Knottin-immunostimulant conjugates and related compositions and methods

Abstract

Provided are conjugates that can be used for insitu cancer vaccination, such as for cancers with limited accessibility (e.g., lung cancer, pancreatic cancer, renal cancer, etc.) and metastatic disease in situations where the primary tumor has been surgically resected.SOLUTION: Provided are complexes comprising a knottin peptide comprising a modified loop that binds to a cell surface molecule and an immunostimulatory agent conjugated to the knottin peptide via a linker. In some embodiments, the immune stimulating agent activates a pathogen recognition receptor (PRR). For example, the immunostimulatory agent can be a toll-like receptor (TLR) agonist, e.g., an agonist of TLR7, TLR8, and / or TLR9. Also provided are compositions (e.g., pharmaceutical compositions) comprising the complexes of the disclosure, as well as kits comprising such compositions and methods of using such compositions, e.g., to treat an individual having cancer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application was filed on September 30, 2019, and is incorporated herein by reference in its entirety. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 908,305. [Background technology]

[0002] In situ cancer vaccination is a promising approach to cancer treatment using immune-enhancing agents such as Toll-like receptor (TLR) agonists. This represents a therapeutic strategy involving intratumoral injection of IgG1, which induces immune activation and targets tumor-associated proteins available at the tumor site. The advantage of this approach is that virtually all types of cancers have specific tumor antigens. The main limitation of this strategy is the need for intratumoral injection. This is because cancers with limited accessibility (e.g., lung, pancreatic, renal) and for metastatic disease in situations where the primary tumor has been surgically resected. It is very difficult to Summary of the Invention

[0003] Provided are knottin peptides containing engineered loops that bind to cell surface molecules. and an immunostimulatory agent conjugated to the knottin peptide via a linker. , complex. According to some embodiments, the immunostimulatory agent is a pathogen recognition receptor (PR) For example, immunostimulants are Toll-like receptor (TLR) agonists, e.g. For example, the compound of the present disclosure may be an agonist of TLR7, TLR8 and / or TLR9. Compositions (e.g., pharmaceutical compositions) containing the bodies, as well as kits containing such compositions, and and methods of using such compositions to treat individuals with, for example, cancer. Methods for making the conjugates of the present disclosure are also provided. [Brief explanation of the drawings]

[0004] [Figure 1] Schematic of tumor-targeting immunostimulatory agent conjugates using integrin-binding knottin peptides and knottin-Fc fusions as targeting agents. [Figure 2] Strategies for functionalizing immunostimulants according to some embodiments of the present disclosure. [Figure 3] 1A-1D are sequences and schematic diagrams of knottin peptides according to some embodiments of the present disclosure. [Figure 4] Strategies for conjugating knottin peptides to functionalized immunostimulatory agents, according to some embodiments of the present disclosure. [Figure 5] According to some embodiments of the present disclosure, knottin-CpG complexes in which the CpGs are incorporated at different sites. [Figure 6] Knottin-CpG conjugates synthesized with different linkers according to some embodiments of the present disclosure. [Figure 7] A knottin-TLR7 / 8 agonist complex, according to some embodiments of the present disclosure. [Figure 8] A knottin-Fc immunostimulatory agent conjugate according to some embodiments of the present disclosure. [Figure 9] A complex comprising a knottin peptide conjugated to a detectable label to enable in vivo fluorescent tracking, according to some embodiments of the present disclosure. [Figure 10] Data showing in vivo fluorescence imaging of a conjugate of the present disclosure administered intratumorally and peritumorally. [Figure 11] 10A-10C show data showing in vivo fluorescence imaging of a conjugate of the present disclosure administered intravenously. [Figure 12] Data showing ex vivo fluorescence imaging of a conjugate of the present disclosure in a resected tumor. [Figure 13]Survival curves demonstrating the therapeutic efficacy of 3CM-CpG in 4T1-Luc breast cancer (n=9–10). [Figure 14] Data showing 4T1-Luc mean tumor growth over time. [Figure 15] Data showing individual 4T1-Luc tumor growth curves. [Figure 16] Immune cell infiltration data are provided. Mice bearing 4T1-luc cell tumors were treated with intravenous (IV) or intratumoral (IT) injection according to the diagram for the following groups: vehicle IV, CpG IV, 3CM-CpG IV, and CpG IT. Tumors were excised after treatment and analyzed for tumor-infiltrating immune cells via FACS. Provided are plots showing the abundance of different immune populations as % of total viable single cells. [Figure 17] Pie chart summarizing the mean abundance (% of total viable single cells) of immune cell populations and two uncharacterized cell populations ("Other") for each treatment group described in Figure 16. DETAILED DESCRIPTION OF THE INVENTION

[0005] Before the conjugates, compositions, kits and methods of the present disclosure are described in further detail, It should be understood that the compositions, kits, and methods are not limited to the specific embodiments described. It should be understood that the terms used herein may be varied in various ways. (The scope of the conjugates, compositions, kits, and methods is limited to the scope of the invention.) It is not intended to be limiting (as it is limited only by the scope of the appended claims). It should also be understood that

[0006] When a range of values ​​is provided, the value between the upper and lower limits of that range (unless the context clearly indicates otherwise) Each intervening value (to the tenth of the lowest unit unless otherwise specified) and its stated range Any other stated or intervening values ​​within the ranges are also applicable to the conjugates, compositions, kits, and It is understood that the upper and lower limits of these smaller ranges are encompassed within the method. Independently, smaller ranges may be included, still subject to the conjugates, compositions, kits and methods. are included within the scope of the invention, subject to any limitations specifically excluded within the scope of the invention. If a range includes one or both of its limits, the range is The scope of the complex, composition, kit, and method also includes the scope excluding both.

[0007] Certain ranges are presented herein with the numerical values ​​preceded by the term "about." A term refers to the exact number it precedes, and to any number that is close or Used herein to provide literal support for approximate numbers. In determining whether a number is close to or approximates an enumerated number, A number not listed is a number that is specifically listed in the context in which it is presented. It may be a substantially equivalent number.

[0008] Unless otherwise defined, all technical and scientific terms used herein are defined by their respective The conjugates, compositions, kits, and methods have the same meaning as commonly understood by those skilled in the art to which they pertain. Any conjugate similar or equivalent to those described herein, The compositions, kits, and methods also include those for practicing the complexes, compositions, kits, and methods. Representative exemplary conjugates, compositions, kits, and methods that can be used in the testing include: It is described here.

[0009] All publications and patents cited herein are to be construed as being incorporated by reference in their entirety. All such references are incorporated herein by reference as if specifically and individually indicated to be incorporated by reference. The methods and / or materials in connection with which the publications are incorporated herein and in which the publications are cited are disclosed or The present invention is incorporated herein by reference for purposes of illustration and example only. The citation of a publication is for illustrative purposes only and is not intended to be limiting unless expressly stated. The publication years provided are for disclosure of the actual publication dates which may need to be independently verified. The date of publication may differ from the date of publication, and the conjugates, compositions, kits, and methods may differ from such publication. This provision should not be construed as an admission that there is no right of antecedence over the law.

[0010] As used in this specification and the appended claims, the singular forms "a," "an," and "an" are used interchangeably. and "the" only include plural referents unless the context clearly dictates otherwise. It is understood that the claims may be drafted to exclude any optional element. Further note: Thus, this statement does not necessarily imply a "solely" context for the enumeration of claim elements. "," "only," etc., or the use of "negative" limitations. It is intended to serve as a basis.

[0011] The conjugates, compositions, kits and It is recognized that certain features of the method may be provided in combination in a single embodiment. Conversely, for the sake of brevity, conjugates, compositions, etc., which are described in the context of a single embodiment, The various features of the kits and methods may be used separately or in any suitable subcombination. All combinations of embodiments are specifically encompassed by this disclosure. To the extent that such combinations encompass operable processes and / or compositions, Each and every combination is treated as though individually or expressly disclosed. Further, such variables may be recited in the embodiments described. All such subcombinations are also specifically contemplated by the present complexes, compositions, kits and methods. and all such subcombinations are individually and expressly disclosed. and all such modifications are herein disclosed as if fully set forth herein.

[0012] As will be apparent to those skilled in the art upon reading this disclosure, the individual components described and illustrated herein may be Each of the embodiments may be combined with any of the other embodiments without departing from the scope or spirit of the disclosed methods. Individual features that can be easily separated from or combined with any of the features of the embodiments of Any recited method has elements and features. or in any other order which is logically possible.

[0013] Complex The present disclosure provides knottin peptide-immunostimulatory agent conjugates. The conjugates are directed against cell surface molecules. a knottin peptide containing a modified loop that binds to the molecule, and a linker that binds to the knottin peptide; Such conjugates include various immunostimulatory agents conjugated to immunopeptides. For example, in the context of cancer therapy, as demonstrated herein, the conjugates Unexpectedly, it is able to localize in solid tumors after systemic administration, achieve substantially greater therapeutic efficacy than unconjugated immunostimulants. The combination resulted in increased percentages of CD8+ T cells, CD4+ T cells, and B cells, as well as bone marrow-derived As shown by the reduction of myocardial infarction suppressor cells (MDSCs), Unexpectedly and significantly altered tumor immune profiles compared with systemic administration of ungated immunostimulants. This study aimed to analyze immune cell profiles using knottin peptide-immunostimulatory agent conjugates. The dramatic changes were distinct from those observed following treatment with intratumoral (IT) administered immune stimulants. Further details regarding the composites of the present disclosure are now described.

[0014] An "immunostimulant" is an agent that directly or indirectly stimulates the activation or maturation of one or more types of cells of the immune system. Various types of immunostimulants are available in combination with the complexes of the present disclosure. Non-limiting examples of immunostimulants that may be employed include polypeptides, nucleic acids, and the like. (e.g., oligonucleotides), carbohydrates, antibodies, ligands, aptamers, nanoparticles, and small molecules. In some embodiments, the immunostimulatory agent is a non-immune cell (e.g., It stimulates cells (e.g., epithelial cells, endothelial cells, and tumor cells) to produce proinflammatory cytokines.

[0015] According to some embodiments, the conjugates of the present disclosure inhibit one or more types of cells of the innate immune system. Immunostimulatory agents include immunostimulatory agents that directly or indirectly induce cell activation or maturation. Non-limiting examples of innate immune system cell types that can be activated directly or indirectly by Phages, dendritic cells, NK cells, neutrophils, basophils, eosinophils, Langerhans cells, In certain embodiments, the conjugates of the present disclosure stimulate adaptive immunity by inhibiting immune responses to immune cells, including inflammatory cytokines, immune cells, and / or monocytes. Immunostimulatory agents include immunostimulatory agents that induce activation or maturation of one or more types of cells in the immune system. Non-limiting examples of adaptive immune system cell types that can be activated by stimulatory agents include T cells and B cells. Examples of T cells include naive T cells (T N ), cytotoxic T cells (T CTL ), memory T-thin Cell (T MEM ), T memory stem cells (T SCM ), central memory T cells (T CM ),effector Memory T cells (T EM ), tissue-resident memory T cells (T RM ), effector T cells (T EFF ), regulatory T cells (T REG ), helper T cells (T H , T H 1. T H 2. T H 17)C D4+ T cells, CD8+ T cells, virus-specific T cells, alpha-beta T cells (T αβ ), and gamma delta T cells (T γδ ) is included.

[0016] In certain embodiments, the immunostimulatory agent is a pathogen-associated molecular pattern (PAMP). PAMPs are part of the pathogen life cycle and Pathogen-specific sugars, lipoproteins, and / or nucleic acids (e.g., disaccharides) expressed in the DNA containing one or more unmethylated repeats of the nucleotide CpG, double-stranded RNA (dsRNA) A), single-stranded RNA (ssRNA), or equivalents). Host proteins that can recognize turns are called pathogen recognition receptors (PRRs). According to some embodiments, the immunostimulatory agent of the conjugate of the present disclosure is a pathogen recognition receptor ( In certain embodiments, the PRR is a Toll-like receptor (TLR), R IG-1-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors NLRs, C-type lectin receptors (CLRs), cytosolic dsDNA sensors (CDSs) ), stimulator of interferon genes (STING), and any combination thereof According to some embodiments, the immunostimulatory agent is selected from the group consisting of: Non-natural PRR activators that can be employed include synthetic small molecule PAMPs. These include, but are not limited to, RR agonists.

[0017] According to some embodiments, the immunostimulatory agent is a Toll-like receptor (TLR) agonist. TLRs sense invading pathogens or endogenous damage signals and mediate innate and adaptive immunity. A family of type I transmembrane PRRs that initiate immune responses. There are 10 functional TLRs in humans. There are 12 functional TLRs in mice (TLRs 1-9, 11-10). 13) Various combinations of TLRs are involved in the regulation of monocytes, macrophages, dendritic cells, and neutrophils. Immune and non-immune cells such as B cells, T cells, fibroblasts, endothelial cells, and epithelial cells Among human TLRs, TLR1, 2, 4, 5, 6 and 10 are expressed on the cell surface and primarily bind to microbial membrane and / or cell wall components. While TLR3, 7, 8, and 9 recognize endolysosomal compartments, TLRs are expressed on the membrane and recognize nucleic acids. They bind different numbers of ligands at their N-terminus. The sensing leucine-rich repeat (LRR) and the cytoplasmic Toll / IL-1 R (TIR) ​​domain The TIR domain is found in MyD88, TRIF, TRAM, and TIRAP. TLRs and adaptor proteins involved in the regulation of TLR signaling, including / MAL, These adaptor molecules mediate the interaction between the signal transduction pathway and the signal transduction pathway activated downstream of these adaptor molecules. The pathways involve inflammatory cytokines, chemokines, and type I and III interferons. Promotes the expression of

[0018] In certain embodiments, the conjugates of the present disclosure are selected from the group consisting of TLR1, TLR2, TLR3, TLR4, One or more of TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 According to some embodiments, the immunostimulatory agent is an agonist of the above. It is a TLR9 agonist. The human TLR9 gene is transcribed into different isoforms. Spliced ​​into five TLR9 isoforms (TLR9A, B, C, D, and E) These TLR9 isoforms are expressed in the spleen, peripheral blood mononuclear cells ( It is differentially expressed in various immune organs and cells, such as PBMCs, and lymph nodes.

[0019] In some embodiments, the immunostimulatory agent is an oligonucleotide-based TLR9 agonist. As used herein, an "oligonucleotide" is a nucleic acid having 5 to 500 nucleotides. Nucleotide, e.g., a single-stranded multinucleotide sequence of 5 to 100 nucleotides Oligonucleotides can be synthetic or enzymatically produced. In some embodiments, the oligonucleotide is 5 to 50 nucleotides in length. A nucleotide contains ribonucleotide monomers (i.e., oligoribonucleotides or are "RNA oligonucleotides"), containing deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides or "DNA oligonucleotides" The oligonucleotide may contain, for example, 10 to 15 nucleotides, or a combination thereof. 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 8 0-100, 100-150, or 150-200, or up to 500 nucleosides The length of the chido may be:

[0020] When the immunostimulatory agent is an oligonucleotide-based TLR9 agonist, may be an oligonucleotide containing one or more unmethylated CpG dinucleotides. Innate immune responses to bacterial and synthetic DNA containing unmethylated CpG motifs The role of TLR9 in this process has been well characterized. kira S. (2006) Journal of Molecular Medici See ne 84(9):712-725. According to some embodiments, immunization When the stimulatory agent is an oligonucleotide-based TLR9 agonist, the immunostimulatory agent is C CpG oligodeoxynucleotides (ODNs) are used in vitro and in Specific sequence contexts are capable of activating leukocytes as demonstrated in vivo and in vivo. Synthetic DNA containing unmethylated CpG dinucleotides in the CpG motif Three major classes of CpG ODNs are distinguished by their structural and biological properties. They are classified based on their physical properties and designated as Class A, Class B, and Class C. The central palindromic CpG-containing phosphodiester (PO) structure is followed by phosphorothioate. Class A oligos are characterized by a ester (PS) homopolymer G stretch followed by It is a potent inducer of interferon-α (IFN-α) production and dendritic cell maturation. Class B oligos, in contrast, usually contain an entirely phosphorothioate (PS) backbone. These oligos also stimulate IFN-α production, but to a lesser extent. They potently activate B cells. Class C oligos combine the properties of classes A and B. It is characterized by a perfect PS backbone and a palindromic CpG-containing motif. pG ODNs contain one or more unmethylated CpG dinucleotides in a specific sequence context. This is likely due to the rarity of this structure in mammalian genomes. In certain embodiments, the present disclosure provides a method for the preparation of a medicament for the treatment of a mammalian cell. The complexes are selected from Class A (Type D), Class B (Type K), and Class C. It contains CpG ODNs from the following classes:

[0021] In some embodiments, the immunostimulatory agent comprises one or more unmethylated CpG dinucleotides. and oligonucleotide-based TLR9 agonists (e.g., CpG ODNs) containing In some embodiments, the immunostimulatory agent comprises at least five nucleotides. The immunostimulant may contain 2 to 100, e.g., about 8 to about 40, nucleotides. In some embodiments, the immunostimulatory agent comprises 10 to 30 nucleotides. In some embodiments, the immunostimulatory agent comprises 15 to 25 nucleotides. The immunostimulatory agent may contain one or more poly-T sequences and / or contain more than about 25% T nucleosides. In some embodiments, the immunoglobulin is a T-rich oligonucleotide having tido residues. The stimulant has a GTC trinucleotide instead of a CG dinucleotide. In embodiments, the immunostimulatory agent has one or more modified cytosines. The immunostimulatory agent is a partially single-chain, dumbbell-shaped, covalently closed, deoxyribonucleotide. In some embodiments, the immunostimulatory agent is an oxyribonucleic acid molecule having the following structure: One or more of the following, i.e., [CGN] x , [N a CG] x、 [N a CGNb] x , [NaC GTTNb] x , and [N a CGN b CGN c ] x where N is any nucleophile. a, b, and c are independently 1 to 15. For example, [N a CGN b ] x The corresponding sequences are ACGT, GTCGTT, and TCGGT. T, TGACGTT, and ACGTACGT.

[0022] CpG motifs exhibit species specificity. For example, the optimal mouse CpG motif is GACG TT, whereas the optimal motif for use in the human context is GTCGTT. CpG ODN 1826, a RasB CpG ODN, is a well-defined mouse This oligo is a TLR9 agonist and is therefore widely used in rodent models. It regulates murine B cell proliferation, maturation of antigen-presenting cells, and induction of polarized Th1-type cell responses. CpG ODN 1826 contains two CpG dinucleotides. Both are flanked by -GA at the 5' end and -TT at the 3' end. It is sulfothioated, in contrast to the native PO backbone found in bacterial or viral genomes. provides nuclease resistance to

[0023] In certain embodiments, the oligonucleotide-based TLR9 agonist is a human CpG Such human CpG ODNs contain the CpG motif GTCGTT. A non-limiting example of such a human CpG ODN is the sequence TCGTCGTTTTGT CGTTTTGTCGTT (SEQ ID NO: 1). The oligonucleotide-based TLR9 agonist is a mouse CpG ODN. Such mouse CpG ODNs may contain the CpG motif GACGTT. A non-limiting example of a CpG ODN is the sequence TCCATGACGTTCCTGACGTT (sequence It has column number 2).

[0024] Oligonucleotide-based immunostimulatory agents, for example, reduce nuclease susceptibility The amino acid sequence may contain one or more modifications to inhibit or prevent the formation of a phosphodiesterase. Examples of such modifications include the natural phosphodiesterase. Modifications to diester oligodeoxyribonucleotide and ribonucleotide polymers For example, the oligonucleotide-based immunostimulatory agent may comprise one or more phosphorothioates. The PS bond may comprise a phosphate bond within the phosphate backbone of the oligonucleotide. The PS modification replaces non-bridging oxygen with a sulfur atom, making the internucleotide bond more resistant to nuclease degradation. Makes it more resistant to

[0025] According to some embodiments, the immunostimulatory agent is a TLR7 agonist, a TLR8 agonist, or a A wide variety of TLR7 and / or TLR8 agonists In certain embodiments, the TLR7 and / or TLR8 agonist is Contains imidazoquinoline (IMZQ) compounds. Imidazoquinolines bind to toll-like receptors, particularly It is a potent immune stimulator, primarily acting through TLR7 and TLR8. In this state, TLR7 and / or TLR8 agonists, including IMZQ compounds, inhibit T78a and its structure is provided in Figure 2. According to some embodiments, IMZQ TLR7 and / or TLR8 agonists, including the hybrid-2(1-(4 -amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)-2-methyl propan-2-ol), XG1-236 (2-butyl-2H-pyrazolo[3,4-c ]quinolin-4-amine), DS802 (2-butyl[1,3]oxazolo[4,5-c ]quinolin-4-amine), CL075 (2-propyl[1,3]thiazolo[4,5-c ]quinolin-4-amine), CL097 (2-(ethoxymethyl)-1H-imidazo[4 ,5-c]quinolin-4-amine), R848 (1-[4-amino-2-(ethoxymethyl) -1H-imidazo[4,5-c]quinolin-1-yl]-2-methylpropane-2-yl ol), methamphetamine, or paraamine. et al.(2017)PLoS ONE 12(6):e0178846, and Ganapathi et al.(2015)PLoS ONE 10(8):e01 See 34640.

[0026] In certain embodiments, the complexes of the present disclosure are conjugated to knottin peptides. The immunostimulatory agent may comprise two or more immunostimulatory agents. For example, the knottin peptide may comprise two or more, three or more, four or more 1 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more immunizations The knottin peptide may be conjugated to two or more immunostimulatory agents. When conjugated, two of the two or more immunostimulatory agents can be the same or different. According to some embodiments, the two or more immunostimulatory agents are independently selected from the group consisting of those described herein. Immunostimulatory agents (e.g., TLR9 agonists, TLR7 and / or 8 agonists, and / or equivalents).

[0027] According to some embodiments, the knottin peptide of the complex of the present disclosure is a detectable target. A "detectable label" is a label that is used to identify a specific molecule that is capable of being detected. can be detected in in vitro and / or in vivo studies and / or clinical applications). The detectable label of interest is a fluorescent label. Labels (e.g., A, such as AlexaFluor 680, as described in the Experimental Section herein) lexaFluor fluorophore), a radioisotope, and an enzyme that produces a detectable product (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, etc.) ), fluorescent proteins, paramagnetic atoms, and the like. The peptide is conjugated to a specific binding partner of the detectable label, e.g., an avidin. Biotin was used so that detection could occur via a detectable label including avidin / streptavidin. Conjugated to Chin.

[0028] According to certain embodiments, the detectable label is a label that can be detected by near-infrared (NIR) optical imaging, single light Electron Emission Computed Tomography (SPECT) / CT Imaging, Positron Emission Tomography (PE T), nuclear magnetic resonance (NMR) spectroscopy, or the like. Detectable labels that find use in such applications include fluorescent labels, radioisotopes, and and the like. In certain embodiments, the detectable label is a Multimodal in vivo imaging that allows in vivo imaging using the above imaging techniques (e.g., Thorp-Greenwood and Coogan (201 1) See Dalton Trans. 40:6129-6143).

[0029] In certain embodiments, the detectable label finds use in near-infrared (NIR) imaging applications. Such agents include Kodak X-SIGHT dyes, P z 247, DyLight 750 and 800 Fluors, Cy 5.5 and 7 Fluors, Alexa Fluor 680 and 750 Dyes, IRDy e including but not limited to 680 and 800 CW Fluors. According to this embodiment, the detectable label is an in vivo label that finds use in SPECT imaging applications. Imaging agents, non-limiting examples of which include: 99m Tc, 111 In, 123 In, 201 T1, and 133 In certain embodiments, the detectable label comprises, for example, 11 C , 13 N, 15 O. 18 F, 64 Cu, 62 Cu, 124 I, 76 Br, 82 Rb, 68 Insulators that find use in positron emission tomography (PET) imaging applications, such as Ga, or equivalents. It is an in vivo imaging agent.

[0030] Knottin peptide The conjugates of the present disclosure comprise a knottin peptide containing an engineered loop that binds to a cell surface molecule. The types of knottin peptides employed in the conjugates of the present disclosure may vary. Non-limiting examples of knottin peptides that can be employed include EETI-II peptide, AgR P peptide, ω-conotoxin peptide, Kalata B1 peptide, MCoTI-II peptide Knotin peptides include peptides, agatoxin peptides, and chlorotoxin peptides. The three-dimensional structure of is minimally defined by the specific arrangement of three disulfide bonds. The characteristic topology is that one disulfide bond is connected to two other intrachain disulfide bridges. The macrocycles formed by the cleavage of the nuclei form molecular knots. Their secondary structure content is Although generally low, knottins contain small molecules stabilized by a disulfide bond framework. The folding and functional activity of knottins are diverse. In most cases, this is mediated by loop regions that are diverse in both length and amino acid composition. Three disulfide bonds is the minimum number that defines the fold of this peptide family. However, knottins can also contain additional cysteine ​​residues, with four or more in their structure. This results in a molecule with the above disulfide bond and an additional constraint loop. The term refers to a Cys residue in which the sulfur group is linked to another amino acid through a disulfide bond. The term "cysteine" refers to the -SH ("half-cystine") form of the residue. The binding loop portion has no other intervening cystine in the primary sequence of the binding loop. Thus, it can be adjacent to a cystine.

[0031] Knottin peptides are a group of several thousand polypeptides that have been identified that contain the cystine knot motif. The online KNOTT database contains detailed amino acid sequences, structures, classification, and functional information of the proteins. The knottins can be peptides listed in the IN database. Found in animals, insects, and fungi.

[0032] Knottin peptides are full-length (i.e., the length of the wild-type peptide / polypeptide). or the knottin peptide is shortened relative to the length of the wild-type peptide / polypeptide. or knotty peptides, the peptides are of the length of the wild-type peptide / polypeptide. It may contain additional amino acids to make it longer than the original.

[0033] According to certain embodiments, the knottin drug conjugate (KDC) of the present disclosure is Ecballium el eterium trypsin inhibitor II (EETI-II) peptide, agouti-related protein ( AgRP) peptide, ω-conotoxin peptide, Kalata B1 peptide, MCoTI-I I peptide, agoutixin peptide, or chlorotoxin peptide In some embodiments, the knottin peptide comprises a knottin peptide based on one of is based on the Ecballium elaterium trypsin inhibitor II (EETI-II) peptide. In some embodiments, the knottin peptide is an agouti-related protein (AgRP). Peptide-based.

[0034] "EETI" stands for Protein Data Bank Entry (PDB) 2E Its entry in the KNOTTIN database is EETI-II. In certain embodiments, the knottin peptide of the complex of the present disclosure has the amino acid sequence: Based on the EETI-II peptide. GCPRILMRCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 3)

[0035] "AGRP" refers to PDB entry 1HYK and KNOTTIN database entry SwissProt AGRP_HUMAN. AGRP is a membrane protein that regulates the function of the human brain. A 132-amino acid neurotransmitter that binds to lanocortin receptors and is involved in the regulation of metabolism and appetite. The biological activity of AgRP is mediated by the C-terminal peptide, which contains five disulfide bonds. It is mediated by a Stain knot domain but contains only four disulfide bonds A fully active 34 amino acid truncated AgRP has been developed. The knottin peptide in the complex is a truncated AGRP peptide with the following amino acid sequence: Based on. CVRLHESCLGQQVPCCDPAATCYCRFFNAFCYCR (Sequence No. No. 4)

[0036] According to some embodiments, the knottin peptide of the complex of the present disclosure has the following amino acid sequence: Based on the Kalata B1 peptide with the amino acid sequence. CGETCVGGTCNTPGCTCSWPVCTRNGLPV (SEQ ID NO: 5)

[0037] In certain embodiments, the knottin peptide of the complex of the present disclosure has the amino acid sequence: Based on the MCoTI-II peptide having the following structure: SGSDGGVCPKILKKCRRDSDCPGACICRGNGYCG (Sequence number No. 6)

[0038] According to certain embodiments, the knottin peptide of the complex of the present disclosure has the following amino acid sequence: Based on the chlorotoxin peptide with sequences. MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR(distribution Column number 7)

[0039] EETI-II, AgRP, ω-conotoxin, Kalata B1, MCoTI-II, Ag Toxins, chlorotoxins, and knottin peptides of the disclosed conjugates may be based Sequence and structure (e.g., loop) information for other knottin peptides is available in the PDB, K They can be found in the NOTTIN database, and other protein databases.

[0040] Knottin peptides contain engineered loops that bind to cell surface molecules, i.e., That is, the loop is engineered to bind to a target molecule on the surface of a cell. Three disulfide bonds interwoven into molecular "knots" that constrain the loop region to the core of the antiparallel β-sheet. Wild-type EETI contains, for example, loop 1 (the trypsin-binding loop) , residues 3-8), loop 2 (residues 10-14), and loop 3 (residues 22-26). It consists of 28 amino acids with three disulfide-constrained loops. Knottin family members, including inhibitors, toxins, and antimicrobial agents, mediate their core functions. Except for the tein residues, they share little sequence homology. The constrained loop allows for a lot of sequence diversity, allowing knottin to collapse without disrupting its tertiary folding. Protein engineering applications that require introducing mutations into proteins without Make it appropriate for your application.

[0041] The modified loops are amino acid substitutions, insertions, or modifications to the existing loops of the knottin peptide. and / or may contain deletions or modified loops in knottin proteins. That is, the knottin peptide of the complex may have an additional loop. In addition to one or more loops present in the target gene, the target gene may contain loops. By combining these, modifications (amino acid sequence and loop) were made to the loop region of the knottin scaffold. Guidelines for introducing the scalability (both in terms of group length) have been developed. For example, Lahti et al. al.(2009)PloS Comput.Biol.5(9):e1000499 Please refer to.

[0042] In some embodiments, the loop of knottin is configured to bind to a cancer cell surface molecule. "Cancer cells" are cells that are altered, for example, by abnormal cell growth, abnormal cell proliferation, density dependence, or the like. Loss of growth inhibition, anchorage-independent growth capacity, and immunocompromised non-human animal models the ability to promote tumor growth and / or development and / or any of the cell transformation "Neoplastic cell phenotype" refers to cells that exhibit a neoplastic cell phenotype, which may be characterized by one or more of the following suitable indicators: "Cancer cells" are used herein to refer to "tumor cells," "malignant cells," or "cancerous cells." " may be used interchangeably with " and refer to solid tumors, semi-solid tumors, liquid tumors, primary tumors, metastatic tumors, and Such modified loops include knottin peptides and their equivalents in cancer cells. In certain embodiments, the peptides confer cancer cell surface molecular recognition properties that are not present in the wild-type peptide. The cancer is one that is known to have one or more tumor antigens. Non-limiting examples of tumor antigens to which the modified loops may bind include 5T4, the AXL receptor tyrosine kinase. AXL, B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonate Carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), cadherin-6, CD1 9, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen ( CEA), cKit, Cripto protein, CS1, Delta-like canonical Notch ligase DLL3, endothelin receptor type B (EDNRB), ephrin A4 (EF NA4), epidermal growth factor receptor (EGFR), EGFRvIII, ectonucleotide pyrophosphate enophosphatase / phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPH A2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (F GFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1), Glycoprotein nontransferase B (GPNMB), guanylate cyclase 2C (GUCY2C), Human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Tegrin alpha, lysosome-associated membrane protein 1 (LAMP-1), Lewis Y, LIV -1, leucine-rich repeat containing 15 (LRRC15), mesothelin (MSLN), mucin 1 (MUC1), mucin 16 (MUC16), sodium-dependent phosphate transport protein 2 B (NaPi2b), nectin-4, NMB, NOTCH3, p-cadherin (p-CA D), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), Solute carrier family 44 member 4 (SLC44A4), a member of the SLIT-like family 6 (SLITRK6), STEAP family member 1 (STEAP1), tissue factor ( TF), T cell immunoglobulin and mucin protein-1 (TIM-1), and trophic factor Contains the trophoblast surface antigen (TROP-2).

[0043] According to some embodiments, the cell surface molecule to which the engineered loop of knottin binds receptors, e.g., cell adhesion receptors, receptors for soluble factors (e.g., growth factors, chemokines, etc.), receptors for soluble factors), immune cell receptors, or equivalents. According to some embodiments, the cell surface molecule to which the engineered loop of knottin binds is a cell surface molecule. Adhesion receptors, e.g., expressed on the surface of cancer cells, expressed on the surface of tumor vasculature cells and / or equivalents of cell adhesion receptors (e.g., integrins). In certain embodiments, when the receptor is a cell adhesion receptor, the receptor is an integrin. For example, the complexes of the present disclosure may be used in combination with αvβ1 integrin, αvβ3 integrin, αvβ 5 integrin, αvβ6 integrin, α5β1 integrin, or any of them A knotting peptide having a loop engineered to bind to any one of the combinations According to certain embodiments, the modified loop may comprise a αvβ1 integrin , αvβ3 integrin, αvβ5 integrin, αvβ6 integrin, and α5β 1 integrin.

[0044] αvβ1 integrin, αvβ3 integrin, αvβ2 integrin, αvβ3 integrin, αvβ1 integrin, αvβ2 integrin, αvβ1 integrin, αvβ3 ... β5 integrin, αvβ6 integrin, and α5β1 integrin, respectively. EETI-based knottin peptides with engineered binding loops (EETI -2.5D) has the following amino acid sequence (the integrin binding loop is underlined): being pulled). GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 8)

[0045] αvβ1 integrin, αvβ3 integrin, αvβ2 integrin, αvβ3 integrin, αvβ1 integrin, αvβ2 integrin, αvβ1 integrin, αvβ3 ... β5 integrin, αvβ6 integrin, and α5β1 integrin, respectively. EETI-based knottin peptides with engineered binding loops (EETI -2.5F) has the following amino acid sequence (the integrin-binding loop is underlined): being pulled). GCPRPRGDNPPLTCSQDSDCLAGCVCGPNGFCG (SEQ ID NO: 9)

[0046] αvβ1 integrin, αvβ3 integrin, αvβ2 integrin, αvβ3 integrin, αvβ1 integrin, αvβ2 integrin, αvβ1 integrin, αvβ3 ... β5 integrin, αvβ6 integrin, and α5β1 integrin, respectively. EETI-based knottin peptides with engineered binding loops (3CM and (designation) has the following amino acid sequence, where Z=5-azido-L-norvaline (indicated): Tegrin-binding loops are underlined). GCPRPRGDNPPLTCZQDSDCLAGCVCGPNGYCG (SEQ ID NO: 10)

[0047] In some embodiments, the knottin peptide of the complex of the present disclosure is listed in Table 1. Such integrin-binding EETI-based knottin peptides. [Table 1]

[0048] In some embodiments, the knottin peptide of the complex of the present disclosure is set forth in Table 2. These are integrin-binding AgRP-based knottin peptides. [Table 2]

[0049] According to some embodiments, the knottin peptide of the complex of the present disclosure inhibits the activity of a protease Non-limiting examples of proteases include membrane proteases, Examples include matriptase.

[0050] In some embodiments, the knottin peptide comprises one or more unnatural amino acids. Such one or more unnatural amino acids may, for example, facilitate the attachment of a drug to a knottin peptide. For example, non- Natural amino acids include azides, alkynes, alkenes, aminooxys, hydrazines, and aldehydes. , nitrones, nitrile oxides, cyclopropenes, norbornenes, isocyanides, halogens aryl borides, and boronic acid functional groups. unnatural amino acids that can be incorporated into the knottin peptide of the knottin drug conjugate of Non-natural amino acids that can be selected to provide desired functional groups are known, e.g. , Maza et al. (2015)Bioconjug.Chem.26(9):1 884-9, Patterson et al. (2014) ACS Chem.Bio l.9:592-605, Adumeau et al. (2016) Mol.Imag ing Biol.(2):153-65, and elsewhere. In embodiments, the knottin peptide contains one or more 5-azido-L-norvaline residues or Derivatives thereof, such as those produced upon conjugation of the residue to a functionalized immunostimulatory agent, This includes derivatives that can be used.

[0051] Conjugates of the present disclosure may be prepared using knottin peptide sequences, such as those provided in Table 1 or Table 2 above. 70 to the amino acid sequence of the knottin peptide described herein, such as any of % or more, 80% or more, 90% or more, 95% or more, or 100% identity The peptide may include a phospholipid.

[0052] According to some embodiments, the knottin peptide of the complex of the present disclosure comprises one or more different The knottin peptide is fused directly to a heterologous polypeptide. In certain embodiments, the knottin peptide is attached to a heterologous polypeptide via a linker. A non-limiting example of a linker that can be employed is the amino acid sequence GGGGSG a serine-glycine linker comprising GGGSGGGGS(G4S)3 (SEQ ID NO: 44), etc. The heterologous polypeptide of interest may comprise an Fc domain (e.g., , human or mouse Fc domain), albumin, transferrin, XTEN, homoa amino acid polymers, proline-alanine-serine polymers, elastin-like peptides, or In some embodiments, the present invention may be used in combination with any of the following: The heterologous polypeptide exhibits a significantly increased activity compared to the same knottin peptide that is not fused to the heterologous polypeptide. In comparison, the stability and / or In certain embodiments, a human Fc domain (e.g., a full-length human Fc domain) is used to increase serum half-life. c domain or fragment thereof) of the present disclosure. According to some embodiments of the present disclosure, a fusion protein is provided, which comprises a fusion protein fused to an Fc domain. Schematic representation of a complex comprising a knottin peptide combined and conjugated to an immunostimulatory agent. This is illustrated schematically in Figure 1. According to some embodiments, such fusion proteins The quality can be, for example, determined by administering to an individual in need thereof (e.g., an individual with cancer) according to the methods of the present disclosure. The conjugates of the present disclosure may be fused to the knottin peptide. Non-limiting examples of human Fc domains include those having the sequence set forth in Table 3 below (SEQ ID NO: 45). It is a human IgG1 Fc domain or a fragment thereof. [Table 3]

[0053] In some embodiments, the knottin peptide is fused to one or more heterologous polypeptides. When used in combination, the knottin peptide is fused to a heterologous polypeptide that is detectable in vivo. Non-limiting examples of in vivo detectable polypeptides include bioluminescent reporters. In certain embodiments, the bioluminescent reporter is a luciferase, e.g., nanoluciferase. It is Ze.

[0054] Knottin peptides with engineered loops that bind to cell surface molecules have been developed. The formula can be varied. Rational and combinatorial approaches can be used to identify novel molecular recognition properties. For example, libraries of knottin proteins have been developed. These have been used in a variety of applications, including bacterial display, phage display, and yeast surface display. ray, fluorescence activated cell sorting (FACS), and / or any other suitable screening The antibodies can be screened by a screening method.

[0055] Yeast surface display has been shown to enhance the expression of novel molecular recognition properties, increased target binding affinity, and proper folding of soluble nucleotides. Potent enzymes that have been used to generate proteins with improved folding and stability This platform allows for the synthesis of desired biochemical and biological products. Libraries of protein variants are screened for high-throughput screening to isolate variants with specific physiological properties. Yeast surface display can be used to generate and screen engineered vectors. This is a successful combinatorial method for generating knottins with enhanced molecular recognition. Yeast surface display has been demonstrated to be a novel method for the expression of proteins via the eukaryotic secretory pathway, with the assistance of chaperones. These proteins benefit from quality control mechanisms for efficient folding and efficient disulfide bond formation.

[0056] Developing knottin peptides with engineered loops that bind to cell surface molecules of interest One exemplary approach for this is to use a yeast cell wall protein bound by two disulfide bonds. The peptide is attached to the yeast mating agglutinin protein Aga2p, which is attached to the protein Aga1p. This Aga2p fusion construct and its integration into the chromosome are then used. The Aga1p expression cassette may be driven by a suitable promoter, such as a galactose-inducible promoter. The N- or C-terminal epitope tag can be expressed under the control of a fluorescently labeled primary or Use a secondary antibody to measure cell surface expression levels by flow cytometry This construct may contain the N-terminus of the knottin (or other protein to be engineered) Aga2, which represents the most widely used display format, but Several alternative variations of the mother surface display plasmid have been described and are included in the complex of the present disclosure. The method may be employed to develop knottin peptides for use in the body. This screening process is superior to the panning-based methods used in mRNA display. One advantage of the platform is that two-color FACS can be used to identify antibodies with binding affinity to desired targets. It is possible to quantitatively distinguish clones that differ in sex by only about two-fold.

[0057] To selectively mutate knotting loop regions at the DNA level, for example, overlap extension Degenerate codons can be introduced by PCR-based oligonucleotide assembly. Then, the vector is sufficiently overlapped with the yeast display vector for homologous recombination in yeast. Genetic material can be amplified using adjacent primers. The method allows the creation of knotting libraries at relatively low cost and effort. Synthetic oligonucleotide libraries allow for clear control over library composition. Braille and modern methods have been developed.

[0058] In certain embodiments, a display library (e.g., a yeast display library) ) are screened for binding to the cell surface molecule of interest by FACS. When screening a knotting library by ACS, typically 4-7 loads are required. Upon sorting of the strands, an enriched pool of binders emerges. For this purpose, two-color FACS may be used, in which one fluorescent label is the c-myc enzyme. The pitope tag is used to detect the target of interest, while the other fluorescent label is used to detect the notch for the target of interest. This can be used to measure the interaction of ting mutants with single cell resolution. To measure the excitation and emission properties of two fluorophores, different instruments laser and / or A filter set can be used to normalize yeast expression levels by binding. This allows for the development of notches that exhibit low yeast expression but bind to highly abundant targets. knottins, which are expressed at high levels but bind weakly to their targets, can be distinguished from knottins, which are expressed at high levels but bind weakly to their targets. Thus, in a two-dimensional flow cytometry plot of binding versus expression, the target appears on the diagonal. A population of yeast cells that bind to the antigen emerges. High affinity binders pass the library selection gate. Alternatively, full length proteins can be isolated in the first selection round. It may be useful to remove unwanted clones from the library that do not express the protein. The targets used in screening must be structurally and functionally relevant to the final application. These molecules are specifically related, for example, to mimic a cell surface molecule of interest.

[0059] Enriching the knottin library for binders to cell surface molecules of interest Yeast plasmids are then recovered and sequenced. Further rounds of FACS can then be performed under the control of the individual yeast. The binding affinity or dynamic dissociation rate of the selected knottin clones can be measured.

[0060] A knottin peptide with an engineered loop that binds to a cell surface molecule of interest is provided on the surface. Once identified by display (e.g., yeast surface display), the modified Knockout Knottins can be produced using suitable methods. Due to their small size, knottins are chemically Production by both synthetic and recombinant expression is possible. The peptides can be produced by solid-phase peptide synthesis followed by in vitro folding. Chemical synthesis can be used to incorporate unnatural amino acids or other chemical handles into knottin peptides. It can be easily incorporated.

[0061] Knottin peptides that are not fused to large heterologous domains can be synthesized on an automated synthesizer. are readily synthesized using solid-phase peptide chemistry. Fmoc-based solid phase peptide chemistry can be utilized. The thiols then promote the oxidation of the cysteine ​​side chain thiols to form disulfide bonds. The resulting product is then folded under conditions, followed by, for example, reversed-phase high-performance liquid chromatography (RP-HPLC). C).

[0062] In certain embodiments, the knottin peptide or a fusion protein comprising the same is prepared by recombinant DNA technology. Knottin can be produced using recombinant methods in a variety of host cell types. Any suitable strategy for producing peptides can be utilized. For example, peptides in E. coli can be produced. Facilitating replasmic folding and serving as a useful purification handle Functional knottins were produced using barnase as a gene fusion partner. According to embodiments, the modified knottin peptide is expressed in yeast. For example, The strain Pichia pastoris produced 2–10 mg / L of purified modified knottin. Yeast expression constructs can be used to express one or more tags (e.g., metal For purification by chelate chromatography (Ni-NTA), e.g., C-terminal hexyl The protein may then encode a sahistadine tag (Sahistadine tag). This will then result in the formation of aggregates, misfolded multimers, etc. Size exclusion chromatography can be used to remove

[0063] Aspects of the present disclosure include knottin peptides and fusion proteins employed in the conjugates of the present disclosure. The nucleic acid encoding the protein is an engineered loop that binds to a cell surface molecule of interest. Any of the knottin peptides and fusion proteins described herein having Nucleic acids encoding the nucleotides are provided. In certain embodiments, such nucleic acids are present in an expression vector. The expression vector contains a nucleic acid encoding a knottin peptide operably linked thereto. a promoter selected to express a knottin peptide. The nucleic acid encoding the knottin peptide of the present disclosure is selected based on the type of host cell. Also provided are host cells comprising any of the above, as well as any expression vectors comprising same.

[0064] Direct or competitive binding assays can be used to identify specific binding sites in cells (e.g., mammalian cancer cells). A method for measuring the affinity of knottins for molecules expressed on the surface of cancer cells is provided. In direct binding assays, the equilibrium binding constant (K D ) is a fluorophore or radioisotope-conjugated knottins, or for detection by labeled antibodies. can be measured using knottins containing N- or C-terminal epitope tags In cases where labels or tags are not feasible or desirable, competitive binding assays can be used. Half-maximal inhibition is the amount of unlabeled knottin that is detectable when 50% of the maximal signal of the labeled competitor is reached. Harmful concentration (IC 50 ) can then be used to determine the measured IC 50 Value to K D Ligand depletion results in high affinity interactors. This is more pronounced when measuring in the low concentration range, and reducing the number of cells added to the experiment This can be avoided or minimized by increasing the volume of the binding reaction. This can be done.

[0065] In certain embodiments, the knottin peptide is present at a concentration of about 0.01 nM to 100 nM, for example about 0. 0.25nM to 75nM, approximately 0.05nM to 50nM, approximately 0.075nM to 25nM, and The equilibrium binding constant (K) for cell surface molecules is approximately 0.1 nM to 10 nM. D ) In some embodiments, the knottin peptide is present at a concentration of about 0.1 nM to 10 nM of a cell surface molecule. The equilibrium binding constant for the molecule (K D In some embodiments, the knottin peptide The ATP has an equilibrium binding constant (K) for cell surface molecules of approximately 0.1 nM. D ) have some In this embodiment, the knottin peptide has an equilibrium binding to cell surface molecules of about 0.5 nM. Coupling constant (K D In some embodiments, the knottin peptide has a , the equilibrium binding constant for cell surface molecules (K D In some embodiments, the notch The tin peptide has an equilibrium binding constant (K ) for cell surface molecules of approximately 5 nM. D ) In some embodiments, the knottin peptide has an affinity for a cell surface molecule of about 10 nM. Equilibrium binding constant (K D )

[0066] Yeast surface display technology, including construction and screening of knottin libraries Engineering knottins through surgery and producing knottins through chemical synthesis and recombinant expression; Furthermore, direct or competitive binding assays were used to identify receptors expressed on cell surfaces. Detailed guidance and tools for cell binding assays measuring the affinity of knottins to target proteins. Moore, S. and Cochran, J. (2012) E ngineering Knottins as Novel Binding Age nts, ​​Methods in Enzymology, 503, 223-251 It is listed.

[0067] Linker The immunostimulatory agents of the present disclosure may be conjugated to the knottin peptide via a variety of suitable linkers. Linkers that find use in the conjugates of the present disclosure include ester linkers, amino linkers, and the like. linker, maleimide or maleimide-based linker, valine-citrulline linker -, hydrazone linker, N-succinimidyl-4-(2-pyridyldithio)butyrate (SPDB) linker, succinimidyl-4-(N-maleimidomethyl)cyclohexane San-1-carboxylate (SMCC) linker, vinyl sulfone-based linker, Linkers containing polyethylene glycol (PEG) (e.g., tetraethylene glycol) linkers containing propanoic acid, linkers containing caproleic acid, and any combination thereof.

[0068] In certain embodiments, the linker is a chemically labile linker, e.g., a linker that is labile at neutral pH (blood It is stable at low pH (pH 7.3-7.5) but does not react with weakly acidic endosomes of target cells (e.g., cancer cells). During internalization into the membrane (pH 5.0-6.5) and lysosome (pH 4.5-5.0) Chemically unstable linkers include acid-cleavable linkers, which are easily hydrolyzed. Dorazone-based linkers, oxime-based linkers, carbonate-based linkers, ester-based linkers According to certain embodiments, the linker is an enzyme A labile linker, e.g., stable in the bloodstream but unstable upon internalization into target cells. , for example, lysosomal proteases (catechumens) in the lysosomes of target cells (e.g., cancer cells). The linker is an enzyme-labile linker that undergoes enzymatic cleavage by enzymes such as phospholipids or plasmin. Enzyme-labile linkers include linkers containing a peptide bond, e.g., valine-citrulline. Linkers (e.g., maleimidocaproyl-valine-citrulline-p-aminobenzyl (M C-vc-PAB) linker), valyl-alanyl-para-aminobenzyloxy (Va Dipeptide-based linkers, such as the l-Ala-PAB linker, and equivalents, are also included. These include, but are not limited to, chemically labile linkers, enzyme-labile linkers, and Non-cleavable linkers are well known and are described, for example, in Ducry & Stump (201 0) Bioconjugate Chem. 21:5-13.

[0069] In certain embodiments, the conjugates of the present disclosure are prepared using methods for making the conjugates and methods described later in this disclosure. and to the knottin peptide via a linker as described in the experimental section and figures. Conjugated immunostimulants are included.

[0070] Method for preparing the complex Methods for producing the complex are also provided. According to some embodiments, the knottin-immunized A method for making a stimulating agent conjugate, comprising attaching an immunostimulating agent to a notching peptide via a linker. In certain embodiments, a method is provided comprising conjugating the conjugate to a Gating involves functionalizing the immunostimulatory agent and notching the functionalized immunostimulatory agent. and conjugating the peptide to a notch peptide. The peptides and / or immunostimulants are described in the preceding specification and in the experimental section below. and / or an immunostimulant selected from any of the knottin peptides and / or immunostimulants described in can be.

[0071] According to some embodiments, the immunostimulatory agent comprises a primary amine, and the immunostimulatory agent is functionalized. The reaction involves reacting a primary amine with an amine-reactive linker. Reactive linkers are employed in practicing the methods for making knottin-immunostimulatory agent conjugates. According to one non-limiting example, the amine-reactive linker can be an amine-reactive NHS In certain embodiments, when an amine-reactive linker is employed, In the present invention, the amine-reactive linker is bicyclo[6.1.0]nonyne (BCN), dibenzyl containing a moiety selected from zocyclooctyne (DBCO), and an azide moiety. When the amine-reactive linker contains such a moiety, the functionalized immunostimulatory agent can be attached to the Conjugating the amine-reactive linker to the peptide converts the portion of the amine-reactive linker to a notch. According to some embodiments, the method may include reacting a portion of the notch peptide with a notch peptide. The knottin peptides include unnatural amino acids that comprise the above-described portions of the knottin peptides. A natural amino acid may provide the azide moiety. In one non-limiting example, the azide moiety is Incorporation of one or more 5-azido-L-norvalines into the desired position of the knottin peptide In certain embodiments, the functionalized immunostimulatory agent is reacted with The amino acid portion of the peptide is the N-terminal amine group.

[0072] In some embodiments, the knottin-immunostimulatory agent conjugates of the present disclosure are and 9, and / or as described in the Experimental Section below. It is made like this.

[0073] Figure 2 provides exemplary strategies for functionalizing immunostimulatory agents. (A) Conjugates Immunostimulants with primary amines available for cleavage are (B) click chemistry handles. Amine-reactive NHS ester linkers (e.g., BCN, DBCO, azide) (C) Click chemistry handle (e.g., BCN, DBCO, azide) This can result in a functionalized immunostimulatory agent having a carboxyl group, which can be used to couple the immunostimulatory agent to a tumor targeting agent. It is used to conjugate (illustrated in Figure 4).

[0074] Figure 3 provides exemplary sequences and illustrations of knottin peptides. (A) Integris The disulfide bond of the cysteine-binding loop (PRPRGDNPPLT) and the cysteine ​​knot scaffold (B) Sequences of knottin peptides 2.5F and 3CM with the N-terminal amine group shown. 3CM with 5-azido-L-norvaline at the 2.5F and X1 positions shown. Schematic diagram of the knottin peptide structure. Knottin peptide 2.5F has a hydroxyl group at the N-terminal amine group. While 3CM can be conjugated to the X1 azide moiety (for this purpose 3CM can be conjugated at the unnatural amino acid incorporated into It also has available N-terminus fragments that can be reacted with probes such as immunostimulants or fluorophores. It also has a terminal amine group (see Figure 9). The amine was replaced by tyrosine in 3CM to facilitate concentration determination by UV absorption. Either amino acid at X2 can be used without compromising binding affinity.

[0075] Shown in Figure 4 is a conjugation of a knottin peptide to a functionalized immunostimulatory agent. (A) The X1 azide of 3CM is a strain-promoting azide. SPAAC was used to generate BCN- or DBCO-functionalized immunosorbent compounds. Immunostimulants (e.g., referred to herein as "C" in the figures, description, and experimental section) An oligonucleotide containing one or more CpG dinucleotides, sometimes referred to as "CpG" Reaction with dopamine-based TLR 9 agonists (e.g., CpG ODN), or T78a (B) The N-terminal amine of 2.5F can be converted to azido- The N-terminal azide can be modified using a PEG4-NHS ester linker. C can be used to react with BCN or DBCO functionalized immunostimulants.

[0076] FIG. 8 (top) shows a knottin-immunostimulatory agent conjugate (in this example, Knottin) according to an embodiment of the present disclosure. 10A-10C are schematic diagrams illustrating how to generate notched Fc-T78a. To conjugate KFc to T78a, To achieve this, BCN-modified KFc and azido-T78a were used.

[0077] Figure 9 (top) shows a method for detecting knottin, in which knottin is further conjugated to a detectable label. 10A and 10B are schematic illustrations of a method for making a chemoattractant-immunostimulatory agent conjugate. In this example, a detectable label is is AlexaFluor 680. To synthesize 3CM-CpG-AF680, According to this exemplary method, 3CM is N-terminally substituted with AF680-NHS ester (AF680-NHS ester). The DNA was modified with a fluorophore and DBCO-CpG at the X1 azide.

[0078] composition As summarized above, the present disclosure provides compositions. The compositions contain the above-described complexes. Section 10.1 of the IEEE International Conference on Computer Vision (ICCV) 2010, which is incorporated herein by reference, but will not be repeated here for brevity. The conjugates may include any of the conjugates of the present disclosure, including conjugates formed by the method of the present invention.

[0079] In certain aspects, the composition comprises a complex of the present disclosure present in a liquid medium. The medium may be an aqueous liquid medium such as water, a buffer solution, and the like. gCl2, KCl, MgSO4), buffer (Tris buffer, N-(2-hydroxyethyl ) piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino ) Ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris[ hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), proteases One or more additives, such as enzyme inhibitors, glycerol, and the like, may be present in such compositions. may exist in

[0080] Pharmaceutical compositions are also provided. The pharmaceutical compositions comprise any of the conjugates of the present disclosure and a drug. The pharmaceutical composition generally contains a therapeutically effective amount of the conjugate. A "therapeutically effective amount" is a dosage sufficient to produce a desired result, e.g., a dose sufficient to induce the formation of a modified loop. Individuals with a cell proliferative disorder (e.g., cancer) associated with the cell surface molecule to which it binds and producing a beneficial or desired therapeutic (including preventative) result, such as a reduction in cell proliferation in a subject. An effective amount can be administered in one or more administrations.

[0081] The conjugates of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. Specifically, the conjugate is prepared by combining it with a suitable pharmaceutically acceptable excipient or diluent. Therefore, it can be formulated into a pharmaceutical composition, such as a tablet, capsule, powder, granule, or ointment. solid, semi-solid, liquid, or gaseous forms, such as solutions, injectables, inhalants, and aerosols The composition may be formulated into the following formulations:

[0082] Formulations of the conjugates of the present disclosure suitable for administration to an individual (e.g., suitable for human administration) generally include: It is sterile and, furthermore, contains no contraindications for administration to an individual according to the chosen route of administration. The composition may be free of exothermic pyrogens or other contaminants.

[0083] In pharmaceutical dosage forms, the conjugates may be used alone or in suitable association with other pharmaceutically active compounds. The following methods and excipients are merely examples, and It is by no means limiting.

[0084] For oral formulations, the complex may be, for example, lactose, mannitol, corn starch, or the like. conventional additives such as cellulose or potato starch, crystalline cellulose, cellulose derivatives, Binders such as gum arabic, cornstarch, or gelatin, corn starch, Disintegrants such as potato starch or sodium carboxymethylcellulose, talc lubricants, such as cellulose or magnesium stearate, and, if desired, diluents, buffers, humectants, Lubricants, preservatives, and flavoring agents are used to prepare tablets, powders, granules, or capsules. These compounds can be used alone or in combination with suitable additives to achieve the above effects.

[0085] The complex may contain vegetable oils or other similar oils, synthetic fatty acid glycerides, and esters of higher fatty acids. Dissolved, suspended, or emulsified in aqueous or non-aqueous solvents such as propylene glycol, and, if desired, adding a solubilizing agent, an isotonic agent, a suspending agent, an emulsifying agent, a stabilizing agent, The composition can be formulated into an injectable preparation using conventional additives such as antiseptics and antiseptics.

[0086] The pharmaceutical composition may be in liquid form, lyophilized form, or in liquid form reconstituted from lyophilized form. The lyophilized preparation should be reconstituted with a sterile solution prior to administration. The standard procedure for reconstituting a lyophilized composition is to add a certain amount of purified water (typically a lyophilized The solution containing the antimicrobial agent is added back into the syringe (a volume equal to the volume removed during the injection), but It may also be used for the production of pharmaceutical compositions for oral administration.

[0087] Aqueous formulations of the complex may have a pH of, for example, about 4.0 to about 8.0, such as about 4.5 to about 7.5, e.g. The compound may be prepared in a pH buffer solution in the range of about 5.0 to about 7.0. Examples of buffers that are phosphate, histidine, citrate, succinate, acetate, and other organic The concentration of the buffer may vary depending, for example, on the buffer and the desired tonicity of the formulation. , about 1 mM to about 100 mM, or about 5 mM to about 50 mM.

[0088] kit Aspects of the present disclosure further include kits. In some embodiments, the subject kits include the Any of the conjugates shown (incorporated herein but not repeated here for brevity, see above) or a pharmaceutical composition containing the same. The pharmaceutical composition includes a pharmaceutical product and instructions for administering the pharmaceutical composition to an individual in need thereof. In embodiments, the complex binds to a cancer cell surface molecule and / or a molecule on the surface of a tumor vasculature cell. an engineered loop that binds to a tumor antigen (e.g., as described elsewhere herein, a modified loop that binds to a cell adhesion receptor (e.g., an integrin), or the like. The pharmaceutical composition comprises a peptide, and instructions are provided for administering the pharmaceutical composition to an individual having cancer to treat the cancer. It is intended to treat.

[0089] In some embodiments, the conjugate or pharmaceutical composition comprises one or more (e.g., two) The term "unit dose" as used herein means a unit dose of a compound of formula (I) or (II). refers to physically discrete units suitable as single dosages for human and animal subjects, Each unit contains a predetermined amount of the complex or composition calculated to be sufficient to produce a desired effect. The amount of the unit dose will depend on the particular complex employed, the effect to be achieved, and It depends on various factors, such as the pharmacodynamics associated with the complex in an individual. In embodiments, the kit may include a single multiple dose of the conjugate or pharmaceutical composition.

[0090] The components of the kit may be in separate containers, or multiple components may be in a single container. may exist in

[0091] The instructions included in the kit may be recorded on a suitable recording medium. For example, the instructions may be written on paper. or printed on a substrate such as plastic. Thus, the instructions may be presented as a package insert. or in the labeling of the kit container or its components (i.e., In other embodiments, the instructions may be present on a suitable computer. computer-readable storage media, such as portable flash drives, DVDs, CD-ROMs, It exists as an electronically stored data file, such as on a diskette. In this form, there are no actual instructions in the kit, but they are available via, for example, the internet. Means are provided for obtaining instructions from a remote source. An example of this embodiment is: A web address where the instructions can be viewed and / or from which the instructions can be downloaded. The kit includes the instructions, as well as the means for obtaining the instructions, which may be written on a suitable substrate. It will be recorded.

[0092] How to use As summarized above, methods of using the conjugates of the present disclosure are also provided. In embodiments, the method is incorporated herein but will not be repeated here for the sake of brevity. , including using any of the conjugates described in the Conjugates section above.

[0093] In some embodiments, a therapeutically effective amount of any of the conjugates of the present disclosure or the medicament of the present disclosure Methods are provided that include administering any of the compositions to an individual in need thereof. In certain embodiments, the individual has cancer and the complex knottin peptide is present in the individual. engineered molecules that bind to cell surface molecules on cancer cells and / or tumor vasculature cells The pharmaceutical composition comprising the complex is administered to an individual in an amount effective to treat cancer. Thus, an aspect of the present disclosure is to administer to an individual with cancer a therapeutically effective amount of a complex of the present disclosure. and a method for treating cancer by administering either one of the compounds or a pharmaceutical composition thereof. include.

[0094] A variety of individuals can be treated according to the subject method. Generally, such individuals are "Mammals" or "mammals" and these terms include carnivores (e.g., dogs and cats). ), rodents (e.g., mice, guinea pigs, and rats), and primates (e.g., to describe organisms within the class of mammals, which includes mammals (humans, chimpanzees, and monkeys) In some embodiments, the individual is a human. In some embodiments, the individual is an animal model, such as a mouse model.

[0095] In some embodiments, an effective amount of the conjugate (or a pharmaceutical composition comprising the conjugate) is alone (e.g., in monotherapy) or in combination with one or more additional therapeutic agents When administered in one or more doses (e.g., in combination therapy), the complex or drug The individual's condition (e.g., a decrease in the severity of a disease) compared to the individual's condition in the absence of treatment with the composition. symptoms of cancer) by at least about 5%, at least about 10%, at least about 15% %, at least about 20%, at least about 25%, at least about 30%, at least about 40 %, at least about 50%, at least about 60%, at least about 70%, at least about 80 %, at least about 90% or more.

[0096] In some embodiments, the individual has cancer and the methods of the disclosure are for treating the cancer in the individual. In certain embodiments, the individual has a solid tumor, a semi-solid tumor, a primary tumor, The presence of metastatic tumors, liquid tumors (e.g., leukemia, lymphoma, etc.), and / or the like In some embodiments, the individual has a cancer characterized by: glioblastoma, neuroblastoma, Blastocysts, head and neck cancer, stomach cancer, ovarian cancer, skin cancer (e.g., basal cell carcinoma, melanoma) , or equivalent), lung cancer, colorectal cancer, prostate cancer, glioma, bladder cancer, intrauterine membrane cancer, kidney cancer, leukemia (e.g., acute myeloid leukemia (AML)), liver cancer (e.g., , hepatocellular carcinoma (HCC), such as primary or recurrent HCC, non-Hodgkin's lymphoma, pancreatic cancer, thyroid cancer, B-cell malignancies, any combination thereof, and any of these According to certain embodiments, the individual has a cancer selected from the subtypes of neoplastic and / or or have a condition characterized by the presence of malignant cells.

[0097] "Treat," "treating," or "treatment" "(Treatment)" refers to the treatment of a disease state (e.g., a cell proliferative disorder, e.g., cancer) in an individual. and improvement in the symptoms associated with the condition being treated. It is used broadly to refer to at least a reduction in the magnitude of a symptom, a parameter that can be measured, for example. Thus, treatment also involves preventing an individual from experiencing a medical condition or at least symptoms that characterize the medical condition. A medical condition (e.g., cancer) or at least a condition associated with it, so that further suffering is prevented. The symptoms that are caused are completely inhibited, e.g., prevented from occurring or stopped. This also includes situations where the project has been discontinued, for example, or has been discontinued.

[0098] The conjugates or pharmaceutical compositions may be used in in vivo and ex vivo methods, as well as in systemic and using any available method and route suitable for drug delivery, including topical administration routes; It can be administered to an individual by conventional pharmaceutically acceptable routes of administration, including intranasal, intramuscular, intratracheal, , subcutaneous, intradermal, topical application, intraocular, intravenous, intraarterial, nasal, oral, and other enteral and Parenteral routes of administration are included. In some embodiments, administration is by parenteral administration. The route of administration may be varied, if desired, in combination, depending on the complex and / or the desired effect. The complex or pharmaceutical composition may be administered in a single dose or in multiple doses. In some embodiments, the conjugate or pharmaceutical composition is administered intravenously. In some embodiments, the conjugate or pharmaceutical composition is administered intravenously, e.g., by systemic delivery (e.g., intravenous delivery). injection) or to a local site, e.g., intratumoral injection, peritumoral injection, and / or is administered by equivalents.

[0099] In some embodiments, the individual has a solid tumor. In the case where the subject has a solid tumor, the method includes administering a knottin-immunostimulatory agent conjugate of the present disclosure to the subject. As demonstrated herein, such conjugates can be administered systemically. showed an unexpected ability to localize to solid tumors following immunostimulation, and According to some embodiments, the individual: The subject has a solid tumor, and the administration is by systemic administration, and the immune cell microenvironment of the solid tumor is Increased immune cell microenvironment in tumors compared to when immune stimulants alone are administered systemically to individuals increased percentage of CD8+ T cells, increased percentage of CD4+ T cells, increased percentage of B cells, and and / or a reduced proportion of myeloid-derived suppressor cells (MDSCs) According to some embodiments, the individual has a solid tumor and the administration is systemic. The percentage of CD8+ T cells, CD4+ T cells, and B cells was determined by the administration. and / or the proportion of myeloid-derived suppressor cells (MDSCs), or any combination thereof. The immune cell microenvironment of solid tumors, as assessed by immunostimulatory agents, is assessed by intratumoral administration of immunostimulants alone. The results were not statistically significantly different compared to the immune cell microenvironment of the tumor when it was treated with HCl.

[0100] In some embodiments, the individual is provided with a therapeutic agent that crosses the blood-brain barrier (BBB). A non-limiting example of such a cancer is a brain tumor, e.g., In some embodiments, the individual is diagnosed with a glioblastoma or an equivalent. In cases where the patient has a cancer that requires the complex to It involves administering a low molecular weight conjugate, such as a drug conjugate, to an individual.

[0101] The following examples are offered by way of illustration and not by way of limitation. [Example]

[0102] experiment Example 1 - Knottin-CpG complexes with CpG incorporated at different sites In this example, immunostimulatory agents incorporated at different sites were used to compare the knottin peptide -immunostimulatory agent conjugates were prepared. This particular example demonstrates the use of Cp CpG ODN conjugated to CpG The left panel of Figure 5 shows the amino-Cp G, 3CM-CpG (X1 azide), and 2.5F-CpG (N-terminal azide) The results of the RAW-Blue NF-κB activation assay are shown. The right panel of Figure 5 shows 3CM, 3C, and 3D conjugated at the sites indicated in parentheses. Competitive results comparing M-CpG (X1 azide) and 2.5F-CpG (N-terminal azide) The results of the combined assay are shown.

[0103] The knottin-CpG complex (at either conjugation site) showed a similar NF-κB activation profile compared to -CpG (positive control), and The binding profile was similar to that of the modified 3CM (positive control). These conjugation sites (X1 azide or N-terminal azide) also confer TLR agonist activity. To synthesize knottin-CpG complexes without adversely affecting the activity or binding affinity of the Amino-CpG has the same NF-κB activation activity as unmodified CpG. The profiles are shown (data not shown).

[0104] Example 2 - Knottin-CpG conjugates synthesized with different linkers In this example, different linkers were used to form knottin peptide-immunostimulatory agent conjugates. This particular example shows the use of different linkers to conjugate to CpG. The left panel of Figure 6 shows the amino-CpG, 3CM knottin peptide. RAW-Blu for CM-CpG (DBCO) and 3CM-CpG (BCN) e Results of NF-κB activation assay are shown. For both complexes, CpG was 3C The right panel of Figure 6 shows 3CM, 3CM- Results of competitive binding assay comparing CpG(DBCO) and 3CM-CpG(BCN) The results are shown.

[0105] The knottin-CpG complexes were either amino-CpG or CpG (positive vs. negative) with either linker. showed a similar NF-κB activation profile compared to unmodified 3CM (positive control) and The binding profiles of both linkers were similar compared to the control. Neither of the treatments (DBCO or BCN) had any adverse effects on TLR agonist activity or binding affinity. It can be used to synthesize knottin-CpG conjugates without any modification.

[0106] Example 3 - Knottin-TLR7 / 8 agonist (T78a) conjugate In this example, knottin peptide-immunostimulating agents were prepared and tested, where knottin peptides were The binding peptide was 3CM, and the immunostimulant was the TLR7 / 8 agonist T78a. The left panel of Figure 7 shows the R The results of the AW-Blue NF-κB activation assay are shown in the right panel of Figure 7. , The results of a competitive binding assay comparing 3CM and 3CM-T78a are shown.

[0107] The TLR7 / 8 agonist (T78a) was tested at the highest concentrations (500–5,000 nM ) activated NF-κB only at a concentration higher than that of 3CM-T78a, whereas the 3CM-T78a complex was active at a lower concentration. Furthermore, 3CM-T78a induced activation of the unconjugated showed a similar binding profile to 3CM-modified integrins, thus demonstrating high binding potential to integrins. Affinity was maintained.

[0108] Example 4 - Knottin-Fc immunostimulatory agent conjugate In this example, a Notin-1 antibody fused to an Fc domain and conjugated to an immunostimulatory agent was Conjugates containing the peptide were synthesized and tested. Figure 8 (top) shows the conjugate (in this example, 10A-10C are schematic diagrams illustrating how to generate Knottin-Fc-T78a. Conjugating KFc-T78a to T78a To achieve this, BCN-modified KFc and azido-T78a were employed. Figure 8 (lower panel) shows RAW-Blue NF for KFc, T78a, and the KFc-T78a complex 1 shows the results of a -κB activation assay.

[0109] KFc-T78a activated NF-κB more effectively than either KFc or T78a at each concentration tested. The synthesis of KFc was significantly higher than that of azide-NHS esters (p<0.0001). modified with a carboxyl linker and conjugated to BCN-T78a (or BCN-CpG) The BCN and azide groups can be interchanged, as shown in Figure 1. DBCO can also be substituted for BCN. can.

[0110] Example 5 - Detectably Labeled Knottin-Immunostimulatory Agent Conjugates In this example, the immunostimulatory agent and the detectable label (here, AlexaFluor A conjugate containing a knottin peptide conjugated to 680 was synthesized and tested. Figure 9 (top) illustrates the methodology employed in this example. To synthesize AF680, 3CM was converted to AF680-NHS ester ( The DNA was modified with a fluorophore (Figure 9) and DBCO-CpG at the X1 azide. (Bottom) Competitive binding assay of 3CM-CpG-AF680 compared to 3CM. The results are shown.

[0111] The binding affinity of 3CM-CpG-AF680 is not significantly different from that of 3CM (unpaired (determined by Student's t-test). Data are the mean (± standard deviation) of three independent experiments. The mean deviation (SD) is shown.

[0112] Example 6 - Non-invasive in vivo and ex vivo fluorescence imaging In this example, the 3CM-CpG-AF680 complex was injected intratumorally and peritumorally. Non-invasive in vivo fluorescence imaging of the body was performed, and the results are shown in Figure 10. Mice bearing two CT26 colon cancer tumors were treated with intratumoral (IT, left intratumor) or intratumoral (IT, left intratumor). 3CM-CpG-AF680 was injected around the tumor (PT, adjacent to the left tumor) at the indicated dose. "Dose" refers to a typical dose (equimolar) for intratumoral CpG therapy = 5.2 nmol 1 (50 μg). The time indicated in hours to the left of the image is the number of hours after injection. The 3CM-CpG-AF680 complex was injected IT or PT followed by 4 h or 26 h After time, it did not localize to non-injected tumor sites.

[0113] This example also demonstrates the non-invasive detection of intravenously injected 3CM-CpG-AF680 complexes. In vivo fluorescence imaging was performed, and the results are shown in Figure 11. Two CT2 Six colon cancer tumor-bearing mice were intravenously injected with 3CM-CpG-AF680 at the indicated doses. (IV, tail vein). "Dose" refers to a typical dose (e.g., The time indicated in hours on the left side of the image is the time of the 3CM-CpG-AF680 (4x dose) was administered within 4 hours after IV injection. The lower dose also localized to both tumors within 24 hours and remained within the tumor site for over 24 hours. This may result in localization to the tumor, but is not measurable by in vivo fluorescence imaging.

[0114] Tumors were excised 26 hours later and injected with 2x and 4x doses (see Figure 12) Ex vivo tumor fluorescence was observed in mice. The results are shown in Figure 12. Intratumoral (IT, left intratumor) tumors were observed in a mouse bearing two CT26 colon cancer tumors on the shoulder. Peritoneal (PT, adjacent to the left tumor) or intravenous (IV, into the tail vein) as indicated The dose of 3CM-CpG-AF680 was injected into the tumor. Typical dose (equimolar) = 5.2 nmol (50 μg). Tumors grew 26 hours after injection. The ex vivo images supported the observations from the in vivo imaging. A) 3CM-CpG-AF680 inhibited the inflammatory response after 26 hours following IT or PT injection. It is not localized in the uninjected tumor (right tumor). B) 3CM-CpG-AF680 delivered via PT exhibited tumor uptake at 26 hours post-injection. C) IV 3CM-CpG-AF680 delivered at 4× the dose significantly increased both tumors at 26 hours post-injection. Localization was observed in one patient receiving 3CM-CpG-AF680 (2x dose) delivered intravenously. This was also observed in tumors of

[0115] Example 7 - Therapeutic efficacy of knottin-immunostimulatory agent conjugates as demonstrated by tumor growth In this example, the therapeutic efficacy of exemplary knottin-immunostimulatory agent conjugates was evaluated. As proof of principle, 3CM-CpG conjugates were employed in this example. Aggressive 4T1 breast cancer A mouse model was used: 3CM-C in 4T1-Luc breast cancer (n=9-10). Survival curves demonstrating the therapeutic efficacy of pG are shown in Figure 13. Three doses of intravenous knotting Mice bearing established tumors received 3 doses of intravenous unmodified CpG or Compared to mice treated with vehicle control, they had significantly prolonged survival.

[0116] Furthermore, knottin-CpG treatment induced complete tumor regression in 6 of 9 mice. The remaining three mice showed delayed tumor growth compared to vehicle-treated mice. Of the mice that showed tumor regression, 50% (3 of 6) of the mice continued to show signs of remission for several months following tumor regression. The patient was cured without any signs of tumor recurrence. These results are significant and unexpected, as achieving a cure through this approach is unprecedented.

[0117] Also, 141 days after inoculation, surviving mice used twice the number of cells used in the original inoculation. A rechallenge experiment was also performed in which 4T1-luc tumor cells were re-injected subcutaneously into the opposite flank. Naive mice were also injected with the same number of tumor cells. 16 days after inoculation, None of the surviving mice had tumors, while all naive mice developed tumors. He was standing.

[0118] Figure 14 shows the 4T1-Luc mean tumor growth over time (from the vehicle group). The mean tumor volume by the time the first mouse was euthanized is plotted for each group. Arrows indicate treatment days (7, 9, 11) for the group receiving 3 doses and 1 for the group receiving 1 dose. Only on day 7. Data represent mean ± SEM (n = 9-10).

[0119] Individual 4T1-Luc tumor growth curves are shown in Figure 15. Complete response without relapse The proportion of mice from each group demonstrating complete recovery (CR) ("long-term survivors") and CR with relapse were calculated. The percentage of mice showing CR is shown on each plot. CR was defined as complete regression of the tumor. CR with recurrence is defined as complete regression of the tumor followed by regrowth at some point after tumor regression. It is defined as a regression.

[0120] Example 8 - Alteration of the tumor immune microenvironment by knottin-immunostimulatory agent complexes In this example, the therapeutic efficacy of an exemplary knottin-immunostimulatory agent conjugate administered intravenously was The mechanism of action was assessed by assaying tumor-infiltrating immune cells. One motivation behind the development of tin-immunostimulatory drug conjugates is targeted delivery to tumor sites. The goal was to enable systemic injection of the immunostimulant Knottin, which reaches the tumor site. When stimulating an immune response, immune cell progenitors within the tumor are required to promote this anti-tumor immune response. It is expected that the file will change (e.g., CD8 + T cell increase). Proof of principle For clarity, 3CM-CpG complexes were employed in this example. As a positive control for the desired change, a 10-mg dose was injected directly into the tumor (and thus reach the tumor site) to detect the immune response. A group of mice treated with intratumoral CpG, known to locally stimulate tumor responses, was included. Ta.

[0121] 4T1-luc cells were subcutaneously implanted into one side of the abdomen of BALB / c mice and allowed to grow for 9 days. Once tumors developed, mice were divided into the following groups according to the schematic diagram (Figure 16, top left): (n = 3 mice per group) twice by intravenous (IV) or intratumoral (IT) injection Treated: Vehicle IV, CpG IV (18.2 nmol), 3CM-CpG IV (18.2 nmol), and CpG IT (5.2 nmol, typical IT dose). On day 3 post-implantation, tumors were excised and analyzed for tumor-infiltrating immune cells via FACS. Figure 16 shows the results of the CD8+ T cells, CD4+ T cells, B cells, and myeloid-derived suppressor cells (MDS). C) Plots of the abundance of different immune populations, including NK cells (total viable single cells) Using one-way ANOVA with Tukey's multiple comparison test, Statistical analysis was performed. Each group was compared with all other groups. Statistically significant differences were found. Group comparisons that were made are marked by a black line drawn between the two groups with an asterisk on the right. *P<0.05, **P<0.01, ***P<0.001, ****P<0.00 01. All other unmarked group comparisons were not significantly different. , the mean abundance of immune cell populations and two uncharacterized cell populations ("others") in each treatment group ( A pie chart summarizing the % of total viable single cells is shown. Uncharacterized cells may contain other immune cells not defined in the analysis. b Divided into two populations to show expression.

[0122] These results demonstrate that 3CM-CpG IV significantly increased the expression of CD8+ T cells, CD4+ T cells, and As indicated by an increased proportion of B cells and a decrease in myeloid-derived suppressor cells (MDSCs), Thus, the tumor immune profile was significantly altered compared to vehicle IV and CpG IV treatments. Furthermore, this dramatic change in immune cell profile by 3CM-CpG IV was demonstrated. These changes were indistinguishable from those observed from treatment with CpG IT. The results showed that systemic injection of the knottin-immunostimulant complex effectively inhibited the growth of the tumor, just as if the immune stimulant had been injected into the tumor. It demonstrates that immune cell populations can be elicited as if injected directly into the site.

[0123] material and method Cell lines and mice B16F10 melanoma and CT26 colon cancer lines were obtained from ATCC, and 4T1- The Luc breast cancer cell line was obtained as a gift. Tumor cells were cultured in 10% fetal bovine serum and 1% erythrocyte colony-stimulating factor (EC) 6.0. Complete medium containing 100% penicillin / streptomycin (4T1-Luc and CT26 For B16-RPMI 1640 with 50 μM 2-mercaptoethanol Cell lines were routinely checked for mycoplasma contamination. Six to eight week old female BALB / c mice were obtained from Charles River It was purchased from the Laboratory.

[0124] NF-κB activation assay Agonist potency was assessed using RAW-B cells derived from murine RAW 264.7 macrophages. TLR agonists were assessed using the lue reporter cell line (Invivogen). Stimulation of reporter cell lines with NF-κB and AP-1 activation, as well as subsequent signaling pathway leading to the subsequent production of secreted embryonic alkaline phosphatase (SEAP) RAW-Blue cells were treated with various concentrations of TLR agonists (free or complex). The SEAP levels in the supernatant were measured according to the manufacturer's instructions. QUANTI-Blue detection medium was used according to the protocol (Invivogen). The data were compared to untreated controls and quantified by colorimetric analysis. Reported as fold change. Error bars represent standard deviation of experiments performed in triplicate. Statistical differences between conditions and untreated controls were analyzed using Prism software (GraphPad ) determined by ordinary two-way ANOVA and Tukey's multiple comparison test. For statistical significance, *p ≤ 0.05, **p ≤ 0.05 compared to untreated controls. 01, ***p≦0.001, and ****p≦0.0001.

[0125] Competitive binding assay The relative binding affinities of unlabeled knottin and knottin-TLR agonist complexes were compared. For comparison, a cell-based competitive binding assay was performed as described by Cox et al. with some modifications. al.(2016)Angew Chem Int Ed.55(34):9894- The assay was performed as previously described. Unlabeled ligand (i.e., non-fluorescent 3CM-immunoglobulin) was used. To compare the binding affinities of the immunostimulatory complexes, Alexa Fluor 488-labeled 3CM (3CM-AF488) was used as a competitor.

[0126] B16F10 melanoma cells (5 × 10 per sample) 4 ) but using cell dissociation buffer The cells were detached by PBS, washed with PBS, and then added to 200 μL of integrin binding buffer (IBB: 25 μL). 100 mM Tris pH 7.4, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl 0.5 nM 3CM-AF488 in 0.2 mM MnCl2, 1 mM MnCl2, and 0.1% BSA and various concentrations of unlabeled ligand were incubated at 4°C for 3 hours. In binding assays involving ligands mediated through cell surface DNA binding interactions, Cells were incubated with integrin-binding agents prior to incubation to reduce depletion of The cells were pretreated with unmodified CpG. The cells were then incubated with a solution of competitor and unlabeled ligand (final volume 2 Before adding 500 nM unmodified CpG (100 μL / sample), The cells were pretreated with HCl (HCl) at room temperature for 10 minutes.

[0127] The cell-bound fluorescence remaining after several washes with PBS + 0.5% BSA was measured using flow cytometry. The geometric mean fluorescence intensity (MFI) from each sample was determined. , the data set is such that the MFI from cells treated with competitor alone is equal to 100% binding. Half-maximal inhibitory concentration (IC50) values ​​were normalized according to the Cheng-Prusoff method. Using the relationship, the equilibrium dissociation constant (K i ) values ​​determined by nonlinear regression analysis It was determined.

[0128] In vivo fluorescence imaging CT26 colon cancer cells (5 × 10 5 ) was injected subcutaneously into both the left and right shoulders. Five days after tumor establishment, mice were injected with intratumoral (IT, left intratumoral), peritumoral (PT, left intratumoral), and peritumoral (PT, left intratumoral) 3cm- at the indicated doses either intravenously (IV) via the tail vein or intravenously (IV) via the tail vein. CpG-AF680 was injected. The "dose" is the equimolar amount of the standard intratumoral dose of CpG (5. 2 nmol, 50 μg). Therefore, 0.2 x dose = 1 nmol, 1 x dose = 5 .2 nmol, 2 x dose = 10.4 nmol, and 4 x dose = 20.8 nmol. .

[0129] At 4 and 26 hours after injection, mice were anesthetized using 2% isoflurane gas. , Spectral Instruments Imaging Ami Imager After the final scan at 26 hours post-injection, one mouse per group was imaged. The animals were sacrificed and tumors were excised and imaged. Fluorescence imaging settings: Excitation / Emission: 64 0 / 730 nm, excitation power = 10, binning = 2, exposure time = 10 seconds, Fstop = 2, FOV=25.

[0130] Tumor inoculation and treatment studies 4T1-Luc tumor cells (1 × 10 4 ) was injected subcutaneously into the right side of the abdomen of BALB / c mice. Seven days after inoculation, mice were randomly assigned to experimental groups (n per group). Mice were administered three doses (days 7, 9, and 11) or one dose via the tail vein. (Day 7 only) CpG (18.2 nmol, 176 μg) or an equimolar amount of 3CM- Mice in the vehicle group were injected with sterile P PBS was injected intravenously (days 7, 9, and 11). All treatments were formulated in sterile PBS. The tumor size was measured using a digital caliper. The volume was monitored every 2-3 days by a microscope (Mitutoyo) and expressed as a volume (length x width x height). Mice were treated when tumor size reached 1.5 cm in maximum diameter or when tumors reached the guideline. If ulceration occurred according to protocol, the mouse was euthanized. The survival days recorded were the number of days a given mouse The time when euthanasia criteria were reached is indicated. The Kaplan-Meier method was used for survival analysis. P values ​​are Calculated using the log-rank (Mantel-Cox) test.

[0131] Synthetic-functionalized immunostimulants Immunostimulants were synthesized using strain-promoted azide-alkyne cycloaddition (SPAAC) Functionalized with a click chemistry handle to enable attachment to targeting drugs A primary alkylamine-modified TLR7 / 8 agonist (designated T78a) and a 5'- Amino-modified class C CpG-C792 (referred to as amino-CpG) are Me Biosciences and Integrated DNA Technology The immunostimulants were purchased from gies. Conjugated with imidazoquinolines, N1-linked, and class C CpG-linked. In this case, it was the 5' phosphate.

[0132] The primary alkylamine of the immunostimulant (amino-CpG or T78a) is Carbohydrates, i.e., BCN-PEG2-NHS ester, DBCO-PEG4-NHS ester ester, or bicyclononyne (BCN) using azido-PEG4-NHS ester, Dibenzocyclooctyne (DBCO) or N-hydroxybenzoates incorporating an azide handle It was functionalized with a hydroxysuccinimide (NHS) ester linker (see Figure 2). Amino-CpG (1 equivalent) was buffered in 25% DMSO / 75% 100 mM sodium borate The mixture was mixed with NHS ester linker (15 equivalents) in HCl (pH 8.5) and stirred overnight at room temperature. The Zeba spin desalting column (7K) was buffer exchanged into PBS before sample loading. Size exclusion using a MWCO (1000 MWCO) allowed functionalized CpG to bind to the remaining NHS ester linker. T78a (1.2 equiv.) was purified from anhydrous HCl with 4.4 equiv. triethylamine. It was mixed with NHS ester linker (1 equivalent) in DMSO and stirred at room temperature for 5-8 hours. The reactions were monitored using analytical HPLC and / or LCMS using an analytical C18 column. HPLC method for monitoring CpG functionalization: 5% to 100% over 30 minutes. A linear gradient of 65% solvent B (solvent A: 100 mM triethylammonium acetate in water, pH 7.0) 7; Solvent B: acetonitrile; 35 °C). HPLC method for monitoring the functionalization of T78a Method: 2 min isocratic hold at 5% solvent B, followed by 15 min of 5% to 75% solvent B. A linear gradient (solvent A: water + 0.1% TFA; solvent B: acetonitrile + 0.1% TFA; room temperature).

[0133] In addition to the synthesis of DBCO-CpG, DBCO-CpG was also synthesized using Integrated Ordered directly from DNA Technologies (IDT).

[0134] Synthetic knottin peptide conjugate Solid phase peptide synthesis (SPPS) was used to synthesize the knotting peptide using standard Fmoc conditions. Peptide synthesis, cleavage, folding, and HPLC purification processes were performed. The protocol is based on Cox et al. (2016) Angew Chem Int Ed. 55(34):9894-7. Knottin peptides have a lysine residue. Therefore, 2.5F was site-specifically conjugated at the N-terminal amine as follows: Added: Azido-PEG4-NHS ester with 4.4 equivalents of TEA in DMSO linker and stirred overnight at room temperature to produce the N-terminal azide-modified 2.5F, which was then purified by R The modified version of 2.5F, designated 3CM, was purified by P-HPLC. 2 to provide a selective conjugation site (shown as X1 azide in Figure 3A). Synthesis using the unnatural amino acid 5-azido-L-norvaline at position 15 of the .5F sequence In addition, the phenylalanine at position 31 (shown as X2 in Figure 3A) In 3CM, it was replaced with tyrosine to facilitate concentration measurement by UV absorption. However, either amino acid at position 31 can be used without compromising binding affinity. This can be done.

[0135] As shown in Figure 4, azide-containing knothin (3CM or azide-2.5F) (1 2 equivalents) was combined with BCN- or DBCO-modified CpG (1 equivalent) in PBS at 30°C. The reaction was continued overnight, and the knottin-CpG complex (3CM-CpG or 2.5F-CpG) The eluate was produced using a Zeba spin desalting column ( The knottin-CpG complex was isolated by size exclusion using a 7K MWCO (1000 MWCO) to separate the unreacted To produce 3CM-T78a, the knottin peptide was purified from BCN-T7. 8a (1.15 equiv.) was mixed with 3CM (1 equiv.) in 1:1 DMSO / PBS overnight at room temperature. The reaction was stirred and allowed to react. The reaction was then purified by RP-MS on a C18 column using the following method: Purification was performed by HPLC: starting with an isocratic hold at 5% solvent B for 2 minutes, followed by A linear gradient of 5% to 75% solvent over 30 min (solvent A: water + 0.1% TFA; solvent B: water + 0.1% TFA) was performed. (Acetonitrile + 0.1% TFA; room temperature). Product fractions were collected, diluted with water, and frozen. The "functionalized immunostimulant" section for CpG and T78a was Analytical HPLC and HPLC were used to monitor the reaction using the same method described in Section 1. and / or LCMS were used.

[0136] Synthesis - Knottin-Fc complex Knottin-Fc (KFc) fusion protein was recombinantly expressed and expressed as previously described. It was purified as follows (BHKwan, et al., J Exp Med. 2017, 2 14(6):1679-90). As shown in Figure 8, BCN-PEG2-NHS esters Functionalizing the KFc fusion with a clickable handle (BCN group) using a tert-linker To achieve this, NHS ester labeling was used. NHS esters are used to label primary amino acids in proteins. It reacts with amines (lysine residues and the N-terminus) to form stable amide bonds. BCN-PEG2-NHS esters in 100 mM sodium bicarbonate buffer, pH 8.3 The resulting mixture was mixed with 100µg of PEG-1000 and 100µg of PEG-1000 linker (6 equivalents) for 2 hours at room temperature. This labeling protocol is typically This yields 2-3 conjugates per KFc. Buffer exchange into PBS was performed before sample loading. BCN was purified by size exclusion using a Zeba spin desalting column (7K MWCO). The decorated KFc was purified from the remaining linker.

[0137] To produce the KFc-immunostimulatory agent complex, BCN-modified KFc (1 equivalent) was added to PBS. The reaction was carried out with azide-T78a (6 equiv.) in 100 ml of water and stirred overnight at room temperature. Zeba spin desalting columns (7K MWCO) were used, which were buffer exchanged into PBS before The KFc-T78a complex was purified from unreacted azido-T78a by size exclusion. Ta.

[0138] For the synthesis, KFc was modified with an azide-NHS ester linker to form BCN-T7. The azide group can be exchanged with BCN to be conjugated to 8a. CpG can also be substituted with functionalized T78a. BCN can also be substituted with DBCO at any time. do.

[0139] immune cell infiltration 4T1-luc cells (2 × 10 4 ) was subcutaneously implanted into one side of the abdomen of BALB / c mice. Once tumors developed (referred to as day 0 post-treatment), the mice were IV treatment on day 0 and on day 2 after treatment for the following groups (n = 3 mice per group): The following were injected intravenously (IV) or intratumorally (IT): vehicle IV (PBS), CpG IV (18.2 nmol), 3CM-CpG IV (18.2 nmol), and CpG IT (5.2 nmol). CpG IT dose (5.2 nmol = 50 μg) was used in mice. Typical IT doses given to patients are lower than IV doses. Three days after the first dose, Tumors were excised and mechanically dissociated into single-cell suspensions.

[0140] Prior to antibody staining, cells were stained with LIVE / DEAD Fixable Aqua Dead The cells were incubated with Cell Stain. Fluorescently labeled antibody in PBS), 1% bovine serum albumin, and 0.01% sodium azide The cells were surface stained with 2% paraformaldehyde and analyzed by flow cytometry. The data were analyzed using Cytobank (www.cytobank.org). The samples were stored and analyzed using the

[0141] For data analysis, cells were gated to include only viable single cells. MDSCs express CD11b + GR1 + NK cells have been characterized as CD3 - CD49b + It was characterized as CD8 + T cells are CD3 + CD8 + CD49b - It was characterized as CD4 + T cells are CD3 + CD4 + CD4 9b - It was characterized as CD8 + B cells are CD3 - B220 + CD49b - was characterized as.

[0142] Thus, the foregoing merely illustrates the principles of the present disclosure. Although not explicitly described or illustrated, they embody the principles of the invention and are included within its spirit and scope. It should be understood that those skilled in the art will be able to devise various configurations for the above-described devices. All examples and conditional language recited herein are intended to be illustrative and not restrictive of the principles and scope of the present invention. To assist the reader in understanding the concepts contributed by the inventors to further the field of technology is intended to be comprehensive and without limitation to such specifically listed examples and conditions. Furthermore, the principles, aspects, and embodiments of the present invention and the related art should be construed as illustrative and not restrictive. All descriptions herein that enumerate specific examples of these include their structural and functional properties. In addition, such equivalents are not intended to encompass any other equivalents that are currently known or otherwise. Both the current product and future equivalents, i.e., products that perform the same function regardless of structure. The scope of the present invention is therefore intended to include any and all elements developed by the present invention. It is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

[Claim 1] The invention described in the specification and drawings of this application.

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