Intracellular delivery of Anti-KRAS antibodies formulated into nanocapsules

Abstract

To provide a composition for intracellular delivery of an anti-KRAS antibody at a clinically appropriate concentration that produces a therapeutic effect.SOLUTION: Provided is a composition comprising a plurality of nanoentities each comprising an inner core surrounded by an outer shell, the outer shell comprising a polymer and the inner core comprising at least one hydrophobic compound, where the nanoentities comprise a pharmaceutical agent, the pharmaceutical agent being an antibody or a fragment thereof, and where the antibody or the fragment thereof binds to an epitope of an activated mutated KRAS protein.SELECTED DRAWING: Figure 8

Description

[Technical Field]

[0001] The present invention relates to the intracellular delivery of antibodies against mutant KRAS oncoproteins, where the antibodies are formulated into particles, nanocapsules or other nanoentities so that they can reach the interior of the cell where they can release their contents. [Background technology]

[0002] Targeted delivery of pharmaceuticals into the body remains a continuing challenge. For example, many drugs are unable to exert their effects effectively because they have difficulty reaching target cells. So far, only monoclonal antibody-based cancer therapeutics have been able to target extracellular proteins, but hundreds of intracellular oncoproteins significantly associated with cancer remain difficult to drug.

[0003] Mutationally activated RAS genes (HRAS, KRAS, and NRAS) comprise a family of genes that are most frequently mutated in cancer. The three RAS genes encode four proteins consisting of 188–189 amino acids, sharing 82–90% amino acid sequence identity. KRAS is a gene that acts as an on / off switch in cell signaling. When functioning normally, KRAS controls cell proliferation. When KRAS is mutated, negative signaling is disrupted, leading to continuous cell proliferation and often to cancer. KRAS proteins are GTPases, normally attached to the plasma membrane by the presence of an isoprene group at their C-terminus. In normal quiescent cells, RAS is primarily in an inactive form bound to GDP. Extracellular stimuli, activation of receptor tyrosine kinases (RTKs) and other cell surface receptors, rapidly and transiently form RAS-GTP, triggering the engagement of effector proteins, which then regulate a variety of intracellular signaling networks and thereby control the mitogenic process.

[0004] Cancer-associated RAS genes are characterized by mutations encoding single amino acid substitutions, primarily at glycine-12 (G12), glycine-13 (G13), or glutamine-61 (Q61) residues, which render RAS GTP-bound and constitutively active, independent of extracellular stimuli, resulting in overstimulation of signaling pathways that lead to cancer growth.

[0005] Mutationally activated RAS genes were first identified in human cancer cells by 1982. More than 30 years later, intensive sequencing of cancer genomes has led to the identification of over 500 validated cancer genes (COSMIC database, catalogue of somatic mutations in cancer), revealing that three RAS genes (HRAS, NRAS, and KRAS) still constitute the most frequently mutated oncogene family in human cancers (Table 1), being detected in 25–30% of all sequenced tumor samples ( Cox et al., 2014 ). [Table 1]

[0006] Although all are widely expressed in adult tissues and tumors, KRAS mutations are much more frequent in human cancers than NRAS or HRAS, with KRAS comprising 85% of all RAS mutations in cancer, followed by NRAS (12%) and less frequently HRAS (3%). One explanation for the higher frequency of KRAS mutations compared with NRAS and HRAS is that the KRAS protein has unique properties that favor oncogenesis.

[0007] Thus, for example, in pancreatic ductal adenocarcinoma (PDAC, 90% of all pancreatic cancers) and lung adenocarcinoma (LAC, 40% of all lung cancers), the frequency of KRAS mutations is nearly 100% and 25-30%, respectively. In colorectal cancer (CRC), KRAS is also the predominant mutated isoform (45%) (Liu et al., 2019; Ferrer et al., 2018; Cox et al., 2014).

[0008] The relative frequency of mutations at positions G12, G13, or Q61 also varies between cancer types. In PDAC, LAC, and CRC, KRAS mutations are primarily found at G12. The substitutions found at a given residue also vary between cancer types. For example, at G12, the predominant substitution is G12D in PDAC and CRC, followed by G12V. In contrast, in LAC, the predominant substitution is G12C, which is rare in PDAC. Genetic experiments have demonstrated the dependence of pancreatic and lung adenocarcinoma on oncogenic KRAS expression (Fisher et al., 2001; Ying et al., 2002). .,2012), highlighting its value as a molecular target. Therefore, KRAS protein plays a very important role in cancer and represents a very well-validated drug target.

[0009] Decades of effort have been devoted to developing small molecules that target oncogenic KRAS. However, direct inhibition of KRAS mutants has proven extremely challenging, primarily due to the difficulty of identifying druggable pockets for small molecules that bind to the surface of RAS, and to date, no clinically approved drugs have been developed. This is due to the "smooth" surface of the protein, with the exception of the GTP-binding pocket, which holds substrates on its surface so tightly that they cannot be moved.

[0010] Various strategies have been proposed for treating cancers that harbor KRAS mutations, including: (1) Inhibition of RAS protein expression by hammerhead ribozymes or antisense oligonucleotides (2) Prevention of membrane localization of RAS by farnesyltransferase inhibitors or acylprotein thioesterase 1 (APT1) inhibitors (3) blocking downstream effectors of the RAS signaling pathway (inhibition of Raf or MEK with antisense oligonucleotides or chemical inhibitors), and (4) Silencing of mutant KRAS transcripts by RNA interference, which distinguishes between a single nucleotide difference between wild-type and mutant KRAS.

[0011] However, most of these strategies have limitations, such as non-selectivity or low therapeutic efficacy. Therefore, more effective strategies that exploit the selectivity of mutant KRAS are needed to treat mutant KRAS-associated cancers.

[0012] Antibodies with large surface area paratopes excel at specifically targeting proteins with high affinity. However, to date, antibody-based cancer therapeutics have been primarily directed at extracellular proteins, while hundreds of intracellular oncoproteins significantly associated with cancer remain challenging to drug discovery. Two major approaches have been used to modify antibodies to confer their ability to cross cell membranes: (i) chemical conjugation with cell-penetrating peptides (CPPs) and (ii) fusion with internalizing autoantibody moieties that contribute to their inherent ability to enter cells.

[0013] However, in most cases, antibody concentrations used in vitro are in the micromolar range (Shin et al., 2017; Akishiba et al., 2017), and in studies reporting in vivo data, the dosing regimens are not clinically translatable (Shin et al., 2017). For CPP-based strategies, the main limitations are related to factors such as low in vivo stability, lack of tissue / cell specificity, inefficient release from intracellular endocytic vesicles, and delayed target binding due to the presence of CPPs. Some of the drawbacks of CPP-based strategies can be circumvented by using the aforementioned fusion protein approach. However, the generation of fusion proteins is relatively laborious and not suitable for rapid screening (Slastnikova et al., 2018; Singh et al., 2019).

[0014] Thus, there is a need for novel intracellular delivery systems that can deliver clinically relevant antibody doses in vivo and serve as versatile and cost-effective platforms for the discovery and development of intracellular antibody-based therapeutics.

[0015] To our knowledge, in the case of anti-KRAS antibodies, they have been used mainly in the context of diagnostic applications and in the treatment of RA. Ras polypeptides have been described as a research tool for investigating Ras polypeptides. However, Shin et al. (2017) reported the development of a human IgG1 antibody, RT11, that internalizes into the cytosol of live cells, selectively binds to the activated GTP-bound form of various oncogenic Ras mutants, blocking their interaction with effector proteins and thereby inhibiting downstream signaling, exerting antiproliferative effects in various tumor cells harboring oncogenic Ras mutants. The RT11 antibody was generated by combining the Ras GTP-specific binding VH domain with a cytosol-permeable VL fragment to form a single IgG antibody. Therefore, this mechanism differs from the encapsulation mechanism of the anti-KRAS antibody described herein.

[0016] Although several companies and scientific papers have emphasized the importance of developing monoclonal antibodies that target intracellular proteins, to our knowledge, no in vivo data has been published demonstrating the efficacy of nanotechnology-based approaches for the intracellular delivery of therapeutic antibodies after systemic IV administration. Therefore, improvements in drug delivery are needed. Summary of the Invention

[0017] One problem to be solved by the present invention is the intracellular delivery of anti-KRAS antibodies at clinically relevant concentrations so that they provide a therapeutic effect.

[0018] This solution is based on the inventors' discovery that anti-KRAS antibodies formulated into specific nano-entities can be delivered intracellularly and further exert their biological activity within the cells.

[0019] As discussed in the Examples herein, the inventors have successfully conjugated anti-KRAS monoclonal antibodies to different polymeric nanocapsule compositions. As shown in Example 5, such nanocapsules can penetrate cell membranes and deliver the antibodies to the interior of the cell membrane. Nanocapsules containing anti-KRAS G12V mAb were efficiently internalized into colon adenocarcinoma cells, and the internalized antibodies showed preferential localization to the target site, i.e., the inner plasma membrane where oncogenic KRAS is localized. Similarly, in Example 6, polymeric nanocapsules loaded with anti-KRAS G12V mAb were efficiently internalized into lung adenocarcinoma cells. Furthermore, in Example 5, it was demonstrated that such nanocapsules could inhibit the in vitro growth of KRAS G12V mutant tumor cells by 25% to 50%. Therefore, it is demonstrated that anti-KRAS antibodies formulated in nanocapsules can be delivered into cells and further exert their biological activity within the cells.

[0020] Remarkably, Example 7 provides the first in vivo evidence of the efficacy of nanocapsules containing anti-KRAS G12V mAb in significantly reducing tumor growth in two mouse models (a pancreatic xenograft model and a colon orthotopic tumor model) without showing signs of therapeutic toxicity.

[0021] As mentioned above, anti-KRAS antibodies themselves cannot penetrate the cell membrane or interact with the KRAS polypeptide, and therefore cannot block the function of the corresponding intracellular target protein to elicit a therapeutic response. We have demonstrated that encapsulating anti-KRAS antibodies in polymeric nanocapsules is an effective approach for delivering these antibodies directly into the cytosol of live cells, thereby eliciting responses in both in vitro and in vivo studies.

[0022] As mentioned previously, despite numerous attempts to develop monoclonal antibodies targeting intracellular proteins, to our knowledge, no in vivo data has been published demonstrating the efficacy of nanotechnology-based delivery systems for intracellular localization after IV administration. Thus, the present invention provides the first approach for intracellular delivery of encapsulated anti-KRAS antibodies, which is believed to produce successful results in vivo. Thus, the present invention solves a technical problem that those skilled in the art have long sought to solve, and also satisfies a long-felt need.

[0023] As discussed herein, the fact that the nanocapsules of the present invention can penetrate cell membranes and deliver suitable clinical doses of antibodies to the inside of the cell membrane, escaping the lysosomal compartment, is generally an important advantage.

[0024] For example, with respect to certain embodiments herein in which the nanocapsules also comprise cell-penetrating peptides and / or tumor / tissue-penetrating peptides, even higher doses of antibodies and better localization to tumor tissues and cancer cells can be obtained.

[0025] Thus, a first aspect of the present invention relates to a composition comprising a plurality of nanoentities comprising an inner core surrounded by an outer shell, the outer shell comprising a polymer, and the inner core comprising at least one hydrophobic compound, wherein the nanoentities comprise a pharmaceutical agent, the pharmaceutical agent being an antibody or fragment thereof, and the antibody or fragment thereof binds to an epitope of an activating mutant KRAS protein.

[0026] A second aspect of the present invention relates to a method for preparing a composition according to the first aspect, comprising the steps of: a) preparing an aqueous phase containing a polymer b) preparing a hydrophobic phase comprising a hydrophobic compound c) adding an antibody or fragment thereof that binds to an epitope of the activating mutant KRAS protein to the aqueous phase, or optionally to the hydrophobic phase if the mAb or fragment is at a high concentration; c) mixing the aqueous phase with the hydrophobic phase.

[0027] A third aspect of the invention relates to a composition for use as a medicament.

[0028] A fourth aspect of the present invention relates to a composition for use in the treatment or prevention of a disease associated with a mutation in the KRAS gene. [Brief explanation of the drawings]

[0029] [Figure 1] This figure shows the stability of anti-KRAS G12V mAb-loaded HA nanocapsules (NCs) (HA 290 kDa NCs; 0.5 mg / mL mAb) in human plasma as measured by dynamic light scattering (DLS). [Figure 2] This figure shows the stability of anti-KRAS G12V mAb-loaded HA nanocapsules (NCs) (HA 290 kDa NCs; 0.5 mg / mL mAb) in human plasma as measured by nanoparticle tracking analysis (NTA). [Figure 3]This figure shows the cellular internalization of anti-KRAS G12V mAb induced by HA nanocapsules (NCs) in SW480 colorectal adenocarcinoma cells expressing the KRAS G12V mutation (labeled with Alexa Fluor® 488) by imaging flow cytometry. Effective internalization was measured by labeling cytoplasmic acidic organelles in live cells with the Lysotracker® fluorescent marker. [Figure 4] This figure shows (A) the inhibition of cell proliferation and (B) the reduction of ERK phosphorylation produced after incubation of H441 lung adenocarcinoma cells expressing the KRAS G12V mutation with C16-HA nanocapsules (NCs) (blank and NCs loaded with anti-KRAS G12V at 166 nM mAb, 0.4 ng mAb / cell dose). [C(-): untreated cells; BL: blank C16-HA NCs; aG12V NCs: C16-HA NCs loaded with anti-KRAS G12V mAb]. [Figure 5] This figure shows the reduction in tumor growth in mice treated with anti-KRAS G12V mAb-loaded PSA-tLyp-1 nanocapsules (NCs) during the treatment period compared to the control group. [C(-): Saline; aG12V NCs: anti-KRAS-loaded tLyp1-PSA NCs; IP administration; pancreatic subcutaneous xenograft tumor model from PA-TU-8902 cells expressing the KRAS G12V mutation]. [Figure 6] This figure shows the reduction in final tumor weight in mice treated with anti-KRAS G12V mAb-loaded HA NCs for 3 weeks compared to the control group. [C(-): PBS; aG12V NCs: anti-KRAS-loaded HA NCs; IV administration; colorectal orthotopic tumor model from subcutaneous tumor fragments derived from the SW480 cell line harboring the KRAS G12V mutation]. [Figure 7]This figure shows significant histological regression in tumors from mice treated with anti-KRAS G12V mAb-loaded HA NCs for 3 weeks compared to the control group. Tumor slides were scanned and analyzed using NDP-View2 software (Hamamatsu). The necrosis rate for each slide was calculated. [C(-): PBS; aG12V NCs: anti-KRAS-loaded HA NCs; IV administration; colorectal orthotopic tumor model from subcutaneous tumor fragments derived from the SW480 cell line harboring the KRAS G12V mutation]. [Figure 8] : This figure shows the weight change of mice treated with HA NCs loaded with anti-KRAS G12V mAb compared to the control group over a 3-week treatment period [C(-): PBS; aG12V NCs: HA NCs loaded with anti-KRAS; colorectal orthotopic tumor model from subcutaneous tumor fragments derived from the SW480 cell line harboring the KRAS G12V mutation]. DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention relates to a composition comprising particles, including nanocapsules or other nanoentities, that comprise an inner core surrounded by an outer shell, the inner core comprising a hydrophobic compound such as an oil, and the outer shell comprising a polymer such as polysialic acid (PSA), hyaluronic acid, polyglutamic acid, polyaspartic acid, polymalic acid, or polylactic acid. The nanoentity further comprises a pharmaceutical compound that is an antibody or fragment thereof directed against a target located intracellularly, where the antibody or fragment thereof binds to an epitope of an activated mutant KRAS protein. The particles of the present invention can reach the interior of cells, where they release the antibody. Furthermore, the polymer coating of the oily core may provide greater stability and protection against aggregation, alter the drug release profile of the associated antibody, increase cellular internalization, and enable specific interactions with specific cell types.

[0031] The term "inner core" may alternatively be referred to herein as "inner portion," and these terms may be used interchangeably herein.

[0032] Antibodies against mutant KRAS polypeptides Some of the first antibodies capable of binding to mutant KRAS polypeptides are described in US5084380 (incorporated herein by reference), which discloses monoclonal antibodies that react with activated KRAS polypeptides containing an amino acid mutation at position 12 of the KRAS polypeptide, but not with proteins containing the normal amino acid glycine at position 12. The inventors of US5084380 identified antibodies E184 and E170 that react with KRAS polypeptides containing glutamic acid at position 12 instead of glycine (G12E), R256 that reacts with KRAS polypeptides containing arginine at position 12 instead of glycine (G12R), and DWP that reacts with KRAS polypeptides containing valine at position 12 instead of glycine (G12V).

[0033] Monoclonal antibodies E170 and E184 substitute glutamine for glycine at position 12. These antibodies were raised against a synthetic peptide corresponding to amino acids 5-16 of a mutant ras gene encoding glutamic acid. These antibodies show specificity for dodecapeptides containing glutamic acid at position 12, but not for dodecapeptides containing Gly, Asp, Ser, Arg, Cys, Ala, or Val at position 12.

[0034] Monoclonal antibody R256 was raised against a synthetic peptide corresponding to amino acids 5–16 of a mutant ras gene encoding arginine instead of glycine at position 12. Monoclonal antibody R256 reacted specifically with dodecapeptides containing Arg at position 12 but did not react with dodecapeptides containing Gly, Glu, Asp, Ser, Cys, Val, or Ala at position 12.

[0035] DWP was generated against a synthetic peptide corresponding to amino acids 5–16 of a mutant ras gene encoding valine instead of glycine at position 12. DWP reacted in competition assays with peptides containing Val or Cys at position 12, but not with peptides containing Gly, Arg, Ser, Ala, Asp, or Glu at position 12 ( Carney et al., 1986 and EP0190033 ).

[0036] Hybridoma cell lines found to secrete monoclonal antibodies have been deposited with the American Type Tissue Culture Collection (ATCC) under the Budapest Treaty and have been assigned accession number HB9194 for E184, accession number HB9195 for E170, accession number HB9196 for R256, and accession number HB8698 for DWP.

[0037] In a particular embodiment of the invention, the antibody is one of the above antibodies disclosed in US Pat. No. 5,084,380 or a fragment thereof.

[0038] Furthermore, US5443956 (incorporated herein by reference) describes that monoclonal antibodies reacting with a mutant ras p21 protein having an aspartic acid at position 12 (G12D) were produced by hybridoma cell lines D113, D205, and D210. These cell lines were deposited with the ATCC under the Budapest Treaty. Hybridoma cell line D113 was assigned ATCC designation number HB10086. Hybridoma cell line D205 was assigned ATCC designation number HB10061. Hybridoma cell line D210 was assigned ATCC designation number HB10083.

[0039] In a particular embodiment of the invention, the antibody is one of the above antibodies disclosed in US Pat. No. 5,443,956 or a fragment thereof.

[0040] Other monoclonal antibodies that bind to mutant KRAS polypeptides include, for example, Abcam's ab221163, ab264094, and ab264095; NewEast Bioscience's catalog numbers 26036 (G12D), 26038 (G12D), 26193 (Q61L), 26474 (G13R), 26192 (Q61R), 26477 (G13V), 26195 (Q61H), 26191 (G13A), and 26186 (G12S); Cell Signaling's Ras (G12D mutant-specific) (D8H7) rabbit mAb 14429 and Ras (G12V mutant-specific) (D2H12) rabbit mAb 14412; and Sigma-Aldrich's G12V Ref. OP38. Anti-Pan-Ras (Ab-1) mouse mAb and others have been developed.

[0041] In one embodiment, the epitope of the activating mutant KRAS protein is the glycine residue at position 12, the glycine residue at position 13, or the glutamine residue at position 61 of the amino acid sequence of SEQ ID NO: 39. This contains mutations in the 2-amino acid residues, which corresponds to the polypeptide sequence of human isoform 2B of the GTPase KRas (the major isoform expressed in human tumors) with UNIPROT reference P01116-2 (RASK_HUMAN).

[0042] In another embodiment, the mutation at position 12 of the amino acid sequence of SEQ ID NO: 39 is selected from the group consisting of arginine, G12R, aspartic acid, G12D, valine G12V, and cysteine ​​G12C. In a further embodiment, the mutation at position 13 of the amino acid sequence of SEQ ID NO: 39 is selected from the group consisting of arginine, G13R, aspartic acid, G13D, and valine G13V. In yet another embodiment, the mutation at position 61 of the amino acid sequence of SEQ ID NO: 39 is selected from the group consisting of glutamine Q61R, leucine Q61L, and histidine Q61H.

[0043] Although one skilled in the art knows how to select antibodies that bind to such described epitopes, in one embodiment the antibody or fragment thereof binds to an epitope of a sequence selected from the group consisting of KLVVVGAVGVGK SEQ ID NO: 40, KLVVVGADGVGK SEQ ID NO: 41, and KLVVVGACGVGK SEQ ID NO: 42.

[0044] In a further embodiment, the antibody or fragment thereof binds to the same epitope of human activating mutant KRAS protein as an antibody obtainable from a hybridoma cell line having a deposit number selected from the group consisting of ATCC-HB-10086 D113, ATCC-HB-10083 D210, and ATCC-HB-8698 DWP.

[0045] In yet another embodiment, the antibody or fragment thereof is obtainable from a hybridoma cell line having a deposit number selected from the group consisting of ATCC-HB-10086 D113, ATCC-HB-10083 D210, and ATCC-HB-8698 DWP.

[0046] The above hybridomas were obtained from ATCC and reactivated to obtain the corresponding antibodies.

[0047] In a further embodiment, the antibody or fragment thereof binds to the same epitope of the human activating mutant KRAS protein as an antibody obtainable from a hybridoma cell line having a deposit number selected from the group consisting of DSM ACC3358, ATCC-HB-10083 D210, ATCC-HB-8698 DWP, ATCC-HB-10086-D113, and DSM ACC3359.

[0048] In yet another embodiment, the antibody or fragment thereof can be obtained from a hybridoma cell line having a deposit number selected from the group consisting of DSM ACC3358, ATCC-HB-10083 D210, ATCC-HB-8698 DWP, ATCC-HB-10086-D113 and DSM ACC3359.

[0049] The hybridoma cell lines DSM ACC3358 and DSM ACC3359 were deposited on the date of deposition, October 16, 2019, at the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (Inhoffenstraße 7B.38124 The mouse hybridoma cell line G12D D113-2-1 was deposited under accession number DSM ACC3358, and the mouse hybridoma cell line G12V DWP-1-1-3 was deposited under accession number DSM ACC3359. The depositor is the Universidade de Santiago de Compostela, Spain.

[0050] The DNA sequence of the anti-KRAS antibody was obtained from the hybridoma cell line deposited as DSM ACC3359. The corresponding antibody is a G12V anti-KRAS monoclonal antibody. In one embodiment, the antibody or fragment thereof comprises a heavy chain variable region CDR1: (SEQ ID NO: 33) SGYYWN, a heavy chain variable region CDR2: (SEQ ID NO: 34) YIGYDGTNNYNPSLKN, a heavy chain variable region CDR3: (SEQ ID NO: 35) LWDY, a light chain variable region CDR1: (SEQ ID NO: 36) RSSQTIVHGNGNTYLE, a light chain variable region CDR2: (SEQ ID NO: 37) TVSNRFS, and a light chain variable region CDR3: (SEQ ID NO: 38) FQGSHAPYT.

[0051] In a specific embodiment, the antibody or fragment thereof comprises a heavy chain region having SEQ ID NO:43 and a light chain region having SEQ ID NO:44.

[0052] The DNA sequence of the anti-KRAS antibody was obtained from the hybridoma cell line deposited as DSM ACC3358. The corresponding antibody is the G12D anti-KRAS monoclonal antibody. In one embodiment, the antibody or fragment thereof comprises a heavy chain variable region CDR1: (SEQ ID NO: 45) SYYMY, a heavy chain variable region CDR2: (SEQ ID NO: 46) EINPSNGGTNFNEKFKS, a heavy chain variable region CDR3: (SEQ ID NO: 47) GGYGY, a light chain variable region CDR1: (SEQ ID NO: 29) RSSKSLLYKDGKTYLN, a light chain variable region CDR2: (SEQ ID NO: 30) LMSTRAS, and a light chain variable region CDR3: (SEQ ID NO: 31) QQVVEYPRT.

[0053] In a specific embodiment, the antibody or fragment thereof comprises a heavy chain region having SEQ ID NO:10 and a light chain region having SEQ ID NO:17.

[0054] In certain embodiments, the antibody is a monoclonal antibody. More particularly, the monoclonal antibody is a humanized monoclonal antibody or a chimeric derivative for human administration.

[0055] Antibodies or fragments thereof The term "antibody" as used herein refers to (a) immunoglobulin polypeptides and immunologically active portions of immunoglobulin polypeptides, i.e., polypeptides of the immunoglobulin family or fragments thereof, that contain an antigen-binding site that immunospecifically binds to a specific antigen, or (b) conservatively substituted derivatives of such immunoglobulin polypeptides or fragments that immunospecifically bind to an antigen. Thus, both antibodies and their fragments have an antigen-binding site, i.e., can bind to a mutant KRAS epitope.

[0056] The term antibody also includes "antibody derivatives," which means antibodies as defined above that have been modified by the covalent attachment of a heterologous molecule, for example, by the attachment of a heterologous polypeptide not normally associated with the antibody, or by glycosylation, acetylation, or phosphorylation, etc.

[0057] In certain embodiments, an antibody is a monoclonal antibody, which refers to an antibody derived from a single cell clone, including any eukaryotic or prokaryotic cell clone, or phage clone, and not the method by which it is produced. Thus, the term "monoclonal antibody," as used herein, is not limited to antibodies produced through hybridoma technology.

[0058] The basic unit of antibody structure is four polypeptides bound together by both non-covalent associations and disulfide bonds: two identical low molecular weight ("light") chains and two Antibodies are complexes of two identical high molecular weight ("heavy") chains. Different antibodies have anywhere from one to five of these basic units. An antibody can be represented diagrammatically as a "Y." Each branch of the "Y" is formed by the amino-terminal portion of a heavy chain and an associated light chain. The base of the "Y" is formed by the carboxy-terminal portions of two heavy chains. The intersection of the "Y" is called the hinge region.

[0059] Five human antibody classes (IgG, IgA, IgM, IgD, and IgE) and various subclasses within these classes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclasses of immunoglobulin molecules are recognized based on structural differences such as the number of immunoglobulin units within a single antibody molecule, the disulfide bridge structure of the individual units, and differences in chain length and sequence. The class and subclass of an antibody is its isotype.

[0060] The antibody may be an intact antibody or an antigen-binding antibody fragment, such as a Fab, F(ab'), F(ab'), Fd chain, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), a fragment comprising any of the VL or VH domains, or a fragment produced by a Fab expression library. Antigen-binding antibody fragments, including single-chain antibodies, can comprise the variable region(s) alone or in combination with all or a portion of the following: hinge region, CH1, CH2, CH3, CH4, and CL domains. Antigen-binding fragments can also comprise any combination of the variable region(s) with the hinge region, CH1, CH2, CH3, CH4, and CL domains. In some embodiments, antibody fragments comprise at least one domain, or a portion of a domain, comprising an interchain disulfide bond.

[0061] Antibodies exist as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests antibodies below the disulfide bond in the hinge region (i.e., toward the Fc domain) to produce a dimer of Fab, F(ab)'2, which is itself a light chain linked to VH-CH1 by a disulfide bond. F(ab)'2 is reduced under mild conditions to cleave the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with part of the hinge region. While various antibody fragments are defined with respect to the digestion of intact antibodies, these and other fragments can also be synthesized de novo, chemically, for example, by recombinant DNA methodologies, such as "phage display." Examples of antibodies include single-chain antibodies, such as single-chain Fv (scFv) antibodies, in which the variable heavy and variable light chains are linked together (directly or via a peptide linker) to form a contiguous polypeptide. Further non-limiting examples of antibodies include nanobodies, antibody fragments, monoclonal antibodies, chimeric antibodies, reverse chimeric antibodies, etc. Antigen-binding fragments include Fab, Fab', F(ab)2, dsFv, sFv, unibodies, minibodies, diabodies, tribodies, tetrabodies, nanobodies, probodies, domain bodies, unibodies, bispecific single-chain variable fragments (bi-scFv), etc.

[0062] An "Fv" is the minimum antibody fragment that contains a complete antigen-recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, noncovalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the complete binding site.

[0063] Antibody affinity can be determined as described in the Examples herein below. A particular antibody binds to the peptide KLVVVGAVGVGK (SEQ ID NO: 40) or KLVVVGADGVGK (SEQ ID NO: 41) at approximately 1 x 10 -7 M or less, specifically about 1 × 10 -8 These antibodies bind with a Kd value of 3.9 nM or less, specifically in the range of 1 to 10 nM, more specifically 3 to 5 nM, and even more specifically 3.9 nM and 5 nM.

[0064] Typically, antibodies are human, rodent (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, a "human" antibody includes antibodies having the amino acid sequence of a human immunoglobulin, including antibodies isolated from human immunoglobulin libraries, human B cells, or animals transgenic for one or more human immunoglobulins. Antibodies can be monospecific, bispecific, trispecific, or of greater multispecificity.

[0065] Particles / Nanocapsules / Nanoentities A "nanoentity," as used herein, typically refers to a nanoentity having an average diameter of less than 1,000 nm, e.g., less than 750 nm, less than 500 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, or less than 100 nm. In some cases, the nanoentity has an average diameter of at least 1 nm, 5 nm, 10 nm, 50 nm, 100 nm, 500 nm, or 1,000 nm. Any combination of these diameters is also possible; for example, the nanoentity has an average diameter range of 100 nm to 300 nm, 1,000 nm to 1 nm, 1,000 nm to 10 nm, 750 nm to 1 nm, 500 nm to 10 nm, 300 nm to 10 nm, 250 nm to 10 nm, 200 nm to 10 nm, 150 nm to 10 nm, 100 nm to 10 nm, etc. In some embodiments, more than one nanoentity is present, and in such cases, the average (arithmetic) diameter of the nanoentities has the dimensions described herein. In some cases, nanoentities have a range of diameters. Such nanoentities are measured by various methods, such as dynamic light scattering or laser light scattering. Examples of nanoentities include nanoparticles, nanocapsules, micelles, or other nanoentities as described herein.

[0066] In some cases, the nanoentity comprises an inner core surrounded by an outer shell, e.g., exposed to the environment surrounding the nanoentity. The inner core may be symmetrically or asymmetrically disposed within the nanoentity. The inner core may comprise, e.g., a liquid (e.g., non-aqueous or aqueous), a solid, and / or a combination thereof. In some embodiments, the inner core comprises one or more pharmaceutical agents or drugs. For example, the inner core may comprise a monoclonal antibody and a small molecule such as docetaxel.

[0067] In some cases, the nanoentity is a capsule (e.g., a nanocapsule). Capsules can be substantially solid or have a rubbery or gel-like shell. Further, in some cases, the nanoentity is a particle, such as a nanoparticle. Particles are solid and have a defined shape. In some cases, particles are nanoentities with an internal core surrounded by an external shell, e.g., a particle is a capsule. Nanocapsules have sizes in the nanometer range. When a nanoparticle is roughly spherical, it can also be referred to as a nanosphere. Nanocapsules are substantially uniform but have additional surface features, such as targeting moieties, permeation enhancers, antibodies, etc., including those described herein.

[0068] In some cases, the particle is a nano-entity having an inner core surrounded by an outer shell, e.g., the particle is a capsule or nanocapsule. In some cases, the nanocapsule has a size in the nanometer range, comprising an inner core and an outer shell having a composition distinguishable from the inner core. The inner core can be, for example, a liquid or solid material. The inner core is often, but not always, an oil. The outer shell is formed of a continuous material and is typically not covalently bonded to the inner core. In some cases, the outer shell has an average thickness of at least 1 nm, at least 2 nm, at least 3 nm, at least 5 nm, at least 10 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, or at least 200 nm.

[0069] In certain embodiments, at least some of the plurality of nanoentities are nanocapsules having an average diameter of less than 1 micrometer.

[0070] Other nanoentities In some cases, the nanoentity is a micelle. Typically, a micelle is formed from multiple surfactant or amphiphilic molecules that define an interior and an exterior. For example, the surfactant molecules are arranged to have a relatively hydrophilic exterior and a relatively hydrophobic interior, formed, for example, from a monolayer of surfactant or amphiphilic molecules. In some cases, the micelle has a size in the nanometer range. In some embodiments, a micelle is formed by amphiphilic molecules at a concentration above the critical micelle concentration (CMC) when the micelle is dispersed in an external phase. When the external liquid phase is aqueous, the hydrophilic portions of the amphiphilic molecules are oriented toward the external phase. Depending on the concentration of the amphiphilic molecules, the micelle can organize itself to form larger structures that are clusters of micelles. For example, a micelle is formed from surfactant molecules with the hydrophilic portions on the surface and the hydrophobic portions facing inward (or vice versa).

[0071] In some cases, the nanoentity is a liposome. Liposomes can have a similar structure, but are typically formed from a bilayer of surfactant or amphiphilic molecules (e.g., a lipid bilayer), thereby defining an interior portion, a middle portion, and an outer shell. For example, the interior portion is relatively hydrophilic, the middle portion (e.g., the outer shell of the liposome formed by the surfactant or amphiphilic molecule bilayer structure) is relatively hydrophobic, and the outside of the liposome is an aqueous or hydrophilic environment.

[0072] When the interior portion of the nanoentity is aqueous, in certain embodiments, the aqueous liquid forming the interior portion can be composed of water containing at least one salt. Additionally, in some embodiments, the aqueous liquid forming the interior portion can include one or more water-soluble stabilizers, preservatives, surfactants, glycols, polyols, sugars, thickeners, gelling agents, and mixtures of these and / or other suitable excipients. These excipients are used, for example, to improve the stability of the formulation, to adjust the viscosity of the final composition, to control the release rate from the internal aqueous phase, etc.

[0073] In some embodiments, the nanoentity is a nanoemulsion. A nanoemulsion is a two-phase dispersion of two immiscible liquids, either water-in-oil (W / O) or oil-in-water (O / W) droplets stabilized by at least one suitable amphiphilic surfactant to form nanometer-sized droplets.

[0074] Inner core; hydrophobic compound As used herein, the property of being "hydrophilic" is understood as the constitutive property of a molecule or functional group to penetrate into or remain in the aqueous phase. Accordingly, the property of being "hydrophobic" is understood as the constitutive property of a molecule or functional group to exhibit behavior toward water that is ecologically irrelevant to humans and the environment, i.e., to not penetrate into water or to exhibit a tendency to leave the aqueous phase. For further details, see Rompp Lexikon Lacke und Druckfarben, Georg Thieme Verlag, Stuttgart, NY, 1998, "Hydrophil See pages 294 and 295 of "Hydrophobicity," "Hydrophobicity." As used herein, the property of being "hydrophobic" can be used interchangeably with the property of being "oleophilic." However, while hydrophobic materials are typically oleophilic, exceptions are known, such as in the case of silicones and fluorocarbons.

[0075] In some embodiments, the inner core comprises at least one hydrophobic compound selected from the group consisting of, for example, an oil, a lipophilic surfactant, a fatty acid, an alkane, a cycloalkane, a bile salt, a bile salt derivative, a terpenoid, a terpene, a terpene-derived moiety, and a fat-soluble vitamin.

[0076] The hydrophobic compound is particularly an oil. The oil can be volatile or non-volatile and, in certain embodiments, is selected from natural oils, semi-synthetic and synthetic pharmaceuticals, or combinations thereof, such as animal oils, vegetable oils, hydrocarbon oils, or silicone oils. These oils can be selected from natural, semi-synthetic, and synthetic pharmaceutical oils, such as vegetable or animal-derived oils, hydrocarbon oils, or silicone oils. Oils suitable for practicing certain embodiments of the present invention include, but are not limited to, caprylic / capric triglycerides, mineral oils, squalene oils, flavor oils, silicone oils, essential oils, water-insoluble vitamins, isopropyl stearate, butyl stearate, octyl palmitate, cetyl palmitate, tridecyl behenate, diisopropyl adipate, dioctyl sebacate, menthyl anthranilate, cetyl octanoate, octyl salicylate, and isopropyl myristate. , neopentyl glycol ketol dicaprate, decyl oleate, alkyl lactate (C12-C15), cetyl lactate, lauryl lactate, isostearyl neopentanoate, myristyl lactate, isocetyl stearoyl stearate, octyldodecyl stearoyl stearate, hydrocarbon oil, isoparaffin, liquid paraffin, isododecane, petroleum jelly, argan oil, rapeseed oil, chili oil, palm oil, corn oil, cottonseed oil, linseed oil, grape seed oil, mustard oil, Olive oil, palm oil, fractionated palm oil, peanut oil, castor oil, pine nut oil, poppy seed oil, pumpkin seed oil, rice bran oil, safflower oil, tea tree oil, truffle oil, vegetable oil, apricot kernel oil, jojoba oil, macadamia nut oil, wheat germ oil, almond oil, soybean oil, sesame seed oil, hazelnut oil, sunflower oil, hemp seed oil, rosewood oil, kukui nut oil, avocado oil, walnut oil, fish oil, berry oil, allspice oil, juniper oil, seed oil, almond seed oil, anise Seed oil, celery seed oil, cumin seed oil, nutmeg seed oil, basil leaf oil, bay leaf oil, cinnamon leaf oil, sage leaf oil, eucalyptus leaf oil, lemon leaf oil, melaleuca leaf oil, oregano oil, patchouli leaf oil, peppermint leaf oil, pine leaf oil, rosemary leaf oil, spearmint oil, tea tree leaf oil, thyme oil, flower essential oils, chamomy oil, clary sage oil, clove oil, geranium flower essential oil, hyssop flower essential oil, jasmine oil, lavender oil, mauka flower essential oil, marjoram flower essential oil, orange flower essential oil,Rose essential oil, ylang-ylang essential oil, bark oil, cassia bark oil, cinnamon bark oil, sassafras bark oil, wood oil, camphor wood oil, cedarwood oil, rosewood oil, sandalwood oil, lily of the valley oil, tall oil, castor oil, myrrh oil, peel oil, bergamot peel oil, grapefruit peel oil, lemon peel oil, lime peel oil, orange peel oil, tangerine peel oil, root oil, valerian oil, oleic acid, linoleic acid, oleyl alcohol, isostearyl alcohol, ethyl oleate, medium-chain triglycerides, e.g., mixtures of decanoyl and octanoyl glycerides (Miglyol® 810N, Miglyol® 812N, Kollisolv® MCT, Captex® 300, Captex® 355, Labrafac® Lipophile WL1349), Labrafil® M 2125 CS (linoleoyl macrogol-6 glycerides), Labrafil® M 2130 CS (lauroyl macrogol-6 glycerides), Labrafil® M 1944 CS (oleoyl polyoxyl-6 glycerides), Labrafac® PG (propylene glycol dicaprylocaprate), Rylo® (mixture of fatty acids), Peceol (registered trademark, (glycerol monooleate) and Maisine® (glycerol monolinoleate), their synthetic or semi-synthetic derivatives, and combinations thereof. In some cases, the oil is one or more of peanut oil, cottonseed oil, olive oil, castor oil, soybean oil, safflower oil, sesame oil, corn oil, palm oil, alpha-tocopherol (vitamin E), isopropyl myristate, squalene, Miglyol®, Labrafil®, Labrafac®, Peceol®, Captex®, Kollisolv® MCT, and Maisine®, or mixtures thereof. Other suitable oils include oils derived from the terpene families formed by isoprene units (2-methylbuta-1,3-diene) and subdivided according to their carbon atoms: hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), sesquiterpenes (C25), triterpenes (C30), tetraterpenes (C40, carotenoids), and polyterpenes; vitamin A; squalene; etc. In some embodiments, the non-aqueous liquid forming the inner portion may contain water-insoluble stabilizers, preservatives, surfactants, organic solvents, and mixtures thereof to provide maximum stability of the formulation. Combinations of one or more of these and / or other oils are also possible in various embodiments.

[0077] In one embodiment, the inner core comprises an oil, particularly a caprylic / capric triglyceride (such as Miglyol® 812N).

[0078] Outer shell: polymer The advantage of nanocapsule systems over emulsion systems is the presence of a polymer coating the oily core, which may impart greater stability and protection against aggregation, altered drug release profiles for associated drugs, increased cellular internalization, and specific interactions with particular cell types. A variety of polymers can be used according to certain embodiments of the present invention.

[0079] In some embodiments, the polymer is selected from the group consisting of polysialic acid (PSA), hyaluronic acid (HA), polyglutamic acid (PGA) and / or pegylated polyglutamic acid (PGA-PEG), polylactic acid (PLA) and / or pegylated polylactic acid (PLA-PEG), poly(aspartic acid) (PASP) and / or pegylated poly(aspartic acid) (PASP-PEG), poly(lactic-co-glycolic acid) (PLGA) and / or pegylated poly(lactic-co-glycolic acid) (PLA-PEG), alginic acid (ALG) and / or pegylated alginic acid (ALG-PEG), polymalic acid (PLMA) and / or pegylated polymalic acid (PLMA-PEG), and mixtures thereof. Combinations of these and / or other polymers are also used in certain embodiments.

[0080] In certain embodiments, the polymer is selected from the group consisting of polysialic acid, hyaluronic acid, polyglutamic acid, polyaspartic acid, polymalic acid, alginic acid, polylactic acid, polylactic-co-glycolic acid, PEGylated forms thereof, and mixtures thereof. In certain embodiments, the polymer is PSA or HA.

[0081] The polymer may be uniformly distributed throughout the entity or may be concentrated within a particular region of the entity, such as the outer shell of a capsule or other outer surface of the entity. In some cases, at least 50% by weight of a portion of the entity, such as the shell, comprises the polymer, and in certain cases, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least The composition may contain up to 99% by weight of polymer.

[0082] Polymer-PSA In certain embodiments, the polymer is PSA.PSA is generally composed of multiple sialic acid units, which are often linked together to form a polymer via 2-->8 and / or 2-->9 bonds, although other linkage arrangements are possible.Typically, there are at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500 sialic acid units linked together to form PSA.In some cases, PSA has 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less sialic acid units linked together to form PSA. Any combination of these is also possible; for example, PSA may have 2 to 100 sialic acid units linked together. Note that the sialic acid units need not be identical and may independently be the same or different, even within the same PSA molecule. Also, note that PSA does not necessarily have to be a linear (linear) chain; various branching arrangements are possible. For example, a sialic acid unit may be linked to three or more different sialic acid units, thereby forming a branch point within the PSA molecule.

[0083] By way of non-limiting example, PSAs have different molecular weights, e.g., 4 kDa, 30 kDa, 95 kDa, etc. In some cases, PSAs comprise more than 300 sialic acid units. By way of further non-limiting example, PSAs have molecular weights of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, the PSA has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less. Any combination of these is also possible, for example, the PSA has a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa, etc. (Unless otherwise specified, molecular weights described herein are number average molecular weights.)

[0084] It should also be noted that the polysialic acids do not necessarily have to be identical. For example, in some embodiments, PSA has a different number of sialic acid units and / or different sialic acid units are present in different PSA molecules present. In some cases, one or more types of PSA molecules may be present, e.g., one or more forms may comprise at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more of the PSA molecules present, i.e., on a molar basis.

[0085] Non-limiting examples of sialic acid units present in PSA include, but are not limited to, N-acetylneuraminic acid (Neu), 2-keto-3-deoxynoic acid (Kdn), lactamic acid, N-sialic acid, and / or O-sialic acid. Other examples include N-glycolylneuraminic acid (Neu5Gc), 9-O-acetyl-8-O-methyl-N-acetylneuraminic acid (Neu5,9Ac28Me), and 7,8,9-tri-O-acetyl-N-glycolylneuraminic acid (Neu5Gc7,8,9Ac3). "Sia" generally refers to any unspecified sialic acid unit. In some embodiments, a sialic acid unit The position includes any derivative of neuraminic acid (a nine-carbon sugar), including the 43 derivatives typically found in nature, including, but not limited to, Neu; Neu5Ac; Neu4,5Ac2; Neu5,7Ac2; Neu5,8Ac2; Neu5,9Ac2; Neu4,5,9Ac3; Neu5,7,9Ac3; Neu5,8,9Ac3; Neu5,7,8,9Ac4; Neu5Ac9Lt; Neu4,5Ac29Lt; Neu5Ac8Me; Neu5,9Ac28Me; Neu5Ac8S; Neu5Ac9P; Neu2en5Ac; Neu2en5,9Ac2; Neu2en5Ac9Lt; Neu2,7an5Ac; Neu5Gc; Neu4Ac5G In one series of embodiments, each of the sialic acid units (prior to polymerization to form PSA) can independently have the following structure: [ka]

[0086] R 1is H; an alpha bond to Gal(3 / 4 / 6), GalNAc(6) (N-acetylgalactosamine), GlcNAc(4 / 6), Sia(8 / 9), or 5-O-Neu5Gc; an oxygen attached to C-7 in a 2,7-anhydride molecule; or an anomeric hydroxyl (double bond to C-3) removed with Neu2en5Ac. 1 is H; an alpha bond to Gal(3 / 4 / 6), GalNAc(6), GlcNAc(4 / 6), Sia(8 / 9), or 5-O-Neu5Gc; an oxygen attached to C-7 in a 2,7-anhydride molecule; or an anomeric hydroxyl (double bond to C-3) removed with Neu2en5Ac. 4 is H; -acetyl; C-8 anhydride; Fuc (fucose); or Gal (galactose). 5 is amino; N-acetyl; N-glycolyl; hydroxyl; N-acetimidoyl; N-glycolyl-O-acetyl; N-glycolyl-O-methyl; or N-glycolyl-O-2-Neu5Gc. 7 is substituted by H; -acetyl; anhydride at C-2; or amino and N-acetyl at Leg (legionaminic acid). 8 is H; -acetyl; C-4 anhydride; -methyl; -sulfate; Sia (sialic acid); or Glc (glucose). 9 is H; -acetyl; -lactyl; -phosphate; -sulfate; Sia; or OH substituted by H in Leg. In some cases, the PSA is colominic acid (when only 2-->8 bonds are present).

[0087] As used herein, sialic acid includes, but is not limited to, water-soluble salts of sialic acid and water-soluble derivatives of sialic acid. For example, sialic acid salts include sodium salts, potassium salts, malate salts, and the like. The sialic acid may be present as a magnesium salt, a calcium salt, or a zinc salt. In one embodiment, at least some of the sialic acids are present as sodium salts. Combinations of multiple types of sialic acids may also be used, for example, as subunits of PSA and / or as different molecules of PSA. In one series of embodiments, at least some of the sialic acids in PSA are modified (although it should be understood that in other embodiments, the PSA is not necessarily modified). For example, in some cases, one or more sialic acid units are modified by conjugation to, for example, polyethylene glycol, alkyl, or other hydrophobic moieties. The hydrophobic moiety includes a hydrophobic molecule or portion thereof, for example, an alkyl group as discussed herein.

[0088] Polymer - Hyaluronic Acid, HA In one series of embodiments, the polymer comprises hyaluronic acid, a linear polymer containing repeating disaccharide structures formed by alternating additions of D-glucuronic acid and D-N-acetylglucosamine linked by alternating beta-1,4 and beta-1,3 glycosidic bonds, as shown in the following formula: [ka] In the formula, the integer n represents the degree of polymerization, i.e., the number of disaccharide units in the hyaluronic acid chain. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Any combination of these is also possible, e.g., n is between 2 and 100. Note that hyaluronic acid units need not be identical and can be independently the same or different, even within the same hyaluronic acid chain. Also, note that hyaluronic acid does not necessarily have to be a linear (linear) chain; various branching arrangements are possible.

[0089] Thus, hyaluronic acid having a wide range of molecular weights can be used. As a non-limiting example, hyaluronic acid can have different molecular weights, such as 4 kDa, 30 kDa, 95 kDa, etc. For example, hyaluronic acid can have a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 75 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. In some cases, hyaluronic acid can be used. Hyaluronic acid has a molecular weight of 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 25 kDa or less, 20 kDa or less, 10 kDa or less, 5 kDa or less, 3 kDa or less, or 1 kDa or less. Any combination of these is also possible. For example, hyaluronic acid has a molecular weight of about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, or about 10 kDa to about 50 kDa.

[0090] Hyaluronic acid as used herein also includes its conjugate base (hyaluronate). This conjugate base can be an alkali salt of hyaluronic acid, including inorganic salts such as sodium salt, potassium salt, calcium salt, ammonium salt, magnesium salt, aluminum salt, and lithium salt, and organic salts such as basic amino acid salts at neutral pH. In some cases, the salt is pharmaceutically acceptable. In one embodiment, the alkali salt is the sodium salt of hyaluronic acid. Combinations of multiple types of hyaluronic acid can also be used, for example, as subunits of hyaluronic acid chains and / or as different molecules of hyaluronic acid.

[0091] Therefore, hyaluronic acid does not necessarily have to be the same.For example, in some embodiments, hyaluronic acid has a different number of hyaluronic acid units (such as those described above) and / or different hyaluronic acid units exist in the different hyaluronic acid chains that exist.In some cases, one or more types of hyaluronic acid molecules exist, and for example, one or more forms comprise at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the hyaluronic acid molecules that exist, i.e., on a molar basis.

[0092] In some embodiments, at least some of the hyaluronic acid units are modified (although it should be understood that in other embodiments, the hyaluronic acid is not necessarily modified). For example, in some cases, one or more hyaluronic acid units are modified by attachment to, for example, polyethylene glycol, alkyl, or other hydrophobic moieties. Hydrophobic moieties include hydrophobic molecules or portions thereof, such as alkyl groups as discussed herein.

[0093] Polymer - Polyglutamic Acid, PGA In one set of embodiments, the polymer comprises polyglutamic acid (PGA). PGA is a polymer of the amino acid glutamic acid (GA). Apart from the molecular weight (Mw), the ratio of D-glutamic acid monomers to L-glutamic acid monomers and the molecular structure are important chemical properties of PGA. PGA exists as two different structures, as shown below: [ka] In the formula, the integer n represents the degree of polymerization, i.e., the number of glutamic acid monomers in the PGA chain. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Therefore, polyglutamic acid having a wide range of molecular weights can be used. By way of non-limiting example, polyglutamic acid can have different molecular weights, for example, polyglutamic acid can have molecular weights of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combinations of these are also possible. It should also be noted that the glutamic acid units do not necessarily have to be in a linear (linear) chain, but various branched arrangements are also possible.

[0094] Poly-α-glutamic acid (α-PGA) can be synthesized chemically, while γ-PGA is only available through microbial production.

[0095] As used herein, polyglutamic acid includes, but is not limited to, water-soluble salts and derivatives of polyglutamic acid (e.g., sodium, potassium, magnesium, calcium salts). In some embodiments, at least some of the glutamic acid units in the PGA are modified (although it should be understood that in other embodiments, the PGA is not necessarily modified). For example, in some cases, one or more glutamic acid units are modified, such as by attachment to polyethylene glycol, alkyl, or other hydrophobic moieties.

[0096] Polymer - Polyaspartic acid, PASP In one series of embodiments, the polymer comprises polyaspartic acid (PASP). PASP is a poly(amino acid) with protein-like amide bonds in its backbone and carboxylic acid pendant groups in each repeat unit. In nature, PASP is found as fragments of larger proteins up to 50 amino acids in length. PASP can be synthesized by different methods. The polymer exists in different forms, such as α, β, and L, D isomers. [ka] where the integer n represents the degree of polymerization, i.e., the number of aspartic acid monomers in the PASP chain. Any number of aspartic acid units may be present within the polymer. For example, n may be at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n may be 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. By way of non-limiting example, polyglutamic acid can have different molecular weights, e.g., polyglutamic acid can have molecular weights of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combination of these is also possible. It should also be noted that other amino acids may be present in the PASP chain, and the polymer may be linear or branched.

[0097] As used herein, polyaspartic acid includes, but is not limited to, water-soluble salts and derivatives of polyaspartic acid (e.g., sodium, potassium, magnesium, and calcium salts). In some embodiments, at least some of the aspartic acids in the PASP are modified (although it should be understood that in other embodiments, the PASP is not necessarily modified). For example, in some cases, one or more aspartic acid units are modified, such as by attachment to polyethylene glycol, alkyl, or other hydrophobic moieties.

[0098] Polymer - Polymalic Acid, PMLA In one set of embodiments, the polymer comprises polymalic acid (PMLA). PLMA is a carboxylic acid-functionalized polyester that can be produced either by chemical synthesis or biological fermentation from the slime mold Physarum polycephalum. PMLA is a completely biodegradable polymer that is metabolized to water and carbon dioxide in the citric acid cycle. While both racemic or optically pure α and β structures can be obtained by chemical methods, microorganisms produce only PMLA with significantly higher optical purity. The pendant carboxylic acid groups can be chemically modified to introduce molecules of interest. The structures of different forms of PLMA are shown below: [ka] where the integer n represents the degree of polymerization, i.e., the number of malic acid units in the PLMA chain. For example, n can be at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n can be 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Thus, polymalic acid having a wide range of molecular weights can be used. As a typical example, polymalic acid can have different molecular weights, for example, polymalic acid can have molecular weights of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combinations of these are also possible. It should also be noted that the malic acid units do not necessarily have to be in a linear chain, but rather various branched arrangements are also possible.

[0099] As used herein, polymalic acid includes, but is not limited to, water-soluble salts and derivatives of polymalic acid (e.g., sodium, potassium, magnesium, calcium salts). In one set of embodiments, at least some of the malic acid units in the PLMA are modified (although it should be understood that in other embodiments, the PLMA is not necessarily modified). For example, in some cases, one or more malic acid units are modified, such as by attachment to polyethylene glycol, alkyl, or other hydrophobic moieties.

[0100] Polymer - Alginic Acid, ALG In one set of embodiments, the polymer comprises alginic acid (ALG). ALG is a hydrophilic polysaccharide widely distributed in the cell walls of brown algae. Its salts are known as alginates. Alginates are actually block copolymers, with the ratio of guluronic acid to mannuronic acid varying depending on the natural source. Alginates are known as a whole family of linear copolymers containing blocks of (1,4)-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. The blocks are composed of consecutive G residues (GGGGGG), consecutive M residues (MMMMMM), and alternating M and G residues (GMGMGM), as shown below (Lee and Mooney, 2012). [ka]

[0101] By way of non-limiting example, alginates can have different molecular weights, for example, alginates having molecular weights of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combinations of these are also possible.

[0102] As used herein, alginic acid includes, but is not limited to, water-soluble salts and derivatives of alginic acid (e.g., sodium, potassium, magnesium, calcium salts). In one set of embodiments, at least some of the guluronic acid and / or mannuronic acid residues in the ALG are modified (although it should be understood that in other embodiments, the ALG is not necessarily modified). For example, in some cases, one or more guluronate and / or mannuronate residues are modified, e.g., by conjugation to polyethylene glycol, alkyl, or other hydrophobic moieties, etc.

[0103] Polymer - Polylactic Acid, PLA In one series of embodiments, the polymer comprises polylactic acid (PLA) polyester, the structure of which is shown below: [ka] where the integer n represents the degree of polymerization, i.e., the number of lactic / lactide units in the PLA chain. For example, n can be at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n can be 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 30 or less, or 10 or less. Thus, PLA having a wide range of molecular weights can be used. By way of non-limiting example, PLA can have different molecular weights, for example, PLA can have a molecular weight of at least 1 kDa, at least 3 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 25 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combination of these is also possible. It should also be noted that the lactic / lactide units do not necessarily have to be in a linear (linear) chain, but rather various branching arrangements are possible.

[0104] Polymer - Polylactic-co-glycolic acid, PLGA In one series of embodiments, the polymer comprises polylactic acid-co-glycol (PLGA) polyester. The structure of PLGA is shown below: [ka] Wherein, integer x represents the number of lactic acid units, and integer y represents the number of glycolic acid units. Depending on the ratio of lactic acid and glycolic acid used in polymerization, different forms of PLGA can be obtained, which are usually distinguished by the molar ratio of the monomers used. Non-limiting examples of PLGA include 75:25 (copolymer with a composition of 75% lactic acid and 25% glycolic acid), 50:50 (copolymer with a composition of 50% lactic acid and 50% glycolic acid), and 25:75 (copolymer with a composition of 25% lactic acid and 75% glycolic acid).

[0105] In some cases, one or more of the lactic acid and / or glycolic acid monomers in the PLA or PLGA are modified, for example, by conjugation to polyethylene glycol, alkyl, or other hydrophobic moieties.

[0106] PEGylated polymer, polymer-PEG In one set of embodiments, the polymer comprises poly(ethylene glycol) (PEG). Thus, in some cases, PEG is conjugated to, for example, HA, PGA, PASP, PLMA, ALG, PLA, or PLGA to form, for example, HA-PEG, PGA-PEG, PASP-PEG, PLMA-PEG, ALG-PEG, PLA-PEG, or PLGA-PEG. However, in other cases, PEG is present, i.e., not conjugated to the polymer. The most common form of PEG is a polymer having the formula: H-(O-CH2-CH2) n -OH, In the formula, n is an integer representing the degree of polymerization of PEG. To form a conjugate polymer-PEG, one or two of the two terminal hydroxyl groups are modified. Modified PEG is, for example, as follows: X 1 -(O-CH2-CH2) n -X 2 , In the formula, X 1 is a hydrogen or hydroxyl protecting group that blocks the OH radical function for subsequent reaction. For example, n is at least 2, at least 4, at least 6, at least 8, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, or at least 500. In some cases, n is 1000 or less, 500 or less, 200 or less, 100 or less, or 50 or less. , 30 or less, or 10 or less. Any combination of these is also possible, for example, n is 2 to 100. Protecting groups for hydroxyl radicals are widely known in the art.

[0107] PEGylation of the polymer can be carried out using any suitable method available in the art. Such polymers are available in a variety of molecular weights. For example, suitable molecular weights for PEG or polymer-PEG are about 1 kDa to about 100 kDa, about 5 kDa to about 80 kDa, and about 10 kDa to about 50 kDa. As further non-limiting examples, PEG or polymer-PEG may have a molecular weight of at least 1 kDa, at least 5 kDa, at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, etc. Any combinations thereof are also possible.

[0108] In some embodiments, the percentage of PEG in a polymer (e.g., HA, PGA, PASP, PLMA, ALG, PLA, PLGA, etc.) can be about 10% to 90% (w / w), about 15% to 80%, about 20% to 70%, or about 20%, about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, or about 60% by weight of the total polymer.

[0109] targeting part In one aspect, the entity also includes a targeting moiety, although it should be noted that in some embodiments, a targeting moiety is not present. The targeting moiety (if present) is used, for example, to target delivery of the entity to a specific cell population within a subject. For example, the targeting moiety facilitates delivery of the nanoentity to one or more types of cells, e.g., cancer cells, endothelial cells, and / or immune cells. In some embodiments, the targeting moiety enables targeting of the entity to a specific location within a subject, e.g., a specific organ or a specific cell type (e.g., tumor or cancer cell). In some cases, the entity is internalized by cells without the need for a targeting moiety, and in some other cases, internalization is facilitated by the targeting moiety. In some embodiments, more than one type of targeting moiety is present. In some embodiments, the targeting moiety comprises a cell and / or tumor / tissue-penetrating peptide. However, it should be understood that in certain embodiments, the targeting moiety does not necessarily also facilitate internalization.

[0110] In various embodiments, a wide variety of targeting moieties can be used. For example, targeting moieties include peptides, proteins, aptamers, antibodies (including monoclonal antibodies, nanobodies, and antibody fragments), nucleic acids, organic molecules, ligands, etc. Some targeting moieties can be found, for example, in Bertrand, et al., 2014, et al., 2016, and Zhou et al., 2016.

[0111] In one series of embodiments, for example, the targeting moiety is a peptide having a length of, for example, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 10 amino acids or less.

[0112] In one set of embodiments, the targeting moiety is a cell-penetrating and / or tumor / tissue-penetrating peptide. Cell-penetrating peptides have the ability to penetrate cell membranes. Tumor-penetrating peptides have the ability to facilitate deep penetration of drug payloads into tumors. In some cases, cell-penetrating and / or tumor / tissue-penetrating peptides also act as nanoentities to cells. Facilitate the targeting of

[0113] As used herein, "naturally occurring amino acids" are the 20 amino acids frequently found in nature, typically as the L-isomer, namely, alanine ("Ala" or "A"), arginine ("Arg" or "R"), asparagine ("Asn" or "N"), aspartic acid ("Asp" or "D"), cysteine ​​("Cys" or "C"), glutamine ("Gln" or "Q"), glutamic acid ("Glu" or "E"), glycine ("Gly" or "G"), histidine ("Hi" or "H"), and ribonucleotides ("RI" or "R"). The amino acids are acetylcholine ("Ile" or "I"), acetylcholine ("Leu" or "L"), lysine ("Lys" or "K"), methionine ("Met" or "M"), phenylalanine ("Phe" or "F"), proline ("Pro" or "P"), serine ("Ser" or "S"), threonine ("Thr" or "T"), tryptophan ("Trp" or "W"), tyrosine ("Tyr" or "Y"), and valine ("Val" or "V").

[0114] Some tumor / tissue-penetrating peptides can be found, for example, in Ruoslahti 2017. For example, a peptide can contain a C-terminal "C-end Rule" (CendR) sequence motif (R / K)XX(R / K), which is associated with the ability to enhance tumor vascular and tumor tissue permeability via binding to receptors such as neuropilin-1 (NRP-1). Each X in this sequence is independently either an amino acid or a non-amino acid.

[0115] In some cases, the targeting moiety is 1 X 1 X 2 Z 2 Contains Z 1 is R or K, and Z 2 is R or K, and X 1 and X 2 are each independently an amino acid residue or a non-amino acid residue. In some cases, one or both ends of a peptide may have, for example, the structure J 1 Z 1 X 1 X 2 Z 2 , Z 1 X 1 X 2 Z 2 J 2 , or J 1 Z 1 X 1 X 2 Z 2 J 2 containing other amino acids, such as in 1 and J. 2 is independently an amino acid sequence (e.g., comprising 1, 2, 3, 4, 5, 6, or more amino acid residues) or an aliphatic carbon. The aliphatic carbon chain comprises carbon and hydrogen atoms in any suitable sequence, e.g., linear or branched, and is saturated or unsaturated. For example, in one set of embodiments, the aliphatic carbon chain has the formula, e.g., -(CH) n -, and n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or another positive integer. 1Z 1 X 1 X 2 Z 2 , CZ 1 X 1 X 2 Z 2 J 2 , or C.J. 1 Z 1 X 1 X 2 Z 2 J 2 As in, it terminates in a cysteine ​​residue.

[0116] Non-limiting examples of CendR peptides include, but are not limited to, Lyp-1, cLyp1, tLyp-1, iNGR, iRGD, RPARPAR, TT1, linear TT1, F3, or CRGRRST. Optionally, other amino acids are present in the peptide. Lyp-1 has the sequence CGNKRTRGC (SEQ ID NO: 1). In some embodiments, two Cys residues are linked to each other via a disulfide bridge, thereby forming a cyclic structure, and is therefore also known as cLyp-1. In some cases, only a portion of the Lyp-1 sequence is present, such as in the case of truncated Lyp-1 or tLyp-1 (CGNKRTR) (SEQ ID NO: 2). iNGR has the sequence CRNGRGPDC (SEQ ID NO: 4), with two cysteines linked together. iRGD has the sequence (CRGDKGPDC) (SEQ ID NO: 5), with two cysteines linked together, and iRGD2 has the sequence CRGDRGPDC (SEQ ID NO: 6). RPARPAR has the sequence RPARPAR (SEQ ID NO: 7). TT1 has the sequence CKRGARSTC (SEQ ID NO: 8), where two cysteines are linked together. Linear TT1 has the sequence AKRGARSTA (SEQ ID NO: 9). F3 has the sequence KDEPQRRSARLSAKPA PPKPEPKPKKAPAKK (SEQ ID NO: 32). CRGRRST has the sequence CRGRRST (SEQ ID NO: 3).

[0117] In some embodiments, the targeting moiety comprises an integrin-binding RGD sequence motif that binds to integrins expressed in tumor endothelial cells. Optionally, other amino acids may also be present in the peptide. Both linear and cyclic RGD peptides can be used as targeting moieties. RGD peptides can be cyclized via linkers such as, for example, S-S disulfide, thioether, and rigid aromatic rings. Some RGD peptides and derivatives can be found, for example, in Kapp, et al., 2017. Non-limiting examples of RGD peptides include, but are not limited to, RGD, RGDS (SEQ ID NO: 14), GRGD (SEQ ID NO: 15), GRGDS (SEQ ID NO: 16), GRGDSP (SEQ ID NO: 13), GRGDSPK (SEQ ID NO: 26), GRGDNP (SEQ ID NO: 27, GRGDTP (SEQ ID NO: 28), RGD-4C (CDCRGDCFC, SEQ ID NO: 11), RGD-10 (GARYCRGDCFDGR, SEQ ID NO: 12), cyclic RGD pentapeptide, cyclic hexapeptide, cilengitide (c(RGDf(NMe)V)), or the aforementioned CendR peptide iRGD. Cyclic RGD pentapeptides can be characterized by the formula c(RGDxX), where the x residue is an aromatic amino acid in the D-configuration (essential for αvβ3-integrin binding affinity), e.g., the sequence c(RGDfV), c(RGDfK), c(RGDyK), or c(RGDfC).

[0118] Peptides comprising any of the sequences disclosed above, in some embodiments, exhibit cell- or tissue-penetrating activity, particularly in tumor tissue. One set of embodiments generally relates to the association of a cell-penetrating peptide without targeting properties to provide cell- or tissue-penetrating activity to at least some of the nanoentities when, for example, non-systemically administered to a subject (e.g., intratumorally, nasally, topically, intraperitoneally, vaginally, rectally, orally, pulmonary, intraocularly, etc.) or when, for example, administered to a living cell or tissue in vitro or ex vivo. In some cases, a portion of the nanocapsule polymer (e.g., PSA, HA, PGA, etc.) is attached to the cell-penetrating peptide, for example, by covalent or non-covalent association.

[0119] Some cell-penetrating peptides can be found, for example, in Zhang et al., 2016, and Regberg et al., 2012.

[0120] Cell-penetrating peptides useful in certain embodiments of the present invention include, but are not limited to, TAT, mTAT (C-5H-TAT-5H-C), G3R6TAT, TAT(49-57), TAT(48-60), MPS, VP22, Antp, gH625, arginine-rich CPPs (e.g., octaarginine, polyarginine, stearyl-polyarginine, HIV-1 Rev34-50, FHVcoat35-49) penetratin, penetratin-Arg, penetratin-Lys, SR9, HR9, PR9, H(7)K(R(2)), Pep-1, Pep-3, transportan, transportan 10, pepFect, pVEC, JB577, TD-1, MPG8, CADY, YTA2, YTA4, SynB1, SynB3, PTD-4, GALA, SPACE, etc.

[0121] Cell-penetrating peptides can also be conjugated to targeting moieties. Some non-limiting examples are PEGA (CPGPEGAGC) (SEQ ID NO: 18), CREKA (SEQ ID NO: 19), RVG (YTIWMPENPRPGTPCDIFTNSRGKRASNG) (SEQ ID NO: 20), DV3 (LGASWHRPDKG) (SEQ ID NO: 21), DEVDG (SEQ ID NO: 22), ACPP-MMP-2 / 9 (PLGLAG) (SEQ ID NO: 23), ACPP-MMP-2 (IAGEDGDEFG) (SEQ ID NO: 24), R8-GRGD (SEQ ID NO: 25), penetratin-RGD, etc.

[0122] Targeting moieties useful in certain embodiments of the present invention are selected from, but are not limited to, CendR peptides (e.g., Lyp1, cLyp1, tLyp1, iRGD, iNGR, TT1, linear TT1, RPARPAR, F3, etc.), RGD peptides, NGR peptides; proteins (e.g., transferrin, ankyrin repeat proteins, insulin); small molecules (e.g., folate, triphenylphosphonium, ACUPA, PSMA); carbohydrate moieties (e.g., mannose, glucose, galactose and derivatives thereof); antibodies (including nanobodies, antibody fragments, monoclonal antibodies, affibodies or other antibodies), and aptamers.

[0123] In one embodiment, the composition comprises a targeting moiety. In another embodiment, the targeting moiety comprises a cell-penetrating peptide and / or a tumor / tissue-penetrating peptide. In a further embodiment, the targeting moiety is selected from the group consisting of Lyp-1, tLyp-1, or cLyp-1. In another embodiment, the polymer is PSA or HA, and the targeting moiety is tLyp-1.

[0124] Conjugation of the polymer to the targeting moiety In certain embodiments, some of the polymers (e.g., PSA, HA, PGA) are bound to the targeting moiety, for example, covalently. The polymer is bound to the targeting moiety directly or indirectly, for example, via a linker such as an aminoalkyl(C1-C4) succinimide linker (including C1, C2, C3, and C4) or an aminoalkyl(C1-C4) amido-isopropyl linker (including C1, C2, C3, and C4). In some cases, other aminoalkyl succinimide or aminoalkyl amido-isopropyl linkers are used. In some embodiments, the targeting moiety includes, for example, a C-terminus for conjugation. In some cases, the aminoalkyl(C1-C4) succinimide linker is an aminoethyl succinimide linker, aminopropyl succinimide, aminobutyl succinimide, etc. Aminoalkyl(C1-C4) succinimide linkers can be prepared, for example, by attaching a maleimide moiety to a carboxylic acid moiety on a monomer unit (e.g., a sialic acid unit) using an EDC / NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / N-hydroxysuccinimide) coupling reaction. In some cases, an N-aminoalkyl(C1-C4) maleimide moiety, such as an N-aminoethylmaleimide moiety, is reacted with a carboxylic acid moiety on a monomer unit to form an amide bond, thereby attaching the maleimide moiety to a polymer (e.g., PSA). Aminoalkyl(C1-C4) amido-isopropyl linkers can be prepared, for example, using aminoethylmethacrylamide or N-(3-aminopropyl)methacrylamide in the presence of BOP / TBA (benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate / tetra-n-butylammonium hydroxide).The maleimide or methacryloyl moiety can then react with a cysteine, thiol group, or other sulfur-containing moiety in the peptide, e.g., by Michael-type addition, to attach the peptide to a polymer (e.g., PSA, HA, PGA) via an aminoalkyl(C1-C4) succinimide, such as an aminoethylsuccinimide linker, or via an aminoalkyl(C1-C4) amido-isopropyl linker.

[0125] In some embodiments, the polymer (e.g., PSA, HA, PGA) is directly attached to the targeting moiety via an amide group. The amide group can be created, for example, by reacting a carboxylic acid moiety on a monomer unit (e.g., a sialic acid unit) with a lysine, arginine, or other primary amine-containing moiety in the peptide, specifically, the primary amine group is on the lysine or arginine amino acid on the targeting moiety. In some embodiments, the amide group is attached to an intermediate, such as a carbodiimide, N-hydroxysuccinimide, or DMTMM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-2-methyl ... An activating agent is present in the reaction to form morpholinium chloride.

[0126] Thus, one set of embodiments generally relates to methods of reacting carboxylate moieties on a polymer (e.g., PSA, HA, PGA) with aminoalkyl(C1-C4)maleimides and / or aminoalkyl(C1-C4)methacrylamides, and reacting the resulting aminoalkyl(C1-C4)maleimides and / or aminoalkyl(C1-C4)methacrylamides with cysteine ​​groups on a peptide to produce polymer-aminoalkyl(C1-C4)succinimide-peptide and / or polymer-aminoalkyl(C1-C4)amide isopropyl-peptide compositions.

[0127] Another set of embodiments relates to methods of reacting a carboxylate moiety on a polymer (e.g., PSA, HA, PGA) with N-hydroxysuccinimide or a carbodiimide and reacting the intermediate formed with a lysine or arginine group on a peptide to produce a polymer-amide-peptide.

[0128] In one embodiment, the targeting moiety is electrostatically bound to the polymer. In another embodiment, the targeting moiety is bound to the polymer via a linker. In a specific embodiment, the polymer is PSA or HA, and the targeting moiety is tLyp-1.

[0129] Polymers attached to hydrophobic moieties For example, the outer shell comprising PSA or HA can be covalently, electrostatically, or by other means bound to the hydrophobic moiety. The hydrophobic moiety can comprise an alkyl group, e.g., a straight-chain alkyl group. In some cases, the hydrophobic moiety comprises at least two carbon atoms. In other cases, the hydrophobic moiety comprises at least three carbon atoms. In some embodiments, the hydrophobic moiety is a C2-C 24 Straight chain alkyl groups (e.g., C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , and / or C 24 In certain embodiments, the hydrophobic moiety comprises a linear C 12 In some cases, the compositions of the present invention further comprise an aliphatic carbon chain covalently bonded to the polymer (e.g., PSA). In other cases, the aliphatic carbon chain is a C-C 24 Aliphatic carbon chains (e.g. C2, C3, C4, C5, C6, C7, C8, C9, C 10 , C11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , C 20 , C 21 , C 22 , C 23 , and / or C 24 ) is included.

[0130] Other examples of hydrophobic moieties include C3, C4, C5, C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19、 C 20 , C 21 , C 22 , C 23 , C 24 or other alkyl groups (e.g., linear or branched alkyl groups, e.g., isoalkyl groups). In some cases, the hydrophobic moiety contains at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, or at least 24 carbon atoms. The hydrophobic moiety is saturated or unsaturated, for example, containing one or more carbon-carbon double or triple bonds. Some techniques for attaching the hydrophobic moiety are described in WO2019 / 086627A1. In some cases, the hydrophobic moiety (e.g., C 12Hydrophobic moieties are attached using activation with quaternary ammonium salts (e.g., tetrabutylammonium hydroxide) and tetrafluoroborate (e.g., 2-bromo-1-ethylpyridinium tetrafluoroborate) before reaction with an alkylamine such as dodecylamine of the formula (I).

[0131] In one embodiment, at least some of the polymers are attached to a hydrophobic moiety. In another embodiment, the hydrophobic moiety is selected from the group consisting of alkyl groups, cycloalkanes, bile salts and derivatives, terpenoids, terpenes, terpene-derived moieties, and fat-soluble vitamins. In a further embodiment, the hydrophobic moiety is a C2-C 24 In yet a further embodiment, the hydrophobic moiety comprises a C 16 Contains straight chain alkyl groups.

[0132] In yet a further embodiment, the polymer is HA and is attached to a hydrophobic moiety. In particular, the hydrophobic moiety is C 16 The nanoentity may optionally comprise a tLyp-1 targeting moiety.

[0133] surfactants The nanoentities may comprise one or more surfactants. Examples of surfactants include, but are not limited to, polyoxyethylene sorbitan monooleate (Polysorbate 80; Tween 80®; HLB 15), polyoxyethylene sorbitan monostearate (Tween® 60, HLB 14.9 and Tween 61®; HLB 9.6), polyoxyethylene sorbitan monooleate (Tween 81®; HLB 10), polyoxyethylene sorbitan tristearate (Tween 65®; HLB 10.5), polyoxyethylene sorbitan trioleate (Tween 85®; HLB 11), polyoxyethylene sorbitan monolaurate (Tween® 20, HLB 16.7 and Tween 21®; HLB 13.3), polyoxyethylene sorbitan monopalmitate (Tween® 40, HLB 14.9 ... monolaurate (Tween® 20, HLB 16.7), polyoxyethylene sorbitan monolaurate (Tween® 20, HLB 16.7), polyoxyethylene sorbitan monolaurate (Tween® 20, HLB 16.7), polyoxyethylene sorbitan 15.6; PEGylated fatty acid esters and mixtures with PEG, polyethylene glycol monostearate (HLB 11.6), polyethylene glycol stearate, polyethylene glycol stearate 40 (HLB 17), polyethylene glycol stearate 100 (HLB 18.8), polyethylene glycol dilaurate 400 (HLB 9.7), polyethylene glycol dilaurate 200 (HLB 5.9), polyethylene glycol monopalmitate (HLB 11.6), macrogol 15 hydroxystearate (Kolliphor HS15®, BASF), polyethylene glycol-15-hydroxystearate (HLB 14-16), D-alpha-tocopheryl polyethylene glycol succinate (TPGS; HLB 13.2), triethanolammonium oleate (HLB 12), sodium oleate (HLB 18), sodium cholate (HLB 18), sodium deoxycholate (HLB 16), sodium lauryl sulfate (HLB 40), sodium glycocholate (HLB 16-18), triethanolamine oleate (HLB 12), tragacanth gum (HLB 11.9) and sodium dodecyl sulfate (HLB 40); poloxamer 124 (HLB 16), poloxamer 188 (HLB 29), poloxamer 237 (HLB 29), poloxamer 238 (HLB 28), poloxamer 278 (HLB 28), poloxamer 338 (HLB 27), and poloxamer 407 (HLB 22), sorbitan monooleate (Span® 80, HLB 4.3), sorbitan monolaurate (Span® 20, HLB 8.6), sorbitan monostearate (Span® 60, HLB 4.7), sorbitan trioleate (Span® 85, HLB 1.8), sorbitan sesquioleate (Span® 83, HLB 3.7), sorbitan monopalmitate (Span® 40, HLB 6.7), sorbitan isostearate (Span® 120, HLB 4.7), lauroyl macrogol glycerides (e.g., Gelucire® 44 / 14, HLB 14 and Labrafil® M2130CS, HLB 4), stearoyl macrogol glycerides (e.g., Gelucire® 50 / 13, HLB 13. ), linoleoyl macrogolglycerides (e.g., Labrafil® M2125CS, HLB 4), oleoyl macrogolglycerides (Labrafil® M1944CS, HLB 4), caprylocaproyl macrogolglycerides (Labrasol®, HLB 14), lecithin (e.g., egg lecithin, soy lecithin, non-GMO lecithin, rapeseed lecithin, sunflower lecithin, lysolecithin, etc.), phospholipids (e.g., egg phospholipids, soy phospholipids, synthetic phospholipids, hydrogenated phospholipids, PEGylated phospholipids, sphingolipids, phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, etc.), Phosal®, Phospholipon®, or any combination of any of these and / or other surfactants. In some cases, the surfactant is cationic, such as benzethonium chloride, benzalkonium chloride, CTAB (hexadecyltrimethylammonium bromide), cetrimide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide, etc. In some cases, the cationic surfactant includes, for example, an ammonium salt as a head group. For example, the head group includes a primary, secondary, tertiary, or quaternary ammonium salt. Furthermore, it should be understood that such surfactants are not required in all embodiments.

[0134] In some embodiments, the surfactant is located primarily between the inner core and the outer shell.

[0135] In certain embodiments, the surfactant is polyoxyethylene sorbitan monooleate (e.g., Tween 80®). In certain embodiments, the nanoentities comprise polyoxyethylene sorbitan monooleate (e.g., Tween 80®) and macrogol 15 hydroxystearate (Kolliphor HS15®) as surfactants.

[0136] Methods for producing nanocapsule compositions Various aspects of the present invention also generally relate to systems and methods for producing compositions, eg, nanocapsules, or other nanoentities, as described herein.

[0137] Polymeric nanocapsules, such as PSA-based nanocapsules, HA-based nanocapsules, and PGA-based nanocapsules, can be produced by a variety of techniques. One of these is the solvent displacement method, which involves mixing a polar solvent with an aqueous phase. Another technique is the self-emulsification method, which does not require the use of organic solvents. In some cases, this involves preparing an aqueous solution containing a polymer (e.g., PSA, HA, PGA, PGA-PEG, PASP, PASP-PEG, PLMA, PLA-PEG, etc.) and, optionally, one or more water-soluble surfactants, preparing an oily solution (e.g., containing oil and one or more surfactants, an organic solvent, etc.), and mixing the solutions together. It is believed that nanocapsules are formed by the interaction of the polymer (e.g., PSA, HA, PGA, PASP) with the positively charged surfactant at the interphase of the oil-in-water emulsion. However, when using hydrophobically modified polymers, the presence of an alkyl chain / hydrophobic moiety may not be necessary, as the presence of the alkyl chain / hydrophobic moiety promotes the binding of the polymer by intercalating into the oily nanodroplets.

[0138] The pharmaceutical agent can be dissolved in the aqueous or oil phase before preparing the nanoentities or incubated with the preformed nanoentities. If the pharmaceutical agent is an antibody, such as a monoclonal antibody, the antibody is specifically encapsulated by dissolving it in the aqueous or oil phase (at high concentrations) before preparing the nanocapsules.

[0139] As an example, in one embodiment, this involves preparing an aqueous phase containing a polymer (e.g., PSA or HA), preparing a hydrophobic phase containing a hydrophobic compound (e.g., containing an oil and one or more surfactants, and optionally an organic solvent, etc.), adding an antibody or fragment thereof that binds to an epitope of an activated mutant KRAS protein to the aqueous phase, or optionally, to the hydrophobic phase if the mAb or fragment is highly concentrated, and mixing the aqueous phase and the hydrophobic phase under stirring. This method can be referred to herein as a "one-step method." In certain embodiments, the hydrophobic compound is oil, and the hydrophobic phase also contains a surfactant. In another embodiment, the aqueous phase further contains a surfactant. In another embodiment, the polymer is bound to the hydrophobic moiety.

[0140] In another embodiment, the method involves preparing an oily solution (e.g., containing an oil and one or more surfactants, and optionally an organic solvent, etc.) and adding it to an aqueous phase (or adding the aqueous phase on top of the oily phase). The aqueous phase contains a polymer (e.g., PSA or HA) and optionally one or more water-soluble surfactants. The solutions are mixed under agitation to form nanocapsules. Once the nanocapsules are formed, an additional aqueous phase containing an antibody or fragment thereof is added under agitation, and incubation produces antibody-loaded nanocapsules. This method may be referred to herein as a "two-step method."

[0141] Once the organic solvent is added, it may be allowed to evaporate completely or partially.

[0142] The polymers can also be functionalized with targeting moieties such as cell-penetrating and / or tumor / tissue-penetrating peptides, or with hydrophobic moieties such as straight-chain alkyl groups.

[0143] As an example, PSA can be conjugated to tLyp-1 through a covalent bond between the thiol group of the peptide tLyp-1 and the carboxylate group of PSA. This synthetic approach uses a heterobifunctional linker, aminoethylmaleimide, which first incorporates it into the carboxylate group of PSA via the amine group of the linker (using carbodiimide chemistry), and then, after a two-step process, allows peptide conjugation by addition of the thiol group (cysteine ​​residue) of the peptide to the maleimide group of the linker (Michael-type addition). This strategy allowed for the preservation of the biologically active groups of the tLyp-1 peptide. Furthermore, the degree of substitution could be easily controlled.

[0144] Typically, purified polypeptides, such as antibodies, are only marginally stable in aqueous conditions and undergo chemical and physical degradation, resulting in loss of biological activity during processing and storage. Furthermore, peptide compositions in the aqueous phase undergo hydrolysis, including deamidation and peptide bond cleavage. These effects pose serious problems for therapeutically active antibodies intended to be administered to humans within a defined dosage range based on biological activity. Therefore, we tested whether the generated nanoentities could maintain functionality after lyophilization.

[0145] Thus, one embodiment of the present invention relates to a method for producing nanoentities, which includes an additional freeze-drying step that can protect them during storage. In some cases, it is not necessary to use a cryoprotectant during freeze-drying. In some embodiments, dilution of the colloidal system prior to freeze-drying is not necessary, as the nanoentities do not form aggregates during reconstitution of the freeze-dried product. In some cases, it is possible to add one or more sugars, e.g., sugars that exert a cryoprotective effect. Examples of cryoprotectants include, but are not limited to, trehalose, glucose, sucrose, mannitol, maltose, polyvinylpyrrolidone (PVP), glycerol, polyethylene glycol (PEG), propylene glycol, 2-methyl-2,4-pentanediol (MPD), raffinose, dextran, fructose, stachyose, etc. In some cases, a cryoprotectant or The additives may have other effects, for example, as buffers to control pH. In lyophilized form, the nanoentities may be stored for long periods and can be reconstituted, for example, by adding water.

[0146] Combinations of antibodies or fragments thereof with other drugs / drugs In certain embodiments, the antibody or fragment thereof is combined with other pharmaceutical agents / drugs, which may be located within and / or on the surface of the nanoentity, depending on the embodiment.

[0147] Pharmaceutical agents may have pharmacological activity and / or may enhance the effect of an antibody or fragment thereof and / or may have other direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease.

[0148] Given the increased metabolic requirements of tumor cells, it is not surprising that certain tumor types have developed mechanisms to remove nutrients from both extracellular and intracellular sources. In particular, RAS-driven cancers have several distinct metabolic adaptations that allow them to recycle a variety of metabolites. This serves two important purposes: 1) providing metabolic flexibility and efficiency, and 2) ensuring sufficient availability of biosynthetic precursors. Importantly, these removal pathways may be crucial for the metabolism of these cancers and offer therapeutic opportunities. Therefore, in certain embodiments, anti-KRAS antibodies are combined with macroautophagy inhibitors, such as chloroquine, glucose transporter (GLUT) inhibitors, lactate dehydrogenase inhibitors, and glycolysis inhibitors.

[0149] KRAS is a central node in a complex network, providing many opportunities for feedback loops that allow cancer growth and survival. Thus, in certain embodiments, anti-KRAS antibodies are combined with other targeted therapies, such as those directed against IGFR, mTOR, RAF, MEK, PIK3, EFGR / ERBB2, SHP2, or immune checkpoint inhibitors, to potentially exert synergistic effects on tumor regression, contribute to the generation of anti-tumor immunity, or overcome resistance compared to each drug alone.

[0150] In certain embodiments, the additional pharmaceutical agent is an anti-cancer agent such as, for example, paclitaxel or docetaxel.

[0151] The pharmaceutical agent is present at up to about 50% by weight, based on the total dry weight of the components of the system. However, the appropriate percentage will depend on various factors, such as the pharmaceutical agent being incorporated, the indication for which it is being used, and the efficiency of administration. For example, in some cases, the pharmaceutical agent may be present at up to about 10% by weight, or up to about 5% by weight, or up to about 2% by weight, or up to about 1% by weight, or up to about 0.5% by weight, or up to about 0.1% by weight. In certain embodiments, more than one pharmaceutical agent is present, which can be dissolved in the same solution or separately, depending on the nature of the active pharmaceutical ingredients being incorporated.

[0152] Pharmaceutical Compositions and Dosage Forms Another aspect provides a method of administering a composition comprising nanoentities, particularly the nanocapsules discussed herein, to an organism. When administered, the compositions of the invention are applied in a therapeutically effective amount as a pharmaceutically acceptable formulation. As used herein, the term "pharmaceutically acceptable" means that the formulation contains agents or excipients that are compatible with the form required for administration to an organism without causing adverse effects. Any composition of the invention is administered to an organism in a therapeutically effective dose. As used herein, "therapeutically effective" or "effective" refers to the ability to delay the onset of, inhibit the progression of, completely halt the onset or progression of, diagnose, or otherwise be medically desirable for the particular condition being treated. It means the amount necessary to achieve the result. When administered to a living body, the effective amount depends on the specific condition to be treated and the desired outcome. The therapeutically effective dose can be determined by those skilled in the art. Some embodiments of the present invention are generally directed to the use of the compositions disclosed herein for the preparation of a medicament.

[0153] Any medically acceptable method can be used to administer the composition to a living organism. Administration can be local (i.e., to a specific region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition being treated. For example, the composition can be administered orally or by other techniques, such as vaginally, rectally, bucally, pulmonary, topically, nasally, transdermally, intratumorally, intraperitoneally, by parenteral injection or implantation, surgically, or by any other administration method that achieves target delivery with the compositions of the present invention. Compositions suitable for oral administration are presented as discrete units, such as hard or soft capsules, pills, sachets, tablets, troches, or lozenges, each containing a predetermined amount of active compound. Other oral compositions suitable for use in the present invention include solutions or suspensions in aqueous or non-aqueous liquids, such as syrups, elixirs, or emulsions. In another series of embodiments, the composition is used to enhance the nutritional value of food or beverages. Rectal administration can be used in some embodiments, for example, in the form of an enema, suppository, or foam.

[0154] In one set of embodiments, administration of the composition is parenteral, intratumoral, or oral. In some embodiments, the composition is administered by injection or infusion. In one embodiment, the injection is selected from intratumoral, intraperitoneal, subcutaneous, intramuscular, or intravenous injection. In another embodiment, the composition is administered by intrathecal injection or infusion.

[0155] In certain embodiments of the present invention, the administration of the composition of the present invention is designed to provide continuous exposure to the composition for a certain period, for example, for several hours, several days, several weeks, several months or several years.This can be achieved, for example, by repeatedly administering the composition of the present invention by one of the above-mentioned methods.The administration of the composition can be alone or in combination with other therapeutic agents and / or compositions.

[0156] In certain embodiments, administration of the composition is intravenous, intratumoral, intraperitoneal, subcutaneous, or by inhalation. In particular, administration is intravenous.

[0157] In certain embodiments of the present invention, the compositions can be combined with a suitable pharmaceutically acceptable carrier, for example, incorporated into a polymeric release system, or suspended in a liquid, for example, in a dissolved or colloidal form. Generally, pharmaceutically acceptable carriers suitable for use in the present invention are well known to those skilled in the art. As used herein, "pharmaceutically acceptable carrier" refers to a non-toxic material that does not significantly interfere with the effectiveness of the biological activity of the administered active compound(s), but is used as a formulation component, for example, to stabilize or protect the active compound(s) in the composition prior to use. The term "carrier," as discussed herein, refers to a natural or synthetic organic or inorganic component with which one or more active compounds of the present invention are combined to facilitate application of the composition. Carriers are mixed or otherwise blended with one or more compositions of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired therapeutic effect. Carriers can be either soluble or insoluble, depending on the intended use. Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The nature of the carrier can be either soluble or insoluble. Those skilled in the art will know of other suitable carriers, or will be able to ascertain them using only routine experimentation.

[0158] In some embodiments, the compositions of the present invention may include a pharmaceutically acceptable carrier along with formulation components such as salts, carriers, buffers, emulsifiers, diluents, excipients, chelating agents, fillers, desiccants, antioxidants, antimicrobial agents, preservatives, binders, extenders, silica, solubilizers, or stabilizers used with the active compound. For example, if the formulation is liquid, the carrier may be a solvent, partial solvent, or non-solvent, and may be aqueous or organic. Examples of suitable formulation ingredients include diluents such as calcium carbonate, sodium carbonate, lactose, kaolin, calcium phosphate, or sodium phosphate; granulating and disintegrating agents such as corn starch or alginic acid; binders such as starch, gelatin, or acacia; lubricants such as magnesium stearate, stearic acid, or talc; retardants such as glycerol monostearate or glycerol distearate; suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, and polyvinylpyrrolidone; dispersing or wetting agents such as lecithin or other naturally occurring phosphatides; thickeners such as cetyl alcohol or beeswax; buffers such as acetic acid and its salts, citric acid and its salts, boric acid and its salts, or phosphoric acid and its salts; or preservatives such as benzalkonium chloride, chlorobutanol, parabens, or thimerosal. Suitable carrier concentrations can be determined by those skilled in the art through simple routine experimentation. The compositions discussed herein can be formulated into solid, semi-solid, liquid, or gaseous form preparations, such as tablets, capsules, elixirs, powders, granules, ointments, solutions, deposits, pills, or injectables, etc. Those skilled in the art will know of other suitable formulation ingredients, or will be able to ascertain such using no more than routine experimentation.

[0159] Preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions, which, in certain embodiments, may be isotonic with the body's blood. Examples of non-aqueous solvents are fixed oils, including polypropylene glycol, polyethylene glycol, vegetable oils such as olive oil, sesame oil, coconut oil, peanut oil, mineral oil, injectable organic esters such as ethyl oleate, or synthetic monoglycerides or diglycerides. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, 1,3-butanediol, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present. Those skilled in the art can readily determine, without undue experimentation, the various parameters for preparing and formulating the compositions as discussed herein.

[0160] The present invention also provides any of the above compositions in a kit, optionally including instructions for using the composition, for example, for the treatment of cancer. Instructions for administering the composition by any suitable technique, for example, orally or intravenously, as previously described, may also be provided. A kit typically defines a package containing any one or combination of the compositions of the present invention and other components as described above. The kit may also include other containers containing one or more solvents, surfactants, preservatives, and / or diluents (e.g., saline (0.9% NaCl) or 5% dextrose), as well as containers for mixing, diluting, or administering the composition to a living organism. The compositions of the kit may be provided as a solution or a dry powder. If the provided composition is a dry powder, the composition can be reconstituted by the addition of a suitable solvent. In embodiments in which a liquid form of the composition is used, the liquid form may be concentrated or ready-to-use. The solvent will depend on the composition and the mode of use or administration.

[0161] medical use Embodiments of the present invention relate to compositions of nanoentities for use in medicine (i.e., as a drug). One embodiment of the present invention relates to a composition comprising a nanoentity for use in the prevention and / or treatment of a disease associated with a mutation in the KRAS gene. Alternatively, the present invention relates to the use of a composition of the present invention for the preparation of a medicament for the prevention and / or treatment of a disease associated with a mutation in the KRAS gene. Alternatively, the present invention relates to a method for preventing and / or treating a disease associated with a mutation in the KRAS gene, comprising administering a composition of the present invention to a subject in need thereof.

[0162] As used herein, the terms "treat," "treatment," or "treatment of" refer to reducing the likelihood of a particular disease or disorder, reducing the occurrence of a particular disease or disorder, and / or reducing the severity of a particular disease or disorder, specifically to the point where the subject no longer suffers from discomfort and / or altered functioning therefrom. For example, "treating" refers to the ability of a therapeutic agent, when administered to a subject, to prevent the occurrence of a particular disease or disorder and / or cure or alleviate the symptoms, signs, or causes of a particular disease. "Treating" also refers to alleviating or reducing at least one clinical symptom, and / or inhibiting or slowing the progression of a condition, and / or preventing or delaying the onset of a disease or condition. Thus, the terms "treat," "treating," or "treatment of" (or grammatical equivalents) refer to both prophylactic and therapeutic treatment regimens. In particular, as used herein, "treatment" refers to the administration of a composition according to the present invention to a subject suffering from a disease associated with a mutation in the KRAS gene, including administration at an early stage of the disease, with the purpose of preventing or slowing (alleviating) undesirable physiological changes or disorders. The present disclosure generally provides methods and compositions that provide a therapeutic benefit or desired clinical outcome. Therapeutic benefit does not necessarily mean a cure for a particular disease or disorder, but rather most typically encompasses outcomes including alleviation of the disease or disorder or prolongation of survival, elimination of the disease or disorder, reduction or alleviation of symptoms associated with the disease or disorder, prevention or mitigation of secondary diseases, disorders, or conditions resulting from the development of the primary disease or disorder, reduction in the extent of the disease, a stable (i.e., non-worsening) state of the disease, delay or slowing of disease progression, improvement or alleviation of the condition, and remission (whether partial or complete), whether detectable or undetectable, and / or prevention of the disease or disorder. Treatment also means extending survival compared to the expected survival if not receiving treatment.

[0163] The terms "prevention," "preventing," or "prevent" as used herein refer to the administration of the combination according to the present invention or the pharmaceutical composition according to the present invention to a subject who has not been diagnosed as possibly having a disease associated with a mutation in the KRAS gene at the time of administration, but who is usually expected to develop the disease or is at high risk of the disease. Prevention aims to avoid the appearance of the disease. Prevention can be complete (e.g., the complete absence of the disease). Prevention can be partial, for example, such that the occurrence of the disease in the subject is less than that which would have occurred without the administration of the composition of the present invention. Prevention also refers to a reduction in susceptibility to a clinical condition.

[0164] The terms "subject" or "individual" or "animal" or "patient" include any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or treatment is desired. Mammalian subjects include humans, domestic animals, livestock, and zoo or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, sheep, cows, etc. In specific embodiments of the invention, the subject is a mammal. In more specific embodiments of the invention, the subject is a human, particularly a human of any race and sex. In some embodiments, the subject is a naive subject. A naive subject is a subject who has not been administered a therapeutic agent. In another embodiment, the subject is a subject who has not been administered a therapeutic agent and / or The patient has been administered one or more doses of a therapeutic agent.

[0165] Oncogenic RAS mutations contribute to the induction of various cancers, including pancreatic ductal adenocarcinoma (PDAC), colorectal adenocarcinoma (CRC), lung adenocarcinoma, and gastric adenocarcinoma. Mutant RAS genes, representing the most frequently mutated oncogene family, are present in approximately 25% of human tumors. Mutations within KRAS account for 85% of all RAS family oncogenic mutations.

[0166] In certain embodiments, the disease associated with a mutation in the KRAS gene is cancer. In more specific embodiments, the cancer is a hematological malignancy such as leukemia. The term "hematological malignancy" refers to a type of cancer that affects the blood, bone marrow, and lymph nodes, including lymphoma, myeloma, and leukemia. Examples of hematological malignancies in which the KRAS gene is mutated include acute myeloid leukemia (AML), core-binding factor acute myeloid leukemia, juvenile myelomonocytic leukemia (JMML), multiple myeloma, myelodysplastic syndrome, and autoimmune lymphoproliferative syndrome.

[0167] In other specific embodiments, the cancer is a solid tumor. A "solid tumor" or solid cancer is a neoplasm (new growth of cells) or lesion (damage to an anatomical structure or impaired physiological function) formed by the abnormal proliferation of body tissue cells other than blood, bone marrow, or lymphocytes. Solid tumors consist of an abnormal mass of cells that may originate from different tissue types, such as the liver, colon, breast, or lung, and initially grow in the organ of their cellular origin. However, such cancers can spread to other organs through metastatic tumor growth in advanced stages of the disease. Examples of solid tumors are carcinomas, sarcomas, germinomas, and blastomas.

[0168] In certain embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is gastric cancer (particularly gastric adenocarcinoma), bile duct cancer, lung cancer (particularly lung adenocarcinoma), colorectal cancer (particularly colorectal adenocarcinoma (CRC)), pancreatic cancer (particularly ductal adenocarcinoma (PDAC)), cutaneous melanoma, uterine endometrial cancer, uterine carcinosarcoma, thyroid cancer, gastric adenocarcinoma, bladder urothelial carcinoma, cervical adenocarcinoma, head and neck squamous cell carcinoma, or esophageal adenocarcinoma.

[0169] G12V and G12D KRAS substitutions are the most commonly observed mutations in pancreatic adenocarcinoma (30% and 51%, respectively) and colorectal adenocarcinoma (27% and 45%, respectively), and are associated with poor prognosis. Thus, in certain embodiments, the cancer is pancreatic adenocarcinoma or colorectal adenocarcinoma.

[0170] In certain embodiments, the disease is associated with a substitution at G12 of the KRAS gene. More specifically, the disease is non-small cell lung cancer, colorectal cancer, malignant solid tumors, acute myeloid leukemia, squamous cell lung cancer, colorectal adenocarcinoma, pancreatic ductal adenocarcinoma, rectal adenocarcinoma, small cell lung cancer, glioma, thyroid cancer, multiple myeloma, and myelodysplastic syndrome.

[0171] In certain embodiments, the substitution is G12V and the disease is colorectal adenocarcinoma, non-small cell lung cancer, pancreatic ductal adenocarcinoma, endometrial neoplasia, and ovarian neoplasia, which are the most prevalent.

[0172] In certain embodiments, the substitution is G12C and the disease is non-small cell lung cancer, colorectal adenocarcinoma, adenocarcinoma of unknown primary, endometrial neoplasia, and carcinoma of unknown primary, the foregoing being the most prevalent.

[0173] In certain embodiments, the substitution is G12D and the disease is colorectal adenocarcinoma, pancreatic ductal adenocarcinoma, non-small cell lung cancer, endometrial neoplasia, and ovarian neoplasia, which are the most prevalent.

[0174] In another embodiment, the disease associated with a KRAS mutation is Noonan syndrome (NS), cardiac These include cardio-facio-cutaneous syndrome (CFC), and epidermal nevi. Germline mutations in the KRAS gene also cause a disorder whose cardinal features overlap with those of cardio-facio-cutaneous syndrome, as well as two related disorders termed Noonan syndrome and Costello syndrome. These conditions have been described as phenotypes associated with KRAS mutations.

[0175] In one aspect, the present invention relates to nanoentities for drug delivery applications. For example, such nanoentities are delivered to a subject to reach a tumor afflicting the subject. The nanoentities are delivered to tumor cells, optionally facilitated by a targeting moiety that also has the ability to act as a cell- or tumor / tissue-penetrating peptide, such as Lyp-1 or tLyp-1, or other peptides discussed herein. Upon delivery, the nanoentities can reach target cells, e.g., cancer cells and metastatic cancer cells, and release the drug (i.e., anti-KRAS antibody) contained therein. Accordingly, one aspect of the present invention relates to a method for delivering an anti-KRAS antibody or a fragment thereof to a target cell, comprising administering to a subject a composition comprising a nanoentity according to the present invention, wherein the nanoentity crosses the cell membrane of the target cell. In particular, the target cell is a cancer cell. According to the examples provided herein, the nanoentity can penetrate the cell and deliver the anti-KRAS antibody or a fragment thereof into the cell. Accordingly, one aspect of the present invention relates to a composition according to the present invention for intracellular delivery, particularly in vivo intracellular delivery. Furthermore, nanoentities comprising antibodies or fragments thereof can perform their biological function within a cell, such as reducing cell proliferation. Thus, the present invention also provides a method for reducing cell proliferation in tumor cells, comprising administering to a subject a composition comprising a nanoentity according to the present invention.

[0176] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the specification and claims, the word "comprises" and variations thereof, such as "comprising," are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of the specification or may be learned by practice of the present invention. Furthermore, the present invention encompasses all possible combinations of the specific preferred embodiments described herein. The following examples and figures are provided herein for illustrative purposes and are not intended to limit the present invention. [Example]

[0177] Example 1: Ability of anti-KRAS mAbs to specifically interact with their target proteins This example demonstrates the ability of anti-KRAS monoclonal antibodies to specifically interact with their target protein. To investigate this ability, two different studies were performed. (i) Interaction of anti-KRAS monoclonal antibodies with synthetic antigen peptides used to generate their predecessor by hybridoma technology. These peptides correspond to the amino acid sequence (positions 5–16) of the KRAS protein. Peptides were synthesized according to the sequences shown in Table 2 (Karebay Biochem, USA; purity by CoA >95%) and, for practical reasons, were further conjugated to the carrier protein bovine serum albumin (BSA) (Biogenes, Germany). The internal codes assigned to the peptides once conjugated to BSA are also shown in Table 2 below. (ii) Interaction of anti-KRAS monoclonal antibodies with different recombinant human mutant KRAS proteins.

[0178] (i) Interaction between anti-KRAS monoclonal antibody and synthetic antigen peptide Preparation of peptide-BSA conjugates for ELISA testing: Terminal cysteine ​​residues was added to the peptide during its synthesis, and the peptide was conjugated to BSA using the sulfhydryl group by the following two-step method. 1. Maleylation of BSA: 1 mg of the cross-linker SMCC (50 mg / ml in N-methyl-2-pyrrolidone, NMP) was added to 1 ml of a BSA solution (10 mg / ml in 0.1 mM NaHCO3, pH 8.3). After incubation at room temperature (RT) for 1 h, the solution was desalted using a column (1.5 × 14 cm) packed with Sephadex G-50 pre-equilibrated with PBS. 2. Conjugation of maleylated BSA: 50 μl of peptide solution (10 mg / ml in double-distilled water) was added to 1 ml of maleylated BSA (1.0 mg / ml in PBS) and incubated at RT for 2 h at 4°C, followed by another 4 h at RT. Unreacted maleimide groups were blocked by adding 2-mercaptoethanol to a final concentration of 10 mM and incubating overnight at 4°C. Finally, the conjugate was dialyzed against 3 x 500 volumes of PBS at 4°C (MW cutoff 10,000). [Table 2]

[0179] ELISA plates were coated with 4 μg / ml (50 μl / well) of antigen peptide 820940-BSA(G12V), antigen peptide 820941-BSA(G12D), and antigen peptide 820942-BSA(G12C) and processed using alkaline phosphatase-mediated indirect ELISA with goat anti-mouse IgG Fc-specific antibody as the secondary antibody. Results were reported as OD after 15 minutes of substrate incubation. 405nm represents.

[0180] All anti-KRAS antibodies (clone DWP, clone D113, and clone D210) interacted with the corresponding antigen peptide. The results obtained for clones D113 and D210 titrated with the corresponding antigen peptide 820941-BSA(G12D) are presented in Table 3 below. [Table 3]

[0181] (ii) Interaction of anti-KRAS monoclonal antibodies with different recombinant human mutant KRAS proteins Next, ELISA plates were coated with 2 μg / ml (50 μl / well) of recombinant KRAS(G12V) mutant protein (SignalChem, Canada, Cat. No. R06-32CH) and processed using alkaline phosphatase-mediated indirect ELISA with goat anti-mouse IgG Fc-specific antibody as the secondary antibody. Results were reported as OD after 15 min of substrate incubation. 405nm represents.

[0182] ELISA plates were also coated with 5 μg / mL (50 μl / well) of recombinant KRAS (G12D) mutant protein (Sino Biological, Cat. No. 12259-H07E1) and processed using horseradish peroxidase-mediated indirect ELISA with goat anti-mouse IgG (H+L) HRP conjugate as the secondary antibody. Results were reported as OD values ​​after 25 minutes of substrate incubation. 405nm represents.

[0183] All anti-KRAS antibodies (clone DWP, clone D113, and clone D210) interacted with the corresponding mutated target proteins.

[0184] Furthermore, the specificity of anti-KRAS antibodies (subclones DWP, D113, and D210) was examined using different antigen peptides corresponding to the G12V, G12D, and G12C KRAS mutations (4 μg / mL), as well as recombinant G12V KRAS protein (5 μg / mL).

[0185] As shown in Table 4 below, each clone was able to interact only with its corresponding target peptide and / or protein. [Table 4]

[0186] conclusion The anti-KRAS antibodies (clones DWP, D113, and D210) could selectively interact with the corresponding antigen peptide (generated by hybridoma technology) and the corresponding recombinant human KRAS target protein. Thus, DWP interacted only with the 820940-BSA peptide (G12V) and recombinant KRAS G12V protein, whereas D113 and D210 interacted only with the 820941-BSA peptide (G12D) and recombinant KRAS G12D protein.

[0187] Example 2: Determination of KD values ​​of two anti-KRAS mabs (D210 and D113) for their soluble antigen (KRAS G12D protein) This example shows the determination of the KD values ​​of two anti-KRAS monoclonal antibodies (D210 and D113) for their soluble antigen (KRAS G12D protein) by surface plasmon resonance (SPR) technology (GE Biacore 8K).

[0188] KD is the equilibrium dissociation constant and is inversely proportional to affinity: the lower the KD value, the higher the affinity of the antibody. Antibody affinity refers to the strength with which an epitope binds to an individual paratope (antigen-binding site) on the antibody. High-affinity antibodies bind antigens rapidly and more easily maintain this binding under different conditions. Most antibodies have low micromolar (10-6 ) to nanomoles (10 -7 ~10 -9 ) range. D High affinity antibodies generally have a value in the low nanomolar range (10 -9 ) is thought to be within

[0189] Briefly, a defined concentration of KRAS G12D protein was splashed onto a sensor chip coated with anti-KRAS antibodies (Protein A sensor chip), and the response was captured over time to indicate the progression of the interaction and association / dissociation cycle. The response measures the change in refractive index and is related to the change in mass near the sensor surface. Thus, the response is proportional to the number of antigen molecules interacting with the antibody. After sequentially testing different concentrations of KRAS G12D protein (performing regeneration from one concentration to another to remove all remaining bound protein from the chip), kinetic parameters and affinity were calculated using BIA evaluation software. The KD (M) of the anti-KRAS antibodies D113 and D210 was 4.98 × 10, respectively. -9 and 3.75 × 10 -9 It was.

[0190] conclusion Both anti-KRAS G12D antibodies, D210 and D113, exhibited similar KD values ​​in the low nanomolar range, suggesting high affinity for the target (KRAS G12D protein).

[0191] Example 3: Formulation of different polymeric nanocapsules for efficient assembly and delivery of anti-KRAS mab This example demonstrates the formulation of different polymeric nanocapsules for efficient assembly and delivery of anti-KRAS monoclonal antibodies (mAbs). The shell-forming polymer can be prepared from a biodegradable polyacid, which can be further functionalized, either covalently or electrostatically, with targeting and / or tumor / tissue-penetrating ligands, such as tLyp-1. Below, we describe the preparation of functionalized polymers, either through covalent attachment of the targeting ligand tLyp-1 or through simple ionic interactions. Next, we detail the preparation and characterization of nanocapsules loaded with anti-KRAS mAbs and made from various polymers.

[0192] Preparation of covalently functionalized PSA polymer (PSA-tLyp-1) Polysialic acid (PSA, 28 kDa, Serum Institute of Industrial PSA (tLyp-1) was modified with N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) / N-(2-aminoethyl)maleimide trifluoroacetate (AEM) using a molar ratio of 11.6 / 2 / 0.40 / 0.0283. For this purpose, PSA was dissolved in 0.1 M MES buffer at pH 6 to a final concentration of 2 mg / mL. Corresponding amounts of EDC, NHS, and AEM were also dissolved in 0.1 M MES buffer and added to the PSA solution. The mixture was then kept under magnetic stirring at room temperature for 4 h. The maleimide-functionalized PSA (PSA-Mal) was purified by dialysis (regenerated cellulose, SnakeSkin 7 KDa MWCO, Thermo Scientific) first against 50 mM NaCl and then against water. For the second reaction, PSA-Mal was dissolved in 0.1 M MES buffer and 50 mM NaCl to a final PSA concentration of 1 mg / mL. The peptide was added to this solution, and the reaction mixture was kept under magnetic stirring at room temperature for 4 hours. The final PSA-tLyp-1 product was purified by dialysis as described above, lyophilized, and stored at 4°C.

[0193] Preparation of electrostatically functionalized PSA and C16-HA polymers (PSA ± tLyp-1; C16-HA ± tLyp-1) Polysialic acid (PSA, 28 kDa, Serum Institute of India) and C16-HA (216 kDa Mw and 5% alkyl substitution, Contipro) were electrostatically modified with the targeting ligand tLyp-1. First, PSA or C16-HA was dissolved in 0.1 M MES buffer at pH 6 to a final concentration of 1 mg / mL, and the corresponding amount of peptide tLyp-1 was added to this solution. The reaction mixture was kept under magnetic stirring at room temperature for 4 hours. The final product was purified by dialysis as described above, lyophilized, and stored at 4°C.

[0194] Preparation of polymeric nanocapsules Nanocapsules coated with PSA (28 kDa, Serum Institute of India), PSA-tLyp-1, HA (290 kDa, Lehvoss Iberica), C16-HA (216 kDa Mw, 5% alkyl substitution, Contipro), or C16-HA + tLyp-1 were prepared by the self-emulsification technique. First, 59 mg of polysorbate 80 (Tween 80®, Merck) and 58 mg of caprylic / capric triglyceride (Mygliol® 812N, IOI Oleochemical GmbH) were weighed into a 2 mL glass vial (oil phase). Next, for formulations containing non-hydrophobically modified polymers or non-amphiphilic polymers as a shell, a cationic surfactant was added to the oil phase (4 microliters of benzethonium chloride, 50 mg / mL, previously solubilized in ethanol). However, it is not essential because the presence of alkyl chains / hydrophobic components promotes the binding of the polymer by inserting it into the oily nanodroplets. All components of the oil phase were maintained under magnetic stirring (500 rpm). In parallel, aqueous phases were prepared by separately solubilizing various concentrations of each polymer (e.g., 3 mg / mL for PSA-based formulations and 0.25–0.5 mg / mL for HA-based formulations) and hydroxystearic acid macrogol 15 (Kolliphor HS15®, BASF) at a concentration of 20 mg / mL in 25 mM PBS (pH 7.3). Then, 0.75 mL of the polymer solution was mixed well with 125 microliters of Kolliphor solution, and this aqueous phase was added on top of the oil phase under magnetic stirring (1100 rpm).

[0195] mAb conjugation process used to conjugate the anti-KRAS mAbs anti-G12V (DWP) and anti-G12D (D113 and D210) The volume of anti-KRAS mAb required to obtain the desired final mAb concentration (eg, 1, 2, or 3 mg / mL) was added to the aqueous phase before mixing with the oil phase.

[0196] Physicochemical properties Nanocapsules were characterized in terms of mean particle size and polydispersity index (PI) by photon correlation spectroscopy (PCS). Samples were diluted with MilliQ water and analyzed at 25°C with an angular detection of 173°C. Zeta potential measurements were performed by laser Doppler anemometry (LDA), with samples diluted with ultrapure water. PCS and LDA analyses were performed in triplicate using NanoZS® (Malvern Instruments, Malvern, UK). Results corresponding to three replicates are shown in Tables 5 and 6, as described elsewhere.

[0197] To determine the assembly efficiency of anti-KRAS mAbs into nanocapsules, an aliquot of each different formulation was first diluted with PBS and then filtered (1 mL) through Amicon Stirred Cells® (polyethersulfone Biomax® 500 KDa ultrafiltration disks, Merck) under 1 bar of nitrogen pressure at 4°C. The assembly efficiency was calculated indirectly as follows: [(total mAb - free mAb) / total mAb] * 100. The results obtained with anti-KRAS G12V and anti-KRAS G12D are shown in Tables 5 and 6 below, respectively. [Table 5] [Table 6]

[0198] Freeze-drying test A preliminary freeze-drying study was performed to evaluate the feasibility of processing the anti-KRAS mAb-containing nanocapsule suspension as a powder for long-term storage. Nanocapsules loaded with PSA anti-KRAS mAb were prepared (10 mL batches) by the method described above. A concentrated solution of trehalose was added to the nanocapsule suspension prior to freeze-drying (final trehalose concentration: 10% w / v). The properties of the freeze-dried nanocapsules were analyzed by measuring particle size, PI, pH, zeta potential, association efficiency, and total mAb content (by ELISA) before and after freeze-drying (FD) at different reconstitution time points during storage at 4°C. Measurements were performed using the same methods as described above. The results, corresponding to three replicates, are shown in Table 7 below and indicate that no significant changes occurred in terms of physicochemical properties and % mAb content, whether associated with the nanocapsules or simply present in the formulation. [Table 7]

[0199] Conclusions: Anti-KRAS monoclonal antibodies were efficiently associated (55-80%) with different polymeric nanocapsule compositions. The nanocapsules exhibited suitable physicochemical properties. The possibility of processing them as powders for long-term storage by lyophilization was also demonstrated.

[0200] Example 4: Plasma stability of polymeric nanocapsules loaded with different anti-KRAS mAbs In many cases, the lack of efficacy of nanocarriers is the result of their aggregation in complex media. This can be due to high ionic strength and / or the presence of proteins in biological media. Therefore, we investigated the stability of polymeric nanocapsules loaded with different anti-KRAS mAbs in plasma. Their stability was used as an indicator of the feasibility of parenteral administration of the mAbs.

[0201] Stability in plasma. Anti-KRAS G12V-loaded nanocapsules prepared as described in Example 3 were incubated in human plasma (dilution 1:10, 37°C) under horizontal shaking (300 rpm, Heidolph Instruments GmbH & Co.). At designated times, samples of the incubation environment were taken for particle size analysis using a Malvern Zeta-Sizer and size and size distribution analysis using nanoparticle tracking analysis (NTA). Samples were analyzed after appropriate further dilutions (1:10,000 in 10 mM PBS (pH 7.4) for NTA and 1:1000 in water for dynamic light scattering (DLS)).

[0202] The stability of one representative formulation (anti-KRAS mAb-loaded HA nanocapsules, 0.5 mg / mL mAb concentration) measured by DLS and NTA is shown in Figures 1 and 2, respectively.

[0203] Conclusion: mAb-loaded nanocapsules exhibit sufficient stability in human plasma for at least 24 hours, representing an important advantage for parenteral administration to subjects.

[0204] Example 5: In vitro cellular internalization of anti-KRAS G12V mAb-loaded nanocapsules in colon adenocarcinoma cells expressing the KRAS G12V mutation This example demonstrates the cellular internalization of anti-KRAS G12V mAb in the SW480 colon adenocarcinoma cell line, which expresses the KRAS G12V mutation. The antibody was first labeled with a fluorescent marker (Alexa Fluor® 488) and then associated with nanocapsules. The retention of nanocapsule properties after loading with the fluorescent antibody was assessed.

[0205] Labeling of anti-KRAS mAb with Alexa Fluor® 488 Briefly, the mAb was diluted to a concentration of 2 mg / mL in 1x PBS (pH 7.2-8.0). Then, 50 μL of 1 M sodium bicarbonate buffer was added to 0.5 mL of the mAb solution (2 mg / mL) (the final pH should be 7.5-8.0). The solution was then added to a vial of the specific reactive dye provided with the Alexa Fluor® 488 Protein Labeling Kit (Thermo-Fisher) and stirred at room temperature for 1 hour. The solution was then stored overnight at 2-8°C. The purification column / resin was prepared by adding resin to the plastic column provided with the kit up to approximately 3 cm from the top. The excess buffer was drained, and the labeled protein was then loaded (at room temperature once). Once all the solution had permeated the resin, 1x PBS was added to elute the protein. The fractions containing the labeled protein (located at the bottom) were then collected, and the protein concentration (M) and degree of labeling were calculated according to Equations 1 and 2, respectively.

number

number

[0206] The degree of labeling achieved was 5 moles of Alexa Fluor® 488 dye per mole of mAb, which is within the optimal range according to the kit specifications (4-9 moles of Alexa Fluor® 488 dye per mole of mAb).

[0207] Preparation of nanocapsules containing fluorescently labeled anti-KRAS G12V mAb For these studies, polymeric nanocapsule formulations were prepared as described above (Example 3) using anti-KRAS G12V mAb previously labeled with Alexa Fluor® 488 (final mAb concentration: 1 mg / mL). Characterization results for size, polydispersity, and zeta potential are shown in Table 8. [Table 8]

[0208] In vitro cellular internalization assay An imaging flow cytometer (ImageStream®) was used to investigate the ability of nanocapsules to induce effective cellular internalization of the associated anti-KRAS antibody. Briefly, nanocapsules loaded with Alexa Fluor® 488 were incubated in a 24-well plate containing SW480 cells, using a separate well for each time point examined (0, 4 h, 8 h, etc.). At each designated time point, cells were trypsinized and images were acquired with the ImageStream® device. Effective internalization was measured by labeling cytoplasmic acidic organelles with the Lysotracker® fluorescent marker in live cells and further confirmed by confocal microscopy (data not shown). Representative images are shown in Figure 3.

[0209] Conclusion: Polymer (HA) NCs enabled efficient internalization of fluorescently labeled anti-KRAS G12V mAb in SW480 cells expressing the KRAS G12V mutation. The internalized antibody showed preferential localization at the target site, i.e., the inner plasma membrane where oncogenic KRAS is localized.

[0210] Example 6: Effect of in vitro cellular internalization of anti-KRAS G12V mAb-loaded nanocapsules in lung adenocarcinoma cells expressing the KRAS G12V mutation This example demonstrates the effect of polymeric nanocapsules loaded with anti-KRAS G12V mAb on the inhibition of cell proliferation and RAS signaling in a cell line (lung adenocarcinoma cell line H441) expressing the KRAS G12V mutation.

[0211] Cell proliferation assay Lung adenocarcinoma cells H441 were seeded at a density of 8,000 cells per well in 96-well plates or 75,000 cells per well in 24-well plates, cultured overnight, and then treated with the antibody at the doses shown in Table 9 below for 3 days. After 3 days of incubation, the cells were harvested and viable cells were measured by MTT (96-well plate test) or trypan blue staining (24-well plate test). Results are expressed as the percentage of viable cells relative to the percentage of controls treated with the respective blank nanocapsules. [Table 9]

[0212] Western Blotting: Cells were seeded in 6-well plates, cultured overnight, and then treated with C16-HA NCs (blank and NCs loaded with anti-KRAS G12V at 166 nM mAb, 0.4 ng mAb / cell dose) for 20 h. Western blotting was performed using specific antibodies according to standard procedures. For quantification of Western blotting data, band intensities were quantified using ImageJ software and normalized to the loading control values. Protein phosphorylation levels were normalized to the total levels of each protein equally loaded on the SDS-PAGE gel. Relative band intensities were expressed as a percentage reduction compared to the corresponding control (blank nanocapsule-treated control). A scan of the Western blot is shown in Figure 4b, along with the corresponding cell proliferation inhibition obtained in a parallel experiment (Figure 4a).

[0213] Figure 4. (A) Inhibition of cell proliferation and (B) reduction of ERK phosphorylation following incubation with C16-HA NCs (blank and nanocapsules loaded with anti-KRAS G12V at 166 nM mAb, 0.4 ng mAb / cell dose) in lung adenocarcinoma cells H441 expressing the KRAS G12V mutation [C(-): untreated cells; BL: blank C16-HA NCs; aG12V NCs: anti-KRAS G12V-loaded C16-HA NCs].

[0214] Conclusions: Polymeric nanocapsules loaded with anti-KRAS G12V mAb inhibited the in vitro growth of KRAS G12V mutant tumor cells by 25-50% or more depending on the experimental conditions. Interference with the RAS signaling pathway was evidenced by a 30-40% reduction in ERK phosphorylation measured by Western blotting.

[0215] Example 7: In vivo assay: Efficacy of nanocapsules containing anti-KRAS G12V mAb in reducing tumor growth in two mouse models: a pancreatic subcutaneous xenograft model and a colon orthotopic tumor model harboring the KRAS G12V mutation This example demonstrates the ability of PSA-tLyp-1 nanocapsules loaded with a polymeric anti-KRAS G12V mAb to reduce tumor growth in two different tumor mouse models: a pancreatic subcutaneous xenograft model and a colon orthotopic tumor model.

[0216] Pancreatic subcutaneous xenograft tumor model Pancreatic xenograft tumor models were generated by subcutaneous inoculation of PA-TU-8902 cells expressing the KRAS G12V mutation into the left and right thighs of Rag2 mice (6 weeks old). Tumor volumes were approximately 150–170 mm. 3When tumor size reached 100 μg / mL, mice were randomly assigned to treatment cohorts and administered PSA-tLyp-1 NC or saline vehicle control intraperitoneally every 3 days (PSA-tLyp-1 NC, 5 mg / kg anti-KRAS G12V mAb dose × 4 doses; 120 μL / dose). Tumor volume and body weight were recorded at regular intervals. At the time of sacrifice, PSA-tLyp-1 loaded with anti-KRAS G12V mAb was administered intraperitoneally. Mice treated with NC showed approximately two-fold lower tumor growth rates than saline controls (Figure 5). No signs of toxicity were recorded in terms of weight loss and animal behavior (data not shown).

[0217] Colon orthotopic tumor model Subcutaneous tumor fragments derived from the SW480 cell line were implanted into the colon of athymic nude Foxn1nu (5-week-old) mice to generate an orthotopic xenograft model derived from the SW480 cell line. Three weeks after implantation, mice were randomized and assigned to different groups to receive different treatments. HA NCs loaded with anti-KRAS G12V mAb at a dose of 10 mg / kg mAb were intravenously administered via the tail vein during the 3-week treatment period (Week 1: 2 doses, i.e., once every 3 days; Week 2: 2 doses, i.e., once every 3 days; Week 3: 4 doses, i.e., once every 2 days, 150 μL / dose). PBS (control) was administered on the same day as untreated controls. Final tumor weights after sacrifice are shown in Figure 6. To quantify the degree of histological regression, tumor slides were scanned and analyzed by NDP. The necrosis rate was calculated in all cases using View2 display software (Hamamatsu). Results from two different tumors per treatment group are shown in Figure 7. The behavior and weight of mice were monitored during treatment to detect signs of treatment toxicity (Figure 8).

[0218] Conclusions: Polymeric nanocapsules loaded with anti-KRAS G12V mAb significantly inhibited tumor growth in vitro in two different KRAS G12V-expressing cell line models: a pancreatic subcutaneous xenograft tumor model (PA-TU-8902 cells) and a colorectal orthotopic tumor model (SW480 cells). Highly significant histological regression in terms of tumor necrosis (>70%) was observed at the time of sacrifice. No weight loss was recorded during the 3-week study, indicating no signs of treatment toxicity. References Non-patent literature: Cox et al.,2014.Drugging the undruggable Ras:mission possible?Nat Rev Drug Discov.2014 November;13(11):828-851 Shin et al.,2017.Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration.Nat Commun.2017 May 10;8:15090 Akishiba et al.,2017.Cytosolic antibody delivery by lipid-sensitive endosomolyti c peptide.Nat Chem.2017;9(8):751 Slastnikova et al.,2018.Targeted intracellular delivery of antibodies:The state of the art,Front.Pharmacol.2018;9:1208 Singh et al.,2019.Antibody delivery for intracellular targets:Emergent therapeutic potential,Bioconjugate Chem.2019;30:1028 Carney et al.,1986.Monoclonal antibody specific for an activated RAS protein.Proc Natl Acad Sci USA.1986 Oct;83(19):7485-9 Rompp Lexikon Lacke und Druckfarben,Georg Thieme Verlag,Stuttgart,N.Y.,1998,“Hydrophilicity”,“Hydrophobicity”,pages 294 and 295. Bertrand et al.,2014.Cancer Nanotechnology:The impact of passive and active targeting in the era of modern cancer biology, Advanced Drug Delivery Reviews 66(2014)2-25 Gilad et al.,2016.Recent innovations in peptide based targeted delivery to cancer cells,Biomedicines,4(2016) Zhou et al.,2016.Aptamers:A promising chemical antibody for cancer therapy,Oncotarget,7(2016)13446-13463 Kapp et al.,2017.A Comprehensive Evaluation of the Activity and Selectivity Profile of Ligands for RGD-binding Integrins.Sci.Rep.7,39805(2017) Ruoslahti,2017.Tumor penetrating peptides for improved drug delivery,Advanced Drug Delivery Reviews,Volumes 110-111(2017)Pages 3-12 Zhang et al.,2016.Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems,Journal of Controlled Release,Volume 229(2016)pages 130-139 Regberg et al.,2012.Applications of cell-penetrating peptides for tumor targeting and future cancer therapies,Pharmaceuticals,5(2012)991-1007 Liu P.et al.,2019.Targeting the untargetable KRAS in cancer therapy,Acta Pharm.Sin.B.,9(2019)871-879 Ferrer et al.,2018.KRAS-Mutant non-small cell lung cancer:From biology to therap y,Lung Cancer,124(2018)53-64 Fisher et al.,2001.Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes,Genes and Development,15(2001)3249-326 Ying et al.,2012.Oncogenic Kras Maintains Pancreatic Tumors through Regulation of Anabolic Glucose Metabolism,149(2012)656-670 Lee and Mooney,2012.Alginate:properties and biomedical applications.Prog Polym Sci.,37(2012)106-126 Patent documents: EP0190033 US5084380 US5443956 WO2019 / 086627A1

Claims

1. A composition comprising a plurality of nanoentities comprising an inner core surrounded by an outer shell, the outer shell comprising a polymer, and the inner core comprising at least one hydrophobic compound, wherein the nanoentities comprise a pharmaceutical agent, the pharmaceutical agent being an antibody or a fragment thereof, and the antibody or fragment thereof binds to an epitope of an activating mutant KRAS protein.

2. 10. The composition of claim 1, wherein the hydrophobic compound of the inner core comprises an oil, a lipophilic surfactant, a fatty acid, an alkane, a cycloalkane, a bile salt, a bile salt derivative, a terpenoid, a terpene, a terpene-derived moiety, or a fat-soluble vitamin.

3. The composition of claim 2 , wherein the hydrophobic compound is an oil.

4. 10. The composition of any one of the preceding claims, wherein the antibody is a humanized antibody.

5. The composition of any one of claims 1 to 4, wherein the epitope of the activating mutant KRAS protein comprises a mutation selected from the group consisting of a glycine residue at position 12, a glycine residue at position 13, or a glutamine residue at position 61 of the amino acid sequence of SEQ ID NO:

39.

6. The composition of claim 5, wherein the mutation at position 12 of the amino acid sequence of SEQ ID NO: 39 is selected from the group consisting of arginine G12R, aspartic acid G12D, valine G12V and cysteine ​​G12C.

7. 7. The composition of any one of claims 1 to 6, wherein the antibody or fragment thereof binds to an epitope comprising a sequence selected from the group consisting of KLVVVGAVGVGK SEQ ID NO:40, KLVVVGADGVGK SEQ ID NO:41, and KLVVVGACGVGK SEQ ID NO:

42.

8. The composition of any one of claims 1 to 6, wherein the antibody or the fragment thereof is obtainable from a hybridoma cell line having a deposit number selected from the group consisting of DSM ACC3358, ATCC-HB-10083 D210, ATCC-HB-8698 DWP, ATCC-HB-10086-D113, and DSM ACC3359.

9. The composition of any one of claims 1 to 6, wherein the antibody or fragment thereof comprises a heavy chain variable region CDR1: (SEQ ID NO: 33) SGYYWN, a heavy chain variable region CDR2: (SEQ ID NO: 34) YIGYDGTNNYNPSLKN, a heavy chain variable region CDR3: (SEQ ID NO: 35) LWDY, a light chain variable region CDR1: (SEQ ID NO: 36) RSSQTIVHGNGNTYLE, a light chain variable region CDR2: (SEQ ID NO: 37) TVSNRFS, and a light chain variable region CDR3: (SEQ ID NO: 38) FQGSHAPYT.

10. The composition of any one of claims 1 to 6, wherein the antibody or fragment thereof comprises a heavy chain variable region CDR1: (SEQ ID NO: 45) SYYMY, a heavy chain variable region CDR2: (SEQ ID NO: 46) EINPSNGGTNFNEKFKS, a heavy chain variable region CDR3: (SEQ ID NO: 47) GGYGY, a light chain variable region CDR1: (SEQ ID NO: 29) RSS KSLLYKDGKTYLN, a light chain variable region CDR2: (SEQ ID NO: 30) LMSTRAS, and a light chain variable region CDR3: (SEQ ID NO: 31) QQVVEYPRT.

11. The polymer may be polysialic acid (PSA), hyaluronic acid (HA), polyglutamic acid (PGA) and / or pegylated polyglutamic acid (PGA-PEG), polylactic acid (PL 11. The composition of any one of claims 1 to 10, wherein the poly(lactic acid-co-glycolic acid) (PLGA) and / or PEGylated poly(lactic acid-co-glycolic acid) (PLA-PEG), poly(aspartic acid) (PASP) and / or PEGylated poly(aspartic acid) (PASP-PEG), poly(lactic-co-glycolic acid) (PLGA) and / or PEGylated poly(lactic-co-glycolic acid) (PLA-PEG), alginic acid (ALG) and / or PEGylated alginic acid (ALG-PEG), polymalic acid (PLMA) and / or PEGylated polymalic acid (PLMA-PEG), and mixtures thereof.

12. The composition of claim 11, wherein the polymer is PSA, HA, or PGA-PEG.

13. The composition of any one of claims 1 to 12, wherein the composition comprises a targeting moiety and / or a cell-penetrating peptide.

14. 14. The composition of claim 13, wherein the composition comprises a targeting moiety that is a CendR peptide selected from the group consisting of Lyp-1, tLyp-1, cLyp-1, and iRGD.

15. The composition of any one of claims 1 to 14, wherein at least some of the polymers are attached to hydrophobic moieties.

16. A composition according to any one of claims 1 to 15 for use as a medicament.

17. A composition according to any one of claims 1 to 15 for use in the treatment or prevention of a disease associated with a mutation in the KRAS gene.

18. 18. The composition for use according to claim 17, wherein the disease is cancer.

19. A pharmaceutical composition comprising a composition according to any one of the preceding claims.

20. The composition of any one of claims 1 to 18 or the pharmaceutical composition of claim 19, wherein said antibody or said fragment thereof is present in combination with at least one other pharmaceutical agent / drug.

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