Protein complexes and methods of using

Abstract

Provided herein are protein complexes comprising lipid nanoparticles and bispecific antibody constructs, wherein the lipid nanoparticles comprise a cargo. Also provided are protein complexes comprising lipid nanoparticles, bispecific antibody constructs, and antibodies, wherein the lipid nanoparticles comprise a cargo. The protein complexes disclosed herein are useful, for example, for delivering the cargo to a cell or a tissue of interest.

Description

PROTEIN COMPLEXES AND METHODS OF USINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 643,862, filed on May 7, 2024; U.S. Provisional Application No. 63 / 644,268, filed May 8, 2024; and U.S. Provisional Application No. 63 / 736,407, filed December 29, 2024, all of which are hereby incorporated by reference in their entireties.FIELD

[0002] The present disclosure relates to the field of molecular biology. Specifically, the disclosure relates to protein complexes comprising lipid nanoparticles and bispecific antibody constructs and methods of use.BACKGROUND

[0003] Lipid nanoparticles (LNP) provide an efficient means for in vivo RNA delivery. BioNTech and Modema’s COVID-19 vaccines both utilize LNP technology for RNA vaccine delivery and have proven the safety' and efficacy of this class of therapeutics to successfully deliver nucleic acids in vivo, overcoming a major barrier in genetic medicines.

[0004] LNPs generally are lipid-based vesicles with a diameter ranging from 50 to 200 nm. They often consist of ionizable lipids, cholesterol, phospholipids, and / or polyethylene glycol (PEG)-lipids. At acidic pH, the tertiary amine of the ionizable lipid can interact with the negatively charged RNA, facilitating its encapsulation in the LNP. Cholesterol can provide structural integrity and stability to the vesicle, while PEG-lipids and phospholipids can form the surface layer. PEG can also create a hydrated layer around the LNP, preventing aggregation and ensuring colloidal stability in vitro and in vivo.

[0005] Upon systemic administration, PEG-lipids may progressively shed from the LNP surface, allowing serum proteins to absorb onto the particles. This process imparts tissue- directed targeting capabilities to the LNPs as the serum proteins influence the tissue tropism of the LNP. Notably, many LNPs become bound by apolipoprotein E and target the liver via the low-density lipoprotein receptor, making LNPs an attractive vehicle for the treatment of hepatic diseases.

[0006] However, many RNA therapeutics require delivery of the RNA to specific (e.g. , nonliver) cell types. For example, to eliminate tumors, an RNA encoding a toxic payload shouldbe targeted predominantly to cancer cells and avoid healthy cells and tissues. Targeted LNPs can also extend the cell and tissue tropism of RNA delivery; for instance, by increasing the efficiency of delivery to otherwise poorly transfected cell types, such as B cells or hematopoietic stem cells (HSC). Targeted delivery has utility not only in vivo, but also in vitro for engineering cells prior to transplantation into a subject.

[0007] Unfortunately, cell-specific delivery’ of LNP therapeutics is still a challenge. Different strategies have been developed for the generation of tissue / cell specific LNPs. For example, modifying the ratio of the different lipids and inclusion of accessory lipids can impact the LNP’s tissue tropism. However, such targeting mechanisms are not very precise.

[0008] Alternatively, attempts have been made to engineer the LNP surface with a ligand that binds specific cell surface molecules. This can be achieved by substituting PEGylated lipids with PEG molecules functionalized with targeting moieties such as peptides or antibodies. Since antibodies usually do not tolerate exposure to the organic solvents used in the LNP preparation, these polypeptides are chemically conjugated to the LNP after RNA encapsulation. The most widely used antibody conjugation method is based on a thiol - maleimide reaction. Maleimide-functionalized LNPs are first prepared using the standard method by incorporating Mal-PEG-DSPE in lipid mixtures. Then, antibodies are subjected to reducing agents to expose free reactive thiol groups for the conjugation step. For antibodies whose thiols are unavailable or absent, a protein modification agent, N-succinimidyl S- acetylthioacetate (SATA), is normally utilized to introduce external thiol groups into protein molecules. Alternative conjugation techniques include modification of the LNP surface with reactive groups, such as carboxyl or amine groups, that can be covalently coupled to a side group on a surface residue of the antibody using standard bioconjugation methods. How ever, these covalent coupling methods have several drawbacks: (1) difficulty of LNP manufacturing at larger scale; (2) high batch-to-batch variability; (3) potential reduction of the antibody’s affinity to its target; (4) requirement for process optimization for each antibody reagent; (5) risk of aggregation and polymerization due to cross-linking at multiple sites on the antibody; (6) need for additional purification steps to remove cross-linking and catalytic reagents (again, resulting in low yield and poor reproducibility): and (7) random orientation of the antibody on the nanoparticle surface such that the antigen binding sites may not be accessible.

[0009] According, new compositions and method for the cell-specific delivery of cargo molecules, including mRNA cargo molecules, using LNP are urgently needed.SUMMARY

[0010] Provided herein are protein complexes comprising lipid nanoparticles and bispecific antibody constructs as well as methods of use.

[0011] Provided herein is a protein complex comprising: (a) a lipid nanoparticle (LNP), the LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen.

[0012] In some embodiments, the first antigen is about 6 to about 20 amino acids in length. In some embodiments, the first antigen comprises a peptide comprising an HA peptide, a FLAG tag, a Pep-1 peptide, or a Pep-2 peptide. In one embodiment, the first antigen comprises an HA peptide (or variant thereof). In some embodiments, the first antigen comprises a peptide comprising SEQ ID NOs:44, 46, 49, 51, or 52. In some embodiments, the first antigen comprises SEQ ID NO:44 or SEQ ID NO:49.

[0013] In one embodiment, the lipid molecule is a PEGylated lipid molecule, wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule. In one embodiment, the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a first linker. In one embodiment, the first linker is a poly glycine linker. In one embodiment, the polyglycine linker essentially consists of GGG. In some embodiments, the lipid molecule comprises a 14C to 20C fatty acid tail. In one embodiment, the lipid molecule comprises a 16C fatty acid tail. In one embodiment, the modified lipid comprises l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-PEG.

[0014] In some embodiments, the bispecific antibody construct comprises: (a) two or more of a Fab, Fab', single domain antibody, (Fab')2 / Fab2, single-chain Fv (scFv), single chain Fab (scFab), minibody, or scFv-Fc; and / or (b) one or more of a (Fab')2 / Fab2, minibody, di-ScFv, scFv-Fc, bi-scFv, Dual-Affinity' Re-Targeting (DART), dual-action Fab (DAF), H-chain heterodimer with orthogonal Fab interfaces, CrossMab, Dual Variable Domain Immunoglobulin G (DVD IgG), bispecific T-cell engager (BiTE), di-diabody, tandem diabody (TandAb), (scFv)2-HSA, or tetravalent-IgG (Tv-IgG). In one embodiment, the bispecific antibody construct comprises a first scFv and a second scFv, wherein the first scFv comprises a binding site for the first antigen and the second scFv comprises a binding site for the second antigen. In one embodiment, the first scFv and the second scFv are connected via a second linker. In one embodiment, the second linker is a polypeptide linker, optionally wherein the polypeptide linker is a flexible linker.

[0015] In one embodiment, the second antigen is presented on the surface of a cell. In one embodiment, the second antigen is presented on the surface of the cell in the context of a multi histocompatibility complex (MHC). In one embodiment, the second antigen is a first polypeptide expressed on the surface of the cell. In some embodiments, the first polypeptide expressed on the surface of the cell is selected from the group consisting ofPD-1, PD-L1, CD3, CD4, CD5. CD8, CD45, and DEC205.

[0016] In one embodiment, the second antigen is a constant region of an antibody that comprises a binding site for a third antigen. In one embodiment, the third antigen is presented on the surface of a cell. In one embodiment, the third antigen is presented on the surface of the cell in the context of an MHC. In one embodiment, the third antigen is a second polypeptide expressed on the surface of the cell. In some embodiments, the second polypeptide expressed on the surface of the cell is selected from the group consisting of PD-1, PD-L1, CD3, CD4, CD5, CD8, CD45, and DEC205.

[0017] In one embodiment, the protein complex further comprising the antibody that comprises a binding site for the third antigen.

[0018] In some embodiments, the cargo comprises a nucleic acid, a polypeptide, or a small molecule. In one embodiment, the nucleic acid comprises a therapeutic RNA molecule. In some embodiments, the therapeutic RNA molecule is a short interfering RNA (siRNA), messenger RNA (mRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin RNA (shRNA).

[0019] Provided herein is a pharmaceutical composition comprising (a) the protein complex disclosed herein and (b) a pharmaceutically acceptable excipient.

[0020] Provided herein is a method of delivering cargo to a cell, the method comprising contacting the cell with a protein complex disclosed herein, wherein the protein complex comprises: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; and (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, and wherein the cell expresses on its surface the second antigen. Provided herein is a method of delivering cargo to a cell, the method comprising contacting the cell with a protein complex disclosed herein, wherein the protein complex comprises: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, wherein the second antigen is a constant region of an antibody that comprises a binding site for a third antigen; and (c) the anantibody that comprises a binding site for a third antigen, and wherein the cell expresses on its surface the third antigen.

[0021] In one embodiment, the cell is a mammalian cell. In some embodiments, the cell is a T cell, a B cell, or a hematopoietic stem cell. In some embodiments, the cell is a T cell, wherein the cargo comprises an mRNA, and wherein the mRNA encodes for a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a cytokine, or a checkpoint inhibitor. In one embodiment, the cell is a human cell.

[0022] Provided is a method of making a protein complex, the method comprising contacting an LNP disclosed herein with a bispecific antibody disclosed herein, wherein the LNP comprises (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen and wherein the bispecific antibody construct comprises a binding site for the first antigen and a binding site for a second antigen.

[0023] Provided is a method of making a protein complex, the method comprising contacting an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen, with (a) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, wherein the second antigen is a constant region of an antibody that comprises a binding site for a third antigen and (b) an antibody that comprises a binding site for a third antigen.BRIEF DESCRIPTION OF THE FIGURES

[0024] Fig. 1A. Schematic of bsAbHA'SunTag. The bispecific antibody construct may be used to promote binding to two antigens in cis or in trans. SP: signal peptide. scFV: single chain variable fragment. H: Heavy variable chain. L: Light variable chain.

[0025] Fig. IB. Schematic of cell-cell aggregation assay used in Example 2. MEL leukemia cells were transduced with a lentiviral vector (LV) expressing mCherry or GFP. MELmCherryand MELGFPcells were subsequently transduced with a LV expressing delta-NGFRHAand delta-NGFRSuntagrespectively. MELmCherry NGFR-HAand MELGFP / NGFR'Suntagcells were mixed in 1 : 1 ratio.

[0026] Fig. 1C. Microscopy image of MELGFP / NGFR’Suntagand MELmCherry / NGFR’HAcell cultures incubated with 1 mg / pl of purified bsAbHA SunTagor unrelated bsAb. Image is representative from three independent cell-cell aggregation experiments.

[0027] Fig. ID. Quantification of clumps of MELeGFP / NGFR’Suntagand MELmn, rrv (il R-”Acells measured by Amnis* Image-Stream analysis. The experiment was repeated three independent times.

[0028] Fig. IE. Graphs showing number and area of clumps cross a dose curve induced by bsAbHA-Suntag, bsAbFLAG-Suntagand bsAbHA FLAGmeasured through by Amnis® Image-Stream analysis.

[0029] Fig IF. Graphs showing number and area of clumps across a dose curve induced by bsAbHA'Suntagwith different protein linkers measured through by Amnis® Image-Stream analysis. 218s linker: (PGGSTSGSGKPGSEGSTKGAS, SEQ ID NO:39); Trilinker: 218s- (GGGGS)2-HMA. (GGGGS)2 = SEQ ID NO:48. HMA: Human Aldolase protein linker = SEQ ID NO:53.

[0030] Fig. 2A. Schematic of the LNPHAwith bound bsAb (top) and depiction of bsAb- mediated uptake through ligand binding (bottom).

[0031] Fig. 2B. Schematic showing how LNPHAcan be redirected to different cell types with different bsAbs that target different cellular antigens.

[0032] Fig. 2C. Schematic showing the bidirectional lentiviral vector (Bid.LV) used to generate PD-LlSuntag / BFP reporter cell line (top). Representative dot-plot of human erythroleukemia cells (K562) transduced with Bid.LV PD-LlSuntag / BFP, K562PD-L1 / SunTag(bottom left). Schematic of bsAbHA’Suntagserving as a bridge between the LNPHAand the PD- LlSuntag exposed on the cell’s surface (bottom right).

[0033] Fig. 2D. Results of flow cytometry' analysis of GFP and PD-L1 expression in K562PD‘L1 / SunTagcells transfected with 10 ng of GFP mRNA in LNP or LNPHAwith or without bsAbIIA‘Suntag. Cells were analyzed 24 hours post LNP treatment. Graphs show the mean ± s.d. percentage of GFP-positive K562PD’L1 SunTagcells. Analysis of GFP expression was performed 24 hours post-transfection. Two-way ANOVA and Tukey’s multiple comparison post-test, **p<0.001; ****p<0.00001 (n=3). Data representative of five independent experiments. Left bars: PD-L1’. Right bars: PD-L1+.

[0034] Fig. 2E. Results of flow cytometry analysis of GFP and PD-L1 expression in 293TPD-Ll / SunTag cells 24 hours post-treatment with 10 ng of GFP mRNA in LNP or LNPHAwith or without bsAbHA‘Suntag. Graph shows fold change of GFP mean florescence intensity (MFI) of PD-L1 Suntag positive cells treated with bsAbHA‘Suntagcompared to untreated cells.

[0035] Fig. 3A. Flow cytometry analysis of GFP and PD-L1 expression in K562PD’L1 / SunTagcells transfected with 10 ng of GFP mRNA in Dlin-MC3 LNPHA or ALC-0315 LNPHA in absence (left column) or presence (right column) of bsAbHA-Suntag. Analysis performed 24 hours post transfection.

[0036] Fig. 3B. Graphs showing percentage (left) and MFI (right) of GFP positive cells from Fig. 3A. Statistical analysis: two-way ANOVA and Tukey’s multiple comparison post-test. ***p<0.0001 (n=3). Data representative of one independent experiment. Left bars: POLL. Right bars: PD-L1+.

[0037] Fig. 3C. Histogram showing GFP MFI of PDL1+cells treated with the indicated bsAb:LNP weight ratio. 10 ng of GFP mRNA LNP was used in this experiment. Analysis of GFP expression was performed 24 hours post-transfection.

[0038] Fig. 3D. Histogram showing GFP MFI of PDL1 positive and negative cells treated with 20 ng of GFP mRNA LNP and LNPHAwith different amount of DSPE-PEG-HA. Analysis of GFP expression was performed 24 hours post-transfection. Left bars: PD-L1". Right bars: PD-L1+.

[0039] Fig. 3E. Representative dot-plots of K562 cell line transfected with LNPHAin absence (top row) or presence (bottom row) of bsAb. Analysis of eGFP expression was performed 24 hr post transfection. Note cells only become GFP-positive when they are transfected with the LNPHA+ bsAb (bottom row ).

[0040] Fig. 4A. Schematic of the bsAbHA'Suntag(upper left) and its binding domain on K562PD-L1-SunTag(bottom left) and bsAbHAPD L1(top right) and its binding domain on K562PD-Li-sunTag (o^omright). Flow cytometry analysis of K562PD'L1‘SunTagtreated with 10 ng of GFP mRNA LNPHAincubated with either bs AbHA'Suntagor bsAbHA'PD'L1. GFP and PD-L 1 levels were measured after 24 hours.

[0041] Fig. 4B. Histogram showing the MFI of GFP expression of K562PDL1'Suntagtreated with eGFP LNPIIAwith bsAbIIA’Suntagand bsAbIIA'PDL1. Cells were analyzed 24 hours post treatment. (n=3 per group, two independent experiments performed). Left bars: PD-LT. Right bars: PD-L1+.

[0042] Fig. 4C. Graphs show' the mean ± s.d. percentage of GFP+K562mPDL1transfected with 20 ng GFP mRNA LNP or LNPHA+ / - bsAbHAPD L1(n=3 biological replicates, 2 independent experiments). Two-way ANOVA and Tukey’s multiple comparison post-test, ***p<0.0001; ****p<0.00001. Left bars: PD-L1". Right bars: PD-L1+.

[0043] Fig. 4D. Histogram showing the MFI of GFP expression of K562mPDL1treated w ith eGFP LNP and LNPHAwith or without bsAbHA’PDL1. Cells were analyzed 24 hours post treatment. (n=3 per group, 2 independent expenments performed). Left bars: PD-L1'. Right bars: PD-L1+.

[0044] Fig. 4E. Schematic of the DsRed-STOP-loxP-eGFP ere recombination system.

[0045] Fig. 5A. Schematic showing the lentiviral transduction strategy to generate tumor reporter cell lines for in vivo studies. B661, ID8 and Bl 6F 10 murine cell line were transduced with a BFP marker, GFPstopLoxP reporter system and mPD-Ll.

[0046] Fig. 5B. Graphs show the percentage of transfected PD-L1+and PD-L1" B16F10dsRed LSL GFPmelanoma cells following intratumoral injection of melanomas with matching concentrations of Cre mRNA encapsulated in LNPHAor LNPHA+ bsAbHA'PD‘L1. Analysis was performed by flow cytometry. Each dot represents a separate mouse. Two-way ANOVA and Tukey's multiple comparison post-test, **p<0.001.

[0047] Fig. 5C. Graphs show the percentage of transfected PD-L1+ and PD-L1- ID8dsRed’ LSL-GFP ovariicancer cells following i.p. injection of ovarian tumor bearing mice with matching concentrations of Cre mRNA encapsulated in LNPHAor LNPHA+ bsAbHA'PD'L1. Analysis was performed by flow' cytometry. Each dot represents a separate mouse. Two-way ANOVA and Tukey's multiple comparison post-test, **p<0.001. Left dots: PD-LT. Right dots: PD-L1+.

[0048] Fig. 5D. Graphs show the percentage of transfected PD-L1+and PD-L1" KCdsRed'LSL"GFPpancreatic cancer cells following i.p. injection of pancreatic tumor bearing mice with matching concentrations of Cre mRNA encapsulated in LNPHAor LNPHA+ bsAbHA'PD'L1. Each dot represents a separate mouse. Two-way ANOVA and Tukey's multiple comparison posttest, **p<0.001. Left dots: PD-L1’. Right dots: PD-L1+.

[0049] Fig. 6A. Schematic of a bispecific antibody construct recognizing HA and CD4.

[0050] Fig. 6B. Flow cytometry analysis of tdTomato and CD4 expression on total splenocytes isolated from Ail4 mice and transfected with 50 ng Cre mRNA encapsulated in LNP. LNPIIA, LNPIIA+ bsAbIIA’CD4. Cultures included IL-2 and anti-CD3 / CD28 beads to activate / expand T cells. Shown are representative dotplots.

[0051] Fig. 6C. Flow' cytometry analysis of splenocytes isolated from Ail4 mice i.v. injected with Cre mRNA encapsulated in LNP or LNPHAand coupled with or without bsAbHA"CD4. Spleens collected 3 days post injection. Dotplots are representative of n=3 mice group, two independent experiments.

[0052] Fig. 6D. Graphs show percent transfection of CD4+and CD4‘ splenocytes from Fig. 6C (n=3 mice, two independent experiments). Left bars: - bsAbHA’CD4. Right bars: + bsAbHA" CD4

[0053] Fig. 6E. Graphs show in vivo targeting efficiency of the different LNP formulations calculated by the ratio of tdTomato CD4+to CD4" splenocytes from Fig. 6D. Left bars: - bsAbHA'CD4. Right bars: + bsAbHA'CD4.

[0054] Fig. 6F. Graphs show the percentage of GFP+resting human T cells treated with 200 ng GFP mRNA encapsulated in LNP or LNPHAwith or without bsAbHA CD5. Expression was measured 24 hours post transfection. Two-way ANOVA and Tukey’s multiple comparisonpost-test, **p<0.001; ***p<0.0001(n=4). Data representative of three independent experiments. Left bars: bsAbCD5 HA. Right bars: + bsAbCD5 HA.

[0055] Fig. 6G. Graphs showing the GFP MFI of resting human T cells. Two-way ANOVA and Tukey’s multiple comparison post-test, ***p<0.0001(n=4). Data representative of three independent experiments. Left bars: bsAbCD5'HA. Right bars: + bsAbCD5'HA.

[0056] Fig. 7A. Schematic of a bispecific antibody construct recognizing HA and DEC205 on GC-B cell.

[0057] Fig. 7B. Expression of DEC205 (RNA-Seq) in mouse B cells upon stimulation with a functional -grade anti-CD40 (HM40-3, 1 pg / mL) antibody and IL-4 (40 ng / mL), 24 hours post-stimulation. Each dot represents one biological replicate. Data are represented as normalized gene counts. An unpaired t-test (* indicates p<0.05).

[0058] Fig. 7C. Histograms showing the percentage of GFP positive cells within the DEC205+and DEC205" GC B cells at the indicated tested doses. Analysis performed 24 hours post LNP treatment. n=3. (Two-way ANOVA and Tukey’s multiple comparison post-test, ***p<0.0001; ****p<0.00001). Left bars: DEC205" cells. Right bars: DEC205+cells.

[0059] Fig. 8A. Results of flow cytometry analysis of GFP and PD-L1 expression in K562mPDL1cells transfected with the indicated LNP. Graph shows the mean ± s.d. of GFP+cells. All groups used matched concentrations of LNP-RNA (10 ng). GFP expression was measured 24 hours post transfection. (n=3 per group, 3 independent experiments performed). Left bars: PD-Ll". Right bars: PD-L11.

[0060] Fig. SB. Histograms showing the percentage of Cre recombination of K562mPD'L1treated with Cre LNP and LNPHAwith or without bsAbHA / PD'L1and with LNP-IgG and LNP- PDL1. GFP expression was measured 72 hours post LNP transfection. Note how treatment with LNPHA / bsAbHA PDL1is more specific than treatment with LNP-PDL1. Left bars: PD-L I . Right bars: PD-L1+.

[0061] Fig. 8C. Flow cytometry analysis of GFP and CD5 expression in K562hCD5transfected with 10 ng GFP mRNA encapsulated in the indicated LNP. Graph show s the mean ± s.d. of GFP mean florescence intensity (MFI) of GFP+cells. (n=3 per group, three independent experiments performed). Left bars: hCD5‘. Right bars: hCD5+.

[0062] Fig. 8D. Results of flow cytometry analysis of GFP and CD5 expression in resting human T cells treated with 200 ng GFP RNA encapsulated in SM-102 LNP, SM-102 LNPHAwith or without bsAbCD5’HAor with LNPMAL’hCD5. Graph showing the mean ± s.d. of the percentage of GFP+resting human T cells. One-way ANOVA and Tukey’s multiplecomparison post-test, ***p<0.0001. Data representative of three independent experiments with n=3 per group.

[0063] Fig. 8E. Results of flow cytometry analysis of GFP and CD5 expression in resting human T cells treated with 200 ng GFP RNA encapsulated in SM-102 LNP, SM-102 LNPHAwith or without bsAbCD5'HAor with LNPAI ,'’I|< I)5. Graph showing the mean ± s.d. GFP MFI of resting human T cells. Statistical analysis: One-way ANOVA and Tukey's multiple comparison post-test. **p<0.001 ***p<0.0001 (n=3) ; Data representative of three independent experiments.

[0064] Fig. 8F. Results of flow cytometry analysis of GFP and CD5 expression in activated human T cells treated with 200 ng GFP mRNA encapsulated in SM-102 LNP or SM-102 LNPHAwith or without bsAbCD5 HA, or LNPMAL hCD5or LNPAIA, A <i. Expression was measured 24 hours post-transfection. Graph showing the mean ± s.d. percentage of GFP+activated human T cells treated. One-way ANOVA and Tukey’s multiple comparison post-test, ***p<0.0001 (n=3); Data representative of 3 independent experiments. (n=3 per group, three independent experiments performed).

[0065] Fig. 8G. Graph showing the mean ± s.d. GFP MFI of activated human T cells treated as in Fig. 8F. (n=3 per group, three independent experiments performed).

[0066] Fig. 8H. Graph showing the mean ± s.d. percent of GFP+activated human T cells treated as in Fig. 8F at 3 days post transfection. (n=3 per group, 3 independent experiments performed).

[0067] Fig. 81. Graph showing the mean ± s.d. GFP MFI in activated human T cells treated as in Fig. 8F at 3 days post transfection. (n=3 per group, 3 independent experiments performed).

[0068] Fig. 9A. Schematic depicting the bsAbHA’ratIgG2btethering antibody binding to LNPHAparticles and a monoclonal antibody with the cognate constant region (Rat IgG2b).

[0069] Fig. 9B. Results from flow cytometry analysis of GFP expression in K562mPDL1cells transfected with 10 ng of GFP mRNA encapsulated in SM-102 LNP or SM-102 LNPHAwith or without bsAbHA / PD'L1or with bsAbIIA ratI=G2bwith or without PD-L1 monoclonal antibody. GFP expression was measured 24 hours post transfection. Graphs show the mean ± s.d. of the percentage of GFP+cells. Left bars: PD-L1’. Right bars: PD-L1L

[0070] Fig. 9C. Overview of illustrative bsAbs that can be used with the compositions and methods disclosed herein.

[0071] Fig. 9D. Left: Schematic of LNPHA / bsAbHA'ratIgG2b / anti-CD20 Ab complex binding to CD20. Right: Histogram showing the percentage of eGFP positive BCL1 cells treated with 50 ng of eGFP SM-102 LNP and LNPHAwith bsAbHAratIgG2b / anti-CD20 Ab complexes. eGFPexpression was measured 24 hours post transfection. (n=3 per group, three independent experiments performed). Left bars: -bsAb. Right bars: + bsAb.

[0072] Fig. 9E. Graphs of total splenocytes treated with Cre mRNA encapsulated in LNP or LNPHAwith or without bsAbHA'IgG2band the indicated monoclonal antibody. Analysis was performed 48 hours after LNP treatment. Data representative of three independent experiments.

[0073] Fig. 10A. Representative dot-plots of 293T (top) and K562 (bottom) cell line transfected with 50 ng of WT D-LIN-MC3 LNP and LNP, in which 5% of the total PEG was substituted with Pep- 1 : PEG and Pep-2:Peg conjugate, respectively. Analysis was performed 24 hours post LNP treatment. FITC-A, FSC-H subsets top row from left to right: 0.36; 3.04; 99.5; 99.5. FITC-A, FSC-H subsets bottom row from left to right: 0.53; 0.235; 38.6; 64.7.

[0074] Fig. 10B. Representative dot-plots of K562 cell line transfected with 10 ng of indicated SM-102 LNP formulation. Analysis was performed 24 hours post LNP treatment.

[0075] Fig. 10C. Bar graphs showing the GFP MFI of cells treated as in Fig. 10B. ALB corresponds to Pep-2.

[0076] Fig. 10D. DLS analysis of standard SM-102 LNP and LNP in which 5% of total peg was substituted with PEP-2 (PEG-conjugated with Pep-2) / WT LNP: left trace. PEP2-LNP: right trace.DETAILED DESCRIPTION

[0077] The development of cell-specific LNPs is a cumbersome task that requires extensive chemical engineering of the LNPs’ lipid components as well as conducting in vivo validation experiments. In contrast, the compositions and methods provided herein provide a person skilled in the art with the flexibility to target an LNP comprising a given cargo to different cell populations in vivo and in vitro without the need for changing the chemical composition of the LNP, but by simply by incubating the LNP with different bispecific antibody constructs. As such, the compositions and methods disclosed can be used to increase the transfection efficiency of specific cell populations in vivo and to increase transfection efficiency of hard to transfect cell population in vitro.

[0078] In the protein complexes disclosed herein, the binding of a bispecific antibody construct to an LNP can be achieved, for example, by presenting a short linear peptide on the surface of the LNP. The LNP can then bind to a variety of different bispecific antibody constructs that comprise a binding site for the shorted linear peptide presented. These LNP- tethered antibody complexes can in turn bind to a second antigen (e.g., a protein presented inthe surface of a target cell) through the second antigen binding site on the bispecific antibody construct. See Fig. 1A, Fig. 2A, and Fig. 2B for a non-limiting illustration of this concept.

[0079] Alternatively, the second binding of the bispecific antibody construct may bind to, e.g., the constant region of another antibody, which in turn has a binding site for a third antigen (e.g., a protein presented in the surface of a target cell). See Fig. 9A for a non-limiting illustration of this concept.

[0080] The approaches presented herein greatly reduce the complexity of LNP generation, which in turns enables large scale manufacturing and enhances particle quality. The compositions and methods disclosed herein are accessible to laboratories that do not have expertise in chemical conjugation of LNPs or in generating bispecific antibody constructs. Of note, the protein complexes disclosed herein can be assembled in physiological conditions without the need for coupling reagents and purification (e.g., to remove cross-linking and catalytic reagents).

[0081] In one embodiment, the LNP comprises a peptide-PEG lipid conjugate. Such conjugates can be easily produced and purified (e.g., up to 98% purity). The conjugates are different from Anchored Secondary scFv Enabling Targeting (ASSET) lipids that can be purified from bacteria at high yield but with moderate purity (47.5%). Kedmi t al., A modular platform for targeted RNAi therapeutics, Nat Nanotechnol. 2018 Mar;13(3):214-219. Bacteria- derived lipids can also be immunogenic. Further, endotoxin contamination could induce unwanted immune responses. The peptide-PEG lipid conjugates disclosed herein work with a variety of species of PEG (with different reactive groups and modification). This is important because unwanted immune reaction against PEG (e.g. , production of anti-PEG antibodies) could limit the efficiency of PEG-containing LNPs in delivering their cargo to a target cells. Further, the compositions and methods disclosed herein allow a precise control of how much peptide- PEG lipid conjugate is incorporated into the LNP and, as a result, how much antibody is conjugated to the LNP.

[0082] Accordingly, provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; and (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen. Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; and (b) a bispecific antibody construct, wherein the bispecific antibody construct comprises a first domain that binds to the first antigen and a second domain that binds to thesecond antigen. Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; wherein the LNP is non-covalently bound to the bispecific antibody construct (through the binding site for the first antigen).

[0083] Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; and (c) an antibody comprising a constant region and a binding site for a third antigen; wherein the second antigen is the constant region. Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a first domain that binds to the first antigen and a second domain that binds to the second antigen; and (c) an antibody comprising a constant region and a domain that binds to a third antigen; wherein the second antigen is the constant region. Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; (c) an antibody comprising a constant region and a binding site for a third antigen, wherein the bispecific antibody construct is non-covalently bound to the LNP through the binding site for the first antigen and wherein the bispecific antibody construct is non-covalently bound to the antibody through the binding site for the second antigen.

[0084] Lipid nanoparticles (LNPs)

[0085] Provided herein is a protein complex comprising an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to an antigen. Also provided are LNP formulations comprising two or more LNPs.

[0086] As used herein, an “LNP” is a complex comprising lipid molecules that form a substantially spheroid nanoparticle with its cargo. An LNP may sen e as a transport vehicle for delivering biologically active substances, including, but not limited to, small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. LNPs are typically sized on the order of micrometers or smaller and may include a lipid bilayer. LNPs, as used herein, unless otherwise specified, also encompass liposomes (e.g., lipid vesicles) andlipoplexes. In some embodiments, a LNP may be a liposome having a lipid bilayer with a diameter of 500 nm or less.

[0087] Lipids

[0088] The totality of the lipids in an LNP may also be referred to herein as the “lipid component” of the LNP.

[0089] The LNP may comprise modified and / or unmodified lipids. The LNP may comprise more than one type of lipid. In addition to the lipid molecule conjugated to an antigen, the LNP may further comprise lipids not conjugated to an antigen.

[0090] The lipids used in an LNP can be cationic / ionizable. PEGylated, structural, phospholipids, and / or other lipids.

[0091] The LNP may include one or more structural lipids. The LNP may include a structural lipid including, but not limited to, cholesterol, fecosterol, sitosterol, campesterol, stigmasterol, brassicasterol, ergosterol, tomatidine, tomatine, ursolic acid, and / or alphatocopherol.

[0092] The LNP may include one or more phospholipids. As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid may be a lipid according to Formula I:(Formula I) in which RPrepresents a phospholipid moiety and Ri and R2 represent fatty acid moieties with or without saturation that may be the same or different.

[0093] The phospholipid moiety may be selected from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0094] The fatty acid moiety may be selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0095] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. A phospholipid or an analog or derivative thereof may include choline. A phospholipid or an analog or derivative thereof can, in some embodiments, not include choline. Phospholipids may be selected from the non-limiting group consisting of l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-gly cero-3-phosphocholine (DLPC), 1 ,2-dimyristoy 1-sn-glycero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1 -hexadecyl-sn- glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, 1.2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinol enoyl -sn-gly cero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l-glycerol) sodium salt (DOPG). and sphingomyelin.

[0096] In one embodiment, the LNP comprises DSPC. In one embodiment, the LNP comprises DOPE. In one embodiment, the LNP comprises DSPC and DOPE.

[0097] In some specific embodiments, a phospholipid can be functionalized with or crosslinked to one or more alkynes, which may undergo a copper-catalyzed cycloaddition upon exposure to an azide.

[0098] In some embodiments, the LNP composition comprises a plurality of different types of phospholipids, for example, at least 2, 3, 4, 5, or more distinct phospholipids.

[0099] Particular phospholipids may facilitate fusion of the LNP to a membrane. In some embodiments, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow' one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0100] In one embodiment, the LNP comprises a PEGylated lipid. As used herein, a “PEG lipid’7or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol (PEG) component.

[0100] A PEG-lipid can comprise one or more ethylene glycol units, for example, at least 1, at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100. at least 120, or at least 150 ethylene glycol units. In some embodiments, a number average molecular weight of the PEG-lipids is from about 200 Da to about 5000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 500 Da to about 3000 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG-lipids is from about 750 Da to about 2500 Da. In some embodiments, a number average molecular weight of the PEG-lipids is about 500 Da, about 750 Da, about 1000 Da, about 1250 Da, about 1500 Da, about 1750 Da, or about 2000 Da. In some embodiments, a poly dispersity index (PDI) of the one or more PEG- lipids is smaller than 2. In some embodiments, a PDI of the one or more PEG-lipids is between about 0.05 and about 2.

[0101] In some embodiments, the LNP comprises a structural lipid. The structural lipid can be selected from steroid, sterol, alkyl resoreinol, cholesterol or derivative thereof, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and a combination thereof. In some embodiments, the structural lipid is a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone.

[0102] In one embodiment, the LNP comprises a cholesterol or a derivative thereof. In some embodiments, the cholesterol or derivative thereof is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate. In some embodiments, the cholesterol or derivative thereof is cholesterol.

[0103] In some embodiments, the cholesterol or derivative thereof is a cholesterol derivative. In some embodiments, the cholesterol derivative is a polar cholesterol analogue. In some embodiments, the polar cholesterol analogue is 5a-cholestanol, 5(3-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether. or 6- ketocholestanol. In some embodiments, the polar cholesterol analogue is cholesteryl-(4'- hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is a non-polar cholesterol analogue. In some embodiments, the non-polar cholesterol analogue is 5a- cholestane. cholestenone, 5a-cholestanone, 5(3-cholestanone, or cholesteryl decanoate.

[0104] In some embodiments, the LNP comprises a helper lipid. In some embodiments, the LNP comprises a neutral lipid. In some embodiments, the LNP comprises a stealth lipid. In some embodiments, the LNP comprises additional lipids.

[0105] As used herein, “neutral lipids” include, for example, a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids include, but are not limited to, 5- heptadecylbenzene-l,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC). dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1,2-distearoyl-sn- glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1- myristoyl-2-palmitoyl phosphatidylcholine (MPPC). 1-palmitoy 1-2 -myristoyl phosphatidylcholine (PMPC), 1 -palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1,2- diarachidoyl-sn-glycero-3-phosphocholine (DBPC), I-stearoyl-2 -palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidyl ethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In some embodiments, the neutral phospholipid may be selected from the group consisting of SPC and dimyristoyl phosphatidyl ethanolamine (DMPE). In some embodiments, the neutral phospholipid is DSPC. Neutral lipids can function to stabilize and improve processing of the LNPs.

[0106] “Helper lipids” can refer to lipids that enhance transfection (e.g. transfection of the LNP including the biologically active cargo agent). The mechanism by which the helper lipid enhances transfection includes enhancing particle stability. In some embodiments, the helper lipid enhances membrane fusogenicity. In some embodiments, the helper lipid is a neutral lipid. A phospholipid can be a helper lipid.

[0107] “Stealth lipids” can refer to lipids that alter the length of time the LNP can exist in vivo (e.g., in the blood). Stealth lipids can assist in the formulation process by. for example, reducing particle aggregation and controlling particle size. Stealth lipids used herein may modulate pharmacokinetic properties of the LNP. Stealth lipids suitable for use in a lipid composition of the disclosure can include, but are not limited to. stealth lipids having a hydrophilic head group linked to a lipid moiety. Examples of stealth lipids and information about the biochemistry of such lipids can be found, e.g., in Romberg et al. PharmaceuticalResearch, Vol. 25, No. 1, 2008, pg. 55-71 and Hoekstra et al, Biochimica et Biophysica Acta 1660 (2004) 41-52.

[0108] In some embodiments, the stealth lipid is a PEG-lipid. In one embodiment, the hydrophilic head group of stealth lipid comprises a polymer moiety selected from polymers based on PEG (sometimes referred to as poly(ethylene oxide)), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), polyaminoacids and poly N-(2- hydroxypropyl)methacrylamide]. Stealth lipids can compnse a lipid moiety. In some embodiments, the lipid moiety of the stealth lipid may be derived from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The di alkydglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups.

[0109] The structures and properties of helper lipids, neutral lipids, stealth lipids, and / or other lipids are further described in WO2017173054A1, WO2019067999A1, US20180290965 Al. US20180147298A1, US20160375134A1. U.S. Pat. Nos. 8,236,770. 8,021,686, 8,236,770B2, 7,371,404B2, 7,780, 983B2, 7,858, 117B2, US20180200186A1, US20070087045A1, WO2018119514A1, and WO2019067992A1, all of which are hereby incorporated by reference in their entireties.

[0110] Other components[OH l] The LNP may comprise one or more small hydrophobic molecules such as a vitamin (e.g. , vitamin A or vitamin E) or a sterol.

[0112] The LNP may comprise one or more permeability' enhancer molecules, carbohydrates, polymers, therapeutic agents, surface altering agents, or other components. A permeability enhancer molecule may be a molecule described by U.S. patent application publication No. 2005 / 0222064, for example. Carbohydrates may include simple sugars (e.g., glucose) and polysaccharides (e.g., glycogen and derivatives and analogs thereof).

[0113] The LNP may comprise one or more antioxidants. In some embodiments, the antioxidant(s) function to reduce a degradation of the cationic lipids, the cargo, or both. In some embodiments, the one or more antioxidants comprise a hydrophilic antioxidant. In some embodiments, the one or more antioxidants is a chelating agent such as ethylenediaminetetraacetic acid (EDTA) and citrate. In some embodiments, the one or more antioxidants comprise a lipophilic antioxidant. In some embodiments, the lipophilic antioxidant comprises a vitamin E isomer or a polyphenol. In some embodiments, the one or moreantioxidants are present in the LNP at a concentration of at least 1 mM, at least 10 mM, at least 20 mM, at least 50 mM, or at least 100 mM. In some embodiments, the one or more antioxidants are present in the LNP at a concentration of about 20 mM.

[0114] The LNP may include one or more surface altering agents. Surface altering agents may include, but are not limited to, anionic proteins (e.g, bovine serum albumin), surfactants (e.g, cationic surfactants such as dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g, cyclodextrin), nucleic acids, polymers (e.g, heparin, polyethylene glycol, and poloxamer), mucolytic agents (e.g, acetylcysteine, mugwort, bromelain, papain, clerodendrum, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin 134, domase alfa, neltenexine. and erdosteine), and DNases (e.g. rhDNase). A surface altering agent may be disposed within an LNP and / or on the surface of an LNP (e.g, by coating, adsorption, covalent linkage, or other process).

[0115] The LNP may comprise a polymer. The polymer can be biodegradable and / or biocompatible. Exemplary polymers include but are not limited to polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. In some embodiments, the LNP comprises poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA). poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA). poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide)(PLLA), poly(D,L-lactide-co- caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co- D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol) (PEG), polyalkylene oxides (PEO). polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly (vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone (PVP), poly siloxanes, polystyrene, polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate)(PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly (isobutyl(meth)acry late), poly(hexyl(meth)acrylate). poly(isodecyl(meth)acrylate). poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxy alkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poloxamines, poly(ortho)esters, poly(butyric acid). poly(valeric acid), poly(lactide-co-caprolactone). trimethylene carbonate, poly(N-acryloylmorpholine) (PAcM). poly(2-methyl-2-oxazoline) (PMOX), poly(2-ethyl-2-oxazoline) (PEOZ), polyglycerol, or a combination thereof.

[0116] Modified lipids

[0117] Provided herein is a protein complex comprising an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to an antigen.

[0118] In one embodiment, the LNP comprises a modified lipid comprising a lipid molecule conjugated to an antigen, wherein the lipid molecule is a PEGylated lipid molecule comprising a PEG portion. In one embodiment, the LNP comprises a modified lipid comprising a lipid molecule conjugated to an antigen, wherein the lipid molecule is a PEGylated lipid molecule comprising a PEG portion and wherein the antigen is conjugated to the PEG portion of the PEGylated lipid molecule. In one embodiment, the antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a linker.

[0119] Methods for conjugating an antigen, including a peptide-based antigen, to a lipid are known in the art.

[0120] In one embodiment, the antigen is linked to the lipid via an amide linkage. For example, an activated carboxyl group of a fatty acid may react with the amino group of a peptide, forming a stable amide bond. Amide conjugations can be performed using conventional amide coupling methods such as carbodiimide-mediated (HATU / HBTU / HOBt) coupling, NHS ester activation or the acid chloride method. Phospholipids with either amine or carboxyl functional groups for conjugation with antigens containing amine, carboxyl, or hydroxy groups are commercially available.

[0121] In one embodiment, an antigen is linked to the lipid via disulfide / thioether conjugation. In one embodiment, this linkage involves a reaction between a thiol group (SH) on a cysteine residue in a peptide and a maleimide group on the lipid. Disulfide linkages can be synthesized by oxidation of two free thiol groups or by modifying one of the thiols to form an activated disulfide. Another disulfide bond formation method uses an S-sulfonate-protectedcysteine of the peptide. S-sulfonates undergo thiolysis to afford disulfide-linked conjugates. Thiol functionalities can be used for preparing maleimide-type linkages, which are widely used for labelling proteins, proceed without a catalyst in aqueous buffers, and result in stable covalent linkages. Thioether bond formation may be achieved via the Michael addition of thiols to maleimides or through the nucleophilic substitution of haloacetamides. The maleimido group can be introduced into antigen using activated esters such as P-maleimidopropionic acid. In the cases of haloacetamides. either of the conjugating partners can be modified with an aminohexyl group using halogenoacetic anhydride treatment.

[0122] In one embodiment, the antigen is linked to the lipid using click chemistry'. A copper- catalyzed azide-alkyne cycloaddition can be employed to form a triazole linkage between a peptide and a lipid. The reaction may be carried out in aqueous buffers at room temperature.

[0123] In one embodiment, the antigen is linked to the lipid via biotin / streptavidin binding. For example, the biotin may be attached to the lipid and the streptavidin to the antigen.

[0124] Alternative linkages include oximes, triazoles, and hydrazides.

[0125] In one embodiment, the modified lipid comprises a maleimide group that reacts with a terminal cysteine of a peptide antigen. In one embodiment, the modified lipid is generated by reacting a peptide comprising an N-terminal cysteine with DSPE-PEG2000 Maleimide. In one embodiment, the modified lipid is generated by reacting a peptide of the sequence SEQ ID NO:47 or SEQ ID NO:50 (or a variant of either) with DSPE-PEG2000 Maleimide. In one embodiment, the modified lipid is generated by reacting a peptide of the sequence SEQ ID NO:47 or SEQ ID NO:50 with DSPE-PEG2000 Maleimide.

[0126] LNP compositions

[0127] The LNP may comprise an ionizable lipid, a phospholipid, a PEGylated lipid, and / or a structural lipid.

[0128] In some embodiments, the ionizable lipid is SM-102, ALC-0315, TT3, or D-Lin- MC3-DMA. In some embodiments, the phospholipid is DSPC or DOPE. In some embodiments, the PEGylated lipid is DMG-PEG2000, ALC-0159 PEG, or DSG-C14-PEG 2K. In one embodiment, the structural lipid is cholesterol.

[0129] The different components of the LNP may be present in different relative ratios. The different components of the LNP may be present at different mol %.

[0130] In some embodiments, the ionizable lipid comprises from about 20 mol % to about 60 mol % of the total lipid present in the LNP. In some embodiments, the ionizable lipid comprises from about 25 mol % to about 55 mol % of the total lipid present in the LNP. In some embodiments, the ionizable lipid comprises from about 30 mol % to about 55 mol % ofthe total lipid present in the LNP. In some embodiments, the ionizable lipid comprises from about 35 mol % to about 55 mol % of the total lipid present in the LNP. In some embodiments, the ionizable lipid comprises from about 40 mol % to about 50 mol % of the total lipid present in the LNP. In some embodiments, the ionizable lipid comprises about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, or about 60 mol % of the total lipid present in the LNP.

[0131] In some embodiments, the phospholipid comprises from about 1 mol % to about 20 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 2 mol % to about 19 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 3 mol % to about 18 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 4 mol % to about 17 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 5 mol % to about 17 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 6 mol % to about 16 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises from about 8 mol % to about 15 mol % of the total lipid present in the LNP. In some embodiments, the phospholipid comprises about 1 mol %, about 2 mol %, about 3 mol %, about 4 mol %, about 5 mol %, about 6 mol %, about 7 mol %, about 8 mol %, about 9 mol %, about 10 mol %, about 11 mol %, about 12 mol %, about 13 mol %. about 14 mol %, about 15 mol %, about 16 mol %, about 17 mol %, about 18 mol %, about 19 mol %, or about 20 mol % of the total lipid present in the LNP.

[0132] In some embodiments, the PEG-lipid comprises from about 0.1 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the PEG-lipid comprises from about 0.3 mol % to about 5 mol % of the total lipid present in the LNP. In some embodiments, the PEG-lipid comprises from about 0.5 mol % to about 2 mol % of the total lipid present in the LNP. In some embodiments, the PEG-lipid comprises from about 0.5 mol % to about 1.5 mol % of the total lipid present in the LNP. In some embodiments, the PEG- lipid comprises about 0.1 mol %, about 0.2 mol %, about 0.3 mol %, about 0.4 mol %, about 0.5 mol %. about 0.6 mol %, about 0.7 mol %. about 0.8 mol %, about 0.9 mol %, about 1.0 mol %, about 1.5 mol %, about 2 mol %, about 2.5 mol %, about 3 mol %, about 3.5 mol %, about 4 mol %, about 4.5 mol %, about 5 mol %, about 5.5 mol %, about 6 mol %, about 6.5 mol %, about 7 mol %, about 7.5 mol %, about 8 mol %, about 8.5 mol %, about 9 mol %, about 9.5 mol %, or about 10 mol % of the total lipid present in the LNP.

[0133] In some embodiments, the cholesterol or the derivative thereof comprises from about 10 mol % to about 60 mol % of the total lipid present in the LNP. In some embodiments, the cholesterol or the derivative thereof comprises from about 20 mol % to about 55 mol % of the total lipid present in the LNP. In some embodiments, the cholesterol or the derivative thereof comprises from about 25 mol % to about 55 mol % of the total lipid present in the LNP. In some embodiments, the cholesterol or the derivative thereof comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP. In some embodiments, the cholesterol or the derivative thereof comprises about 10 mol %, about 15 mol %, about 20 mol %, about 25 mol %, about 30 mol %, about 35 mol %, about 40 mol %, about 45 mol %, about 50 mol %, about 55 mol %, or about 60 mol % of the total lipid present in the LNP.

[0134] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a PEGylated lipid, and a structural lipid at a ratio of about 20 to about 55 : about 5 to about 30 : about 35 to about 45 : about 0.5 to about 2. In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a PEGylated lipid, and a structural lipid at a ratio of about 45 to about 55 : about 8 to about 12 : about 38 to about 43 : about 1.3 to about 1.7. In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a PEGylated lipid, and a structural lipid at a ratio of about 50 : about 10 : about 30 : about 1.5. In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a PEGylated lipid, and a structural lipid at a ratio of about 50 : about 10 : about 38.5 : about 1.5.

[0135] The amount of cargo in / on an LNP may depend on the size, sequence, and other characteristics of the cargo and on the size, composition, desired target, and other characteristics of the LNP.

[0136] In some embodiments, the wt / wt ratio of the lipid component to an mRNA cargo may be from about 5 : 1 to about 50: 1. such as 5: 1, 6: 1, 7: 1, 8: 1. 9: 1, 10: 1, 11: 1. 12: 1. 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, and 50: 1. For example, the wt / wt ratio of the lipid component to an mRNA may be from about 10: 1 to about 40: 1.

[0137] The amount of mRNA in an LNP may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0138] In some embodiments, the one or more mRNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an mRNA. The one or more mRNA, lipids, and amounts thereof may be selected to provide an N:P ratio from about 2: 1 to about 8: 1, such as 2: 1, 3: 1, 4: 1. 5: 1, 6: 1, 7: 1, and 8: 1. In certain embodiments, the N:P ratio may be from about 2: 1 to about 5: 1. In preferred embodiments, the N:P ratio maybe about 4: 1. In other embodiments, the N:P ratio is from about 5: 1 to about 8: 1. For example, the N:P ratio may be about 5.0: 1, about 5.5: 1. about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1.

[0139] Physical and chemiccd properties of an LNP

[0140] The physical properties of an LNP may be characterized by a variety of methods. In some embodiments, microscopy (e.g.. transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distnbution of an LNP. Dynamic light scattering or potentiometry (e.g, potentiometric titrations) may be used to measure zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of an LNP, such as particle size, poly dispersity index, and zeta potential.

[0141] Also provided herein are LNP formulations that comprise two or more LNPs.

[0142] The average LNP diameter of an LNP formulation may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). In some embodiments, the average LNP diameter of the LNP formulation may be from about 40 nm to about 150 nm. such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 50 nm to about 100 nm. from about 50 nm to about 90 nm. from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In some embodiments, the average LNP diameter of the LNP formulation may be from about 70 nm to about 100 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 70 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 80 nm. In a particular embodiment, the average LNP diameter of the LNP formulation may be about 90 nm. In some embodiments, the average LNP diameter of the LNP formulation may be about 100 nm.

[0143] A LNP may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of an LNP, e.g., the particle size distribution of the LNPs. A small (e.g, less than 0.3) poly dispersity index generally indicates a narrow particle size distribution.A LNP may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10. 0.11. 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18. 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.

[0144] The zeta potential of an LNP may be used to indicate the electrokinetic potential of the composition. In some embodiments, the zeta potential may describe the surface charge of an LNP. Lipid nanoparticles with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the zeta potential of an LNP may be from about -10 mV to about +20 mV, from about -10 mV to about +15 mV, from about -10 mV to about +10 mV, from about -10 mV to about +5 mV, from about -10 mV to about 0 mV, from about -10 mV to about -5 mV. from about -5 mV to about +20 mV, from about -5 mV to about + 15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV. from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0145] The chemical properties of an LNP may be characterized by a variety of methods known to a person skilled in the art. In some embodiments, electrophoresis (e.g., capillary electrophoresis) or chromatography (e.g., reverse phase liquid chromatography) may be used to examine the RNA integrity (e.g., if the cargo is an RNA molecule).

[0146] Methods of making, LNPs

[0147] A process for making LNPs can comprise several general steps: (i) providing a first solution, such as citrate or phosphate buffer, comprising one or more nucleic acid molecular entities in a first reservoir; (ii) providing a second solution comprising one or more lipids and an organic solvent, such as an alcohol (e.g., ethanol) in a second reservoir; and (iii) mixing the first solution with the second solution. The first reservoir is optionally in fluid communication with the second reservoir.

[0148] The process can optionally comprise one or more dilution steps, one or more incubation steps, one or more buffer exchange steps, one or more concentration steps, and / or one or more filtrations steps. In some embodiments, the dilution step involves dilution by adding a dilution buffer. In some embodiments, the dilution step involves dilution with aqueous buffer (e.g. citrate buffer or pure water) e.g., using a pumping apparatus (e.g. a peristaltic pump). In some embodiments, the dilution buffer is an organic solution such as alcohol. The dilution step can comprise a dilution that is 1 to 20 times of the initial volume, or any numbers or ranges therebetween. In some embodiments, the dilution step comprises a dilution that is 1to 10 times of the initial volume. In some embodiments, the dilution step is followed by the buffer exchange step or the incubation step.

[0149] The incubation step comprises allowing a solution from the mixing step to stand in a vessel for about 0 to about 100 hours at about room temperature and optionally protected from light. In some embodiments, the incubation step runs from 0 to 24 hours, 1 minute to 2 hours, or 1 minute to 60 minutes. In some embodiments, the incubation step runs from 1 minutes to 120 minutes. In some embodiments, the incubation step is followed by the buffer exchange step. In some embodiments, the incubation step follows the buffer exchange step.

[0150] In some embodiments, the buffer exchange step comprises a solvent exchange that results in a higher concentration of phosphate buffered saline (PBS) buffer. In some embodiments, the buffer exchange step comprises removing all or a portion of organic solvent. In some embodiments, the buffer exchange step comprises dialysis through a suitable membrane (e.g. 10,000 mwc snakeskin membrane). In some embodiments, the buffer exchange step comprises filtration such as tangential flow filtration (TFF). In some embodiments, the buffer exchange step comprises chromatography such as using a desalting column, e g, PD10 column. In some embodiments, the buffer exchange step comprises ultrafiltration. Ultrafiltration comprises concentration of the diluted solution followed by diafiltration, e.g., using a suitable pumping system (e.g. pumping apparatus such as a peristaltic pump or equivalent thereof) in conjunction with a suitable ultrafiltration membrane (e.g. GE Hollow fiber cartridges or equivalent).

[0151] In some embodiments, the mixing step provides a clear single phase. In some embodiments, after the mixing step, the organic solvent is removed to provide a suspension of particles, wherein the one or more nucleic acid molecular entities are encapsulated by the lipid(s). The selection of an organic solvent can involve consideration of solvent polarity and the ease with which the solvent can be removed at the later stages of particle formation. The organic solvent, which can serve as a solubilizing agent, can be in an amount sufficient to provide a clear single phase mixture of the one or more nucleic acid molecular entities and lipid(s). The organic solvent may be selected from one or more (e.g, two) of chloroform, dichloromethane, diethylether, cyclohexane, cyclopentane, benzene, toluene, methanol, and other aliphatic alcohols (e.g. Ci to Cs) such as ethanol, propanol, isopropanol, butanol, tertbutanol, iso-butanol, pentanol and hexanol. The methods used to remove the organic solvent can involve diafiltration or dialysis or evaporation at reduced pressures or blowing a stream of inert gas (e.g. nitrogen or argon) across the mixture.

[0152] In some embodiments, the method further comprises adding nonlipid polycations which are useful to effect the transformation of cells using the present compositions. Examples of suitable nonlipid poly cations include, but are limited to, hexadimethrine bromide (sold under the brand name POLYB RENE®, from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts of hexadimethrine. Other suitable poly cations include, e.g., salts of poly-L-omithine, poly-L-arginine, poly-L-lysine, poly -D-ly sine, polyallylamine and polyethyleneimine. In certain embodiments, the formation of the LNPs can be carried out either in a mono-phase system (e.g. a Bligh and Dyer monophase or similar mixture of aqueous and organic solvents) or in a two-phase system with suitable mixing.

[0153] The LNPs can be formed in a mono- or a bi-phase system. In some embodiments, in a mono-phase system, the amino lipid(s) and one or more nucleic acid molecular entities are each dissolved in a volume of the mono-phase mixture. Combining the two solutions provides a single mixture in which the complexes form. In some embodiments, in a bi-phase system, the amino lipids bind to the one or more nucleic acid molecular entities (which is present in the aqueous phase) and thus increasing the solubility in organic phase.

[0154] In some embodiments, the LNPs are prepared in an apparatus comprising a first reservoir for holding an aqueous solution and a second reservoir for holding an organic lipid solution. In some embodiments, the apparatus comprises additional reservoirs for holding an aqueous solution (such as for a portion of the one or more nucleic acid molecular entities) and / or an organic solution. The apparatus can include a pump mechanism configured to pump the aqueous and the organic lipid solutions into a mixing region or mixing chamber at substantially equal flow rates. In some embodiments, the mixing region or mixing chamber comprises a T coupling or equivalent thereof, which allows the aqueous and organic fluid streams to combine as input into the T connector and the resulting combined aqueous and organic solutions to exit out of the T connector into a collection reservoir or equivalent thereof.

[0155] In some embodiments, the first solution comprises an aqueous buffer. In some embodiments, the first solution comprises a mixture of an aqueous buffer mixed with an organic solvent. In some embodiments, the organic solvent present in the aqueous buffer is ethanol. In some embodiments, the second solution comprises a mixture of an aqueous buffer mixed with an organic solvent. In some embodiments, the second solution comprises ethanol. In some embodiments, the second solution comprises ethanol and water. In some embodiments, the ethanol percentage in the aqueous buffer ranges from 0.1% to 50%, or any numbers or ranges therebetween. In some embodiments, the dilution buffer comprises an aqueous buffer. In some embodiments, the dilution buffer comprises an organic solvent. In some embodiments,the dilution buffer comprises ethanol and water. In some embodiments, the dilution buffer comprises 10% to 20% of ethanol in PBS buffer.

[0156] In some embodiments, the mixing comprises laminar mixing, vortex mixing, turbulent mixing, or a combination thereof. In some embodiments, the mixing comprises crossmixing. In some embodiments, the mixing comprises inline mixing. In some embodiments, the mixing comprises introducing at least a portion of the first solution through a first inlet channel and at least a portion of the second solution through a second inlet channel, and wherein an angle between the first inlet channel and the second inlet channel is from about 0 to 180 degrees. In some embodiments, the angle between the first inlet channel and the second inlet channel is from about 15 to 180 degrees, from about 30 to 180 degrees, from about 45 to 180 degrees, from about 60 to 180 degrees, from about 90 to 180 degrees, or any numbers or ranges therebetween. In some embodiments, the mixing comprises introducing a portion of the first solution through a third inlet channel. The mixing step can take place by any number of methods, e.g., by mechanical means such as a vortex mixer. In some embodiments, the mixing step comprises inline mixing.

[0157] In some embodiments, a method of making a formulation comprising the LNPs comprises a filtration step. In some embodiments, a method of making a formulation comprising the LNPs comprises buffer exchange. In some embodiments, the buffer exchange comprises dialysis, chromatography, or TFF.

[0158] Bispecific antibody constructs

[0159] The term “antibody construct” refers to a molecule in which the structure and / or function is / are based on or derived from the structure and / or function of an antibody, e.g, of a full-length or whole immunoglobulin molecule. An antibody construct is hence capable of binding to its specific target(s) or antigen(s).

[0160] The term “bispecific” as used herein refers to an antibody construct which is “at least bispecific,” i.e., the antibody construct comprises at least a first binding domain and a second binding domain. Accordingly, antibody constructs disclosed herein comprise specificities for at least two different antigens or targets. The term “bispecific antibody construct” also encompasses multispecific antibody constructs such as trispecific antibody constructs, the latter ones including three binding domains, or constructs having more than three (e.g. four, five...) specificities.

[0161] As used herein, the “bispecific antibody construct” refers to a construct that has (at least) two binding sites for different antigens and comprises one or more antibodies or antigen-binding fragments thereof. The term “antibody” is used in the broadest sense and includes monoclonal antibodies (including full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and antigen-binding fragments of antibodies, as well as combinations and fusions of any of these, so long as they exhibit the desired biological activity and specificity. A bispecific antibody construct usually comprises the minimum structural requirements of an antibody which allow for antigen binding. This minimum requirement may, e.g. , be defined by the presence of at least the three light chain CDRs (i.e. CDR1 , CDR2 and CDR3 of the VL region) and / or the three heavy’ chain CDRs (i.e. CDR1 , CDR2 and CDR3 of the VH region), preferably of all six CDRs. Some antibodies and fragments thereof, such as single domain antibodies, bind with only three CDRs. Within the definition of “antibody” are full-length or whole antibodies, single chain antibodies, and immunoglobulin-based antibodies, and antibodies generated by biotechnological or protein engineering methods or processes. Full- length antibodies may be for example monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies. Also, within the definition of “antibody” are fragments of full-length antibodies, including, but not limited to. VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab')2 constructs. An antibody fragment does not need to be a consecutive fragment of an antibody. An antibody may also refer to a modified fragment of an antibody or might refer to an antibody variant. The term “antibody construct” includes molecules consisting of only one polypeptide chain as well as molecules consisting of more than one polypeptide chain, which chains can be either identical (homodimers, homotrimers or homo oligomers) or different (heterodimer, heterotrimer or heterooligomer). Given that the antibody constructs disclosed herein are (at least) bispecific, they do not occur naturally, and they are markedly different from naturally occurring products.

[0162] The terms “antigen-binding portion” or “antigen-binding fragment” as used herein may refer to a region on an antibody or antibody construct that binds to its antigen.

[0163] Bispecific antibody constructs can be produced by a variety of methods known in the art. See, e.g., Songsivilai & Lachmann, Bispecific antibody: a tool for diagnosis and treatment of disease, Clin Exp Immunol. 1990 Mar;79(3):315-21; Ma et al.. Bispecific Antibodies: From Research to Clinical Application, Front Immunol. 2021 May 5;12:626616; Suurs et al., A review of bispecific antibodies and antibody constructs in oncology’ and clinical challenges, Pharmacol Ther. 2019 Sep;201: 103-119, all of which are incorporated herein by reference in their entireties.

[0164] In one embodiment, the bispecific antibody construct comprises two or more monospecific antibodies or antibody fragments. As used herein, a “monospecific" antibody or antigen-binding fragment thereof binds a single target or antigen. The two or more monospecific antibodies may be joined with a linker. In one embodiment, the bispecific antibody construct comprises one or more monospecific antibodies or antibody fragments and one or more bispecific antibodies or antibody fragments.

[0165] In one embodiment, the monospecific antibody or antibody fragment is a Fab fragment, which comprises or consists essentially of a variable (VL) and constant (CL) domain of the light chain and a variable domain (VH) and the first constant domain (Cnl) of the heavy chain. Usually, the CHI domain and the CL domain are linked.

[0166] In one embodiment, the monospecific antibody or antibody fragment is a Fab’ fragment, which comprises or consists essentially of a Fab fragment having one or more cysteine residues at the C-terminus of the Cnl domain.

[0167] In one embodiment, the monospecific antibody or antibody fragment is a “single domain antibody” or “sdAb,” which refers to an antibody fragment consisting or consisting essentially of a single monomeric variable antibody domain. sdAbs can be readily produced and engineered against a variety of targets. In one embodiment, the sdAb is derived from the antigen-binding portion of a camelid heavy-chain-only antibody. Such an sdAb is also referred to as a VHH fragment or a nanobody (Nb). Nanobodies (Nbs) are single-domain antigenbinding fragments that are fairly small (often around 15 kDa). Despite their small size, Nbs bind antigens with high affinity and specificity. In one embodiment, the sdAb is derived from the antigen-binding portion of a cartilaginous fish heavy-chain-only antibodies (IgNAR, immunoglobulin new antigen receptor). Such an sdAb is called a VNAR fragment. Alternatively, an sdAb can be generated from conventional IgGs by obtaining or engineering monomeric, stable VH or VL domains.

[0168] In one embodiment, the monospecific antibody or antibody fragment is a (Fab’)2 / Fab2, which comprises or consists essentially of two Fab regions with hinge. A (Fab')2 / Fab2 can be monospecific or bispecific.

[0169] In one embodiment, the monospecific antibody or antibody fragment is a singlechain Fv (scFv), which comprises or consists essentially of the VH and VL domains of antibody, such that these domains are present in a single polypeptide chain. Generally, an Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen-binding. See, e.g.. Pluckthun, 113 Pharmacology Monoclonal Antibodies 269 (Rosenburg & Moore, eds., Springer-Verlag, New York, 1994).Accordingly, in some embodiments, the monospecific antibody fragment is a Fv fragment comprising or consisting essentially of the VL and VH domains of a single arm of an antibody.

[0170] In one embodiment, the monospecific antibody or antibody fragment is a single chain Fab (scFab), which comprises or consists essentially of a Fav, wherein one of the constant regions is connected to the variable region of the respective other chain via a linker. For example, the CL domain may be linked to the VH domain via a linker.

[0171] In one embodiment, the monospecific antibody or antibody fragment is a minibody, which comprises or consists essentially of two scFv fragments linked to two CH3 regions. A minibody can be monospecific or bispecific.

[0172] In one embodiment, the monospecific antibody or antibody fragment is an scFv-Fc, which comprises or consists essentially of two scFv fragments linked to an Fc region (comprising CH2 and CH2 domains). An scFv-Fc is around 50 kDa lighter than a full-size IgG Ab. An scFv-Fc can be monospecific or bispecific.

[0173] In one embodiment, the monospecific antibody or antibody fragment is linear antibody. Linear antibodies refer to the antibodies as described in Zapata et al.. Engineering linear F(ab’)2 fragments for efficient production in Escherichia coli and enhanced antiproliferative activity. Protein Eng. 1995 Oct;8(10): 1057-62, which is incorporated herein in this entirety. Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH- CHI), which, together with complementary light chain polypeptides, form a pair of antigenbinding regions. Linear antibodies can be bispecific or monospecific. In one embodiment, the antibody fragment is a linear antibody comprising a pair of tandem Fd segments (VH-CH1 -VH- Cnl) which, together with complementary light chain polypeptides, form a pair of antigenbinding regions.

[0174] In one embodiment, the bispecific antibody construct comprises one or more bispecific antibodies or antibody fragments. As used herein, a bispecific antibody or antigenbinding fragment thereof binds to two targets or antigens. In one embodiment, the bispecific antibody construct comprises one or more trispecific antibodies or antibody fragments.

[0175] In one embodiment, the bispecific antibody or antibody fragment is a (Fab')2 / Fab2, which comprises or consists essentially two Fab regions with hinge. A (Fab’)2 I Fab2 can be monospecific or bispecific.

[0176] In one embodiment, the bispecific antibody or antibody fragment is a di-ScFv (also referred to as dscFv, diabody, or divalent scFV), which refers to two scFv fragments joined via a linker. In one embodiment, the bispecific antibody construct comprises a tri-scFv (triabody), which comprises or consists essentially of three scFv fragments that are linked to each other.

[0177] In one embodiment, the bispecific antibody or antibody fragment is a minibody, which comprises or consists essentially of two scFv fragments linked to two CH3 regions. A minibody can be monospecific or bispecific.

[0178] In one embodiment, the bispecific antibody or antibody fragment is an scFv-Fc, which comprises or consists essentially of two scFv fragments linked to an Fc region (comprising CH2 and CH2 domains). An scFv-Fc is around 50 kDa lighter than a full-size IgG Ab. An scFv-Fc can be monospecific or bispecific.

[0179] In one embodiment, the bispecific antibody or antibody fragment is a bi-scFv (also referred to as bis-scFv or bispecific-scFv), which comprises or consists essentially of two scFv fragments, wherein each scFv fragments binds to a different antigen.

[0180] In one embodiment, the bispecific antibody or antibody fragment is a Dual-Affinity Re-Targeting (DART). DART molecules generally comprise or consist essentially of two distinct polypeptides that are co-expressed to generate a covalently linked heterodimeric complex with one binding site for each of 2 specificities. In a DART, each Fv may be formed by the association of a VL partner on one chain with a VH partner on the second chain in a VLA-VHB + VLB-VHA configuration. DART molecules may different carboxyterminal heterodimerization domains, e.g, a pairing of VEPKSC on one chain and FNRGEC on the other or a pairing of oppositely charged, coiled-coil domains.

[0181] In one embodiment, the bispecific antibody or antibody fragment is a dual-action Fab (DAF), which comprises two different binding sites in each Fab arm.

[0182] In one embodiment, the bispecific antibody or antibody fragment is a H-chain heterodimer with orthogonal Fab interfaces, a bispecific antibody format that utilizes forced heterodimerization of heavy chains and distinct Fab domains to ensure correct pairing of heavy and light chains.

[0183] In one embodiment, the bispecific antibody or antibody fragment is a CrossMab. CrossMab antibodies are based on the crossover of the antibody domain within one Fab arm of a bispecific IgG antibody to enable correct chain association, whereby the correct association of heavy chains can be enforced by the knobs in holes, electrostatic steering, or alternative technologies.

[0184] In one embodiment, the bispecific antibody or antibody fragment is a Dual Variable Domain Immunoglobulin G (DVD IgG), which refers to a tetravalent antibody structure that comprises or consists essentially of a full-length IgG with additional VH and VL fragments at the end of each arm.

[0185] In one embodiment, the bispecific antibody or antibody fragment is a bispecific T- cell engager (BiTE), which comprises or consists essentially of two scFvs connected by a flexible linker. One of the scFvs may target a tumor antigen, the other an antigen located on a T cell.

[0186] In one embodiment, the bispecific antibody or antibody fragment is a di-diabody, which comprises or consists essentially of two diabodies connected to a Fc domain.

[0187] In one embodiment, the bispecific antibody or antibody fragment is a tandem diabody (TandAb), which has a tetravalent property and comprises two chains in VHA-VLB - VHLB-VLA and VLA-VHB - VLB-VHA configurations.

[0188] In one embodiment, the bispecific antibody or antibody fragment is a (scFv)2-HSA which comprises or consists essentially of two scFvs connected by human serum albumin (HSA).

[0189] In one embodiment, the bispecific antibody or antibody fragment is a tetraval ent- IgG (Tv-IgG). For example, the VH / VI domains of an antibody may be fused to the C terminus of the light chain of another IgG antibody.

[0190] Other bispecific antibody constructs can be envisioned. See. e.g., Madsen et al. Design and engineering of bispecific antibodies: insights and practical considerations, Front Bioeng Biotechnol. 2024 Jan 25;12: 1352014, incorporated herein by reference in its entirety7.

[0191] An antibody disclosed wherein may have any constant region, including IgAl, IgA2, IgM. IgG, IgD, and IgE. and any isotype, including IgGl, IgG2, IgG3, and IgG4. In one embodiment, the human isotype IgGl is used. In one embodiment, the human isotype IgG4 is used. Light chain constant regions can be X or K. The antibody or antigen-binding fragment thereof may comprise sequences from more than one class or isotype.

[0192] In one embodiment, the bispecific antibody construct comprises a sequence that is 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% identical to any one of SEQ ID NOs: 1-16. In one embodiment, the bispecific antibody construct comprises any one of SEQ ID NOs: 1-16. The bispecific antibody construct may optionally comprise a purification or detection tag, such as a His tag.

[0193] In one embodiment, the bispecific antibody construct comprises a heavy7variable chain sequence that is 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% identical to any one of SEQ ID NOs: 18. 20. 22. 24. 26, 28, 30, or 32. In one embodiment, thebispecific antibody construct comprises a heavy variable chain sequence comprising any one of SEQ ID NOs : 18, 20, 22, 24, 26, 28. 30. or 32.

[0194] In one embodiment, the bispecific antibody construct comprises a light variable chain sequence that is 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% identical to any one of SEQ ID NOs: 19. 21, 23, 25, 27, 29, 31, or 33. In one embodiment, the bispecific antibody construct comprises a light variable chain sequence comprising any one of SEQ ID NOs: 19, 21, 23, 25, 27, 29, 31, or 33.

[0195] In one embodiment, the bispecific antibody construct comprises:(a) a heavy variable chain sequence that is 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% identical to any one of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, or 32; and / or(b) a light variable chain sequence that is 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% identical to any one of SEQ ID NOs: 19. 21. 23. 25, 27, 29, 31, or 33.

[0196] In one embodiment, the bispecific antibody construct comprises(a) a heavy variable chain sequence comprising any one of SEQ ID NOs: 18, 20, 22, 24, 26, 28, 30, or 32; and / or(b) light variable chain sequence comprising any one of SEQ ID NOs: 19. 21. 23. 25. 27, 29, 31, or 33.

[0197] In one embodiment, the bispecific antibody construct comprises(a) a heavy variable chain sequence that is 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% identical to SEQ ID NO: 18 and a light variable chain sequence that is 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% identical to SEQ ID NO: 19;(b) a heavy variable chain sequence that is 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% identical to SEQ ID NO:20 and a light variable chain sequence that is 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% identical to SEQ ID NO:21;(c) a heavy variable chain sequence that is 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% identical to SEQ ID NO:22 and a light variable chain sequence that is 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% identical to SEQ ID NO:23;(d) a heavy variable chain sequence that is 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% identical to SEQ ID NO:24 and a light variable chain sequence that is 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% identical to SEQ ID NO:25;(e) a heavy variable chain sequence that is 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% identical to SEQ ID NO:26 and a light variable chain sequence that is 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% identical to SEQ ID NO:27;(f) a heavy variable chain sequence that is 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% identical to SEQ ID NO:28 and a light variable chain sequence that is 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% identical to SEQ ID NO:29;(g) a heavy variable chain sequence that is 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% identical to SEQ ID NO:30 and a light variable chain sequence that is 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% identical to SEQ ID NO:31; or(h) a heavy variable chain sequence that is 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% identical to SEQ ID NO:32 and a light variable chain sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:33.

[0198] In one embodiment, the bispecific antibody construct comprises:(a) a heavy variable chain sequence comprising SEQ ID NO: 18 and a light variable chain sequence comprising SEQ ID NO: 19;(b) a heavy variable chain sequence comprising SEQ ID NO:20 and a light variable chain sequence comprising SEQ ID NO:21;(c) a heavy variable chain sequence comprising SEQ ID NO: 22 and a light variable chain sequence comprising SEQ ID NO: 23;(d) a heavy variable chain sequence comprising SEQ ID NO:24 and a light variable chain sequence comprising SEQ ID NO:25;(e) a heavy variable chain sequence comprising SEQ ID NO: 26 and a light variable chain sequence comprising SEQ ID NO:27;(f) a heavy variable chain sequence comprising SEQ ID NO:28 and a light variable chain sequence comprising SEQ ID NO:29;(g) a heavy variable chain sequence comprising SEQ ID NO: 30 and a light variable chain sequence comprising SEQ ID NO:31; or(h) a heavy variable chain sequence comprising SEQ ID NO: 32 and a light variable chain sequence comprising SEQ ID NO:33.

[0199] In one embodiment, the bispecific antibody construct comprises a signal peptide (also referred to as a signal sequence).

[0200] A person skilled in the art may employ any signal sequence compatible with the compositions and methods disclosed herein. See, e.g., Owji et al.. A comprehensive review of signal peptides: Structure, roles, and applications, Eur J Cell Biol. 2018 Aug;97(6):422-441; O'Neill P, Mistry RK, Brown AJ, James DC. Protein-Specific Signal Peptides for Mammalian Vector Engineering, ACS Synth Biol. 2023 Aug 18;12(8):2339-2352, which are incorporated herein by reference in their entireties.

[0201] In one embodiment, the signal peptide comprises SEQ ID NO: 17 or a variant thereof. In some embodiments, the signal peptide comprises a sequence that has 1, 2, 3. 4, 5, 6. 7. 8, or 9 amino acid substitutions as compared to SEQ ID NO: 17.

[0202] Linkers

[0203] Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid moleculeconjugated to a first antigen, optionally, via a linker; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, optionally, wherein the binding site for the first antigen and the binding site for a second antigen are connected via a linker.

[0204] In one embodiment, the antigen is conjugated to the lipid molecule via linker.

[0205] In one embodiment, the LNP comprises a modified lipid comprising a lipid molecule conjugated to a first antigen, wherein the modified lipid is a PEGylated lipid molecule (i.e.. comprises a PEG portion) and wherein the modified lipid comprises a linker (1) between the lipid and the PEG portion and / or (2) between the PEG portion and the antigen. In one embodiment, the antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a linker.

[0206] In one embodiment, the bispecific antibody construct comprises a binding site for the first antigen and a binding site for a second antigen, wherein the binding site for the first antigen and the binding site for a second antigen are connected via a linker. In one embodiment, the bispecific antibody construct comprises two monospecific antibodies (or antigen-binding fragments thereof) connected via a linker. In one embodiment, the bispecific antibody construct comprises two scFvs connected via a linker.

[0207] More than one type of linker can be used in a protein complex.

[0208] Suitable linkers used herein can be of any of a number of suitable lengths, such as from 1 amino acid to 25 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids. A linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, or 25 aa in length. In some cases, a linker has a length of from 25 aa to 50 aa, e.g., from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, or from 45 to 50 aa in length.

[0209] In one embodiment, the linker is a flexible linker. Non-limiting examples of linkers include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n(where n is an integer of at least one)), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art (see. e.g., Chichili et al., Linkers in the structural biology of protein-protein interactions. Protein Sci. 2013 Feb;22(2): 153-67, which is incorporated herein by reference in its entirety). Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga,Predicting Three-Dimensional Structures of Oligopeptides, In Reviews in Computational Chemistry. John Wiley & Sons, Ltd., 1992, Vol 3; pp 73-142).

[0210] In one embodiment, the linker is a polyglycine linker. In one embodiment, the linker is a polyserine linker. In one embodiment, the linker primarily consists of glycines and serines. In one embodiment, the linker essentially consists of GGG. In one embodiment, the linker essentially consists of GGGG (SEQ ID NO:42). In one embodiment, the linker essentially consists of GGGGG (SEQ ID NO:43).

[0211] In one embodiment, the linker is a rigid linker. Rigid polypeptide linkers comprise a sequence of amino acids that effectively separates protein domains by maintaining a substantially fixed distance / spatial separation between the domains, thereby reducing or substantially eliminating unfavorable interactions between such domains. Rigid polypeptide linkers thus may be employed where it is desired to minimize the interaction between the different domains of a fusion protein. Rigid peptide linkers include, but are not limited to, peptide linkers rich in proline, and peptide linkers having an inflexible helical structure, such as an a-helical structure.

[0212] Flexible or rigid linkers can be used, or a combination thereof. Provided herein are also linkers that are combinations or that contain repeats of any of the linkers disclosed herein.

[0213] Other linkers that can be used with the compositions and methods disclosed herein include non-peptide linkers, such as polymers comprising, for example, polyethylene glycol (PEG). Other linkers, including those resulting from coupling with a bifunctional crosslinking agent, may also be utilized.

[0214] In some embodiments, the linker comprises a sequence selected from any one of SEQ ID NOs:34-43, 48, or 53, or variants thereof. In some embodiments, the tinker comprises a sequence that has 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions as compared to any one of SEQ ID NOs: 34-43, 48, or 53.

[0215] Antigens

[0216] Provided herein is a protein complex comprising: (a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; (b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen.

[0217] As used herein, an “antigen’" is a target molecule that an antigen-binding protein (such as an antibody or antibody construct) binds to. An antigen may be. for example, a peptide, a protein, a polysaccharide, a lipid, or a nucleic acid. An antigen may be found on the surfaceof a pathogen such as a bacterium, virus, or fungus. Antigens can be derived from healthy cells or diseased cells (e.g., a cancer cell). Antigens can be naturally occurring or artificial. An antigen can be located on a target cell that a person skilled in the art wishes to target using the compositions and methods disclosed herein. Encompassed within the definition of an antigen are fragments of the antigen that an antigen-binding protein binds to. An epitope is an antigen.

[0218] An antigen may be presented in the context of an MHC complex. The MHC may be a class I and class II MHC. MHC class I molecules present peptides from proteins synthesized within cells, allowing CD8+T cells to detect and destroy abnormal cells expressing mutant sequences, microbial genes, or foreign polymorphic genes. Generally, class I molecules bind peptides of about 8-12 amino acids in length. Humans have between three and six different class I molecules, which can each bind many different types of peptides. The polymorphic nature of MHC class I molecules, with over 10,000 different alleles, allows for a diverse repertoire of peptides to be presented. MHC II molecules are found on immune cells such as B cells, monocytes, macrophages, dendritic cells, and on epithelial cells when triggered by inflammatory’ signals. In contrast, MHC I molecules are expressed more ubiquitously. Dendritic cells use MHC II molecules to present antigens to naive CD4+T cells, causing CD4+T cell activation. Peptide fragments presented by MHC class II complexes are generally larger than those presented by MHC class I complexes, since the peptide-binding groove of MHC class II is open, allowing peptides to extend out of this site. The MHC class II associated peptides are usually derived from extracellular proteins and from self-proteins that are degraded in the endosomal pathway. The MHC-molecules of the human are also designated as human leukocyte-antigens (HLA).

[0219] As used herein, an “epitope"’ is the specific part of the antigen that the antigenbinding protein binds to. As used herein, an “epitope” can be formed both from contiguous amino acids, or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary' folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5, about 9, or about 8-10 amino acids in a particular spatial conformation. Epitopes define the minimum binding site for an antigen-binding protein or antigen-binding fragment thereof and thus can represent the target of specificity’ of an antigen-binding protein or antigen-binding fragment thereof.

[0220] The bispecific antibody construct disclosed herein binds to at least two antigens. In one aspect, one antigen is located on the LNP. One binding site of the bispecific antibody construct binds to this first antigen. The second antigen can be located on a target cell (allowing,e.g., for the delivery of a cargo encapsulated in (or associated with) the LNP to the target cell). See Fig. 2B, Fig. 9A, and Fig. 9C (top) for a non-limiting illustration of this concept.

[0221] The antigen may be a peptide that is between about 5 amino acids to about 35 amino acids in length. In some embodiments, the antigen is about 6 to about 20 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 6 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 7 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 8 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 9 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 10 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 11 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 12 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 13 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 14 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 15 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 16 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 17 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 18 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 19 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 20 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 21 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 22 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 23 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 24 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 25 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 26 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 27 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 28 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 29 amino acids in length. Insome embodiments, the antigen (by itself or in combination with one or more linkers) is 30 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 31 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 32 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 33 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 34 amino acids in length. In some embodiments, the antigen (by itself or in combination with one or more linkers) is 35 amino acids in length.

[0222] The first antigen may a peptide that allows the non-covalent conjugation of the bispecific antibody construct to the LNP. In some embodiments, the antigen comprises one or more of AU1 epitope, AU5 epitope. Bacteriophage T7 epitope (T7-tag). Bacteriophage V5 epitope (V5-tag), Bluetongue virus tag (B-tag), Calmodulin binding peptide (CBP), E2 epitope, FLAG epitope, E2 epitope Glu-Glu (EE-tag), Human influenza hemagglutinin (HA), HaloTag®, Histidine affinity tag, HSV epitope, KT3 epitope, Myc epitope, Polyarginine (Argtag), Polyaspartate (Asp-tag), Polycysteine (Cys-tag), Polyhistidine (His-tag), Polyphenylalanine (Phe-tag), SI -tag, S-tag, Tandem Affinity Purification (TAP), T7 epitope, and VSV-G. Other peptide antigens designed for detection or purification may be used as well.

[0223] In some embodiments, the antigen comprises an HA peptide, a FLAG tag, a Pep-1 peptide, or a Pep-2 peptide.

[0224] In some embodiments, the antigen comprises any one of SEQ ID NOs:44, 46, 49, 51 , or 52. See Table 1. In some embodiments, the antigen comprises 1 , 2, 3, 4, 5, or 6 amino acid substitutions as compared to an antigen disclosed herein.Table 1. Illustrative antigen sequences. Two variants of the HA tags were used, see Table 1. No difference in transfection efficiency was observed for these two variants. *Canonical HA sequence. See, e.g. Schembri et al., The HA tag is cleaved and loses immunoreactivity during apoptosis, Nat Methods. 2007 Feb;4(2): 107-8,

[0225] The second antigen may be located on a target cell that the person skilled in the art wishes to target with a protein complex disclosed herein.

[0226] In one embodiment, the antigen is a post-translationally modified (PTM) peptide. PTMs are well known neo-antigens in infectious diseases (e.g., HIV), autoimmune diseases and oncology’ indications.

[0227] In one embodiment, the antigen is derived from a bacterial pathogen. Non-limiting examples of bacterial molecules which may be a source of bacterial antigen, e.g, antigenic determinants, include lipopolysaccharides isolated from gram-negative bacterial cell walls and staphylococcus-specific, streptococcus-specific, pneumococcus-specific (e.g., PspA; see PCT Publication No. WO 1992 / 14488. which is incorporated herein by reference in its entirety), Neisseria gonorrhea-specific, Borrelia-specific (e.g., OspA, OspB, OspC of Borrelia associated with Lyme disease such as Borrelia burgdorferi, Borrelia afzelli, and Borrelia garinii (see, e.g., U.S. Pat. No. 5,523,089; PCT Publication Nos. WO 90 / 04411, WO 91 / 09870, WO 93 / 04175, WO 96 / 06165, W093 / 08306; PCT / US92 / 08697; Jonsson et al., Heterogeneity of outer membrane proteins in Borrelia burgdorferi: comparison of osp operons of three isolates of different geographic origins. Infect Immun. 1992 May;60(5): 1845-53; Johnson et al., Incomplete protection of hamsters vaccinated with unlipidated OspA from Borrelia burgdorferi infection is associated with low levels of antibody to an epitope defined by mAb LA-2. Vaccine. 1995 Aug;13(12): 1086-94; Edelman, The Sixth International Conference on Lyme Borreliosis: progress on the development of Lyme disease vaccines. 19-22 June 1994, Bologna, Italy. Vaccine. 1995 Jan;13(l): 133-5, all of which are incorporated herein by reference in their entireties), and pseudomonas-specific proteins or peptides. Additional non-limiting examples of bacterial antigens include, e.g., antigens from Neisseria gonorrhea, Mycobacterium tuberculosis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasma hominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus. Brucella melitensis, Brucella suis, Brucella canis, Campylobacter fetus, Campylobacter fetus intestinalis, Leptospira pomona. Listeria monocytogenes, Brucella ovis, Chlamydia psittaci. Escherichia coh. Actinobacillus equuli. Salmonella abortus ovis. Salmonella abortus equi. Pseudomonas aeruginosa, Corynebacterium equi, Corynebacterium pyogenes, and Actinobaccilus seminis.

[0228] In one embodiment, the antigen is derived from a viral pathogen. In one embodiment, the antigen is derived from a viral protein, including, but not limited to, LCMV gp33, CMV pp65, HIV gag, HIV reverse transcriptase, SARS-CoV-2 spike protein, and EBVBMLF1. In some embodiments, the antigen is derived from influenza virus (e.g, surface glycoproteins hemagluttinin (HA) and neuramimidase (NA)); immunodeficiency virus (e.g, a human immunodeficiency virus antigens (HIV) such as gp!20, gpl60, pl 8 antigen Gag p!7 / p24, Tat, Pol, Nef, and Env); herpesvirus (e.g., a glycoprotein from herpes simplex virus (HSV), Marek's Disease Virus, cytomegalovirus (CMV), or Epstein-Barr virus); hepatitis virus (e.g, Hepatitis B surface antigen (HBsAg)); papilloma virus; rous associated virus (e.g., RAV- 1 env); infectious bronchitis virus (e.g. matrix and / or preplomer); flavivirus (e.g. a Japanese encephalitis virus (JEV) antigen, a Yellow Fever antigen, or a Dengue virus antigen); Morbillivirus (e.g, a canine distemper virus antigen, a measles antigen, or rinderpest antigen such as HA or F); rabies (e.g., rabies glycoprotein G); parvovirus (e.g, a canine parvovirus antigen); poxvirus (e.g, an ectromelia antigen, a canary poxvirus antigen, or a fowl poxvirus antigen); chicken pox virus (varicella zoster antigen); infectious bursal disease virus (e.g, VP2, VP3, or VP4); Hantaan virus, and mumps virus. In some embodiments, the antigen is derived from CMV, HIV, hepatitis virus, or SARS-CoV-2 virus.

[0229] In one embodiment, the antigen is derived from a pathogenic protozoon. In one embodiment, the antigen is derived from Plasmodium, such as P. falciparum, P. vivax, P. ovale or P. malariae.

[0230] In one embodiment, the antigen is derived from a malaria-specific protein, including, but not limited to, circumsporozoite (CS) protein, Thrombospondin Related Adhesion (Anonymous) protein (TRAP), also called Sporozoite Surface Protein 2 (SSP2), LSA I. hsp70, SALSA, STARP, Hepl 7, MSA, RAP-1 , RAP-2.

[0231] In one embodiment, the antigen is derived from a fungal pathogen. Non-limiting examples of fungal proteins from which antigenic determinants may be isolated include those isolated from Candida (e.g, MP65 from Candida albicans), trichophyton, and ptyrosporum.

[0232] In some embodiments, the antigen is a helminth parasite peptide or an ectoparasite peptide.

[0233] Neoantigens, also known as tumor-specific antigens (TSAs), are peptides that arise from DNA mutations in tumor cells. Unlike other tumor antigens, neoantigens are usually not present in normal tissues, making them fairly specific to cancer cells. This specificity allows the immune system to target and attack cancer cells with minimal impact on healthy cells. In one embodiment, the antigen is a neoantigen.

[0234] In one embodiment, the antigen is a cancer antigen, also referred to herein as a tumor antigen. Non-limiting examples of tumor-associated proteins from which antigenic determinants may be isolated include, e.g, adipophilin, AIM-2, ALDH1A1, alpha-actinin-4,alpha-fetoprotein (“AFP”), ARTCI, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2. beta-catenin. BING-4. CA-125, CALCA, carcinoembryonic antigen (“CEA”), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin DI, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen (“ETA”). ETV6-AML1 fusion protein, EZH2, FGF5, FLT3-ITD, FN1. G250 / MN / CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7, GAS7, glypican-3, GnTV, gplOO / Pmel l7, GPNMB. HAUS3. Hepsin, HER-2 / neu, HERV-K- MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1. KMHN1 also known as CCDC110, LAGE-1, LDLR- fucosyltransferaseAS fusion protein. Lengsin, M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE- A9, MAGE-CI, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, MEI, Melan-A / MART- 1, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR- 1, NY-ESO-l / LAGE-2, OAL OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB38 / NY-MEL-1, RAGE-1, RBAF600, RGS5, RhoC, RNF43, RU2AS, SAGE, secemin 1, SIRT2, SNRPD1, SOXIO. Spl7, SPA17, SSX-2, SSX-4. STEAP1, survivin, SYT-SSX1 or - SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG. TRAG- 3, Triosephosphate isomerase, TRP-l / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase (“TYR”), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-ESO1, MAGE-A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MARTI], gp 100, tyrosinase, TRP-l / gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC- 1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2. MAGE-4, MAGE-6, MAGE-10, MAGE-12, BAGE- 1. CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO- l / LAGE-2, NA-88, GnTV, TRP2-INT2. In some embodiments, the antigen is a tumor specific antigen.

[0235] In some embodiments, the antigen is a phosphopeptide, lipopeptide, or glycopeptide. In some embodiments, the antigen is modified with fatty acids, isoprenoids, sterols, phospholipids, or glycosylphosphatidyl inositol.

[0236] In some embodiments, the antigen is an autoimmune peptide. The autoimmune peptide can act as an antigen, which means it can trigger an immune response. In autoimmune diseases, the immune system mistakenly targets the body’s own tissues, and autoimmune peptides may be involved in this process by being recognized as targets by the immune system. Similar to cancer antigens, autoimmune peptides are recognized by the immune system but are part of the body’s own cells. This misrecognition can lead to the immune system attacking healthy tissues, contributing to autoimmune conditions like rheumatoid arthritis, type I diabetes, and multiple sclerosis.

[0237] In some embodiments, the antigen may be PD-1, PD-L1, CD3, CD4, CD5, CD8, CD45, DEC205, or a portion of PD-1, PD-L1, CD3, CD4, CD5, CD8, CD45, or DEC205.

[0238] The bispecific antibody construct disclosed herein binds to at least two antigens. In one aspect, one antigen is located on the LNP. One binding site of the bispecific antibody construct bind to this first antigen. The second antigen can be a second antigen-binding protein, such as an antibody or antigen-binding fragment thereof. For example, the bispecific antibodyconstruct may bind with its second binding site to the immunoglobulin constant region of an antibody or antigen-binding fragment thereof that also comprises a binding site for a third antigen. This third antigen can be located on a target cell (allowing, e g., for the delivery of a cargo encapsulated in (or associated with) the LNP to the target cell). See Fig. 9C (bottom) for anon-limiting illustration of this concept. Thus, in this embodiment, the composition disclosed herein allows to quickly conjugate a given LNP to a variety of off-the-shelf antibodies. This approach minimizes the need for development of bispecific antibodies.

[0239] Accordingly, provided herein is a protein complex comprising:(a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen;(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; and(c) an antibody comprising a constant region and a binding site for a third antigen; wherein the second antigen is the constant region.

[0240] Cargo

[0241] Provided herein is an LNP comprising one or more cargo molecules. The cargo molecule(s) may be encapsulated in the LNP or may be covalently or non-covalently associated with the surface of the LNP. An LNP may comprise more than one type of cargo.

[0242] The cargo can be purified or partially purified, and can be naturally occurring or synthetic, or chemically modified. Illustrative cargo molecules include, but are not limited to, antibodies (e.g, monoclonal, chimeric, humanized, nanobodies, and fragments thereof etc.), hormones, peptides, proteins, chemotherapeutics and other types of antineoplastic agents, low molecular weight drugs, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, antisense DNA or RNA compositions, chimeric DNA:RNA compositions, allozymes. aptamers, ribozyme, decoys and analogs thereof, plasmids and other types of expression vectors, and small nucleic acid molecules, RNAi agents, short interfering nucleic acid (siRNA), messenger ribonucleic acid (messenger RNA, mRNA). short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), siRNA (small internally segmented interfering RNA), aiRNA (asymmetric interfering RNA), and siRNA with 1, 2 or more mismatches between the sense and anti-sense strand to relevant cells and / or tissues, such as in a cell culture, subject or organism.

[0243] The efficiency of encapsulation of a cargo into an LNP (or the efficiency of association of the cargo with the surface of the LNP), describes the amount of cargo that is encapsulated or otherwise associated with the LNP after preparation, relative to the initial amount provided. The encapsulation / association efficiency is desirably high (e.g. close to 100%). The encapsulation / association efficiency may be measured, for example, by comparing the amount of cargo in a solution containing the LNP before and after breaking up the LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free cargo (e g, RNA) in a solution. Fluorescence may be used to measure the amount of free cargo (e.g. , RNA) in a solution. For the LNPs described herein, the encapsulation / association efficiency of a cargo may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation / association efficiency may be at least 80%. In some embodiments, the encapsulation / association efficiency may be at least 90%. In some embodiments, the encapsulation / association efficiency may be at least 95%.

[0244] Nucleic acid cargo molecules

[0245] In one embodiment, the cargo is a nucleic acid molecule. The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- stranded form and includes DNA and RNA. DNA may be in theform of, e.g. , antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (PI, PAC, BAC. YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, aiRNA, miRNA, mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'- 0-methyl ribonucleotides, and PNAs. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al.. Nucleic Acid Res., 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et ah, Mol. Cell. Probes, 8:91- 98 (1994)). “Nucleotides'’ contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0246] In some embodiments, the cargo is selected from a group consisting of an siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, shRNA, dsRNA, mRNA, self- amplifying RNA, and a plasmid, including plasmids from which an interfering RNA or mRNA is transcribed.

[0247] In one embodiment, the cargo is an RNA molecule. In one embodiment, the cargo is a DNA molecule.

[0248] Messenger RNA

[0249] In one embodiment, the cargo is an mRNA. An mRNA may be a naturally or non- naturally occurring mRNA. An mRNA may include one or more modified nucleobases, nucleosides, or nucleotides. A nucleobase of an mRNA is an organic base such as a purine orpyrimidine or a derivative thereof. A nucleobase may be a canonical base (e.g, adenine, guanine, uracil, and cytosine) or a non-canonical or modified base including one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction. Thus, a nucleobase may be selected from the non-limiting group consisting of adenine, guanine. uracil, cytosine. 7-methylguanine, 5-methylcytosine, 5- hydroxymethylcytosine, thymine, pseudouracil, dihydrouracil, hypoxanthine, and xanthine.

[0250] A nucleoside of an mRNA is a compound including a sugar molecule (e.g., a 5- carbon or 6-carbon sugar, such as pentose, ribose, arabinose, xylose, glucose, galactose, or a deoxy derivative thereof) in combination with a nucleobase. A nucleoside may be a canonical nucleoside (e.g.. adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxy adenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine) or an analog thereof and may include one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction of the nucleobase and / or sugar component.

[0251] A nucleotide of an mRNA is a compound containing a nucleoside and a phosphate group or alternative group (e.g., boranophosphate, thiophosphate, selenophosphate, phosphonate, alkyl group, amidate, and glycerol). A nucleotide may be a canonical nucleotide (e.g, adenosine, guanosine, cytidine, uridine, 5-methyluridine, deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and thymidine monophosphates) or an analog thereof and may include one or more substitutions or modifications including but not limited to alkyd, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction of the nucleobase, sugar, and / or phosphate or alternative component. A nucleotide may include one or more phosphate or alternative groups. For example, a nucleotide may include a nucleoside and a triphosphate group. A ‘'nucleoside triphosphate” (e.g., guanosine triphosphate, adenosine triphosphate, cytidine triphosphate, and uridine triphosphate) may refer to the canonical nucleoside triphosphate or an analog or derivative thereof and may include one or more substitutions or modifications as described herein. For example, “guanosine triphosphate” is understood to include the canonical guanosine triphosphate, 7-methylguanosine triphosphate, or any other definition encompassed herein.

[0252] An mRNA may include a 5' untranslated region, a 3' untranslated region, and / or a coding or translating sequence. An mRNA may include any number of base pairs, including tens, hundreds, or thousands of base pairs. Any number (e.g., all, some, or none) ofnucleobases, nucleosides, or nucleotides may be an analog of a canonical species, substituted, modified, or otherwise non-naturally occurring. In certain embodiments, all of a particular nucleobase type may be modified. For example, all cytosine in an mRNA may be 5- methylcytosine.

[0253] In some embodiments, an mRNA may include a 5' cap structure, a chain terminating nucleotide, a stem loop, a polyA sequence, and / or a polyadenylation signal.

[0254] A cap structure or cap species is a compound including two nucleoside moieties joined by a linker and may be selected from a naturally occurring cap, a non-naturally occurring cap or cap analog, or an anti-reverse cap analog (ARCA). A cap species may include one or more modified nucleosides and / or linker moieties. For example, a natural mRNA cap may include a guanine nucleotide and a guanine (G) nucleotide methylated at the 7 position joined by a triphosphate linkage at their 5' positions, e.g, m7G(5')ppp(5')G, commonly written as m7GpppG. A cap species may also be an anti-reverse cap analog. Cap species include m7GpppG, m7Gpppm7G, m73'dGpppG, m27 C)3'GpppG, m27 O3'GppppG, m27,O2'GppppG, m7Gpppm7G, m73'dGpppG, m27,O3'GpppG. m27 < l3GppppG. and m27’02'GppppG.

[0255] An mRNA may instead or additionally include a chain terminating nucleoside. For example, a chain terminating nucleoside may include those nucleosides deoxygenated at the 2' and / or 3’ positions of their sugar group. Such species may include 3 '-deoxy adenosine (cordycepin), 3 '-deoxy uridine, 3 '-deoxy cytosine, 3'-deoxyguanosine, 3'-deoxythymine, and 2',3'-dideoxynucleosides, such as 2'.3 '-di deoxy adenosine. 2',3'-dideoxyuridine. 2', 3'- dideoxy cytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymine.

[0256] An mRNA may instead or additionally include a stem loop, such as a histone stem loop. A stem loop may include 1, 2, 3, 4, 5, 6, 7, 8, or more nucleotide base pairs. For example, a stem loop may include 4, 5, 6. 7, or 8 nucleotide base pairs. A stem loop may be located in any region of an mRNA. For example, a stem loop may be located in, before, or after an untranslated region (a 5' untranslated region or a 3' untranslated region), a coding region, or a polyA sequence or tail.

[0257] An mRNA may instead or additionally include a polyA sequence and / or polyadenylation signal. A polyA sequence may be comprised entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3' untranslated region of an mRNA.

[0258] An mRNA may encode any polypeptide of interest, including any naturally or non- naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNAmay be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell.

[0259] In one embodiment, the protein complex is used to express an mRNA in a T cell. In one embodiment, the mRNA encodes for a chimeric antigen receptor (CAR), which enables T cells to recognize and eliminate cancer cells. In one embodiment, the mRNA encodes for a modified T-cell receptor (TCR). which can enhance antigen specificity. In one embodiment, the mRNA encodes for a cytokine such as IL-2, IL-15, and IL-21, which promote T-cell survival and function. In one embodiment, the mRNA encodes for a checkpoint inhibitor, such as an inhibitor targeting PD-1, CTLA-4, or LAG-3 to help prevent immune suppression. In one embodiment, the mRNA is an anti-apoptotic gene like BCL-2 to improve T-cell persistence. In one embodiment, the mRNA encodes for a metabolic regulator, such as AMPK or mTOR, to optimize cellular metabolism for sustained activity.

[0260] In one embodiment, the cargo comprises an mRNA molecule encoding a CAR or a cytokine.

[0261] In some embodiments, the cargo comprises an mRNA molecule encoding a Cas nuclease, z.e., a Cas nuclease mRNA. In some embodiments, the cargo comprises one or more guide RNAs or nucleic acids encoding guide RNAs. In some embodiments, the cargo comprises a template nucleic acid for repair or recombination. In some embodiments, the cargo comprises an mRNA encoding a gene editor nuclease. In some embodiments, the cargo comprises an mRNA encoding a base editor nuclease. In some embodiments, the cargo comprises an mRNA encoding a restriction enzyme. In some embodiments, the cargo comprises zine-finger nuclease or TALEN nuclease.

[0262] In some embodiments, the Cas nuclease mRNA encodes a Cas protein from a CRISPR / Cas system. In some embodiments, the Cas protein comprises at least one domain that interacts with a guide RNA ('‘gRNA”). In some embodiments, the Cas protein is directed to a target sequence by a guide RNA. The guide RNA can interact with the Cas protein as well as the target sequence such that, it can direct binding to the target sequence. In some embodiments, the guide RNA provides the specificity for the targeted cleavage, and the Cas protein may be universal and paired with different guide RNAs to cleave different target sequences. In certain embodiments, the Cas protein may cleave single or double-stranded DNA. In certain embodiments, the Cas protein may cleave RNA. In certain embodiments, the Cas protein may nick RNA. In some embodiments, the Cas protein comprises at least one DNA binding domain and at least one nuclease domain. In some embodiments, the nuclease domain may be heterologous to the DNA binding domain. In certain embodiments, the Cas protein may bemodified to reduce or eliminate nuclease activity. The Cas protein may be used to bind to and modulate the expression or activity of a DNA sequence.

[0263] Interfering RNA

[0264] In one embodiment, the cargo is an interfering RNA. The term “interfering RNA” or “RNAi” or “interfering RNA sequence” refers to single- stranded RNA (e.g., mature miRNA) or double-stranded RNA (z.e., duplex RNA such as siRNA. aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g.. by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to the single-stranded RNA that is complementary' to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity' to the target gene or sequence, or may comprise a region of mismatch (z. e. , a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.

[0265] Interfering RNA includes “small-interfering RNA” or "siRNA,” e.g.. interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15- 30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21- 23 (duplex) nucleotides in length (e.g, each complementary sequence of the double-stranded siRNA is 15- 60. 15-50, 15-40, 15-30. 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21 -23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19- 21 base pairs in length). siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini. Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary' antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acidbased or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary' sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0266] siRNA can be chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g. , dsRNA greater than about 25 nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA. A dsRNA can be at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain instances, siRNA may be encoded by a plasmid (e.g.. transcribed as sequences that automatically fold into duplexes with hairpin loops).

[0267] Therapeutic agents

[0268] In one embodiment, the cargo is a therapeutic agent or therapeutic moiety. The definition of therapeutic agent can include a nucleic acid cargo.

[0269] Therapeutic agents may include, but are not limited to, cytotoxic, chemotherapeutic, and other therapeutic agents.

[0270] Examples of therapeutic moieties include, but are not limited to, anti-inflammatory agents, anti-cancer agents, anti-neurodegenerative agents, or anti-infective agents.

[0271] Non-limiting examples of therapeutic moieties include radionuclides with high- energy ionizing radiation that are capable of causing multiple strand breaks in nuclear DNA, and therefore suitable for inducing cell death (e.g., of a cancer cell). Non-limiting examples of high-energy radionuclides include:90Y,125I,131I,123I,luIn,105Rh,153Sm,67Cu.67Ga,166Ho,177LU,186Re and188Re. These isotopes typically produce high-energy a- or [3-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells and are essentially non-immunogenic.

[0272] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as cytostatics (e.g., alkylating agents, DNA synthesis inhibitors, DNA-intercalators or crosslinkers, or DNA-RNA transcription regulators), enzyme inhibitors, gene regulators, cytotoxic nucleosides, tubulin binding agents, hormones and hormone antagonists, anti-angiogenesis agents, and the like.

[0273] Non-limiting examples of therapeutic moieties also include alkylating agents such as the anthracy cline family of drugs (e.g., 52axotere52e, carminomycin, cyclosporin- A, chloroquine, methopterin, mithramycin, porfiromycin, streptonigrin, anthracenediones, and aziridines). In another embodiment, the chemotherapeutic moiety is a cytostatic agent such as a DNA synthesis inhibitor. Examples of DNA synthesis inhibitors include, but are not limited to, methotrexate and di chloromethotrexate, 3-amino-l,2,4-benzotriazine 1,4-di oxide,aminopterin, cytosine 0-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, actinomycin-D. and mitomycin C. Non-limiting examples of DNA- intercalators or cross-linkers include, but are not limited to, bleomycin, carboplatin, carmustine, chlorambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin.

[0274] Non-limiting examples of therapeutic moieties also include transcription regulators such as actinomycin D, daunorubicin. doxorubicin, homoharringtonine, and idarubicin. Other non-limiting examples of cytostatic agents that are compatible with the embodiments disclosed herein include ansamycin benzoquinones, quinonoid derivatives (e.g., quinolones, genistein, bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone, diaziquone, carbazilquinone, indoloquinone EO9, diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, and nitrosourea compounds (e.g., carmustine, lomustine, semustine).

[0275] Non-limiting examples of therapeutic moieties also include cytotoxic nucleosides such as, for example, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridme, ftorafur, and 6-mercaptopurine; tubulin binding agents such as 53axoter (e.g., paclitaxel, docetaxel, taxane), nocodazole, rhizoxin, dolastatins (e.g., Dolastatin-10, -11, or -15), colchicine and colchicinoids (e.g., ZD6126), combretastatins (e.g, C ombre tastatin A-4, AVE-6032), and vinca alkaloids (e.g., vinblastine, vincristine, vindesine, and vinorelbine (navelbine)); anti-angiogenesis compounds such as Angiostatin KI -3, DL-a- difluoromethyl -ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

[0276] Non-limiting examples of therapeutic moieties also include hormones and hormone antagonists, such as corticosteroids (e.g., prednisone), progestins (e.g., hydroxy progesterone or medroprogesterone), estrogens, (e.g., diethylstilbestrol), antiestrogens (e.g., tamoxifen), androgens (e.g., testosterone), aromatase inhibitors (e.g., aminogluthetimide), 17-(allylamino)- 17-demethoxygeldanamycin, 4-amino-l,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-a. rapamycin, sex hormone-binding globulin, and thapsigargin.

[0277] Non-limiting examples of therapeutic moieties also include enzyme inhibitors such as, S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenz-imidazole 1-0-D- ribofuranoside. etoposide, formestane, fostriecin, hispidin, 2-imino-l-imidazolidineacetic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

[0278] Non-limiting examples of therapeutic moieties also include gene regulators such as 5-aza-2'-deoxycytidine, 5 -azacytidine, cholecalciferol (vitamin D3). 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal (vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoic acid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone.

[0279] Non-limiting examples of therapeutic moieties also include cytotoxic agents such as, for example, the pteridine family of drugs, diynenes. and the podophyllotoxins. Particularly useful members of those classes include, for example, methopterin, podophyllotoxin, or podophyllotoxin derivatives such as etoposide or etoposide phosphate, leurosidine, vindesine, leurosine and the like.

[0280] Still other cytotoxins that are compatible with the teachings herein include auristatins (e.g., auristatin E and monomethyl auristan E), calicheamicin, gramicidin D, maytansanoids (e.g., maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B, ethidium bromide, emetine, tenoposide, 54axotere54e, dihydroxy anthracindione, mitoxantrone, procaine, tetracaine, lidocaine, propranolol, puromycin, and analogs or homologs thereof.

[0281] Cytotoxic agents may include, for example, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin. dihydroxy anthracinedione, mitoxantrone, mithramycin, actinomycin D. 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, rachelmycin, and analogs thereof.

[0282] Radioactive ions may also be used as therapeutic agents and may include, for example, radioactive iodine, strontium, phosphorous, palladium, cesium, iridium, cobalt, yttrium, samarium, and praseodymium.

[0283] Other therapeutic agents may include, for example, antimetabolites (e.g, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracil, and decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa, chlorambucil, rachelmycin, melphalan, carmustine, lomustine, cyclophosphamide, busulfan. dibromomannitol, streptozotocin. mitomycin C, and cis-dichlorodiamine platinum (II) (DDP), and cisplatin), anthracy clines (e.g., daunorubicin and doxorubicin), antibiotics (e.g., dactinomycin, bleomycin, mithramycin, and anthramycin), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol, and maytansinoids).

[0284] In one embodiment, the cargo is a detectable moiety. Examples of detectable moieties include fluorescent moieties or labels, imaging agents, radioisotopic moieties,radiopaque moi eties, and the like, e.g., detectable labels such as biotin, fluorophores, chromophores, spin resonance probes, or radiolabels. Illustrative fluorophores include fluorescent dyes (e.g. fluorescein, rhodamine, and the like) and other luminescent molecules (e.g. luminal). A fluorophore may be environmentally-sensitive, such that its fluorescence changes if it is located close to one or more residues in the modified protein that undergo structural changes upon binding a substrate (e.g., dansyl probes). Illustrative radiolabels include small molecules containing atoms with one or more low sensitivity nuclei (13C,15N.2H,125I,123I, "Tc,43K,52Fe,67Ga,68Ga,mIn and the like). Other useful moieties are known in the art.

[0285] In one embodiment, the cargo is a diagnostic moiety. Examples of diagnostic moieties include detectable moieties suitable for revealing the presence of a disease or disorder. Typically, a diagnostic moiety allows for determining the presence, absence, or level of a molecule, for example, a target peptide, protein, or proteins, that is associated with a disease or disorder. Such diagnostics are also suitable for prognosing and / or diagnosing a disease or disorder and its progression.

[0286] A cargo molecule may also have one or more of the herein-mentioned functions.

[0287] Nucleic acids and cells

[0288] Provided herein are nucleic acids encoding any peptide (including a peptide-based antigen) or polypeptide (including a bispecific antibody construct) disclosed herein.

[0289] Provided herein are vectors comprising one or more nucleic acids encoding a peptide or polypeptide disclosed herein. “Vector,” as used herein, means a vehicle that comprises a polynucleotide to be delivered into a host cell, either in vitro or in vivo. Non-limiting examples of vectors include a recombinant plasmid, yeast artificial chromosome (YAC), mini chromosome, DNA mini-circle, or a virus (including virus-derived sequences). A viral particle is a vector herein. Also, provided herein are viral genomes comprising one or more nucleic acids encoding a peptide or polypeptide disclosed herein.

[0290] Provided herein is an expression construct comprising a nucleic acid sequence encoding a peptide or polypeptide disclosed herein. The nucleic acid sequence encoding the peptide or polypeptide is usually operatively linked to an expression control sequence. As used herein, “operatively linked” refers to a first molecule joined to a second molecule, wherein the molecules are so arranged that the first molecule affects the function of the second molecule. The two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent. For example, an expression control sequence is operatively linked to atranscribable polynucleotide molecule if the expression control sequence modulates transcription of the transcribable polynucleotide molecule of interest in a cell. Additionally, two portions of an expression control sequence are operatively linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion. Two sequences may be operatively linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operatively linked to one another with no intervening nucleotides present

[0291] Expression control sequences can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e.. Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion.

[0292] A great number of expression control sequences, e.g. , native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized to drive expression of the transgene, depending upon the type of expression desired. For eukaryotic cells, expression control sequences commonly used include a promoter, an enhancer, and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene.

[0293] As used herein, the term “promoter"’ or “transcription regulatory sequence"’ refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter.

[0294] A “constitutive"’ promoter is a promoter that is active in most tissues under most physiological and developmental conditions.

[0295] An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g., by the application of a chemical inducer.

[0296] Provided are cells that comprise a nucleic acid or a vector disclosed herein.

[0297] In some embodiments, the cell is a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the cell is a human cell. The cell may be isolated.

[0298] Purification

[0299] In some embodiments, an LNP, a bispecific antibody construct, a protein complex, a nucleic acid, or a vector disclosed herein is an isolated LNP, bispecific antibody construct, protein complex, nucleic acid, or vector.

[0300] The terms "‘purified" or ‘“isolated” LNP, bispecific antibody construct, protein complex, nucleic acid, or vector refer to an LNP. bispecific antibody construct, protein complex, nucleic acid, or vector that has been separated from other proteins, lipids, and / or nucleic acids with which it may be naturally associated. The LNP, bispecific antibody construct, protein complex, nucleic acid, or vector can constitute at least 10% (z.e., any percentage between 10% and 100%. e. , 20%, 30%, 40%, 50%, 60%, 70 %, 80%, 85%, 90%, 95%, and 99%) by dry weight of the purified preparation.

[0301] Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0302] Modifications

[0303] In some embodiments, amino acid sequence modification(s) of antigens or bispecific antibody constructs disclosed herein are contemplated. Amino acid sequence variants of antigens or bispecific antibody constructs can be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the antigens or bispecific antibody constructs, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antigen or bispecific antibody construct. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., with respect to binding specificity, binding affinity, biological activity, etc.

[0304] One ty pe of variant is a conservative amino acid substitution variant. These variants have at least one amino acid residue in the antigen-binding protein or antigen-binding fragment thereof replaced by a different residue that has similar side chain properties. Amino acids can be grouped according to similarities in the properties of their side chains (see Lehninger, BIOCHEMISTRY (2nded., Worth Publishers, New York, 1975)):

[0305] (1) non-polar: Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), Trp (W), Met (M);

[0306] (2) uncharged polar: Gly (G). Ser (S), Thr (T), Cys (c). Tyr (Y), Asn (N), Gin (Q);

[0307] (3) acidic: Asp (D), Glu (E);

[0308] (4) basic: Lys (K), Arg (c), His (H).

[0309] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a non-polar amino acid with another non-polar amino acid.

[0310] Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties:

[0311] (1) hydrophobic: Ala (A), Vai (V), Leu (L), He (I), Met (M);

[0312] (2) neutral hydrophilic: Ser (S). Thr (T), Cys (C), Asn (N). Gin (Q);

[0313] (3) acidic: Asp (D), Glu (E);

[0314] (4) basic: Lys (K), Arg (R), His (H);

[0315] (5) residues that influence chain orientation: Gly (G), Pro (P);

[0316] (6) aromatic: Phe (F), Trp (W), Tyr (Y).

[0317] As such, a non-limiting example for a conservative amino acid substitution is one that replaces a hydrophobic amino acid with another hydrophobic amino acid.

[0318] In some embodiments, one or more of the CDRs of a bispecific antibody construct or antigen-binding fragment thereof disclosed herein have a conservative amino acid substitution.

[0319] Provided herein is a bispecific antibody construct or antigen-binding fragment thereof that comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitution as compared to another bispecific antibody construct or antigen-binding fragment thereof disclosed herein.

[0320] Provided herein is a bispecific antibody construct or antigen-binding fragment thereof that comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared to another bispecific antibody construct or antigen-binding fragment thereof disclosed herein.

[0321] Further contemplated are amino acid sequence insertions, which can include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a dozen or more residues, as well as intrasequence insertions of single or multiple amino acid residues.

[0322] Any cysteine residue not involved in maintaining the proper conformation of the antigen or the bispecific antibody construct or antigen-binding fragments thereof also can be substituted, for example with a serine or an alanine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.

[0323] Conversely, cysteine bond(s) can be added to the antigen or the bispecific antibody construct or antigen-binding fragments thereof to improve its stability (particularly where thebispecific antibody construct or antigen-binding fragment thereof comprises an antibody fragment such as an Fv fragment).

[0324] In some embodiments, the antigen or the bispecific antibody construct or antigenbinding fragments thereof has one or more amino acid alterations that alter the original glycosylation pattern of the antigen or the bispecific antibody construct or antigen-binding fragments thereof. By “altering the original glycosylation pattern” is meant deleting one or more carbohydrate moieties found in the antigen or the bispecific antibody construct or antigenbinding fragments thereof, and / or adding one or more glycosylation sites that are not present in the antigen or the bispecific antibody construct or antigen-binding fragments thereof disclosed herein. Glycosylation of antigen-binding proteins is typically either N-linked or O- linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine. galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyproline or 5-hydroxylysine can also be used. Addition of glycosylation sites to the antigen or the bispecific antibody construct or antigen-binding fragments thereof is accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration can also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody or antigen-binding fragment thereof (for O-linked glycosylation sites).

[0325] In some embodiments, the antigen or the bispecific antibody construct or antigenbinding fragments thereof are deglycosylated or aglycosylated.

[0326] The contemplated bispecific antibody constructs and antigen-binding fragments thereof also feature humanized frameworks for reduced immunogenicity. In certain embodiments, the CDRs of the contemplated bispecific antibody construct or antigen-binding fragment thereof are located in frameworks obtained from a human antigen-binding protein or antigen-binding fragment thereof. In other embodiments, surface-exposed framework residues of the contemplated bispecific antibody construct or antigen-binding fragment thereof are replaced with framework residues of a human antigen-binding protein or antigen-bindingfragment thereof. The CDRs may also be located in rabbit or humanized frameworks linked to human constant regions (z.e., chimeric antigen-binding proteins).

[0327] Pharmaceutical compositions

[0328] In one embodiment, provided herein is a pharmaceutical composition comprising (a) an LNP disclosed herein, a bispecific antibody construct disclosed herein, a protein complex disclosed herein, a nucleic acid disclosed herein, or a vector disclosed herein and (b) a pharmaceutically acceptable carrier.

[0329] Pharmaceutically-acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0330] The terms “pharmaceutically acceptable,'’ “phy siologically tolerable,” as referred to compositions, carriers, diluents, and reagents, are used interchangeably and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[0331] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well know n in the art. Except insofar as any conventional media or compound is incompatible with the compositions disclosed herein, use of the media or compound in the compositions disclosed herein is contemplated. In some embodiments, a second therapeutic agent, such as an anti-cancer or anti-tumor agent, can also be incorporated into pharmaceutical compositions.

[0332] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed hereinare stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0333] In some embodiments, the pharmaceutical composition further includes a cryoprotectant (e.g, glycerol, DMSO, PEG).

[0334] In some embodiments, the pharmaceutical composition will be suitable for administration to a subject, e.g, will be sterile. For example, in some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g, where the composition is sterile and is free of detectable pyrogens and / or other toxins and / or such detectable pyrogens and / or other toxins are below permissible limits.

[0335] Where a composition disclosed herein is administered as an injectable (e.g, subcutaneously, intraperitoneally, intramuscularly, and / or intravenously) directly into a tissue, a formulation can be provided as a ready -to-use dosage form, a non-aqueous form (e.g, a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients. The formulations comprising an antigenbinding protein or antigen-binding fragment thereof disclosed herein may also be provided so as to enhance serum half-life of the subject protein following administration. For example, the antigen-binding protein or antigen-binding fragment thereof may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum halflife. A variety of methods are available for preparing liposomes are known in the art. The preparations may also be provided in controlled release or slow-release forms.

[0336] Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. For example, a pharmaceutical composition can be present in a container, e.g.. a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0337] The concentration of an LNP disclosed herein, a bispecific antibody construct disclosed herein, a protein complex disclosed herein, a nucleic acid disclosed herein, or a vector disclosed herein in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient’s needs.

[0338] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0339] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0340] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetylalcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glycery l monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatly acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil. polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPEIOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68. POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0341] A binding agent may be starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol. mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®). and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

[0342] Preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butyl at ed hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Chelating agents include ethylenediaminetetraacetic acid (EDTA). citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodiumedetate, tartaric acid, and / or trisodium edetate. Antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, benzy l alcohol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to. vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butyl ated hydroxy toluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES). sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMAB EN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0343] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g. HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline. Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0344] Examples of oils include, but are not limited to. almond, apricot kernel, avocado, babassu, bergamot, black cunent seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademianut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury7, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils as well as butyl stearate, capry lic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil. octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0345] Methods

[0346] Provided herein are methods of making and using the compositions disclosed herein. Methods can be performed, in vitro, in vivo, or ex vivo, as appropnate and / or desired.

[0347] For example, provided is a method of producing a polypeptide of interest in a cell that involves contacting a cell with a protein complex comprising an RNA cargo disclosed herein, whereby the RNA is translated to produce the polypeptide of interest.

[0348] Provided is a method of delivering a cargo to a cell, the method comprising contacting the cell with a protein complex comprising:(a) an LNP comprising (i) the cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; and(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, wherein the cell expresses on its surface the second antigen.

[0349] Provided is a method of delivering a cargo to a cell, the method comprising contacting the cell with a protein complex comprising:(a) an LNP comprising (i) the cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen;(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; and(c) an antibody comprising a constant region and a binding site for a third antigen; wherein the second antigen is the constant region; and wherein the cell expresses on its surface the third antigen.

[0350] In one embodiment, the LNPs are preincubated with the bispecific antibody construct before contacting the cell. In one embodiment, the LNP and the bispecific antibody construct are added simultaneously to the cell culture.

[0351] Provided is a method of delivering a cargo to a cell, the method comprising contacting the cell with:(a) an LNP comprising (i) the cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen; and(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen, wherein the cell expresses on its surface the second antigen.

[0352] Provided is a method of delivering a cargo to a cell, the method comprising contacting the cell with:(a) an LNP comprising (i) the cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen;(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen; and(c) an antibody comprising a constant region and a binding site for a third antigen; wherein the second antigen is the constant region; and wherein the cell expresses on its surface the third antigen.

[0353] The methods provided herein may result an enhanced delivery of the cargo to a cell. As used herein, the term “enhanced delivery’7means delivery of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3-fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10- fold more) of a cargo by a protein complex to a target cell type / tissue of interest compared to the level of delivery of the cargo by a control nanoparticle to the target cell type / tissue of interest. It will be understood that the enhanced delivery of a cargo to a target cell t pe / tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g., a rat model).

[0354] The methods provided allow for the specific delivery’ of a cargo to a target cell type / tissue. As used herein, the term “specific delivery,” “specifically deliver.” or “specifically delivering” means delivery’ of more (e.g., at least 1.5 fold more, at least 2-fold more, at least 3- fold more, at least 4-fold more, at least 5-fold more, at least 6-fold more, at least 7-fold more, at least 8-fold more, at least 9-fold more, at least 10-fold more) of a cargo by a protein complex to a target cell type / tissue of interest (e.g., mammalian liver) compared to an off-target tissue e.g., mammalian spleen). It will be understood that the ability of a protein complex tospecifically deliver a cargo to a target cell type / tissue need not be determined in a subject being treated, it may be determined in a surrogate such as an animal model (e.g, a rat model).

[0355] The cell may be a bacterial cell, a yeast cell, an insect cell, or a mammalian cell. In one embodiment, the cell is a human cell. The cell may be isolated.

[0356] In one embodiment, the cell is an immune cell. In one embodiment, the cell is a lymphocyte. Lymphocytes are one of the subtypes of a white blood cell in a vertebrate’s immune system and include T cells. B cells, and natural killer (NK) cells. In one embodiment, the cell is a “tumor infiltrating lymphocyte (TIL)”. In one embodiment, the cell is a T cell. An illustrative antigen on a T cell that can be targeted by a protein complex disclosed herein is CD5. In one embodiment, the cell is a B cell. An illustrative antigen on a T cell that can be targeted by a protein complex disclosed herein is DEC205.

[0357] In one embodiment, the cell is a cancer cell. In one embodiment, the cell is an endothelial cell. In one embodiment, the cell is a hematopoietic stem cell.

[0358] In one embodiment, the cell is a cell that is usually difficult to transfect with a nanoparticle, such as a quiescent T cell.

[0359] The protein complexes disclosed herein can be used as a research tool. For example, the protein complex may comprise an LNP comprising an RNA encoding a reporter protein (e.g, a fluorescent protein). Cells targeted by the protein complex can be readily identified by the expression of the report protein.

[0360] Certain antigens, such as the SunTag, may be fused to different cell surface proteins. A protein complex comprising a bispecific antibody construct binding to the SunTag may be used to screen the targetability of different receptors for enhancing LNP uptake. See Fig. 2C for a non-limiting illustration.

[0361] In some embodiments, the method involves administration of a protein complex disclosed herein to a subject.

[0362] Provided is a method of making a protein complex, the method comprising contacting an LNP disclosed herein with a bispecific antibody construct disclosed herein, whereby the bispecific antibody construct non-covalently binds to the LNP.

[0363] Provided are methods of treating a subject having a disease or disorder by administering a therapeutically effective amount of a protein complex disclosed herein.

[0364] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological condition, disorder or disease, or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited toalleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (z.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms "‘prevent”, ‘'prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0365] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0366] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past, is currently undergoing any form of treatment, or is receiving treatment in the future. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to. a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In some embodiments, the subject is a human. In some embodiments, the subject is immune-depleted.

[0367] A person skilled in the art can readily choose an appropriate protein complex for the treatment of a specific disease. The cargo to be delivered by the protein complex and the antigen to be targeted by the protein complex will differ for different diseases.

[0368] For example, the protein complex can be used to deliver a therapeutic or a toxic agent to a target cell. As a specific example, the protein complex can be used to deliver an anticancer agent to a cancer cell. Alternatively, the protein complex may be used to deliver an antiviral agent to a cell infected by a virus (e.g., the cell displays a viral peptide on its surface in an MHC). In another embodiment, the protein complex can be used to delivery RNAi therapeutics to a target cell reduce the expression of disease-causing genes.

[0369] The compositions and methods provided herein are also useful for creating vaccines. For example, in the case of mRNA vaccines, the mRNA is taken up by cells in a subject’s body. The cell expresses the polypeptide encoded by the mRNA (e.g., the spike protein found on the surface of the coronavirus). The subject’s immune system recognizes the polypeptide as foreign and mounts an immune response against it, producing antibodies and memory cells. As such, the protein complex disclosed herein maybe be used to deliver a nucleic acid encoding for a polypeptide derived from a pathogen to a cell in a subject.

[0370] A person skilled in the art will appreciate that the protein complexes disclosed herein are suitable for a magnitude of applications, both in the research and in the therapeutic context.

[0371] Kits

[0372] Further embodiments disclosed herein can concern kits for use with methods and compositions. Kits can also include a suitable container, for example, vials, tubes, mini- or microfuge tubes, test tube, flask, bottle, syringe, or other containers. The disclosed kits can optionally include pharmaceutically acceptable carriers and / or diluents. In one embodiment, a kit includes one or more other components, adjuncts, or adjuvants as described herein. In one embodiment, a kit includes instructions or packaging materials that describe how to administer a compound or composition of the kit. Containers of the kit can be of any suitable material, e.g., glass, plastic, metal, etc., and of any suitable size, shape, or configuration. Where an additional component or agent is provided, the kit can contain one or more additional containers into which this agent or component may be placed. The kit may include instructions.

[0373] Kits herein will also typically include a means for containing a protein complex, an LNP, or a bispecific antibody construct disclosed herein and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.

[0374] It is contemplated that the disclosed kits can be used in connection with the disclosed methods of making, the disclosed methods of using, and / or the disclosed compositions.

[0375] Illustrative embodiments

[0376] Provided herein are illustrative embodiments that do not limit the claimed invention.

[0377] Embodiment 1. A method of making a protein complex, the method comprising contacting an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen, with a bispecificantibody construct comprising a binding site for the first antigen and a binding site for a second antigen.

[0378] Embodiment 2. The method of embodiment 1, wherein the first antigen is about 6 to about 20 amino acids in length.

[0379] Embodiment 3. The method of embodiment 1, wherein the first antigen comprises a peptide comprising an HA peptide, a FLAG tag, a Pep-1 peptide, or a Pep-2 peptide, preferably an HA peptide.

[0380] Embodiment 4. The method of embodiment 1, wherein the first antigen comprises a peptide comprising any one of SEQ ID NOs:44, 46, 49, 51, or 52, preferably, SEQ ID NO:44 or 49.

[0381] Embodiment 5. The method of any one of embodiments 1-4, wherein the wherein the lipid molecule is a PEGylated lipid molecule and wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule.

[0382] Embodiment 6. The method of embodiment 5, wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a linker.

[0383] Embodiment 7. The method of embodiment 6. wherein the linker is a polyglycine linker.

[0384] Embodiment 8. The method of embodiment 7, wherein the polyglycine linker essentially consists of GGG.

[0385] Embodiment 9. The method of any one of embodiments 1-8. wherein the lipid molecule comprises a 14C to 20C fatty acid tail.

[0386] Embodiment 10. The method of embodiment 9, wherein the lipid molecule comprises a 16C fatty' acid tail.

[0387] Embodiment 11. The method of embodiment 10, wherein the modified lipid comprises 1 ,2-distearoyl-sn-gly cero-3-phosphoethanolamine-N-PEG.

[0388] Embodiment 12. The method of any one of embodiments 1-11, wherein the bispecific antibody construct comprises:(a) two or more of a Fab, Fab', single domain antibody, (Fab')2 / Fab2, single-chain Fv (scFv), single chain Fab (scFab), minibody. scFv-Fc; and / or(b) one or more of a (Fab')2 / Fab2, minibody, di-ScFv, scFv-Fc, bi-scFv, Dual-Affinity ReTargeting (DART), dual-action Fab (DAF), H-chain heterodimer with orthogonal Fab interfaces, CrossMab, Dual Variable Domain Immunoglobulin G (DVD IgG), bispecific T-cell engager (BiTE), di-diabody, tandem diabody (TandAb). (scFv)2-HSA, tetravalent-IgG (Tv- IgG).

[0389] Embodiment 13. The method of any one of embodiments 1-11, wherein the bispecific antibody construct comprises a first scFv and a second scFv. wherein the first scFv comprises a binding site for the first antigen and the second scFv comprises a binding site for the second antigen.

[0390] Embodiment 14. The method of embodiment 13, wherein the first scFv and the second scFv are connected via a linker.

[0391] Embodiment 15. The method of embodiment 14, wherein the linker is a polypeptide linker, optionally wherein the polypeptide linker is a flexible linker.

[0392] Embodiment 16. The method of any one of embodiments 1-15, wherein the second antigen is presented on the surface of a cell.

[0393] Embodiment 17. The method of embodiment 16, wherein the second antigen is presented on the surface of the cell in the context of an MHC.

[0394] Embodiment 18. The method of embodiment 16, wherein the second antigen is a polypeptide expressed on the surface of the cell.

[0395] Embodiment 19. The method of any one of embodiments 16-18. wherein the cell is a mammalian cell.

[0396] Embodiment 20. The method of any one of embodiments 18 or 19, wherein the cell is a T cell, a B cell, or a hematopoietic stem cell.

[0397] Embodiment 21. The method of any one of embodiments 16-20, wherein the cell is a human cell.

[0398] Embodiment 22. The method of embodiment 18 or 21, wherein the polypeptide expressed on the surface of the cell is selected from the group consisting of PD-1, PD-L1, CD3, CD4, CD5. CD8, CD45, and DEC205.

[0399] Embodiment 23. A method of making a protein complex, the method comprising contacting a bispecific antibody construct comprising a binding site for a first antigen and a binding site for a second antigen with:(a) an LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to the first antigen; and(b) an antibody that comprises a constant region and a binding site for a third antigen, wherein the second antigen is the constant region.

[0400] Embodiment 24. The method of embodiment 23, wherein the first antigen is about 6 to about 20 amino acids in length.

[0401] Embodiment 25. The method of embodiment 23, wherein the first antigen comprises a peptide comprising an HA peptide, a FLAG tag, a Pep-1 peptide, or a Pep-2 peptide, preferably an HA peptide.

[0402] Embodiment 26. The method of embodiment 23, wherein the first antigen comprises a peptide comprising any one of SEQ ID NOs:44, 46, 49, 51, or 52, preferably, SEQ ID NO:44 or SEQ ID NO:49.

[0403] Embodiment 27. The method of any one of embodiments 23-26, wherein the wherein the lipid molecule is a PEGylated lipid molecule and wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule.

[0404] Embodiment 28. The method of embodiment 1 , wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a linker.

[0405] Embodiment 29. The method of embodiment 28, wherein the linker is a polyglycine linker.

[0406] Embodiment 30. The method of embodiment 29, wherein the polyglycine linker essentially consists of GGG.

[0407] Embodiment 31. The method of any one of embodiments 23-30, wherein the lipid molecule comprises a 14C to 20C fatty acid tail.

[0408] Embodiment 32. The method of embodiment 31, wherein the lipid molecule comprises a 16C fatty acid tail.

[0409] Embodiment 33. The method of embodiment 31, wherein the modified lipid comprises l ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG.

[0410] Embodiment 34. The method of any one of embodiments 23-33, wherein the bispecific antibody construct comprises:(a) two or more of a Fab, Fab', single domain antibody, (Fab')2 / Fab2, single-chain Fv (scFv), single chain Fab (scFab), minibody, scFv-Fc; and / or(b) one or more of a (Fab')2 / Fab2, minibody, di-ScFv, scFv-Fc, bi-scFv, Dual-Affinity ReTargeting (DART), dual-action Fab (DAF), H-chain heterodimer with orthogonal Fab interfaces, CrossMab, Dual Variable Domain Immunoglobulin G (DVD IgG), bispecific T-cell engager (BiTE), di-diabody. tandem diabody (TandAb). (scFv)2-HSA, tetravalent-IgG (Tv- IgG).

[0411] Embodiment 35. The method of any one of embodiments 23-33, wherein the bispecific antibody construct comprises a first scFv and a second scFv. wherein the first scFv comprises a binding site for the first antigen and the second scFv comprises a binding site for the second antigen.

[0412] Embodiment 36. The method of embodiment 35, wherein the first scFv and the second scFv are connected via a linker.

[0413] Embodiment 37. The method of embodiment 36, wherein the linker is a polypeptide linker, optionally wherein the polypeptide linker is a flexible linker.

[0414] Embodiment 38. The method of any one of embodiments 23-37, wherein the third antigen is presented on the surface of a cell.

[0415] Embodiment 39. The method of embodiment 38. wherein the third antigen is presented on the surface of the cell in the context of an MHC.

[0416] Embodiment 40. The method of embodiment 38, wherein the third antigen is a polypeptide expressed on the surface of the cell.

[0417] Embodiment 41. The method of any one of embodiments 38-40. wherein the cell is a mammalian cell.

[0418] Embodiment 42. The method of any one of embodiments 40 or 41, wherein the cell is a T cell, a B cell, or a hematopoietic stem cell.

[0419] Embodiment 43. The method of any one of embodiments 38-42. wherein the cell is a human cell.

[0420] Embodiment 44. The method of embodiment 40 or 43, wherein the polypeptide expressed on the surface of the cell is selected from the group consisting of PD-1, PD-L1, CD3, CD4, CD5. CD8, CD45, and DEC205.

[0421] Embodiment 45. The method of any one of embodiments 23-44, wherein the cargo comprises a nucleic acid, a polypeptide, or a small molecule.

[0422] Embodiment 46. The method of embodiment 45, wherein the nucleic acid comprises a therapeutic RNA molecule.

[0423] Embodiment 47. The method of embodiment 46, wherein the therapeutic RNA molecule is an siRNA, mRNA, siRNA, dsRNA, miRNA, or shRNA.

[0424] It is to be understood that this disclosure is not limited to the particular molecules, compositions, methodologies, or protocols described, as these may vary. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments disclosed herein. It is further to be understood that this disclosure includes all possible combinations of such particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the disclosure, or a particular claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other particular aspects and embodiments disclosed herein.

[0425] Where reference is made herein to a method comprising two or more defined steps, the defined steps can be carried out in any order or simultaneously (except where the context excludes that possibility), and the method can include one or more other steps which are carried out before any of the defined steps, between two of the defined steps, or after all the defined steps (except where the context excludes those possibilities).

[0426] All other referenced patents and applications, scientific articles, book chapters, etc. are incorporated herein by reference in their entireties. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0427] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. The following examples should not be read to limit or define the entire scope of the disclosure.EXAMPLES

[0428] Example 1: Materials and Methods for Examples 2-11

[0429] Lipids

[0430] SM-102 (CAS# 2089251-47-6), ALC-0315 (CAS#: 2036272-55-4) and ALC-0159(CAS#: 1849616-42-7) were purchased from MedKoo Biosciences. (6Z,9Z,28Z,31Z)- Heptatriaconta-6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (D-Lin-MC3-DMA) (Cat# 1224606-06-7) was purchased from Broad Pharm. Cholesterol (CAS# 57-88-5) was purchased from Millipore Sigma. 1.2-dimyristoyl-rac-glycero-3-methypolyoxyethylene (DMG-PEG2000) was purchased from NOF America Corporation. 1.2-disteraroyl-sn-glycero- 3-phosphocholine (DSPC) (SKU# 850365P-25mg), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine 18: 1 (A9-Cis) PE (DOPE) and l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000 Maleimide, PEGNlal) (SKU# 880126P-10mg), were purchased from Avanti Polar Lipids. Two variants of the HA tags were used (C-GGG-YPYDVPDYA, SEQ ID NO:47) and C-GGG- YPDDVPDYA (SEQ ID NO:50) No difference in transfection efficiency was observed. The HA tags were synthesized as peptide-DSPE-PEG2000-Mal conjugation on Cys (PEGIIA).

[0431] Chemicals

[0432] Pierce® SATA (N-succinimidyl S-acetylthioacetate) (Cat#26102) and Pierce® Hydroxylamine-HCl (Cat#26103) were purchased from ThermoFisher Scientific. Buffered formaldehyde (4%, J60401-AK) was purchased from Thermo Fischer Scientific. O. lx TE was obtained from Maxi Qiagen kit #12362 purchased from Qiagen. 0.25M CaC12 (Cat#C7902- 1KG) was purchased from Sigma Aldrich. 2x HBS was prepared in house. For 500mL: IM HEPES (50ml Coming #25-060-Cl), 2M NaCl (70.25ml Fisher Bioreagents #BP358-1); 0.5M Na2HPO4 (1.5ml # BP332-500), 378.25ml Tissue Culture Tested Water (Coming #46-000- CV), 5M NaOH to pH. Quant-it™ RiboGreen RNA Assay Kit and RiboGreen RNA Reagent was purchased from ThermoFisher Scientific (Cat# R11491).

[0433] Cells and cell culture conditions

[0434] 293T cells (embryonic kidney; human) and ID8 Brcal -deficient cells (ovarian adenocarcinoma; C57BL / 6 mice origin) were grown in IMDM with 10% heat-inactivated fetal bovine serum (FBS), lOO U / ml penicillin / streptomycin and 2 mM L-Glutamine. 293T cells were purchased from ATCC (#CRL-3216). ID8 Brcal -deficient cell line was provided by Jean J. Zhao (Harvard). B16-F10 cells (C57BL / 6 mouse; ATCC #CRL-6475) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing heat-inactivated 10% FBS, 100 U / mlpenicillin / streptomycin and 2 mM L-Glutamine. K562 (human; ATCC #CCL-243™) and BCL-1 (BALB / c mice; ATCC #TIB-197 ™) were cultured in Roswell Park Memorial Institute (RPMI) medium containing 10% heat-inactivated FBS, 100 U / ml penicillin / streptomycin and 2 mM L-Glutamine. The KC pancreatic cancer cell line was cultured in DMEM containing heat-inactivated 10% FBS, 100 U / ml penicillin / streptomycin and 2 mM L-Glutamine. MEL cell line was cultured in RPMI containing heat-inactivated 10% FBS, 100 U / ml penicillin / streptomycin and 2 mM L-Glutamine.

[0435] Ail 4 splenocytes were cultured in RPMI 10% FBS with 500 U ml-l IL-2, 2- Mercaptoethanol (Thermo Fisher Scientific, Cat # 21985023), and activated with Gibco™ Dynabeads™ Mouse T-Activator CD3 / CD28 (Fisher Scientific. Cat # 11452-D) for 2 days prior LNP treatment. Human primary T lymphocytes were isolated from peripheral blood mononuclear cells of healthy donors by leukapheresis and Ficoll-Hypaque gradient separation. Cells were enriched to purity using EasySep™ Human T Cell Isolation Kit (Cat# 17951) according to manufacturer instructions. Unstimulated T cells were cultured at a concentration of 1 * 106cells / ml in RPMI supplemented with penicillin, streptomycin. 10% FBS and and 2-Mercaptoethanol. Stimulated T cells were cultured in presence of recombinant human IL-2 500 U ml-l (Peprotech) and Gibco™ Dynabeads™ Human T-Activator CD3 / CD28 (Fisher Scientific, Cat # 11161-D) for 2 days before LNP treatment.

[0436] Murine B cells were isolated from the spleens of donor mice using a negative selection kit (EasySep Mouse B cell Isolation Kit. StemCell Technologies cat # 19854) according to the manufacturer’s instructions. No ACK lysis was performed. The enriched B cell fractions were then used to prepare a 1 : 1 mix of DEC-205 + CD45.2 B cells and DEC -205 KO B cells, and 2.5x105 total B cells were plated into each well (48-well plates) with 1 mL of growth medium (Advanced RPMI + 10% FBS + Glutamax + 2-Mercaptoethanol). along with recombinant BAFF (R&D Systems cat #8876-BF-010) or anti-CD40 antibody (clone HM40- 31, 1 microgram / mL, Thermofisher Scientific cat #16-0402-85) and recombinant murine interleukin 4 (mIL-4, 40 ng / mL, Peprotech cat #214-14). LNP (wildtype or HA) coated or uncoated with anti-DEC-205-anti-HA bsAB were then added to the culture. The cells were incubated in a controlled environment (5% CO2) and analyzed 24 hours later via flow cytometry.

[0437] Mouse Strains

[0438] In vivo studies were performed using C57BL / 6 (J AX# 000664) and Ail 4 (J AX# 007914) mice obtained from Jackson laboratories and housed in the Mount Sinai vivarium during use. Experiments targeting B cells were performed using Bl-8hi (CD45.2) (JAX#007594) and Ly75- / - (also known as DEC-205 KO)(CD45.1) (J AX# 005528) congenic mice, all backcrossed for more than 10 generations on a C57B1 / 6 background. Bl-8hi and Ly75- / - mice provided by Michel C. Nussenzweig and Gabriel D. Victora (The Rockefeller University). All mouse experiments were carried out under institutional IACUC approval (IACUC-2014- 0015; IACUC-2018-0070). All mice were randomized before experimentation.

[0439] Lentiviral vector production

[0440] Briefly, HEK293T cells were seeded in 15 cm tissue culture plates (Thermo Scientific Nunclon #168381) 24 hours prior to achieve an approximate cell density of 70% at the time of transfection. Transfection was carried out using the calcium-phosphate method. Reporter plasmid constructs were mixed with packaging plasmids (Gag, Rev, Pol) and suspended in 0. lx TE and 0.25M CaC12; one volume of 2x EIBS was added in a dropwise fashion while continually vortexing, and the resulting solution was immediately added onto HEK293T cells and allowed to sit overnight. IMDM medium was replaced the next morning and supernatants collected and 0.2 pm-filtered 24-30h after that. Lentivirus supernatant aliquots were stored at -80°C until use.

[0441] Bispecific antibody construct (bsAb) production and purification

[0442] All bsAbs were produced using the Expi293T™ expression Kit (Gibco, Cat #A14635) according to the manufacturer instructions. For all transfections a ratio of 1.0 ug plasmid DNA / mL of transfection culture was used. Transfections w ere performed on Expi293 cells in a final volume of 30 mL in 125 mL non-baffled flasks (Greiner Bio-One, Cat. 679501). Cultures were maintained on a CO2 resistant shaker with an orbit of 19 mm (Thermo Fisher, Cat. 88881101). Expi293F cells were cultured in Expi293 Expression Medium (Gibco, Cat. A1435101) supplemented with 1% penicillin-streptomycin (Gibco, 15140-122) in ahumidified 8% CO2 incubator at 37°C and 125 RPM. Supernatant containing bsAb was harvested 4 days post-transfection, centrifuged at 3000 x g for 25 minutes at 4°C and filtered through a 0.2 pM PES filter (Thermo Scientific, Cat. 564-0020).

[0443] bsAb possessing a His tag were purified using Ni-NTA agarose (QIAGEN, Cat. 30210) with gravity flow columns (Marvelgent Biosciences Inc., Cat 12-0278-050). 2.5 mL of Ni-NTA agarose were used for every 30 ml supernatant. Briefly, Ni-NTA agarose was washed 3 times with molecular biology grade water (Coming, Cat. 46-000-CV). The supernatant from the transfection was incubated with the Ni-NTA agarose in slow agitation for at least 60 minutes at 4°C. 1 L of binding buffer was prepared by mixing 25 mL of 1 M Tris-HCL with a pH of 7.5 (Fisher Bioreagents. Cat. BP1757-500). 200 mL of 2.5 M NaCl (Fisher Bioreagents, Cat. BP358-1), 50 mL of glycerol (Research Products International, Cat. G22025-0.5), 70 pLof 2-mercaptoethanol (Gibco, Cat. 21985023), 1 mL of 5 M imidazole (Fisher Bioreagents, 03196-500), and 723.93 mL of molecular biology grade water. 1 L of 1 M imidazole elution buffer was prepared by mixing 25 mL of 1 M Tris-HCL with a pH of 7.5, 200 mL of 2.5 M NaCl, 50 mL of glycerol, 70 pL of 2-mercaptoethanol, 200 mL of 5 M imidazole, and 524.93 mL of molecular biology' grade water. All solid reagents had been resuspended in molecular biology grade water to reach the indicated concentration. The binding and elution buffers were mixed to create elution buffers with varying imidazole concentrations as follows: 10 mM. 15 mM, 50 mM, 250 mM, and 500 mM. A gravity flow column was equilibrated with 20 mL of binding buffer. The supematant / agarose mixture was passed through the column and the flow through was collected. The column was then washed twice with 10 mL of binding buffer; 10 mL each of 10 mM and 15 mM imidazole elution buffers; and 6 mL each of 250 mM, 500 mM, and 1 M imidazole elution buffers. Each wash fraction was collected in separate conical tubes and was analyzed by loading a sample onto an SDS-PAGE gel. Fractions that contained the bsAb were combined and concentrated to a final volume of 1 mL by centrifuging at 6,000 x g at 4°C in a Vivaspin 20 Centrifugal Concentrator (Sartorius, Cat. VS2002). The concentrated bsAb was placed in an equilibrated dialysis cassette (Thermo Scientific. Cat. A52966) and dialyzed in 500 mL of PBS (Coming, Cat. 21-040-CV) overnight at 4°C. Dialyzed bsAb were then quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific) and stored in 10% glycerol at -80°C.

[0444] Cloning of DNA template for RNA in vitro transcription

[0445] Templates for in vitro transcription (IVT) were generated by restriction cloning using a T7 promoter sequence containing 5’UTR, an open reading frame, and 3’UTR. Our construct employed NASAR UTRs for enhanced gene expression. The addition of a 120- nulcleotide poly-A tail to the mRNAs was encoded on the PCR template with a reverse primer containing a 120-nucleotide poly-T. To generate IVT template from plasmid, we amplified DNA with Q5® High-Fidelity Master Mix (NEB, M0492). Following PCR, RNA was incubated at 37°C for 30 minutes with Dpnl (NEB, R0176) to remove plasmid contaminants and PCR product was cleaned up with QIAquick® PCR Purification Kit (Qiagen). Concentration was measured using the NanoDrop™ 2000 (Thermo Scientific) for application in IVT.

[0446] Synthesis and purification of IVT generated modified mRNA

[0447] All mRNAs were synthesized using HiScribe® T7 High Yield RNA Synthesis Kit (NEB). UTP was fully substituted with Nlm (Nl-methylpseudouridine-5'-triphosphate) (Crystal Chem Cat# 13946). mRNA was capped by inclusion of 1 :4 premix of CleanCap®reagent AG (TriLink) and capping occurred co-transcriptionally. The reaction mixtures were incubated at 37°C for 6 hours and further incubated at 37°C for 30 min in the presence of TURBO DNasel (Thermo Scientific). RNA products were purified with Monarch® RNA Cleanup Kit (NEB, T2050). Concentration of RNA was measured using the NanoDrop™ 2000 (Thermo Scientific) and species purity and size was assessed on a denaturing RNA gel.

[0448] Lipid nanoparticle formulation and modified mRNA encapsulation

[0449] mRNA encapsulation in LNP was performed using the NanoAssembler™Ignite™ microfluidic mixing device (Cytiva).

[0450] SM-102-based LNPs were formulated with the helper lipid DSPC, cholesterol, DMG-PEG2000 (molar ratio 50:10:38.5: 1.5) and mRNA dissolved in a citrate buffer.

[0451] ALC-0315 -based LNPs were formulated with ALC-0315. DOPE, cholesterol, and ALC-0159 PEG (molar ratio 46.3:9.4:42.7: 1.6) and mRNA dissolved in a citrate buffer.

[0452] TT3-based LNPs were formulated with TT3, DOPE, cholesterol, and DSG-C14- PEG 2K (molar ratio 20:30:40:0.75) and mRNA dissolved in a citrate buffer.

[0453] D-Lin-MC3-DMA-based LNPs were formulated with D-Lin-MC3-DMA, DSPC, cholesterol, DMG-PEG2000 (molar ratio 50: 10:38.5: 1.5) and mRNA dissolved in a citrate buffer.

[0454] LNPHAvariants of these particles were also modified to include 15% PEGHA(CGGGYPYDVPDYA (SEQ ID NO:47) DSPE-PEG2000-Mal conjugation on Cys or CGGGYPDDVPDYA (SEQ ID NO:50) DSPE-PEG2000-Mal conjugation on Cys). PEG-HA contains a 16C fatty acid tail, the HA-peptide sequence and a small poly-glycine linker sequence (GGG).Table 2. Illustrative LNP compositions.

[0455] After formulation, the freshly formed RNA-LNPs were dialyzed overnight against PBS buffer using Slide-A-Lyzer dialysis cassettes (3.5K MWCO, Life Technologies) and subsequently concentrated in Amicon® Ultra Centrifugal Filters (10 kDa MWCO, Sigma) to desired concentration. Particle size and zeta potential of LNPs were measured using a Zetasizer Advance (Malvern Panalytical) at a scattering angle of 173° and a temperature of 25°C. All formulations were within the following parameters: 60-100 nm average size, >90% encapsulation, polydispersity <0.2.

[0456] Encapsulation efficiency of LNPs was determined using Quant-it™ RiboGreen RNA Assay Kit according to manufacturer protocols. The concentration of LNP-mRNA was defined by the amount of mRNA encapsulated in the LNP. In general, we prepared LNP- mRNA to have a final concentration of mRNA of 0.1 mg / ml.

[0457] Generation ofLNPHA / bsAb complex

[0458] mRNA-loaded LNPHAand the corresponding bsAb were either pre-incubated together for 15 minutes or simultaneously added to the cell culture at a 1 :4 weight ratio (e.g. for 1 pg LNP-mRNA (i.e. 1 pg RNA) we incubated with 4 pg of bsAb). For in vivo studies, LNPHAand bsAb were pre-incubated for 15 minutes before injection at a 1: 1 weight ratio (e.g. for 1 pg of LNP-mRNA we incubated with 1 pg of bsAb).

[0459] Generation ofLNPMal'Ab

[0460] For LNPMalpreparation, 10% of total PEG in SM-102 LNP was substituted with PEGMal. Targeting antibodies or control isotype-matched IgG was functionalized with SATA to introduce sulfhydryl groups allowing conjugation to maleimide. SATA was deprotected using 0.5 M hydroxylamine followed by purification by removing the unreacted components by Amicon® Ultra Centrifugal Filters (10 kDa MWCO, Sigma). The reactive sulfhydryl group on the antibody was then conjugated to LNP'',althrough thioether conjugation chemistry by overnight incubation at 4°C.

[0461] In vitro cell transfection

[0462] K562 and BCL-1 cells were plated at 103cells / well in a clear, flat-bottom 96-well plate. Cells were treated with lOng / well LNP-mRNA and 40 ng / well bsAb. if not otherwise specified. 293T cells were plated at 105cells / well in 24 well plate. Cells were treated with Ing / well LNP-mRNA and 4 ng / well bsAb. Isolated murine T cells were plated at 2x105cells / well in a clear, flat-bottom 96-well plate and treated with 50 ng / well LNP-mRNA and 200 ng / w-ell bsAb if not otherwise specified. Isolated human T cells were plated at 2x105cells / well in a clear, flat-bottom 96-well plate. For both unstimulated and stimulated T cells, cells were treated with 200ng / well LNP-mRNA and 800ng / well bsAb. For experiments withmurine B cells 2.5xl05cells (1 : 1 mix of DEC-205 + CD45.2 B cells and DEC-205 KO B cells), were plated onto individual wells (clear, flat bottom 48-well plates) in 1 mL of growth medium and cytokines. LNP (WT or HA) coated or uncoated with anti-DEC-205-anti-HA bsAB were added to each well (0.5-2 micrograms of LNP according to the experiment). All cells were harvested and analyzed 24 hours post treatment via flow cytometry.

[0463] Animal experimental

[0464] For in vivo experiments, Ail4 mice were i.v injected with 1 mg / kg Cre mRNA encapsulated in LNP, which was pre-incubated with bsAbHA'CD4at ratio 1 : 1 (weight / weight). At experimental endpoints, mice were sacrificed, and spleens were collected for flow cytometry analysis.

[0465] To generate disseminated ovarian tumors, 5xl06ID8 cells were injected intraperitoneally (i.p.) into 8-10 weeks old female C57BL / 6J mice. At 14 days post-tumor injection, mice were i.p. injected with Img / kg Cre mRNA encapsulated in LNPHAplus or minus bsAb. Mice were euthanized upon ascites development and tumor tissue from the omentum was collected and processed for flow cytometry.

[0466] For the melanoma model, 105B16F10 melanoma cellswere subcutaneously (s.c.) injected into -10 weeks old female C57BL / 6 mice. Mice were randomly grouped and when the tumor reached ~50 mm3- (~1 week), 10 ug Cre mRNA encapsulated in LNPHAwere injected intratumorally plus or minus bsAb. At 2 days after LNP -mRNA injection, tumor tissue was collected and processed for flow cytometry.

[0467] For the orthotopic pancreatic cancer model, tumor cells derived from KrasLSL"Gi2D / +;ptfla&e / + (KC) mice were used. In brief, 5xl04were orthotopically grafted into the pancreas of wildtype C57BL / 6J mice. One week after tumor injection mice were i.p. injected with 1 mg / kg Cre mRNA encapsulated in LNP or LNPHAplus / minus bsAb. At 2 days after LNP injection, mice were euthanized and tumor tissue collected and processed for flow cytometry as described in the "Flow Cytometry” section.

[0468] For experiments targeting murine germinal center B cells, splenic B cells were isolated from donor mouse spleens (Bl-8hi, CD45.2, DEC -205+) using a negative selection kit (Easy Sep Mouse B cell Isolation Kit, StemCell Technologies cat # 19854). No ACK lysis was performed. 106 total B cells were w ashed, resuspended in 150 microliters of sterile PBS, and adoptively transferred into recipient CD45.1 DEC-KO mice via retroorbital injection under isoflurane anesthesia. The next day, each mouse was immunized with 25 micrograms of NP- OVA (BioSearch Technologies cat# N-5051-10) in PBS and precipitated in alum adjuvant 1 : 1 (Alhydrogel 2%, Invivogen cat# vac-alu-50) to induce germinal center formation in thedraining lymph nodes. While this strategy' recruits Bl-8hi B cells (DEC -205+) into germinal centers, a significant fraction of CD45.1 DEC-KO cells was also detected (-20%). Ten days post-immunization, we injected LNP-bsAb complexes diluted in PBS into the same sites (50 microliters total volume). LNP and bsAb had been preincubated for 10 minutes at room temperature before injection. 24 hours later, we terminated all mice and isolated the draining lymph nodes for analysis.

[0469] Flow cytometry

[0470] For flow cytometry analysis of ID8 tumors in the omentum and B16F10 melanomas, the tumor-bearing tissue was collected from mice and chopped into small pieces wjith scissors in digest media containing collagenase and DNAase (Sigma Aldrich) and then incubated in the same media with 500 RPM lateral shaking at 37°C for 45 min. The single cell suspension was obtained by passaging the digested tumors through a 70 pm filter and washing with PBS. Cells were centrifuged at 350g for 5 minutes at 4°C and cell pellets were resuspended with ACK lysis buffer (Life Technologies) to lyse red blood cells at room temperature for 10 minure and washed with cold PBS. Flow cytometry samples were acquired with BD LSRFortessa II (BD Biosciences) and cell sorting was performed with BD FACSAria III Sorter (BD Biosciences). Pancreatic tumors were chopped into small pieces and processed.

[0471] For analysis of tdTomato expression in T cells from Ail4 mice injected with bsAbHA"CD4 / LNP, spleens were collected and minced using a sterile blade, homogenized by smashing, and filtered through a 70-pm cell strainer. The tissue solution was washed once with PBS and pelleted by centrifuging for 5 minutes at 350g. The supernatant was removed, and the cell pellet was resuspended in 1.5mL of lx RBC lysis buffer. After incubation, 15 rnL of PBS was added to stop red blood cell lysis. The solution was centrifuged again at 350g for 5 minute to obtain a cell pellet. The single cells were suspended in a cell staining buffer and added to flow tubes that contained antibodies (total volume 100 pL). The spleen cells were analyzed using a LSRFortessa X-20 (BD Biosciences) for tdTomato.

[0472] For analysis or sorting of cells in vitro, adherent cells were detached with 0.05% tiypsin-EDTA and resuspended in the flow buffer (PBS with 2% BSA).

[0473] For the analysis of B cell populations in draining lymph nodes, mononuclear cell suspensions were isolated in cell isolation buffer (PBS+2% FBS+lmM EDTA) and filtered through 70-micron cell strainers, then incubated for 2 minutes in ACK lysing buffer (ThermoFisher Scientific) to eliminate residual RBCs. Cells from each pair of draining lymph nodes (popliteal+inguinal) were resuspended in 50 microliters of buffer containing Fc block (1 microgram / mL), incubated for 10 minutes on ice, and then we added a 2X primary antibodycocktail resuspended in 50 microliters. The cells were then incubated for 30 minutes on ice before washing and resuspending for flow analysis. All centrifugations were perfomed at 350xg for 5 minutes at 4 degrees.

[0474] Germinal center B cells were identified at B220+, IgD-, CD381o, CD95hi. Naive B cells as B220+, IgD+. DEC-205+ and DEC-205 KO B cells were distinguished based on their expression of congenic markers (CD45.1 vs CD45.2).

[0475] Quantification and statistical analysis

[0476] Statistical values including the number of replicates and statistical significance are reported in the figure or figure legends when appropriate. For the majority of in vivo experiments, the experiments were repeated at least two separate times with different cohorts of mice, synthesized RNA. and encapsulated and quantified RNA-LNP. Statistical analysis was performed using Microsoft Excel or GraphPad Prism 8 (GraphPad Software Inc). Flow cytometry analysis was performed using FlowJo software. The levels of significant (unpaired two-tailed student’s t-test, one-way and two-way ANOVA) are denoted as *p<0.05, **p<0.01, ***p<0 001 and ****p<0.001.

[0477] Example 2: Generation of linear epitope binding bispecific antibody as a tool compound

[0478] A bispecific antibody construct (bsAb) was generated that recognized linear epitopes from an HA tag and a SunTag. Both are short linear epitopes that can easily be fused to other molecules, including potentially PEG. The HA-tag is a protein tag derived from amino acids 98-106 of the human influenza hemagglutinin (HA) protein. See Table 1.

[0479] The bsAb (z.e., bsAbHA’SunTag) was created by cloning nucleic acid sequences encoding for two single chain variable fragments (scFv) derived from the antigen binding domain (Fab) portions of anti-SunTag and anti-HA antibodies, separated by a 218s linker variant (PGGSTSGSGKPGSEGSTKGAS, SEQ ID NO:39) (Fig. 1A and Table 3). See Whitlow et al., An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability, Protein Eng. 1993 Nov;6(8):989-95. The anti-HA scFv was derived from Zhao et al.. A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo, Nat Commun. 2019 Jul 3;10(l):2947. A mouse IgG signal peptide was included forthe secretion of the bsAb. A histidine tag was included for purification.

[0480] Expi293 cells were transfected with the expression plasmids and cultured for 72 hours to allow for sufficient production of the construct. The cell supernatant was collected andconcentrated by nickel column purification. Protein integrity was verified by SDS-PAGE and Westem-Blot (data not shown).Table 3. Illustrative amino acid sequences.

[0481] To test the functionality of both binding arms of the bsAb, a cell-cell aggregation assay was developed to assess the ability of the bsAbIIA‘SunTagto bring HA and SunTag expressing cells together. To that end, two lines of Mouse Erythroleukemia (MEL) cells were engineered. The first cell line expressed enhanced green fluorescent protein (GFP) and a truncated neurotrophic growth factor receptor (ANGFR) with SunTag fused to the N-terminus (MELGFP SunTag). The other cell line expressed mCherry fluorescent protein (mCherry) and ANGFR with HA fused to the N-terminus (MELcheny,HA) (Fig. IB). 105cells of each line were co-cultured at a 1: 1 ratio. bsAbHA'SunTagwas added at a dose ranging from 0.065 to 128 ng / ul. Cells were then incubated for 48 hours, after which they were analyzed by fluorescent microscopy and Amnis® ImageStream Mkll flow cytometry.

[0482] Visual inspection revealed that in sham treated conditions, GFP+and mCherry+cells were mostly separated single cells. In contrast, in cultures cultured with the bsAbHA'SunTag, there was a noticeable degree of mCherry and GFP+cell-cell aggregation (data not shown). A significant increase in cell aggregation in the bsAbHA'SunTagcultures was confirmed using ImageStream analysis, by quantifying the total area of GFP and mCherry double positive clusters (Fig. 1C and Fig. ID). The bsAbHA'SunTagworked particularly well within a range from 0.065 ng / ul to 1.875 ng / ul, with a predictable reduction in efficiency at higher doses (>3.75 ng / ul). This was indicative of a Hook effect, in which all antigen was bound by either antigenbinding domain of an antibody, which prevents cells from coming into contact with one another.

[0483] To test how other linear epitopes might perform as a bsAb target, the scFv of SunTag was substituted with the scFv of a Flag Tag antibody (hybridoma clone 4E11) (bsAbHA'Flag). Results from the cell aggregation assay showed that bsAbHA'Flagalso mediated an increase in aggregation of target expressing cells, in this case MELGFP,Flagand MELcherry HAcells (Fig. IE and Fig. IF).

[0484] These results demonstrate the generation and validation of linear epitope-targeting bsAbs capable of binding their targets and promoting cell -cell clustering.

[0485] Example 3: BsAb mediate cell-specific targeting of LNPs

[0486] Next, it was tested whether the bsAbHA‘SunTagcould be attached to the surface of an LNP modified to present the HA peptide and whether these particles could be used to enhance targeting to and transfection of SunTag expressing cell (Fig. 2A and Fig. 2B).

[0487] HEK293T cells and K562 leukemia cells were engineered to co-express a blue fluorescent protein (BFP) reporter and a recombinant fusion of human PD-L1 with the SunTag in the N-terminal (PD-L1 -SunTag) (Fig. 2C). PD-L1 was chosen because it is a molecule that is highly upregulated on cells within tumors, and sen es to promote T cell exhaustion and immune evasion. However, alternative fusion proteins to present an antigen recognized by the bsAb could have been used. BFP was used as a reporter to enable monitoring LNPIIAtransfection of PD-Ll / SunTag positive cells without the need for staining for PD-L1 , whose signal can be impaired by endocytosis of the receptor and / or steric hindrance of the bound bsAbHA'SunTag. It was confirmed that the cells co-expressed BFP and PD-L1 (Fig. 2C).

[0488] Next, in vitro transcribed (IVT) modified mRNA was generated encoding for GFP. The mRNA was encapsulated in an SM- 102-based LNP in which 15% of total PEG was substituted with PEGHA. PEGHAis a PEG lipid with the short HA peptide covalently attached to the terminal end of the PEG chain. Then, the LNPHAwas incubated with bsAbHA'SunTag. The resulting particles were only slightly bigger in size compared to standard LNP (84.3 ± 8.8 nm and 73.80 ± 0.628 nm respectively) and showed similar PDI (0. 11 ± 0.03 and 0. 15 ± 0.05) and mRNA encapsulation efficiency (data not shown). K562PUL1’Sunlagand K562 expressing cells were mixed in a 1: 1 ratio, producing a culture comprising both target+ and target- cells.

[0489] Transfection with LNP or LNPHAresulted in little to no GFP expression in the cells, consistent with the fact that at the utilized LNP-RNA dose. K562 cells are poorly transfected by LNPs (Fig. 2D). Instead, when the cells w ere transfected with LNPHAthat was pre-incubatedwith bsAbHA'SunTag, >90% of PD-Ll+ / SunTag+cells became GFP+, and at much higher GFP levels than control -treated cells (Fig. 2D). Transfection was remarkably specific, since only ~1% of PD-Ll-negative / SunTag-negative cells became GFP+in the same culture. Target cell transfection was dependent on bsAb being bound to the LNPHAparticle, since the K562PDL1‘ SunTagwere noj transfected by SM-102 LNP (no PEGHA) that had been pre-incubated with bsAbHA-SunTag.

[0490] Since it was found that the bsAb-coupled LNP not only increased specificity but also delivered more RNA per cell (as indicated by the higher GFP MFI in transfected cells (Fig. 2D), it was determined whether the bsAb / LNP would enhance transfection of cells already permissive to LNP-RNA delivery. To test this, 293TPDI'1'SunTagcells were transfected. Once again, a 1: 1 mix of target expressing and non-expressing cells (i.e. 293T : 293TPDL1-SunTag) was used.

[0491] With LNPHA, -90% of cells were transfected and expressed GFP at an MFI of 2,100. When the cells were transfected with LNPHA / bsAbHA’SunTag, a similar percent of cells were transfected. Cells expressing PD-LlSunTaghad a 3 fold increase in GFP MFI compared to the PD-LlSunTagnegative cells (Fig. 2E).

[0492] This data indicated that the bsAb / LNP -targeting technology allows transfection of otherwise refractory cell lines and increases the level of mRNA expression at saturating doses in transfection-permissive cells.

[0493] Example 4: Impact of LNP chemical composition on LNPHAtargeting efficacy

[0494] Next, it was determined whether LNPHAtargeting was compatible with different LNP chemical compositions.

[0495] To that end, LNPHAwere generated using different sets of lipids, based on different FDA approved LNP formulations. Specifically, GFP-encoding was encapsulated in mRNA in LNPHAcontaining Dlin-MC3-DMA (mimicking ONPATTRO" (patisiran) produced by Alnylam Pharmaceuticals) and ALC-0315 (mimicking Pfizer’s mRNA vaccine nanoparticle composition). When K.562PDL1-SunTagcells were transfected with either of the additional LNPHAformulations coupled with bsAbHA‘SunTag, once again it was found that there was efficient and specific delivery to target-expressing cells, as indicated by GFP-positivity in cells expressing PD-LlSunTag(Fig. 3A and Fig. 3B).

[0496] Similar results were obtained when eGFP mRNA was encapsulated in LNPHAcontaining Dlin-MC3-DMA, Sm-102 (mimicking Modema’s mRNA vaccine nanoparticle composition), ALC-0315, and TT3 ionizable lipids. TT3 ionizable lipid was provided fromDong lab at Mount Sinai. A human leukemia cell line was transfected with LNPHAin presence of bsAb. eGFP expression detected measured in cells expressing the target of the bsAb (PDL1). In contrast, cells that did not express the bsAb target were not transfected (Fig. 3E).

[0497] This data showed that bsAb coupling can be used to enhance the targeting of different LNPs formulations.

[0498] To determine whether the targeting technique was compatible with different bsAb amounts used for coupling with the LNP, 10 ng of LNPHAwas incubated bsAbHA / SunTagat concentrations ranging from 1 ng up to 200 ng, respectively, and then transfected K562PDL1‘ SunTagcel]s

[0499] Particularly high activity', measured as GFP MFI of PD-L1+cells, was observed with50 ng of bsAbHA,SunTag(Fig. 3C).

[0500] At 1 : 1 weight ratio and lower bsAb:LNPHAratios, there was a modest increase in LNPHAsize (~10 nm) indicating binding of bsAb to the HA tag on the surface of the LNPHA. At highest bsAb concentration (100 and 40 ng) there was a 3-fold and 2-fold increase in LNPHAsize, respectively, most likely reflecting aggregation of LNPHAdue to an excess of bsAb (Table 4).Table 4. DLS data for LNPHAbefore and after incubation with different ratio of bsAb:LNPIIA.

[0501] A range of PEGHAthat can be incorporated in the LNP preparation was titrated, evaluating a gradient in which PEGHAaccounts from 7.5% to 30% of total PEG. The higher the PEGHAcontent, the bigger the size and PDI values of the LNPHA(Table 4) with a concomitant decrease in mRNA encapsulation efficiency and recovery rate (data not shown).

[0502] 15% PEGHAsubstitution rate and 1 : 1 bsAb: LNP weight ratio was selected for future experiments, since at this dose particles retained PDI values and mRNA encapsulation efficiency similar to standard LNP and showed high transfection efficiency with a modest size increase (~10 nm) (Fig. 3D). Other PEGHAsubstitution rates and bsAb:LNP weight ratios could have been used, depending on the desired application.

[0503] Of note, no difference in delivery efficiencies were observed regardless of whether the LNPs were preincubated with the bsAb before cell treatment or if the LNPHAand bsAb w ere added simultaneously to the cell culture.Table 5. DLS data for LNPHA with different amount of total DSPE-PEG-HA.

[0504] Example 5: LNPHA / bsAb complexes increase targeting of PD-L1 positive cancer cells

[0505] To assess the efficiency of LNPHA / bsAb targeting to a therapeutically relevant molecule, a different bsAb was generated in which the anti-SunTag scFv was substituted with the scFV of TECENTRIQ® (atezolizumab), a monoclonal antibody that recognizes both human and murine PD-L1. The resulting construct was referred to as bsAbHA’PDL1. BsAbHA-SunTag and bsAbHA-PDLl bind to different epitopes of PD-Ll -SunTag (Fig. 3A). GFP mRNA was encapsulated in SM-102 LNP. LNPHAwas incubated with or without bsAbHA'SunTagand bsAbHA-pDL1.

[0506] As before. LNPHA / bsAbHA SunTagmediated specific and efficient delivery to SunTag expressing K562PDL1'SunTagcells (Fig. 4A). Similarly, bsAbHA'PDL1promoted cell-specific transfection, with -95% of PD-L1+ cells becoming GFP+when treated with LNPHA / BsAbHA"PDL1and only low levels of off-target transfection of PD-L1 -negative cells (Fig. 4A). There was a 1.4-fold increase in GFP MFI of cells treated with LNPHA / bsAbHA’PDL1compared to LNPHA / bsAbHA‘SunTag, showing that targeting a natural ligand. PD-L1, did not negatively impact targeting activity and even improved transfection efficiency of LNPHA(Fig. 4B).

[0507] To verify the ability of bsAbHA'PDL1to also recognize mouse PD-L1, K562 cells expressing murine PD-L1 (K562mPDL1) were generated. When LNPHAand bsAbHA’PDL1were coupled and added to cultures of K562mPDL1in which -45% of cells expressed mPD-Ll on their surface, there was a major increase in LNP transfection of the mPDLl+cells (Fig. 4C). As in the previous experiments conducted on K562PDL1'SunTag, LNPHAparticle alone were less effective than standard SM-102 LNP in transfecting K562mPDL1(-5% vs -20% respectively) (Fig. 4C). However, when LNPHAwas coupled with BsAbHA PDL1, >90% of mPD-Ll+ cellswere transfected, and there was a 3-fold increase in GFP expression levels compared to SM- 102 LNPs (Fig. 4C and Fig. 4D).

[0508] GFP mRNA provides cellular resolution of transfection efficiency. However, the instability of mRNA results in GFP expression decreasing over time. As a more sensitive system for assessing transfection efficiency, a Cre-loxP recombination system was used. Using a lentiviral vector, ID8 ovarian cancer cells were engineered to encode a dsRed-STOP-loxP- GFP expression cassette. This resulted in cells stably expressing dsRed fluorescent protein. However, when cells expressed Cre recombinase, the dsRed was excised and GFP was expressed (Fig. 4E).

[0509] Cre encoding mRNA was encapsulated into SM-102 LNP or SM-102 LNPHA+ / - bsAbHA PDL1. In vitro transfection of the ID8 cells with SM-102 LNP (+ / -bsAb) resulted in 25% of the PD-L1+ cells becoming dsRed negative. In contrast, cells treated with SM-102 LNPHA / bsAbHA'PDL1were -97% dsRed negative, indicating the LNPHA / bsAb complexes efficiently directed the particles to the mPD-Ll expressing cells (data not shown).

[0510] Example 6: LNPHA / bsAb enhances in vivo transfection of targeted cells

[0511] The efficiency of targeted delivery was assessed in vivo. For these experiments, the dsRed-STOP-loxP-GFP ID8 was utilized in ovarian cancer cells. Further, KPC pancreatic cancer cells and B16F10 melanoma cells were engineered ith the same reporter construct. The cells were engineered to express blue fluorescent protein (BFP) so they could be tracked in vivo and mPD-Ll (Fig. 5A).

[0512] To assess targeted transfection in a model of melanoma, the B16F 10 melanoma cells (BFP dsRed+GFP mPD-Ll+) were injected subcutaneously (s.c.) into wildtype mice. Cre mRNA was encapsulated into the LNPHAparticle and incubated + / - bsAbHA PDL1. When tumor size reached 5 mm in diameter, the LNPs were injected into the tumor. After 2 days, the tumors were harvested. Flow cytometry was performed to assess the percent of BFP+, GFP+, and mPDLl+cells.

[0513] In mice that received LNPHAwith no bsAb. <4% of cells were GFP+. In contrast, in tumors injected with LNPHA / bsAbHA‘PDL1, >20% of mPDLU cancer cells were GFP+(Fig. 5B).

[0514] As a more challenging delivety context, bsAb-mediated LNP targeting to disseminated tumors was assessed. To that end, the ID8 ovarian cancer model was employed. The dsRed+BFP+mPD-Ll+ID8 cancer cells were mixed with the parent ID8 cancer cells (no BFP, no dsRed, no mPD-Ll), in a 1: 1 ratio, and then intraperitoneally (i.p.) injected into immunocompetent mice. This results in disseminated tumors throughout the peritoneal cavityand models metastatic ovarian cancer. After 10 days, when tumors had engrafted and formed macroscopic lesions, LNPHA+ / - bsAbHA PDL1carrying Cre mRNA was injected into the peritoneal cavity. Once the mice developed ascites (indicative of large tumors), the animals were sacrificed.

[0515] The BFP+cancer cells were analyzed for expression of GFP by flow-cy tometry (Fig. 5C). Without the bsAb, the LNPHAmediated -5% transfection of the cancer cells and this was similar between PD-L1- and PD-L1+ cancer cells. Impressively, with LNPHA / bsAbHA‘PDL1. -20% of PD-L1+ cancer cells were transfected, whereas only -6% of PD-L1 -negative cancer cells were transfected in the same animals, indicating that the bsAb coupled LNP mediated a 3-fold increase in transfection efficiency specifically to target expressing cells in vivo (Fig. 5C).

[0516] Lastly, targeted delivery to pancreatic tumors was tested using an orthotopic model of the disease. KC pancreatic cancer cells, which carry an activating KrasG12Dmutation, were injected into the pancreas of mice. Tumors were allowed to grow. After 2 weeks, when tumors were significant size, SM-102 LNP or LNPHA / bsAbHA PDL1encapsulating Cre mRNA were injected into the peritoneal cavity, which led to vascular dissemination of the particles. Two days later, the tumors were harvested. BFP and GFP expression was analyzed by flow cytometry.

[0517] Though overall transfection efficiency was modest (unsurprisingly since these tumors grow in the pancreas and are ven’ poorly vascularized) the bsAb coupled LNP was able to mediate a 3-fold increase in cancer cell delivery. Importantly, transfection was specific for PD-L1+cancer cells (Fig. 5D), with as many as 5% of PD-L1+pancreatic cancer cells transfected from sy stemic delivery of the LNP.

[0518] Altogether, these data demonstrate that the binding of the bsAb to LNPHAis preserved in vivo, and that this non-chemical coupling approach is able to target the LNP to cancer cells expressing a specific molecule, in this case PD-L1, and enhance uptake and expression of a locally or systemically delivered mRNA to cells in vivo.

[0519] Example 7: BsAb-LNPs enhance efficiency and specificity of RNA delivery to primary mouse and human T cells

[0520] T cells have emerged as an important therapeutic target of LNP-RNA. As such, it was determined whether bsAb coupled LNPs could also mediate delivery to T cells. To that end, a bsAb was generated that comprised an HA-targeting scFv and an scFv specific for murine CD4 (bsAbHA'CD4, Fig. 6A). Cre mRNA was encapsulated into LNPHA. The particlewas incubated with bsAbHA’CD4to decorate the LNP particle. Total splenocytes were isolated from Ail4 mice, which encode a STOPfl fl-tdTomato cassette in which tdTomato is only produced upon Cre-mediated excision of the premature STOP codon. The cells were cultured for two days with IL-2 and anti-CD3 / CD28 beads to activate and expand the T cells. The cells were then treated with LNPHA+ / - bsAbHA'CD4. After three days, tdTomato expression was measured in CD4+ and CD4- T cells by flow cytometry.

[0521] With SM-102-based LNP. -10% of CD4+ and CD4- T cells were tdTomato+. indicating no specific tropism of the particles (Fig. 6B). With SM-102-based LNPHAwithout the bsAb, there was no tdTomato+ cells, further demonstrating that the modified particle has a reduced transfection efficiency. However, when the LNPHAwas coupled with bsAbHA’CD4, CD4+ T cell transfection was made possible, with an efficiency similar to SM-102-LNP. Importantly though, there was negligible transfection of CD4-negative T cells (Fig. 6B).

[0522] This further demonstrated the ability' of bs Ab-conjugated LNPs to mediate target cell-specific delivery of RNA.

[0523] Next, in vivo delivery’ of the particles to T cells was evaluated. Cre mRNA was encapsulated into SM-102-based LNP and LNPHA, incubated with or without bsAbHA'CD4, and intravenous injected into Ail 4 mice. After 3 days, mice were sacrificed, and splenocytes analyzed for tdTomato expression by flow cytometr .

[0524] With the standard SM-102-based LNP, there was splenocyte transfection, but the majority of cells were CD4-negative and virtually no CD4 cells were transfected (Fig. 6C). The transfected cells were likely mononuclear phagocytic cells, such as monocytes and DCs, which are more efficiently transfected by LNPs than lymphocytes. With the standard LNP or LNPHAwithout the bsAb, CD4+cells were not tdTomato+, indicating they were not transfected. Instead, when mice were injected with LNPHA / bsAbHA CD4, -10% of the CD4+ cells were tdTomato+ (1% of the 9% of splenocytes that were CD4+), which is a 16-fold increase over the standard particle (Fig. 6D and Fig. 6E). When the CD4-negative compartment was analyzed, reduced Cre recombination in mice treated with the LNPHA / bsAbHA'CD4complexes was observed. This shoyvs a higher degree of specificity of LNPHA / bsAbHA’CD4compared to standard LNP formulation or LNPHAwithout bsAb (Fig. 6D and Fig. 6E).

[0525] CD5 is an endocytic receptor expressed by all T cells, which has been used as a target of antibody-targeted LNPs. To see if CD5 could serve as a target for the bsAb-coupled LNP, a bsAb composed of the scFV of human CD5 antibody to create bsAbhCD5’HAwas generated. For cell engineering, T cells are normally activated ex vivo to enhance theirtransduction, however this has the caveat of producing more exhausted T cells. Thus, it is beneficial if LNP transfection of naive or quiescent T cells can be enhanced.

[0526] GFP mRNA was encapsulated in SM-102 LNP or LNPHAwith or without bsAbhCD5‘HA, and transfected unstimulated human T cells isolated from peripheral blood.

[0527] Treatment with standard LNP formulation was associated with very low levels of GFP expression, whereas with LNPHA / bsAbhCD5'HA. -70% of T cells became GFP+(Fig. 6F). In addition to a higher frequency of transfected T cells, the absolute expression of GFP in the cells increased by ~4-fold with the bsAb-coupled LNP compared to the standard LNP formulation (Fig. 6G). These results further demonstrate that bsAb-coupled LNPs can achieve efficient cell-specific RNA delivery and reduce off-target transfection in vitro and in vivo, including enhancing transfection of unstimulated / quiescent human T cells.

[0528] Example 8: Germinal Center B cells are efficiently transfected in vivo by targeted bsAb-LNP

[0529] B cells are generally not efficiently transduced or transfected, which makes their manipulation challenging. Based on the finding that the LNPHA / bsAb enhanced transfection of unstimulated T cells, it was determined whether a similar strategy could be employed to target B cells in vivo.

[0530] DEC205 is an endocytic receptor encoded by the Ly75 gene that is upregulated on germinal center (GC) B cells, which are responsible for generating high-affinity antibodies. An HAxDEC205 bsAb (bsAbHA'DEC205) was generated and conjugated to LNPI 1Aparticles encapsulating GFP mRNA (Fig. 7A, top schematic). To assess the specificity of bsAbHA'DEC205in vitro, a 1 : 1 mixture of freshly isolated DEC205 KO (CD45.1) and DEC205 WT (CD45.2) B cells was treated with LNPHA / bsAbHA DEC205. The cells were cultured either with B-cell Activating Factor (BAFF) to keep them in a resting state in which they do not express DEC205, or with anti-CD40 antibody and IL-4 to activate them and upregulate DEC205. Resting B cells (DEC205") were completely refractory to LNP treatment. Activated B cells were also refractory to transfection by standard LNP, whereas DEC205+B cells were highly transfected (up to 60%) by LNPHA / bsAbHA'DEC205(data not shown). This aligned with differences in DEC205 levels between culture conditions (Fig. 7B).

[0531] Next, the activity of LNPHA / bsAbHA'DEC205was evaluated in vivo. Bl-8hlB cells (CD45.1), which harbor a rearranged VDJ region conferring specificity for the 4-hydroxy-3- nitrophenylacetyl (NP) hapten, were isolated and transplanted into DEC205-deficient mice (Ly75 KO, CD45.2). The mice were immunized with NP-OVA to promote the differentiationof B cells into GC B cells and trigger DEC205 upregulation. Ten days post-immunization, mice received either 10 or 20 big of GFP RNA encapsulated in LNPHAcoupled with bsAbHA DEC205. The draining lymph nodes were collected the next day for analysis of GFP expression within the different B cell populations (Fig. 7C). There was highly efficient targeting of DEC205 GC B cells, and negligible transfection of DEC205 GC B cells or DEC205lownaive B cells. Remarkably, in animals treated with 10 pg of the formulation, nearly all DEC205+GC B cells - in average 92% - were transfected, and at a relatively high intensity of GFP expression (Fig. 7C).

[0532] These results further establish the ability of bs Ab-coupled LNPs to mediate targeted RNA delivery in vivo and provide a novel approach for the genetic manipulation of germinal center B cells.

[0533] There is a chance that even with chemical conjugation, PEG shedding from the LNP, which occurs naturally, results in some loss of antibody decoration (monoclonal or bispecific). Nonetheless, the data provided herein shows that bs Ab-conjugated LNPs were effective in vivo. This included increased targeting of PD-L1 -expressing cancer cells within pancreatic tumors and DEC205+germinal center B cells within lymph nodes, of which up to 92% were transfected, with little to no transfection of DEC205-negative GC B cells. Thus, even though there might be some loss of bsAb from the LNP, the coupling was sufficiently maintained to enable targeted cell delivery.

[0534] Example 9: bsAb-LNP mediate show comparable or enhanced transfection as compared to chemically conjugated Ab-LNP

[0535]

[0536] Next, the efficiency of LNP targeted-delivery using the bsAb platform and using maleimide-thiol chemistry was compared. Maleimide-thiol chemistry is commonly used to conjugate antibodies to LNPs. GFP mRNA was encapsulated in LNPHAor in malemide-PEG containing LNP (LNPMAL). In both cases, the same lipid formulas were used and only the PEG differed.

[0537] To attach an antibody to the LNPMAL, the targeting antibody (anti-mPD-Ll) or control isotype-matched IgG was functionalized with N-succinimidyl S-acetylthioacetate (SATA) to introduce sulfhydryl groups. The reactive sulfhydry l group on the antibodies were then conjugated to LNPMalthrough thioether conjugation chemistry by overnight incubation. For each antibody, a titration of the maleimide-to-mAb ratio was performed to identify the preferred working conditions. In parallel, LNPHAwere conjugated by incubating with bsAbHA'mPDLi y0 comparedelivery efficiency, K562mPDL1cells were transfected with the different formulations.

[0538] Flow cytometry showed similarly high levels of targeted cell transfection by both conjugation technologies, with ~97% of mPD-Ll+ K562 cells becoming GFP+(Fig. 8A). Notably, there was a higher degree of specificity with the bsAb / LNPHA, in which only -4% of PD-L 1 -negative cells were GFP+. compared with LNPKIal’mPDL1. which resulted in -37% of GFP+PD-L 1 -negative cells. Similar results were obtained when Cre mRNA was delivered to K562LoxP-GFP-mPDL1with LNPHA / bsAbHA’PDL1and LNPMal’mPDL1(Fig. 8B).

[0539] LNPsMalchemically-conjugated with anti-CD5 mAb have been used to enhance transfection of primary' human T cells. To see how bsAb non-chemical conjugation would compare. GFP mRNA was encapsulated in LNPMal. The particles were then conjugated with different molar ratios of maleimide: Ab. Tests with K562 cells expressing human CD5 (hCD5) were performed to find a preferred mal-PEG: Ab ratio. The highest transfection efficiency was achieved with LNP made with a 4:1 molar ratio of PEGIal:Ab. There was reduced transfection efficiency with lower and higher PEGMal:Ab ratios, indicating that insufficient and saturating doses of Ab were used in these treatment conditions respectively (Fig. 8C).

[0540] Fluman T cells were isolated from peripheral blood. The cells were left unstimulated and transfected with concentration matched GFP mRNA encapsulated in the different conjugated particles (bsAb or covalent chemistry). The bsAb approached was considerably more efficient, with >80% of unstimulated T cells becoming GFP1compared to -40% GFP1T cells with the LNPMal'hCD5. This was also reflected in the level of GFP in the cells, which more than doubled with the bsAb coupled particle compared to the chemically conjugated LNP (Fig. 8D and Fig. 8E).

[0541] Transfections were repeated using primary T cells activated with anti-CD3 / CD28 magnetic beads and IL-2 for 2 days. In this setting, the same bsAb / LNPHAand LN?^1 10135particles had similar transfection efficiencies. In the context of activated T cells, even the unconjugated LNPs resulted in high transfection. Of note how ever, the anti-CD5 decorated particles (bsAb or Mai) resulted in higher GFP MFI, indicating the antibody did enhance transfection (Fig. 8F and Fig. 8G). Differences across conditions were even more evident when cells were analyzed at 4 days post transfection (Fig. 8H and Fig. 81).

[0542] Overall, these results indicate that chemical-free conjugation of LNPs using a bsAb is as efficient as the covalently conjugated particles, and that the bsAb approach can even enhance LNP transfection of unstimulated T cells.

[0543] Example 10:A tethering bsAb enables non-chemical conjugation of off-the-shelf antibodies with LNPs

[0544] LNPHA / bsAb complexes enable targeted LNP cell transfection without chemical conjugation of the LNP but require the production of a new bsAb for each molecular target. To minimize the need for bsAb development, an approach was tested in which the non-LNP binding portion of the bsAb was modified to recognize the constant region of an antibody. This bsAb was used to tether the LNP to any antibody with the corresponding constant region (Fig. 9A). To this end, a bsAb was generated with the scFV for HA and a second scFv that binds the constant region of rat IgG2b to create bsAbHA4gG2b.

[0545] To test the bsAbHA4gG2btethering system, a rat anti-murine PD-L1 antibody with an IgG2b constant region was selected. GFP mRNA was encapsulated in LNPHA. The particles were incubated with either bsAbHA'PDL1or with bsAbHA4gG2bplus anti-PD-Ll (tethered LNP). K562mPDL1were treated with equal concentrations of parental LNP formulation and LNPHAas well as with control conditions that did not include one or another antibody (bsAbHA4gG2bor anti-PD-Ll antibody respectively). bsAbHA PDL1was used as positive control.

[0546] Transfection with the tethered LNP resulted in -90% of the mPD-Ll+ K562 cells being GFP+(Fig. 9B). This was similar in efficiency to LNPHA / bsAbHA'PDL1. Importantly, this was specific, as LNPHA / bsAbHA4gG2balone (z.e. without anti-PD-Ll tethered) resulted in <5% of GFP+PD-L1+cells. The tethered LNP was also far more efficient than the standard LNP at transfecting the K562 cells (Fig. 9B). Similar results were obtained when Cre mRNA was delivered to K562LoxP-GFP-mPDL1cells using the tethered LNP (Fig. 9C).

[0547] This data shows that the dual component bsAb tethering system is as efficient as the single bsAb in targeting LNP-RNA to PD-L1+ cells.

[0548] To further assess the approach, LNPHA / bsAbHA4gG2bparticles (GFP mRNA encapsulated) were incubated with or without a rat anti-CD20 antibody that has an IgG2b constant region, in order to tether the anti-CD20 antibody to the LNP. Then, BCL leukemic B cells, which express CD20. At the tested dose, w ere transfected.

[0549] Very low levels of transfection were observed with standard SM-102 LNP as well as with SM-102 LNPHAin absence of the bsAb (Fig. 9D). However, with LNPs in which anti- CD20 was tethered via bsAbHA4gG2b, -60% of BCL1 cells became GFP+(Fig. 9D).

[0550] This data show s that the bsAbHA4gG2bw as able to tether the anti-CD20 antibody to the LNP and enhance transfection.

[0551] The bsAbHA4gG2benables a person skilled in the art to easily test many different antibodies for targeted LNP delivery of RNA. Cre mRNA w as encapsulated in LNP or LNPHA.An aliquot of LNPHAwas incubated with bsAbHA'IgG2b. All three formulations (LNP, LNPHA, LNPHA / bsAbHA IgG2b) were then incubated with anti-CD4. anti-CD8, anti-CD3, and anti-CD45 monoclonal antibodies with rat IgG2b constant regions. As all four antibody’s target molecules are expressed by T cells, primary murine T cells were isolated from the spleens of Ail4 mice and transfected them with each formulation.

[0552] In all cases, the bsAbHAJgG2bwas able to tether the monoclonal antibody to the LNP and mediate enhanced delivery to the T cells expressing the targeted molecule, as indicated by induction of tdTomato expression (Fig. 9E).

[0553] This data demonstrates that bsAbHA4gG2bcan be used with different rat IgG2b monoclonal antibodies to direct RNA-LNP to cells of interest and highlight the flexibility' of our conjugation approach in generating antibody-directed LNPs.

[0554] Example 11: Conjugation of additional peptides to the LNP surface

[0555] As shown in the above Examples, linear epitopes such as an HA peptide and a FLAG tag can be used in the compositions and methods disclosed herein. However, other epitopes can be used. See. e.g., Fig. 10A, Fig. 10B, Fig. 10C., and Fig. 10D.

[0556] In sum, the platform technology' described herein provides a means to decorate an LNP with a moiety for the targeting of specific cells without the need for chemical coupling. Importantly, the approach was effective across all FDA-approved LNP formulations, on primary human cells of clinical relevance, and for targeted in vivo delivery. LNP can be produced at scale and retain the same biophysical characteristics of standard LNP formulations, including PDI, charge and mRNA encapsulation efficiency. The LNPs disclosed herein have the capacity’ to enable cell-targeted delivery, which can be critical when the RNA cargo encodes a protein with toxic potential, such as a chimeric antigen receptor (CAR) or immunostimulatory cytokines like IL- 12.

[0557] Further, the LNPs disclosed herein have utility for enhancing transfection of target expressing cells, even in vitro, and are therefore a valuable research tool.

Claims

CLAIMSWe claim:1 . A protein complex comprising:(a) a lipid nanoparticle (LNP), the LNP comprising (i) a cargo and (ii) at least one modified lipid, wherein the modified lipid comprises a lipid molecule conjugated to a first antigen;(b) a bispecific antibody construct comprising a binding site for the first antigen and a binding site for a second antigen.

2. The protein complex of claim 1, wherein the first antigen is about 6 to about 20 amino acids in length.

3. The protein complex of claim 1, wherein the first antigen comprises a peptide comprising an HA peptide, a FLAG tag, a Pep-1 peptide, or a Pep-2 peptide, preferably, an HA peptide.

4. The protein complex of claim 1, wherein the first antigen comprises a peptide comprising SEQ ID NOs:44, 46, 49, 51, or 52, preferably, SEQ ID NO:44 or SEQ ID NO:49.

5. The protein complex of any one of claims 1-4, wherein the wherein the lipid molecule is a PEGylated lipid molecule and wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule.

6. The protein complex of claim 5, wherein the first antigen is conjugated to the PEG portion of the PEGylated lipid molecule via a first linker.

7. The protein complex of claim 6, wherein the first linker is a polyglycine linker.

8. The protein complex of claim 7. wherein the polyglycine linker essentially consists of GGG.

9. The protein complex of any one of claims 1-8, wherein the lipid molecule comprises a 14C to 20C fatty acid tail.

10. The protein complex of claim 9, wherein the lipid molecule comprises a 16C fatty acid tail.

11. The protein complex of claim 10, wherein the modified lipid comprises 1,2-distearoyl-sn- gly cero-3 -phosphoethanolamine-N-PEG.

12. The protein complex of any one of claims 1-11, wherein the bispecific antibody construct comprises:(a) two or more of a Fab, Fab’, single domain antibody, (Fab’)2 / Fab2, single-chain Fv (scFv), single chain Fab (scFab), minibody, or scFv-Fc; and / or(b) one or more of a (Fab’)2 / Fab2, minibody, di-ScFv, scFv-Fc, bi-scFv, Dual-Affinity ReTargeting (DART), dual-action Fab (DAF), H-chain heterodimer with orthogonal Fab interfaces, CrossMab, Dual Variable Domain Immunoglobulin G (DVD IgG). bispecific T-cell engager (BiTE), di-diabody. tandem diabody (TandAb). (scFv)2-HSA, or tetravalent-IgG (Tv- IgG).

13. The protein complex of any one of claims 1-11, wherein the bispecific antibody construct comprises a first scFv and a second scFv. wherein the first scFv comprises a binding site for the first antigen and the second scFv comprises a binding site for the second antigen.

14. The protein complex of claim 13, wherein the first scFv and the second scFv are connected via a second linker.

15. The protein complex of claim 14, wherein the second linker is a polypeptide linker, optionally wherein the polypeptide linker is a flexible linker.

16. The protein complex of any one of claims 1-15, wherein the second antigen is presented on the surface of a cell.

17. The protein complex of claim 16, wherein the second antigen is presented on the surface of the cell in the context of a multi histocompatibility complex (MHC).

18. The protein complex of claim 16. wherein the second antigen is a first polypeptide expressed on the surface of the cell.

19. The protein complex of claim 18, wherein the first polypeptide expressed on the surface ofthe cell is selected from the group consisting of PD-1, PD-L1, CD3, CD4, CD5, CD8, CD45, and DEC205.

20. The protein complex of any one of claims 1-15, wherein the second antigen is a constant region of an antibody that comprises a binding site for a third antigen.

21. The protein complex of claim 20, wherein the third antigen is presented on the surface of a cell.

22. The protein complex of claim 21, wherein the third antigen is presented on the surface of the cell in the context of an MHC.

23. The protein complex of claim 21, wherein the third antigen is a second polypeptide expressed on the surface of the cell.

24. The protein complex of claim 23, wherein the second polypeptide expressed on the surface of the cell is selected from the group consisting of PD-1, PD-L1, CD3, CD4, CD5, CD8, CD45, and DEC205.

25. The protein complex of any one of claims 20-24, the protein complex further comprising the antibody that comprises a binding site for the third antigen.

26. The protein complex of any one of claims 1-25, wherein the cargo comprises a nucleic acid, a polypeptide, or a small molecule.

27. The protein complex of claim 26, wherein the nucleic acid comprises a therapeutic RNA molecule.

28. The protein complex of claim 27, wherein the therapeutic RNA molecule is a short interfering RNA (siRNA), messenger RNA (mRNA), short interfering RNA (siRNA), doublestranded RNA (dsRNA). micro-RNA (miRNA). or short hairpin RNA (shRNA).

29. A pharmaceutical composition comprising (a) the protein complex of any one of claims 1-28 and (b) a pharmaceutically acceptable excipient.

30. A method of delivering a cargo to a cell, the method comprising contacting the cell with the protein complex of any one of claims 1-19 or 26-30, wherein the cell expresses on its surface the second antigen.

31. A method of delivering a cargo to a cell, the method comprising contacting the cell with the protein complex of claim 25, wherein the cell expresses on its surface the third antigen.

32. The method of claim 30 or 31, wherein the cell is a mammalian cell.

33. The method of claim 32, wherein the cell is a T cell, a B cell, or a hematopoietic stem cell.

34. The method of any one of claims 1-25, wherein the cell is a T cell, wherein the cargo comprises an mRNA, and wherein the mRNA encodes for a chimeric antigen receptor (CAR), a T-cell receptor (TCR), a cytokine, or a checkpoint inhibitor.

35. The method of any one of claims 31-34, wherein the cell is a human cell.

36. A method of making a protein complex, the method comprising contacting the LNP as defined in any one of claims 1-11, 26-28, or 34 with the bispecific antibody construct as defined in any one of claims 1 or 12-24.

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