WO2018022957A1

WO2018022957A1 - T cell binding conjugates and methods of use

Info

Publication number: WO2018022957A1
Application number: PCT/US2017/044289
Authority: WO
Inventors: Richard Wooster; Mary SIMCOX; Mark T. Bilodeau; Sudhakar Kadiyala; Benoît MOREAU
Original assignee: Tarveda Therapeutics, Inc.
Priority date: 2016-07-29
Filing date: 2017-07-28
Publication date: 2018-02-01

Abstract

The present invention relates to cancer immunotherapy. Conjugates and nanoparticles comprising T cell binding moieties that can elicit a tumor specific immune response are provided. The conjugates comprise one or more tumor cell binding moieties that are connected to the T cell binding moieties. Nanoparticles comprising the conjugates of the present invention are also provided to improve the delivery of the conjugates, and increase immunogenicity and lower toxicity.

Description

T CELL BINDING CONJUGATES AND METHODS OF USE REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No. 62/368,632 filed July 29, 2016, the contents of which are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The sequence listing filed, entitled 2065_1052USPCT_SL.txt, was created on July 28, 2017 and is 4648 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of immuno-oncology therapy. In particular, the present invention relates to T cell binding conjugates and particles comprising such conjugates.

BACKGROUND OF THE INVENTION

[0004] T cell-mediated tumor killing is suppressed through multiple mechanisms in the tumor microenvironment. Enhancement of effector T cell mediated cytotoxicity to induce an antitumor immune response is an attractive therapeutic concept for the treatment of cancer. Clinical proof of concept for overcoming tumor immune evasion has been demonstrated with immune check point inhibitors, antibodies that block inhibitory receptors on T cells: CTLA-4 and PD-1. Significant antitumor responses and long-term remissions have been observed in patients with metastatic melanoma, renal cell carcinoma and non-small cell lung cancer (Hodi et al., J Immunol, vol.170:4397 (2010); Lynch et al., J Clin Oncol, vol.30(17): 2046 (2012); Yang et al., J Immunother., vol.30:825 (2007)). Another approach to increase T cell mediated tumor eradication in patients is treatment with bispecific T cell engager molecules that direct cytolytic T cell activity against tumor cells through co-localization of cells to create an immune synapse between the T cell and the cancer cell. One such bispecific T cell engager molecule, blinatumomab, which binds CD3 on T cells and CD 19 on cancer cells was recently approved for the treatment of relapsed/refractory B cell acute lymphoblastic leukemia (Topp et al., J Clin Oncol, vol.29:2493 (2011); Topp et al., Blood, vol. l20:5185 (2012); Klinger et al., Blood, vol.119:6226 (2012)).

[0005] An issue with existing T cell engaging and activating anti-tumor therapies is toxicity related to non-tumor cell mediated toxicity (Portell et al. Clin Pharmacol., vol.5(Suppl 1):5- 11 (2013); Naidoo et al, Ann Oncol 26:2375 (2015); Champiat et al., Ann Oncol 27:559 (2016); Horvat et al., J Clin Oncol., 33 :3193 (2105).). Further, most of the bispecific T cell engager molecules employ antibodies or antibody fragments on one side or both sides of the molecules. There remains an unmet need for improved tumor targeted immunotherapy to induce tumor-specific T cell cytotoxity.

SUMMARY OF THE INVENTION

[0006] The present invention provides conjugates comprising a T cell binding moiety and a tumor cell binding moiety. These conjugates are designed to optimize the tumor-directed cytolytic activity of T cells by effective T cell-tumor cell interaction through enhancement of the pharmacokinetic behavior including plasma half life and tumor distribution, optimizing the geometry of the immune synapse and fine tuning the binding affinities of the T cell binding componnets and tumor antigen binding moieties of the conjugates.

[0007] In some embodiments, the conjugates of the present invention comprise a single chain variable fragment (scFv) CD3 binder that recognizes an epitope on CD3s that is an invariable part of the T cell receptor complex and is expressed on all T cells. The conjugates also comprise a tumor cell binding moiety, such as a tumor associated antigen binding moiety. In one example, the conjugates comprise a somatostatin receptor type 2 (SSTR2) binding ligand as a tumor cell binding moiety. In another example, the conjugates comprise a luteinizing hormone-releasing hormone receptor (LHRHR or G RHR) binding ligand as a tumor cell binding moiety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Fig. 1 shows purified, recombinant CD3 -binding scFv preapared in Example 1 binds to cellular CD3 as confirmed by FACS.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.

[0010] A variety of strategies have been developed to elicit cancer specific immune responses. The present conjugates provide platforms for targeted cancer immunotherapy modalities. The conjugate comprise the following moieties: a T cell binding moiety, a tumor cell binding moiety and an optional linker that connects the T cell binding agent and the tumor cell binding moiety. The T cell binding moiety may be an agent that can

stimulate/increase a cancer specific immune response. The tumor cell binding moiety of the conjugate can function to deliver the T cell binding moiety of the conjugate to a targeted area such as a tumor tissue. In some cases, the tumor cell binding moiety itself may have an immune stimulating activity or an immune checkpoint inhibiting activity. The optional linker of the conjugate connects the T cell binding moiety and the tumor cell binding moiety.

[0011] Design of the present conjugates is flexible and may be configured in various combinations depending on types, origins, metastatic status, and other clinical and

pathological status of the cancers to be related. In some embodiments, one or more tumor cell binding agents from the same category such as different tumor antigen binding peptides that bind to one common tumor associated antigen protein, or peptides that bind to different tumor associated antigen proteins but associated with one type of tumor; or peptides that bind a combination of tumor associated antigens isolated from a single patient, i.e. personalized, may be connected to a T cell binding moiety through one or more linkers in a conjugate.

[0012] In addition to the conjugate itself, the present invention also provide particles, nanoparticles and/or polymeric nanoparticles that can encapsulate one or more conjugates of the present invention, providing an improved nanodelivery system. The present nano-delivery system improves pharmacokinetics, targeting of tissues and cells to enhance efficacy, specificity and lower toxicity. The present conjugates designed for increasing immune response, and particles comprising such conjugates provide more specific compositions and methods to treat cancer. In some embodiments, the congugates are only released within certain environments, such as with the presence of lysozymes. In some embodiments, particles, nanoparticles and/or polymeric nanoparticles target bone marrow and delivers conjugates to bone marrow. Such solid polymeric nanoparticles and their preparation are taught in, for example, WO2014/106208 to Bilobeau et al., the contents of which are incorporated herein in their entirety.

COMPOSITIONS OF THE INVENTION

[0013] Compositions of the present inventions include conjugates comprising a T cell binding moiety, an optional linker, and a tumor cell binding moiety. Nanoparticles that package one or more such conjugates are also provided. The conjugates can be

incorporated/dispersed into nanoparticles or disposed on the surface of the particles. In particular, conjugates of the present invention and nanoparticles comprising such conjugates may be used as immuno-oncological agents to enhance anti-cancer immune responses alone or in combination with other immunotherapies. The conjugates, nanoparticles comprising the conjugates, and/or formulations thereof can provide improved temporospatial delivery of the T cell binding moiety and/or improved biodistribution compared to delivery of the T cell binding moiety alone.

[0014] Conjugates, nanoparticles and other compositions of the present invention provide a system that is flexible in tailoring the composition and numbers of T cell binding moieties (e.g., flexible addition and subtraction of T cell binding moieties connected to the tumor cell binding moiety) important for harnessing an anti-tumor immune response, for example, antigen specific T cell activation and response.

[0015] Conjugates, nanoparticles and other compositions of the present invention may provide increased targeting properties since the tumor cell binding moieties of the conjugates specifically target to a selected tumor tissue and/or certain types of tumor cells of interest.

[0016] Conjugates, nanoparticles and other compositions of the present invention may coordinate action of the innate and adaptive phases of the immune system to produce an effective anti-cancer immune response.

[0017] In further another embodiment, conjugates, nanoparticles and other compositions of the present invention may also be used for in vivo and ex vivo activation and expansion of lymphocytes including T cells to elicit an anti-tumor immune response.

[0018] Therefore, conjugates, nanoparticles and other compositions of the present invention optimize the tumor-directed cytolytic activity of T cells by effective T cell-tumor cell interaction through enhancement of the pharmacokinetic behavior including plasma half life and tumor distribution, optimizing the geometry of the immune synapse and fine tuning the binding affinities of the T cell binding and tumor cell binding moieties of the conjugates.

I. Conjugates of the Invention

[0019] In accordance with the present invention, conjugates comprise at least three moieties: a tumor cell binding moiety (or ligand), an optional linker, and a T cell binding agent that is connected to the tumor cell binding moiety via a covalent bond or the optional linker. In some embodiments, the conjugate may be a conjugate between a single tumor cell binding agent and a single T cell binding moiety with the formula: X-Y-Z, wherein X is the tumor cell binding moiety; Y is an optional linker; and Z is the T cell binding agent. In certain embodiments, One tumor cell binding moiety can be conjugated to two or more T cell binding moieties wherein the conjugate has the formula: X-(Y-Z)_n. In certain embodiments, one T cell binding moiety can be linked to two or more tumor binding moieties wherein the conjugate has the formula: (X-Y)n-Z. In other embodiments, one or more tumor cell binding agents may be connected to one or more T cell binding moieties wherein the conjugate formula may be (X-Y-Z)n. In various combinations, the formula of the conjugates maybe, for example, X-Y-Z-Y-X, (X-Y-Z)n-Y-Z, or X-Y-(X-Y-Z)_n, wherein X is a tumor cell binding moiety; Y is an optional linker; Z is a T cell binding moiety. The number of each moiety in the conjugate may vary depending on types of agents, sizes of the conjugate, delivery targets, particles used to packaging the conjugate, other active agents (e.g., immunologic adjuvants) and routes of administration. Each occurrence of X, Y, and Z can be the same or different, e.g. the conjugate can contain more than one type of tumor cell binding moiety, more than one type of linker, and/or more than one type of T cell binding moiety, n is an integer equal to or greater than 1. In some embodiments, n is an integer between 1 and 50, or between 2 and 20, or between 5 and 40. In some embodiments, n may be an integer of 2, 3, 4, 5, 6, 7, 8. 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49 or 50.

[0020] In some embodiments, the conjugate may comprise pendent or terminal functional groups that allow further modification or conjugation. The pendent or terminal functional groups may be protected with any suitable protecting groups.

[0021] Conjugates of the present invention may target discrete pathways involved in critical processes of anti-cancer immune responses. These critical processes may include migration of activated T cells to the tumor microenvironment in response to chemokines and homing receptor expression, or having effector T cells gain access to antigen expressing tumor cells and maintenance of sufficient functionality of effector T cell to destroy tumor cells.

[0022] In some embodiments, the tumor cell is a tumor epithelial cell. In some

embodiments, the tumor cell is a tumor associated endothelial cell. In some other

embodiments, the tumor cell is a tumor associated fibroblast.

[0023] In one embodiment, the conjugate comprises a single chain variable fragment (scFv) CD3 binding agent that recognizes an epitope on CD3s (CD3 epsilon chain) that is an invariable part of the T cell receptor complex and is expressed on all T cells. The conjugate may further comprise a tumor associated antigen binding moiety, such as a somatostatin receptor type 2 (SSTR2)-binding agent. The conjugate binds to CD3 on T cells and binds to tumor cells expressing SSTR2 and can induce T cell-mediated tumor cytotoxicity against the tumor cells expressing SSTR2.

[0024] In some embodiments, the conjugate comprises a payload that binds to a chimeric antigen receptor (CAR) T cell, a linker, and a targeting moiety that binds to a tumor cell. For example, the targeting moiety may bind to a cell surface protein on tumor cells, such as but not limited to a folate receptor, a somatostatin receptor (SSTR), or a luteinizing hormone- releasing hormone receptor (LHRHR). The payload may be a single chain variable fragment (scFV) that binds to a cell surface protein on CAR T cells.

[0025] In some instances, a conjugate may have a molecular weight of less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da. In some cases, the conjugate may have a molecular weight of between about 1,000 Da and about 50,000 Da, in some embodiments between about 1,000 Da and about 40,000 Da, in some embodiments between about 1,000 Da and about 30,000 Da, in some embodiments bout 1,000 Da and about 50,000 Da, between about 1,000 Da and about 20,000 Da, in some embodiments between about 1,000 Da and about 15,000 Da, in some embodiments between about 1,000 Da and about 10,000 Da, in some embodiments between about 1,000 Da and about 8,000 Da, in some embodiments between about 1,000 Da and about 5,000 Da, and in some embodiments between about 1,000 Da and about 3,000 Da. The molecular weight of the conjugate may be calculated as the sum of the atomic weight of each atom in the formula of the conjugate multiplied by the number of each atom. It may also be measured by mass spectrometry, MR, chromatography, light scattering, viscosity, and/or any other methods known in the art. It is known in the art that the unit of molecular weight may be g/mol, Dalton (Da), or atomic mass unit (amu), wherein 1 g/mol = 1 Da = 1 amu.

A. T cell binding moiety

[0026] Conjugates of the present invention comprise T cell binding moieties (may also be refered to payload of the conjugates), Z, wherein Z can be polypeptides (e.g., antibodies), peptides, antibody mimetics, nucleic acids (e.g., aptamers), glycoproteins, small molecules, carbohydrates, or lipids.

[0027] In some embodiments, the T cell binding moiety, Z, may be a T cell receptor (TCR) activator. As used herein, a TCR activator can activate T cells in the absence of specific antigens. Suitable T cell activators include the mitogenic lectins concanavalin-A (ConA), phytohemagglutinin (PHA) and pokeweed mitogen (PWM), and antibodies that crosslink the T cell receptor/CD3 complex. Exemplary antibodies that crosslink the T cell receptor include the HIT3a, UCHT1 and OKT3 monoclonal antibodies.

[0028] In some embodiments, the T cell binding moiety of the conjugate is a CD3-binding agent, such as a peptide or derivative that binds to CD3, a CD3 antibody or a CD3 -binding fragment thereof. Activation of cytotoxic T cell may occur via binding of the CD3 antigen as effector antigen on the surface of the cytotoxic T cell by the conjugates of the present invention. CD3 (cluster of differentiation 3) complex, or CD3 antigen, is a T cell co- receoptor that helps to activate T cells. CD3 complex may comprise several chians: CD3D (CD3 delta chain), CD3G (CD3 gamma chain), CD3E (CD3 epsilon chain) and/or CD247 (CD3 zeta chain). The CD3-binding agent, CD3 antibody or the CD3-binding fragment may bind to any epitope on any of the chains.

[0029] As non-limiting examples, the T cell binding moiety of the conjugate may bind to any epitope on any of the CD3 chains in the table:

[0030] CD3 antigens are cell-surface proteins and are bound to the membrances of all mature T cells. Conjugates of the present invention comprising CD3 binding agents may bind to and activate T cells in the absence of independent TCR/MHC binding. The activated T cell can then exert a cytotoxic effect on tumor cells. In one embodiment, CD3 antigens do not internalize upon binding of the conjugates.

[0031] The CD3 binding agent may be a Fab fragment of a CD3 antibody, a single CDR CD3 antibody, a single chain variable fragment (scFv) of a CD3 antibody, a single-chain antibody mimic that is much smaller than an antibody such as nanofitin® (Affilogic). Non- limiting examples of CD3 antibodies or fragments thereof include, a humanized CD3-specific scFv disclosed by Liddy et al. {Nature Medicine, vol. l8(6):980 (2012)), a single-chain anti- CD3 antibody derived from UCHT1 disclosed by Kuo et al. {Protein Engineering, Design & Selection, vol.25(10):561 (2012)), an anti-CD3 scFv comprising an amino acid sequence of SEQ ID No.2 in CA2561826 to Wang et al., an anti-CD3 portion of an anti-CD3&anti- EpCAM bispecific antibody (SEQ ID No. l) disclosed in WO2005061547 to Baeuerle et al., a reshaped Fab antibody against human CD3, a reshaped single-domain antibody against human CD3 or a reshaped scFv against human CD3 disclosed in US20050175606 to Huang et al., anti-CD3 VH disclosed in US20050079170 to Gall et al., any CD3-binding scFv including scFv(UCHT-l)-PE38 disclosed in US20020142000 to Digan et al., the contents of each of which are incorporated herein by reference in their entirety.

[0032] In one embodiment, the CD3 binding moiety may comprise an anti-CD3 single chain Fv antibody with a sequence of Accession No. ABN79462. In another embodiment, the CD3 binding moiety may comprise any anti-human CD3 single chain Fv antibody disclosed in Kipriyanov et al. (Kipriyanov et al., Protein Engineering, vol. l0(4):445 (1997), the contents of which are incorporated herein by reference in their entirety).

[0033] Other non-limiting examples of CD3 binding moiety include any CD3 binding agent disclosed in Dreier et al. {J Immunol, vol.170:4397 (2003)), in Klinger et al. {Blood, vol.119:6226 (2012)), or blinatumomab, a bispecific single-chain antibody targeting CD3 and CD19 antigen disclosed in Topp et al. {J Clin Oncol, vol.29:2493 (2011)), anti-CD20/CD3 T cell-dependent bispecific antibody disclosed in Sun et al. {Sci Transl Med, vol.7:287 (2015)), anti-CD3 x anti-CD20 bispecific antibody disclosed in Gall et al. {Exp Hematol,

vol.33(4):452 (2005)), CEA/CD3-bispecific T cell-engaging antibody disclosed in Osada et al. {Cancer Immunol Immunother., vol.64(6):677 (2015)), or EpCAM/CD3-bispecific T-cell engaging antibody MT110 disclosed in Cioffi et al. {Clin. Cancer Res., vol. l8(2):465

(2012)).

[0034] In some embodiments, the T cell binding moiety (e.g., CD3 binding moeity) may have a molecular weight of less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.

[0035] The T cell binding moiety may be an antibody mimetic such as a monobody, e.g., an AD ECTIN™ (Bristol-Myers Squibb, New York, New York) , an Affibody® (Affibody AB, Stockholm, Sweden), Affilin, nanofitin (affitin, such as those described in WO 2012/085861, an Anticalin™, an avimers (avidity multimers), a DARPin™, a Fynomer™, Centyrin™, and a Kunitz domain peptide. In certain cases, such mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules may be antibody mimetic.

[0036] In some embodiments, the tumor cell binding moiety is a small molecule, a protein scaffold, a stabilized peptide, a nanotifin, a bipodal peptide, an aptide as described herein.

[0037] In certain embodiments, the T cell binding moiety or moieties of the conjugate comprise a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%), or about 50% to about 60%>, or about 60%> to about 70%, or about 70% to about 80%), or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of T cell binding moiety of the conjugate may also be expressed in terms of proportion to the tumor cell binding moiety. For example, the present teachings provide a ratio of T cell binding moiety to tumor cell binding moiety of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.

B. Tumor cell binding moiety

[0038] In accordance with the present invention, a conjugate can contain one or more tumor cell binding moieties (may also be refered to as ligands of the conjugates). Tumor cell binding moieties, X, can be polypeptides (e.g., antibodies), peptides, antibody mimetics, nucleic acids (e.g., aptamers), glycoproteins, small molecules, carbohydrates, lipids, or nanoparticles. X may bind to any cell surface protein on tumor cells. In some embodiments, the cell surface protein is tumor associated antigens (TAA). [0039] In some embodiments, the tumor cell binding moiety may have a molecular weight of less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.

[0040] In some embodiments, the tumor cell binding moiety, X, may be peptides such as somatostatin, octeotide, LHRH (luteinizing hormone releasing hormone), epidermal growth factor receptor (EGFR) binding peptide, aptide or bipodal peptide, RGD-containing peptides, a protein scaffold such as a fibronectin domain, a single domain antibody, a stable scFv, or other homing peptides, or derivatives thereof.

[0041] As non-limiting examples, the tumor cell binding moiety, X, can be an aptamer being either RNA or DNA or an artificial nucleic acid; small molecules; carbohydrates such as mannose, galactose and arabinose; vitamins such as ascorbic acid, niacin, pantothenic acid, carnitine, inositol, pyridoxal, lipoic acid, folic acid (folate), riboflavin, biotin, vitamin B 12, vitamin A, E, and K; a protein or peptide that binds to a cell-surface receptor such as a receptor for thrombospondin, tumor necrosis factors (TNF), annexin V, interferons, cytokines, transferrin, GM-CSF (granulocyte-macrophage colony-stimulating factor), or growth factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), (platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF).

[0042] In some embodiments, the tumor cell binding moiety binds to a somatostatin receptor (SSTR) such as SSTR2 or luteinizing hormone releasing hormone receptor (LHRHR or G RHR) such as G RHR1.

[0043] In some embodiments, the tumor cell binding moiety binds to to a cell surface protein selected from the group consisting of CD20, carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), and CD 19. Non-limiting examples of CD 19 binding agents that may be used as a tumor cell binding moiety in the conjugates include any CD19 binding agent disclosed in Dreier et al. J Immunol, vol.170:4397 (2003)), in Klinger et al. {Blood, vol.119:6226 (2012)), or blinatumomab, a bispecific single-chain antibody targeting CD3 and CD19 antigen disclosed in Topp et al. (J Clin Oncol, vol.29:2493 (2011)). Non-limiting examples of CD20 binding agents include anti-CD20/CD3 T cell-dependent bispecific antibody disclosed in Sun et al. (Sci Transl Med., vol.7:287 (2015)) or anti-CD3 x anti-CD20 bispecific antibody disclosed in Gall et al. (Exp Hematol, vol.33(4):452 (2005)). Non-limiting examples of CEA binding agents include CEA/CD3-bispecific T cell-engaging antibody disclosed in Osada et al. {Cancer Immunol Immunother., vol.64(6):677 (2015)). Non-limiting examples of EpCAM binding agents include EpCAM/CD3-bispecific T-cell engaging antibody MT110 disclosed in Cioffi et al. (Clin. Cancer Res., vol. l8(2):465 (2012)).

[0044] In some embodiments, the tumor cell binding moiety is a protein scaffold. The protein scaffold may be an antibody-derived protein scaffold. Non-limiting examples include single domain antibody (dAbs), nanobody, single-chain variable fragment (scFv), antigen-binding fragment (Fab), Avibody, minibody, CH2D domain, and Fcab. In some embodiments, scFv is a stable scFv, wherein the scFv has hyperstable properties. In some embodiments, the nanobody may be derived from the single variable domain (VHH) of camelidae antibody.

[0045] In some embodiments, the protein scaffold may be a non-antibody-derived protein scaffold, wherein the protein scaffold is based on nonantibody binding proteins. The protein scaffold may be based on engineered Kunitz domains of human serine protease inhibitors (e.g., LAC1-D1), DARPins (designed ankyrin repeat domains), avimers created from multimerized low-density lipoprotein receptor class A (LDLR-A), anticalins derived from lipocalins, knottins constructed from cysteine-rich knottin peptides, affibodies that are based on the Z-domain of staphylococcal protein A, adnectins or monobodies and pronectins based on the 10^th or 14^th extracellular domain of human fibronectin III, Fynomers derived from SH3 domains of human Fyn tyrosine kinase, or nanofitins (formerly Affitins) derived from the DNA binding protein Sac7d.

[0046] In some embodiments, the protein scaffold may be based on a fibronectin domain. In some embodiments, the protein scaffold may be based on fibronectin type III (FN3) repeat protein. In some embodiments, the protein scaffold may be based on a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter "Tenascin"). Any protein scaffold based on a fibronectin domain disclosed in US Pat. No. 8569227 to Jacobs et al., the content of which is incorporated herein by reference in its entirety; may be used as a tumor cell binding moiety of the conjugate of the invention.

[0047] In some embodiments, the protein scaffold may be any protein scaffold disclosed in Mintz and Crea, BioProcess, vol.11(2):40-48 (2013), the contents of which are incorporated herein by reference in their entirety. Any of the protein scaffolds disclosed in Tables 2-4 of Mintz and Crea may be used as a tumor cell binding moiety of the conjugate of the invention. [0048] In some embodiments, the tumor cell binding moiety is an arginylglycylaspartic acid (RGD) peptide, a tripeptide composed of L-arginine, glucine and L-aspartic acid, which is a common cell targeting element for cellular attachment via integrins.

[0049] In some embodiments, a tumor cell binding moiety may be an antibody that specifically binds to a tumor associated antigen (TAA) and/or an antigenic peptide (epitope). As one skilled in the art can envision, an antibody fragment (e.g., an Fc fragment of an antibody) may be used for the same purpose.

[0050] In addition to tumor cells specific antigen or antigen epitopes, antibodies may be specific to a ubiquitous antigenic site on various cancers. Many studies have revealed that cancer cells share certain common characteristics. Many types of human cancer cells are characterized by substantial abnormalities in the glycosylation patterns of their cell-surface proteins and lipids (e.g., Hakomori et. al., 1996, Cancer Res. 56:5309-18; and Springer et al., 1997, JMolMed 75:594-602). These differences have led to the identification of antigenic determinants on cancer cells. Natural IgM antibodies to these epitopes are present in the circulation and can be used as a tumor cell binding moiety of a conjugate of the present invention.

[0051] In some embodiments, the tumor cell binding moiety is an antibody mimetic such as a monobody, e.g., an ADNECTIN™ (Bristol-Myers Squibb, New York, New York) , an Affibody® (Affibody AB, Stockholm, Sweden), Affilin, nanofitin (affitin, such as those described in WO 2012/085861, an Anticalin™, an avimers (avidity multimers), a DARPin™, a Fynomer™, Centyrin™, and a Kunitz domain peptide. In certain cases, such mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa. Nucleic acids and small molecules may be antibody mimetic.

[0052] In some embodiments, the tumor cell binding moiety is a stabilized peptide.

Intramolecular crosslinkers are used to maintain the peptide in the desired configuration, for example using disulfide bonds, amide bonds, or carbon-carbon bonds to link amino acid side chains. Such peptides which are conformationally stabilized by means of intramolecular cross-linkers are sometimes referred to as "stapled" peptides. The cross-linkers connect at least two amino acids of the peptide. The cross-linkers may comprise at least 5, 6, 7, 8, 9, 10, 11, or 12 consecutive carbon-carbon bonds. The cross-linkers may comprise at least 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms. Stapled peptides may penetrate cell membranes and bind to an intracellular receptor. [0053] In one non-limiting example, the stapled peptide is a cross-linked alpha-helical polypeptide comprising a crosslinker wherein a hydrogen atom attached to an a-carbon atom of an amino acid of the peptide is replaced with a substituent of formula R-, wherein R- is alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroalkyl, or heterocycloalkyl, unsubstituted or substituted with halo-, as disclosed in US 20140323701 to Nash et al., the contents of which are incorporated herein by reference in their entirety. In another example, the stapled peptides have improved in vivo half life such as any stapled peptide disclosed in US 20100298201 to Nash et al., the contents of which are incorporated herein by reference in their entirety. In another example, the tumor cell binding moiety may be any stapled peptide disclosed in US 9175045 to Nash et al., the contents of which are incorporated herein by reference in their entirety, wherein the stapled peptide possesses reduced affinity to serum proteins while still remaining sufficient affinity to cell membranes. In another example, the cross-linker of the stapled peptide links the a-positions of at least two amino acids, such as any stapled peptide disclosed in US 9175047 to Nash et al., the contents of which are incorporated herein by reference in their entirety. In another example, the tumor cell binding moiety comprise any stapled peptide disclosed in US 8927500 to Guerlavais et al., the contents of which are incorporated herein by reference in their entirety, wherein the stapled peptide has homology to p53 protein and can bind to the MDM2 and/or MDMX proteins. In another example, the stapled peptide generates a reduced antibody response. Any stapled peptide disclosed in US 8808694 to Nash et al., the contents of which are incorporated herein by reference in their entirety, may be used as a tumor cell binding moiety. In another example, the stapled peptide may be any polypeptide with optimized protease stability disclosed in US 20110223149 to Nash et al., the contents of which are incorporated herein by reference in their entirety.

[0054] In some embodiments, the tumor cell binding moiety is a nanofitin® (Affilogic). Nanofitin, as used as herein, refers to a single-chain antibody mimic that are much smaller than antibodies. Nanofitins are small and stable, lack disulfide bridges, and can be produced at high levels. The molecular weight of nanofitins are below lOKDa, preferably around 7KDa. Because of their small size and short half-life, nanofitins may both accumulate specifically at the site of the tumor and be cleared from the serum rapidly, therefore reducing off-target toxicity compared to long lasting antibodies. Conjugates comprise nanofitins may deliver an active agent deeper into a tumor. Nanofitins may bind intracellular targets and affect intracellular protein-protein interaction. [0055] Nanofitins are derived from scaffold proteins engineered via combinational mutation/selection process disclosed in WO 2008/068637 (page 2, line 16 to page 9, line 2; examples page 12 to page 34; figures 1 to 21 and sequence listing), the contents of which are incorporated herein by reference in their entirety. The scaffold proteins are chosen from proteins that bind a wide range of structurally different ligands. One such family is proteins with oligonucleotide/oligosaccharide binding fold (OB-fold), a five-stranded β-barrel capped by an amphiphilic a-helix. OB-fold proteins recognize nucleic acids, oligosaccharides, proteins, and metallic ions. Combinatorial mutation/selection process to generate nanofitins comprises obtaining a combinatorial library corresponding to the randomization of a number of residues involved in the binding of a starting OB-fold protein with its native ligand, followed by a selection, in said library, of variants which bind specifically to a target of interest.

[0056] Non-limitative examples of OB-fold proteins which can be used in producing nanofitins include: Sac7d from Sulfolobus acidocaldarius (PDB lazp; UNIPROT or

GenBank P13123 or Q4JC17) and the truncated form of Sac7d, and Sac7b, Sac7e from Sulfolobus acidocaldarius (UNIPROT or GenBank P13125), Sso7d from Sulfolobus solfataricus (PDB M4; UNIPROT or GenBank P39476 or P81550), DBP 7 from Sulfolobus tokodaii (UNIPROT or GenBank Q96X56), Ssh7b from Sulfolobus shibatae (UNIPROT or GenBank 059632), Ssh7a from Sulfolobus shibatae (UNIPROT or GenBank P61990, 059631, P80170, Q9UW18), p7ss from Sulfolobus solfataricus (UNIPROT or GenBank P61991, 059631, P80170, Q9UW18), the N-terminal domain of SEB, the chain A of the Shiga-like toxin He of Escherichia coli (PDB 2bosA or lr4pB), the human Neutrophil Activating Peptide-2 (NAP-2, PDB ltvxA), the Molybdenum Binding Protein (modg) of Azotobacter vinelandii (PDB lh9j), the N-terminal domain of SPE-C, the B5 subunit of E. coli Shiga-like toxin, Cdcl3, the cold-shock DNA-binding domain of the human Y-box protein YB-1, the E. coli inorganic pyrophosphatase EPPase, any of the proteins listed in Table 3 of the article by Arcus et al., Curr Opin Struct Biol, vol.12(6): 794-801, (2002), the contents of which are incorporated herein by reference in their entirety), any protein in Tables 1, 2 and 3 of WO2008068637 to Pecorari et al., the contents of which are incorporated herein by reference in their entirety, the Lysyl-tRNA synthetase LysS of E.coli (PDB lkrs;

UNIPROT or GenBank B7UHT9), the Asp-tRNA synthetase of E.coli (PDB IcOaA;

UNIPROT or GenBank P21889), the Asp-tRNA synthetase of Thermococcus kodakaraensis (PDB lb8aA; UNIPROT or GenBank Q52428), the Lysyl-tRNA synthetase LysU of E.coli (PDB llylA; UNIPROT or GenBank P0A8N6), the human Replication protein A, 32kDa subunit (PDB lquqA; UNIPROT or GenBank PI 5927), the human Replication protein A, 14kDa subunit (PDB IquqB; UNIPROT or GenBank P35244), the human Replication protein A, 70kDa subunit (RPA70) fragment (PDB ljmcA; UNIPROT or GenBank P27694), the Telomere-end-binding protein of Oxytricha nova (PDB lotcA and lOtcB: UNIPROT or GenBank P29549 and P16458), the human mitochondrial ssDNA-binding protein (PDB 3ullA; UNIPROT or GenBank Q567R6), the Pertussis toxin S5 subunit of Bordetella pertussis (PDB lprtF; UNIPROT or GenBank P04981), the Pertussis toxin S5 subunit (ATP bound) of B. pertussis (PDB IbcpD; UNIPROT or GenBank P0A3R5), the Cholera Toxin of Vibrio cholera (PDB 3chbD; UNIPROT or GenBank D0UTQ9), the Heat-labile toxin of E. coli (PDB ItiiD; UNIPROT or GenBank P43529), the Verotoxin-1 /Shiga toxin, B-pentamer of E. coli (PDB 2bosA; UNIPROT or GenBank Q9MBZ7), the human TIMP-2 (PDB lbr9; UNIPROT or GenBank PI 6035), the Superantigen SPE-C of Streptococcus pyogenes (PDB lan8 UNIPROT or GenBank Q8NKX2), the Superantigen SPE of Staphylococcus aureus (PDB 3seb; UNIPROT or GenBank Q5MAA8), the Toxic shock syndrome toxin of

Staphylococcus aureus (PDB law7A; UNIPROT or GenBank A0FIN2), the Major cold- shock protein of E.coli (PDB ljmc; UNIPROT or GenBank P0A9Y1), the Initiation translation factor 5a of Pyrobaculum aerophylum (PDB lbkb; UNIPROT or GenBank P56635), the SI RNA-binding domain of PNPase of E.coli (PDB lsro; UNIPROT or GenBank P05055), the human Initiation translation factor 1, elFla (PDB ld7qA; UNIPROT or GenBank P47813), the Initiation translation factor 1, IF1 of E.coli (PDB lah9; UNIPROT or GenBank P69224), , the RNA guanylyltransferase of Chlorella virus, PBCV-1 (PDB lckmA; UNIPROT or GenBank Q84424), the ATP-dependent DNA ligase, of Bacteriophage T7 (PDB laOi; UNIPROT or GenBank P00969), the Staphylococcal nuclease,

Staphylococcus aureus (PDB lsnc; UNIPROT or GenBank gi/224650), the DNA helicase RuvA subunit, N-terminal domain of E.coli (PDB lhjp; UNIPROT or GenBank P0A811), the Gene V protein of Pseudomonas bacteriophage pf3 (PDB lpfsA; UNIPROT or GenBank P03672), the Gene V protein of Filamentous bacteriophage fl, Ml 3 (PDB lgvp; UNIPROT or GenBank D0U157), the Gene 32 protein (gp32) core of Bacteriophage T4 (PDB lgpc; UNIPROT or GenBank B3IYU0), and the Inorganic pyrophosphatase of Thermus thermophilus (PDB 2prd; UNIPROT or GenBank Q72H95). OB-folds domains originating from toxins may be used as starting OB-fold protein even for purposes in which toxicity is to be avoided, since mutations in their binding site, and hence change in their binding specificity, can completely abolish their toxicity.

[0057] By superimposing several sequences and 3D-structures of OB-fold domains using the web sites WU-Blast2, T-COFFEE, DALI lite, and DALI, the positions of the residues involved in the binding of the OB-fold protein can be identified, which can be substituted for obtaining Nanofitins according the method taught in WO 2008/068637 to Pecorari et al. and EP 2469278 to BEDOUELLE et al., the contents of each of which are incorporated herein by reference in their entirety. These residues are often located in β-strands β3, β4 and β5 and in loops 1, 3 and 4 of the OB-fold (Fig. lb and Fig. 2 of WO 2008/068637). For example, the online tool "DaliLite pairwise comparison of protein structures" was used to superimpose the structures of the protein under consideration on the structure of Sac7d chain A by using their PDB coordinates (PDB code lAZP for Sac7d chain A). A visual analysis of the

superimposed structures with the Pymol software (Pymol) was used to identify the correspondences between residues.

[0058] The same approach can be used to engineer a different binding site using the residues from another side of the OB-fold.

[0059] In some embodiments, nanofitins comprise 66 amino acid residue and are derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius. Sac7d is chemically and thermally stable and is resistant to extreme pH. Its molecular organization is simple, comprising only 66 amino acids, lacking a disulfide bridge, and possessing only one structural domain (the OB-fold). The binding face of Sac7d can be modified to recognize various targets without chaning its favorable biophysical properties.

[0060] The binding area of nanofitins that are Sac7d derivatives is located on the surface and is composed of 14 residues (i.e., residues 7-9, 21, 22, 24, 26, 29, 31, 33, 40, 42, 44, and 46), which can be modified to alter specificity (Mouratou, et al., Proc. Natl. Acad. Sci. USA 104: 17983-8 (2007), the contents of which are incorporated herein by reference in their entirety).

[0061] As identified in Fig. l b and Fig. 2 of WO 2008/068637, the residues of Sac7d which can be substituted are V2, K3, K5, K7, Y8, K9, G10, E14, T17, K21, K22, W24, V26, G27, K28, M29, S31, T33, D36, N37, G38, K39, T40, R42, A44, S46, E47, K48, D49, A50 and P51. The residues of Sac7d which can be deleted are: A59, R60, A61 and E64. Insertions of 1 to 15 amino acid residues can be performed in loop 3, for example in the region of residues 25 to 30 of Sac7d, preferably between residues 27 and 28. Insertions of 1 to 15 amino acid residues can be performed in loop 4, for example in the region of residues 35 to 40 of Sac7d, preferably between residues 37 and 38. Insertions of 1 to 20 amino acid residues can also be performed in loop 1, for example in the region of residues 7 to 12 of Sac7d, preferably between residues 9 and 10.

[0062] In one example, the tumor cell binding moiety of conjugates of the present invention comprises any nanofitin disclosed in WO2012085861 to BEDOUELLE et al., the contents of which are incorporated herein by reference in their entirety, such as a nanofitin which binds human immunoglobulins of class G (IgG) with high affinity ( d < 100 uM) (SEQ ID NO: 46- 58).

[0063] In another example, the tumor cell binding moiety of conjugates of the present invention comprises any nanofitin disclosed in WO2008068637 to Pecorari et al., the contents of which are incorporated herein by reference in their entirety, such as nanofitins that bind to PulD-N, PulDNl, NGF, PknG, Gar A, lysozyme, or human IgG.

[0064] In some embodiments, the tumor cell binding moiety X may be an aptide or bipodal peptide. X may be any D-Aptamer-Like Peptide (D-Aptide) or retro-inverso Aptide which specifically binds to a target comprising: (a) a structure stabilizing region comprising parallel, antiparallel or parallel and antiparallel D-amino acid strands with interstrand noncovalent bonds; and (b) a target binding region I and a target binding region II comprising randomly selected n and m D-amino acids, respectively, and coupled to both ends of the structure stabilizing region, as disclosed in US Pat. Application No. 20140296479 to Jon et al., the content of which is incorporated herein by reference in its entirety. X may be any bipodal peptide binder (BPB) comprising a structure stabilizing region of parallel or antiparallel amino acid strands or a combination of these strands to induce interstrand non-covalent bonds, and target binding regions I and II, each binding to each of both termini of the structure stabilizing region, as disclosed in US Pat. Application No. 20120321697 to Jon et al., the content of which is incorporated herein by reference in its entirety. X may be any bipodal peptide binder comprising a β-hairpin motif or a leucine-zipper motif as a structure stabilizing region comprising two parallel amino acid strands or two antiparallel amino acid strands, and a target binding region I linked to one terminus of the first of the strands of the structure stabilizing region, and a target binding region II linked to the terminus of the second of the strands of the structure stabilizing region, as disclosed in US Pat. Application No. 20110152500 to Jon et al., the content of which is incorporated herein by reference in its entirety. X may be any bipodal peptide binder targeting G protein-coupled receptor as disclosed in WO2011132938 to Jon et al., any bipodal peptide binder targeting receptor tyrosine kinase as disclosed in WO2011132940 to Jon et al., the content of each of which is incorporated herein by reference in their entirety. X may also be bipodal peptide binders targeting cluster differentiation (CD7) or an ion channel.

[0065] In some embodiments, the tumor cell binding moiety may comprise a nucleic acid targeting moiety. In general, a nucleic acid targeting moiety is any nucleic acid that binds to an organ, tissue, cell, or a component associated therewith such as extracellular matrix component. In some embodiments, the tumor cell binding moiety may be an aptamer, which is generally an oligonucleotide (e.g., DNA, RNA, or an analog or derivative thereof) that binds to a particular target, such as a polypeptide.

[0066] In some embodiments, the tumor cell binding moiety may be a non-immunoreactive ligand. For example, the non-immunoreactive ligand may be insulin, insulin-like growth factors I and II, lectins, apoprotein from low density lipoprotein, etc. as disclosed in US 20140031535 to Jeffrey, the content of which is incorporated herein by reference in its entirety. Any protein or peptide comprising a lectin disclosed in WO2013181454 to Radin, the content of which is incorporated herein by reference in its entirety, may be used as a tumor cell binding moiety.

[0067] In some embodiments, the conjugate may have a terminal half-life of longer than about 72 hours and the tumor cell binding moiety may be selected from Table 1 or 2 of US 20130165389 to Schellenberger et al., the contents of which are incorporated herein by reference in their entirety. The tumor cell binding moiety may be an antibody targeting deltalike protein 3 (DLL3) in disease tissues such as lung cancer, pancreatic cancer, skin cancer, etc., as disclosed in WO2014125273 to Hudson, the contents of which are incorporated herein by reference in their entirety. The tumor cell binding moiety may also any tumor cell binding moiety in WO2007137170 to Smith, the contents of which are incorporated herein by reference in their entirety. The tumor cell binding moiety binds to glypican-3 (GPC-3) and directs the conjugate to cells expressing GPC-3, such as hepatocellular carcinoma cells.

[0068] In certain embodiments, the tumor cell binding moiety or moieties of the conjugate are present at a predetermined molar weight percentage from about 1% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%, or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the molar weight percentages of the components of the conjugate is 100%. The amount of tumor cell binding moieties of the conjugate may also be expressed in terms of proportion to the T cell binding agent(s), for example, in a ratio of tumor cell binding moieties to T cell binding moieties of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5 : 1, 4: 1, 3 : 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4; 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.

C. Linkers

[0069] The T cell binding moiety may be connected to the tumor cell binding moiety via covalent bonds or a linker moieity. The linker moiety, Y, can be a Ci-Cio straight chain alkyl, Ci-Cio straight chain O-alkyl, Ci-Cio straight chain substituted alkyl, Ci-Cio straight chain substituted O-alkyl, C4-C13 branched chain alkyl, C4-C13 branched chain O-alkyl, C2- C12 straight chain alkenyl, C2-C12 straight chain O-alkenyl, C3-C12 straight chain substituted alkenyl, C3-C12 straight chain substituted O-alkenyl, polyethylene glycol, polylactic acid, polyglycolic acid, poly(lactide-co-glycolide), polycarprolactone, polycyanoacrylate, ketone, aryl, heterocyclic, succinic ester, amino acid, aromatic group, ether, crown ether, urea, thiourea, amide, purine, pyrimidine, bypiridine, indole derivative acting as a cross linker, chelator, aldehyde, ketone, bisamine, bis alcohol, heterocyclic ring structure, azirine, disulfide, thioether, hydrazone and combinations thereof. For example, the linker can be a C3 straight chain alkyl or a ketone. The alkyl chain of the linker can be substituted with one or more substituents or heteroatoms. In some embodiments the linker contains one or more atoms or groups selected from -0-, -C(=0)-, - R, -0-C(=0)- R-, -S-, -S-S-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid.

[0070] In some embodiments, the alkyl chain of the linker may optionally be interrupted by one or more atoms or groups selected from -0-, -C(=0)-, -NR, -0-C(=0)-NR-, -S-, -S-S-. The linker may be selected from dicarboxylate derivatives of succinic acid, glutaric acid or diglycolic acid.

[0071] In some embodiments, the linker is stable in tumor microenvironment. In some embodiments, the linker is not a cleavable linker.

[0072] In one embodiment, the linker may be a beta-glucuronide linker disclosed in US 20140031535 to Jeffrey, the contents of which are incorporated herein by reference in their entirety. In another embodiment, the linker may be a self-stabilizing linker such as a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring, disclosed in US20130309256 to Lyon et al., the contents of which are incorporated herein by reference in their entirety. In another embodiment, the linker may be a human serum albumin (HAS) linker disclosed in US 20120003221 to McDonagh et al., the contents of which are incorporated herein by reference in their entirety. In another embodiment, the linker may comprise a fullerene, e.g., C₆o, as disclosed in US 20040241173 to Wilson et al., the contents of which are incorporated herein by reference in their entirety. In another embodiment, the linker may be a recombinant albumin fused with polycysteine peptide as disclosed in US 8541378 to Ahn et al., the contents of which are incorporated herein by reference in their entirety. In another embodiment, the linker comprises a heterocycle ring. For example, the linker may be any heterocyclic 1,3 -substituted five- or six-member ring, such as thiazolidine, disclosed in US 20130309257 to Giulio, the content of which is incorporated herein by reference in its entirety.

[0073] In some embodiments, the linker may be used with compositions of the invention are well known in the art, and include, e.g., thyrogiobulin, albumins such as human seruni albumin, tetanus toxoid, polyamino acid residues such as poly L-lysine, poly L-glutamic acid, influenza virus proteins, hepatitis B virus core protein, and the like.

[0074] In some embodiments, the linker may be a hydrophilic linker as disclosed by Zhao et al. in PCT patent publication NO., WO2014/080251; the content of which is incorporated by reference in its entirety. The hydrophilic linkers may contain phosphinate, sulfonyl, and/or sulfoxide groups to link T cell binding agents to a tumor cell binding moiety.

[0075] In some embodiments, the linker may comprise a maleimide group.

[0076] In some embodiments, the linker of the conjugate may be optional. In this context, the T cell binding agent and the tumor cell binding moiety of the conjugated are directly connected to each other, such as via covalent bonds.

D. Masking Moiety

[0077] The disclosure also provides activatable compositions that include conjugates that are coupled to a masking moiety where the ability of the conjugate to bind to T cells and/or the tumor cells is reduced. Such conjugates are refered to as masked conjugates. The binding of the T cell binding moiety to target T cells or the binding of the tumor cell binding moiety to target tumor cells may be inhibited or hindered by the masking moiety. For example, the binding may be sterically hindered by the presence of the masking moiety or may be inhibited by the charge of the masking moiety. [0078] Cleavage of the masking moiety, a conformation change, or a chemical transformation may unmask/activate the conjugate. The masking/unmasking process may be reversible or irreversible. When the masked conjugates are activated, the ability to bind to T cells and/or the targeted tumor cells is at least comparable to the corresponding, un-masked conjugate.

[0079] In some embodiments, the masking moiety contains a peptide sequence that includes a substrate for a protease. The protease may be produced by a tumor that is in proximity to T cells or by a tumor that expresses the targeted tumor cell. Once the masking moiety is cleaved by the protease, the masking moiety no longer interferes with the binding of the conjugate to target T cells or target tumor cells, thereby activating the conjugates of the present invention. The masking moiety prevents binding of the conjugates of the present invention at nontreatment sites. Such conjugates can further provide improved biodistribution characteristics.

[0080] In one embodiment, the conjugate comprises a T cell binding moiety that binds to CD3s, and thereby binds to T cells that express CD3s. The conjugate further comprises a masking moiety, wherein the masking moiety has a substrate for a protease that is produced by a tumor that is in proximity to T cells that express CD3s. The protease may also be produced by a tumor that is co-localized with cells that express CD3s. Therefore, the conjugate is only activated when it gets to the tumor site that is in proximity to T cells that express CD3s.

[0081] In some embodiments, the masking moiety comprises a peptide that may be a substrate for an enzyme selected from the group consisting of MMPl, MMP2, MMP3, MMP8, MMP9, MMPl 4, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K,

CATHEPSIN S, ADAM 10, ADAM 12, ADAMTS, Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase- 11, Caspase-12, Caspase-13, Caspase-14, and TACE. For example, the masking moiety may comprise a protease substrate such as a plasmin substrate, a caspase substrate or a matrix metalloprotease (MMP) substrate (e.g., a substrate of MMP-1, MMP-2, MMP-9, or MMP- 14).

[0082] In some embodiments, the masking moiety is connected to the T cell binding moiety, the linker, or the tumor cell binidng moiety of the conjugate by a cleavable linker that is cleaved in the chemical environment of the tumor, for example in the acidic or reducing environment of a tumor. The masked conjugates are stable in circulation, activated at intended sites of therapy and/or diagnosis, but not in normal tissues. For example, the cleavable linker may comprise a cysteine-cysteine pair capable of forming a reducible disulfide bond, which may be cleaved by a reducing agent. Reducing agents of particular interest include cellular reducing agents such as proteins or other agents that are capable of reducing a disulfide bond under physiological conditions, e.g., glutathione, thioredoxin, NADPH, flavins, and ascorbate.

E. Pharmacokinetic Modulating Unit

[0083] The conjugates of the present invention may further comprise at least one external linker connected to a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof, or comprise at least one external linker connected to a pharmacokinetic modulating unit. The external linkers connecting the conjugates and the reacting group or the pharmacokinetic modulating units may be cleavable linkers that allow release of the conjugates. Hence, the conjugates may be separated from the protein or pharmacokinetic modulating units as needed.

[0084] In some embodiments, the conjugates comprise at least one reacting group that reacts with a functional group on a protein or an engineered protein or

derivatives/analogs/mimics thereof. The reaction between the reacting group and the functional group may happen in vivo after administration or is performed prior to

administration. The protein may be a naturally occurring protein such as a serum or plasma protein, or a fragment thereof. Particular examples include thyroxine-binding protein, transthyretin, αΐ-acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an

immunoglobulin, α-2-macroglobulin, a lipoprotein, or fragments thereof. The reaction between the reacting group and the functional group may be reversible.

[0085] In one example, the functional group is on human serum albumin (HSA or albumin) or its derivative/analog/mimic. Albumin is the most abundant plasma protein (35-50 g/L in human serum) with a molecular weight of 66.5 KDa and an effective diameter of 7.2 nm (Kratz, J. of Controlled Release, vol.132: 171, (2008), the contents of which are incorporated herein by reference in their entirety). Albumin has a half-life of about 19 days. Albumin preferentially accumulates in malignant and inflamed tissues due to a leaky capillary and an absent or defective lymphatic drainage system. Albumin accumulates in tumors such as solid tumors also because albumin is a major energy and nutrition source for turmor growth. The function group may be the cysteine-34 position of albumin that has an accessible free thiol group. Reacting groups that react with a functional group on albumin or it

derivative/analog/mimic may be selected from a disulfide group, a vinylcarbonyl group, a inyl acetylene group, an aziridine group, an acetylene group or any of the following groups:

where R is CI, Br, F, mesylate, tosylate, 0-(4-nitrophenyl), O-pentafluorophenyl, and wherein optionally the activated disulfide group, the vinylcarbonyl group, the vinyl acetylene group, the aziridine group, and the acetylene group may be substituted. The reacting group may also be any protein-binding moiety disclosed in US 9216228 to Kratz et al., the contents of which are incorporated herein by reference in their entirety, selected from the group consisting of a maleinimide group, a halogenacetamide group, a halogenacetate group, a pyridylthio group, a vinylcarbonyl group, an aziridine group, a disulfide group, a substituted or unsubstituted acetylene group, and a hydroxysuccinimide ester group. In some cases, the reacting group is a disulfide group. The disulfide group undergoes an exchange with a thiol group on a protein or an engineered protein or a polymer or derivatives/analogs/mimics thereof, such as albumin, to form a disulfide between the conjugate and the protein or an engineered protein or a polymer or derivatives/analogs/mimics thereof.

[0086] In another example, the functional group is on transthyretin or its

derivative/analog/mimic. Transthyretin is a 55 KDa serum protein that has an in vivo half-life of around 48 h. Reacting groups that react with a functional group on transthyretin or it derivative/analog/mimic may be selected from AGIO (structure shown below) or its derivative disclosed by Penchala et al. in Nature Chemical Biology, vol.11 :793, (2015) or formula (I), (II), (III) or (IV) (structures shown below) disclosed in US Pat. No. 5714142 to Blaney et al., the contents of each of which are incorporated herein by reference in their entirety. Any transthyretin-selective ligand disclosed on pages 5-8 of Blaney et al. or their derivatives may be used as a reacting group, such as but not limited to, tetraiodothyroacetic acid, 2,4,6-triiodophenol, flufenamic acid, diflunisal, milrinone, EMD 21388.

[0087] In some cases, the reacting group may be any protein binding moiety may be any protein binding moiety disclosed in US 9216228 to Kratz, the contents of which are incorporated herein by reference in their entirety, such as a maleimide group, a

halogenacetamide group, a halogenacetate group, a pyridylthio group, a vinylcarbonyl group, an aziridin group, a disulfide group, a substituted or unsubstituted acetylene group, and a hydroxysuccinimide ester group.

[0088] In some embodiments, the conjugates comprise at least one pharmacokinetic modulating unit. The pharmacokinetic modulating unit may be a natural or synthetic protein or fragment thereof. For example, it may be a serum protein such as thyroxine-binding protein, transthyretin, a 1 -acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an immunoglobulin, α-2-macroglobulin, a lipoprotein, or fragments thereof. The

pharmacokinetic modulating unit may also be a natural or synthetic polymer, such as polysialic acid unit, a hydroxyethyl starch (HES) unit, or a polyethylene glycol (PEG) unit. Further, the pharmacokinetic modulating unit may be a particle, such as dendrimers, inorganic nanoparticles, organic nanoparticles, and liposomes.

[0089] The pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight of at least about 10 KDa, at least about 20 KDa, at least about 30 KDa, at least about 40 KDa or at least about 50 KDa. Generally, the pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight between about 10 KDa and about 70 KDa. Preferably, the pharmacokinetic modulating unit or pharmacokinetic modulating units have a total molecular weight between about 30 KDa and about 70 KDa, between about 40 KDa and about 70 KDa, between about 50 KDa and about 70 KDa, between about 60 KDa and about 70 KDa. II. Particles and nanoparticles

[0090] Particles comprising one or more conjugates can be polymeric particles, lipid particles, solid lipid particles, solid lipid nanoparticles, solid nanoparticles, inorganic particles, or combinations thereof (e.g., lipid stabilized polymeric particles). In some embodiments, the conjugates are incorporated, dispersed, or distributed throughout the particles. In some embodiments, the conjugates are substantially encapsulated or particularly encapsulated in the particles. In some embodiments, the conjugates are disposed on the surface of the particles. The conjugates may be attached to the surface of the particles with covalent bonds, or non-covalent interactions. In some embodiments, the conjugates of the present invention self-assemble into a particle.

[0091] As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the conjugates of the invention, encapsulation may be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.999% of conjugate of the invention may be enclosed, surrounded or encased within the particle.

"Partially encapsulation" means that less than 10, 10, 20, 30, 40 50 or less of the conjugate of the invention may be enclosed, surrounded or encased within the particle. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the particle. Encapsulation may be determined by any known method. In some embodiments, the particles are polymeric particles or contain a polymeric matrix. The particles can contain any of the polymers described herein or derivatives or copolymers thereof. The particles will generally contain one or more biocompatible polymers. The polymers can be biodegradable polymers. The polymers can be hydrophobic polymers, hydrophilic polymers, or amphiphilic polymers. In some embodiments, the particles contain one or more polymers having an additional targeting moiety attached thereto. In some embodiments, the particles are inorganic particles, such as but not limited to, gold nanoparticles and iron oxide

nanoparticles.

[0092] The size of the particles can be adjusted for the intended application. The particles can be nanoparticles or microparticles. The particle can have a diameter of about 10 nm to about 10 microns, about 10 nm to about 1 micron, about 10 nm to about 500 nm, about 20 nm to about 500 nm, or about 25 nm to about 250 nm. In some embodiments the particle is a nanoparticle having a diameter from about 25 nm to about 250 nm. In some embodiments, the particle is a nanoparticle having a diameter from about 50 nm to about 150 nm. In some embodiments, the particle is a nanoparticle having a diameter from about 70 nm to about 130 nm. In some embodiments, the particle is a nanoparticle having a diameter of about 100 nm. It is understood by those in the art that a plurality of particles will have a range of sizes and the diameter is understood to be the median diameter of the particle size distribution.

Polydispersity index (PDI) of the particles may be < about 0.5, < about 0.2, or < about 0.1. Drug loading may be > about 1%, > about 5%, > about 10%, or > out 20%. Drug loading, as used herein, refers to the weight ratio of the conjugates of the invention and depends on maximum tolerated dose (MTD) of free drug conjugate. Particle ζ-potential (in l/lO^ PBS) may be <0 mV or from about -10 to 0 mV. Drug released in vitro from the particle at 2h may be less than about 60%, less than about 40%, or less than about 20%. Regarding

pharmacokinetics, plasma area under the curve (AUC) in a plot of concentration of drug in blood plasma against time may be at least 2 fold greater than free drug conjugate, at least 4 fold greater than free drug conjugate, at least 5 fold greater than free drug conjugate, at least 8 fold greater than free drug conjugate, or at least 10 fold greater than free drug conjugate. Tumor PK/PD of the particle may be at least 5 fold greater than free drug conjugate, at least 8 fold greater than free drug conjugate, at least 10 fold greater than free drug conjugate, or at least 15 fold greater than free drug conjugate. The ratio of Cmax of the particle to Cmax of free drug conjugate may be at least about 2, at least about 4, at least about 5, or at least about 10. Cmax, as used herein, refers to the maximum or peak serum concentration that a drug achieves in a specified compartment or test area of the body after the drug has been administrated and prior to the administration of a second dose. The ratio of MTD of a particle to MTD of free drug conjugate may be at least about 0.5, at least about 1, at least about 2, or at least about 5. Efficacy in tumor models, e.g., TGI%, of a particle is better than free drug conjugate.

Toxicity of a particle is lower than free drug conjugate.

[0093] In various embodiments, a particle may be a nanoparticle, i.e., the particle has a characteristic dimension of less than about 1 micrometer, where the characteristic dimension of a particle is the diameter of a perfect sphere having the same volume as the particle. The plurality of particles can be characterized by an average diameter (e.g., the average diameter for the plurality of particles). In some embodiments, the diameter of the particles may have a Gaussian-type distribution. In some embodiments, the plurality of particles have an average diameter of less than about 300 nm, less than about 250 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 50 nm, less than about 30 nm, less than about 10 nm, less than about 3 nm, or less than about 1 nm. In some embodiments, the particles have an average diameter of at least about 5 nm, at least about 10 nm, at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 150 nm, or greater. In certain embodiments, the plurality of the particles have an average diameter of about 10 nm, about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 500 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 50 nm and about 400 nm, between about 100 nm and about 300 nm, between about 150 nm and about 250 nm, between about 175 nm and about 225 nm, or the like. In some embodiments, the plurality of particles have an average diameter between about 10 nm and about 500 nm, between about 20 nm and about 400 nm, between about 30 nm and about 300 nm, between about 40 nm and about 200 nm, between about 50 nm and about 175 nm, between about 60 nm and about 150 nm, between about 70 nm and about 130 nm, or the like. For example, the average diameter can be between about 70 nm and 130 nm. In some embodiments, the plurality of particles have an average diameter between about 20 nm and about 220 nm, between about 30 nm and about 200 nm, between about 40 nm and about 180 nm, between about 50 nm and about 170 nm, between about 60 nm and about 150 nm, or between about 70 nm and about 130 nm. In one embodiment, the particles have a size of 40 to 120 nm with a zeta potential close to 0 mV at low to zero ionic strengths (1 to 10 mM), with zeta potential values between + 5 to - 5 mV, and a zero/neutral or a small -ve surface charge.

[0094] In some embodiments, the particles of the invention may comprise more than one conjugates. The conjugates may be different, e.g., comprising different T cell binding moieties or tumor cell binding moieties. In some embodiments, the particles of the invention may comprises conjugates having different PK values. Conjugates in the same particle are protected by the particle and are released at the same time.

[0095] In some embodiments, the weight percentage of the conjugate in the particles is at least about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% such that the sum of the weight percentages of the components of the particles is 100%. In some embodiments, the weight percentage of the conjugate in the particles is from about 0.5% to about 10%, or about 10% to about 20%, or about 20% to about 30%, or about 30% to about 40%), or about 40% to about 50%, or about 50% to about 60%, or about 60% to about 70%, or about 70% to about 80%, or about 80% to about 90%, or about 90% to about 99% such that the sum of the weight percentages of the components of the particles is 100%. A. Polymers

[0096] The particles of the invention may contain one or more polymers. Polymers may contain one more of the following polyesters: homopolymers including glycolic acid units, referred to herein as "PGA", and lactic acid units, such as poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as "PLA", and caprolactone units, such as poly(s-caprolactone), collectively referred to herein as "PCL"; and copolymers including lactic acid and glycolic acid units, such as various forms of poly(lactic acid-co-glycolic acid) and poly(lactide-co- glycolide) characterized by the ratio of lactic acid:gly colic acid, collectively referred to herein as "PLGA"; and polyacrylates, and derivatives thereof. Exemplary polymers also include copolymers of polyethylene glycol (PEG) and the aforementioned polyesters, such as various forms of PLGA-PEG or PLA-PEG copolymers, collectively referred to herein as "PEGylated polymers". In certain embodiments, the PEG region can be covalently associated with polymer to yield "PEGylated polymers" by a cleavable linker.

[0097] The particles may contain one or more hydrophilic polymers. Hydrophilic polymers include cellulosic polymers such as starch and polysaccharides; hydrophilic polypeptides; poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamic acid, poly-L- aspartic acid, poly-L-serine, or poly-L-lysine; polyalkylene glycols and polyalkylene oxides such as polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO); poly(oxyethylated polyol); poly(olefinic alcohol); polyvinylpyrrolidone);

poly(hydroxyalkylmethacrylamide); poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids); poly(vinyl alcohol); polyoxazoline; and copolymers thereof.

[0098] The particles may contain one or more hydrophobic polymers. Examples of suitable hydrophobic polymers include polyhydroxyacids such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acids); polyhydroxyalkanoates such as poly3- hydroxybutyrate or poly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);

polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones); polycarbonates such as tyrosine polycarbonates; polyamides (including synthetic and natural polyamides), polypeptides, and poly(amino acids); polyesteramides; polyesters; poly(dioxanones);

poly(alkylene alkylates); hydrophobic polyethers; polyurethanes; polyetheresters;

polyacetals; polycyanoacrylates; polyacrylates; polymethylmethacrylates; polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers; polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates; polyalkylene succinates; poly(maleic acids), as well as copolymers thereof.

[0099] In certain embodiments, the hydrophobic polymer is an aliphatic polyester. In some embodiments, the hydrophobic polymer is poly(lactic acid), poly(glycolic acid), or poly(lactic acid-co-glycolic acid).

[00100] The particles can contain one or more biodegradable polymers. Biodegradable polymers can include polymers that are insoluble or sparingly soluble in water that are converted chemically or enzymatically in the body into water-soluble materials.

Biodegradable polymers can include soluble polymers crosslinked by hydolyzable cross- linking groups to render the crosslinked polymer insoluble or sparingly soluble in water.

[00101] Biodegradable polymers in the particle can include polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,

polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, alkyl cellulose such as methyl cellulose and ethyl cellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, and hydroxybutyl methyl cellulose, cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, polymers of acrylic and methacrylic esters such as poly (methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly (isobutylmethacry late), poly(hexlmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinyl chloride polystyrene and polyvinylpyrrolidone, derivatives thereof, linear and branched copolymers and block copolymers thereof, and blends thereof. Exemplary biodegradable polymers include polyesters, poly(ortho esters), poly(ethylene imines), poly(caprolactones), poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides, poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates, polyphosphate esters, polyphosphazenes, derivatives thereof, linear and branched copolymers and block

copolymers thereof, and blends thereof. In some embodiments the particle contains biodegradable polyesters or polyanhydrides such as poly(lactic acid), poly(glycolic acid), and poly(lactic-co-glycolic acid). [00102] The particles can contain one or more amphiphilic polymers. Amphiphilic polymers can be polymers containing a hydrophobic polymer block and a hydrophilic polymer block. The hydrophobic polymer block can contain one or more of the hydrophobic polymers above or a derivative or copolymer thereof. The hydrophilic polymer block can contain one or more of the hydrophilic polymers above or a derivative or copolymer thereof. In some

embodiments the amphiphilic polymer is a di -block polymer containing a hydrophobic end formed from a hydrophobic polymer and a hydrophilic end formed of a hydrophilic polymer. In some embodiments, a moiety can be attached to the hydrophobic end, to the hydrophilic end, or both. The particle can contain two or more amphiphilic polymers.

B. Lipids

[00103] The particles may contain one or more lipids or amphiphilic compounds. For example, the particles can be liposomes, lipid micelles, solid lipid particles, or lipid-stabilized polymeric particles. The lipid particle can be made from one or a mixture of different lipids. Lipid particles are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. The lipid particle is preferably made from one or more biocompatible lipids. The lipid particles may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH.

[00104] The particle can be a lipid micelle. Lipid micelles for drug delivery are known in the art. Lipid micelles can be formed, for instance, as a water-in-oil emulsion with a lipid surfactant. An emulsion is a blend of two immiscible phases wherein a surfactant is added to stabilize the dispersed droplets. In some embodiments the lipid micelle is a microemulsion. A microemulsion is a thermodynamically stable system composed of at least water, oil and a lipid surfactant producing a transparent and thermodynamically stable system whose droplet size is less than 1 micron, from about 10 nm to about 500 nm, or from about 10 nm to about 250 nm. Lipid micelles are generally useful for encapsulating hydrophobic active agents, including hydrophobic therapeutic agents, hydrophobic prophylactic agents, or hydrophobic diagnostic agents.

[00105] The particle can be a liposome. Liposomes are small vesicles composed of an aqueous medium surrounded by lipids arranged in spherical bilayers. Liposomes can be classified as small unilamellar vesicles, large unilamellar vesicles, or multi-lamellar vesicles. Multi-lamellar liposomes contain multiple concentric lipid bilayers. Liposomes can be used to encapsulate agents, by trapping hydrophilic agents in the aqueous interior or between bilayers, or by trapping hydrophobic agents within the bilayer.

[00106] The lipid micelles and liposomes typically have an aqueous center. The aqueous center can contain water or a mixture of water and alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as «-butanol, isobutanol, sec-butanol, tert-butanol, pentanol (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1-hexanol, 2-hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3 -heptanol and 4-heptanol) or octanol (such as 1-octanol) or a combination thereof.

[00107] The particle can be a solid lipid particle. Solid lipid particles present an alternative to the colloidal micelles and liposomes. Solid lipid particles are typically submicron in size, i.e. from about 10 nm to about 1 micron, from 10 nm to about 500 nm, or from 10 nm to about 250 nm. Solid lipid particles are formed of lipids that are solids at room temperature. They are derived from oil-in-water emulsions, by replacing the liquid oil by a solid lipid.

[00108] Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including 1 ,2-diacyl-glycero-3-phosphocholines;

phosphatidyl serine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids;

sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1- ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl- sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated l,2-diacyl-s«-glycero-3-phosphocholines, l-acyl-2-acyl-s«-glycero- 3-phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids.

[00109] Suitable cationic lipids include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-).

Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[l-(2,3- dioloyloxy)propyl]-N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dialkyloxy-3-dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 - [N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Choi); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl-l -propanaminium trifluoro-acetate (DOSPA), β-alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diCi4-amidine, N-ferf- butyl-N'-tetradecyl-3-tetradecylamino-propionamidine, N-(alpha- trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N- (trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)- propylamide (DOSPER), and N , N , N' , N'-tetramethyl- , N'-bis(2-hydroxylethyl)-2,3- dioleoyloxy-1 ,4-butanediammonium iodide. In one embodiment, the cationic lipids can be l-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, for example, l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2-(hexadecanoyloxy)ethyl]-2- pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTEVI). In one embodiment, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3- dimethyl-hydroxy ethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3-dimethyl- hydroxy ethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DSRIE).

[00110] Suitable solid lipids include, but are not limited to, higher saturated alcohols, higher fatty acids, sphingolipids, synthetic esters, and mono-, di-, and triglycerides of higher saturated fatty acids. Solid lipids can include aliphatic alcohols having 10-40, preferably 12- 30 carbon atoms, such as cetostearyl alcohol. Solid lipids can include higher fatty acids of 10- 40, preferably 12-30 carbon atoms, such as stearic acid, palmitic acid, decanoic acid, and behenic acid. Solid lipids can include glycerides, including monoglycerides, diglycerides, and triglycerides, of higher saturated fatty acids having 10-40, preferably 12-30 carbon atoms, such as glyceryl monostearate, glycerol behenate, glycerol palmitostearate, glycerol trilaurate, tricaprin, trilaurin, trimyristin, tripalmitin, tristearin, and hydrogenated castor oil. Suitable solid lipids can include cetyl palmitate, beeswax, or cyclodextrin.

[00111] Amphiphilic compounds include, but are not limited to, phospholipids, such as 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), and dilignoceroylphatidylcholine (DLPC), incorporated at a ratio of between 0.01-60 (weight lipid/w polymer), for example, between 0.1-30 (weight lipid/w polymer). Phospholipids which may be used include, but are not limited to, phosphatidic acids, phosphatidyl cholines with both saturated and unsaturated lipids, phosphatidyl ethanolamines,

phosphatidylglycerols, phosphatidyl serines, phosphatidylinositols, lysophosphatidyl derivatives, cardiolipin, and β-acyl-y-alkyl phospholipids. Examples of phospholipids include, but are not limited to, phosphatidylcholines such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine

dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC),

distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcho- line (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC); and phosphatidylethanolamines such as

dioleoylphosphatidylethanolamine or 1 -hexadecyl-2-palmitoylglycerophos-phoethanolamine. Synthetic phospholipids with asymmetric acyl chains (e.g., with one acyl chain of 6 carbons and another acyl chain of 12 carbons) may also be used.

C. Hydrophobic ion-pairing complexes

[00112] The particles may comprise hydrophobic ion-pairing complexes or hydrophobic ioin-pairs formed by one or more conjugates described above and counterions.

[00113] Hydrophobic ion-pairing (HIP) is the interaction between a pair of oppositely charged ions held together by Coulombic attraction. HIP, as used here in, refers to the interaction between the conjugate of the present invention and its counterions, wherein the counterion is not H⁺ or HO^" ions. Hydrophobic ion-pairing complex or hydrophobic ion-pair, as used herein, refers to the complex formed by the conjugate of the present invention and its counterions. In some embodiments, the counterions are hydrophobic. In some embodiments, the counterions are provided by a hydrophobic acid or a salt of a hydrophobic acid. In some embodiments, the counterions are provided by bile acids or salts, fatty acids or salts, lipids, or amino acids. In some embodiments, the counterions are negatively charged (anionic). Non- limited examples of negative charged counterions include the counterions sodium

sulfosuccinate (AOT), sodium oleate, sodium dodecyl sulfate (SDS), human serum albumin (HSA), dextran sulphate, sodium deoxycholate, sodium cholate, anionic lipids, amino acids, or any combination thereof. Non-limited examples of positively charged counterions include l,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), cetrimonium bromide (CTAB), quaternary ammonium salt didodecyl dimethylammonium bromide (DMAB) or Didodecyldimethylammonium bromide (DDAB). Without wishing to be bound by any theory, in some embodiments, HIP may increase the hydrophobicity and/or lipophilicity of the conjugate of the present invention. In some embodiments, increasing the hydrophobicity and/or lipophilicity of the conjugate of the present invention may be beneficial for particle formulations and may provide higher solubility of the conjugate of the present invention in organic solvents. Without wishing to be bound by any theory, it is believed that particle formulations that include HIP pairs have improved formulation properties, such as drug loading and/or release profile. Without wishing to be bound by any theory, in some embodiments, slow release of the conjugate of the invention from the particles may occur, due to a decrease in the conjugate's solubility in aqueous solution. In addition, without wishing to be bound by any theory, complexing the conjugate with large hydrophobic counterions may slow diffusion of the conjugate within a polymeric matrix. In some emobodiments, HIP occurs without covalent conjuatation of the counterion to the conjugate of the present invention.

[00114] Without wishing to be bound by any theory, the strength of HIP may impact the drug load and release rate of the particles of the invention. In some embodiments, the strength of the HIP may be increased by increasing the magnitude of the difference between the pKa of the conjugate of the present invention and the pKa of the agent providing the counterion. Also without wishing to be bound by any theory, the conditions for ion pair formation may impact the drug load and release rate of the particles of the invention.

[00115] In some embodiments, any suitable hydrophobic acid or a combination thereof may form a HIP pair with the conjugate of the present invention. In some embodiments, the hydrophobic acid may be a carboxylic acid (such as but not limited to a monocarboxylic acid, dicarboxylic acid, tricarboxylic acid), a sulfinic acid, a sulfenic acid, or a sulfonic acid. In some embodiments, a salt of a suitable hydrophobic acid or a combination thereof may be used to form a HIP pair with the conjugate of the present invention. Examples of hydrophobic acids, saturated fatty acids, unsaturated fatty acids, aromatic acids, bile acid, polyelectrolyte, their dissociation constant in water (pKa) and logP values were disclosed in

WO2014/043,625, the content of which is incorporated herein by reference in its entirety. The strength of the hydrophobic acid, the difference between the pKa of the hydrophobic acid and the pKa of the conjuagate of the present invention, logP of the hydrophobic acid, the phase transition temperature of the hydrophobic acid, the molar ratio of the hydrophobic acid to the conjugate of the present invention, and the concentration of the hydrophobic acid were also disclosed in WO2014/043,625, the content of which is incorporated herein by reference in its entirety.

[00116] In some embodiments, particles of the present invention comprising a HIP complex and/or prepared by a process that provides a counterion to form HIP complex with the conjugate may have a highter drug loading than particles without a HIP complex or prepared by a process that does not provide any counterion to form HIP complex with the conjugate. In some embodiments, drug loading may increase 50%, 100%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times.

[00117] In some embodiments, the particles of the invention may retain the conjugate for at least about 1 minute, at least about 15 minutes, at least about 1 hour, when placed in a phosphate buffer solution at 37°C

D. Additional active agents

[00118] The particles can contain one or more additional active agents in addition to those in the conjugates. The additional active agents can be therapeutic, prophylactic, diagnostic, or nutritional agents. The additional active agents can be present in any amount, e.g. from about 1%) to about 90%), from about 1%> to about 50%, from about 1%> to about 25%, from about 1% to about 20%), from about 1%> to about 10%>, or from about 5% to about 10%> (w/w) based upon the weight of the particle. In one embodiment, the agents are incorporated in about 1%> to about 10%) loading w/w.

E. Additional targeting moieties

[00119] The particles can contain one or more targeting moieties targeting the particle to a specific organ, tissue, cell type, or subcellular compartment in addition to the targeting moieties of the conjugate. The additional targeting moieties can be present on the surface of the particle, on the interior of the particle, or both. The additional targeting moieties can be immobilized on the surface of the particle, e.g., can be covalently attached to polymer or lipid in the particle. In preferred embodiments, the additional targeting moieties are covalently attached to an amphiphilic polymer or a lipid such that the targeting moieties are oriented on the surface of the particle.

III. Pharmaceutical formulations

[00120] In some embodiments, conjugates, particles of the present invention may be formulated as vaccines, provided as liquid suspensions or as freeze-dried products. Suitable liquid preparations may include, but are not limited to, isotonic aqueous solutions, suspensions, emulsions, or viscous compositions that are buffered to a selected pH.

[00121] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit. As used herein, the term "active ingredient" refers to any chemical and biological substance that has a physiological effect in human or in animals, when exposed to it. In the context of the present invention, the active ingredient in the formulations may be any conjugates and particles as discussed herein above.

[00122] A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[00123] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between .5 and 50%), between 1-30%, between 5-80%>, at least 80%> (w/w) active ingredient.

[00124] The conjugates or particles of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) permit the sustained or delayed release (e.g., from a depot formulation of the monomaleimide); (3) alter the biodistribution (e.g., target the monomaleimide compounds to specific tissues or cell types); (4) alter the release profile of the monomaleimide compounds in vivo. Non-limiting examples of the excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, and

preservatives. Excipients of the present invention may also include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics and combinations thereof.

Accordingly, the formulations of the invention may include one or more excipients, each in an amount that together increases the stability of the monomaleimide compounds.

[00125] In some embodiments, the particles comprise biocompatible polymers. In some embodiments, the particles comprise about 0.2 to about 35 weight percent of a therapeutic agent; and about 10 to about 99 weight percent of a biocompatible polymer such as a diblock poly(lactic) acid-poly(ethylene)glycol as disclosed in US 20140356444 to Troiano et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety. Any therapeutically particle composition in US 8663700, 8652528, 8609142, 8293276 and 8420123, the contents of each of which are incorporated herein by reference in their entirety, may also be used.

[00126] In some embodiments, the particles comprise a hydrophobic acid. In some embodiments, the particles comprise about 0.05 to about 30 weight percent of a substantially hydrophobic acid; about 0.2 to about 20 weight percent of a basic therapeutic agent having a protonatable nitrogen; wherein the pKa of the basic therapeutic agent is at least about 1.0 pKa units greater than the pKa of the hydrophobic acid; and about 50 to about 99.75 weight percent of a diblock poly(lactic) acid-poly(ethylene)glycol copolymer or a diblock poly(lactic acid-co-glycolic acid)-poly(ethylene)glycol copolymer, wherein the therapeutic nanoparticle comprises about 10 to about 30 weight percent poly(ethylene)glycol as disclosed in

WO2014043625 to Figueiredo et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety. Any therapeutical particle composition in US 20140149158, 20140248358, 20140178475 to Figueiredo et al., the contents of each of which are incorporated herein by reference in their entirety, may also be used.

[00127] In some embodiments, the particles comprise a chemotherapeutic agent; a diblock copolymer of poly(ethylene)glycol and polylactic acid; and a ligand conjugate, as disclosed in US 20140235706 to Zale et al. (BIND Therapeutics), the contents of which are

incorporated herein by reference in their entirety. Any of the particle compositions in US 8603501, 8603500, 8603499, 8273363, 8246968, 20130172406 to Zale et al., may also be used.

[00128] In some embodiments, the particles comprise a targeting moiety. As a non-limiting example, the particles may comprise about 1 to about 20 mole percent PLA-PEG-basement vascular membrane targeting peptide, wherein the targeting peptide comprises PLA having a number average molecular weight of about 15 to about 20 kDa and PEG having a number average molecular weight of about 4 to about 6 kDa; about 10 to about 25 weight percent anti-neointimal hyperplasia (NIH) agent; and about 50 to about 90 weight percent non- targeted poly-lactic acid-PEG, wherein the therapeutic particle is capable of releasing the anti-NIH agent to a basement vascular membrane of a blood vessel for at least about 8 hours when the therapeutic particle is placed in the blood vessel as disclosed in US 8563041 to Grayson et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.

[00129] In some embodiments, the particles comprise about 4 to about 25% by weight of an anti-cancer agent; about 40 to about 99% by weight of poly(D,L-lactic)acid- poly(ethylene)glycol copolymer; and about 0.2 to about 10 mole percent PLA-PEG-ligand; wherein the pharmaceutical aqueous suspension have a glass transition temperature between about 39 and 41°C, as disclosed in US 8518963 to Ali et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.

[00130] In some embodiments, the particles comprise about 0.2 to about 35 weight percent of a therapeutic agent; about 10 to about 99 weight percent of a diblock poly(lactic) acid- poly(ethylene)glycol copolymer or a diblock poly(lactic)-co-poly (glycolic) acid- poly(ethylene)glycol copolymer; and about 0 to about 75 weight percent poly(lactic) acid or poly(lactic) acid-co-poly (glycolic) acid as disclosed in WO2012166923 to Zale et al. (BIND Therapeutics), the contents of which are incorporated herein by reference in their entirety.

[00131] In some embodiments, the particles are long circulating and may be formulated in a biocompatible and injectable formulation. For example, the particles may be a sterile, biocompatible and injectable nanoparticle composition comprising a plurality of long circulating nanoparticles having a diameter of about 70 to about 130 nm, each of the plurality of the long circulating nanoparticles comprising about 70 to about 90 weight percent poly(lactic) acid-co-poly(ethylene) glycol, wherein the weight ratio of poly(lactic) acid to poly(ethylene) glycol is about 15 kDa/2 kDa to about 20 kDa/10 kDa, and a therapeutic agent encapsulated in the nanoparticles as disclosed in US 20140093579 to Zale et al. (BIND Therapeutics), the content of which is incorporated herein by reference in its entirety.

[00132] In some embodiments, provided is a reconstituted lyophilized pharmaceutical composition suitable for parenteral administration comprising the particles of the present invention. For example, the reconstituted lyophilized pharmaceutical composition may comprise a 10-100 mg/mL concentration of polymeric nanoparticles in an aqueous medium; wherein the polymeric nanoparticles comprise: a poly(lactic) acid-block-poly(ethylene)glycol copolymer or poly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycol copolymer, and a taxane agent; 4 to 6 weight percent sucrose or trehalose; and 7 to 12 weight percent hydroxypropyl β-cyclodextrin, as disclosed in US 8637083 to Troiano et al. (BIND

Therapeutics), the contents of which are incorporated herein by reference in their entirety. Any pharmaceutical composition in US 8603535, 8357401, 20130230568, 20130243863 to Troiano et al. may also be used.

[00133] In some embodiments, the conjugates and/or particles of the invention may be delivered with a bacteriophage. For example, a bacteriophage may be conjugated through a labile/non labile linker or directly to at least 1,000 therapeutic drug molecules such that the drug molecules are conjugated to the outer surface of the bacteriophage as disclosed in US 20110286971 to Yacoby et al., the content of which is incorporated herein by reference in its entirety. According to Yacoby et al., the bacteriophage may comprise an exogenous targeting moiety that binds a cell surface molecule on a target cell.

[00134] In some embodiments, the conjugates and/or particles of the invention may be delivered with a dendrimer. The conjugates may be encapsulated in a dendrimer, or disposed on the surface of a dendrimer. For example, the conjugates may bind to a scaffold for dendritic encapsulation, wherein the scaffold is covalently or non-covalently attached to a polysaccharide, as disclosed in US 20090036553 to Piccariello et al., the content of which is incorporated herein by reference in its entirety. The scaffold may be any peptide or oligonucleotide scaffold disclosed by Piccariello et al.

[00135] In some embodiments, the conjugates and/or particles of the invention may be delivered by a cyclodextrin. In one embodiment, the conjugates may be formulated with a polymer comprising a cyclodextrin moiety and a linker moiety as disclosed in US

20130288986 to Davis et al., the content of which is incorporated herein by reference in its entirety. Davis et al. also teaches that the conjugate may be covalently attached to a polymer through a tether, wherein the tether comprises a self-cyclizing moiety. [00136] In some embodiments, the conjugates and/or particles of the invention may be delivered with an aliphatic polymer. For example, the aliphatic polymer may comprise polyesters with grafted zwitterions, such as polyester-graft-phosphorylcholine polymers prepared by ring-opening polymerization and click chemistry as disclosed in US 8802738 to Emrick; the content of which is incorporated herein by reference in its entirety.

A. Excipients

[00137] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[00138] In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

[00139] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions.

[00140] Exemplary diluents 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, etc., and/or combinations thereof.

[00141] Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn 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 polyvinylpyrrolidone) (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, etc., and/or combinations thereof.

[00142] Exemplary surface active agents and/or emulsifiers 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, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl 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 fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate

[TWEEN®20], polyoxyethylene sorbitan [TWEENn®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. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether

[BRIJ®30]), poly(vinyl-pyrrolidone), di ethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

[00143] Exemplary binding agents include, but are not limited to, 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, polyvinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid;

polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.

[00144] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabi sulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabi sulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary 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. Exemplary 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.

Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabi sulfite, potassium sulfite, potassium metabi sulfite, GLYDANT PLUS®, PHENONIP®, methylparaben,

GERMALL® 115, GERMABEN®II, EOLO E™, KATHON™, and/or EUXYL®.

[00145] Exemplary 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, 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, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.

[00146] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[00147] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl my ri state, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, 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, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof. [00148] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.

B. Lipidoids

[00149] Lipidoids may be used to deliver conjugates of the present invention. Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the conjugates of the present invention, for a variety of therapeutic indications including vaccine adjuvants, following the injection of a lipidoid formulation via localized and/or systemic routes of administration. Lipidoid complexes of conjugates of the present invention can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.

[00150] The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to conjugates of the present invention.

[00151] The use of lipidoid formulations for the localized delivery of conjugates to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the conjugates.

C. Liposomes, Lipid Nanopar tides and Lipoplexes

[00152] The conjugates of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of the conjugates of the invention include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. [00153] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to- batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[00154] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from l,2-dioleyloxy-N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), l,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety).

[00155] In one embodiment, the conjugates of the invention may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.

[00156] In one embodiment, the conjugates of the invention may be formulated in a lipid- polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S. Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine,

polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. WO2012013326; herein incorporated by reference in its entirety. In another

embodiment, the conjugates of the invention may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[00157] The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the

PEGylation, ratio of all components and biophysical parameters such as size.

[00158] In one embodiment, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US Patent Publication No.

US20100036115 and US20120202871; each of which is herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. WO2012040184,

WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365 and WO2012044638; each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected from, but not limited to, formula CLI-CLXXIX of International Publication No.

WO2008103276, formula CLI-CLXXIX of US Patent No. 7,893,302, formula CLI- CLXXXXII of US Patent No. 7,404,969 and formula I- VI of US Patent Publication No. US20100036115; the contents of each of which are herein incorporated by reference in their entirety.

[00159] In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865; each of which is herein incorporated by reference in their entirety.

[00160] In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. WO201 1127255 or WO2008103276, each of which is herein incorporated by reference in their entirety. As a non-limiting example, conjugates described herein may be encapsulated in LNP formulations as described in WO2011127255 and/or WO2008103276; each of which is herein incorporated by reference in their entirety. As another non-limiting example, conjugates described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S. Pub. No. 20120207845; herein incorporated by reference in its entirety.

[00161] The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a conjugate. As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121; herein incorporated by reference in its entirety).

[00162] Nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosa tissue within seconds or within a few hours. Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photo bleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S. Pat. No. 8,241,670, herein incorporated by reference in its entirety.

[00163] Nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines,

polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (See e.g., International App. No. WO201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include 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 cyanoacralate, 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 poly(ethylene 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, polysiloxanes, polystyrene (PS), 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)acrylate), 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, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), and trimethylene carbonate, polyvinylpyrrolidone. The nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer, and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).

[00164] The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains). [00165] In one embodiment, the conjugate of the invention is formulated as a lipoplex, such as, without limitation, the ATUPLEX™ system, the DACC system, the DBTC system and other conjugate-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of therapeutic agents (Aleku et al.

Cancer Res. 2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13 : 1222-1234; Santel et al., Gene Ther 2006 13 : 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23 :334-344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. JImmunother. 2009 32:498-507; Weide et al. JImmunother. 2008 31 : 180-188; Pascolo, Expert Opin. Biol. Ther. 4: 1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34: 1-15; Song et al., Nature Biotechnol. 2005, 23 :709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008 19: 125- 132; all of which are incorporated herein by reference in its entirety).

[00166] In one embodiment such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells (e.g., antigen presenting cells, dendritic cells, T lymphocytes, B lymphocytes, natural killer cells and leukocytes), tumor cells and endothelial cells, (Akinc et al. Mol Ther. 2010 18: 1357-1364; Song et al., Nat Biotechnol. 2005 23 :709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13 : 1222-1234; Santel et al., Gene Ther 2006 13 : 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23 :334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin DrugDeliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of which are incorporated herein by reference in its entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al., Biomacromolecules. 2011, 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008, 5:309- 319; Akinc et al., Mol Ther. 2010 18: 1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820: 105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer, J Control Release . 2010, 20:63-68; Peer et al., Proc Natl Acad Sci USA. 2007, 104:4095-4100; Kim et al., Methods Mol Biol. 2011, 721 :339-353; Subramany a et al., Mol Ther. 2010, 18:2028-2037; Song et al., Nat Biotechnol. 2005, 23 :709-717; Peer et al., Science. 2008, 319:627-630; Peer and Lieberman, Gene Ther. 2011, 18: 1127-1133; all of which are incorporated herein by reference in its entirety).

[00167] In one embodiment, the conjugates of the invention are formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).

[00168] In one embodiment, the conjugates of the invention can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In one embodiment, the conjugates of the invention may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate" means to enclose, surround or encase. As it relates to the formulation of the conjugates of the invention, encapsulation may be substantial, complete or partial. The term "substantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of conjugate of the invention may be enclosed, surrounded or encased within the particle. "Partially encapsulation" means that less than 10, 10, 20, 30, 40 50 or less of the conjugate of the invention may be enclosed, surrounded or encased within the particle. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the particle.

[00169] In another embodiment, the conjugates of the invention may be encapsulated into a nanoparticle or a rapidly eliminated nanoparticle and the nanoparticles or a rapidly eliminated nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).

[00170] In another embodiment, the nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As a non-limiting example, the nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.

[00171] In one embodiment, the conjugate formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OP ADR Y®, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® and cellulose derivatives such as

ethylcellulose aqueous dispersions (AQUACOAT® and SURELEASE®).

[00172] In one embodiment, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains.

Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L- lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[00173] In one embodiment, the conjugate of the present invention may be encapsulated in a therapeutic nanoparticle. Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos.

WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, US Pub. Nos. US20110262491, US20100104645, US20100087337, US20100068285,

US20110274759, US20100068286 and US20120288541, and US Pat No. 8,206,747, 8,293,276 8,318,208 and 8,318,211; each of which is herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.

[00174] In one embodiment, the therapeutic nanoparticle may be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time. The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the conjugate of the present invention (see International Pub No. 2010075072 and US Pub No. US20100216804, US20110217377 and US20120201859, each of which is herein incorporated by reference in their entirety).

[00175] In one embodiment, the therapeutic nanoparticles may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticles may include a

corticosteroid (see International Pub. No. WO2011084518 herein incorporated by reference in its entirety). In one embodiment, the therapeutic nanoparticles of the present invention may be formulated to be antiviral immunotherapeutics or vaccine adjuvants. As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and US Pub No. US20100069426, US20120004293 and US20100104655, each of which is herein incorporated by reference in their entirety.

[00176] In one embodiment, the nanoparticles of the present invention may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,

polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.

[00177] In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. In one embodiment, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester) or combinations thereof.

[00178] As a non-limiting example the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety). [00179] In one embodiment, the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910; each of which is herein incorporated by reference in its entirety).

[00180] In one embodiment, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g.,

U.S. Pub. No. 20120076836; herein incorporated by reference in its entirety).

[00181] In one embodiment, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.

[00182] In one embodiment, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.

[00183] In one embodiment, the therapeutic nanoparticles may comprise at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,

poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No. 8,287,849; herein incorporated by reference in its entirety) and combinations thereof.

[00184] In one embodiment, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4- hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[00185] In another embodiment, the therapeutic nanoparticle may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its entirety).

[00186] In one embodiment, the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No. WO2011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).

[00187] In one embodiment, the conjugates of the invention may be encapsulated in, linked to and/or associated with synthetic nanocarriers. Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149411 and WO2012149454 and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US20120244222, each of which is herein incorporated by reference in their entirety. The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. WO2010005740,

WO2010030763 and WO201213501 and US Pub. Nos. US20110262491, US20100104645, US20100087337 and US20120244222, each of which is herein incorporated by reference in their entirety. In another embodiment, the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub. No. WO2011072218 and US Pat No. 8,211,473; each of which is herein incorporated by reference in their entirety.

[00188] In one embodiment, the synthetic nanocarriers may contain reactive groups to release the conjugates described herein (see International Pub. No. WO20120952552 and US Pub No. US20120171229, each of which is herein incorporated by reference in their entirety).

[00189] In one embodiment, the synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarrier is formulated to release the conjugates at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the conjugates after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. WO2010138193 and WO2010138194 and US Pub Nos. US20110020388 and US20110027217, each of which is herein incorporated by reference in their entirety).

[00190] In one embodiment, the synthetic nanocarriers may be formulated for controlled and/or sustained release of conjugates described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. WO2010138192 and US Pub No. 20100303850, each of which is herein incorporated by reference in their entirety.

[00191] In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof. As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No. 20120282343; herein incorporated by reference in its entirety. D. Polymers, Biodegradable Nanopar tides, and Core-Shell Nanopar tides

[00192] The conjugates of the invention can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC POLYCONJUGATE® (Arrowhead Research Corp.,

Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) and Roche Madison

(Madison, WI), PHASERX™ polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGY™ (Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers,

RONDEL™ (RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERX™ (Seattle, WA).

[00193] A non-limiting example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S. Pub. No.

20120258176; herein incorporated by reference in its entirety). Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof.

[00194] In one embodiment, the polymers used in the present invention have undergone processing to reduce and/or inhibit the attachment of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in International Pub. No.

WO2012150467, herein incorporated by reference in its entirety.

[00195] A non-limiting example of PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARD® which is formed by dissolving PLGA in 66% N- methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide. Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).

[00196] In one embodiment, the pharmaceutical compositions may be sustained release formulations. In a further embodiment, the sustained release formulations may be for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITE®

(Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).

[00197] As a non-limiting example modified mRNA may be formulated in PLGA microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the conjugate in the PLGA microspheres while maintaining the integrity of the conjugate during the encapsulation process. EVAc are non-biodegradable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine device; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407 F is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene- polyoxy ethylene having a low viscosity at temperatures less than 5°C and forms a solid gel at temperatures greater than 15°C. PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITE® and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic interaction to provide a stabilizing effect.

[00198] Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N- acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al., Proc Natl Acad Sci U S A. 2007 104: 12982-12887; Davis, Mol Pharm. 2009, 6:659-668; Davis, Nature, 2010, 464: 1067-1070; each of which is herein incorporated by reference in its entirety).

[00199] The conjugates of the invention may be formulated with or in a polymeric compound. The polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethylenimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine- containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof.

[00200] As a non-limiting example, the conjugates of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6, 177,274; herein incorporated by reference in its entirety. In another example, the conjugate may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.

[00201] As another non-limiting example the conjugate of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which are herein incorporated by reference in their entireties) or PLGA- PEG-PLGA block copolymers (See U.S. Pat. No. 6,004,573, herein incorporated by reference in its entirety). As a non-limiting example, the conjugate of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).

[00202] A polyamine derivative may be used to deliver conjugates of the invention or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No. 20100260817 herein incorporated by reference in its entirety). As a non-limiting example, a pharmaceutical composition may include the conjugates of the invention and the polyamine derivative described in U.S. Pub. No. 20100260817 (the contents of which are incorporated herein by reference in its entirety). As a non-limiting example the conjugates of the invention may be delivered using a polyamide polymer such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280; herein incorporated by reference in its entirety). [00203] The conjugate of the invention may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl

methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof.

[00204] In one embodiment, the conjugates of the invention may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos.

WO2011115862, WO2012082574 and WO2012068187 and U.S. Pub. No. 20120283427, each of which are herein incorporated by reference in their entireties. In another

embodiment, the conjugates of the invention may be formulated with a polymer of formula Z as described in WO2011115862, herein incorporated by reference in its entirety. In yet another embodiment, the conjugates of the invention may be formulated with a polymer of formula Z, Z' or Z" as described in International Pub. Nos. WO2012082574 or

WO2012068187, each of which are herein incorporated by reference in their entireties. The polymers formulated with the conjugates of the present invention may be synthesized by the methods described in International Pub. Nos. WO2012082574 or WO2012068187, each of which are herein incorporated by reference in their entireties.

[00205] Formulations of conjugates of the invention may include at least one amine- containing polymer such as, but not limited to polylysine, polyethylene imine,

poly(amidoamine) dendrimers or combinations thereof.

[00206] For example, the conjugate of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof. The biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S. Pat. No. 6,696,038, U.S. App. Nos. 20030073619 and 20040142474 each of which is herein incorporated by reference in their entireties. The poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety. The biodegradable polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat. Nos. 6,517,869 and 6,267,987, the contents of which are each incorporated herein by reference in their entirety. The linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886. The PAGA polymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,217,912 herein incorporated by reference in its entirety. The PAGA polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyarginine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides). The biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are herein incorporated by reference in their entireties. For example, the multi- block copolymers may be synthesized using linear polyethylenimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines. Further, the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos. 6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.

[00207] The conjugates of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.

[00208] The conjugate of the invention may be formulated with at least one cross linkable polyester. Cross linkable polyesters include those known in the art and described in US Pub. No. 20120269761, herein incorporated by reference in its entirety.

[00209] In one embodiment, the polymers described herein may be conjugated to a lipid- terminating PEG. As a non-limiting example, PLGA may be conjugated to a lipid- terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG conjugates for use with the present invention are described in International Publication No. WO2008103276, herein incorporated by reference in its entirety. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its entirety.

[00210] In one embodiment, the conjugates of the invention may be conjugated with another compound. Non-limiting examples of conjugates are described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. In another embodiment, the conjugates of the invention may be conjugated with conjugates of formula 1-122 as described in US Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. The modified RNA described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al. Journ. Amer. Chem. Soc. 2009 131(6): 2072-2073; herein incorporated by reference in its entirety). In another embodiment, the conjugates of the invention may be conjugated and/or

encapsulated in gold-nanoparticles. (Interantional Pub. No. WO201216269 and U.S. Pub. No. 20120302940; each of which is herein incorporated by reference in its entirety).

[00211] In one embodiment, the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein incorporated by reference in its entirety. The cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside- polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2- dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, l,2-Dioleoyl-3-Trimethylammonium -Propane (DOTAP), N-[l- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l-[2- (oleoyloxy)ethyl]-2-oleyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTEVI), 2,3- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium

trifluoroacetate (DOSPA), 3B-[N— (N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1) diheptadecylamidoglycyl spermidine (DOGS), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N- dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N- dimethylammonium chloride DODAC) and combinations thereof.

[00212] The conjugates of the invention may be formulated in a polyplex of one or more polymers (U.S. Pub. No. 20120237565 and 20120270927; each of which is herein

incorporated by reference in its entirety). In one embodiment, the polyplex comprises two or more cationic polymers. The catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI. [00213] The conjugates of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate. Components may be combined in a core-shell, hybrid, and/or layer- by-layer architecture, to allow for fine-tuning of the nanoparticle so that delivery of the conjugates of the invention may be enhanced (Wang et al., Nat Mater. 2006, 5:791-796; Fuller et al., Biomaterials. 2008, 29: 1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011, 63 :748-761; Endres et al., Biomaterials. 2011, 32:7721-7731; Su et al., o/ Pharm. 2011, Jun 6;8(3):774-87; each of which is herein incorporated by reference in its entirety). As a non-limiting example, the nanoparticle may comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (International Pub. No. WO20120225129; herein incorporated by reference in its entirety).

[00214] Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver therapeutic agents in vivo. In one embodiment, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the conjugate of the present invention. For example, to effectively deliver a therapeutic agent in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010, 142: 416-421; Li et al., J Contr Rel. 2012, 158: 108-114; Yang et al., Mo/ Ther. 2012, 20:609-615; herein

incorporated by refereince in its entirety). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the therapeutic agent.

[00215] In one embodiment, a PEG-charge-conversional polymer (Pitella et al.,

Biomaterials. 2011, 32:3106-3114) may be used to form a nanoparticle to deliver the conjugate of the present invention. The PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.

[00216] The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011, 108: 12996-13001). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver a therapeutic agent to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.

[00217] The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011, 108: 12996-13001). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver a therapeutic agent to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.

[00218] In one embodiment, the lipid nanoparticles may comprise a core of the conjugates disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.

[00219] Core-shell nanoparticles for use with the conjugates of the present invention are described and may be formed by the methods described in U.S. Pat. No. 8,313,777 herein incorporated by reference in its entirety.

[00220] In one embodiment, the core-shell nanoparticles may comprise a core of the conjugates disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acid molecules in the core.

E. Inorganic nanoparticles

[00221] Inorganic nanoparticles exhibit a combination of physical, chemical, optical and electronic properties and provide a highly multifunctional platform to image and diagnose diseases, to selectively deliver therapeutic agens, and to sensitive cells and tissues to treatment regiments. Not wishing to be bound to any theory, enhanced permeability and retention (EPR) effect provides a basis for the selective accumulation of many high- molecular-weight drugs. Circulating inorganic nanoparticles preferentially accumulate at tumor sites and in inflamed tissues (Yuan et al., Cancer Res., vol.55(17):3752-6, 1995, the contents of which are incorporated herein by reference in their entirety) and remain lodged due to their low diffusivity (Pluen et al., PNAS, vol.98(8):4628-4633, 2001, the contents of which are incorporated herein by reference in their entirety). The size of the inorganic nanoparticles may be 10 nm - 500 nm, 10 nm - 100 nm or 100 nm - 500 nm. The inorganic nanoparticles may comprise metal (gold, iron, silver, copper, nickel, etc.), oxides (ZnO, T1O2, AI2O3, S1O2, iron oxide, copper oxide, nickel oxide, etc.), or semiconductor (CdS, CdSe, etc.). The inorganic nanoparticles may also be perfluorocarbon or FeCo.

[00222] Inorganic nanoparticles have high surface area per unit volume. Therefore, they may be loaded with therapeutic drugs and imaging agents at high densitives. A variety of methods may be used to load therapeutic drugs into/onto the inorganic nanoparticles, including but not limited to, colvalent bonds, electrostatic interactions, entrapment, and encapsulation. In addition to therapeutic agent drug loads, the inorganic nanoparticles may be funcationalized with targeting moieties, such as tumor-targeting ligands, on the surface. Formulating therapeutic agents with inorganic nanoparticles allows imaging, detection and monitoring of the therapeutic agents.

[00223] In one embodiment, the conjugate of the invention is hydrophobic and may be form a kinetically stable complex with gold nanoparticles funcationalized with water-soluble zwitterionic ligands disclosed by Kim et al. (Kim et al., JACS, vol.131(4): 1360-1361, 2009, the contents of which are incorporated herein by reference in their entirety). Kim et al.

demonstrated that hydrophobic drugs carried by the gold nanoparticles are efficiently released into cells with little or no cellular uptake of the gold nanoparticles.

[00224] In one embodiment, the conjugates of the invention may be formulated with gold nanoshells. As a non-limiting example, the conjugates may be delivered with a temperature sensitive system comprising polymers and gold nanoshells and may be released

photothermally. Sershen et al. designed a delivery vehicle comprising hydrogel and gold nanoshells, wherein the hydrogels are made of copolymers of N-isopropyl aery 1 amide

(NIPAAm) and acrylamide (AAm) and the gold nanoshells are made of gold and gold sulfide (Sershen et al., J Biomed Mater, vol.51 :293-8, 2000, the contents of which are incorporated herein by reference in their entirety). Irradiation at 1064 nm was absorbed by the nanoshells and converted to heat, which led to the collapse of the hydrogen and release of the drug. The conjugate of the invention may also be encapsulated inside hollow gold nanoshells.

[00225] In some embodiments, the conjugates of the invention may be attached to gold nanoparticles via covalent bonds. Covalent attachment to gold nanoparticles may be achieved through a linker, such as a free thiol, amine or carboxylate functional group. In some embodiments, the linkers are located on the surface of the gold nanoparticles. In some embodiments, the conjugates of the invention may be modified to comprise the linkers. The linkers may comprise a PEG or oligoethylene glycol moiety with varying length to increase the particles' stability in biological environment and to control the density of the drug loads. PEG or oligoethylene glycol moieties also minimize nonspecific adsorption of undesired biomolecules. PEG or oligoethylene gycol moieties may be branched or linear. Tong et al. disclosed that branched PEG moieties on the surface of gold nanoparticles increase circulatory half-life of the gold nanoparticles and reduced serum protein binding (Tong et al., Langmuir, vol.25(21): 12454-9, 2009, the contents of which are incorporated herein by reference in their entirety).

[00226] In one embodiment, the conjugate of the invention may comprise PEG-thiol groups and may attach to gold nanoparticles via the thiol group. The synthesis of thiol -PEGylated conjugates and the attachment to gold nanoparticles may follow the method disclosed by El- Sayed et al. (El-Sayed et al., Bioconjug. Chem., vol.20(12):2247-2253, 2010, the contents of which are incorporated herein by reference in their entirety).

[00227] In another embodiment, the conjugate of the invention may be tethered to an amine- functionalized gold nanoparticles. Lippard et al. disclosed that Pt(IV) prodrugs may be delivered with amine-functionalized polyvalent oligonucleotide gold nanoparticles and are only activated into their active Pt(II) forms after crossing the cell membrane and undergoing intracellular reduction (Lippard et al., JACS, vol.131(41): 14652-14653, 2009, the contents of which are incorporated herein by reference in their entirety). The cytotoxic effects for the Pt(IV)-gold nanoparticle complex are higher than the free Pt(IV) drugs and free cisplatin.

[00228] In some embodiments, conjugates of the invention are formulated with magnetic nanoparticle such as iron, cobalt, nickel and oxides thereof, or iron hydroxide nanoparticles. Localized magnetic field gradients may be used to attract magnetic nanoparticles to a chosen site, to hold them until the therapy is complete, and then to remove them. Magnetic nanoparticles may also be heated by magnetic fields. Alexiou et al. prepared an injection of magnetic particle, Ferro fluids (FFs), bound to anticancer agents and then concentrated the particles in the desired tumor area by an external magnetic field (Alexiou et al., Cancer Res. vol.60(23):6641-6648, 2000, the contents of which are incorporated herein by reference in their entirety). The desorption of the anticancer agent took place within 60 min to make sure that the drug can act freely once localized to the tumor by the magnetic field.

[00229] In some embodiments, the conjugates of the invention are loaded onto iron oxide nanoparticles. In some embodiments, the conjugates of the invention are formulated with super paramagnetic nanoparticles based on a core consisting of iron oxides (SPION). SPION are coated with inorganic materials (silica, gold, etc.) or organic materials (phospholipids, fatty acids, polysaccharides, peptides or other surfactants and polymers) and can be further functionalized with drugs, proteins or plasmids.

[00230] In one embodiment, water-dispersible oleic acid (OA)-poloxamer-coated iron oxide magnetic nanoparticles disclosed by Jain et al. (Jain, Mol. Pharm., vol.2(3): 194-205, 2005, the contents of which are incorporated herein by reference in their entirety) may be used to deliver the conjugates of the invention. Therapeutic drugs partition into the OA shell surrounding the iron oxide nanoparticles and the poloxamer copolymers (i.e., Pluronics) confers aqueous dispersity to the formulation. According to Jain et al., neither the formulation components nor the drug loading affected the magnetic properties of the core iron oxide nanoparticles. Sustained release of the therapeutic drugs was achieved.

[00231] In one embodiment, the conjugates of the invention are bonded to magnetic nanoparticles with a linker. The linker may be a linker capable of undergoing an

intramolecular cyclization to release the conjugates of the invention. Any linker and nanoparticles disclosed in WO2014124329 to Knipp et al., the contents of which are incorporated herein by reference in their entirety, may be used. The cyclization may be induced by heating the magnetic nanoparticle or by application of an alternating

electromagnetic field to the magnetic nanoparticle.

[00232] In one embodiment, the conjugates of the invention may be delivered with a drug delivery system disclosed in US 7329638 to Yang et al., the contents of which are incorporated herein by reference in their entirety. The drug delivery system comprises a magnetic nanoparticle associated with a positively charged cationic molecule, at least one therapeutic agent and a molecular recognition element.

[00233] In one embodiment, nanoparticles having a phosphate moiety are used to deliver the conjugates of the invention. The phosphate-containing nanoparticle disclosed in US 8828975 to Hwu et al., the contents of which are incorporated herein by reference in their entirety, may be used. The nanoparticles may comprise gold, iron oxide, titanium dioxide, zinc oxide, tin dioxide, copper, aluminum, cadmium selenide, silicon dioxide or diamond. The nanoparticles may contain a PEG moiety on the surface.

F. Peptides and Proteins

[00234] The conjugate of the invention can be formulated with peptides and/or proteins in order to increase peneration of cells by the conjugates of the invention. In one embodiment, peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations. A non- limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention include a cell-penetrating peptide sequence attached to poly cations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr. Pharm. Des. 2003,

11(28):3597-611; and Deshayes et al., Cell. Mol. Life Sci. 2005, 62(16): 1839-49, all of which are incorporated herein by reference). The compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space. The conjugates of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologies (Cambridge, MA) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol. 2010, 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009, 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009, 73 :3-6; Verdine and Hilinski, Methods Enzymol. 2012, 503 :3-33; all of which are herein incorporated by reference in its entirety). In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner" includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where conjugates of the invention may be introduced.

IV. Administration, Dose and Dosage form

[00235] Administration: Compositions and formulations containing an effective amount of conjugates or particles of the present invention may be administered to a subject in need thereof by any route which results in a therapeutically effective outcome in said subject. These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal),

intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intraci sternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube),

intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

[00236] In some embodiments, particles, nanoparticles and/or polymeric nanoparticles are administered to bone marrow. In some embodiments, particles, nanoparticles and/or polymeric nanoparticles are administered to areas having a lot of dendritic cells, such as subcutaneous space.

[00237] Dose and Dosage forms : Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[00238] In some embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In some embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple

administrations are employed, split dosing regimens such as those described herein may be used.

[00239] As used herein, a "split dose" is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a "single unit dose" is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a "total daily dose" is an amount given or prescribed in 24 hr. period. It may be administered as a single unit dose.

[00240] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, and subcutaneous).

[00241] In some embodiments, the dosage forms may be liquid dosage forms. Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof. [00242] In certain embodiments, the dosages forms may be injectable. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[00243] In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compounds then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Injectable depot forms are made by forming microencapsule matrices of the conjugates in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of conjugates to polymer and the nature of the particular polymer employed, the rate of active agents in the conjugates can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the conjugates in liposomes or microemulsions which are compatible with body tissues.

[00244] In some embodiments, solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.

Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

METHODS OF USE

[00245] Compositions of the present invention may be used to harness the immune system to eliminate tumor cells. In accordance with the present invention, conjugates, particles and formulations comprising conjugates and particles may be used to induce tumor specific T cell cytotoxicity against tumor cells, wherein the conjugates bind to such tumor cells.

[00246] The conjugates or particles as described herein or formulations containing the conjugates or particles as described herein can be administered to treat any hyperproliferative disease, tumor, cancer, or any other disease, as appropriate. The formulations may be delivered to various body parts, such as but not limited to, brain and central nervous system, eyes, ears, lungs, bone, heart, kidney, liver, spleen, breast, ovary, colon, pancreas, muscles, gastrointestinal tract, mouth, skin, to treat disesase associated with such body parts.

Formulations may be administered by injection, orally, or topically, typically to a mucosal surface (lung, nasal, oral, buccal, sublingual, vaginally, rectally) or to the eye (intraocularly or transocularly).

[00247] The conjugates or particles as described herein or formulations containing the conjugates or particles as described herein can be administered to treat cancer; the cancer may be any cancer, including but not limited to any of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureter cancer, urinary bladder cancer, and digestive tract cancer such as, e.g., esophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumor, cancer of the oral cavity, colon cancer, and hepatobiliary cancer. [00248] In some embodiments, the conjugates or particles of the present invention may be combined with at least one other active agent to form a composition. The at least one active agent may be a therapeutic, prophylactic, diagnostic, or nutritional agent. It may be a small molecule, protein, peptide, lipid, glycolipid, glycoprotein, lipoprotein, carbohydrate, sugar, or nucleic acid. The conjugates or particles of the present invention and the at least one other active agent may have the same target and/or treat the same disease.

[00249] The particles of the present invention and the at least one other active agent may be administered semitaneously or sequentially. They may be present as a mixture for

simultaneous administration, or may each be present in separate containers for sequential administration.

[00250] The term "simultaneous administration", as used herein, is not specifically restricted and means that the particles and the at least one other active agent are substantially administered at the same time, e.g. as a mixture or in immediate subsequent sequence.

[00251] The term "sequential administration", as used herein, is not specifically restricted and means that the particles and the at least one other active agent are not administered at the same time but one after the other, or in groups, with a specific time interval between administrations. The time interval may be the same or different between the respective administrations of the particles and the at least one other active agent and may be selected, for example, from the range of 2 minutes to 96 hours, 1 to 7 days or one, two or three weeks. Generally, the time interval between the administrations may be in the range of a few minutes to hours, such as in the range of 2 minutes to 72 hours, 30 minutes to 24 hours, or 1 to 12 hours. Further examples include time intervals in the range of 24 to 96 hours, 12 to 36 hours, 8 to 24 hours, and 6 to 12 hours.

[00252] In some embodiments, an effective immunotherapy with conjugates or particles of the present invention may be combined with different interventions including strategies to increase systemically the frequency of anti-cancer T cells, strategies to overcome distinct immune suppressive pathways within the tumor microenvironment and strategies to trigger innate immune activation and inflammation in tumor sites.

DEFINITIONS

[00253] The terms used in this invention are, in general, expected to adhere to standard definitions generally accepted by those having ordinary skill in the relevant art. [00254] About: As used herein, the term "about" means a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within

50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value.

[00255] Administration: As used herein, the term "administration" means the actual physical introduction of the composition into or onto (as appropriate) the host. Any and all methods of introducing the composition into the host are contemplated according to the invention; the method is not dependent on any particular means of introduction and is not to be so construed. Means of introduction are well-known to those skilled in the art, and also are exemplified herein

[00256] Adoptive cellular immunotherapy : As used herein, the terms "adoptive cellular immunotherapy" or "adoptive immunotherapy ' or "J cell immunotherapy", or "Adoptive T cell therapy (ACT)", are used interchangeably. Adoptive immunotherapy uses T cells that a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient. The injection of a large number of activated tumor specific T cells can induce complete and durable regression of cancers.

[00257] Agonist: As used herein, the term "agonist" refers to any substance that binds to a target (e.g. a receptor); and activates or increases the biological activity of the target. For example, an "agonist" antibody is an antibody that activates or increases the biological activity of the antigen(s) it binds.

[00258] Antagonist: As used herein, the term "antagonist" refers to any agent that inhibits or reduces the biological activity of the target(s) it binds. For example, an "antagonist" antibody is an antibody that inhibits or reduces biological activity of the antigen(s) it binds.

[00259] Antigen: As used herein, the terms "antigen "or "immunogen," as being used interchangeably, is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itself. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells such as cytotoxic T lymphocytes and T helper cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. The term "antigenic" or "immunogenic" refers to a structure that is an antigen. These terms are used interchangeably.

[00260] Antigen presenting cells (APCs) : As used herein, the term "antigen presenting cells" refers to cells that process antigens and present peptide epitopes on the cell surface via MHC molecules; APCs include dendritic cells (DCs), Langerhans cells, macrophages, B cells, and activated T cells. Dendritic cells (DCs) and macrophages are antigen presenting cells in vivo. The dendritic cells are more efficient APCs than macrophages. These cells are usually found in structural compartments of the lymphoid organs such as the thymus, lymph nodes and spleen, and in the bloodstream and other tissues of the body as well.

[00261] Antibodies: As used herein, "antibodies" are specialized proteins called

immunoglobulins (Igs) that specifically recognize and bind to specific antigens that caused their stimulation. Antibody production by B lymphocytes in vivo and binding to foreign antigens is often critical as a means of signaling other cells to engulf, kill or remove that substance that contains the foreign antigens from the body. An immunoglobulin is a protein comprising one or more polypeptides substantially encoded by the immunoglobulin kappa and lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Also subclasses of the heavy chain are known. For example, IgG heavy chains in humans can be any of IgGl, IgG2, IgG3 and IgG4 subclass.

[00262] Antibodies may exist as full length intact antibodies or as a number of well- characterized fragments produced by digestion with various peptidases or chemicals, such as F(ab')2, a dimer of Fab which itself is a light chain joined to VH-CHl by a disulfide bond; an Fab' monomer, a Fab fragment with the hinge region; and a Fc fragment, a portion of the constant region of an immunoglobulin.

[00263] While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that any of a variety of antibody fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein also includes antibody fragments either produced by the modification of whole antibodies or synthesized de novo or antibodies and fragments obtained by using recombinant DNA methodologies. Recombinant antibodies may be conventional full length antibodies, antibody fragments known from proteolytic digestion, unique antibody fragments such as Fv or single chain Fv (scFv), domain deleted antibodies, and the like. An Fv antibody is about 50 Kd in size and comprises the variable regions of the light and heavy chain. A single chain Fv ("scFv") polypeptide is a covalently linked VH: :VL heterodimer. [00264] An antibody may be a non-human antibody, a human antibody, a humanized antibody or a chimeric antibody. The "chimeric antibody" means a genetically engineered fusion of parts of a non-human (e.g., mouse) antibody with parts of a human antibody.

Generally, chimeric antibodies contain approximately 33% non-human protein and 67% human protein. Developed to reduce the HAMA response elicited by non-human antibodies, they combine the specificity of the non-human antibody with the efficient human immune system interaction of a human antibody. A human antibody may be a "fully human" antibody. The terms "human" and 'fully human" is used to label those antibodies derived from transgenic mice carrying human antibody genes or from human cells. To the human immune system, however, the difference between "fully human" "humanized", and "chimeric" antibodies may be negligible or nonexistent and as such all three may be of equal efficacy and safety.

[00265] Autologous: As used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[00266] Cancer : As used herein, the term "cancer" refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and grow results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream.

[00267] Combination therapy: As used herein, the term "combination therapy" means a therapy strategy that embraces the administration of therapeutic compositions of the present invention (e.g., conjugates comprising one or more neoantigens) and one or more additional therapeutic agents as part of a specific treatment regimen intended to provide a beneficial (additive or synergistic) effect from the co-action of these therapeutic agents. Administration of these therapeutic agents in combination may be carried out over a defined time period (usually minutes, hours, days, or weeks depending upon the combination selected). In combination therapy, combined therapeutic agent may be administered in a sequential manner, or by substantially simultaneous administration.

[00268] Compound: As used herein, the term "" compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. In the present application, compound is used interchangeably with conjugate.

Therefore, conjugate, as used herein, is also meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. [00269] The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

[00270] Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, IH- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[00271] Compounds of the present disclosure also include all of the isotopes of the atoms occurring in the intermediate or final compounds. "Isotopes" refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium.

[00272] The compounds and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.

[00273] Copolymer: As used herein, the term "copolymer" generally refers to a single polymeric material that is comprised of two or more different monomers. The copolymer can be of any form, such as random, block, graft, etc. The copolymers can have any end-group, including capped or acid end groups.

[00274] Cytokine: As used herein, the term "cytokine" refers to a substance secreted by certain cells of the immune system and has a biological effect on other cells. Cytokines can be a number of different substances such as interferons, interleukins and growth factors. [00275] Cytotoxic agent: As used herein, the term "cytotoxic agenf means a substance that inhibits or prevents the function of cells and/or causes destruction of cells, such as radioactive isotopes, chemotherapeutic agents, and toxins.

[00276] Cytotoxic T cell: As used herein, the terms "cytotoxic T cell (TCf or "cytotoxic T lymphocyte (CTL)", or "T-killer cells", or "CD8+ T-cell" or "killer T cell" are used interchangeably. This type of white blood cells are T lymphocytes that can recognize abnormal cells including cancer cells, cells that are infected particularly by viruses, and cells that are damaged in other ways and induce the death of such cells.

[00277] Epitope: As used herein, the term "epitope" means a small peptide structure formed by 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 about 9, or about 8-15 amino acids. A T cell epitope means a peptide which can be bound by the MHC molecules of class I or II in the form of a peptide-presenting MHC molecule or MHC complex and then, in this form, be recognized and bound by native T cells, cytotoxic T- lymphocytes or T-helper cells, respectively.

[00278] Human Leukocyte Antigen (HLA): As used herein, the terms "Human Leokocyte Antigen (HLA)", " HLA proteins", "HLA antigens", "Major Histocompatibility Complex (MHC)", "MHC molecules", or "MHC proteins" all refer to proteins capable of binding peptides resulting from the proteolytic cleavage of protein antigens and representing potential T-cell epitopes, transporting them to the cell surface and presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T-helper cells. The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and/or foreign antigens and thus for regulating immunological processes. The major histocompatibility complex is classified into two gene groups coding for different proteins, namely molecules of MHC class I and molecules of MHC class II. The molecules of the two MHC classes are specialized for different antigen sources. The molecules of MHC class I present endogenously synthesized antigens, for example viral proteins and tumor antigens. The molecules of MHC class II present protein antigens originating from exogenous sources, for example bacterial products. The cellular biology and the expression patterns of the two MHC classes are adapted to these different roles. [00279] MHC class I molecules (called HLA class I in human) consist of a heavy chain and a light chain and are capable of binding a short peptide with suitable binding motifs, and presenting it to cytotoxic T-lymphocytes. The peptide bound by the MHC molecules of class I originates from an endogenous protein antigen. The heavy chain of the MHC molecules of class I is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is β-2- microglobulin.

[00280] MHC class II molecules (called HLA class II in human) consist of an a-chain and a β-chain and are capable of binding a short peptide with suitable binding motifs, and presenting it to T-helper cells. The peptide bound by the MHC molecules of class II usually originates from an extracellular of exogenous protein antigen. The a-chain and the β-chain are in particular HLA-DR, HLA-DQ, HLA-DP, HLA-DO and HLA-DM monomers.

[00281] Immune cell: As used herein, the term "immune celF refers to a cell that is capable of participating, directly or indirectly, in an immune response. Immune cells include, but are not limited to T-cells, B-cells, antigen presenting cells, dendritic cells, natural killer (NK) cells, natural killer T (NK) cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, neutrophils, granulocytes, mast cells, platelets, Langerhan's cells, stem cells, peripheral blood mononuclear cells, cytotoxic T-cells, tumor infiltrating lymphocytes (TIL), etc. "An antigen presenting cell" (APC) is a cell that are capable of activating T cells, and includes, but is not limited to, monocytes/macrophages, B cells and dendritic cells (DCs). "Dendritic cell" or "DC" refers to any member of a diverse population of morphologically similar cell types found in lymphoid or non-lymphoid tissues. These cells are characterized by their distinctive morphology, high levels of surface MHC-class II expression. DCs can be isolated from a number of tissue sources. DCs have a high capacity for sensitizing MHC- restricted T cells and are very effective at presenting antigens to T cells in situ. The antigens may be self-antigens that are expressed during T cell development and tolerance, and foreign antigens that are present during normal immune processes. As used herein, an "activated DC" is a DC that has been pulsed with an antigen and capable of activating an immune cell. "T- cell" as used herein, is defined as a thymus-derived cell that participates in a variety of cell- mediated immune reactions, including CD8+ T cell and CD4+ T cell. "B-cell" as used herein, is defined as a cell derived from the bone marrow and/or spleen. B cells can develop into plasma cells which produce antibodies.

[00282] Immune response: As used herein, the term "immune response" means a defensive response a body develops against "foreigner" such as bacteria, viruses and substances that appear foreign and harmful. An anti-cancer immune response refers to an immune surveillance mechanism by which a body recognizes abnormal tumor cells and initiates both the innate and adaptive of the immune system to eliminate dangerous cancer cells.

[00283] The innate immune system is a non-specific immune system that comprises the cells (e.g., Natural killer cells, mast cells, eosinophils, basophils; and the phagocytic cells including macrophages, neutrophils, and dendritic cells) and mechanisms that defend the host from infection by other organisms. An innate immune response can initiate the productions of cytokines, and active complement cascade and adaptive immune response. The adaptive immune system is specific immune system that is required and involved in highly specialized systemic cell activation and processes, such as antigen presentation by an antigen presenting cell; antigen specific T cell activation and cytotoxic effect.

[00284] Linker: As used herein, the term "linker" refers to a carbon chain that can contain heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.) and which 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, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 atoms long. Linkers may be substituted with various substituents including, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, and ureido groups. Those of skill in the art will recognize that each of these groups may in turn be substituted. Examples of linkers include, but are not limited to, pH-sensitive linkers, protease cleavable peptide linkers, nuclease sensitive nucleic acid linkers, lipase sensitive lipid linkers, glycosidase sensitive carbohydrate linkers, hypoxia sensitive linkers, photo-cleavable linkers, heat-labile linkers, enzyme cleavable linkers (e.g., esterase cleavable linker), ultrasound-sensitive linkers, and x-ray cleavable linkers. Linkers may include any of those taught in, for example, WO2014/10628, the contents of which are incorporated herein by reference in their entirety.

[00285] Mean particle size: As used herein, the term "mean particle size" generally refers to the statistical mean particle size (diameter) of the particles in the composition. The diameter of an essentially spherical particle may be referred to as the physical or hydrodynamic diameter. The diameter of a non-spherical particle may refer to the hydrodynamic diameter. As used herein, the diameter of a non-spherical particle may refer to the largest linear distance between two points on the surface of the particle. Mean particle size can be measured using methods known in the art such as dynamic light scattering. Two populations can be said to have a "substantially equivalent mean particle size" when the statistical mean particle size of the first population of particles is within 20% of the statistical mean particle size of the second population of particles; for example, within 15%, or within 10%.

[00286] The terms "monodisperse" and "homogeneous size distribution,^'" as used

interchangeably herein, describe a population of particles, microparticles, or nanoparticles all having the same or nearly the same size. As used herein, a monodisperse distribution refers to particle distributions in which 90% of the distribution lies within 5% of the mean particle size.

[00287] Peptide: As used herein, the term "peptide" refers to a molecule composed of a series of residues, typically L-amino acids, connected one to the other, typically by peptide bonds between the a-amino and carboxyl groups of adjacent amino acids. Peptide sometimes is used interchangeably with the term "polypeptide," Polypeptides or peptides can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification not destroy the biological activity of the polypeptides as herein described. In some embodiments, peptides are less than 50 amino acids in length.

[00288] Tumor associated antigen (TAA): As used herein, the term "tumor associated antigen (TAA)" refers to an antigenic substance produced in tumor cells. Tumor associated antigens may be encoded by a primary open reading frame of gene products that are differentially expressed by tumors, and not by normal tissues. They may also be encoded by mutated genes, intronic sequences, or translated alternative open reading frames,

pseudogenes, antisense strands, or represent the products of gene translocation events.

Tumor-associated antigens (TAA) can derive from any protein or glycoprotein synthesized by the tumor cell. TAA proteins can reside in any subcellular compartment of the tumor cell; i.e., they may be membrane-bound, cytoplasmic, nuclear-localized, or even secreted by the tumor cells. A TAA may allow for a preferential recognition of tumor cells by specific T cells or immunoglobulins, therefore activate an anti-tumor immune response to kill tumor cells.

[00289] Vaccine: As used herein, the term "vaccine" refers to a composition for generating immunity for the prophylaxis and/or treatment of diseases. Examples

Example 1. Preparation of Purified Recombinant scFV CD3 Binding Moiety

[00290] Single chain variable fragment (scFV) CD3 binding construct (SEQ ID No. 1) was prepared. The sequence encoding ScFV CD3 binding protein (SEQ ID No. 3) was cloned and expressed in E coli. Recombinant scFV was purified and the ScFv binding to cell surface CD3 was assessed by FACS.

[00291] The scFV sequences are listed in Table 1. Amino acids 1 to 22 are the pelB leader sequence MKYLLPTAAAGLLLLAAQPAMA (SEQ ID No. 2). Carboxyl terminal amino acids 293 to 298 are the histidine tag HHHHHH.

Table 1. Sequences of scF V CD3 binding moiety

Anti-CD3 scFV MKYLLPTAAAGLLLLAAQPAMAQPAMAQVQLQQSGAEL SEQ ID sequence ARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIG No. 1

YINPSRGYTNYNQKFKDKATLTTDKS S ST AYMQLS SLTSE

D S AVYYCARYYDDHYSLD YWGQGTTLTVS S VEGGSGGS

GGSGGSGGVDDIQLTQSPAIMSASPGEKVTMTCSASSSVS

YMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTS

YSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRAD

TAPTGSEQKLISEEDLNSCHHHHHH

(the underlined and bold C (cysteine) is the attachment point of

linker and ligand of the conjugates)

pelB leader sequence MKYLLPTAAAGLLLLAAQPAMA SEQ ID for bacterial No. 2 expression

DNA sequence ATGAAGTATCTGCTGCCGACCGCGGCGGCGGGCCTGCT SEQ ID encoding anti- GCTGCTGGCGGCGCAACCGGCGATGGCGCAACCGGCG No. 3 CD3 scFV ATGGCGCAAGTGCAGCTGCAGCAAAGCGGTGCGGAGC

TGGCGCGTCCGGGTGCGAGCGTGAAAATGAGCTGCAAG

GCGAGCGGCTACACCTTCACCCGTTATACCATGCACTG

GGTTAAACAGCGTCCGGGTCAAGGCCTGGAATGGATCG

GCTACATTAACCCGAGCCGTGGTTACACCAACTATAAC

CAGAAGTTTAAAGACAAGGCGACCCTGACCACCGATAA

GAGCAGCAGCACCGCGTACATGCAACTGAGCAGCCTGA

CCAGCGAGGACAGCGCGGTGTACTATTGCGCGCGTTAC

TATGACGATCACTACAGCCTGGATTATTGGGGTCAAGG

CACCACCCTGACCGTGAGCAGCGTTGAAGGTGGCAGCG

GTGGCAGCGGTGGCAGCGGTGGCAGCGGTGGCGTTGAC

GATATCCAGCTGACCCAAAGCCCGGCGATTATGAGCGC

GAGCCCGGGCGAGAAAGTGACCATGACCTGCAGCGCG

AGCAGCAGCGTGAGCTACATGAACTGGTATCAGCAGAA

GAGCGGCACCAGCCCGAAGCGTTGGATCTACGACACCA

GCAAACTGGCGAGCGGTGTGCCGGCGCACTTCCGTGGT

AGCGGTAGCGGTACCAGCTATAGCCTGACCATTAGCGG

TATGGAGGCGGAAGATGCGGCGACCTACTATTGCCAGC

AATGGAGCAGCAACCCGTTCACCTTTGGCAGCGGTACC

AAACTGGAAATCAACCGTGCGGATACCGCGCCGACCGG

TAGCGAACAAAAACTGATTAGCGAAGAAGACCTGAAT

AGCTGCCACCACCACCACCACCATTAA Experimental details:

[00292] Primary peripheral blood mononuclear cells (PBMC) were purchased from ATCC (Cat#PCS-800-011). PBMC were removed from liquid nitrogen storage and rapidly thawed in a 37°C water bath. The contents of the vial were transferred to a 15mL tube and rinsed with lOmL of Dulbecco's phosphate buffered saline (DPBS) without Ca2+ or Mg2+

(ThermoFisher Cat# 14190144) supplemented with 10% fetal bovine serum (ThermoFisher Cat#10437028). PMBCs were then centrifuged at 4°C at lOOOrpm for 5 minutes. The wash was aspirated and the PBMCs were re-suspended in 2mLs of DPBS with 5% bovine serum albumin (BSA) (ThermoFisher Cat# 37525). Cells were then counted and re-suspended in DPBS with 5% BSA to a final concentration of 10χ10^Λ6 cells per mL. 200uL of the PBMC solution was then transferred to a 96-well v-bottom plate (VWR Cat# 82050-656) and spun at 4°C at lOOOrpm for 5 minutes. The liquid was removed using an 8-channel aspirator and cells were re-suspended in lOOuL of DPBS with 5% BSA. The following antibodies or controls were then added:

1. 20uL of DPB S with 5% B S A

2. 20uL FITC Mouse IgG2a Isotype Control (BD Cat# 555573)

3. 20uL FITC Mouse Anti-Human CD3 (BD Cat# 555339)

4. 20uL of DPBS with 5% BSA

5. 20ug of CD3 scFV

[00293] Samples were incubated for 1 hour protected from light at 4°C. Cells were then centrifuged at 4°C at lOOOrpm for 5 minutes. The liquid was removed using an 8-channel aspirator and cells were re-suspended in lOOuL of DPBS with 5% BSA. This washing process was repeated twice for a total of three washes. Samples 1 through 3 were then re- suspended in lOOuL of FluoroFix (Biolegand Cat# 422101) and incubated for 2 hours at 4°C the dark. After this incubation cells were washed twice, re-suspended in DPBS plus 5% BSA and stored at 4°C, protected from light, until ready to be analyzed.

[00294] Sample 4 was re-suspended in lOOuL of a 1 :50 dilution of FITC Mouse IgG2b isotype Control (BD Cat# 555742). Sample 5 was re-suspended in lOOuL of a 1 :50 dilution of FITC 6x-His Epitope Tag Antibody (ThermoFisher Cat# R933-25). Samples were then incubated for 1 hour protected from light at 4°C. After this incubation, cells were washed three times, and re-suspended in lOOuL of Flurofix. Samples were incubated for 2 hours at 4°C the dark. After this incubation cells were washed twice, re-suspended in DPBS plus 5% BSA and stored at 4°C, protected from light until ready to be analyzed. The following day all samples were analyzed on a BD LSR II Flow Cytometer.

[00295] Purified, recombinant CD3 -binding scFV has been prepared and the binding to cellular CD3 has been confirmed by fluorescence-activated cell sorting (FACS) (Fig. 2).

Example 2. Preparation of Conjugates comprising CD3 Binding Moiety

[00296] A tumor cell binding moiety may be attached to the CD3 -binding scFV prepared in Example 1 at its cysteine unit. Conjugate preparation: CD3 scFv (1 mg/mL in PBS buffer pH = 7.2) was provided as a dimer and was first reduced using a TCEP (10 equiv) solution in PBS pH 7.2. The solution was shaken for 16 hours, then the tumor cell binding moiety (ligand) with a cysteine reactive group for conjugation (such as maleimide) (10 equivalents) was dissolved in a PBS pH 7.2 buffer and then added to the CD3 scFv monomer. The solution was shaken for 16 hours.

[00297] Non-limiting examples of ligands and linkers with maleimide attached for linking to the scFV CD3 binding protein include:

1. SSTR2 binding moieties:

1

- 85 -

[00298] Cellular models for assessing binding and T cell-mediated cell killing with SSTR2 and GNRHR conjugates are identified. Conjugates with different SSTR2 -binding moieties or GNRHR-binding moieties and/or different linkers are tested to improve affinity, PK or T cell mediated cytotoxicity against SSTR2 or GNRHR-expressing tumor cells.

[00299] Conjugates with which in vitro T cell mediated cytotoxicity can be demonstrated are advanced for in vivo testing, including determination of pharmacokinetic properties and antitumor efficacy. Initial efficacy testing are conducted in immunocompromised mice with co-injection of human T cells (PBMCs, peripheral blood mononuclear cells) and tumor cells followed by dosing with the conjugate of the present invention, following a protocol established for bispecific single-chain antibody molecules by Dreier et al. (Dreier et al., J Immunol., vol.170:4397 (2003), the contents of which are incorporated herein by reference in their entirety).

EQUIVALENTS AND SCOPE

[00300] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[00301] In the claims, articles such as "a," "an," and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.

[00302] It is also noted that the term "comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of is thus also encompassed and disclosed.

[00303] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. [00304] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[00305] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

[00306] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A conjugate for eliciting a tumor specific immune response comprising a T cell binding moiety and a tumor cell binding moiety.

2. The conjugate of claim 1 having a structure of the formula X-Y-Z, wherein X is the tumor cell binding moiety; Y is an optional linker; and Z is the T cell binding moiety.

3. The conjugate of claim 1, wherein the T cell binindg moiety is selected from the group consisting of polypeptides, peptides, antibody mimetics, nucleic acids, glycoproteins, small molecules, carbohydrates, or lipids.

4. The conjugate of claim 3, wherein the T cell binding moiety is an antibody or an antibody fragment.

5. The conjugate of claim 4, wherein the T cell binding moiety is a single chain variable fragment of an antibody (scFv).

6. The conjugate of claim 3, wherein the T cell binding moiety is an aptamer.

7. The conjugate of claim 3, wherein the T cell binding moiety is a small molecule.

8. The conjugate of claim 1, wherein the T cell binding moiety binds to CD3 on a T cell.

9. The conjugate of claim 8, wherein the T cell binding moiety binds to CD3s chain.

10. The conjugate of claim 9, wherein the T cell binding moiety comprises a peptide of SEQ ID No. 3.

11. The conjugate of claim 1, wherein the T cell binding moiety has a molecular weight of less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.

12. The conjugate of claim 1, wherein the tumor cell binding moiety is selected from the group consisting of polypeptides, peptides, antibody mimetics, nucleic acids, glycoproteins, small molecules, carbohydrates, or lipids.

13. The conjugate of claim 12, wherein the tumor cell binding moiety is an antibody or an antibody fragment.

14. The conjugate of claim 13, wherein the T cell binding moiety is a single chain variable fragment of an antibody (scFv).

15. The conjugate of claim 12, wherein the T cell binding moiety is an aptamer.

16. The conjugate of claim 12, wherein the T cell binding moiety is a small molecule.

17. The conjugate of claim 1, wherein the tumor cell binding moiety binds to a cell surface protein on a tumor cell.

18. The conjugate of claim 17, wherein the cell surface protein is a tumor associated antigen (TAA).

19. The conjugate of claim 17, wherein the cell surface protein is a somatostatin receptor (SSTR) or a luteinizing hormone-releasing hormone receptor (LHRHR or GNRHR).

20. The conjugate of claim 19, wherein the cell surface protein is SSTR2.

21. The conjugate of claim 20, wherein the conjugate comprises Compound 1, 2, 3, or 4.

22. The conjugate of claim 19, wherein the cell surface protein is GNRHRl .

23. The conjugate of claim 22, wherein the conjugate comprises Compound 5, 6, 7, or 8.

24. The conjugate of claim 1, wherein the tumor cell binding moiety has a molecular weight of less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.

25. The conjugate of claim 1, wherein the linker is stable in tumor environment.

26. The conjugate of claim 1, wherein the linker is a non-cleavable linker.

27. The conjugate of claim 1, further comprising a masking moiety.

28. The conjugate of claim 27, wherein the masking moiety contains a peptide sequence that includes a substrate for a protease.

29. The conjugate of claim 1, further comprising a reacting group that reacts with a functional group on a protein or an engineered protein or derivatives/analogs/mimics thereof.

30. The conjugate of claim 29, wherein the protein is a naturally occurring protein such as a serum or plasma protein, or a fragment thereof.

31. The conjugate of claim 30, wherein the protein is thyroxine-binding protein, transthyretin, αΐ-acid glycoprotein (AAG), transferrin, fibrinogen, albumin, an

immunoglobulin, α-2-macroglobulin, a lipoprotein, or a fragment thereof.

32. The conjugate of claim 1, further comprising a pharmacokinetic modulating unit.

33. The conjugate of claim 32, wherein the pharmacokinetic modulating unit is a natural or synthetic protein or fragment thereof, a natural or synthetic polymer, or a particle.

34. The conjugate of claim 33, wherein the pharmacokinetic modulating unit comprises a polysialic acid unit, a hydroxyethyl starch (HES) unit, or a polyethylene glycol (PEG) unit.

35. The conjugate of claim 33, wherein the pharmacokinetic modulating unit comprises dendrimers, inorganic nanoparticles, organic nanoparticles, or liposomes.

36. The conjugate of claim 1, wherein the conjugate has a molecular weight of less than about 50,000 Da, less than about 40,000 Da, less than about 30,000 Da, less than about 20,000 Da, less than about 15,000 Da, less than about 10,000 Da, less than about 8,000 Da, less than about 5,000 Da, or less than about 3,000 Da.

37. A nanoparticle for eliciting a tumor specific immune response comprising the conjugate of claim 1.

38. The nanoparticle of claim 37, wherein the nanoparticle comprises a polymeric matrix.

39. The nanoparticle of claim 38, wherein the polymeric matrix comprises one or more polymers selected from the group consisting of hydrophobic polymers, hydrophilic polymers, and copolymers thereof.

40. The nanoparticle of claim 39, wherein the hydrophobic polymers are selected from the group consisting of polyhydroxyacids, polyhydroxyalkanoates, polycaprolactones, poly(orthoesters), polyanhydrides, poly(phosphazenes), poly(lactide-co-caprolactones), polycarbonates, polyesteramides, polyesters, and copolymers thereof.

41. The nanoparticle of claim 39, wherein the hydrophilic polymers are selected from the group consisting of polyalkylene glycols, polyalkylene oxides, poly(oxyethylated polyol), poly(olefinic alcohol), polyvinylpyrrolidone), poly(hydroxyalkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(hydroxy acids), poly(vinyl alcohol), and copolymers thereof.

42. The nanoparticle of claim 38, wherein the polymeric matrix comprises one or more polymers selected from the group consisting of poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(ethylene oxide), poly(ethylene glycol), poly(propylene glycol), and copolymers thereof.

43. The nanoparticle of claim 37, wherein the size of the nanoparticle is between 10 nm and 5000 nm.

44. The nanoparticle of claim 43, wherein the particle has a diameter between 30-70 nm, 70 - 120 nm, 120-200 nm, 200-5000 nm, or 500 - 1000 nm.

45. The nanoparticle of claim 44, wherein the weight percentage of the conjugate is between 0.1 % and 35 %.

46. A pharmaceutical formulation for eliciting a tumor specific immune response comprising the conjugate of claim 1 or the nanoparticle of claim 37 and at least one excipient.

47. A method for inducing tumor specific T cell cytotoxicity against a tumor cell comprising administering the conjugate of claim 1, the nanoparticle of claim 37, or the pharamceutical formulation of claim 46.

48. The method of claim 47, wherein the tumor cell expresses SSTR2.

49. The method of claim 47, wherein the tumor cell expresses G RH1.

50. A method for treating a cancer in a subject comprising administering to the subject a pharmaceutically effective amount of the conjugate of claim 1, the nanoparticle of claim 37, or the pharmaceutical formulation of claim 46.

51. The method of claim 48, wherein the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, soft tissue cancer, testicular cancer, thyroid cancer, ureter cancer, urinary bladder cancer, and digestive tract cancer such as, e.g., esophageal cancer, gastric cancer, pancreatic cancer, stomach cancer, small intestine cancer, gastrointestinal carcinoid tumor, cancer of the oral cavity, colon cancer, and hepatobiliary cancer.