQ ID NO.43)
  • CDRH3 SHYGLDWNFDV (SEQ ID NO.44)
  • CDRLl HASQNIYVLN (SEQ ID NO.45)
  • CDRL2 KASNLHT (SEQ ID NO.46)
  • CDRL3 QQGQTYPYT (SEQ ID NO.47) [0139]
  • CD80, CD86, and their derivatives as the costimulatory molecule and CD28 as the cognate molecule may be extrapolated to the other costimulatory molecules described herein, including but not limited to those listed in Table 4.
  • the costimulatory molecule (or the immune cel engager) may comprise any polypeptide sequence of in Table 4, to an extracellular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 4, to an extracellular portion thereof, or to a functional fragment thereof.
  • the costimulatory molecule is a VHH, an scFv, DARPin or any other antigen binding protein.
  • Immune Cel Binding Domains [0140] Disclosed herein, in some embodiments, are immune cel binding domains.
  • the immune cel binding domain may be included as part of an immune cel engager.
  • An example of an immune cel binding domain may include a TCR-binding molecule.
  • the immune cel engager may include an immune cel binding domain (e.g., a TCR-binding molecule) or other subunit that provides an activation signal to a target cel. However, in some embodiments, the immune cel engager does not include a TCR-binding molecule or other activation domain.
  • the immune cel engager may incorporate a TCR-binding molecule as a separate molecule, Atorney Docket No.061479-511001WO or the immune cel engager may lack any TCR-binding molecule.
  • the TCR-binding molecule may be a protein, termed herein a “TCR-binding protein.”
  • the immune cel binding domain may be or include an activation protein.
  • TCR-binding molecule refers to a molecule capable of directly binding the extracellular portion of the T cel receptor (TCR) by contacting one or more components of the TCR or otherwise providing a primary or “signal 1” activation signal to a target cel (e.g., a T cel or NK cel).
  • TCR T cel receptor
  • a target cel e.g., a T cel or NK cel
  • TCR-binding molecules may include an antibody, or antigen binding domain, that specificaly binds CD3 (an anti-CD3 monoclonal antibody, or antigen binding fragment thereof).
  • the immune cel binding domain comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3.
  • Ilustrative antibodies include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab and visilizumab.
  • the complementarity determining regions of OKT3 are as folows in Table 5.
  • the immune cel binding domain (e.g., TCR-binding molecule) may be a single chain variable fragment (scFv) incorporated into the fusion protein (or immune cel engager). OKT3 in scFv format may be used.
  • the immune cel binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a heavy chain variable domain (VH) domain.
  • the VH domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 312.
  • the immune cel binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a light chain variable domain (VL) domain.
  • the VL domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 313.
  • the immune cel binding domain e.g.
  • TCR-binding molecule is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% Atorney Docket No.061479-511001WO identical to the anti-CD3 scFv of SEQ ID NO: 31, which includes a variable light (VL) and variable heavy (VH) domain with a 3 ⁇ GGGS (SEQ ID NO: 307) linker: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSG TDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGG VVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPS
  • TCR-binding molecule is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 249, which includes a variable light (VL) and variable heavy (VH) domain with a 3 ⁇ GGGS (SEQ ID NO: 307) linker: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSG TDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGG VVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRD
  • CD3, OX40, CD27, ICOS, 41BB, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC.
  • the immune cel engager disclosed herein may comprise portions of these proteins or domains that bind to these proteins.
  • T cels may express one or both of CD4 and/or CD8 and the fusion molecules disclosed herein may comprise domains that engage with either or both of CD4 and/or CD8.
  • other binding domains may be more appropriate.
  • an immune cel engager targeting NK cels may comprise domains that engage with proteins found on NK cels.
  • these proteins include CD2, CD16, NKp46, NKp30, and NKG2D.
  • immune cel engager intended to target and/or activate NK cels may comprise domains that bind to CD2, CD16, NKp46, NKG2D, etc. Domains that bind to NKG2D may be derived from NKG2D ligands including, but not limited to: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6.
  • the immune cel engager described herein Atorney Docket No.061479-511001WO comprises a CD58 domain, a domain that binds NKG2D, and optionaly a third domain which enhances activation of the target NK cel.
  • the immune cel binding domain may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide).
  • Tumor cel binding domains [0158] Some embodiments include a tumor cel binding domain.
  • an engager such as an immune cel engager may include a tumor cel binding domain.
  • the tumor cel binding domain may contain any polypeptide that binds a desired antigen (e.g. antigen expressed on a tumor of interest).
  • the domain may comprise a scFv, a portion of an antibody or an alternative scafold.
  • scFvs lama VHH antibodies, other VH only antibody fragments, peptides, or smal protein binding domains (e.g. D domains) may be utilized.
  • the tumor cel binding domain may comprise an scFv or VHH that binds to a target tumor antigen or the tumor cel binding domain may comprise a natural ligand, for example, folate.
  • the antigen to which the tumor cel binding domain binds can be any antigen of interest, e.g., can be an antigen on a tumor cel.
  • the tumor cel may be, e.g., a cel in a solid tumor, or a cel of a blood cancer.
  • the antigen can be any antigen that is expressed on a cel of any tumor or cancer type, e.g., cels of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic smal round cel tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cel tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma,
  • said lymphoma can be chronic lymphocytic leukemia (smal lymphocytic lymphoma), B-cel prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasma cel myeloma, plasmacytoma, extranodal marginal zone B cel lymphoma, MALT lymphoma, nodal marginal zone B cel lymphoma, folicular lymphoma, mantle cel lymphoma, difuse large B cel lymphoma, mediastinal (thymic) large B cel lymphoma, intravascular large B cel lymphoma, primary effusion lymphoma, Burkit's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cel leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/
  • the B cels of the CLL have a normal karyotype.
  • the cancer is chronic lymphocytic leukemia (CLL)
  • the B cels of the CLL cary a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion or a p53 deletion.
  • the antigen is expressed on a B-cel malignancy cel, relapsed/refractory CD19-expressing malignancy cel, diffuse large B-cel lymphoma (DLBCL) cel, Burkit’s type large B-cel lymphoma (B-LBL) cel, folicular lymphoma (FL) cel, chronic lymphocytic leukemia (CLL) cel, acute lymphocytic leukemia (ALL) cel, mantle cel lymphoma (MCL) cel, hematological malignancy cel, colon cancer cel, lung cancer cel, liver cancer cel, breast cancer cel, renal cancer cel, prostate cancer cel, ovarian cancer cel, skin cancer cel, melanoma cel, bone cancer cel, brain cancer cel, squamous cel carcinoma cel, leukemia cel, myeloma cel, B cel lymphoma cel, kidney cancer cel, uterine cancer cel, adenocarcinoma cel, pancreatic cancer cel, chronic myelogenous le
  • the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).
  • TAA tumor-associated antigen
  • TSA tumor-specific antigen
  • the tumor-associated antigen or tumor- specific antigen is B cel maturation antigen (BCMA), B cel Activating Factor (BAFF), GPRC5D, FCRL5/FCRH5, ROR1, L1-CAM, CD22, folate receptor, carboxy anhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL-13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4 L six family member 1 (TM4SF1), epithelial cel adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, glypican-3 (GPC3), CD133, CD147, EGFR, MUC1, GD
  • Some example antibodies useful for target cel-binding domains or tumor cel-binding domains may include anti-mucin monoclonal antibody CAM 17.1, and MOv18 which is an IgE antibody that targets folate receptor alpha.
  • the tumor cel binding domain comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD19 and CD22, BCMA and GPRC5D, or CD20 and CD22.
  • the tumor cel binding domain comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA.
  • Exemplary binding domains include scFvs derived from the anti-CD19 antibody FMC63, which are known in the art. Binding domains targeting CD19 are described, for example, in U.S. Pat. Pub. No.20160152723; and U.S. Pat. Nos.10,736,918; 10,357,514; and 7,446,190.
  • Exemplary binding domains include VH domains that bind BCMA and scFvs that bind BCMA.
  • Binding domains targeting BCMA are described, for example, in U.S. Pat. Pub. Nos.2020/0246381 and 2019/0161553; and U.S. Pat. Nos.10,918,665. Binding domains targeting GPRC5D may be found in U.S. Pat. Pub. Nos.2018/0118803 and 2021/10393689. Binding domains targeting FCRL5 may be found in U.S. Pat. Pub. No. US 2017/0275362. Binding domains targeting ROR1 may be found in U.S. Pat. Pub. No.2022/0096651.
  • the tumor cel binding domain binds to an antigen selected from the group consisting of: CD19, EpCAM, Her2/neu, EGFR, CD66e, CD33, EphA2, or MCSP. In some embodiments, the tumor cel binding domain binds to an antigen selected from the group consisting of: CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. In some embodiments, the tumor cel binding domain binds to a myeloid cel or dendritic cel antigen. In some embodiments, the tumor cel binding domain binds to CD33, DC-SIGN, CDlb, CDlc, or CD18.
  • a DUPA derivative can be the ligand of the smal molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015/057852, incorporated herein by reference.
  • the tumor cel binding domain comprises CCK2R ligand, a ligand bound by CCK2R-positive cancer cels (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics.2013. ePublication).
  • CCK2R-positive cancer cels e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics.2013. ePublication).
  • the tumor cel binding domain comprises folate, folic acid, or an analogue thereof, a ligand bound by the folate receptor on cels of cancers that include cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, E.I. et al., Cancer Metastasis Rev.2008.27(4):655-64).
  • the tumor cel binding domain comprises an NK-1R ligand. Receptors for NK-1R the ligand are found, for example, on cancers of the colon and pancreas.
  • the NK-1R ligand may be synthesized according the method disclosed in Int’l Patent Appl. No. PCT/US2015/044229, incorporated herein by reference.
  • the tumor cel binding domain comprises a peptide ligand, for example, the ligand may be a peptide ligand that is the endogenous ligand for the NK1 receptor.
  • the ligand may be a regulatory peptide that belongs to the family of tachykinins Atorney Docket No.061479-511001WO which target tachykinin receptors.
  • the tumor cel binding domain comprises a CAIX ligand.
  • Receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers.
  • the CAIX ligand may also be refered to herein as CA9.
  • the tumor cel binding domain comprises a ligand of gamma glutamyl transpeptidase.
  • the tumor cel binding domain comprises a CCK2R ligand.
  • Receptors for the CCK2R ligand found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, among others.
  • Multimerization Domains [0172] Polypeptides described herein may form multimers such as dimers, trimers and higher oligomers, e.g., consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc.
  • a multimerization polypeptide when fused to a second molecule, such as a heterologous polypeptide sequence, facilitates the formation of dimers, trimers, tetramers, pentamers or higher order oligomers or mixtures thereof among the polypeptides.
  • Fusing a multimerization polypeptide to an antigen binding protein, such as an antigen binding polypeptide (e.g., antibody) can be used to increase the number of antigen binding sites via oligomer formation.
  • the immune cel engager further comprises a multimerization domain.
  • the multimerization domain is a Fc domain from an IgG.
  • the multimerization domain is a Fc domain from IgA.
  • the multimerization domain is a Fc domain from IgD.
  • the multimerization domain is a Fc domain from IgE. In some embodiments, the multimerization domain is a Fc domain from IgM. In some embodiments, the multimerization domain is a Fc domain from IgG1. In some embodiments, the multimerization domain is a Fc domain from IgG2. In some embodiments, the multimerization domain is a Fc domain from IgG3. In some embodiments, the multimerization domain is a Fc domain from IgG4 Atorney Docket No.061479-511001WO [0175] In some embodiments, the multimerization domain is the self-assembling tetramerization domain (TD) from the tumor suppressor gene p53.
  • TD tetramerization domain
  • the multimerization domain is the SpyCather-SpyTag system. [0176] In some embodiments, the multimerization domain is a self-associating peptide. In some embodiments, the self-associating peptide is RHCC derived from a right-handed coiled-coil peptide of an archaebacterium. In some embodiments, the self-associating peptide is COMPcc from human cartilage oligomeric matrix protein. In some embodiments, the self-associating peptide is C4bp ⁇ derived from human plasma C4-binding protein ⁇ -chain.
  • the disclosure provides a pharmaceutical composition comprising an immune cel engager according to the disclosure and a pharmaceuticaly acceptable carier.
  • the formulations and compositions of the present disclosure may comprise a combination of any number of immune cel engagers, and optionaly one or more additional pharmaceutical agents (viral particles, polypeptides, polynucleotides, compounds etc.) formulated in pharmaceuticaly acceptable or physiologicaly-acceptable compositions for administration to a cel, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy.
  • the one or more additional pharmaceutical agents further increases transduction efficiency of viral particles.
  • the formulations and compositions of the present disclosure may comprise a combination of any number of immune cel engagers, and optionaly one or more nanocarriers.
  • Ilustrative nanocariers include, but are not limited to, miceles, polymers, liposomes, and lipid nanoparticles (LNPs).
  • the present disclosure provides compositions comprising a therapeuticaly-effective amount of an immune cel engager, as described herein, formulated together with one or more pharmaceuticaly acceptable cariers (additives) and/or diluents.
  • the composition further comprises other agents, such as, e.g., cytokines, growth factors, hormones, smal molecules, or various pharmaceuticaly active agents.
  • compositions and formulations of the immune cel engager used in accordance with the present disclosure may be prepared for storage by mixing an immune cel engager having the desired degree of purity with optional pharmaceuticaly acceptable cariers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Acceptable cariers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
  • one or more pharmaceuticaly acceptable surface-active agents surfactant
  • bufers isotonicity agents
  • salts amino acids
  • sugars stabilizers and/or antioxidant
  • Suitable pharmaceuticaly acceptable surfactants comprise but are not limited to polyethylen-sorbitan-faty acid esters, polyethylene-polypropylene glycols, polyoxyethylene- stearates, and sodium dodecyl sulphates.
  • Suitable buffers comprise but are not limited to histidine- buffers, citrate-buffers, succinate-bufers, acetate-bufers, and phosphate-buffers.
  • Isotonicity agents are used to provide an isotonic formulation.
  • An isotonic formulation is liquid, or liquid reconstituted from a solid form, e.g., a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum.
  • Suitable isotonicity agents comprise but are not limited to salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars including but not limited to glucose, sucrose, trehalose or and any component from the group of amino acids, sugars, salts, and combinations thereof.
  • isotonicity agents are generaly used in a total amount of about 5 mM to about 350 mM.
  • Non-limiting examples of salts include salts of any combinations of the cations sodium potassium, calcium or magnesium with anions chloride, phosphate, citrate, succinate, sulphate, or mixtures thereof.
  • Non-limiting examples of amino acids comprise arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, or proline.
  • Non-limiting examples of sugars according to the disclosure include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, rafinose, glucosamine, N-methylglucosamine (also refered to as “meglumine”), galactosamine and neuraminic acid and combinations thereof.
  • Non-limiting examples of stabilizer includes amino acids and sugars as described above as wel as commercialy available cyclodextrins and dextrans of any useful kind and molecular weight.
  • Non-limiting examples of antioxidants include excipients such as methionine, benzylalcohol or any other excipient used to minimize oxidation.
  • pharmaceuticalaly acceptable refers to molecular entities and compositions that do not produce an an organic or similar untoward reaction when administered to a human.
  • the preparation of an aqueous composition that contains a protein as an active ingredient is wel understood in the art. Typicaly, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.
  • carrier includes any and al solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, bufers, carrier solutions, suspensions, coloids, and the like. Except insofar as any media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • compositions comprising a carier are suitable for parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration.
  • pharmaceuticaly acceptable carriers may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • compositions may further comprise one or more polypeptides, polynucleotides, vector genomes comprising same, compounds that increase the transduction eficiency of vector genomes, formulated in pharmaceuticaly acceptable or physiologicaly-acceptable solutions for administration to a cel or an animal, either alone, or in combination with one or more other modalities of therapy.
  • compositions of the present disclosure may be administered in combination with other agents as wel, such as, e.g., cytokines, growth factors, hormones, smal molecules, or various pharmaceuticaly active agents.
  • compositions comprising an expression cassete or vector (e.g., therapeutic vector) and one or more pharmaceuticaly acceptable cariers, diluents, or excipients.
  • the pharmaceutical composition comprises an immune cel engager and a lentiviral vector comprising an expression cassete disclosed herein, e.g., wherein the expression cassete comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptor (CARs) and variants thereof.
  • CARs chimeric antigen receptor
  • the pharmaceutical compositions that contain immune cel engagers may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration.
  • the immune cel engager can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with pharmaceutical supports, to animals and human beings.
  • the pharmaceutical composition comprises cels transduced ex vivo with immune cel engagers and any of the vector genomes according to the present disclosure.
  • the immune cel engagers, or a pharmaceutical composition comprising immune cel engagers is effective when administered systemicaly.
  • the immune cel engagers of the disclosure in some cases, may be administered intravenously to subject (e.g., a primate, such as a non-human primate or a human).
  • the immune cel engagers administered in combination with the viral vectors of the disclosure are capable of inducing Atorney Docket No.061479-511001WO expression of CARs in various immune cels when administered systemicaly (e.g., in T-cels, dendritic cels, NK cels).
  • the pharmaceutical compositions contain vehicles (e.g., cariers, diluents, and excipients) that are pharmaceuticaly acceptable for a formulation capable of being injected.
  • vehicles e.g., cariers, diluents, and excipients
  • Ilustrative excipients include a poloxamer.
  • Formulation bufers for immune cel engagers may contain salts to prevent aggregation and other excipients (e.g., poloxamer) to reduce stickiness of the immune cel engagers.
  • the formulation is stable for storage and use when frozen (e.g., at less than 0 °C, about -60 °C, or about -72 °C). In some embodiments, the formulation is a cryopreserved solution.
  • compositions of the present disclosure formulation of pharmaceuticaly acceptable excipients and carier solutions may be useful to those of skil in the art, such as for development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.
  • suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation.
  • Solutions of the active compounds as free base or pharmacologicaly acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcelluloseose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • a surfactant such as hydroxypropylcelluloseose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No.5,466,468, incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Fluidity may be maintained, for example, by use of a coating, such as lecithin, by maintenance of a useful particle size in the case of dispersion and by the use of surfactants.
  • microorganisms can be facilitated by various antibacterial and antifungal agents, for Atorney Docket No.061479-511001WO example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride, are added.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if useful or necessary and the liquid diluent first rendered isotonic with adequate saline or glucose.
  • aqueous solutions are especialy suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the solution intended for subcutaneous administration includes hyaluronidase.
  • a sterile aqueous medium that can be employed may be useful.
  • One dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincot Wiliams & Wilkins, 2005). Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration wil, in any event, determine the appropriate dose for the individual subject. In some embodiments, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards set by the FDA Office of Biologics standards.
  • the present disclosure provides formulations or compositions suitable for the delivery of immune cel engagers and viral vector systems (e.g., viral- mediated transduction) including, but not limited to, retroviral (e.g., lentiviral) vectors.
  • Gene Therapy Vectors [0198] Some embodiments include a composition that includes a gene therapy vector. The composition may further include an immune cel engager herein. The gene therapy vector may be included in a method herein, such as a method of treatment or use. The method may include use of a gene therapy vector with an immune cel engager. The method may include use of a particle with an immune cel engager, or administration of a gene therapy vector and an immune cel engager. A gene therapy vector may be included in or with a particle.
  • the immune cel engager is administered with a gene therapy vector. In some embodiments, the immune cel engager is administered before the gene therapy vector. In some embodiments, the immune cel engager is administered after the gene therapy vector. In some embodiments, the immune cel engager is co-administered with a gene therapy vector.
  • gene therapy vector refers to gene therapy delivery vehicles, or cariers, that deliver therapeutic genes to cels.
  • a gene therapy vector is any vector suitable for use in gene therapy, e.g., any vector suitable for the therapeutic delivery of nucleic acid polymers (encoding a polypeptide or a variant thereof) into target cels (e.g., sensory neurons) of a patient.
  • the gene therapy vector delivers the nucleic acid encoding a CAR.
  • the vector may be Atorney Docket No.061479-511001WO of any type, for example it may be a plasmid vector or a minicircle DNA.
  • the vector is a viral vector. These include both geneticaly disabled viruses such as adenovirus and nonviral vectors such as liposomes.
  • the viral vector may for example be derived from an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus.
  • AAV derived vectors may comprise an AAV genome or a derivative thereof.
  • Some embodiments include a composition that includes a particle.
  • the composition may further include an immune cel engager herein.
  • the particle may be included in a method herein, such as a method of treatment or use.
  • the method may include use of a particle with an immune cel engager, or administration of a particle and an immune cel engager.
  • a particle may include a vector such as a gene therapy vector.
  • a viral particle such as a lentiviral particle may be used as or include a gene therapy vector.
  • the disclosure provides particles of various types, including but not limited to lentiviral particles (e.g., a virion), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers.
  • the particle may be loaded with an adhesion molecule, a costimulatory molecule, or a combination thereof.
  • the particle may be loaded with the immune cel engager. Any of the adhesion molecule, costimulatory molecule, or activation molecules may be included in a fusion molecule.
  • the adhesion molecule, costimulatory molecule, activation molecule or combination thereof may be loaded onto a surface of the particle.
  • the fusion molecule may be loaded onto a surface of the particle.
  • the immune cel engager is administered with a particle.
  • the particle may be co-administered with the immune cel engager to increase transduction.
  • the immune cel engager is administered at the same time as the particle.
  • the immune cel engager is administered before the particle.
  • the immune cel engager is administered after the particle [0203]
  • the particle may be a lipid nanoparticle (LNP) or a poly(beta-amino) esters (PBAE) nanocarriers, both of which have been shown to transduce T cels when administered to a subject in vivo or contacted with T cels ex vivo.
  • LNP lipid nanoparticle
  • PBAE poly(beta-amino) esters
  • the particle is a viral particle.
  • Methods for generating viral vectors from various virus types are known in the art.
  • Exemplary types of viral particles that may be recombinantly engineered as delivery vehicles include retroviruses, lentivirus (e.g. HIV and its derivatives and SIV), adeno-associated virus, adenovirus, MMLV retrovirus, MSCV retrovirus, baculovirus, vesicular stomatitis virus, herpes simplex virus, and vaccinia virus.
  • retroviruses e.g. HIV and its derivatives and SIV
  • adeno-associated virus e.g. HIV and its derivatives and SIV
  • adeno-associated virus e.g. HIV and its derivatives and SIV
  • adeno-associated virus e.g. HIV and its derivatives and SIV
  • adeno-associated virus e.g. HIV and its derivatives and SIV
  • adeno-associated virus e.g. HIV
  • the particle is a retroviral particle.
  • the particle is a lentiviral particle.
  • Atorney Docket No.061479-511001WO Lentiviral particles may be made using packaging cel lines as described in WO 2016/139463 or by using a polycistronic vector as described in Int’l Pat. Pub. No. WO 2020/106992 A1.
  • Each of the foregoing specificaly describes methods for making lentiviral particles. Their disclosures are incorporated by reference herein. Numerous other methods for making viral particles, including lentiviral particles, may be useful.
  • Retroviruses a group that includes lentiviruses, are enveloped viruses.
  • Lentiviral particles generaly package a vector genome and, incidentaly or intentionaly, may package other molecules present in the producer cel.
  • the vector genome may be an artificial vector genome engineered to encode a heterologous protein or polynucleotide.
  • Lentiviral particles may contain structural and/or functional genetic elements that are primarily from a virus. Lentiviral particles are characterized by the predominant source of genetic or structural material in the lentiviral particle.
  • the term “retroviral particle” refers to a viral particle containing structural proteins and vector genome elements, primarily from a retrovirus.
  • lentiviral particle refers to a viral particle containing structural proteins and vector genome elements, primarily from a lentivirus. To package its vector genome, a lentiviral particle may generaly require at least one copy of the long-terminal repeats (LTRs) that flank a native lentiviral vector genome, or functional variants thereof.
  • LTRs long-terminal repeats
  • a viral particle comprises a viral glycoprotein. In some embodiments, the viral particle comprises a viral glycoprotein different from the native viral glycoprotein. When the viral glycoprotein is heterologous to the vector genome, the viral particle is termed a “pseudotyped” viral particle.
  • the viral particle is derived from HIV, which typicaly includes the glycoprotein gp120.
  • HIV-based particles may be “pseudotyped” and, instead of expressing their native glycoprotein, express a glycoprotein from a diferent virus.
  • the viral glycoprotein may be a portion of RD114 or one of its variants, VSV-G, Gibbon-ape leukemia virus (GALV), the Amphotropic envelope glycoprotein, Measles envelope glycoprotein, or baboon retroviral envelope glycoprotein.
  • the viral envelope glycoprotein is a G protein from the Cocal strain (Cocal G), or a functional variant thereof.
  • Ilustrative viral glycoproteins include the VSV G protein, the Cocal G protein, and variants thereof. Ilustrative viral glycoproteins may be expressed as a single protein or in multiple subunits or parts. The viral glycoprotein may serve as ligand for cel-surface receptors on a target cel, and thereby promote transduction or the target cel. The viral glycoprotein may be engineered to lack LDLR binding affinity—for example, by mutation at positions 47 (e.g., K47Q) and/or 354 (e.g., R354A). This may be termed a “blinded” viral glycoprotein. Ilustrative envelope variants are provided in, e.g., US 2020/0216502 A1, which is incorporated herein by reference in its entirety.
  • the immune cel engager as described herein may permit use of a viral Atorney Docket No.061479-511001WO glycoprotein that does not, by itself, cause transduction of target cel. Without being bound by theory, it is believed that the immune cel engager may serve as a ligand for cel-surface receptor while the viral glycoprotein retains a structural function, but not a function as a ligand for a cel- surface receptor.
  • the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 47. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 354.
  • the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a R354A mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q and a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 47. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 354. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a R354A mutation.
  • the viral glycoprotein is a cocal glycoprotein that comprises a K47Q and a R354A mutation.
  • the viral glycoprotein comprises a mutation at position 47.
  • the viral glycoprotein comprises a mutation at position 354.
  • the viral glycoprotein comprises a K47Q mutation.
  • the viral glycoprotein comprises a R354A mutation.
  • the viral glycoprotein comprises a K47Q and a R354A mutation.
  • the Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the folowing sequence: NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHK AIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGY ATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATL VDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYA AAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGP
  • the Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the folowing sequence: MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTH KAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCG YATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDA TLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDV YAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNP
  • the backbones are HIV-based vector backbones (e.g., HIV cis-acting sequence elements).
  • Retroviral particles have been generated by multiply atenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologicaly safe.
  • Ilustrative lentiviral particles and methods for making them are described in Naldini et al. Science 272:263-7 (1996); Zuferey et al. J. Virol.72:9873-9880 (1998); Dul et al. J. Virol. 72:8463-8471 (1998); Miyoshi et al. J.
  • Protocols for producing replication-defective recombinant viruses are provided in W095/14785, W096/22378, U.S. Pat. No.5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No.5,278,056, and W094/19478.
  • Viral particles may be assessed in various ways, including, for example, measuring the vector copy number (VCN) or vector genomes (vg) in a sample of viral particle by quantitative polymerase chain reaction (qPCR) or digital droplet PCR (ddPCR), or testing to the viral particles on target cels to measure a “titer” of the virus in, e.g., infectious units per mililiter (IU/mL).
  • VCN vector copy number
  • vg vector genomes
  • ddPCR digital droplet PCR
  • the titer may be assessed using a functional assay performed on the cultured tumor cel line HT1080 as described in Humbert et al. Molecular Therapy 24:1237–1246 (2016).
  • the method of use may be with regard to an immune cel engager, or a composition or formulation.
  • a method may include immune cel manufacturing, virus manufacturing, or nanoparticle manufacturing.
  • a method may include an in vivo method, an in vitro method, or an ex vivo method.
  • a method may be performed in vivo or with a subject, may be performed in vitro, or may be performed ex vivo.
  • a method may include delivery of exogenous proteins, or may include use of a gene therapy vector.
  • Immune cel engagers of the present disclosure may enhance particle to T cel binding. Immune cel engagers of the present disclosure may enhance T cel activation. Immune cel engagers Atorney Docket No.061479-511001WO of the present disclosure may enhance immune cel expansion. Immune cel engagers of the present disclosure may enhance immune cel transduction.
  • Cel Manufacturing [0220] Disclosed herein, in some embodiments, are methods for use in immune cel manufacturing.
  • An immune cel manufacturing method may include an ex vivo method such as an ex vivo manufacturing method.
  • the immune cel engager can be used for immune cel manufacturing. In some embodiments, the immune cel engager can be used for activation of immune cels during immune cel manufacturing. In some embodiments, the immune cel engager can be used for expansion of immune cels during immune cel manufacturing. In some embodiments, immune cel manufacturing includes engineering immune cels (e.g. T cels and/or NK cels) to express one or more non-endogenous proteins.
  • immune cel manufacturing includes engineering immune cels (e.g. T cels and/or NK cels) to express one or more non-endogenous proteins.
  • Incorporation of one or more ligands for a co-receptor, such as CD28, a molecule expressed by T cels may cause the T cels to activate via a secondary activation signal.
  • SMAC supramolecular activation cluster
  • the immune cel engager By engaging multiple proteins typicaly found within the SMAC on the surface of the T cel, the immune cel engager can induce increased T cel activation and proliferation.
  • engagement of CD2 by CD58 provides an additional signal that may enhance activation and effector functions.
  • Other immune cel engagers may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cels.
  • SMAC supramolecular activation complex
  • CD3, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC.
  • the immune cel engager can be used for concomitant selection, activation, and expansion of T cels in a one step process. Selection, activation, and expansion of T cels during preparation of T cels for immunotherapy can be limited by three things: 1) generating immune cels with desired characteristics, 2) scale of operation needed to generate T cels for immunotherapy and 3) resource availability.
  • the immune cel engager describe herein alows for the generation of immune cels with desired characteristics through switching domains in the fusion protein.
  • the CD58 binding domain can be replaced with a CD16 binding domain to activate NK cels.
  • the disclosure provides a method of delivering a nucleic acid to a cel ex vivo. In some embodiments, the disclosure provides a method of delivering a nucleic acid to an immune cel ex vivo. In some embodiments, the immune cel engagers of the disclosure activate and increase transduction of an immune cel ex vivo.
  • the disclosure provides a method of delivering an immune cel engager and a nucleic acid to a cel in an ex vivo CAR T manufacturing process.
  • Such methods typicaly involve the isolation of peripheral blood mononuclear cels (PBMCs) from a patient via leukapheresis.
  • PBMCs peripheral blood mononuclear cels
  • such methods involve obtaining whole blood from a patient without isolation of PBMCs and forward processing the whole blood.
  • the PBMCs may be washed and optionaly further purified via one or more selection steps to isolate particular T cel populations of interest. In some aspects, these might include CD4+ and/or CD8+ T cels.
  • the washed and/or purified cels may be optionaly activated and then transduced using an immune cel engager and a lentiviral vector.
  • the activation step may comprise contacting the cels with an exogenous activation agent such as the immune cel engagers described herein, anti-CD3 and anti-CD28 antibodies bound to a substrate or using unbound antibodies.
  • the immune engagers described herein may be immobilized on a substrate – e.g. a resin, bead, polymer, expamer, or other substrate described herein – and used to isolate and activate target cels simultaneously.
  • an immune engager as described herein may be useful to engage with (e.g.
  • an immune cel engager as described herein may eliminate one or more separate unit operations traditionaly present in ex vivo immune cel manufacturing. Additionaly, the immune cel engagers described herein need not be immobilized on a substrate and instead may be provided in soluble form to activate the target immune cels.
  • Transduction may be accomplished by contacting the patient’s PBMCs, isolated cels, or, in some cases, whole blood with immune cel engagers and/or lentiviral particles described herein. After transduction, the cels may be optionaly further washed and cultured until harvest.
  • Methods of manufacturing engineered cel therapies including CAR T cels (see e.g., Abou-el-Enein, M. et al. Blood Cancer Discov (2021), Vol 2(5): 408-422; Arcangeli, S. et al. Front. Immunol (19 Jun 2020), Vol.11 (1217) 1-13; Ghassemi, S.
  • the present disclosure provides an ex vivo method of generating an engineered cel comprising contacting a target cel with a particle and an immune cel engager comprising an adhesion molecule linked to a costimulatory molecule, immune cel engager comprising an adhesion molecule linked to an immune cel binding domain, or an immune cel engager comprising an adhesion molecule linked to a costimulatory molecule and an immune cel binding domain wherein the contacting step is performed for approximately one hour, for approximately two hours, approximately three hours, approximately four hours, approximately five hours, approximately six hours, approximately 12 hours, approximately 24 hours, approximately 12- 24 hours (inclusive of endpoints), or longer.
  • This method may require the contacting step to be performed in a closed-loop manufacturing or extracorporeal process as described herein.
  • this method may require the contacting step to be performed in a traditional ex-vivo engineered cel manufacturing process.
  • a perfusion incubator or a centrifuge such as a SepaxTM or RoteaTM machine.
  • the compositions described herein such as immune cel engagers described may be used in vitro or ex vivo.
  • the immune cel engagers described may be used ex vivo, in a cel manufacturing process or at a bedside as described, e.g., in Int’l Pat. Pub. No. WO 2022/072885, Int’l Pat. Pub. No.
  • the disclosure provides an ex vivo method of transducing target cels, comprising contacting the target cels with the immune cel engager and a delivery vehicle (such as viral particle according to the present disclosure, a lipid nanoparticle, or other means of delivering a polynucleotide to a cel).
  • a delivery vehicle such as viral particle according to the present disclosure, a lipid nanoparticle, or other means of delivering a polynucleotide to a cel.
  • the immune cel engager described herein may be used to increase transduction of cels that have not been previously activated.
  • the immune cel engager described herein may be useful for increasing transduction of cels that have not been previously contacted with cel activation beads or activation reagents (e.g., DynabeadsTM or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof).
  • cel activation beads or activation reagents e.g., DynabeadsTM or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof.
  • a method herein describes use of a lentiviral particle with immune cel engager, use of another particle is contemplated where appropriate and feasible.
  • use of an immune cel engager with a lentiviral particle use of a composition or fusion molecule is also contemplated where appropriate and feasible.
  • an immune cel engager bound to the surface of a lentiviral particle, or a pharmaceutical composition may be administered to or contacted with a cel such as an immune cel (e.g., T cel).
  • a cel such as an immune cel (e.g., T cel).
  • an immune cel e.g., T cel.
  • Atorney Docket No.061479-511001WO Non-limiting examples of cels that can be the target of the immune cel engager described herein include T lymphocytes, dendritic cels (DC), Treg cels, B cels, Natural Kiler cels, and macrophages.
  • an immune cel engager described herein is used to enhance transduction of a nucleic acid sequence (polynucleotide) encoding one or more exogenous proteins into a cel (e.g., a T lymphocyte, NK cel, or other target cel) in vivo.
  • a cel e.g., a T lymphocyte, NK cel, or other target cel
  • the transduction of the lentiviral particle results in expression of one or more exogenous proteins in the transduced cels.
  • the immune cel expressing the exogenous protein is generated by co-administration of the immune cel engager described herein and a particle comprising a nucleic acid encoding the exogenous protein.
  • the particle is a virus particle.
  • the particle is a lentivirus particle. In some embodiments, the particle is a nanoparticle. In some embodiments, the particle is a lipid nanoparticle. In some embodiments the immune cel engager and particle are administered to a subject simultaneously. In some embodiments, the immune cel engager is administered to a subject prior to the particle. In some embodiments, the immune cel engager is administered to a subject after the particle.
  • An exemplary exogenous protein is a chimeric antigen receptor (CAR).
  • CARs are artificial membrane-bound proteins that direct a T lymphocyte to an antigen and stimulate the T lymphocyte to kil cels displaying the antigen. See, e.g., Eshhar, U.S. Pat.
  • CARs are geneticaly engineered receptors comprising an extracellular domain that binds to an antigen, e.g., an antigen on a cel, an optional linker, a transmembrane domain, and an intracellular (cytoplasmic) domain comprising a costimulatory domain and/or a signaling domain that transmits an activation signal to an immune cel.
  • an antigen e.g., an antigen on a cel
  • an optional linker e.g., a transmembrane domain
  • an intracellular (cytoplasmic) domain comprising a costimulatory domain and/or a signaling domain that transmits an activation signal to an immune cel.
  • a CAR when expressed on the surface of, e.g., a T lymphocyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cel surface, to kil the cel expressing the antigen.
  • T lymphocytes may require two signals, a primary activation signal and a costimulatory signal, in order to maximaly activate
  • CARs can comprise a stimulatory and a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal.
  • Ilustrative CARs may be designed in a modular fashion, e.g., as described in (see, e.g., Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy - Methods & Clinical Development.2019; 12: 145-156), incorporated by reference. Atorney Docket No.061479-511001WO [0233]
  • the immune cel engager herein activates target cels in vivo.
  • the target cels are immune cels.
  • the immune cels are T cels.
  • the immune cels are NK cels.
  • the immune cel engager described herein increase transduction T cels in vivo.
  • the immune cel engager and a viral particle comprising a polynucleotide encoding a CAR described herein transduce T cels in vivo generating CAR T cels.
  • the immune cel engager described herein comprise a CD58-CD80-anti-CD3 scFv tri-fusion polypeptide and increase transduction of T cels in vivo generating CAR T cels.
  • the immune cel engagers are administered via a route selected from the group consisting of extracorporeal, parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the immune cel engagers are administered multiple times. In some embodiments, the immune cel engagers are administered by intralymphatic injection of the viral particle. In some embodiments, the immune cel engagers are administered by intraperitoneal injection of the macromolecular complexes. In some embodiments, the immune cel engagers are administered by intra-nodal injection – that is, the immune cel engagers may be administered via injection into one or more lymph nodes.
  • the lymph nodes for administration are the inguinal lymph nodes.
  • the immune cel engagers are administered by injection of the immune cel engagers into tumor sites (e.g., intratumoral). In some embodiments, the immune cel engagers are administered subcutaneously. In some embodiments, the immune cel engagers are administered systemicaly. In some embodiments, the immune cel engagers are administered intravenously. In some embodiments, the immune cel engagers are administered intra-arterialy. In some embodiments, the immune cel engagers are co-administered with a viral particle. [0235] In some embodiments, the immune cel engagers are administered by intraperitoneal, subcutaneous, or intranodal injection.
  • the immune cel engagers are administered by intraperitoneal injection. In some embodiments, the immune cel engagers are administered by subcutaneous injection. In some embodiments, the immune cel engagers are administered by intranodal injection. [0236] In some embodiments, the transduced immune cels comprising a polynucleotide of the present disclosure are administered to the subject. [0237] The disclosure provides a method of treating a condition in a subject comprising administering to the subject the immune cel engager or the pharmaceutical composition of the disclosure.
  • the disclosure provides a method of treating a malignancy in a subject, comprising administering to the subject the immune cel engagers and a viral particle comprising a nucleic acid encoding a CAR or pharmaceutical composition of the disclosure.
  • the malignancy is a B-cel malignancy, a myeloma, or a solid tumor malignancy.
  • the disclosure Atorney Docket No.061479-511001WO provides a method of treating diffuse large B-cel lymphoma (DLBCL), Burkit’s type large B-cel lymphoma (B-LBL), folicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cel lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cel carcinoma, leukemia, myeloma, B cel lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, meduloblastoma, or sarcoma in a subject, comprising administering to the subject the immune cel engager and
  • Various disease or disorders may be treated using an immune cel engager and particles comprising a nucleic acid encoding a CAR as disclosed herein, or pharmaceutical composition comprising them.
  • the immune cel engager and particles may be administered to a subject comprising from or at risk for a B-cel malignancy, relapsed/refractory malignancy, difuse large B-cel lymphoma (DLBCL), Burkit’s type large B-cel lymphoma (B-LBL), folicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cel lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cel carcinoma, leukemia, myeloma, B cel lymphoma, kidney cancer, uterine cancer, aden
  • the condition is an autoimmune disease or disorder.
  • the disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject immune cel engager and viral particles or pharmaceutical composition of the disclosure.
  • an autoimmune disease may include systemic lupus erythematosus, Sjögren’s Syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Stil’s disease, inflammatory bowel disease, type 1 diabetes melitus, graft vs.
  • the immune cel engager disclosed herein may be used with viral particles in a method to generate engineered cels in vivo.
  • the lentiviral particles incorporating the immune cel engager e.g., a fusion protein as disclosed herein
  • administering lentiviral particles via one or more lymph nodes may contribute to generation of a predominately TCM engineered cel phenotype.
  • TCM may be characterized by expression of certain surface markers, for example, TCM may be CD62L+. TCM may also be CCR7+. TCM may also be Atorney Docket No.061479-511001WO characterized as CD45RA-, CD45RO+, and/or CD27+. In some embodiments, TCM are characterized as CCR7+, CD45RA-, CD45RO+, CD62L+, and CD27+. In some embodiments, TCM are characterized as CD45RA-, CCR7+. Without wishing to be bound by theory, it is contemplated that engineered TCM may persist for a longer time in vivo and may show improved effector function compared with engineered effector memory (TEM) or similar effector cel types.
  • TEM engineered effector memory
  • the present disclosure further provides a method of generating predominately engineered TCM in vivo. Similar observations may be found in the ex-vivo seting, so the present disclosure further provides a method of generating predominately engineered TCM ex vivo, using one or more of the methods disclosed here. For example, via extracorporeal delivery or traditional ex-vivo manufacturing.
  • the method of treatment may include administering an engager herein to a subject in need of treatment for a disease.
  • the method of treatment may include administering a pharmaceutical composition (e.g. comprising an engager) to a subject in need of treatment for a disease.
  • the disease comprises cancer. In some embodiments, the disease comprises an autoimmune disease.
  • Making Immune Cel Engagers [0243] Some embodiments include a method of making an immune cel engager. Such methods may include an aspect shown in FIG.4. [0244] In some embodiments, the disclosure provides a method of making an immune cel engager, comprising introducing a polynucleotide encoding an immune cel engager into a host cel comprising a polynucleotide encoding an immune cel engager (or fusion protein) as described herein. The immune cel engager (or fusion protein) is expressed by the host cel.
  • the disclosure provides an in vivo method of transducing target cels in a subject in need thereof, comprising administering to the subject an immune cel engager and a particle or pharmaceutical composition of the disclosure.
  • the immune cel engager and particle may be administered by intranodal, intravenous, or subcutaneous injection.
  • Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an immune cel binding domain, or a fusion protein (e.g., immune cel engager).
  • the method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion molecule, costimulatory molecule, immune cel binding domain, or fusion molecule (e.g., immune cel engager).
  • a nucleic acid such as a DNA or RNA
  • a protein comprising the adhesion molecule, costimulatory molecule, immune cel binding domain, or fusion molecule (e.g., immune cel engager).
  • Virus or Nanoparticle Manufacturing [0247] Disclosed herein, in some embodiments, are methods for use in virus manufacturing or nanoparticle manufacturing.
  • a virus may include a lentivirus.
  • the immune Atorney Docket No.061479-511001WO cel engager can be used for quantifying the purity of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus.
  • the immune cel engager can be used for determining the concentration of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus. In some embodiments, the immune cel engager can be used for determining activity of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus.
  • the immune cel engager described presently can also be incorporated onto the surface of viral particles.
  • the immune cel engager can also be caled a fusion protein when incorporated onto the surface of viral particles. One chalenge when working with viral particles is determining an accurate concentration of viral particles present in each production lot. Viral concentration can be measured by standard molecular techniques including qPCR, PCR, ELISA, functional readouts (expression of reporter protein) and protein concentration.
  • the immune cel engager described herein may be used to generate a standard curve of protein concentrations that can be used to determine the concentration of viral particles that incorporate the fusion protein on to their surface.
  • the immune cel engager described herein may be used to determine the purity of viral particles that incorporate the fusion protein on their surface.
  • Immune cel engagers described herein may be used to determine functional activity of viral particles that incorporate the fusion protein on to their surface. [0249] In some embodiments, the immune cel engager described herein can be used to purify viral particles that incorporate the fusion protein on to their surface.
  • the immune cel engager described herein can be loaded on to a solid support.
  • the solid support is a resin.
  • the resin is a chromatography resin.
  • the resin loaded with the immune cel engager can be used to purify lentivirus that have the fusion protein incorporated onto their surface.
  • the immune cel engager described herein can be used as a reagent to screen new lentivirus that have incorporated the fusion protein onto their surface. The screen may alow for the testing of other adhesion molecules, costimulatory molecules, or immune cel binding domains that have been incorporated into the fusion protein on the particle surface.
  • kits Disclosed herein, in some embodiments, are kits.
  • the kit includes an adhesion molecule.
  • the kit includes a costimulatory molecule.
  • the kit includes an immune cel binding domain.
  • the kit includes a fusion molecule.
  • the kit includes an immune cel engager.
  • the kit includes a particle.
  • the kit includes a composition Atorney Docket No.061479-511001WO described herein.
  • the kit may include instructions for use, such as instructions for use in a method herein.
  • the disclosure provides a kit comprising the immune cel engager and instructions for use in treatment of a subject.
  • the kit may include a pharmaceuticaly acceptable carier and/or an injection device.
  • the kit may further include suitable tubing for administering the immune cel engager.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specificaly disclosed al the possible subranges as wel as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specificaly disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as wel as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a sample includes a plurality of samples, including mixtures thereof.
  • and/or refers to and encompasses any and al possible combinations of one or more of the associated listed items, as wel as the lack of combinations when interpreted in the alternative (or).
  • determining may be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations.
  • Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.
  • a sequence herein may include a conservative amino acid substitution.
  • a “conservative amino acid substitution” may refer to a substitution of an amino acid residue with another amino acid residue without abolishing a protein's desired properties. Such a substitution may be with an Atorney Docket No.061479-511001WO amino acid of a same amino acid class.
  • amino acid class examples include amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, or tyrosine; amino acids having basic side chains, such as lysine, arginine, or histidine; amino acids having acidic side chains, such as asparatic acid or glutamic acid; or amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine.
  • a conservative amino acid substitution may include substitution with a chemicaly derivatized residue or non-natural amino acid that does not abolish a protein's desired properties.
  • a percent sequence identity may be determined by comparing two optimaly aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the % sequence identity.
  • a sequence identity may include a sequence identity to a reverse complement.
  • thymine (T) and uracil (U) may be interchangeable. T and U may be interchangeable when describing an oligonucleotide. In some embodiments, Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T.
  • Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T.
  • Any discrepancies between the writen description and a sequence listing submited herein may typicaly be resolved in favor of the writen description.
  • the terms “subject,” “individual,” or “patient” may be used interchangeably herein.
  • a subject can be an animal.
  • a subject can be a mammal.
  • a subject can be a primate.
  • the subject can be a human.
  • a subject may be healthy.
  • a subject may have a disease.
  • a subject may be diagnosed or suspected of being at high risk for a disease.
  • the term “about” a number refers to that number plus or minus 15% of that number.
  • the term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value.
  • administering may include introducing a compound or a composition into a subject.
  • the term may encompasses external or internal administration. Internal administration can refer to, for example, where a molecule is generated inside the subject’s body.
  • administering an RNA may include administering a plasmid DNA encoding the RNA, or a vehicle comprising said plasmid DNA, upon which the RNA may be expressed from the plasmid DNA in a cel of the subject.
  • treatment or “treating” may be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient.
  • Beneficial or desired Atorney Docket No.061479-511001WO results may include a therapeutic benefit and/or a prophylactic benefit.
  • a therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated.
  • a therapeutic benefit may be achieved by eradication or amelioration of one or more of the physiological symptoms associated with an underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may stil be aflicted with the underlying disorder.
  • a prophylactic effect may includes delaying, preventing, or eliminating appearance of a disease or condition, delaying or eliminating onset of a symptom, slowing, halting, or reversing progression of a disease or condition, or a combination thereof.
  • a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.
  • FIG.4 summarizes example production, purification, and analysis workflows for generation of soluble MDFs (sMDFs). Briefly, the transmembrane/cytosolic domain was replaced with an IgG1 Fc domain or 8XHistag (monomer MDF1) (FIG.5). The sMDF proteins were purified from the supernatant of lentiviral vector producing cel line 293T after the transfection of sMDF encoding plasmids.
  • This example shows that a dimer form of sMDF was more potent at activating T cels than a similar monomer Atorney Docket No.061479-511001WO form.
  • This example also shows sMDF2 was more potent at activating T cels than sMDF1.
  • the T cel NFAT activation assay was used to test the ability of monomer and dimer sMDFs to activate T cels. T cels were treated with purified monomer or dimer sMDFs in the T cel activation assay. Dimer sMDF2 reached an activation plateau level after 1 ng, while dimer sMDF1 showed a similar level of activation at 100 ng.
  • CD58 protein (smaler size than MDF) can be used as a standard for quantification, however the comparison of diferent sized protein may cause the inaccurate quantification of the protein.
  • the standard curve from using the sMDF generated a R2 of 0.9485. This illustrateates that purified sMDF can be used for the quantification of the protein in MDF containing particles.
  • Example 4. T cel activation test in PBMCs [0272] This example shows activation of primary T cels with example immune cel engagers. This example shows that dimerized versions of the sMDFs were more potent at stimulating primary T cel activation than monomer forms of the sMDFs.
  • the purified sMDFs (monomer and dimers) were tested on primary human PBMCs (FIG.12).1 milion human PBMCs were incubated with diferent amounts of sMDFs, and the activation was evaluated by the expression of CD25. In both CD4+ and CD8+ cels, sMDF2-treated cels showed higher activation compared to sMDF1-treated cels. Quantification of percentage of CD25 positive cels (marker of activation) at day 3 and day 7 (post sMDF treatment) found a similar trend of activation profile as above, which is, higher potency of sMDF2 than sMDF1. Almost no T cel activation from monomeric sMDF1 was noted (FIGs.12- 14).
  • Example 5 PBMC binding assay [0273] This example shows immune cel binding affinities of example immune cel engagers. This example shows that the higher T cel activation associated with MDF2 was due to higher binding affinity of MDF2. To test whether there was a binding affinity difference between sMDF1 and sMDF2, primary human PBMCs were incubated with sMDF1 or sMDF2 for 1.5 hrs and then washed. The amount of bound sMDF1 or sMDF2 was assessed by FACS using an Fc antibody.
  • the solid phase material such as beads provide similar environment of in vivo T cel activation like dendritic cels. Therefore, the solid phase form of antibody or activators are generaly used for the T cel activation. Thus, the immune cel engager described herein may be used as a new T cel activation reagent. * * * [0275] Al publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specificaly and individualy indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, controls.

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Abstract

Disclosed herein are immune cell engagers. The immune cell engager may include a cell binding domain, an immune cell adhesion molecule, an immune cell costimulatory molecule, or a combination thereof. Methods of use and production are further provided.

Description

Atorney Docket No.061479-511001WO FUSION PROTEIN FOR USE AS IMMUNE CELL ENGAGER CROSS-REFERENCE [0001] This application claims the benefit of U.S. Provisional Application No.63/644,321, filed May 8, 2024, which application is incorporated herein by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0002] The instant application contains a Sequence Listing which has been submited electronicaly in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 21, 2025, is named 061479-511001WO_seqs.xml and is 208,402 bytes in size. FIELD [0003] This disclosure relates generaly to cel biology, immunology, and medicine - more particularly to immune cel engagers for use in cel therapy manufacturing and in medical treatments. BACKGROUND [0004] T cels may be geneticaly engineered for use as therapeutic agents. For example, some chimeric antigen receptor (CAR) T cels have been approved as treatments for liquid tumors. Improved methods and compositions for enhancing T cels are needed. Genetic engineering of T cels may require delivery of polynucleotides into the T cels selected for engineering, a procedure termed transduction. In one example, an immune cel engager is used to enhance activation of immune cels. The immune cel engager may be designed to display molecules that enhance transduction. An antibody or antibody fragment against a component of a T cel receptor, such as CD3, may be incorporated into the immune cel engager to target the engager to T cels. Additionaly, engagement of CD3 by an immune cel engager comprising a binding domain targeting CD3 may cause the T cels to activate via a primary activation signal. Incorporation of one or more ligands for a co-receptor into the immune cel engager, such as CD28, a molecule expressed by T cels, may cause the T cels to activate via a secondary activation signal. Activation of a T cel, via the primary and optionaly secondary activation signals, may make them more susceptible to transduction. Transduction of T cels may be achieved using various viral and non-viral delivery vehicles. A ligand for CD28 may include a B7 protein. Ligands for CD28 may include, for example, CD80 and CD86. [0005] There remains a need in the art for new compositions and methods that further improve activation of T cels and/or transduction of T cels by fusion proteins or immune cel engagers described herein. The present disclosure addresses these needs and others. Atorney Docket No.061479-511001WO SUMMARY [0006] The present disclosure relates, in part, to the recognition by the present inventors that an immune cel engager that includes an adhesion molecule, such as a binding domain that binds CD2 and/or activates CD2 signaling in a target cel (e.g., a T cel or other immune cel) may be used to activate cels (e.g., T cels or other immune cels), and may, for example, be used as a reagent alone or in combination with a polynucleotide delivery vehicle. In some embodiments, an immune cel engager described herein is able to bind and activate T cels independent of a virus particle. Additionaly, transduction of target cels (such as T cels) may be enhanced by co-administering the immune cel engager with viral particles. [0007] The present inventors have further recognized that the immune cel engager may be enhanced by fusing an adhesion molecule to a costimulatory molecule, an immune cel binding domain, or both. Surprisingly, the present inventors discovered that, in some embodiments, an immune cel engager as disclosed herein, e.g. comprising adhesion molecule domain(s), costimulatory molecule domain(s), and optionaly immune cel binding domain(s) was able to bind and activate target cels indicating that each domain was not only appropriately positioned to enable binding its cognate ligand, but maintained its functional characteristics (e.g. folding and structure). [0008] In one aspect, the present disclosure provides an engager, comprising: an cel binding domain that specificaly binds a receptor on a target cel comprising a target immune cel or a target tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target cel or on an immune cel, wherein concurent binding of the cel binding domain and the adhesion molecule to the target cel forms a supramolecular adhesion complex on the target cel. The target cel may be an immune cel. An example immune cel is a T cel. The target cel may be a B cel. The target cel may be a cancer cel. The target cel may be a tumor cel. The target cel may be a malignant immune cel. The target cel may be a malignant B cel. [0009] In one aspect, the present disclosure provides an immune cel engager for activating a target immune cel, comprising: an cel binding domain that specificaly binds a receptor on a target immune cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cel forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel. [0010] In some embodiments, the engager further comprises a multimerization domain. In some embodiments, the engager further comprises a dimerization domain. In some embodiments, the engager is a homodimer. [0011] In some embodiments, the target immune cel is a T cel. In some embodiments, the cel binding domain is a binding domain that specificaly binds CD3. In some embodiments, the cel binding domain is a binding domain that comprises complementarity determining regions of an anti- CD3 antibody. Atorney Docket No.061479-511001WO [0012] In some embodiments, the adhesion molecule is a binding domain that specificaly binds CD2. In some embodiments, the adhesion molecule is CD58, CD48, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112, or a functional fragment thereof. [0013] In some embodiments, engager further comprises an immune cel costimulatory molecule that provide a costimulatory signal to the immune cel. In some embodiments, the costimulatory molecule is a binding domain that specificaly binds CD28.In some embodiments, the costimulatory molecule is CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD70, CD30L, LIGHT, LTalpha, MICA, or MICB, or a functional fragment thereof. [0014] The present disclosure provides an immune cel engager comprising a fusion molecule, the fusion molecule comprising an antigen-binding domain of an anti-CD3 antibody, optionaly a single- chain variable fragment (CD3scFv); a CD58 extracelular domain; a CD80 extracelular domain or a CD86 extracelular domain, wherein the fusion molecule forms an artificial supramolecular adhesion complex with T cels. [0015] In some embodiments, the fusion molecule further comprises a dimerization domain and the complex is a homodimer. In some embodiments, the dimerization domain is a crystalization fragment (Fc) of an immunoglobulin. In some embodiments, the domains of the fusion molecule are fused via polypeptide linkers to form a single polypeptide chain. In some embodiments, the domains are linked in N- to C-terminal order as CD58-CD3scFv-CD80/CD86-Fc. In some embodiments, the domains are linked in N- to C-terminal order as CD3scFv-CD58-CD80/CD86-Fc. [0016] In some embodiments, the engager is a monomer. In some embodiments, the domains of the fusion molecule are fused via polypeptide linkers to form a single polypeptide chain. In some embodiments, the domains are linked in N- to C-terminal order as CD58-CD3scFv-CD80/CD86. In some embodiments, the domains are linked in N- to C-terminal order as CD3scFv-CD58- CD80/CD86. [0017] In some embodiments, the engager is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% to a sequence in Table 1. In some embodiments, the engager is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% to a sequence in Table 2. [0018] In some embodiments, the fusion molecule forms an artificial supramolecular adhesion complex with T cels and specificaly activates T cels by engaging, adhering to, and/or costimulating T cels via the CD3scFv, CD58, and CD80/CD86 domains, respectively. [0019] In some embodiments, the engager is isolated. In some embodiments, the engager is soluble. In some embodiments, the engager lacks a transmembrane or a lipid associating domain. In some embodiments, the engager is not associated with a viral particle. [0020] Disclosed herein, in some embodiments, are cel engagers, comprising: a cel binding domain that specificaly binds an antigen on a target cel; and an immune cel adhesion molecule Atorney Docket No.061479-511001WO that specificaly binds a reciprocal adhesion molecule of an immune cel, wherein binding of the immune cel adhesion molecule to the immune cel activates the immune cel, and wherein the cel engager is soluble. In some embodiments, the target cel is a tumor cel. In some embodiments, the antigen comprises CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, BCMA, ACTN4, BAGE-1, BCR-ABL, beta-catenin, CA 125, CA 15-3, CA 195, CA 242, CA 50, CAM43, Casp-8, CDC27, CDK4, CDKN2A, CEA, COA-1, DEK-CAN fusion protein, EBNA1, EF2, an Epstein Bar virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, HSP70-2, KIAA0205, MART2, MUM-1, MUM-2, MUM-3, neo-PAP, myosin class I proteins, OS-9, PML-RARα fusion protein, PTPRK, K-RAS, N-RAS, TPI, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GNT-V, HERV-K- MEL, LAGE-1, NA-88, LAGE-2, SP17, SSX-2, TRP2-INT2, PMEL, TYR, TRP-1, TRP-2, MAGE- 1, MAGE-3, RAGE, GAGE-1, GAGE-2, CDKN2B, SCP-1, PRAME, p53, H-RAS, HER2/neu, E2A-PRL, H4-RET, IGH-IGK, HPV E6 antigen, HPV E7 antigen, TSP-180, MAGE-4, MAGE-5, MAGE-6, p180-erbB-3, c-Met, nm-23H1, PSA, TAG-72-4, CA 19-9, mucin, NuMa, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, Beta-HCG, BCA225, BTAA, CD68\KP1, CO- 029, FGF5, G250, Ga733, HTgp-175, M344, FLT3-ITD, MG7-Ag, FOLR1, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CS1, GPCR5, ganglioside G2, EGFRvII MSLN, PAP, prostein, TARP, Trp-p8, STEAP1, an abnormal ras protein, an abnormal p53 protein, CD51, CD61, galectin, or RalB. In some embodiments, the target cel is the immune cel or another immune cel. In some embodiments, the antigen comprises CD3. In some embodiments, the reciprocal adhesion molecule comprises CD2. In some embodiments, the immune cel adhesion molecule comprises a CD58 protein. Some embodiments include an immune cel costimulatory molecule. In some embodiments, the immune cel costimulatory molecule binds CD28. In some embodiments, the immune cel costimulatory molecule comprises a CD80 or CD86 protein. [0021] Disclosed herein, in some embodiments, are pharmaceutical compositions comprising an engager herein and a pharmaceuticaly acceptable carier. Disclosed herein, in some embodiments, are methods of treatment, comprising administering the pharmaceutical composition to a subject in need of treatment for a disease. Disclosed herein, in some embodiments, are methods of treatment, comprising administering an engager herein to a subject in need of treatment for a disease. In some embodiments, the disease comprises cancer. In some embodiments, the disease comprises an autoimmune disease. [0022] In some embodiments, the engager is associated with a reagent comprising a substrate. In some embodiments, the substrate is a solid support. In some embodiments, the substrate is a bead. In some embodiments, the substrate is a resin. In some embodiments, the substrate is a chromatography resin. In some embodiments, engager is atached to a solid support. In some embodiments, the engager is atached to an expamer. Atorney Docket No.061479-511001WO [0023] The present disclosure provides an in vivo method of generating engineered immune cels in a patient, comprising: a. administering to the patient an immune cel engager, or a vector encoding said immune cel engager, wherein the immune cel engager forms an artificial supramolecular adhesion complex with target immune cels and specificaly activates target immune cels by engaging, adhering to, and costimulating target immune cels, and c. administering a gene therapy vector capable of transducing the target immune cels, wherein the gene therapy vector transduces target immune cels thereby generating engineered immune cels, and wherein transduction of the immune cels and/or generation of engineered immune cels is increased compared to administering only the gene therapy vector. [0024] The present disclosure provides a method of treating or preventing cancer, comprising administering an immune cel engager and a gene therapy vector that transduces target immune cels to generate, in vivo, engineered immune cels capable of kiling cancer cels, wherein the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector. [0025] The present disclosure provides a method of making engineered immune cels, comprising contacting target immune cels with an immune cel engager and with a gene therapy vector that generates, ex vivo, engineered immune cels, wherein the engager comprises: an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurrent binding of the cel binding domain and the adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector. [0026] The present disclosure provides a method of activating immune cels, comprising: a. selecting an immune cel binding domain that specificaly binds a receptor on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cel, and optionaly an immune cel costimulatory molecule; Atorney Docket No.061479-511001WO b. preparing an immune cel engager comprising functional fragments of the immune cel binding domain, immune cel adhesion molecule, and optionaly the immune cel costimulatory molecule; c. contacting target immune cels with the immune cel engager. [0027] In some embodiments, the contacting comprises administering the immune cel engager to a patient, thereby contacting the immune cel engager with target immune cels in vivo and activating the immune cels. [0028] In some embodiments, the method further comprises administering a gene therapy vector specific to the target immune cels, wherein co-administration of the immune cel engager with the vector increases transduction of target immune cels by the vector in the patient. [0029] In some embodiments, the target immune cels are T cels. BRIEF DESCRIPTION OF THE DRAWINGS [0030] FIG.1 includes diagrams ilustrating examples of immune cel engagers that include a soluble multi-domain fusion 1 (sMDF1) monomer, a sMDF1 dimer, and a soluble multi-domain fusion 2 (sMDF2) dimer. [0031] FIG.2A is a schematic that shows an ilustrative immune cel engager comprising a CD58 extracelular region and anti-CD3 scFv fused to the N-terminus of a CD80 protein via a linker The construct is termed “498.” The schematic may include an N terminus on the left side, and a C terminus on the right side. [0032] FIG.2B is a schematic that shows an ilustrative immune cel engager comprising a CD58 extracelular region fused to an N-terminus of a CD80 protein via a linker. The construct is termed “455.” anti-CD3 scFv is expressed as a separate polypeptide in the producer cels. The schematic may include an N terminus on the left side, and a C terminus on the right side. [0033] FIG.3A includes diagrams ilustrating example immune cel engagers. [0034] FIG.3B includes diagrams ilustrating example immune cel engagers. [0035] FIG.4 is a diagram summarizing an example production, purification, and analysis workflow for generation of some immune cels engagers (e.g. sMDF1 and sMDF2). [0036] FIG.5 includes diagrams ilustrating example immune cel engagers that include soluble multi-domain fusion 1 (sMDF1) and soluble multi-domain fusion 2 (sMDF2). A transmembrane domain (TM_CT) was removed and replaced with IgG1 Fc to produce the example immune cel engagers shown in the figure. [0037] FIG.6 includes an image of an SDS-PAGE gel that shows sMDF1 and sMDF2 produced in 293T cels have structural and post-translational modifications (e.g., glycosylation). Isolated sMDF proteins were treated with the indicated enzymes and run on a non-reducing SDS page gel. Atorney Docket No.061479-511001WO [0038] FIG.7 summarizes example parameters for isolating sMDF1 on a His-Tag column or sMDF1 IgG Fc or sMDF2 IgG Fc on a Protein A Sepharose column. Example buffers and flow rates for each type of column are listed. [0039] FIG.8 includes SDS-PAGE gel images showing successful isolation of example immune cel engagers that included sMDF variants: sMDF1 (HisTag), sMDF1 IgG Fc and sMDF2 IgG Fc. The sMDF1 (HisTag) gel indicates the volume loaded, purity and calculated concentration. The sMDF1 IgG Fc and sMDF2 IgG Fc gel indicates the column elution fraction, the purity, and the calculated concentration. [0040] FIG.9 shows a western blot of purified sMDF1 HisTag (monomer) and Fc (Dimer) run on the SDS-PAGE. sMDF was detected with an anti-CD58 antibody. In the non-reducing condition, dimer MDF1 showed expected 200 kD size (2X 905 aa), while monomer MDF1 showed expected 75 kD (674 aa). In the reducing gel, the dimer was reduced to monomer form (100 kD, 905 aa). The diferent size of monomer in the reducing gel is the diferent Tag (HisTag vs. Fc tag). [0041] FIG.10 shows a graph that quantifies T cel activation after purified sMDFs were incubated with NFAT reporter cels. The sMDFs and cels were incubated for 2 hours and then NFAT activation was measured. sMDF was added at the listed concentrations. In each panel (left and right), the line at the botom under the header, “Monomer,” represents data for sMDF1 (663HisTag), the middle line represents data for sMDF1 (664 IgG-Fc), and the line with the greatest slope that appears at the top of the graph represents data for MDF2 (702 IgG-Fc). [0042] FIG.11 shows a capilary electrophoresis (Jess) analysis of different amounts of purified sMDFs and lentivirus particles containing the MDF. A standard curve of sMDF concentration was generated and was used to quantify the amount of virus expressing the MDF (VPL 128 and VPL 390). [0043] FIG.12 shows percentages of CD25+ PBMCs incubated with the indicated amount of sMDF after 3 days of culture. CD4+ and CD8+ T cels were evaluated separately for their activation after treatment with the immune cel engagers (sMDF1 and sMDF2). [0044] FIG.13 shows percentages of CD25+ PBMCs incubated with the indicated amount of sMDF after 7 days of culture. CD4+ and CD8+ T cels were evaluated separately for their activation after treatment with sMDF. [0045] FIG.14 shows percentages of CD8+ PBMCs incubated with the indicated amount of sMDF after 7 days of culture. [0046] FIG.15 shows percentages of sMDF bound to CD3+ PMBCs. PBMCs were incubated with sMDF for 1.5 hours and washed. The presence of sMDFs on the surface of the PBMCS was assessed by FACS using Fc antibody Atorney Docket No.061479-511001WO DETAILED DESCRIPTION [0047] The present disclosure relates generaly to an immune cel engager for use in the binding and activation of target cels, such as immune cels, or specificaly T cels. In one aspect, the disclosure provides, an immune cel engager for binding and activation of target cels, comprising an adhesion molecule linked to a costimulatory molecule, an immune cel binding domain, or both. [0048] In some embodiments, the immune cel engager of the present disclosure combines an adhesion molecule with a costimulatory molecule or an immune cel binding domain, or both. [0049] The term “transduction” is used in its broadest sense to mean delivery of an agent to a cel, such as a therapeutic agent. The agent may be a smal molecule, polynucleotide, or polypeptide. A combination of agents may be delivered, such as several polynucleotides or a protein-nucleic acid complex (e.g., a gene-editing nuclease in complex with guide nucleic acid). The term “particle” includes but is not limited to viral particles (e.g., a virion), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers. [0050] As contemplated by the present disclosure, an immune cel engager may be engineered to incorporate any of the adhesion molecules or costimulatory molecules; extracelular fragments thereof; or functional fragments thereof discussed in this application. Extracelular portions of these molecules may be identified in databases such as UniProt, which is available at www.uniprot.org, or may be predicted using methods, such as a method implemented by the TMHMM 2.0 program available at services.healthtech.dtu.dk. Furthermore, in some cases, functional fragments of each are identified in scientific literature or they may be identified using laboratory methods. For example, one may identify the fragments of a protein likely to form wel-folded domains. Fragments may be tested in binding assays against a cognate molecule or used in pul-down assays compared to the ful molecule. Functional assays, such as expression of a fluorescence reporter under the control of a promoter activated by T-cel signaling (e.g., the NKkB promoter) when a T cel is contacted with a cel, an immune cel engager or particle expressing a putative functional fragment. The sequence of the adhesion molecule, costimulatory molecule, or immune cel binding domain may be varied to identify and use variants that retain function. For example, conservative mutations may be made to a molecule, or a molecule may be randomly mutated with the function of the variant confirmed experimentaly. [0051] In each case, a molecule may be incorporated as a ful-length form, without its native transmembrane domain. Alternatively, the extracelular portion of the molecule may be replaced with a heterologous protein. Each immune cel engager may be a monomer, bound to another engager to form a dimer or bound to an additional antigen binding domain. For example, an adhesion molecule, costimulatory molecule, and immune cel binding domain may be linked in any order with only the most N-terminal or C-terminal of the molecules. In a variant, the immune cel engager comprises or is associated with another membrane-associated molecule. The term “display” is used, in a broad sense, to mean positioned on the surface of the immune cel engager such that the molecule Atorney Docket No.061479-511001WO may contact cognate molecules on the target cel. It is further contemplated that the immune cel engager may be loaded onto a solid support either by association with a component of the particle (e.g., a capsid protein) or by the addition of an additional molecule with afinity for the solid support (e.g., as an immune cel engager comprising a capsid protein). Immune Cel Engagers [0052] Disclosed herein, in some embodiments, are immune cel engagers. As used herein, an “engager” refers to a binding molecule or a complex of binding molecules capable a binding to a target cel or target cels. The target cel may be an immune cel, in which case the engager may be termed an “immune cel engager.” The engager may be a T-cel engager. The target cel may be an tumor cel, in which case the engager may be termed a “tumor cel engager.” In some embodiments, the immune cel engager may include a fusion molecule. The fusion molecule may include an adhesion molecule, a costimulatory molecule, or a cel binding molecule. The fusion molecule may include an adhesion molecule. The fusion molecule may include a costimulatory molecule. The fusion molecule may include an immune cel binding domain. The fusion molecule may include a tumor cel binding domain. The fusion molecule may include an adhesion molecule, a costimulatory molecule, and an immune cel binding domain. The fusion molecule may include an adhesion molecule, a costimulatory molecule, and a tumor cel binding domain. The fusion molecule may include an adhesion molecule and an immune cel binding domain. The fusion molecule may include an adhesion molecule and a tumor immune cel binding domain. The fusion molecule may include a costimulatory molecule and an immune cel binding domain. The fusion molecule may include a costimulatory molecule and a tumor cel binding domain. The fusion molecule may be or include a fusion protein. The immune cel engager may be isolated. The immune cel engager may be soluble. The immune cel engager may be or include a soluble fusion protein. The immune cel engager (or the multispecific immune cel engager) may be used in a method described herein. [0053] In some embodiments, the disclosure provides an immune cel engager comprising a combination of an adhesion molecule, a costimulatory molecule, and an immune cel binding domain (e.g., a TCR-binding molecule), thereof each component linked directly or indirectly to the other components. In some embodiments, the immune cel engager comprises adhesion molecule, a costimulatory molecule, and an immune cel binding domain (e.g., a TCR-binding molecule). In some embodiments, the immune cel engager comprises adhesion molecule and a costimulatory molecule, but not a TCR-binding molecule. In some embodiments, the immune cel engager comprises an adhesion molecule and an immune cel binding domain (e.g., a TCR-binding molecule), but not a costimulatory molecule. The immune cel engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cel binding domains (e.g., TCR-binding molecules). Atorney Docket No.061479-511001WO [0054] In some embodiments, the disclosure provides a tumor cel engager comprising a combination of an adhesion molecule, a costimulatory molecule, and a tumor cel binding domain, thereof each component linked directly or indirectly to the other components. In some embodiments, the tumor cel engager comprises adhesion molecule, a costimulatory molecule, and a tumor cel binding domain. In some embodiments, the immune cel engager comprises an adhesion molecule and a tumor cel binding domain, but not a costimulatory molecule. The tumor cel engager may further comprise one or more additional adhesion molecules, costimulatory molecules, or immune cel binding domains (e.g., TCR-binding molecules). [0055] An immune cel engager may be soluble. An immune cel engager may exclude a transmembrane domain. As used herein, the term “soluble” refers to any molecule that does not contain a transmembrane domain. In some embodiments, the engager is a soluble macromolecule, substantialy free of lipids. In some embodiments, the soluble engager is not part of a viral particle. [0056] As used herein, the term “macromolecular complex” refers to a stable assembly of two or more fusion proteins. The immune cel engager may be a monomer. The immune cel engager may comprise an Fc domain. The immune cel engager may comprise an Fc domain on the C terminus. The immune cel engager may comprise an Fc domain on the N terminus. The Fc domain of the immune cel engager may facilitate dimer formation. In some embodiments, the dimer is a homodimer. [0057] As used herein, the term “transmembrane domain” refers to the portion of a protein that spans the phospholipid bilayer of a biological membrane, such as the plasma membrane of a cel. In some embodiments, the immune cel engager may be comprised of proteins without their transmembrane domain. In some embodiments, the immune cel engager may be comprised of proteins without their transmembrane domain. In some embodiments, the transmembrane domain is replaced with a heterologous protein. [0058] As used herein, the term “fusion molecule” refers to any molecule having multiple components linked together, directly or indirectly, covalently or non-covalently. The fusion molecule may be soluble. The fusion molecule may be made up of more than one protein. The immune cel engager may be made up of one or more fusion molecules. When those proteins are linked together into a single molecule by peptide bonds, the fusion molecule is termed a “fusion protein.” [0059] The fusion molecule may be made using various linkers, including chemical (covalent) bonds (e.g., by click chemistry) or by peptide bounds. When the fusion molecule is a fusion protein, the linker between each component of the fusion protein may be a single peptide bound (e.g., a direct C- to N- peptide bound in a polypeptide chain) or via a polypeptide linker. Ilustrative polypeptide linkers may include, but are not limited to, the glycine-serine linkers, such as GGSGGS (SEQ ID NO.304), GSSGSS (SEQ ID NO.305), or others. Atorney Docket No.061479-511001WO [0060] In some embodiments, the fusion molecule is or includes a fusion protein. The fusion protein may comprise an adhesion protein, a polypeptide linker, and a costimulatory portion. In some embodiments, the fusion protein comprises an adhesion molecule, a costimulatory molecule, and an immune cel binding domain. [0061] In some embodiments of the fusion protein, the adhesion molecule is N-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is N-terminal to the immune cel binding domain. In some embodiments, the adhesion molecule is C-terminal to the costimulatory molecule. In some embodiments, the adhesion molecule is C-terminal to the immune cel binding domain. [0062] In some embodiments of the fusion protein, the immune cel binding domain is N-terminal to the costimulatory molecule. In some embodiments, the immune cel binding domain is N-terminal to the adhesion molecule. In some embodiments, the immune cel binding domain is C-terminal to the costimulatory molecule. In some embodiments, the immune cel binding domain is C-terminal to the adhesion molecule. [0063] In some embodiments of the fusion protein, the costimulatory molecule is N-terminal to the immune cel binding domain. In some embodiments, the costimulatory molecule is N-terminal to the adhesion molecule. In some embodiments, the costimulatory molecule is C-terminal to the immune cel binding domain. In some embodiments, the costimulatory molecule is C-terminal to the adhesion molecule. [0064] Some embodiments of the fusion protein include a linker. Some embodiments include multiple linkers. In some embodiments, a linker directly connects the costimulatory molecule with the adhesion molecule. In some embodiments, a linker directly connects the costimulatory molecule with the immune cel binding domain. In some embodiments, a linker directly connects the adhesion molecule with the immune cel binding domain. [0065] In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed (via a polypeptide bond or a polypeptide linker sequence) with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the costimulatory molecule is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the costimulatory molecule is juxtaposed with an end of the immune cel binding domain. In some embodiments of the fusion protein, a C terminal end of the costimulatory molecule is juxtaposed with an end of the immune cel binding domain. [0066] In some embodiments of the fusion protein, an N terminal end of the immune cel binding domain is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, a C terminal end of the immune cel binding domain is juxtaposed with an end of the adhesion molecule. In some embodiments of the fusion protein, an N terminal end of the immune cel binding domain is juxtaposed with an end of the costimulatory molecule. In some embodiments Atorney Docket No.061479-511001WO of the fusion protein, a C terminal end of the immune cel binding domain is juxtaposed with an end of the costimulatory molecule. [0067] In some embodiments of the fusion protein, an N terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule. In some embodiments of the fusion protein, a C terminal end of the adhesion molecule is juxtaposed with an end of the costimulatory molecule. In some embodiments of the fusion protein, an N terminal end of the adhesion molecule is juxtaposed with an end of the immune cel binding domain. In some embodiments of the fusion protein, a C terminal end of the adhesion molecule is juxtaposed with an end of the immune cel binding domain. [0068] In some embodiments of the fusion protein, the fusion protein comprises a Fc fusion. Fc- based fusion proteins are composed of an immunoglobin Fc domain that is directly linked to another peptide. The fused partner can be any other proteinaceous molecule of interest, such as a ligand that activates upon interaction with a cel-surface receptor, a peptide antigen (Ag) against a chalenging pathogen or a ‘bait’ protein to identify binding partners assembled in a protein microarray. In some embodiments of the fusion protein, if a component protein naturaly contains a transmembrane domain, the transmembrane domain is replaced with another protein of interest. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc fusion. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgA. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgE. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgM. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG1. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG2. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG3. In some embodiments of the fusion protein, the transmembrane domain is replaced with a Fc domain from IgG4. In some embodiments, the Fc domain alows the fusion protein to form a monomer. In some embodiments, the Fc domain alows the fusion protein to form a dimer. In some embodiments, the Fc domain alows the fusion protein to form a multimer. In some embodiments, the Fc domain alows the fusion protein to form an oligomer. [0069] In some embodiments of the fusion protein, the transmembrane domain is replaced with a protein tag. In some embodiments of the fusion protein, the transmembrane domain is replaced with a streptavidin tag. Protein tags can be used to afinity purify proteins of interest or eliminate specific proteins from a solution (negative selection), to help solubilize proteins through maintaining proper protein folding and keep them from aggregating, to alter chromatographic properties of the protein to afford different resolution across a particular separation technique, to add an additional epitope to the protein to alow its purification, to add a fluorescent tag, for enzymatic modification (biotinylation) or chemical modification. Atorney Docket No.061479-511001WO [0070] In some embodiments of the fusion protein, the transmembrane domain is replaced with an additional binding domain. The additional binding domain can be an antibody or scFv. Incorporation of the antibody or scFv into the fusion protein may alow for targeting the fusion protein to specific cel types. [0071] In some embodiments of the fusion protein, the transmembrane domain is replaced with a cytokine. Cytokines are smal proteins that are crucial in controling the growth and activity of immune system cels and bloods cels. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is an interferon. In some embodiments, the cytokine is a tumor necrosis factor. In some embodiments, the cytokine is a growth factor. Linking the fusion protein (or immune cel engager) to a cytokine may alow the cytokine to bind to its cognate receptor upon binding of the fusion protein to a cel. The addition of the cytokine to the fusion protein may alow for the fusion protein to further activate immune cels in vivo and in vitro. [0072] In some embodiments of the fusion protein, the transmembrane domain is replaced with a protein that binds viral particles. In some embodiments of the fusion protein, the transmembrane domain is replaced with heparin. Heparin is a naturaly occuring glycosaminoglycan that can be used an anticoagulant. Heparin has a natural affinity for viral particles and has been used in chromatography to purify viruses. Incorporating a heparin molecule into the fusion protein may alow for the fusion protein to coat the outside of the viral particle with the fusion protein. The viral particle generated in this protocol can be used in any method or composition described herein. [0073] In some embodiments, the fusion protein is atached to a substrate. In some embodiments of the fusion protein, the fusion protein is atached to a solid surface. In some embodiments of the fusion protein, the fusion protein is atached to a nanoparticle. In some embodiments, the fusion protein is atached to a lipid nanoparticle. In some embodiments, the fusion protein is atached to a bead. A bead can be a particle comprising polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, celulose, nylon, silicone, silica gel, controled pore glass, Dynabead, Wang resin, Merrifield resin, Sephadex/Sepharose, celulose, or a combination thereof. In some embodiments, the bead is a streptavidin bead. In some embodiments, the bead is an agarose bead. In some embodiments, the bead is a magnetic bead. In some embodiments, the bead is a hydrogel bead. In some embodiments, the bead is an antibody conjugated bead. In some embodiments, the bead is a protein A conjugated bead. In some embodiments, the bead is a protein G conjugated bead. In some embodiments, the bead is a protein NG conjugated bead. In some embodiments, the bead is a protein L conjugated bead. In some embodiments, the bead is an oligodT conjugated bear. In some embodiments, the bead is a silica bead. In some embodiments, the bead is a silica like bead. In some embodiments, the bead is an anti-biotin microbead. In some embodiments, the bead is an anti-fluorochrome microbead. Atorney Docket No.061479-511001WO [0074] In some embodiments, the fusion protein is atached to a resin. In some embodiments, the fusion protein is atached to a chromatography resin. In some embodiments, the resin is a protein G resin. In some embodiments, the resin is a protein A resin. In some embodiments, the resin is a protein L resin. In some embodiments, the resin is glutathione resin. In some embodiments, the resin is a GST fusion protein resin. In some embodiments, the resin is a nickel charge resin. In some embodiments, the resin is a streptavidin resin. In some embodiments, the resin is an anti- DYKDDDDK (Flag Tag) (SEQ ID NO.306) resin. In some embodiments, the resin is a NI-IDS resin. In some embodiments, the resin is an Iodoacetyl resin. In some embodiments, the resin is an anti-HIS affinity resin. [0075] In some embodiments, the fusion protein atached to a resin is used in a column. In some embodiments, the fusion protein atached to a resin is used in a column for protein purification. In some embodiments, the fusion protein is atached to a polymer. In some embodiments, the fusion protein atached to expamer. Expamers are proteinaceous multimers that enable precise regulation of T cel stimulation duration and provide promise of control over T cel profiles in future products. The expamer with a fusion protein atached may be used for immune cel activation. In some embodiments, the fusion protein is atached to a hydrogel. In some embodiments, the fusion protein is atached via covalent bond. In some embodiments, the fusion protein is atached via a non-covalent bond. [0076] As used herein, the term “substrate” refers, in a broad sense, to a material which provides the surface on which something is deposited. The fusion protein may be associated with the substrate by covalent or non-covalent interactions. The substrate may be a solid support (e.g., a microtiter plate), a bead, a resin, a column or any other commonly used substrate in protein biology, cel biology, or virology. [0077] The fusion protein may comprise, in any order, a cluster of differentiation 80 (CD80), a CD80 extracelular domain, or a functional fragment of CD80; a cluster of diferentiation 58 (CD58), a CD58 extracelular domain; or a functional fragment of CD58; an immune cel binding domain (e.g., a TCR-binding molecule); and polypeptide linkers. The fusion protein may comprise, in any order, a cluster of differentiation 58 (CD58), a CD58 extracelular domain; or a functional fragment of CD58; a tumor cel binding domain; and polypeptide linkers. [0078] The fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracelular domain, or a functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracelular domain; or a functional fragment of CD58. [0079] The fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracelular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracelular domain, or a functional fragment of CD80. [0080] The fusion protein may comprise, in N- to C-terminal order, an immune cel binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD80, a CD80 extracelular domain, or a Atorney Docket No.061479-511001WO functional fragment of CD80; a polypeptide linker; and CD58, a CD58 extracelular domain; or a functional fragment of CD58. [0081] The fusion protein may comprise, in N- to C-terminal order, CD80, a CD80 extracelular domain, or a functional fragment of CD80; a polypeptide linker; CD58, a CD58 extracelular domain; or a functional fragment of CD58; a polypeptide linker; and an immune cel binding domain (e.g., a TCR-binding protein). [0082] The fusion protein may comprise, in N- to C-terminal order, an immune cel binding domain (e.g., a TCR-binding protein); a polypeptide linker; CD58, a CD58 extracelular domain; or a functional fragment of CD58; a polypeptide linker; and CD80, a CD80 extracelular domain, or a functional fragment of CD80. [0083] The fusion protein may comprise, in N- to C-terminal order, CD58, a CD58 extracelular domain; or a functional fragment of CD58; a polypeptide linker; CD80, a CD80 extracelular domain, or a functional fragment of CD80; a polypeptide linker; and an immune cel binding domain (e.g., a TCR-binding protein). [0084] An ilustrative fusion protein comprises a CD58 extracelular domain and anti-cluster of diferentiation 3 (CD3) scFv fused to the N-terminus of a CD80 via a linker; this construct is termed a tri-fusion polypeptide and/or termed “498.” [0085] An ilustrative fusion protein comprises a CD58 extracelular domain fused to the N- terminus of CD80 via a linker; this construct is termed a bi-fusion polypeptide and/or termed “455.” In this construct, an anti-CD3 scFv is expressed as a separate polypeptide in the producer cels. [0086] In each case, the polypeptide linker may be optional. It may be omited by directly linking a protein molecule to the next via a peptide bound. Although one may generate fusion proteins through chemical synthesis, fusion proteins are made by expressing the fusion protein from a single polynucleotide comprising a polynucleotide sequence encoding the entire fusion protein. Methods for designing and cloning polynucleotides are known in the art. [0087] The fusion molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). In some embodiments, the disclosure provides polynucleotides encoding such fusion proteins. The polynucleotide may be an isolated polynucleotide, or it may be part of a vector (e.g., a plasmid) or it may be introduced into and propagated in a host cel. [0088] Some example immune cel engagers are shown in FIGs.1-3B. An immune cel engager may include any aspect in any of these figures. Some aspects of such figures may be optional, such as a tag or linker. [0089] Polypeptide sequences of ilustrative dual CD58+CD80 fusion proteins are provided in Table 1. In each case, an optional signal peptide is shown in parentheses. In each case, the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 1. In some embodiments, the fusion protein may comprise a polypeptide Atorney Docket No.061479-511001WO having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 1. In each case, an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Diagrams of some example fusions are provided in FIG.2. Table 1 Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO [0090] Polypeptide sequences of ilustrative triple CD58+CD80+anti-CD3scFv fusion proteins are provided in Table 2. In each case, the fusion protein may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 2. In some embodiments, the fusion protein may comprise a polypeptide less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 99%, or less than 100% sequence identity to any sequence in Table 2. In each case, an optional signal peptide is shown in parentheses. The signal peptide is cleaved during expression of the sequence. Sequence identity to a reference sequence is determined without the optional residues. Table 2 Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO [0091] In one aspect, the present disclosure provides an engager, comprising: a cel binding domain that specificaly binds a receptor on a target cel comprising a target immune cel or a target tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target cel or on an immune cel. Concurent binding of the cel binding domain and the adhesion molecule may form a supramolecular adhesion complex. The supramolecular adhesion complex may be formed on the target cel. The supramolecular adhesion complex may be formed on an immune cel. The target cel may be an immune cel. An example immune cel is a T cel. An example immune cel is a natural kiler (NK) cel. The target cel may be a B cel. The target cel may be a cancer cel. The target cel may be a tumor cel. For example, some embodiments relate to or include an engager, comprising: a cel binding domain that specificaly binds a receptor on a target Atorney Docket No.061479-511001WO tumor cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on an immune cel. The target cel may be a malignant immune cel. The target cel may be a malignant B cel. [0092] Disclosed herein, in some embodiments, are cel engagers. The engager may include a cel binding domain that specificaly binds an antigen on a target cel. The engager may include an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of an immune cel. Binding of the cel binding domain to the immune cel may activate the immune cel. Binding of the cel binding domain to the antigen on the target cel and binding of the immune cel adhesion molecule to the immune cel may activate the immune cel. The engager may be soluble. Disclosed herein, in some embodiments, are cel engagers, comprising: a cel binding domain that specificaly binds an antigen on a target cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of an immune cel, wherein binding of the immune cel adhesion molecule to the immune cel activates the immune cel, and wherein the cel engager is soluble. In some embodiments, the target cel is a tumor cel. In some embodiments, the antigen comprises cluster of differentiation 19 (CD19), cluster of differentiation 20 (CD20), cluster of diferentiation 22 (CD22), cluster of differentiation 27(CD27), cluster of differentiation 30 (CD30), cluster of diferentiation 70 (CD70), cluster of diferentiation 123 (CD123), cluster of differentiation (CD133), B-cel maturation antigen (BCMA), alpha-actinin-4 (ACTN4), B melanoma antigen 1 (BAGE-1), breakpoint cluster region (BCR) – Abelson murine leukemia (ABL) fusion protein (BCR-ABL), beta-catenin, cancer antigen 125 (CA 125), cancer antigen 15-3 (CA 15-3, also known as CA 27.29), cancer antigen 195 (CA195), cancer antigen 242 (CA 242), cancer antigen 50 (CA 50), tumor associated antigen defined by CT43 monoclonal antibody (CAM43), Caspase-8 (Casp-8), cel division cycle protein 27 (CDC27), cyclin-dependent kinase (CDK4), cyclin-dependent kinase inhibitor 2A (CDKN2A), carcinoembryonic antigen (CEA), colorectal tumor-associated antigen-1 (COA-1), DEK-CAN fusion protein, Epstein-Barr virus nuclear antigen 1 (EBNA1), elongation factor (EF2), an Epstein Bar virus antigen, erythroblast transformation specific variant transcription factor 6 (ETV6) – acute myeloid leukemia 1 protein (AML1) fusion protein (ETV6-AML1), human leukocyte antigen-A2 (HLA-A2), human leukocyte antigen-A11 (HLA-A11), heat shock protein 70- 2 (HSP70-2), lysophosphatidylglycerol acyltransferase 1 (LPGAT1 also known as KIAA0205), melanoma antigen recognized by T cels 2 (MART2), multiple myeloma oncogene 1 (MUM-1), multiple myeloma oncogene 2 (MUM-2), multiple myeloma oncogene 3 (MUM-3), polynucleotide adenylyltransferase gamma (neo-PAP), myosin class I proteins, osteosarcoma amplified 9 (OS-9), promyelocytic leukemia (PML) - retinoic acid receptor alpha (RARα) fusion protein (PML-RARα), protein tyrosine phosphatase receptor type K (PTPRK), Kirsten rat sarcoma viral oncogene (K- RAS), neuroblastoma rat sarcoma viral oncogene (N-RAS), triose-phosphate isomerase (TPI), G antigen 3 (GAGE3), G antigen 4 (GAGE4), G antigen 5 (GAGE5), G antigen 6 (GAGE6), G antigen 7 (GAGE7), N-Acetylglucosaminyl-Transferase V (GNT-V), human endogenous retrovirus-K Atorney Docket No.061479-511001WO (HERV-K-MEL), L antigen family member (LAGE-1), human putative tumor antigen NA88-A (NA-88), L antigen family member 2 (NY-Eso-2/LAGE2), sperm protein (SP17), synovial sarcoma X breakpoint 2B (SSX2, also known as HOM-MEL-40), tyrosine-related protein 2 containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP2-INT2), premelanosome protein (PMEL ), tyrosinase (TYR), tyrosine related protein 1 (TRP-1), tyrosine related protein 2 (TRP-2), melanoma antigen 1 (MAGE-1), melanoma antigen 3 (MAGE-3), receptor for advanced glycation endproducts (RAGE), G antigen 1 (GAGE-1), G antigen 2 (GAGE-2), cyclin dependent kinase inhibitor 2B (CDKN2B also known as p15), synaptonemal complex protein 1 (SCP-1), preferentialy expressed antigen of melanoma (PRAME), tumor protein P53 (p53), Harvey rat sarcoma viral oncogene (H- RAS), human epidermal growth factor receptor 2 (HER2/neu), enhancer binding transcription factors E12 and E47 (E2A) pre-B cel leukemia (PRL) fusion protein (E2A-PRL), H4(D10S170) and rearanged during transfection (RET) fusion protein (H4-RET), immunoglobulin heave chain and kappa light chain (IGH-IGK), human papilomavirus E6 (HPV E6), human papilomavirus protein E7 (HPV E7), tumor surface protein (TSP-180), melanoma antigen 4 (MAGE-4), melanoma antigen 5 (MAGE-5), melanoma antigen 6 (MAGE-6), erb-b2 receptor tyrosine kinase 3 (p180- erbB-3 also known as HER-3), met proto-oncogene (c-Met), nonmetastatic clone 23 Type 1 (nm- 23H1), prostate-specific antigen (PSA), tumor-associated glycoprotein 72 (TAG-72-4), cancer antigen 19-9 (CA 19-9), mucin, nuclear mitotic apparatus protein (NuMa), cyclin-dependent kinase inhibitor 2A (CDKN2A also known as p16), toxic advanced glycation end-product (TAGE), prostate-specific membrane antigen (PSMA), cancer/testis antigen 7 (CT7), telomerase, tumor- associated antigen 43-9F (43-9F), trophoblast glycoprotein (TPBG also known as 5T4), tumor associated antigen 791Tgp72 (CD55), beta subunit human chorionic gonadotropin (Beta-HCG), breast cancer antigen 225 (BCA225), breast tumor associated antigen (BTAA), cluster of diferentiation (CD68), tetraspanin 8 (TSPAN8 also known as CO-029), fibroblast growth factor (FGF5), renal cel carcinoma-associated antigen G250 (G250), epithelial cel adhesion molecule (EpCAM/Ga733), tumor associated antigen HTgp-175 (HTgp-175), M344 tumor associated antigen (M344), FMS-like tyrosine kinase 3 (FLT3-ITD), monoclonal gastric cancer 7 antigen (MG7-Ag), folate receptor alpha (FOLR1), human ovarian tumor-associated antigen NB\70K (NB\70K), New- York Colon 1 (NY-CO-1), receptor binding cancer antigen expressed on SiSo cels (RCAS1), serologicaly defined colon cancer antigen 16 (SDCCAG16), 90kD glycoprotein tumor-associated antigen (TA-90), tumor-associated antigen L6 (TAAL6), tumor-associated glycoprotein 72 (TAG72), tumor liberated protein (TLP), tissue polypeptide specific antigen (TPS), CD2 Subset 1 (CS1), G protein coupled receptor family group 5 member D (GPCR5D), ganglioside G2 (GD2), epidermal growth factor receptor variant II (EGFRvII), mesothelin (MSLN), prostatic acid phosphatase (PAP), prostate cancer-associated protein 6 (prostein), T cel receptor gamma alternate reading frame protein (TARP), transient receptor potential channel prostate-specific expression patern 8 (Trp-p8), six-transmembrane epithelial antigen of the prostate 1 (STEAP1), an abnormal Atorney Docket No.061479-511001WO ras protein, an abnormal p53 protein, integrin αvβ3 (CD51/CD61), galectin, or Ras-like protooncogene B (RalB). In some embodiments, the target cel is the immune cel or another immune cel. In some embodiments, the target cel is the immune cel. In some embodiments, the target cel is another immune cel. In some embodiments, the antigen comprises cluster of differentiation 3 (CD3). In some embodiments, the reciprocal adhesion molecule comprises cluster of differentiation 2 (CD2). In some embodiments, the immune cel adhesion molecule comprises a cluster of diferentiation 58 (CD58) protein. Some embodiments include an immune cel costimulatory molecule. In some embodiments, the immune cel costimulatory molecule binds cluster of diferentiation 28 (CD28). In some embodiments, the immune cel costimulatory molecule comprises a cluster of diferentiation 80 (CD80) or a cluster of differentiation 86 (CD86) protein. Adhesion Molecules [0093] Disclosed herein, in some embodiments, are adhesion molecules. The adhesion molecule(s) may be included as part of a fusion protein. The adhesion molecule may be included as part of an immune cel engager. [0094] As used herein, the term “adhesion molecule” refers, in a broad sense, to a molecular component of a SMAC or other immune synapse, other than an immune cel binding domain (e.g., TCR-binding agent) or a costimulatory molecule, which contributes to adhesion of a particle to target cels. Adhesion molecules from natural sources may be molecules expressed, natively, on antigen- presenting cels and adapted for use here on particles. Both naturaly occuring adhesion molecules, and their variants, and artificial adhesion molecules, such as antibodies, or fragments thereof, are contemplated. An adhesion molecule, as the term is used herein, specificaly binds a conjugate molecule with affinity suficient to cause increased adhesion between the immune cel engager and the target cel compared to the adhesion of a reference immune cel engager lacking the adhesion molecule to the same or similar target cel. The term adhesion molecule includes but is not limited to CD58, a CD58 extracelular domain, and functional fragments of CD58. As described above, the term “functional fragment” is used herein to a fragment of a polypeptide, or other molecule, that retains the desired function of the polypeptide. For example, a functional fragment of CD58 is a fragment of CD58 that specificaly binds cluster of differentiation 2 (CD2). The adhesion molecule may be a protein, termed herein an “adhesion protein.” [0095] In some embodiments, the adhesion molecule is or includes a binding domain that specificaly binds CD2. In some embodiments, the adhesion molecule is or includes CD58, cluster of diferentiation 48 (CD48), intercelular adhesion molecule 1 (ICAM-1), intercelular adhesion molecule 2 (ICAM-2), intercelular adhesion molecule 3 (ICAM-3), intercelular adhesion molecule 4 (ICAM-4), intercelular adhesion molecule 5 (ICAM-5), junctional adhesion molecule A (JAM- A), cluster of differentiation 155 (CD155) or cluster of differentiation 112 (CD112), or a functional fragment thereof. Atorney Docket No.061479-511001WO [0096] In some embodiments, the costimulatory and/or adhesion molecule comprises an amino acid sequence 100% identical to a sequence in Table 3A or Table 3B. In some embodiments, the costimulatory and/or adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 3A or Table 3B. In some embodiments, the costimulatory and/or adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 3A or Table 3B. [0097] Polypeptide sequences of ilustrative adhesion molecules are provided in Table 3A, with the “start” and “end” positions of the extracelular portion of each. In each case, the adhesion molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to any sequence in Table 3A, or functional fragments thereof. Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 (or any range thereof) amino acid portion that retains binding affinity to its cognate molecule, when measured using afinity assays such as biolayer interferometry or other assays that may be known in the art. [0098] Some embodiments include an immune cel adhesion molecule. Some examples of immune cel adhesion molecule that include a CD58 protein fragment are provided in Table 3C. A CD58 protein may bind CD2. A CD58 protein that binds CD2 may be or include a CD58 protein fragment. The examples In some embodiments, an adhesion molecule shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence in Table 3C. In some embodiments, an adhesion molecule shares less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to a sequence in Table 3C. In some embodiments, the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 309. In some embodiments, the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 310. In some embodiments, the immune cel adhesion molecule includes an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 311. Atorney Docket No.061479-511001WO Table 3A Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Table 3B Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Table 3C [0099] In some embodiments, the adhesion molecule is CD58. CD58 is also known as lymphocyte function-associated antigen 3 (LFA-3). CD58 binds to CD2 (LFA-2) on T cels. The extracelular portion of CD58 is residues 29-215 of SEQ ID NO: 1 (SEQ ID NO: 10): FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYN LTSSDEDEYEMESPNITDTMKFFLYVLESLPSPTLTCALTNGSIEVQCMIPEHYNSHRGLIMYSWDCP MEQCKRNSTSIYFKMENDLPQKIQCTLSNPLFNTTSSILTTCIPSSGHSRHR (SEQ ID NO: 10) [0100] In some embodiments, the polypeptide sequence of CD58 shares at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 248: FSQQIYGVVYGNVTFHVPSNVPLKEVLWKKQKDKVAELENSEFRAFSSFKNRVYLDTVSGSLTIYN LTSSDEDEYEMESPNITDTMKFFLYVLESL (SEQ ID NO: 248) Atorney Docket No.061479-511001WO [0101] A crystal structure of CD58 is described in Ikemizu et al. PNAS USA 96(8):4289-94 (1999). The extracelular portion of CD58 has a ligand-binding domain and a second extracelular domain. In some embodiments, the ligand-binding domain may be used as the functional fragment of CD58—e.g., without the second extracelular domain. [0102] In some embodiments, the adhesion molecule (or immune cel engager) comprises the polypeptide sequence of SEQ ID NO: 1 or 10, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 1 or 10. In some embodiments, the adhesion molecule (or immune cel engager) comprises a sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to SEQ ID NO: 1 or 10. The adhesion molecule may encoded by a polynucleotide (e.g. a DNA or RNA polynucleotide). [0103] The adhesion molecule may be encoded by the polynucleotide sequence of CD58, SEQ ID NO: 11, or by a subsequence encoding the extracelular portion or a functional fragment. SEQ ID NO: 11 (5’ to 3’): ATGGTTGCTGGGAGCGACGCGGGGCGGGCCCTGGGGGTCCTCAGCGTGGTCTGCCTGCTGCAC TGCTTTGGTTTCATCAGCTGTTTTTCCCAACAAATATATGGTGTTGTGTATGGGAATGTAACTTT CCATGTACCAAGCAATGTGCCTTTAAAAGAGGTCCTATGGAAAAAACAAAAGGATAAAGTTGC AGAACTGGAAAATTCTGAGTTCAGAGCTTTCTCATCTTTTAAAAATAGGGTTTATTTAGACACT GTGTCAGGTAGCCTCACTATCTACAACTTAACATCATCAGATGAAGATGAGTATGAAATGGAA TCGCCAAATATTACTGATACCATGAAGTTCTTTCTTTATGTGCTTGAGTCTCTTCCATCTCCCAC ACTAACTTGTGCATTGACTAATGGAAGCATTGAAGTCCAATGCATGATACCAGAGCATTACAA CAGCCATCGAGGACTTATAATGTACTCATGGGATTGTCCTATGGAGCAATGTAAACGTAACTCA ACCAGTATATATTTTAAGATGGAAAATGATCTTCCACAAAAAATACAGTGTACTCTTAGCAATC CATTATTTAATACAACATCATCAATCATTTTGACAACCTGTATCCCAAGCAGCGGTCATTCAAG ACACAGATATGCACTTATACCCATACCATTAGCAGTAATTACAACATGTATTGTGCTGTATATG AATGGTATTCTGAAATGTGACAGAAAACCAGACAGAACCAACTCCAAT. [0104] The polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 11, or a suitable subsequence, which may be used to express the adhesion molecule. [0105] It wil be appreciated that further variants of CD58 may be used. For example, homologs of CD58 from other species (mice, ape, horse, etc.) may be identified and tested for use in transducing human, or non-human, target cels. It is expected that at least some non-human homologs wil retain adhesion molecule function when used with human target cels. [0106] Further adhesion molecules useful in the practice of an immune cel engager may include any molecule that specificaly binds CD2, LFA-1, or DNAM-1. For example, the adhesion molecule may be a molecule that comprises an antibody, or antigen-binding domain thereof, specific to CD2, Atorney Docket No.061479-511001WO LFA-1, or DNAM-1. In some embodiments, the adhesion molecule is a VHH, an scFv, DARPin or any other antigen binding proteins. [0107] In some embodiments, the adhesion molecule binds to CD2. CD2 is also known as T11, LFA-2, and the erythrocyte rosete receptor. In its native state, CD2 is a surface protein expressed on T lymphocytes and NK cels. CD2 is a natural ligand for CD58. In addition to performing adhesion functions, engagement of CD2 by CD58 provides a costimulatory signal that may enhance activation and efector functions. In some embodiments, the immune cel engager comprises an adhesion molecule that binds to CD2, which may be CD58 or a fragment thereof. In some embodiments, the immune cel engager comprises an antibody, single domain antibody, antibody fragment, and/or nanobody specific for CD2. [0108] The foregoing description of CD58 and its derivatives as the adhesion molecule and CD2 as the cognate molecule may be extrapolated to the other adhesion molecules described herein. The adhesion molecule (or immune cel engager) may comprise any polypeptide sequence in Table 1, to an extracelular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 1, to an extracelular portion thereof, or to a functional fragment thereof. Costimulatory Molecules [0109] Disclosed herein, in some embodiments, are costimulatory molecules. The costimulatory molecule may be included as part of a fusion molecule. The costimulatory molecule may be included as part of an immune cel engager. [0110] The fusion molecule comprising the immune cel engager may include a costimulatory molecule. However, in some embodiments, the fusion molecule does not include a costimulatory molecule. The fusion molecule may incorporate a costimulatory molecule as a separate molecule, or the fusion molecule may lack any costimulatory molecule. The costimulatory molecule may be a protein, termed herein a “costimulatory protein.” [0111] As used herein, the term “costimulatory molecule” refers to a molecule capable of providing a costimulatory signal to target cels. In T cel biology, the binding of the T cel receptor by an antigen can provide the primary stimulatory signal to the cel. So-caled costimulatory signals are provided by accessory molecules. An example costimulatory signal is the signal provided by binding of cluster of differentiation 28 (CD28) on T cels by a ligand. Some examples of ligands of CD28 include CD80 and cluster of differentiation 86 (CD86). [0112] Ilustrative costimulatory molecules include, but are not limited to, CD80, CD86, CD40L (also known as CD154), GITRL, OX40L, 41BBL, ICOSL, CD27, CD30L, LIGHT, LTalpha, MICA, MICB and anti-CD28. Each of the foregoing may be employed as a costimulatory molecule as a ful-length protein, an extracelular domain, or functional fragment. In some embodiments, engager Atorney Docket No.061479-511001WO includes an immune cel costimulatory molecule that provide a costimulatory signal to the immune cel. In some embodiments, the costimulatory molecule is or includes a binding domain that specificaly binds CD28. In some embodiments, the costimulatory molecule is or includes cluster of diferentiation 80 (CD80), cluster of diferentiation 86 (CD86), CD40 ligand (CD40L also known as CD154), glucocorticoid-induced TNF receptor ligand (GITRL), tumor necrosis factor superfamily member 4 (TNFSF4 also known as OX40L), tumor necrosis factor superfamily member 9 (TNFSF9 also known as 41BBL), inducible T cel costimulatory ligand (ICOSL), cluster of diferentiation 70 (CD70), CD30 ligand (CD30L), tumor necrosis factor superfamily member 14 (TNFSF14 also known as LIGHT), lymphotoxin-alpha (LTalpha), MHC class I polypeptide-related sequence A (MICA), or MHC class I polypeptide-related sequence B (MICB), or a functional fragment thereof. [0113] Polypeptide sequences of ilustrative costimulatory molecules are provided in Table 4, with the “start” and “end” positions of the extracelular portion of each. In each case, the costimulatory molecule may comprise a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any sequence in Table 4, or functional fragments thereof. In some embodiments, the costimulatory molecule comprises a polypeptide having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or 100% sequence identity to any sequence in Table 4, or a functional fragment thereof. Functional fragments may be or include any 10, 20, 30, 40, 50, 75, 100, 200, 300, 400, 500, or 600 amino acid portion that retains binding affinity to its cognate molecule, when measured using affinity assays such as biolayer interferometry or other assays known in the art. Table 4 Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO Atorney Docket No.061479-511001WO [0114] In some embodiments, the costimulatory molecule is or includes CD80. In some embodiments, the costimulatory molecule is or includes a molecule that binds CD28. CD80 binds to CD28. The extracelular portion of CD80 includes residues 35-230 of SEQ ID NO: 12, which includes an Ig-like V-type domain (SEQ ID NO: 25) and an Ig-like C2-type domain (SEQ ID NO: 26), either or both of which may be included to form the costimulatory molecule. VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNL SIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVT (SEQ ID NO: 25) PSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSF MCLIKYGHLRVNQTFN (SEQ ID NO: 26) [0115] The crystal structure of CD80 (also known as B7-1) is described in Ikemizu et al. Immunity 12:51-60 (2000). The extracelular portion of CD80 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD80. [0116] In some embodiments, the costimulatory molecule is or includes CD86. CD86 binds to CD28. The extracelular portion of CD86 includes residues 33-225 of SEQ ID NO: 13, which includes an Ig-like V-type domain (SEQ ID NO: 27) and an Ig-like C2-type domain (SEQ ID NO: 28), either or both of which may be included to form the costimulatory molecule. NETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRL HNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELS (SEQ ID NO: 27) NVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIF CILETDKT (SEQ ID NO: 28) [0117] The crystal structure of CD86 (also known as B7-1) is described in Schwartz et al. Nature 410: 604-608 (2001). The extracelular portion of CD86 has two domains, described above. In embodiments, one or both of the domains may be used as the functional fragment of CD86. [0118] It wil be appreciated that further variants of CD80 or CD86 may be used. For example, homologs of CD80 or CD86 from other species (mice, ape, horse, etc.) may be identified and tested for use in transducing human, or non-human, target cels. It is expected that at least some non-human homologs wil retain costimulatory molecule function when used with human target cels. [0119] In some embodiments, the costimulatory molecule (or immune cel engager) comprises the polypeptide sequence of one or more of SEQ ID NO: 12-13 and 25-28, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to one or more of SEQ ID NO: 12-13 and 25-28. [0120] In some embodiments, the costimulatory molecule comprises a CD80 protein. The CD80 protein may be or include the polypeptide sequence of SEQ ID NO: 250, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 250. Atorney Docket No.061479-511001WO [0121] In some embodiments, the costimulatory molecule (or immune cel engager) comprises a polypeptide sequence having less than 75%, less than 80%, less than 85%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, or less than 100% identity to one or more of SEQ ID NO: 12-13 and 25- 28. The costimulatory molecule may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). The costimulatory molecule may be encoded by the polynucleotide sequence of CD80 (SEQ ID NO: 29) or CD86 (SEQ ID NO: 30), or by a subsequence encoding the extracelular domain or a functional fragment. ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGC TCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAA AGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATC TACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCC GAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGC GCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGC GGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGA CTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAG CCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAA GATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACA GCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAAC CAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAAT GGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGG AATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA (SEQ ID NO: 29) ATGGATCCCCAGTGCACTATGGGACTGAGTAACATTCTCTTTGTGATGGCCTTCCTGCTCTCTGG TGCTGCTCCTCTGAAGATTCAAGCTTATTTCAATGAGACTGCAGACCTGCCATGCCAATTTGCA AACTCTCAAAACCAAAGCCTGAGTGAGCTAGTAGTATTTTGGCAGGACCAGGAAAACTTGGTT CTGAATGAGGTATACTTAGGCAAAGAGAAATTTGACAGTGTTCATTCCAAGTATATGGGCCGC ACAAGTTTTGATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATCAAGGACAAGGGCT TGTATCAATGTATCATCCATCACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAACTC TGAACTGTCAGTGCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATTTCTAATATAACAGAA AATGTGTACATAAATTTGACCTGCTCATCTATACACGGTTACCCAGAACCTAAGAAGATGAGTG TTTTGCTAAGAACCAAGAACTCAACTATCGAGTATGATGGTGTTATGCAGAAATCTCAAGATAA TGTCACAGAACTGTACGACGTTTCCATCAGCTTGTCTGTTTCATTCCCTGATGTTACGAGCAATA TGACCATCTTCTGTATTCTGGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAG CTTGAGGACCCTCAGCCTCCCCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTA TTATATGTGTGATGGTTTTCTGTCTAATTCTATGGAAATGGAAGAAGAAGAAGCGGCCTCGCAA CTCTTATAAATGTGGAACCAACACAATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAG AAAAAATCCATATACCTGAAAGGTCTGATGAAGCCCAGCGTGTTTTTAAAAGTTCGAAGACAT CTTCATGCGACAAAAGTGATACATGTTTT (SEQ ID NO: 30) [0122] The polynucleotide sequence may be varied by codon-optimization or other methods to generate polynucleotide sequences having at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO: 29 or 30, or a suitable subsequence, which may be used to express the costimulatory molecule. [0123] Further costimulatory molecules useful in the practice of the present disclosure may include any molecule that specificaly binds CD28. For example, the costimulatory molecule may be a molecule that comprises an antibody, or antigen-binding fragment thereof, specific to CD28. [0124] CD28 is a receptor expressed on T cels that provide costimulatory signal. T cel co- stimulation through CD28, resulting in, for example, the production of various interleukins (in Atorney Docket No.061479-511001WO particular IL-6). In some embodiments, the costimulatory molecule is an antibody, or fragment thereof, that specificaly binds to CD28. Examples of such antibodies include 15E8, TGN1412, CD28.2, and 10F3, as wel as humanized variants thereof. [0125] 15E8 is a mouse monoclonal antibody to human CD28. Its complementarity determining regions (CDRs) are as folows: [0126] CDRH1: GFSLTSY (SEQ ID NO: 36) [0127] CDRH2: WAGGS (SEQ ID NO.37) [0128] CDRH3: DKRAPGKLYYGYPDY (SEQ ID NO.38) [0129] CDRL1: RASESVEYYVTSLMQ (SEQ ID NO.39) [0130] CDRL2: AASNYES (SEQ ID NO.40) [0131] CDRL3: QQTRKVPST (SEQ ID NO.41) [0132] TGN1412 (also known as CD28-SuperMAB) is a humanized monoclonal antibody that not only binds to, but also is a strong agonist for, the CD28 receptor. Its CDRs are as folows. [0133] CDRHl: GYTFSY (SEQ ID NO.42) [0134] CDRH2: YPGNVN (SEQ ID NO.43) [0135] CDRH3: SHYGLDWNFDV (SEQ ID NO.44) [0136] CDRLl: HASQNIYVLN (SEQ ID NO.45) [0137] CDRL2: KASNLHT (SEQ ID NO.46) [0138] CDRL3: QQGQTYPYT (SEQ ID NO.47) [0139] The foregoing description of CD80, CD86, and their derivatives as the costimulatory molecule and CD28 as the cognate molecule may be extrapolated to the other costimulatory molecules described herein, including but not limited to those listed in Table 4. The costimulatory molecule (or the immune cel engager) may comprise any polypeptide sequence of in Table 4, to an extracelular portion thereof, or to a functional fragment thereof, or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a sequence in Table 4, to an extracelular portion thereof, or to a functional fragment thereof. In some embodiments, the costimulatory molecule is a VHH, an scFv, DARPin or any other antigen binding protein. Immune Cel Binding Domains [0140] Disclosed herein, in some embodiments, are immune cel binding domains. The immune cel binding domain may be included as part of an immune cel engager. An example of an immune cel binding domain may include a TCR-binding molecule. [0141] The immune cel engager may include an immune cel binding domain (e.g., a TCR-binding molecule) or other subunit that provides an activation signal to a target cel. However, in some embodiments, the immune cel engager does not include a TCR-binding molecule or other activation domain. The immune cel engager may incorporate a TCR-binding molecule as a separate molecule, Atorney Docket No.061479-511001WO or the immune cel engager may lack any TCR-binding molecule. The TCR-binding molecule may be a protein, termed herein a “TCR-binding protein.” The immune cel binding domain may be or include an activation protein. [0142] As used herein, the term “TCR-binding molecule” refers to a molecule capable of directly binding the extracelular portion of the T cel receptor (TCR) by contacting one or more components of the TCR or otherwise providing a primary or “signal 1” activation signal to a target cel (e.g., a T cel or NK cel). The structure of the TCR, its components, and function is described in Sušac et al. Cel 185(17):3201-3213.e19 (2022). Some examples of TCR-binding molecules may include an antibody, or antigen binding domain, that specificaly binds CD3 (an anti-CD3 monoclonal antibody, or antigen binding fragment thereof). In some embodiments, the immune cel binding domain comprises an antibody, single domain antibody, antibody fragment, nanobody, or other binding protein specific for CD3. Ilustrative antibodies include OKT3 (also known as Muromonab-CD3), otelixizumab, teplizumab and visilizumab. The complementarity determining regions of OKT3 are as folows in Table 5. Table 5 [0143] The immune cel binding domain (e.g., TCR-binding molecule) may be a single chain variable fragment (scFv) incorporated into the fusion protein (or immune cel engager). OKT3 in scFv format may be used. [0144] The immune cel binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a heavy chain variable domain (VH) domain. The VH domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 312. [0145] The immune cel binding domain may include an anti-CD3 antibody or antibody fragment (such as a scFv) that includes a light chain variable domain (VL) domain. The VL domain may be or include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 313. [0146] In some embodiments, the immune cel binding domain (e.g. TCR-binding molecule) is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% Atorney Docket No.061479-511001WO identical to the anti-CD3 scFv of SEQ ID NO: 31, which includes a variable light (VL) and variable heavy (VH) domain with a 3 × GGGS (SEQ ID NO: 307) linker: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSG TDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGG VVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNS KNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSAAAKP (SEQ ID NO: 31) [0147] In some embodiments, the immune cel binding domain (e.g. TCR-binding molecule) is or includes an scFv comprising a polypeptide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 99%, or 100% identical to the anti-CD3 scFv of SEQ ID NO: 249, which includes a variable light (VL) and variable heavy (VH) domain with a 3 × GGGS (SEQ ID NO: 307) linker: DIQMTQSPSSLSASVGDRVTITCSASSSVSYMNWYQQTPGKAPKRWIYDTSKLASGVPSRFSGSGSG TDYTFTISSLQPEDIATYYCQQWSSNPFTFGQGTKLQITRTSGGGGSGGGGSGGGGSQVQLVQSGGG VVQPGRSLRLSCKASGYTFTRYTMHWVRQAPGKGLEWIGYINPSRGYTNYNQKVKDRFTISRDNS KNTAFLQMDSLRPEDTGVYFCARYYDDHYCLDYWGQGTPVTVSSA (SEQ ID NO: 249) [0148] The complementarity determining regions of this scFv are as folows: [0149] CDRH1: RYTMH (SEQ ID NO: 54) [0150] CDRH2: YINPSRGYTNYNQKVKD (SEQ ID NO: 55) [0151] CDRH3: YYDDHYCLDY (SEQ ID NO: 56) [0152] CDRL1: SASSSVSYMN (SEQ ID NO: 57) [0153] CDRL2: DTSKLASG (SEQ ID NO: 58) [0154] CDRL3: QQWSSNPFT (SEQ ID NO: 59) [0155] Other immune cel binding domains may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cels. For example, CD3, OX40, CD27, ICOS, 41BB, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC. The immune cel engager disclosed herein may comprise portions of these proteins or domains that bind to these proteins. For example, without wishing to be bound by theory, T cels may express one or both of CD4 and/or CD8 and the fusion molecules disclosed herein may comprise domains that engage with either or both of CD4 and/or CD8. [0156] When cels other than T cels are the intended target of the immune cel engager as disclosed herein, other binding domains may be more appropriate. For example, an immune cel engager targeting NK cels may comprise domains that engage with proteins found on NK cels. In some embodiments, these proteins include CD2, CD16, NKp46, NKp30, and NKG2D. In some such embodiments, immune cel engager intended to target and/or activate NK cels may comprise domains that bind to CD2, CD16, NKp46, NKG2D, etc. Domains that bind to NKG2D may be derived from NKG2D ligands including, but not limited to: MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. In some embodiments, the immune cel engager described herein Atorney Docket No.061479-511001WO comprises a CD58 domain, a domain that binds NKG2D, and optionaly a third domain which enhances activation of the target NK cel. [0157] The immune cel binding domain may be encoded by a polynucleotide (e.g., a DNA or RNA polynucleotide). Tumor cel binding domains [0158] Some embodiments include a tumor cel binding domain. For example, an engager such as an immune cel engager may include a tumor cel binding domain. In some embodiments, the tumor cel binding domain may contain any polypeptide that binds a desired antigen (e.g. antigen expressed on a tumor of interest). The domain may comprise a scFv, a portion of an antibody or an alternative scafold. For example, scFvs, lama VHH antibodies, other VH only antibody fragments, peptides, or smal protein binding domains (e.g. D domains) may be utilized. For example, the tumor cel binding domain may comprise an scFv or VHH that binds to a target tumor antigen or the tumor cel binding domain may comprise a natural ligand, for example, folate. [0159] The antigen to which the tumor cel binding domain binds can be any antigen of interest, e.g., can be an antigen on a tumor cel. The tumor cel may be, e.g., a cel in a solid tumor, or a cel of a blood cancer. The antigen can be any antigen that is expressed on a cel of any tumor or cancer type, e.g., cels of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic smal round cel tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cel tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a fibroma, a lipoma, or the like. In some embodiments, said lymphoma can be chronic lymphocytic leukemia (smal lymphocytic lymphoma), B-cel prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasma cel myeloma, plasmacytoma, extranodal marginal zone B cel lymphoma, MALT lymphoma, nodal marginal zone B cel lymphoma, folicular lymphoma, mantle cel lymphoma, difuse large B cel lymphoma, mediastinal (thymic) large B cel lymphoma, intravascular large B cel lymphoma, primary effusion lymphoma, Burkit's lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cel leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastic NK cel lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cel lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cel lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma. In some embodiments, in which the Atorney Docket No.061479-511001WO cancer is chronic lymphocytic leukemia (CLL), the B cels of the CLL have a normal karyotype. In some embodiments, in which the cancer is chronic lymphocytic leukemia (CLL), the B cels of the CLL cary a 17p deletion, an 11q deletion, a 12q trisomy, a 13q deletion or a p53 deletion. [0160] In some embodiments, the antigen is expressed on a B-cel malignancy cel, relapsed/refractory CD19-expressing malignancy cel, diffuse large B-cel lymphoma (DLBCL) cel, Burkit’s type large B-cel lymphoma (B-LBL) cel, folicular lymphoma (FL) cel, chronic lymphocytic leukemia (CLL) cel, acute lymphocytic leukemia (ALL) cel, mantle cel lymphoma (MCL) cel, hematological malignancy cel, colon cancer cel, lung cancer cel, liver cancer cel, breast cancer cel, renal cancer cel, prostate cancer cel, ovarian cancer cel, skin cancer cel, melanoma cel, bone cancer cel, brain cancer cel, squamous cel carcinoma cel, leukemia cel, myeloma cel, B cel lymphoma cel, kidney cancer cel, uterine cancer cel, adenocarcinoma cel, pancreatic cancer cel, chronic myelogenous leukemia cel, glioblastoma cel, neuroblastoma cel, meduloblastoma cel, or a sarcoma cel. [0161] In some embodiments, the antigen is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In some embodiments, without limitation, the tumor-associated antigen or tumor- specific antigen is B cel maturation antigen (BCMA), B cel Activating Factor (BAFF), GPRC5D, FCRL5/FCRH5, ROR1, L1-CAM, CD22, folate receptor, carboxy anhydrase IX (CAIX), claudin 18.2, FAP, mesothelin, IL-13Ra2, Lewis Y, CCNA1, WT-1, TACI, CD38, SLAMF7, CD138, DLL3, transmembrane 4 L six family member 1 (TM4SF1), epithelial cel adhesion molecule (EpCAM), PD-1, PD-L1, CTLA-4, AXL, ROR2, glypican-3 (GPC3), CD133, CD147, EGFR, MUC1, GD2, Her2, prostate stem cel antigen (PSCA), prostate-specific membrane antigen (PSMA) alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), EGFRvII, cancer antigen-125 (CA- 125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrilary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, vascular endothelial growth factor receptor (VEGFR), the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein. Some example antibodies useful for target cel-binding domains or tumor cel-binding domains may include anti-mucin monoclonal antibody CAM 17.1, and MOv18 which is an IgE antibody that targets folate receptor alpha. In some embodiments, the tumor cel binding domain comprises binding domains that target two or more antigens as disclosed herein, in any combination. For example: CD19 and CD3, BCMA and CD3, GPRC5D and CD3, FCRL5 and CD3, CD38 and CD3, CD19 and CD20, CD19 and CD22, BCMA and GPRC5D, or CD20 and CD22. In Atorney Docket No.061479-511001WO some embodiments, the tumor cel binding domain comprises binding domains that target two or more antigens on the same target protein, for example two epitopes in BCMA. [0162] Exemplary binding domains include scFvs derived from the anti-CD19 antibody FMC63, which are known in the art. Binding domains targeting CD19 are described, for example, in U.S. Pat. Pub. No.20160152723; and U.S. Pat. Nos.10,736,918; 10,357,514; and 7,446,190. Exemplary binding domains include VH domains that bind BCMA and scFvs that bind BCMA. Binding domains targeting BCMA are described, for example, in U.S. Pat. Pub. Nos.2020/0246381 and 2019/0161553; and U.S. Pat. Nos.10,918,665. Binding domains targeting GPRC5D may be found in U.S. Pat. Pub. Nos.2018/0118803 and 2021/10393689. Binding domains targeting FCRL5 may be found in U.S. Pat. Pub. No. US 2017/0275362. Binding domains targeting ROR1 may be found in U.S. Pat. Pub. No.2022/0096651. [0163] In some embodiments, the tumor cel binding domain binds to an antigen selected from the group consisting of: CD19, EpCAM, Her2/neu, EGFR, CD66e, CD33, EphA2, or MCSP. In some embodiments, the tumor cel binding domain binds to an antigen selected from the group consisting of: CD19, EpCAM, CD20, CD123, BCMA, B7-H3, CDE, or PSMA. In some embodiments, the tumor cel binding domain binds to a myeloid cel or dendritic cel antigen. In some embodiments, the tumor cel binding domain binds to CD33, DC-SIGN, CDlb, CDlc, or CD18. [0164] In some embodiments, the tumor cel binding domain comprises DUPA (DUPA-(99m) Tc), a ligand bound by PSMA-positive human prostate cancer cels with nanomolar affinity (KD = 14 nM; see Kularatne, S.A. et al., Mol Pharm.2009.6(3):780-9). In some embodiments, a DUPA derivative can be the ligand of the smal molecule ligand linked to a targeting moiety, and DUPA derivatives are described in WO 2015/057852, incorporated herein by reference. [0165] In some embodiments, the tumor cel binding domain comprises CCK2R ligand, a ligand bound by CCK2R-positive cancer cels (e.g., cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon; see Wayua. C. et al., Molecular Pharmaceutics.2013. ePublication). [0166] In some embodiments, the tumor cel binding domain comprises folate, folic acid, or an analogue thereof, a ligand bound by the folate receptor on cels of cancers that include cancers of the ovary, cervix, endometrium, lung, kidney, brain, breast, colon, and head and neck cancers; see Sega, E.I. et al., Cancer Metastasis Rev.2008.27(4):655-64). [0167] In some embodiments, the tumor cel binding domain comprises an NK-1R ligand. Receptors for NK-1R the ligand are found, for example, on cancers of the colon and pancreas. In some embodiments, the NK-1R ligand may be synthesized according the method disclosed in Int’l Patent Appl. No. PCT/US2015/044229, incorporated herein by reference. [0168] In some embodiments, the tumor cel binding domain comprises a peptide ligand, for example, the ligand may be a peptide ligand that is the endogenous ligand for the NK1 receptor. In some embodiments, the ligand may be a regulatory peptide that belongs to the family of tachykinins Atorney Docket No.061479-511001WO which target tachykinin receptors. Such regulatory peptides include Substance P (SP), neurokinin A (substance K), and neurokinin B (neuromedin K), (see Hennig et al., International Journal of Cancer: 61, 786-792). [0169] In some embodiments, the tumor cel binding domain comprises a CAIX ligand. Receptors for the CAIX ligand found, for example, on renal, ovarian, vulvar, and breast cancers. The CAIX ligand may also be refered to herein as CA9. [0170] In some embodiments, the tumor cel binding domain comprises a ligand of gamma glutamyl transpeptidase. The transpeptidase is overexpressed, for example, in ovarian cancer, colon cancer, liver cancer, astrocytic gliomas, melanomas, and leukemias. [0171] In some embodiments, the tumor cel binding domain comprises a CCK2R ligand. Receptors for the CCK2R ligand found on cancers of the thyroid, lung, pancreas, ovary, brain, stomach, gastrointestinal stroma, and colon, among others. Multimerization Domains [0172] Polypeptides described herein may form multimers such as dimers, trimers and higher oligomers, e.g., consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. For example, a multimerization polypeptide, when fused to a second molecule, such as a heterologous polypeptide sequence, facilitates the formation of dimers, trimers, tetramers, pentamers or higher order oligomers or mixtures thereof among the polypeptides. Fusing a multimerization polypeptide to an antigen binding protein, such as an antigen binding polypeptide (e.g., antibody) can be used to increase the number of antigen binding sites via oligomer formation. Increasing the number of antigen binding sites can in turn increase antibody avidity for the antigen, which is useful in any therapeutic or diagnostic antibody application, particularly where it is desirable or advantageous to increase antibody avidity. [0173] As used here in, “multimerization domains” refers to protein domains that alow for higher order protein structure to form consisting of multiple protein molecules. [0174] In some embodiments, the immune cel engager further comprises a multimerization domain. In some embodiments, the multimerization domain is a Fc domain from an IgG. In some embodiments, the multimerization domain is a Fc domain from IgA. In some embodiments, the multimerization domain is a Fc domain from IgD. In some embodiments, the multimerization domain is a Fc domain from IgE. In some embodiments, the multimerization domain is a Fc domain from IgM. In some embodiments, the multimerization domain is a Fc domain from IgG1. In some embodiments, the multimerization domain is a Fc domain from IgG2. In some embodiments, the multimerization domain is a Fc domain from IgG3. In some embodiments, the multimerization domain is a Fc domain from IgG4 Atorney Docket No.061479-511001WO [0175] In some embodiments, the multimerization domain is the self-assembling tetramerization domain (TD) from the tumor suppressor gene p53. In some embodiments, the multimerization domain is the SpyCather-SpyTag system. [0176] In some embodiments, the multimerization domain is a self-associating peptide. In some embodiments, the self-associating peptide is RHCC derived from a right-handed coiled-coil peptide of an archaebacterium. In some embodiments, the self-associating peptide is COMPcc from human cartilage oligomeric matrix protein. In some embodiments, the self-associating peptide is C4bpα derived from human plasma C4-binding protein α-chain. Pharmaceutical Compositions and Formulations [0177] In some embodiments, the disclosure provides a pharmaceutical composition comprising an immune cel engager according to the disclosure and a pharmaceuticaly acceptable carier. [0178] The formulations and compositions of the present disclosure may comprise a combination of any number of immune cel engagers, and optionaly one or more additional pharmaceutical agents (viral particles, polypeptides, polynucleotides, compounds etc.) formulated in pharmaceuticaly acceptable or physiologicaly-acceptable compositions for administration to a cel, tissue, organ, or an animal, either alone, or in combination with one or more other modalities of therapy. In some embodiments, the one or more additional pharmaceutical agents further increases transduction efficiency of viral particles. [0179] In some embodiments, the formulations and compositions of the present disclosure may comprise a combination of any number of immune cel engagers, and optionaly one or more nanocarriers. Ilustrative nanocariers include, but are not limited to, miceles, polymers, liposomes, and lipid nanoparticles (LNPs). [0180] In some embodiments, the present disclosure provides compositions comprising a therapeuticaly-effective amount of an immune cel engager, as described herein, formulated together with one or more pharmaceuticaly acceptable cariers (additives) and/or diluents. In some embodiments, the composition further comprises other agents, such as, e.g., cytokines, growth factors, hormones, smal molecules, or various pharmaceuticaly active agents. [0181] In some embodiments, compositions and formulations of the immune cel engager used in accordance with the present disclosure may be prepared for storage by mixing an immune cel engager having the desired degree of purity with optional pharmaceuticaly acceptable cariers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable cariers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed. In some embodiments, one or more pharmaceuticaly acceptable surface-active agents (surfactant), bufers, isotonicity agents, salts, amino acids, sugars, stabilizers and/or antioxidant are used in the formulation. Atorney Docket No.061479-511001WO [0182] Suitable pharmaceuticaly acceptable surfactants comprise but are not limited to polyethylen-sorbitan-faty acid esters, polyethylene-polypropylene glycols, polyoxyethylene- stearates, and sodium dodecyl sulphates. Suitable buffers comprise but are not limited to histidine- buffers, citrate-buffers, succinate-bufers, acetate-bufers, and phosphate-buffers. [0183] Isotonicity agents are used to provide an isotonic formulation. An isotonic formulation is liquid, or liquid reconstituted from a solid form, e.g., a lyophilized form and denotes a solution having the same tonicity as some other solution with which it is compared, such as physiologic salt solution and the blood serum. Suitable isotonicity agents comprise but are not limited to salts, including but not limited to sodium chloride (NaCl) or potassium chloride, sugars including but not limited to glucose, sucrose, trehalose or and any component from the group of amino acids, sugars, salts, and combinations thereof. In some embodiments, isotonicity agents are generaly used in a total amount of about 5 mM to about 350 mM. [0184] Non-limiting examples of salts include salts of any combinations of the cations sodium potassium, calcium or magnesium with anions chloride, phosphate, citrate, succinate, sulphate, or mixtures thereof. Non-limiting examples of amino acids comprise arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, or proline. Non-limiting examples of sugars according to the disclosure include trehalose, sucrose, mannitol, sorbitol, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, rafinose, glucosamine, N-methylglucosamine (also refered to as “meglumine”), galactosamine and neuraminic acid and combinations thereof. Non-limiting examples of stabilizer includes amino acids and sugars as described above as wel as commercialy available cyclodextrins and dextrans of any useful kind and molecular weight. Non-limiting examples of antioxidants include excipients such as methionine, benzylalcohol or any other excipient used to minimize oxidation. [0185] The phrase “pharmaceuticaly acceptable” refers to molecular entities and compositions that do not produce an alergic or similar untoward reaction when administered to a human. The preparation of an aqueous composition that contains a protein as an active ingredient is wel understood in the art. Typicaly, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. [0186] As used herein, “carrier” includes any and al solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, bufers, carrier solutions, suspensions, coloids, and the like. Except insofar as any media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. [0187] As used herein “pharmaceuticaly acceptable carrier” includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like Atorney Docket No.061479-511001WO that are physiologicaly compatible, including pharmaceuticaly acceptable cel culture media. In some embodiments, a composition comprising a carier is suitable for parenteral administration, e.g., intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration. Pharmaceuticaly acceptable carriers may include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. Except insofar as any media or agent is incompatible with the transduced cels, use thereof in the pharmaceutical compositions of the present disclosure is contemplated. [0188] The compositions may further comprise one or more polypeptides, polynucleotides, vector genomes comprising same, compounds that increase the transduction eficiency of vector genomes, formulated in pharmaceuticaly acceptable or physiologicaly-acceptable solutions for administration to a cel or an animal, either alone, or in combination with one or more other modalities of therapy. It wil also be understood that, if desired, the compositions of the present disclosure may be administered in combination with other agents as wel, such as, e.g., cytokines, growth factors, hormones, smal molecules, or various pharmaceuticaly active agents. There is virtualy no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely afect the ability of the composition to deliver the intended therapy. [0189] The present disclosure also provides pharmaceutical compositions comprising an expression cassete or vector (e.g., therapeutic vector) and one or more pharmaceuticaly acceptable cariers, diluents, or excipients. In some embodiments, the pharmaceutical composition comprises an immune cel engager and a lentiviral vector comprising an expression cassete disclosed herein, e.g., wherein the expression cassete comprises one or more polynucleotide sequences encoding one or more chimeric antigen receptor (CARs) and variants thereof. [0190] The pharmaceutical compositions that contain immune cel engagers may be in any form that is suitable for the selected mode of administration, for example, for intraventricular, intramyocardial, intracoronary, intravenous, intra-arterial, intra-renal, intraurethral, epidural, intrathecal, intraperitoneal, or intramuscular administration. The immune cel engager can be administered, as sole active agent, or in combination with other active agents, in a unit administration form, as a mixture with pharmaceutical supports, to animals and human beings. In some embodiments, the pharmaceutical composition comprises cels transduced ex vivo with immune cel engagers and any of the vector genomes according to the present disclosure. [0191] In some embodiments, the immune cel engagers, or a pharmaceutical composition comprising immune cel engagers, is effective when administered systemicaly. For example, the immune cel engagers of the disclosure, in some cases, may be administered intravenously to subject (e.g., a primate, such as a non-human primate or a human). In some embodiments, the immune cel engagers administered in combination with the viral vectors of the disclosure are capable of inducing Atorney Docket No.061479-511001WO expression of CARs in various immune cels when administered systemicaly (e.g., in T-cels, dendritic cels, NK cels). [0192] In various embodiments, the pharmaceutical compositions contain vehicles (e.g., cariers, diluents, and excipients) that are pharmaceuticaly acceptable for a formulation capable of being injected. Ilustrative excipients include a poloxamer. Formulation bufers for immune cel engagers may contain salts to prevent aggregation and other excipients (e.g., poloxamer) to reduce stickiness of the immune cel engagers. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especialy freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. In some embodiments, the formulation is stable for storage and use when frozen (e.g., at less than 0 °C, about -60 °C, or about -72 °C). In some embodiments, the formulation is a cryopreserved solution. [0193] The pharmaceutical compositions of the present disclosure, formulation of pharmaceuticaly acceptable excipients and carier solutions may be useful to those of skil in the art, such as for development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intraperitoneal, and intramuscular administration and formulation. [0194] In certain circumstances, it may be desirable to deliver the compositions disclosed herein parenteraly, intravenously, intramuscularly, or intraperitonealy, for example, in U.S. Pat. Nos. 5,543,158; 5,641,515 and 5,399,363 (each incorporated herein by reference in its entirety). Solutions of the active compounds as free base or pharmacologicaly acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcelulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. [0195] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No.5,466,468, incorporated herein by reference in its entirety). In al cases, the form should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Fluidity may be maintained, for example, by use of a coating, such as lecithin, by maintenance of a useful particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be facilitated by various antibacterial and antifungal agents, for Atorney Docket No.061479-511001WO example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In some embodiments, isotonic agents, for example, sugars or sodium chloride, are added. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. [0196] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if useful or necessary and the liquid diluent first rendered isotonic with suficient saline or glucose. These particular aqueous solutions are especialy suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In some embodiments, the solution intended for subcutaneous administration includes hyaluronidase. In this condition, a sterile aqueous medium that can be employed may be useful. One dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincot Wiliams & Wilkins, 2005). Some variation in dosage may occur depending on the condition of the subject being treated. The person responsible for administration wil, in any event, determine the appropriate dose for the individual subject. In some embodiments, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards set by the FDA Office of Biologics standards. [0197] In some embodiments, the present disclosure provides formulations or compositions suitable for the delivery of immune cel engagers and viral vector systems (e.g., viral- mediated transduction) including, but not limited to, retroviral (e.g., lentiviral) vectors. Gene Therapy Vectors [0198] Some embodiments include a composition that includes a gene therapy vector. The composition may further include an immune cel engager herein. The gene therapy vector may be included in a method herein, such as a method of treatment or use. The method may include use of a gene therapy vector with an immune cel engager. The method may include use of a particle with an immune cel engager, or administration of a gene therapy vector and an immune cel engager. A gene therapy vector may be included in or with a particle. [0199] In some embodiments, the immune cel engager is administered with a gene therapy vector. In some embodiments, the immune cel engager is administered before the gene therapy vector. In some embodiments, the immune cel engager is administered after the gene therapy vector. In some embodiments, the immune cel engager is co-administered with a gene therapy vector. [0200] As used herein “gene therapy vector” refers to gene therapy delivery vehicles, or cariers, that deliver therapeutic genes to cels. A gene therapy vector is any vector suitable for use in gene therapy, e.g., any vector suitable for the therapeutic delivery of nucleic acid polymers (encoding a polypeptide or a variant thereof) into target cels (e.g., sensory neurons) of a patient. In some embodiments, the gene therapy vector delivers the nucleic acid encoding a CAR. The vector may be Atorney Docket No.061479-511001WO of any type, for example it may be a plasmid vector or a minicircle DNA. Typicaly, the vector is a viral vector. These include both geneticaly disabled viruses such as adenovirus and nonviral vectors such as liposomes. The viral vector may for example be derived from an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus. AAV derived vectors. The vector may comprise an AAV genome or a derivative thereof. Particles [0201] Some embodiments include a composition that includes a particle. The composition may further include an immune cel engager herein. The particle may be included in a method herein, such as a method of treatment or use. The method may include use of a particle with an immune cel engager, or administration of a particle and an immune cel engager. A particle may include a vector such as a gene therapy vector. For example, a viral particle such as a lentiviral particle may be used as or include a gene therapy vector. [0202] In some embodiments, the disclosure provides particles of various types, including but not limited to lentiviral particles (e.g., a virion), lipid nanoparticles (LNPs), lipoplexes, liposomes, and nanocarriers. The particle may be loaded with an adhesion molecule, a costimulatory molecule, or a combination thereof. The particle may be loaded with the immune cel engager. Any of the adhesion molecule, costimulatory molecule, or activation molecules may be included in a fusion molecule. The adhesion molecule, costimulatory molecule, activation molecule or combination thereof may be loaded onto a surface of the particle. The fusion molecule may be loaded onto a surface of the particle. In some embodiments, the immune cel engager is administered with a particle. The particle may be co-administered with the immune cel engager to increase transduction. In some embodiments, the immune cel engager is administered at the same time as the particle. In some embodiments the immune cel engager is administered before the particle. In some embodiments the immune cel engager is administered after the particle [0203] The particle may be a lipid nanoparticle (LNP) or a poly(beta-amino) esters (PBAE) nanocarriers, both of which have been shown to transduce T cels when administered to a subject in vivo or contacted with T cels ex vivo. Using the compositions and methods described herein, transduction of T cels with LNPs and PBAE-based nanocariers may be increased. [0204] In some embodiments, the particle is a viral particle. Methods for generating viral vectors from various virus types are known in the art. Exemplary types of viral particles that may be recombinantly engineered as delivery vehicles include retroviruses, lentivirus (e.g. HIV and its derivatives and SIV), adeno-associated virus, adenovirus, MMLV retrovirus, MSCV retrovirus, baculovirus, vesicular stomatitis virus, herpes simplex virus, and vaccinia virus. Examples include adeno-associated virus (AAV) particles used for gene therapy. In a prefered embodiment, the particle is a retroviral particle. In a particularly prefered embodiment, the particle is a lentiviral particle. Atorney Docket No.061479-511001WO [0205] Lentiviral particles may be made using packaging cel lines as described in WO 2016/139463 or by using a polycistronic vector as described in Int’l Pat. Pub. No. WO 2020/106992 A1. Each of the foregoing specificaly describes methods for making lentiviral particles. Their disclosures are incorporated by reference herein. Numerous other methods for making viral particles, including lentiviral particles, may be useful. [0206] Retroviruses, a group that includes lentiviruses, are enveloped viruses. The immune cel engager described herein may be displayed on such enveloped viruses by linking the immune cel engager, or its various components, through covalent or non-covalent interactions. [0207] Lentiviral particles generaly package a vector genome and, incidentaly or intentionaly, may package other molecules present in the producer cel. The vector genome may be an artificial vector genome engineered to encode a heterologous protein or polynucleotide. [0208] Lentiviral particles may contain structural and/or functional genetic elements that are primarily from a virus. Lentiviral particles are characterized by the predominant source of genetic or structural material in the lentiviral particle. Thus, the term “retroviral particle” refers to a viral particle containing structural proteins and vector genome elements, primarily from a retrovirus. Likewise, the term “lentiviral particle” refers to a viral particle containing structural proteins and vector genome elements, primarily from a lentivirus. To package its vector genome, a lentiviral particle may generaly require at least one copy of the long-terminal repeats (LTRs) that flank a native lentiviral vector genome, or functional variants thereof. [0209] In some embodiments, a viral particle comprises a viral glycoprotein. In some embodiments, the viral particle comprises a viral glycoprotein different from the native viral glycoprotein. When the viral glycoprotein is heterologous to the vector genome, the viral particle is termed a “pseudotyped” viral particle. For example, in some embodiments, the viral particle is derived from HIV, which typicaly includes the glycoprotein gp120. However, such HIV-based particles may be “pseudotyped” and, instead of expressing their native glycoprotein, express a glycoprotein from a diferent virus. For example, the viral glycoprotein may be a portion of RD114 or one of its variants, VSV-G, Gibbon-ape leukemia virus (GALV), the Amphotropic envelope glycoprotein, Measles envelope glycoprotein, or baboon retroviral envelope glycoprotein. In some embodiments, the viral envelope glycoprotein is a G protein from the Cocal strain (Cocal G), or a functional variant thereof. Ilustrative viral glycoproteins include the VSV G protein, the Cocal G protein, and variants thereof. Ilustrative viral glycoproteins may be expressed as a single protein or in multiple subunits or parts. The viral glycoprotein may serve as ligand for cel-surface receptors on a target cel, and thereby promote transduction or the target cel. The viral glycoprotein may be engineered to lack LDLR binding affinity—for example, by mutation at positions 47 (e.g., K47Q) and/or 354 (e.g., R354A). This may be termed a “blinded” viral glycoprotein. Ilustrative envelope variants are provided in, e.g., US 2020/0216502 A1, which is incorporated herein by reference in its entirety. Surprisingly, in some embodiments, the immune cel engager as described herein may permit use of a viral Atorney Docket No.061479-511001WO glycoprotein that does not, by itself, cause transduction of target cel. Without being bound by theory, it is believed that the immune cel engager may serve as a ligand for cel-surface receptor while the viral glycoprotein retains a structural function, but not a function as a ligand for a cel- surface receptor. [0210] In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 47. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a mutation at position 354. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a R354A mutation. In some embodiments, the viral glycoprotein is a VSV-G glycoprotein that comprises a K47Q and a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 47. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a mutation at position 354. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a K47Q mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a R354A mutation. In some embodiments, the viral glycoprotein is a cocal glycoprotein that comprises a K47Q and a R354A mutation. [0211] In some embodiments, the viral glycoprotein comprises a mutation at position 47. In some embodiments, the viral glycoprotein comprises a mutation at position 354. In some embodiments, the viral glycoprotein comprises a K47Q mutation. In some embodiments, the viral glycoprotein comprises a R354A mutation. In some embodiments, the viral glycoprotein comprises a K47Q and a R354A mutation. [0212] The Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the folowing sequence: NFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTHK AIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCGY ATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDATL VDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDVYA AAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGTGP AFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKFPL FMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTVVT FFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 74). [0213] The Cocal G protein may have a polypeptide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity to the folowing sequence: MNFLLLTFIVLPLCSHAKFSIVFPQSQKGNWKNVPSSYHYCPSSSDQNWHNDLLGITMKVKMPKTH KAIQADGWMCHAAKWITTCDFRWYGPKYITHSIHSIQPTSEQCKESIKQTKQGTWMSPGFPPQNCG YATVTDSVAVVVQATPHHVLVDEYTGEWIDSQFPNGKCETEECETVHNSTVWYSDYKVTGLCDA TLVDTEITFFSEDGKKESIGKPNTGYRSNYFAYEKGDKVCKMNYCKHAGVRLPSGVWFEFVDQDV YAAAKLPECPVGATISAPTQTSVDVSLILDVERILDYSLCQETWSKIRSKQPVSPVDLSYLAPKNPGT GPAFTIINGTLKYFETRYIRIDIDNPIISKMVGKISGSQTERELWTEWFPYEGVEIGPNGILKTPTGYKF Atorney Docket No.061479-511001WO PLFMIGHGMLDSDLHKTSQAEVFEHPHLAEAPKQLPEEETLFFGDTGISKNPVELIEGWFSSWKSTV VTFFFAIGVFILLYVVARIVIAVRYRYQGSNNKRIYNDIEMSRFRK (SEQ ID NO: 247) [0214] Ilustrative lentiviruses include but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2; visna-maedi virus (VMV) virus; the caprine arthritis- encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, the backbones are HIV-based vector backbones (e.g., HIV cis-acting sequence elements). Retroviral particles have been generated by multiply atenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector biologicaly safe. [0215] Ilustrative lentiviral particles and methods for making them are described in Naldini et al. Science 272:263-7 (1996); Zuferey et al. J. Virol.72:9873-9880 (1998); Dul et al. J. Virol. 72:8463-8471 (1998); Miyoshi et al. J. Virol.72:8150-57 (1998); U.S. Pat. No.6,013,516; and U.S. Pat. No.5,994,136. [0216] Protocols for producing replication-defective recombinant viruses are provided in W095/14785, W096/22378, U.S. Pat. No.5,882,877, U.S. Pat. No.6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No.5,278,056, and W094/19478. [0217] Viral particles may be assessed in various ways, including, for example, measuring the vector copy number (VCN) or vector genomes (vg) in a sample of viral particle by quantitative polymerase chain reaction (qPCR) or digital droplet PCR (ddPCR), or testing to the viral particles on target cels to measure a “titer” of the virus in, e.g., infectious units per mililiter (IU/mL). For example, the titer may be assessed using a functional assay performed on the cultured tumor cel line HT1080 as described in Humbert et al. Molecular Therapy 24:1237–1246 (2016). When titer is assessed on a cultured cel line that is continualy dividing, stimulation may be unnecessary, and hence the measured titer may be uninfluenced by surface engineering of the retroviral particle. Other methods for assessing the eficiency of retroviral vector systems are provided in Gaererts et al. BMC Biotechnol.6:34 (2006). Methods [0218] Provided herein are methods of use. The method of use may be with regard to an immune cel engager, or a composition or formulation. A method may include immune cel manufacturing, virus manufacturing, or nanoparticle manufacturing. A method may include an in vivo method, an in vitro method, or an ex vivo method. A method may be performed in vivo or with a subject, may be performed in vitro, or may be performed ex vivo. A method may include delivery of exogenous proteins, or may include use of a gene therapy vector. [0219] Immune cel engagers of the present disclosure may enhance particle to T cel binding. Immune cel engagers of the present disclosure may enhance T cel activation. Immune cel engagers Atorney Docket No.061479-511001WO of the present disclosure may enhance immune cel expansion. Immune cel engagers of the present disclosure may enhance immune cel transduction. Cel Manufacturing [0220] Disclosed herein, in some embodiments, are methods for use in immune cel manufacturing. An immune cel manufacturing method may include an ex vivo method such as an ex vivo manufacturing method. In some embodiments, the immune cel engager can be used for immune cel manufacturing. In some embodiments, the immune cel engager can be used for activation of immune cels during immune cel manufacturing. In some embodiments, the immune cel engager can be used for expansion of immune cels during immune cel manufacturing. In some embodiments, immune cel manufacturing includes engineering immune cels (e.g. T cels and/or NK cels) to express one or more non-endogenous proteins. [0221] Engagement of CD3 by an immune cel engager comprising a binding domain targeting CD3 may cause the T cels to activate via a primary activation signal. Incorporation of one or more ligands for a co-receptor, such as CD28, a molecule expressed by T cels, may cause the T cels to activate via a secondary activation signal. Activation of a T cel, via the primary and optionaly secondary activation signals, may make them more proliferative and susceptible to transduction. More specificaly, without being bound by theory, contacting an immune cel to be engineered with an immune cel engager comprising an adhesion molecule, a co-stimulation molecule, and optionaly an immune cel binding domain (such as an anti-CD3 antibody fragment) may generate a macromolecular complex on the surface of the immune cel that may replicate signaling caused by a supramolecular activation cluster (SMAC). By engaging multiple proteins typicaly found within the SMAC on the surface of the T cel, the immune cel engager can induce increased T cel activation and proliferation. In addition to performing adhesion functions, engagement of CD2 by CD58 provides an additional signal that may enhance activation and effector functions. Other immune cel engagers may comprise the binding regions of other proteins commonly found in the supramolecular activation complex (SMAC) between T lymphocytes and antigen presenting cels. For example, CD3, CD2, CD4, CD8, CD28, LFA-1, CD45, CD43, CD40, ICAM-1, CTLA-4, CD80, CD86, MHC, LFA-3, AND CD40L are proteins that may be present within the SMAC. [0222] In some embodiments, the immune cel engager can be used for concomitant selection, activation, and expansion of T cels in a one step process. Selection, activation, and expansion of T cels during preparation of T cels for immunotherapy can be limited by three things: 1) generating immune cels with desired characteristics, 2) scale of operation needed to generate T cels for immunotherapy and 3) resource availability. The immune cel engager describe herein alows for the generation of immune cels with desired characteristics through switching domains in the fusion protein. For example, the CD58 binding domain can be replaced with a CD16 binding domain to activate NK cels. The production of the immune cel engager can be scaled to activate large Atorney Docket No.061479-511001WO numbers of immune cels and the cel culture material needed to make immune cel engager are readily available. Thus, the immune cel engager described herein can be used for large scale concomitant selection, activation, and expansion of T cels in a one step process. [0223] In some aspects, the disclosure provides a method of delivering a nucleic acid to a cel ex vivo. In some embodiments, the disclosure provides a method of delivering a nucleic acid to an immune cel ex vivo. In some embodiments, the immune cel engagers of the disclosure activate and increase transduction of an immune cel ex vivo. [0224] In some embodiments, the disclosure provides a method of delivering an immune cel engager and a nucleic acid to a cel in an ex vivo CAR T manufacturing process. Such methods typicaly involve the isolation of peripheral blood mononuclear cels (PBMCs) from a patient via leukapheresis. In some embodiments, such methods involve obtaining whole blood from a patient without isolation of PBMCs and forward processing the whole blood. The PBMCs may be washed and optionaly further purified via one or more selection steps to isolate particular T cel populations of interest. In some aspects, these might include CD4+ and/or CD8+ T cels. [0225] The washed and/or purified cels may be optionaly activated and then transduced using an immune cel engager and a lentiviral vector. The activation step may comprise contacting the cels with an exogenous activation agent such as the immune cel engagers described herein, anti-CD3 and anti-CD28 antibodies bound to a substrate or using unbound antibodies. [0226] In some embodiments, the immune engagers described herein may be immobilized on a substrate – e.g. a resin, bead, polymer, expamer, or other substrate described herein – and used to isolate and activate target cels simultaneously. For example, an immune engager as described herein may be useful to engage with (e.g. bind and isolate) a T cel via the CD3 binder present in the engager as wel as activate the T cel via the CD3 binder and costimulatory and adhesion domains. This isolation and activation step may be performed in a column or other purification device, or in solution such that the bound cels and bound immune engagers are able to be removed from the unbound cels and other materials. Thus, as part of an ex vivo manufacturing process, an immune cel engager as described herein may eliminate one or more separate unit operations traditionaly present in ex vivo immune cel manufacturing. Additionaly, the immune cel engagers described herein need not be immobilized on a substrate and instead may be provided in soluble form to activate the target immune cels. In such embodiments, activation and transduction may be accomplished via separate steps, or in the same step, as described below. [0227] Transduction may be accomplished by contacting the patient’s PBMCs, isolated cels, or, in some cases, whole blood with immune cel engagers and/or lentiviral particles described herein. After transduction, the cels may be optionaly further washed and cultured until harvest. Methods of manufacturing engineered cel therapies, including CAR T cels (see e.g., Abou-el-Enein, M. et al. Blood Cancer Discov (2021), Vol 2(5): 408-422; Arcangeli, S. et al. Front. Immunol (19 Jun 2020), Vol.11 (1217) 1-13; Ghassemi, S. et al. Nat Biomed Eng (Feb 2022), Vol 6(2): 118-128; Atorney Docket No.061479-511001WO Vormitag, P. et al. Curr Opin Biotechnol (Oct 2018), Vol.54: 164-181; each of which is herein incorporated by reference), may be useful. Ilustrative methods of autologous CAR T manufacturing are disclosed in US Patent Publication nos. US 2019/0269727, US 2016/0122782, US 2021/0163893, and US 2017/0037369, each of which is incorporated herein in its entirety. [0228] It is contemplated that the present disclosure provides an ex vivo method of generating an engineered cel comprising contacting a target cel with a particle and an immune cel engager comprising an adhesion molecule linked to a costimulatory molecule, immune cel engager comprising an adhesion molecule linked to an immune cel binding domain, or an immune cel engager comprising an adhesion molecule linked to a costimulatory molecule and an immune cel binding domain wherein the contacting step is performed for approximately one hour, for approximately two hours, approximately three hours, approximately four hours, approximately five hours, approximately six hours, approximately 12 hours, approximately 24 hours, approximately 12- 24 hours (inclusive of endpoints), or longer. This method may require the contacting step to be performed in a closed-loop manufacturing or extracorporeal process as described herein. Alternatively, this method may require the contacting step to be performed in a traditional ex-vivo engineered cel manufacturing process. For example, in a perfusion incubator or a centrifuge (such as a SepaxTM or RoteaTM machine). [0229] The compositions described herein such as immune cel engagers described may be used in vitro or ex vivo. The immune cel engagers described may be used ex vivo, in a cel manufacturing process or at a bedside as described, e.g., in Int’l Pat. Pub. No. WO 2022/072885, Int’l Pat. Pub. No. 2019/217954, Int’l Pat. Pub. No.2020/123649, and Int’l Pat. Pub. No.2009/072003. In some embodiments, the disclosure provides an ex vivo method of transducing target cels, comprising contacting the target cels with the immune cel engager and a delivery vehicle (such as viral particle according to the present disclosure, a lipid nanoparticle, or other means of delivering a polynucleotide to a cel). In some embodiments, the immune cel engager described herein may be used to increase transduction of cels that have not been previously activated. For example, the immune cel engager described herein may be useful for increasing transduction of cels that have not been previously contacted with cel activation beads or activation reagents (e.g., DynabeadsTM or other reagents comprising anti-CD3 and/or anti-CD28 antibodies or binding fragments thereof). Where a method herein describes use of a lentiviral particle with immune cel engager, use of another particle is contemplated where appropriate and feasible. Where a method herein describes use of an immune cel engager with a lentiviral particle, use of a composition or fusion molecule is also contemplated where appropriate and feasible. For example, an immune cel engager, bound to the surface of a lentiviral particle, or a pharmaceutical composition may be administered to or contacted with a cel such as an immune cel (e.g., T cel). Atorney Docket No.061479-511001WO [0230] Non-limiting examples of cels that can be the target of the immune cel engager described herein include T lymphocytes, dendritic cels (DC), Treg cels, B cels, Natural Kiler cels, and macrophages. In Vivo Methods [0231] In some embodiments, an immune cel engager described herein is used to enhance transduction of a nucleic acid sequence (polynucleotide) encoding one or more exogenous proteins into a cel (e.g., a T lymphocyte, NK cel, or other target cel) in vivo. In some embodiments, the transduction of the lentiviral particle results in expression of one or more exogenous proteins in the transduced cels. In some embodiments, the immune cel expressing the exogenous protein is generated by co-administration of the immune cel engager described herein and a particle comprising a nucleic acid encoding the exogenous protein. In some embodiments, the particle is a virus particle. In some embodiments, the particle is a lentivirus particle. In some embodiments, the particle is a nanoparticle. In some embodiments, the particle is a lipid nanoparticle. In some embodiments the immune cel engager and particle are administered to a subject simultaneously. In some embodiments, the immune cel engager is administered to a subject prior to the particle. In some embodiments, the immune cel engager is administered to a subject after the particle. [0232] An exemplary exogenous protein is a chimeric antigen receptor (CAR). CARs are artificial membrane-bound proteins that direct a T lymphocyte to an antigen and stimulate the T lymphocyte to kil cels displaying the antigen. See, e.g., Eshhar, U.S. Pat. No.7,741,465. Generaly, CARs are geneticaly engineered receptors comprising an extracelular domain that binds to an antigen, e.g., an antigen on a cel, an optional linker, a transmembrane domain, and an intracelular (cytoplasmic) domain comprising a costimulatory domain and/or a signaling domain that transmits an activation signal to an immune cel. With a CAR, a single receptor can be programmed to both recognize a specific antigen and, when bound to that antigen, activate the immune cel to atack and destroy the cel bearing that antigen. When these antigens exist on tumor cels, an immune cel that expresses the CAR can target and kil the tumor cel. Al other conditions being satisfied, when a CAR is expressed on the surface of, e.g., a T lymphocyte, and the extracelular domain of the CAR binds to an antigen, the intracelular signaling domain transmits a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cel surface, to kil the cel expressing the antigen. Because T lymphocytes may require two signals, a primary activation signal and a costimulatory signal, in order to maximaly activate, CARs can comprise a stimulatory and a costimulatory domain such that binding of the antigen to the extracelular domain results in transmission of both a primary activation signal and a costimulatory signal. Ilustrative CARs may be designed in a modular fashion, e.g., as described in (see, e.g., Guedan S, Calderon H, Posey AD, Maus MV, Molecular Therapy - Methods & Clinical Development.2019; 12: 145-156), incorporated by reference. Atorney Docket No.061479-511001WO [0233] In some embodiments, the immune cel engager herein activates target cels in vivo. In some embodiments, the target cels are immune cels. In some embodiments, the immune cels are T cels. In some embodiments, the immune cels are NK cels. In some embodiments, the immune cel engager described herein increase transduction T cels in vivo. In some embodiments, the immune cel engager and a viral particle comprising a polynucleotide encoding a CAR described herein transduce T cels in vivo generating CAR T cels. In some embodiments, the immune cel engager described herein comprise a CD58-CD80-anti-CD3 scFv tri-fusion polypeptide and increase transduction of T cels in vivo generating CAR T cels. Where a method herein describes use of an immune cel engager, use of another fusion protein is contemplated where feasible. [0234] In some embodiments, the immune cel engagers are administered via a route selected from the group consisting of extracorporeal, parenteral, intravenous, intramuscular, subcutaneous, intratumoral, intraperitoneal, and intralymphatic. In some embodiments, the immune cel engagers are administered multiple times. In some embodiments, the immune cel engagers are administered by intralymphatic injection of the viral particle. In some embodiments, the immune cel engagers are administered by intraperitoneal injection of the macromolecular complexes. In some embodiments, the immune cel engagers are administered by intra-nodal injection – that is, the immune cel engagers may be administered via injection into one or more lymph nodes. In some embodiments, the lymph nodes for administration are the inguinal lymph nodes. In some embodiments, the immune cel engagers are administered by injection of the immune cel engagers into tumor sites (e.g., intratumoral). In some embodiments, the immune cel engagers are administered subcutaneously. In some embodiments, the immune cel engagers are administered systemicaly. In some embodiments, the immune cel engagers are administered intravenously. In some embodiments, the immune cel engagers are administered intra-arterialy. In some embodiments, the immune cel engagers are co-administered with a viral particle. [0235] In some embodiments, the immune cel engagers are administered by intraperitoneal, subcutaneous, or intranodal injection. In some embodiments, the immune cel engagers are administered by intraperitoneal injection. In some embodiments, the immune cel engagers are administered by subcutaneous injection. In some embodiments, the immune cel engagers are administered by intranodal injection. [0236] In some embodiments, the transduced immune cels comprising a polynucleotide of the present disclosure are administered to the subject. [0237] The disclosure provides a method of treating a condition in a subject comprising administering to the subject the immune cel engager or the pharmaceutical composition of the disclosure. The disclosure provides a method of treating a malignancy in a subject, comprising administering to the subject the immune cel engagers and a viral particle comprising a nucleic acid encoding a CAR or pharmaceutical composition of the disclosure. In some embodiments, the malignancy is a B-cel malignancy, a myeloma, or a solid tumor malignancy. The disclosure Atorney Docket No.061479-511001WO provides a method of treating diffuse large B-cel lymphoma (DLBCL), Burkit’s type large B-cel lymphoma (B-LBL), folicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cel lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cel carcinoma, leukemia, myeloma, B cel lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, meduloblastoma, or sarcoma in a subject, comprising administering to the subject the immune cel engager and viral particles or pharmaceutical composition of the disclosure. [0238] Various disease or disorders may be treated using an immune cel engager and particles comprising a nucleic acid encoding a CAR as disclosed herein, or pharmaceutical composition comprising them. The immune cel engager and particles may be administered to a subject sufering from or at risk for a B-cel malignancy, relapsed/refractory malignancy, difuse large B-cel lymphoma (DLBCL), Burkit’s type large B-cel lymphoma (B-LBL), folicular lymphoma (FL), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), mantle cel lymphoma (MCL), hematological malignancy, colon cancer, lung cancer, liver cancer, breast cancer, renal cancer, prostate cancer, ovarian cancer, skin cancer, melanoma, bone cancer, brain cancer, squamous cel carcinoma, leukemia, myeloma, B cel lymphoma, kidney cancer, uterine cancer, adenocarcinoma, pancreatic cancer, chronic myelogenous leukemia, glioblastoma, neuroblastoma, meduloblastoma, or sarcoma. [0239] In some embodiments, the condition is an autoimmune disease or disorder. The disclosure also provides a method of treating an autoimmune disease in a subject, comprising administering to the subject immune cel engager and viral particles or pharmaceutical composition of the disclosure. Some examples of an autoimmune disease may include systemic lupus erythematosus, Sjögren’s Syndrome, ANCA-associated vasculitis and autoimmune hemolytic anemia, rheumatoid arthritis, systemic sclerosis, multiple sclerosis, neuromyelitis optica spectrum disorder, chronic inflammatory demyelinating polyradiculoneuropathy, immune-mediated necrotizing myopathy, pemphigus vulgaris, dermatomyositis, adult-onset Stil’s disease, inflammatory bowel disease, type 1 diabetes melitus, graft vs. host disease, a myasthenia gravis, multiple sclerosis, Immune dysregulation, Polyendocrinopathy Enteropathy X-linked (IPEX) or autoimmune arthritis. [0240] In some embodiments, the immune cel engager disclosed herein may be used with viral particles in a method to generate engineered cels in vivo. In some embodiments, the lentiviral particles incorporating the immune cel engager (e.g., a fusion protein as disclosed herein) may preferentialy generate engineered central memory T cels (TCM). In some embodiments, administering lentiviral particles via one or more lymph nodes may contribute to generation of a predominately TCM engineered cel phenotype. TCM may be characterized by expression of certain surface markers, for example, TCM may be CD62L+. TCM may also be CCR7+. TCM may also be Atorney Docket No.061479-511001WO characterized as CD45RA-, CD45RO+, and/or CD27+. In some embodiments, TCM are characterized as CCR7+, CD45RA-, CD45RO+, CD62L+, and CD27+. In some embodiments, TCM are characterized as CD45RA-, CCR7+. Without wishing to be bound by theory, it is contemplated that engineered TCM may persist for a longer time in vivo and may show improved effector function compared with engineered effector memory (TEM) or similar efector cel types. [0241] Thus, it is contemplated that the present disclosure further provides a method of generating predominately engineered TCM in vivo. Similar observations may be found in the ex-vivo seting, so the present disclosure further provides a method of generating predominately engineered TCM ex vivo, using one or more of the methods disclosed here. For example, via extracorporeal delivery or traditional ex-vivo manufacturing. [0242] Disclosed herein, in some embodiments, are methods of treatment. The method of treatment may include administering an engager herein to a subject in need of treatment for a disease. The method of treatment may include administering a pharmaceutical composition (e.g. comprising an engager) to a subject in need of treatment for a disease. In some embodiments, the disease comprises cancer. In some embodiments, the disease comprises an autoimmune disease. Making Immune Cel Engagers [0243] Some embodiments include a method of making an immune cel engager. Such methods may include an aspect shown in FIG.4. [0244] In some embodiments, the disclosure provides a method of making an immune cel engager, comprising introducing a polynucleotide encoding an immune cel engager into a host cel comprising a polynucleotide encoding an immune cel engager (or fusion protein) as described herein. The immune cel engager (or fusion protein) is expressed by the host cel. [0245] In some embodiments, the disclosure provides an in vivo method of transducing target cels in a subject in need thereof, comprising administering to the subject an immune cel engager and a particle or pharmaceutical composition of the disclosure. The immune cel engager and particle may be administered by intranodal, intravenous, or subcutaneous injection. [0246] Some embodiments include a method of making an adhesion molecule, a costimulatory molecule, an immune cel binding domain, or a fusion protein (e.g., immune cel engager). The method may include transcribing or translating a nucleic acid (such as a DNA or RNA) that encodes a protein comprising the adhesion molecule, costimulatory molecule, immune cel binding domain, or fusion molecule (e.g., immune cel engager). Virus or Nanoparticle Manufacturing [0247] Disclosed herein, in some embodiments, are methods for use in virus manufacturing or nanoparticle manufacturing. A virus may include a lentivirus. Disclosed herein, in some embodiments, are methods for use in lentivirus manufacturing. In some embodiments, the immune Atorney Docket No.061479-511001WO cel engager can be used for quantifying the purity of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus. In some embodiments, the immune cel engager can be used for determining the concentration of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus. In some embodiments, the immune cel engager can be used for determining activity of a lentivirus preparation that incorporates the fusion protein onto the surface of the virus. [0248] The immune cel engager described presently can also be incorporated onto the surface of viral particles. The immune cel engager can also be caled a fusion protein when incorporated onto the surface of viral particles. One chalenge when working with viral particles is determining an accurate concentration of viral particles present in each production lot. Viral concentration can be measured by standard molecular techniques including qPCR, PCR, ELISA, functional readouts (expression of reporter protein) and protein concentration. Commercialy available standards can be used for quantification of viral protein concentration, but the comparison of different sized proteins may cause the inaccurate quantification of the protein. The immune cel engager described herein may be used to generate a standard curve of protein concentrations that can be used to determine the concentration of viral particles that incorporate the fusion protein on to their surface. The immune cel engager described herein may be used to determine the purity of viral particles that incorporate the fusion protein on their surface. Immune cel engagers described herein may be used to determine functional activity of viral particles that incorporate the fusion protein on to their surface. [0249] In some embodiments, the immune cel engager described herein can be used to purify viral particles that incorporate the fusion protein on to their surface. In some embodiments, the immune cel engager described herein can be loaded on to a solid support. In some embodiments, the solid support is a resin. In some embodiments, the resin is a chromatography resin. In some embodiments, the resin loaded with the immune cel engager can be used to purify lentivirus that have the fusion protein incorporated onto their surface. [0250] In some embodiments, the immune cel engager described herein can be used as a reagent to screen new lentivirus that have incorporated the fusion protein onto their surface. The screen may alow for the testing of other adhesion molecules, costimulatory molecules, or immune cel binding domains that have been incorporated into the fusion protein on the particle surface. The screen may alow for the testing of new orientations of the diferent domains in the fusion protein. Kits [0251] Disclosed herein, in some embodiments, are kits. In some embodiments, the kit includes an adhesion molecule. In some embodiments, the kit includes a costimulatory molecule. In some embodiments, the kit includes an immune cel binding domain. In some embodiments, the kit includes a fusion molecule. In some embodiments, the kit includes an immune cel engager. In some embodiments, the kit includes a particle. In some embodiments, the kit includes a composition Atorney Docket No.061479-511001WO described herein. The kit may include instructions for use, such as instructions for use in a method herein. [0252] In some embodiments, the disclosure provides a kit comprising the immune cel engager and instructions for use in treatment of a subject. The kit may include a pharmaceuticaly acceptable carier and/or an injection device. The kit may further include suitable tubing for administering the immune cel engager. Definitions [0253] Unless defined otherwise, al terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skil in the art to which the claimed subject mater pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial diference over what is generaly understood in the art. [0254] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specificaly disclosed al the possible subranges as wel as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specificaly disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as wel as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0255] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof. [0256] As used herein, “and/or” refers to and encompasses any and al possible combinations of one or more of the associated listed items, as wel as the lack of combinations when interpreted in the alternative (or). [0257] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” may be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context. [0258] A sequence herein may include a conservative amino acid substitution. A “conservative amino acid substitution” may refer to a substitution of an amino acid residue with another amino acid residue without abolishing a protein's desired properties. Such a substitution may be with an Atorney Docket No.061479-511001WO amino acid of a same amino acid class. Some examples of an amino acid class include amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, or tyrosine; amino acids having basic side chains, such as lysine, arginine, or histidine; amino acids having acidic side chains, such as asparatic acid or glutamic acid; or amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan or cysteine. A conservative amino acid substitution may include substitution with a chemicaly derivatized residue or non-natural amino acid that does not abolish a protein's desired properties. [0259] A percent sequence identity may be determined by comparing two optimaly aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the % sequence identity. A sequence identity may include a sequence identity to a reverse complement. [0260] In determining a sequence identity, thymine (T) and uracil (U) may be interchangeable. T and U may be interchangeable when describing an oligonucleotide. In some embodiments, Ts and Us are interchangeable depending on whether the oligonucleotide is an RNA or DNA, where RNA includes U and DNA includes T. [0261] Any discrepancies between the writen description and a sequence listing submited herein may typicaly be resolved in favor of the writen description. [0262] The terms “subject,” “individual,” or “patient” may be used interchangeably herein. A subject can be an animal. A subject can be a mammal. A subject can be a primate. The subject can be a human. A subject may be healthy. A subject may have a disease. A subject may be diagnosed or suspected of being at high risk for a disease. [0263] As used herein, the term “about” a number refers to that number plus or minus 15% of that number. The term “about” a range refers to that range minus 15% of its lowest value and plus 15% of its greatest value. [0264] “Administering” or “administer” may include introducing a compound or a composition into a subject. The term may encompasses external or internal administration. Internal administration can refer to, for example, where a molecule is generated inside the subject’s body. For example, administering an RNA may include administering a plasmid DNA encoding the RNA, or a vehicle comprising said plasmid DNA, upon which the RNA may be expressed from the plasmid DNA in a cel of the subject. [0265] The terms “treatment” or “treating” may be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired Atorney Docket No.061479-511001WO results may include a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. A therapeutic benefit may be achieved by eradication or amelioration of one or more of the physiological symptoms associated with an underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may stil be aflicted with the underlying disorder. A prophylactic efect may includes delaying, preventing, or eliminating appearance of a disease or condition, delaying or eliminating onset of a symptom, slowing, halting, or reversing progression of a disease or condition, or a combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made. [0266] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject mater described. Specificaly, features described in one section may be combined with features in any other section of the description. EXAMPLES [0267] The folowing Examples describe how embodiments of the invention may be made, evaluated, and used. The Examples are intended to be ilustrative and non-limiting. Example 1. Generation of soluble MDF1 and MDF2 [0268] This example describes generation, production, and purification of example immune cel engagers referred to as multi-domain fusion 1 (MDF1) and multi-domain fusion 2 (MDF2). MDF1 has the arangement of CD58-CD3scFv-CD80 and MDF2 has the arrangement of CD3scFv-CD58- CD80. [0269] FIG.4 summarizes example production, purification, and analysis workflows for generation of soluble MDFs (sMDFs). Briefly, the transmembrane/cytosolic domain was replaced with an IgG1 Fc domain or 8XHistag (monomer MDF1) (FIG.5). The sMDF proteins were purified from the supernatant of lentiviral vector producing cel line 293T after the transfection of sMDF encoding plasmids. Production of the sMDF in 293T cels alows for structural and post-translational modifications (e.g., glycosylation) on the sMDF (FIG 6). Dimer sMDFs were purified by protein A column and monomer sMDF was purified by HisTag column (FIG.7-8). Dimer forms of sMDFs were confirmed in the comparison of reducing/non-reducing gel (FIG.9). Example 2. T cel activation bioassay (NFAT activation assay) [0270] This example shows activation of T cels by example immune cel engagers. This example shows that a dimer form of sMDF was more potent at activating T cels than a similar monomer Atorney Docket No.061479-511001WO form. This example also shows sMDF2 was more potent at activating T cels than sMDF1. The T cel NFAT activation assay was used to test the ability of monomer and dimer sMDFs to activate T cels. T cels were treated with purified monomer or dimer sMDFs in the T cel activation assay. Dimer sMDF2 reached an activation plateau level after 1 ng, while dimer sMDF1 showed a similar level of activation at 100 ng. This shows the high potency of dimer sMDF2 compared to dimer sMDF1 in terms of T cel activation. Additionaly, the monomer form of sMDF1 didn’t show the activation by 100 ng, which shows that the T cel activation is done through the interaction of the dimer form of sMDFs with T cels (FIG.10). Example 3. Application of purified MDFs for the quantification of incorporated MDF on particles [0271] This example shows that purified sMDFs can be used as a standard for quantification of particles that incorporate the MDF. Purified sMDFs were applied as a standard material for the quantification of the MDF level in a particle (FIG.11). Commercialy available CD58 protein (smaler size than MDF) can be used as a standard for quantification, however the comparison of diferent sized protein may cause the inaccurate quantification of the protein. The standard curve from using the sMDF generated a R2 of 0.9485. This ilustrates that purified sMDF can be used for the quantification of the protein in MDF containing particles. Example 4. T cel activation test in PBMCs [0272] This example shows activation of primary T cels with example immune cel engagers. This example shows that dimerized versions of the sMDFs were more potent at stimulating primary T cel activation than monomer forms of the sMDFs. The purified sMDFs (monomer and dimers) were tested on primary human PBMCs (FIG.12).1 milion human PBMCs were incubated with diferent amounts of sMDFs, and the activation was evaluated by the expression of CD25. In both CD4+ and CD8+ cels, sMDF2-treated cels showed higher activation compared to sMDF1-treated cels. Quantification of percentage of CD25 positive cels (marker of activation) at day 3 and day 7 (post sMDF treatment) found a similar trend of activation profile as above, which is, higher potency of sMDF2 than sMDF1. Almost no T cel activation from monomeric sMDF1 was noted (FIGs.12- 14). Example 5. PBMC binding assay [0273] This example shows immune cel binding affinities of example immune cel engagers. This example shows that the higher T cel activation associated with MDF2 was due to higher binding affinity of MDF2. To test whether there was a binding affinity difference between sMDF1 and sMDF2, primary human PBMCs were incubated with sMDF1 or sMDF2 for 1.5 hrs and then washed. The amount of bound sMDF1 or sMDF2 was assessed by FACS using an Fc antibody. Atorney Docket No.061479-511001WO sMDF2 showed higher binding affinity than sMDF1, which is consistent with the previous T cel activation results and showed the higher activation was due to the higher binding affinity of MDF2 (FIG.15). Example 6. Immobilization of soluble macromolecular complex to the solid phase [0274] This example wil show the use of the soluble immune cel engager immobilized to a solid support can be used to activate T cels. Currently, commercial DynabeadTM human T-cel activators have been widely used for the T cel activation/proliferation or purification. The beads are paramagnetic spherical polymer particles with the adsorption or coupling of CD3 and CD28 antibody. The solid phase material such as beads provide similar environment of in vivo T cel activation like dendritic cels. Therefore, the solid phase form of antibody or activators are generaly used for the T cel activation. Thus, the immune cel engager described herein may be used as a new T cel activation reagent. * * * [0275] Al publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specificaly and individualy indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world. [0276] While ilustrative embodiments have been described and depicted, it wil be appreciated that various changes can be made to these ilustrative embodiments without departing from the spirit and scope of the invention.

Claims

Atorney Docket No.061479-511001WO CLAIMS 1. An immune cel engager for activating a target immune cel, comprising: a cel binding domain that specificaly binds an antigen on the target immune cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of the target immune cel, wherein concurent binding of the cel binding domain and the immune cel adhesion molecule to the target immune cel forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, and wherein the immune cel engager is soluble. 2. The engager of claim 1, further comprising a multimerization domain. 3. The engager of claim 2, wherein the multimerization domain comprises a dimerization domain. 4. The engager of claim 3, wherein the engager is part of a homodimer that includes a second immune cel engager comprising a second dimerization domain bound to the dimerization domain. 5. The engager of claim 1, wherein the target immune cel is a T cel. 6. The engager of claim 5, wherein the antigen comprises CD3. 7. The engager of claim 6, wherein the cel binding domain comprises complementarity determining regions of an anti-CD3 antibody. 8. The engager of claim 5, wherein the reciprocal adhesion molecule comprises CD2. 9. The engager of claim 8, wherein the immune cel adhesion molecule comprises CD58, ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, JAM-A, CD155 or CD112, or a functional fragment thereof. 10. The engager of claim 8, wherein the immune cel adhesion molecule comprises a CD2-binding fragment of a CD58 protein. 11. The engager of claim 1, further comprising an immune cel costimulatory molecule that provides a costimulatory signal to the target immune cel. 12. The engager of claim 11, wherein the immune cel costimulatory molecule comprises a binding domain that specificaly binds CD28. 13. The engager of claim 11, wherein the immune cel costimulatory molecule comprises CD80, CD86, CD40L, GITRL, OX40L, 41BBL, ICOSL, CD27, CD30L, LIGHT, LTalpha, MICA, or MICB, or a functional fragment thereof. 14. The engager of claim 12, wherein the immune cel costimulatory molecule comprises a CD28-binding fragment of a CD80 protein. 15. An immune cel engager comprising a fusion protein that comprises: an antigen-binding domain of an anti-CD3 antibody; a CD58 extracelular domain; and a CD80 extracelular domain or a CD86 extracelular domain, Atorney Docket No.061479-511001WO wherein the fusion protein is soluble and forms an artificial supramolecular adhesion complex with a T cel. 16. The engager of claim 15, wherein the antigen-binding domain of the anti-CD3 antibody comprises an anti-CD3 single-chain variable fragment (CD3scFv). 17. The engager of claim 15, wherein the fusion protein further comprises a first dimerization domain that binds with a second dimerization domain of a second fusion protein comprising a second antigen-binding domain of an anti-CD3 antibody, a second CD58 extracelular domain, and a second CD80 extracelular domain or CD86 extracelular domain. 18. The engager of claim 17, wherein the first and second dimerization domains comprises a crystalization fragment (Fc) of an immunoglobulin. 19. The engager of claim 15, wherein the engager is a monomer. 20. The engager of claim 15, wherein the antigen-binding domain, the CD58 extracelular domain, and the CD80 extracelular domain or CD86 extracelular domain are fused via polypeptide linkers to form a single polypeptide chain. 21. The engager of claim 20, wherein the antigen-binding domain comprises a CD3scFv, and wherein the single polypeptide chain comprises the folowing N to C order of components: CD58-CD3scFv-CD80/CD86 or CD3scFv-CD58-CD80/CD86. 22. The engager of claim 20, further comprising an Fc of an immunoglobulin, wherein the antigen-binding domain comprises a CD3scFv, and wherein the single polypeptide chain comprises the folowing N to C order of components: CD58-CD3scFv-CD80/CD86-Fc or CD3scFv-CD58-CD80/CD86-Fc. 23. The engager of claim 16, wherein the supramolecular adhesion complex specificaly activates the T cel by engaging, adhering to, and/or costimulating the T cel via the CD3scFv, CD58, and CD80/CD86 domains, respectively. 24. The engager of any one of claims 1-23, wherein the engager is isolated. 25. The engager any one of claims 1-23, wherein the engager lacks a transmembrane or a lipid associating domain. 26. The engager of any one of claims 1-23, wherein the engager is not associated with a viral particle. 27. The engager of any one of claims 1-23, atached to a substrate. 28. The engager of claim 27, wherein the substrate comprises a bead or resin. 29. The engager of claim 27, wherein the substrate comprises a chromatography resin. 30. The engager of any one of claims 1-23, atached to an expamer. 31. An in vivo method of generating engineered immune cels in a patient, comprising: a. administering to the patient an immune cel engager, or a vector encoding said immune cel engager, wherein the immune cel engager forms an artificial supramolecular Atorney Docket No.061479-511001WO adhesion complex with target immune cels and specificaly activates target immune cels by engaging, adhering to, and costimulating target immune cels, and b. administering a gene therapy vector capable of transducing the target immune cels, wherein the gene therapy vector transduces target immune cels thereby generating engineered immune cels, and wherein transduction of the immune cels and/or generation of engineered immune cels is increased compared to administering only the gene therapy vector. 32. A method of treating or preventing cancer, comprising administering an immune cel engager and a gene therapy vector that transduces target immune cels to generate, in vivo, engineered immune cels capable of kiling cancer cels, wherein the complex comprises: a cel binding domain that specificaly binds an antigen on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurent binding of the cel binding domain and the immune cel adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector. 33. A method of making engineered immune cels, comprising contacting target immune cels with an immune cel engager and with a gene therapy vector that generates, ex vivo, engineered immune cels, wherein the engager comprises: a cel binding domain that specificaly binds an antigen on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule on the target immune cels, and optionaly an immune cel costimulatory molecule that provides a costimulatory signal to the immune cel, wherein concurent binding of the cel binding domain and the immune cel adhesion molecule to the target immune cels forms a supramolecular adhesion complex on the target immune cel and activates the target immune cel, thereby increasing transduction of the target immune cels by the vector. 34. A method of activating immune cels, comprising: a. selecting a cel binding domain that specificaly binds an antigen on a target immune cel, an immune cel adhesion molecule that specificaly binds a reciprocal Atorney Docket No.061479-511001WO adhesion molecule on the target immune cel, and optionaly an immune cel costimulatory molecule; b. preparing an immune cel engager comprising functional fragments of the cel binding domain, immune cel adhesion molecule, and optionaly the immune cel costimulatory molecule; c. contacting target immune cels with the immune cel engager. 35. The method of claim 34, wherein the contacting comprises administering the T cel engager to a patient, thereby contacting the engager with target immune cels in vivo and activating the immune cels. 36. The method of claim 35, further comprising administering a gene therapy vector specific to the target immune cels, wherein co-administration of the T cel engager with the vector increases transduction of target immune cels by the vector in the patient. 37. The method of any one of claims 31-36, wherein the target immune cels are T cels. 38. The method of any one of claims 31-36, wherein the engager is a soluble macromolecule substantialy free of lipids. 39. A cel engager, comprising: a cel binding domain that specificaly binds an antigen on a target cel; and an immune cel adhesion molecule that specificaly binds a reciprocal adhesion molecule of an immune cel, wherein binding of the immune cel adhesion molecule to the immune cel activates the immune cel, and wherein the cel engager is soluble. 40. The cel engager of claim 39, wherein the target cel is a tumor cel. 41. The cel engager of claim 39, wherein the target cel is the immune cel or is another immune cel. 42. The cel engager of claim 40, wherein the antigen comprises CD19, CD20, CD22, CD27, CD30, CD70, CD123, CD133, BCMA, ACTN4, BAGE-1, BCR-ABL, beta-catenin, CA 125, CA 15-3, CA 195, CA 242, CA 50, CAM43, Casp-8, CDC27, CDK4, CDKN2A, CEA, COA- 1, DEK-CAN fusion protein, EBNA1, EF2, an Epstein Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, HSP70-2, KIAA0205, MART2, MUM-1, MUM-2, MUM-3, neo- PAP, myosin class I proteins, OS-9, PML-RARα fusion protein, PTPRK, K-RAS, N-RAS, TPI, GAGE3, GAGE4, GAGE5, GAGE6, GAGE7, GNT-V, HERV-K-MEL, LAGE-1, NA-88, LAGE- 2, SP17, SSX-2, TRP2-INT2, PMEL, TYR, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE- 1, GAGE-2, CDKN2B, SCP-1, PRAME, p53, H-RAS, HER2/neu, E2A-PRL, H4-RET, IGH-IGK, HPV E6 antigen, HPV E7 antigen, TSP-180, MAGE-4, MAGE-5, MAGE-6, p180-erbB-3, c-Met, nm-23H1, PSA, TAG-72-4, CA 19-9, mucin, NuMa, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, Beta-HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF5, G250, Ga733, HTgp- Atorney Docket No.061479-511001WO 175, M344, FLT3-ITD, MG7-Ag, FOLR1, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, TLP, TPS, CS1, GPCR5, ganglioside G2, EGFRvII MSLN, PAP, prostein, TARP, Trp-p8, STEAP1, an abnormal ras protein, an abnormal p53 protein, CD51, CD61, galectin, or RalB. 43. The cel engager of claim 41, wherein the antigen comprises CD3. 44. The cel engager of claim 39, wherein the reciprocal adhesion molecule comprises CD2. 45. The cel engager of claim 44, wherein the immune cel adhesion molecule comprises a CD58 protein. 46. The cel engager of claim 39, further comprising an immune cel costimulatory molecule. 47. The cel engager of claim 46, wherein the immune cel costimulatory molecule binds CD28. 48. The cel engager of claim 47, wherein the immune cel costimulatory molecule comprises a CD80 or CD86 protein. 49. A pharmaceutical composition comprising the engager of any one of claims 1-23 or 39-48 and a pharmaceuticaly acceptable carier. 50. A treatment method, comprising administering the pharmaceutical composition of claim 49 to a subject in need of treatment for a disease. 51. The method of claim 50, wherein the disease comprises cancer or an autoimmune disease.
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