[go: up one dir, main page]

WO2025083276A1 - Système d'administration ciblée isolé amélioré - Google Patents

Système d'administration ciblée isolé amélioré Download PDF

Info

Publication number
WO2025083276A1
WO2025083276A1 PCT/EP2024/079642 EP2024079642W WO2025083276A1 WO 2025083276 A1 WO2025083276 A1 WO 2025083276A1 EP 2024079642 W EP2024079642 W EP 2024079642W WO 2025083276 A1 WO2025083276 A1 WO 2025083276A1
Authority
WO
WIPO (PCT)
Prior art keywords
macrophages
monocytes
msr
population
ferritin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2024/079642
Other languages
English (en)
Inventor
Malgorzata Maria KUBIAK
Paulina Barbara KUCHARZEWSKA-SIEMBIEDA
Bartlomiej Taciak
Magdalena Krol
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellis AG
Original Assignee
Cellis AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cellis AG filed Critical Cellis AG
Publication of WO2025083276A1 publication Critical patent/WO2025083276A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • the present invention relates to an isolated targeted delivery system comprising an immune cell (monocyte or macrophage) comprising a complex of ferritin and a pharmaceutically active substance and/or label, wherein the cell (monocyte or macrophage) is characterized by a high expression of MSR- 1.
  • Tumorigenesis is a complex and multistep process, in which accumulation of mutations leads to the development of cancer cells.
  • Cancer cells are able to modify their surroundings through the secretion of various factors, which results in the formation of a specific tumour microenvironment consisting of cells that support the tumour growth.
  • tumour-associated macrophages One of the most abundant cells in the tumour microenvironment are tumour-associated macrophages.
  • Most current anti-cancer therapies are based on highly cytotoxic and non-selective substances, which are dangerous also for healthy cells and organs. There is thus a high interest and unmet need in targeted delivery systems capable of delivering cytotoxic cargo directly into tumour cells, thereby avoiding or reducing side effects. Due to the physiological interaction between cancer cells and macrophages, macrophages are promising candidates as carrier cells allowing active tumour targeting and controlled drug release.
  • Protein nanocarriers are investigated as part of targeted delivery systems.
  • the encapsulation of cytotoxic drugs would protect healthy cells and lead to reduction of side effects.
  • Protein nanocarriers are promising due to their biocompatibility, biodegradability, and low toxicity.
  • Ferritin a highly conserved, major iron-storage protein ubiquitously expressed among almost all living species, seems to be an ideal candidate for a protein nanocarrier.
  • One of its unique attributes is the nanocage structure built of H- and L-subunits with a large, highly symmetrical cavity. This endogenous protein can be easily disassembled and reassembled, which makes the encapsulation of the drug molecules relatively easy.
  • the surface of the 24-mer can be modified chemically or genetically to facilitate targeted delivery.
  • the ferritin nanocages comprising a cytotoxic drug need to be loaded into carrier cells, in particular macrophages (or their precursors - monocytes), in a way that is both specific and efficient.
  • carrier cells in particular macrophages (or their precursors - monocytes)
  • ferritin and transferrin receptor 1 TfR-1
  • MSR-1 transferrin receptor 1
  • MSR-1 another receptor
  • the inventive targeted delivery system comprising macrophages characterized by a high expression of MSR-1 comprising a complex of ferritin and an active ingredient provides inter alia the following advantages over the prior art: (i) higher concentration of ferritin nanocages and active ingredient within the macrophages or monocytes; (ii) higher treatment efficacy with lower doses of loaded macrophages or monocytes, (iii) lower variability of the final product due to highly specific loading.
  • the inventive method of preparing a targeted delivery system provides inter alia the following advantages over the prior art: (i) easy, fast and efficient loading of macrophages or monocytes with ferritin nanocages; (ii) highly specific loading of macrophages or monocytes with ferritin nanocages; (iii) easier and faster production of the final product.
  • the inventive method of selecting a monocyte donor provides inter alia the following advantages over the prior art: (i) reliable, fast and easy selection of suitable donors for allogeneic therapy; (ii) reliable, fast and easy patient stratification.
  • the present invention relates to an isolated targeted delivery system comprising a population of macrophages and/or monocytes comprising a complex of ferritin and a pharmaceutically active substance, label or pharmaceutically active substance and label, wherein the population of macrophages and/or monocytes is characterized by a high expression of MSR-1.
  • the invention provides a pharmaceutical composition comprising the isolated targeted delivery system according to the first aspect of the invention.
  • the invention provides a pharmaceutical comprising a plurality of macrophages comprising a complex of ferritin and a pharmaceutically active substance, label or pharmaceutically active substance and label, wherein at least 5%, at least 10%, at least 15%, or at least 20% of the macrophages express MSR-1.
  • the invention provides the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention for use in medicine.
  • the invention provides the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention for use in the treatment of cancer.
  • the invention provides a method of preparing an isolated targeted delivery system, in particular the isolated targeted delivery system according to the first aspect of the invention, comprising the steps of a) providing purified ferritin; b) covalently or non-covalently linking a pharmaceutically active substance, label or pharmaceutically active substance and label to and/or encapsulating a pharmaceutically active substance, label or pharmaceutically active substance and label in an iron binding protein; c) providing a population of macrophages and/or monocytes characterized by a high expression of MSR-1, preferably at least 5%, at least 10%, at least 15%, at least 20% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes express MSR-1; and d) incubating the population of macrophages in the presence of the iron binding protein produced in step b) until the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes are at least partially loaded with the complex of the iron
  • the invention provides a method of selecting a monocyte donor, comprising a) generating a population of macrophages from a population of monocytes obtained from a donor; b) determining the percentage of
  • the percentage of MSR-1 -expressing monocytes is high, preferably at least 2%, at least 3%, or at least 4%,
  • the percentage of MSR-1 -expressing macrophages is high, preferably at least 5%, at least 10%, at least 15%, or at least 20%, and/or
  • the invention provides a method of providing macrophages or monocytes with high MSR-1 expression.
  • the cells provided by this method are able to take up more ferritin in complex with an active ingredient than other cells.
  • amino acid encompasses naturally occurring amino acids as well as amino acid derivatives.
  • amino acids are identified using the 1 -letter code (Hausman RE, Cooper GM (2004). The cell: a molecular approach. Washington, D.C: ASM Press, p. 51. ISBN 978-0-87893-214-6).
  • An amino acid identified with the letter X corresponds to any amino acid.
  • An amino acid identified with the letter B corresponds to either D (asparagine) or N (aspartic acid).
  • An amino acid identified with the letter Z corresponds to either E (glutamine) or Q (glutamic acid).
  • nucleic acid and “nucleic acid” are used interchangeably herein and are understood as a polymeric or oligomeric macromolecule made from nucleotide monomers.
  • Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups.
  • a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers.
  • nucleic acid molecules include but are not limited to ribonucleic acid (RNA) and its various forms (e.g.
  • nucleic acids can e.g. be synthesized chemically, e.g. in accordance with the phosphotriester method (see, for example, Uhlmann, E. &Peyman, A. (1990) Chemical Reviews, 90, 543-584).
  • Aptamers are nucleic acids which bind with high affinity to a polypeptide. Aptamers can be isolated by selection methods such as SELEmirl46-a (see e.g. Jayasena (1999) Clin.
  • RNA molecules from a large pool of different single-stranded RNA molecules.
  • Aptamers can also be synthesized and selected in their mirror-image form, for example as the L-ribonucleotide (Nolte et al. (1996) Nat. Biotechnol., 14, 1116- 9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5).
  • L-ribonucleotide Nolte et al. (1996) Nat. Biotechnol., 14, 1116- 9; Klussmann et al. (1996) Nat. Biotechnol., 14, 1112-5.
  • sequence identity is used throughout the specification with regard to polypeptide and nucleotide sequence comparisons. In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise. If the reference sequence is indicated, the sequence identity is determined on the basis of the full length of the reference sequence indicated by SEQ ID, if not specifically indicated otherwise.
  • a polypeptide sequence consisting of 200 amino acids compared to a reference 300 amino acid long polypeptide sequence may exhibit a maximum percentage of sequence identity of 66.6 % (200/300) while a sequence with a length of 150 amino acids may exhibit a maximum percentage of sequence identity of 50 % (150/300). If 15 out of those 150 amino acids are different from the respective amino acids of the 300 amino acid long reference sequence, the level of sequence identity decreases to 45 %.
  • the similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin&Altschul (1993) Proc.
  • HMMER package http://hmmer.wustl.edu/
  • CLUSTAL algorithm Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80
  • sequence identity may be calculated using e.g.
  • BLAST, BLAT or BlastZ or BlastX
  • Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
  • sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields.
  • antibody refers to a glycoprotein belonging to the immunoglobulin superfamily; the terms antibody and immunoglobulin are often used interchangeably.
  • An antibody refers to a protein molecule produced by plasma cells and is used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, its antigen.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen.
  • binding fragments encompassed within the term “antibody fragment” include a fragment antigen binding (Lab) fragment, a Lab’ fragment, a E(ab’)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scLv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.
  • sdAb single-domain antibody
  • scLv single-chain fragment variable
  • Fv fragment variable
  • antigen is used to refer to a substance, preferably an immunogenic peptide that comprises at least one epitope, preferably an epitope that elicits a B or T cell response or B cell and T cell response.
  • an “epitope”, also known as antigenic determinant, is that part of a substance, e.g. an immunogenic polypeptide, which is recognized by the immune system. Preferably, this recognition is mediated by the binding of antibodies, B cells, or T cells to the epitope in question.
  • binding preferably relates to a specific binding. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the term “epitope” comprises both conformational and non-conformational epitopes. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the present invention relates to an isolated targeted delivery system comprising a population of macrophages and/or monocytes comprising a complex of ferritin and a pharmaceutically active substance, label or pharmaceutically active substance and label, wherein the population of macrophages and/or monocytes is characterized by a high expression of MSR-1.
  • the macrophages and/or monocytes comprise ferritin, wherein the ferritin is in a complex with a pharmaceutically active substance and/or label.
  • the population of macrophages and/or monocytes is characterized by a high endogenous expression of MSR-1.
  • the isolated targeted delivery system comprises monocytes. In some embodiments, the isolated targeted delivery system comprises monocytes and macrophages. In some embodiments, the isolated targeted delivery system comprises macrophages. In some embodiments, the isolated targeted delivery system comprises only macrophages, but no monocytes.
  • MSR-1 belongs to the class A scavenger receptors (SR-A) described in Becker et al., Eur. J. Immunol. 36, 950-960 (2006). Alternative names for MSR-1 include: SCARA1, CD204, MSR1, MSR- Al, SRA1, SR- Al and SR-A1.
  • MSR-1 is a type II trimeric transmembrane glycoproteins built of a N- terminal cytoplasmic domain, transmembrane part, collagenous domain and a large extracellular C- terminal positively charged domain, which comprises a scavenger receptor cysteine rich (SRCR) domain and is involved in polyanionic ligand identification and binding. MSR-1 is expressed predominantly on macrophages.
  • the population of macrophages and/or monocytes is genetically modified to overexpress MSR-1.
  • the overexpression is transient or stable, preferably transient. Exemplary methods of overexpressing MSR-1 expression in macrophages and/or monocytes are described below and in the examples section.
  • the term “expression” indicates that the surface antigen MSR-1 is produced within the cell and detectably exposed on the surface of a cell.
  • the level of expression and, thus the number of surface antigens detectably exposed on the surface of a cell can vary greatly among different cells.
  • a cell is considered to express MSR-1, in other words to be positive, i.e. indicated to be “ + ”, for MSR-1, if at least 5, preferably at least 10 copies of MSR-1 are detectably exposed on the surface of the cell.
  • Preferred methods include Fluorescence Activated Cell Sorting (FACS).
  • fluorescently labelled antibodies are used to bind to cellular surface antigens of a population of cells, the cells are subsequently isolated into single cells and based on fluorescence intensity measured for the single cell, characterized as being positive or negative for the given cellular surface antigen.
  • Exemplary methods of detecting MSR-1 expression by FACS are described in the examples section.
  • expression of MSR-1 can be analyzed by assessing the presence of mRNA encoding for MSR-1 within the cell. It is well known in the art how to determine expression of a given protein or its encoding mRNA in a population of cells or even in single cells, e g.
  • At least 10% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes express MSR-1. In some embodiments, at least 15% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes express MSR-1. In some embodiments, at least 20% of the macrophages and/or monocytes compnsed in the population of macrophages and/or monocytes express MSR-1. The percentage of macrophages and/or monocytes comprised in the population of macrophages and/or monocytes expressing MSR-1 can be assessed by flow cytometry using standard protocols.
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the macrophages and/or monocytes comprise on average at least a predetermined number of MSR-1 molecules per cell, such as at least (for a monocyte) 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 or 1 x 10 4 , or at least (for a macrophage) 1 x 10 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 or 1
  • the number of MSR- 1 molecules per cell may be (for a monocyte) less than 1 x 10 4 , 5 x 10 3 , 1000, 900, 800, 700, 600, 500, 400, 300, 200 or 150.
  • the number of MSR-1 molecules per cell may be (for a macrophage) less than 1 x 10 6 , 5 x 10 5 or 1 x 10 5 .
  • the number of MSR-1 molecules per cell may be even higher, such as at least 5 x 10 5 , 1 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , or 1 x 10 8 , and/or less than 5 x 10 9 , 1 x 10 9 , 5 x 10 8 , 1 x 10 8 , 5 x 10 6 , or 1 x 10 6 .
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the macrophages and/or monocytes comprise on average at least a predetermined number of MSR-1 -encoding mRNA molecules per cell (as determined e g.
  • single cell sequencing such as (for a monocyte) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 290, 300, or at least (for a macrophage) 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 480, 490 or 500.
  • a monocyte at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150,
  • the number of MSR-1 -encoding mRNA molecules per cell may be (for a monocyte) less than 300, 200 or 100.
  • the number of MSR-1- encoding mRNA molecules per cell may be (for a macrophage) less than 1000, 800, 600 or 500.
  • the number of MSR-1 -encoding mRNA molecules per cell may be even higher, such as at least 750, 1000, 1500, 2000, 3000, or 5000, and/or less than 10000, 8000, 6000, 5000, 3000, or 2000.
  • the number of MSR-1 -encoding mRNA molecules per cell can also be determined by qPCR (quantitative PCR) combined with determination of the cell number per sample.
  • the Cq value also known as the Ct value or quantification cycle
  • the Cq value in qPCR refers to the cycle number at which the fluorescence signal of the amplified DNA crosses a set threshold.
  • the difference in Cq values between MSR-1 and the housekeeping gene 0-actin averages 7.39 ⁇ 0.64 cycles. This indicates that the amount of MSR-1 mRNA is lower than that of fyactin mRNA, requiring approximately 7 additional PCR cycles for MSR-1 to become detectable compared to P-actin.
  • the difference in Cq values between MSR-1 and the housekeeping gene p-actin is 7.4 or less, 7.3 or less, 7.2 or less, 7. 1 or less, 7.0 or less, 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less or 6.0 or less.
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the MSR-1 mRNA comprised in the macrophages and/or monocytes is on average at least a predetermined percentage of the total mRNA comprised in the macrophages and/or monocytes, such as at least 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.06%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5%. The percentage may be less than 10% or less than 5%.
  • the expression “the population of macrophages is characterized by a high expression of MSR-1” denotes that the MSR-1 protein comprised in the macrophages is on average at least a predetermined percentage of the total protein comprised in the macrophages, such as at least 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.06%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5%. The percentage may be less than 10% or less than 5%.
  • the expression “the population of macrophages is characterized by a high expression of MSR-1” denotes that the MSR-1 mRNA comprised in the macrophages is on average at least a predetermined percentage of the total mRNA comprised in the macrophages, such as at least 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.06%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% or 5%. The percentage may be less than 10% or less than 5%.
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the MSR-1 expression normalized to the beta-actin expression in the macrophages and/or monocytes is on average at least a predetermined value, e.g. 0.5, 1.0, 1.5, 2.0 or 2.5.
  • the expression “the population of macrophages is characterized by a high expression of MSR-1” denotes that the MSR-1 expression normalized to the beta-actin expression in the macrophages is on average at least a predetermined value, such as 0.5, 1.0, 1.5, 2.0 or 2.5 or the MSR-1 expression normalized to the lamin Bl expression in the macrophages is on average at least a predetermined value, such as 6, 6.5, 7, 7.5, 7.6, or 7.7.
  • the MSR-1 expression normalized to the lamin Bl expression may be less than 10.
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the mean fluorescence intensity (MFI) units, when detecting MSR-1 expression in the macrophage and/or monocyte population as described in the examples, is at least a predetermined value, e.g. 200, 300, 400, 5000, 600, 700, 800, 900, or 1000.
  • MFI mean fluorescence intensity
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the mean MSR-1 expression in the macrophage and/or monocyte population is increased by at least a predetermined amount, e.g. 1 ,5x, 2x, 3x, 4x, or 5x compared to the mean MSR-1 expression in an average macrophage and/or monocyte population.
  • the mean MSR-1 expression may be increased by less than lOx.
  • the expression “the population of macrophages and/or monocytes is characterized by a high expression of MSR-1” denotes that the mean MSR-1 expression in the macrophage and/or monocyte population is increased by at least a predetermined amount, e.g. 1 ,5x, 2x, 3x, 4x, or 5x compared to the average mean MSR-1 expression in a macrophage and/or monocyte population.
  • the average mean MSR-1 expression may be increased by less than lOx.
  • Mean MSR-1 positive cells (percentage of cells on which SR-A1 is detectable assessed by flow cytometry) is 12.32% ⁇ 12.40% (range 0.98% - 62.3%) for monocytes and mean MSR-1 positive cells (assessed by flow cytometry) is 23.07% ⁇ 19.44% (range 0.73% - 73%) for macrophages.
  • Mean MFI signal of anti-MSR-l-BV241 is 86.17 ⁇ 33.92% (range 19 - 198.3) for monocytes and mean MFI signal of anti-MSR-l-BV241 is 847.82 ⁇ 520.27 (range 116 - 2557) for macrophages.
  • Mean fold change of MSR-1 expression between macrophages and monocytes is 12.2 ⁇ 11.9 (12.2 times higher on macrophages than on monocytes), in the range from 1.19 to 59.5.
  • the above values are preferably determined for monocytes on day 0 (directly after isolation) and for macrophages after 7 days of culture with differentiating agents.
  • the differentiating agents are agents that cause differentiation of monocytes into macrophages, in particular M-CSF.
  • the percentage of macrophages and/or monocytes comprised in the population of macrophages and/or monocytes that express MSR-1 is high, but also the level of MSR-1 expression by a single macrophage and/or monocyte is high.
  • at least 10, preferably at least 15 copies of MSR-1 are detectably exposed on the surface of the cell.
  • the MSR-1 expression of a macrophage population is at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 20-fold, the MSR-1 expression of the monocyte population from which the macrophage population was generated.
  • the increase corresponds to an increase after 7 days of culture.
  • the MSR-1 expression of a macrophage population generated from a monocyte population isolated from PBMCs may be at least 2-fold, at least 4-fold, at least 6-fold, at least 8-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 20-fold, the MSR-1 expression of the monocyte population from which the macrophage population was generated.
  • the macrophage population is generated from a monocyte population isolated from PBMCs via incubation with M-CSF for 7 days.
  • MSR-1 is responsible for ferritin uptake into human macrophages and monocytes and that ferritin uptake can be increased by increasing MSR-1 expression on macrophages or monocytes or selecting for high MSR-1 expression on macrophages or monocytes. This also applies if the ferritin has an increased affinity for TfR-1, such as the ferritin according to SEQ ID NO: 4.
  • the term “active ingredient” is used to refer to the at least one pharmaceutically active substance and/or at least one label.
  • the active ingredient is a pharmaceutically active substance.
  • targeted delivery refers to the delivery of a therapeutic or diagnostic agent (herein together referred to also as “active ingredient”) to a subject, e g. patient, in particular to a cell, more particular directly into a cell within the body of a patient.
  • active ingredient a therapeutic or diagnostic agent
  • the targeted delivery results in an increased concentration of the active ingredient in a particular region of the body when compared to administration of the active ingredient alone, administration of a complex of an iron binding protein and the active ingredient, or administration of other delivery systems.
  • the targeted delivery results in an increased concentration of the active ingredient in tumor tissue, in particular ovarian cancer, more particularly inside ovarian cancer cells, when compared to administration of the active ingredient alone, administration of a complex of an iron binding protein and the active ingredient, or administration of other delivery systems.
  • Targeted delivery also includes “targeted theragnostic delivery”, meaning that both a therapeutic and a diagnostic agent are delivered concomitantly, preferably to a diseased region, thus allowing simultaneous treatment and diagnosis and/or treatment monitoring.
  • the active ingredient is delivered directly into the cancer cells, preferably via direct transfer from the CD45 + leukocyte to the cancer cell (direct cell-cell transfer).
  • CD45+ leukocyte refers to a macrophage or monocyte.
  • the direct transfer is preferably via a mechanism involving cell-cell contact and/or fusion of cell membranes.
  • targeted label delivery system is used in the present application to refer to a system that is capable of delivering a label to the targeted region, i.e. capable of targeted delivery within the body of a patient, preferably to a diseased region.
  • targeted theragnostic delivery system is used in the present application to refer to a system that is capable of delivenng a complex of a pharmaceutically active substance and at the same time a label to the targeted region, i.e. capable of targeted delivery within the body of a patient, preferably to a diseased region and thus allows simultaneous treatment and diagnosis and/or treatment monitoring.
  • targeted delivery system is used to commonly refer to “targeted pharmaceutically active substance delivery system”, “targeted label delivery system” and “targeted theragnostic delivery system”.
  • Targeted delivery systems have been described in WO 2016/207257 Al, WO 2016/207256 Al and WO 2017/222398 Al, which are incorporated herein by reference.
  • the active ingredient may be covalently or non-covalently bound to ferritin.
  • the term “complex” also encompasses the enclosure of an active ingredient multimers of ferritin forming a “ferritin cage”. In instance where the active ingredient is encapsulated within a ferritin multimer, the encapsulation may occur in the presence or absence of a covalent or non-covalent bond.
  • active ingredients can be encapsulated within the internal cavity of a ferritin oligomer (physical confinement) by exploiting the association/dissociation properties of the ferritin macromolecule itself. In such embodiments, the active ingredients are held in place by non-covalent interactions with amino acid residues within the cavity internal surface.
  • the formation of the complex allows the transport of the active ingredients into the macrophage when the macrophage is internalizing the ferritin.
  • the active ingredients are bound to the ferritin in a way that does not interfere with the transport mechanism. This can be easily tested by the skilled person using uptake assays known in the art and described in WO 2016207257 Al, WO 2016207256 Al and WO 2017222398 Al.
  • the complex comprising an active ingredient is taken up by a macrophage and transported to a target cell within the body, it is preferred that the complex is sufficiently stable to survive the transport within the cell to the target region within the body.
  • the complex rather than the active ingredient alone is delivered to, preferably into the target cells in the target region. This property also reduces possible deleterious effects, e.g. cytotoxicity, of the active ingredient to the macrophage delivering the active ingredient or to other cells of the body that are not the target cells.
  • the active ingredient and the ferritin are covalently and/or non- covalently linked, preferably covalently linked. If active ingredients are covalently linked to the iron binding proteins such coupling is preferably through amino acids residues known to be located in surface areas that are not involved in binding of the ferritin to a receptor involved in endocytosis. In particular, the coupling is through amino acids residues known to be located in surface areas that are not involved in binding of the ferritin to MSR-1.
  • ferritin and the active ingredient are covalently linked, they may be linked directly or via a linker, i.e. indirectly. In preferred embodiments, the ferritin and the active ingredient are covalently linked via a linker.
  • Linkers are known to the skilled artisan, such as polyalanine, polyglycm, carbohydrates, (CH 2 ) n groups or polypeptide linkers.
  • the linker may be biodegradable or non-biodegradable, preferably biodegradable.
  • the linker is cleavable.
  • the linker may be a peptide, disulfide, or hydrazone linker or a linker comprising carbohydrates . It is preferred that the linker is a peptide linker.
  • the peptide linker is cleavable by a protease.
  • a linker comprising carbohydrates maybe cleavable by 0-glucuronidase.
  • a hydrazone linker may be cleaved by acid hydrolysis.
  • a disulfide linker may be cleaved by cytosolic reductive cleavage.
  • the linker preferably a peptide linker, is cleavable by lysosomal proteases, more preferably lysosomal cysteine proteases, even more preferably cathepsins, most preferably cathepsin B.
  • the linker comprises a reactive group that, when activated by a defined stimulus, effects cleavage of a covalent bond within linker.
  • Suitable reactive groups are e.g. light- activatable groups (i.e. groups that can be activated by UV light, such as 3-amino-3-(-2-nitro)phenyl- propionic acid or a light-activatable structural equivalent thereof), dithionite-activatable groups (such as an azobenzene moiety), or periodate-activatable groups (such as a 1,2-dihydroxy moiety, a l-amino-2- hydroxy moiety or 4-amino-4-deoxy-L-threonic acid).
  • the linker is cleavable via a pH shift.
  • compositions or labels may also be covalently bound to ferritin amino acid side chains (lysines or cysteines) by appropriate choice of phenylhydrazone, succinimide or maleimide activated drugs.
  • a phenylhydrazone derivative may break and liberate the drug from the iron binding protein, a lysine bound derivative may become active after full protein degradation into amino acids, or a cysteine bound derivative may be liberated within the cell through reductive hydrolysis of the maleimede thioether link.
  • the linker is a dipeptide linker, in particular Val-Cit, Val-Ala, or Ala-Ala, or a tripeptide and tetrapeptide linker.
  • the linker is a hetero-bifunctional crosslinker that contains N- hydroxysuccinimide (NHS) ester and maleimide groups that allow covalent conjugation of amine- and sulfhydryl -containing molecules.
  • the linker is succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (SMCC) or Sulfo-SMCC.
  • the cleavable linker is cleaved within the lysosomal compartment.
  • the linker is a maleimidocaproyl-valine-citrulline-para- aminobenzoyloxycarbonyl (mc-vc-PAB) linker.
  • the ferritin is conjugated to the active ingredient or linker via a cysteine residue or a lysine residue, preferably a cysteine residue.
  • relevant thiol, amino or carboxyl groups of the ferritin are used to covalently couple active ingredients reactive towards thiol or amino groups directly or indirectly to the ferritin.
  • the active ingredients may be modified by specific active moieties, i.e. linkers.
  • ferritin may be linked to cysteine thiol reactive pharmaceutically active substances and/or labels bearing a peptide based cleavable linker (e.g. cathepsin sensitive valine-citrulline sequence and para-aminobenzylcarbamate spacer).
  • a peptide based cleavable linker e.g. cathepsin sensitive valine-citrulline sequence and para-aminobenzylcarbamate spacer.
  • MMAE monomethyl auristatin E
  • the peptide-based linker binds the protein to the cytotoxic compound in a stable manner, so the drug is not easily released from the ferritin under physiologic conditions, which helps prevent toxicity to healthy cells and ensures dosage efficiency.
  • the ferritin pharmaceutically active substance and/or label adduct thus generated is capable of attaching to the selected receptor types.
  • DM1 -SMCC is an efficient mertansine derivative bearing a linker that specifically binds to lysine residues generating a covalent complex with ferritin in a reaction that has been successfully described for antibodies.
  • ferritin can be reacted with DM1-SMCC thus providing a covalent protein-drug adduct that can be cleaved inside cells and releases the active drug in a time-dependent manner.
  • the suppression of microtubule dynamics by DM1 induces mitotic arrest and cell death.
  • active ingredients are comprised in the isolated targeted delivery system.
  • one type of active ingredient may be bound to a covalently bound to a ferritin polypeptide, while another type is encapsulated in the complex.
  • This approach utilizes different release rates of the active ingredients from the complex once delivered to the targeted tissue and/or cells.
  • an active ingredient can be covalently attached to a ferritin molecule either on the surface of the 24-mer or within the internal cavity by exploiting the reactivity of relevant thiol, amino or carboxyl groups.
  • the types of such useful reactions are well known in the art and can be adopted by the person skilled in the art to the particular active ingredient without any additional work. Examples of such reactions are described in Behrens CR, Liu B. Methods for site-specific drug conjugation to antibodies. MAbs. 2014 Jan-Feb;6(l):46-53.
  • the complex comprises both a label and a pharmaceutically active substance
  • the label is covalently attached to ferritin and the pharmaceutically active substance is non-covalently bound to the ferritin and/or entrapped in the internal cavity formed upon assembly of the multimer of ferritin polypeptides.
  • Ferritin (Ft) is a highly conserved major iron-storage protein, ubiquitously expressed among almost all living species.
  • Ferritin is a hollow globular protein complex consisting of 24 ferritin monomer subunits assembled into a cage-like structure.
  • Ferritin is the primary intracellular iron-storage protein. It is produced by almost all living organisms and is present in every cell type. Ferritin genes are highly conserved among species. In vertebrates, two ferritin monomers exist: the light (L) chain and the heavy (H) chain type with a molecular weight of 19 kDa or 21 kDa respectively.
  • Vertebrate ferritin 24-mers can be homo-oligomers consisting of either L or H chains, or hetero-oligomers consisting of both L and H chains (Theil EC, 1987, Annual Review of Biochemistry. 56 (1): 289-315): Typically, ferritin complexes have internal and external diameters of about 8 and 12 nm, respectively. Ferritin was shown to be internalized by endocytosis upon binding to CD71. Interaction of ferritin and CD71 is mediated via ferritin-H chains (Li L et al, Proc. Natl. Acad. Sci. USA 107 (8) (2010) 3505-3510) Ferritins are not abundant in plasma but can be readily produced in high yield as recombinant proteins in common protein expression systems such as Escherichia coli cells.
  • the ferritin is selected from heavy (H) type ferritin, light (L) ferritin and/or mitochondrial ferritin.
  • the term ferritin encompasses structural variants of the naturally occurring protein and, thus relates to proteins that have at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% more preferably at least 100% of the ability of the respective wild-type ferritin to bind iron ion(s).
  • the ferritin used in the context of the present invention is preferably of mammalian, more preferably mouse, rat, dog, ape, in particular, chimpanzee, or human, most preferably of human origin. Consensus sequences of the preferred ferritins and preferred structural variants are disclosed in WO 2016/207257 Al and WO 2022/195092 Al.
  • the ferritin is a mammalian ferritin.
  • the mammalian ferritin may be a mouse, rat, dog, ape, in particular chimpanzee, or human ferritin.
  • the mammalian ferritin is a mouse, rabbit, rat or human ferritin, preferably human ferritin.
  • the human ferritin is a human heavy chain ferritin.
  • Amino acid substitutions within the iron binding proteins are preferably selected in a way that they do not unduly change the conformation of the polypeptide.
  • a “small amino acid” should be substituted with another small amino acid.
  • a “small amino acid” in the context of the present invention is preferably an amino acid having a molecular weight of less than 125 Dalton.
  • a small amino acid in the context of the present invention is selected from the group consisting of the amino acids' glycine, alanine, serine, cysteine, threonine, and valine, or derivatives thereof.
  • an amino acid having a hydrophobic side chain should be substituted with another amino acid having a hydrophobic side chain.
  • Any ferritin used in the isolated targeted delivery system for use according to the invention has to retain the properties of a wild-type ferritin with regard to complex formation (cage-like structure consisting of 24 ferritin monomer subunits, i.e. ferritin 24-mer formation) and iron binding, i.e. uptake of iron into the ferritin 24-mer.
  • the ferritin comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NO: 1-5 and has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the ability of wild-type ferritin, in particular human wild type ferritin, to bind iron ion(s) and/or form ferritin 24-mers.
  • SEQ ID NO: 3 is a mammalian consensus sequence.
  • X at position 6 can be any naturally occurring amino acid, preferably Pro X at position 14 can be any naturally occurring amino acid, preferably His, X at position 16 can be any naturally occurring amino acid, preferably Asp, X at position 21 may be present or absent, if present it means any amino acid, preferably He, X at position 29 can be any naturally occurring amino acid, preferably Tyr, X at position 81 can be any naturally occurring amino acid, preferably Phe, X at position 83 can be any naturally occurring amino acid, preferably Gin, X at position 105 can be any naturally occurring amino acid, preferably His, X at position 144 can be any naturally occurring amino acid, preferably Ala or Ser, more preferably Ala, X at position 180 is absent or any naturally occurring amino acid, preferably Asn, X at position 181 is absent or any naturally occurring amino acid, preferably Glu, X at position 182 is absent or any naturally occurring amino acid, preferably Ser.
  • SEQ ID NO: 5 is a mammalian consensus sequence.
  • X at position 6 can be any naturally occurring amino acid, preferably Pro
  • X at position 14 can be any naturally occurring amino acid, preferably His
  • X at position 16 can be any naturally occurring amino acid, preferably Asp
  • X at position 21 may be present or absent, if present it means any amino acid, preferably He
  • X at position 22 means any amino acid, preferably Asn
  • X at position 30 can be any naturally occurring amino acid, preferably Tyr
  • X at position 40 can be any naturally occurring amino acid, preferably Tyr or Cys, more preferably Tyr
  • X at position 82 can be any naturally occurring amino acid, preferably Phe
  • X at position 84 can be any naturally occurring amino acid, preferably Gin
  • X at position 91 can be any naturally occurring amino acid, preferably Arg or Cys, more preferably Cys
  • X at position 106 can be any naturally occurring amino acid, preferably His
  • X at position 110 can be any naturally
  • the ferritin comprises an amino acid sequence according to SEQ ID NO: 4, optionally comprising 1-5, 1-10, 1-15, 1-20 or 1-25 amino acid mutations outside position 54, 72, 87 and/or 144, in particular outside position 54, and having at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the ability of wild-type ferritin, in particular human wild type ferritin, to bind iron ion(s) and/or form ferritin 24-mers, in particular to form ferritin 24-mers.
  • the ferritin according to SEQ ID NO: 4 has a particularly high affinity for TfR-1 due to the presence of isosteric mutations.
  • ferritin has an increased affinity for TfR-1.
  • the ferritin may comprise a transferrin receptor binding domain (TRBD) that comprises one or more glutamine residues mutated into glutamic acid residues and/or one or more asparagine residues mutated into aspartic acid residues.
  • TRBD transferrin receptor binding domain
  • Such isosteric mutations increase the affinity for TfR-1.
  • active substance is used to refer to a “drug” or a “label”.
  • drug or “pharmaceutically active substance” are used synonymously in the context of the present invention and refer to any compound that modifies or modulates cell activity or is capable of being activated, i.e. a prodrug, to modify or modulate cell activity, preferably in the body of a patient.
  • active ingredients include so called “small molecules” and peptides.
  • small molecule is used in the context of the present invention to refer to a hydrocarbon with a molecular mass of below 1.500 g/mol or to pharmaceutically active radioactive isotopes.
  • the pharmaceutically active substance is an anticancer drug selected from the group consisting of a protein, a peptide, a nucleic acid, a non-protein non-nucleic acid compound with a molecular weight of less than 1.5 kD, a photosensitizing compound, a virus, and pharmaceutically active radioactive isotope.
  • a preferred pharmaceutically active radioactive isotope is an a or B radiation emitting radioisotope, which also emits a cell damaging amount of y radiation is selected from the group consisting of hitetium- 177, ytterbium-90, iodine-131, samarium-153, phosphorus-32, caesium-131, palladium- 103, radium- 233, iodine-125, and boron-10 or a cell damaging amount of a radiation, preferably selected from the group consisting of actinium-225, bismuth-213, lead-212, and polonium-212. Also preferred is a complex of above-mentioned compounds and isotopes linked to the nanoparticles (e.g. gold, argentum, graphen) or these nanoparticles.
  • nanoparticles e.g. gold, argentum, graphen
  • the pharmaceutically active substance is a virus, it is preferably an oncolytic virus.
  • the drug is a nucleic acid
  • the anticancer drug is a cytostatic drug, cytotoxic drug or prodrug thereof.
  • Preferred anticancer drugs are selected from an apoptosis/autophagy or necrosis-inducing drug.
  • An apoptosis/autophagy or necrosis-inducing drug can be any drug that is able to induce apoptosis/autophagy or necrosis effectively even in cells having an abnormality in cell proliferation.
  • These drugs are preferably used in complexes with one or more ferritins.
  • the anti-cancer drug is selected from the group consisting of an apoptosis-inducing drug, an alkylating substance, anti-metabolites, antibiotics, antimitotic agent, a DNA-modifying drug, a DNA minor groove interstrand crosslinking drug, an inhibitor of DNA synthesis, an inhibitor of RNA synthesis, epothilones, nuclear receptor agonists and antagonists, an anti- androgene, an anti-estrogen, a platinum compound, a hormone, a antihormone, an interferon, an inhibitor of cell cycle-dependent protein kinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, a biogenic fatty acid, a biogenic fatty acid derivative, including prostanoids and leukotrienes, an inhibitor of protein kinases, an inhibitor of protein phosphatases, an inhibitor of lipid kinases, a platinum coordination complex, an ethyleneimine, a methylmelamine, a triazin
  • the anti-cancer drug is selected from the group consisting of acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chaliceamycin, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycindapsone, daunorubicin, deruxtecan, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, dolastatin 10, dolastatin 15, dynemycinA, enediynes, epirubicin, epothilone B, epothilone
  • the anti-cancer drug is selected from the group consisting of auristatin, banoxantrone, bendamustine, chlorambucil, chaliceamycin, dynemycin A, maytansine, melphalan, mertansine, neocazinostatin and pyrrolobenzodiazepine.
  • the anti-cancer drug is auristatin, in particular monomethyl auristatin E or monomethyl auristatin F.
  • the anti -cancer drug is deruxtecan. The examples were performed using the drug monomethyl auristatin E.
  • the anti-cancer drug is an immunomodulatory drug that activates or inhibits an activity of an immune cell
  • the immunomodulatory drug is a ligand or antagonist of Pattern Recognition Receptors, particularly Toll-like Receptors, NOD-like receptors (NLR), RIG-I-like receptors (RLR) or Stimulator of interferon genes (STING) protein.
  • Pattern Recognition Receptors particularly Toll-like Receptors, NOD-like receptors (NLR), RIG-I-like receptors (RLR) or Stimulator of interferon genes (STING) protein.
  • NLR NOD-like receptors
  • RLR RIG-I-like receptors
  • STING Stimulator of interferon genes
  • the anti-cancer drug is a proliferation inhibiting protein or peptide, preferably a cell cycle inhibitor or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a nucleic acid, preferably encoding a proliferation inhibiting protein or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a siRNA, oligonucleotide, LNA, or DNAzyme.
  • prodrug refers to any active ingredient that, after administration, is metabolized or otherwise converted to a biologically active or more active ingredient (or drug) with respect to at least one property.
  • a prodrug is modified chemically in a manner that makes it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered to the patient.
  • a prodrug may for example have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavor (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference).
  • a prodrug may be synthesized using reactants other than the corresponding drug.
  • the pharmaceutically active substance is a hypoxia-activated prodrug, preferably selected from the group consisting of benzotriazine N-oxides, apaziquone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH-302, TH-4000 and AQ4N.
  • EO9 apaziquone
  • TPN tirapazamine
  • SN30000 PR-104A
  • TH-302, TH-4000 AQ4N.
  • the active ingredient is a hypoxia-activated prodrug.
  • hypoxia-activated prodrugs are any prodrug that is less active or inactive, relative to the corresponding drug, and comprises the drug and one or more bioreducible groups.
  • Such hypoxia-activated prodrugs include all prodrugs activated by a variety of reducing agents and reducing enzymes, including without limitation single electron transferring enzymes (such as cytochrome P450 reductases) and two electron transferring (or hydride transferring) enzymes.
  • hypoxia-activated prodrug is TH-302. Methods of synthesizing TH-302 are described in PCT application WO 07/002931 and WO 08/083101.
  • the pharmaceutically active substance is an antigen or a nucleic acid encoding an antigen.
  • the terms “label” or “diagnostic agent” are used interchangeably herein and refer to any kind of compound being suitable for diagnostic purposes.
  • the label is selected from the group consisting of a fluorescent dye, a radioisotope/fluorescence emitting isotope, a detectable polypeptide or nucleic acid encoding a detectable polypeptide, and a contrast agent or the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations. More preferably, the label is selected from a fluorescent dye, a radioisotope and a contrast agent.
  • a contrast agent is a dye or other substance that helps to show abnormal areas inside the body.
  • Preferred fluorescent dyes are selected from the group consisting of the following classes of fluorescent dyes: xanthens (e.g. fluorescein), acridines (e.g. acridine yellow), oxazines (e.g. oxazine 1), cynines (e.g. Cy7 / Cy 3), styryl dyes (e.g. dye-28), coumarines (e.g. Alexa Fluor 350), porphines (e.g. chlorophyll B), metal-ligand-complexes (e.g. PtOEPK), fluorescent proteins (e.g. APC, R- phycoerythrin), nanocrystals (e.g. QuantumDot 705), perylenes (e.g. Lumogen red F300) and phtalocyanines (e.g. IRDYETM700DX) as well as conjugates and combinations of these classes of dyes.
  • xanthens e.g
  • Preferred radioisotopes/fluorescence emitting isotopes are selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65Tb, fluorescence emitting isotopes, such as 18F, 51Cr, 67Ga, 68Ga, 89Zr, U lin, 99mTc, 140La, 175Yb, 153Sm, 166Ho,88Y, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101ml5Rh, 119Sb, 128Ba, 1231, 1241, 1311, 197Hg, 211At, 169Eu, 203Pb, 212
  • Preferred detectable polypeptides are an autofluorescent protein, preferably green fluorescent protein or any structural variant thereof with an altered adsorption and/or emission spectrum.
  • Preferred contrast agents are selected from paramagnetic agents, e.g. Gd, Eu, W and Mn, preferably complexed with a chelating agent. Further options are superparamagnetic iron (Fe) complexes and particles, compounds containing atoms of high atomic number, i.e. iodine for computer tomography (CT), microbubbles and carriers such as liposomes that contain these contrast agents.
  • the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations.
  • Preferred chelating agents are selected from the group consisting of 1,4,7,10- tetraazacyclododecane-N,N',N,N' -tetraacetic acid (DOTA), ethylenediaminetetraacetic acid (EDTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, Diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTP A) and 6-Hydrazinopyridine-3 -carboxylic acid (HYNIC).
  • DOTA Diethylenetriamine-N,N,N',N',N',N"-pentaacetic acid
  • HYNIC 6-Hydrazinopyridine-3 -carboxylic acid
  • the ability of a given cell or of a population thereof to internalize ferritin depends on the expression of receptors involved in this internalization process.
  • the inventors show thatMSR-1 is the main receptor responsible for ferritin uptake into human macrophages.
  • the inventors show that macrophages express high amounts of MSR-1, while monocytes express only low amounts of MSR-1 or no MSR-1 (Fig. IE). Macrophages also take up more ferritin than monocytes (Fig. 1 A and C).
  • M2 macrophages show higher MSR-1 expression than Ml macrophages (Fig. 2 B).
  • M2 macrophages take up more ferritin than Ml macrophages (Fig. 2 C and D).
  • the skilled person is well aware of how to measure the amount of ferritin uptake and preferred methods of measuring the uptake are described in the Example Section below.
  • the inventors developed a method which allows for improving the efficiency and specificity of ferritin uptake into monocytes and macrophages, in particular ferritin in a complex with an active ingredient. This can be achieved by selecting macrophages or monocytes that have a high MSR-1 expression or by increasing the expression of MSR-1 on macrophages or monocytes.
  • Selection of macrophages and monocytes can be performed using a FACS sorter which sorts a heterogeneous mixture of cells based upon the specific fluorescent characteristics of each cell. Cells are first tagged using fluorescent antibodies targeting MSR-1 receptor on target cells.
  • MSR-1 positive cells can be done for example by DNA plasmid transfection using a plasmid encoding human MSR1 (CD204/MSR1 cDNA ORF Clone).
  • the plasmid is first multiplied in Subcloning Efficiency DH5a Competent Cells (Thermo Fisher Scientific) and purified using EndoFree Plasmid Maxi Kit (Qiagen). It can be used plasmids coding wild-type MSR1. Day before transfection cells are seeded in 24-well plates for flow cytometry analysis or 12-well plates for protein isolation and western blot analysis and grown in their respective medium at 37 °C in a humidified 5% CO2 atmosphere.
  • Cells are transfected with plasmids using lipofection method with commercially available kit LipofectammeTM LTX Reagent with PLUSTM Reagent (Invitrogen) according to the transfection protocol provided by the manufacturer. Cells are incubated for 24 h at 37 °C in a humidified 5% CO2 atmosphere before further experiments.
  • the macrophages and/or monocytes comprised in the targeted delivery system are further characterized as follows:
  • the targeted delivery system comprises a macrophage.
  • the macrophage is an activated macrophage, preferably a CDl lb+ macrophage, more preferably a CDl lb+ CD16+ macrophage, CDl lb+ CD32+ macrophage, CDl lb+ CD64+ macrophage, CDl lb+ CD68+ macrophage, preferably a CDl lb+ CD86+ Ml macrophage, preferably producing iNOS and/or secreting interleukin 12 (IL-12) or preferably CDl lb+ CCR2+ M2 macrophage, CD 1 lb+ CD204+ M2 macrophage, CD 1 lb+ CD206+ M2 macrophage, CD 1 lb+ CD204+ CD206+ M2 macrophage, CDl lb+ Mayor Histocompatibility Complex 11+ (MHCII+) (low or hi expression) M2 macrophage, CD1 lb+ CD200R+ M2 macrophage, CD1 lb+ CD163+ M
  • the macrophage expresses at least one chemokine receptor, preferably selected from the group consisting of CCR1, CCR2+, CXCR4+, and CXCR6+, or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony stimulating factor Receptor (CD 115), granulocyte colony stimulating factor Receptor (CD 114), and granulocytemacrophage colony stimulating factor Receptor (consisting of CD 116 and CD 131 ) .
  • chemokine receptor preferably selected from the group consisting of CCR1, CCR2+, CXCR4+, and CXCR6+
  • growth factor receptor preferably selected from the group consisting of macrophage colony stimulating factor Receptor (CD 115), granulocyte colony stimulating factor Receptor (CD 114), and granulocytemacrophage colony stimulating factor Receptor (consisting of CD 116 and CD 131 ) .
  • cytokines preferably IL-10 and IL-12, chemokines and/or to produce iNOS, arginase or other immunomodulating enzymes
  • (ii) is characterized by expression of at least one of following antigens: CD64, CD86, CD 16, CD32 HLA-DR, and/or production of iNOS and/or IL-12;
  • (iv) is characterized by expression of at least one of following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4 (CXCR4), CD163, and/or show low expression of HLA-DR;
  • cytokine secretion preferably of IL-12, or IL-10, or production of inducible nitric oxide synthetase (iNOS), pro-inflammatory compounds, arginase immunosuppressive compounds or anti-inflammatory compounds, preferably wherein
  • iNOS inducible nitric oxide synthetase
  • the Ml inducer is selected from the group consisting of LPS, INF-y, GM-CSF, and viral or bacterial proteins or products; or
  • the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody, IgG, heat activated gamma-globulin, glucocorticosteroid, TGF-p, IL-1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, adenosine, helminth or fungal proteins or products.
  • the macrophage is an undifferentiated macrophage. In some embodiments, the macrophage is a naive macrophage. In some embodiments, the macrophage is an MO macrophage. In some embodiments, the macrophage is an M2 macrophage. In some embodiments, the macrophage is mildly polarized towards M2. The skilled person is aware of surface markers expressed by M2 macrophages or macrophages that are mildly polarized towards M2.
  • the macrophage is a human macrophage.
  • Macrophages are tissue-resident professional phagocytes and antigen-presenting cells (APC), which differentiate from circulating peripheral blood monocytes (PBMs).
  • APC antigen-presenting cells
  • PBMs peripheral blood monocytes
  • the term “activated macrophage” is used in the context of the present invention to refer to any macrophage that is polarized. Macrophage activation is in general achieved by incubation with interleukins, cytokines and/or growth factors. In particular IL-4 and M-CSF can be used as activating agents.
  • Ml -macrophages classically activated macrophages (CAM) and M2-macrophages, alternatively activated macrophages (AAM)
  • CAM classically activated macrophages
  • AAM alternatively activated macrophages
  • the classically activated Ml- macrophages comprise immune effector cells with an acute inflammatory phenotype. These are highly aggressive against bacteria and produce large amounts of lymphokines (Murray, and Wynn, 2011, J LeukocBiol, 89(4):557-63).
  • the alternatively activated, anti-inflammatory M2-macrophages can be separated into at least three subgroups. These subtypes have various different functions, including regulation of immunity, maintenance of tolerance and tissue repair/wound healing.
  • Ml inducer is used in the context of the present invention to refer to a compound that directs differentiation of PBMs to macrophages of the Ml type.
  • M2 inducer is used in the context of the present invention to refer to a compound that directs differentiation of PBMs to macrophages of the M2 type.
  • the skilled person is aware of a large number of ways to promote differentiation into either Ml or M2 macrophages.
  • phagocytosis by macrophages is the process by which a macrophage engulfs a solid particle to form an internal vesicle known as a phagosome.
  • viral/bacterial/fungal/helminth proteins or products refers to molecules produced by or originating from viruses, bacteria, fungi or helminths during a viral/bacterial/fungal/helminth infection.
  • CDl lb+ indicates that the majority of cells within a population of cells or essentially all cells express the surface antigen CD 11b, CD 16 or CD64, respectively.
  • the term “high expression” of a given protein refers to detectable expression of that protein that is at least 70% of the highest expression level found, i.e. number of copies per cell, in a population of healthy cells, in particular macrophages.
  • the term “low expression” of a given protein refers to detectable expression of that protein that is 30% or less of the highest expression level found, i.e. number of copies of that protein per cell, in a population of healthy cells, in particular macrophages.
  • the “highest expression level” is determined as the average of the highest expression levels found in healthy cells, in particular macrophages of different subjects.
  • preferred subpopulations of cells are characterized as “producing” a given protein. This is understood to mean that the protein is not necessarily detectable on the surface of the cell but may only be present inside the cell. The skilled person is well aware how to detect and/or quantify production of a protein inside a cell and/or select cells producing such proteins.
  • the macrophage comprised in the targeted delivery system for use according to the invention is producible from a CD34 + hematopoietic precursor cell via a monocyte precursor. Macrophages are differentiated from monocytes. Macrophages can thus also be referred to a “differentiated monocytes”.
  • the term “differentiated monocyte” is used in the context of the present invention to refer to a monocyte differentiated from the committed precursor termed macrophage-DC precursor (MDP) mainly resident in bone marrow (but could be also in the spleen) and differentiate into either dendritic cells or macrophages.
  • MDP macrophage-DC precursor
  • mice consist of two main subpopulations: (i) CD1 lb + cell with high expression of CX3CR1, low expression of CCR2 and Ly6C- and (ii) CD1 lb + cell with low expression of CX3CR1, high expression of CCR2 and Ly6C + .
  • mouse Ly6C + monocytes differentiate into Ly6C- monocytes in circulation.
  • CD 14 ++ classical monocytes leave bone marrow and differentiate into CD 14 ++ CD16 + intermediate monocytes and sequentially to CD I4 CD I6 iion-classical monocytes in peripheral blood circulation (Y ang et al. 2014; Biomark Res 2(1) doi. 10.1186/2050-7771-2-1).
  • the targeted delivery system comprises a monocyte.
  • monocytes are a mixed population of different monocytes.
  • the monocyte comprised in the targeted delivery system is a CDl lb + monocyte, preferably selected from the group consisting of a CDl lb + CD14 + monocyte, a CDl lb + CD16 + monocyte, a CD1 lb + CD14 + CD16 + monocyte, a CDl lb + CD 14 + HLA-DR monocyte, a CDl lb + CD14 + CD115 + monocyte, a CDl lb + CD14 + monocyte, a CD1 lb + CD16 + monocyte, a CDl lb + CCR1 + monocyte, a CD1 lb + CCR2 + monocyte, a CD1 lb + CX3CR + monocyte, a CD1 lb + CXR4 + monocyte, a CD1 lb + CXR6 + monocyte and a CD1 lb + CD14 + CD
  • the monocyte comprised in the targeted delivery system, or the monocyte from which the macrophage comprised in the targeted delivery system is differentiated is a differentiated monocyte.
  • the differentiated monocyte is selected from the group consisting of a macrophage, an activated macrophage, preferably a CDl lb + macrophage, more preferably a CDl lb + CD16 + macrophage, a CDl lb + CD32 + macrophage, a CDl lb + CD64 + macrophage, a CDl lb + CD68 + macrophage, preferably a CD1 lb + CD86 + Ml macrophage, preferably producing iNOS and/or secreting interleukin 12 (IL-12) or preferably a CDl lb + CCR2 + M2 macrophage, a CDl lb + CD204 + M2 macrophage, a CDl lb + CD206 + M2 macrophage, a CDl lb + CD204 + CD206 + M2 macrophage, a CDl lb + HLA-DR + M2 macrophage, a CDl lb + CD200R + M2
  • the differentiated monocyte expresses at least one chemokine receptor, preferably selected from the group consisting of CCR1, CCR2, CXCR4, and CXCR6, or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony-stimulating factor receptor (CD115), granulocyte colony-stimulating factor receptor (CD114), and granulocytemacrophage colony stimulating factor receptor (CD 116 and CD 131).
  • the differentiated monocyte is not a dendritic cell.
  • the monocyte or differentiated monocyte is derived from a monocyte or differentiated monocyte.
  • (ii) is producible by in vitro incubation of monocytes with at least one inducer, preferably Ml or M2 inducer, more preferably at least one M2 inducer; (iii) is characterized by expression of at least one of the following antigens: TfR, CD163, CD14, CD16, CD33, CXCR4, 25f9, HLA-DR and/or CD115 and optionally CD172a and/orCXCR4, in particular at least one of TfR, CD163, CD14, CD16, CD33, 25f9, CD172a and/or CD115 or at least one of TfR, CD163, CD14, CD16, CXCR4, 2519, and/or CXCR1; and/or
  • the Ml inducer is selected from the group consisting of LPS, GM-CSF, INF-y, viral or bacterial proteins or products;
  • the M2 inducer is selected from the group consisting of IL-4, IL- 10, IL-13, an immune complex of an antigen and antibody, IgG, heat activated gamma-globulins, glucocorticosteroids, TGF-0, IL-1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, cancer-conditioned medium, cancer cells, adenosine and helminth or fungal proteins or products.
  • the M2 inducer is selected from the group consisting of IL-4, IL- 10, IL-13, an immune complex of an antigen and antibody, IgG, heat activated gamma-globulins, glucocorticosteroids, TGF-0, IL-1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, cancer-conditioned medium, cancer cells, adenosine and helminth or fungal proteins or products.
  • the differentiated monocyte or the macrophage is characterized by expression of at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven or all of TfR, CD163, CD14, CD16, CD33, 25f9, CD172a and/or CD115 or at least one, at least two, at least three, preferably at least four, at least five, more preferably at least six, at least seven or all of TfR, CD163, CD14, CD16, CXCR4, 25f9, and/or CXCRL
  • the targeted delivery system for use of the present invention still provides the outlined advantages, if in a population of cells not every cell has a particular property in as long as the majority of cells within that population has that property. Thus, in the following the property of one preferred cell of the targeted delivery system for use of the present invention is described.
  • the macrophages or monocytes comprised in the targeted delivery system are derived from isolated peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the macrophages or monocytes comprised in the targeted delivery system are primary cells, i.e. cells isolated directly from human tissues, in particular peripheral blood. It is preferred that macrophages or monocytes comprised in the targeted delivery system, are not cells of an immortalized cell line.
  • the macrophages are bone marrow derived macrophages (BMDM) or monocyte-derived macrophages (MDM).
  • the macrophages or monocytes comprised in the targeted delivery system originate from the patient to be treated.
  • the cell loaded with the complex would be autologous to the patient.
  • patients are HLA typed prior to treatment with the targeted delivery system of the present invention and that the cell type used for a given patient is HLA matched to the patient.
  • the cell is a primary cell or derived by a low number of differentiation steps from a primary cell.
  • the cell may be from an immortalized but preferably non-transformed cell line.
  • the blood used for isolation of CD45 + leukocyte cells is preferably obtained from the patient to be treated or from a healthy donor. Alternatively, the blood can be obtained from the blood bank. Use of umbilical cord blood is also considered herein.
  • the invention provides a pharmaceutical composition comprising the isolated targeted delivery system according to the first aspect of the invention.
  • the pharmaceutical composition according to the invention comprises a pharmaceutically acceptable carrier and/or suitable excipient(s).
  • the isolated targeted delivery system comprises living cells, it is preferred that carriers and excipients are chosen in such to keep the cells alive.
  • “Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • carrier refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, surfactants, stabilizers, physiological buffer solutions or vehicles with which the pharmaceutically active substance is administered.
  • Such pharmaceutical carriers can be liquid or solid.
  • Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • a saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Suitable pharmaceutical “excipients” include starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • “Surfactants” include anionic, cationic, and non-ionic surfactants such as but not limited to sodium deoxycholate, sodium dodecylsulfate, Triton X-100, and polysorbates such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80.
  • “Stabilizers” include but are not limited to mannitol, sucrose, trehalose, albumin, as well as protease and/or nuclease antagonists.
  • “Physiological buffer solution” include but are not limited to sodium chloride solution, demineralized water, as well as suitable organic or inorganic buffer solutions such as but not limited to phosphate buffer, citrate buffer, tris buffer (tris(hydroxymethyl)aminomethane), HEPES buffer ([4 (2 hydroxyethyl)piperazino]ethanesulphonic acid) or MOPS buffer (3 morpholino- 1 propane sulphonic acid). The choice of the respective buffer in general depends on the desired buffer molarity. Phosphate buffer are suitable, for example, for injection and infusion solutions.
  • the invention provides the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention for use in medicine.
  • the invention provides the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention for use in the treatment of cancer, an inflammatory disease or ischemic areas; or for prophylactic or therapeutic vaccination, in particular to prevent or treat an infectious disease or cancer.
  • the cancer is selected from breast cancer, pancreatic cancer, bladder cancer, lung cancer, colon cancer, ovarian cancer, liver cancer, glioma/glioblastoma or a tumour having hypoxic areas.
  • the ischemic areas are skm wounds or ischemic retina or tissue after organ infarctus, in particular heart infarctus.
  • the aspect relating to vaccination also includes targeted delivery of antigens to physiological or non-physiological lymph nodes in order to vaccinate an individual or to induce immune memory.
  • treatment includes all types of preventive and/or therapeutic interventions medically allowed for the purpose of cure, temporary remission, prevention, etc. for different purposes including delaying or stopping the progress of a disease, making a lesion regress or disappear, preventing onset, or inhibiting recurrence.
  • the isolated targeted delivery system comprises a pharmaceutically active substance.
  • the isolated targeted delivery system is provided for use in a method of diagnosis of ovarian cancer, it comprises a label.
  • the method of diagnosis is an in vivo method of diagnosis.
  • the isolated targeted delivery system is provided for use in a method of treatment of ovarian cancer.
  • the treatment comprises or consists of the killing of ovarian cancer cells.
  • the treatment comprises or consists of the prevention of metastatic spread.
  • the invention provides a method of treatment or diagnosis of cancer, an inflammatory disease or ischemic areas comprising administration of isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention in an effective amount to a patient in need thereof.
  • the invention provides a method of prophylactic or therapeutic vaccination comprising administration of isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical compositions of the invention in an effective amount to a patient.
  • the invention provides a method of preparing an isolated targeted delivery system, in particular the isolated targeted delivery system according to the first aspect of the invention, comprising the steps of a) providing purified ferritin; b) covalently or non-covalently linking a pharmaceutically active substance, label or pharmaceutically active substance and label to and/or encapsulating a pharmaceutically active substance, label or pharmaceutically active substance and label in ferritin; c) providing a population of macrophages and/or monocytes characterized by a high expression of MSR-1, wherein preferably at least 5%, at least 10%, at least 15%, at least 20% of the macrophages express MSR-1; and d) incubating the population of macrophages and/or monocytes in the presence of the ferritin produced in step b) until the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes are at least partially loaded with the complex of the ferritin and the a pharmaceutically active substance, label or pharmaceutically
  • At least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 4% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes provided in step c) express MSR-1.
  • At least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes provided in step c) express MSR-1.
  • the population of macrophages and/or monocytes is characterized by a high endogenous expression of MSR-1. In some embodiments, a population of monocytes is provided. In some embodiments, a population of macrophages and monocytes is provided. In some embodiments, a population of macrophages is provided.
  • the macrophages are undifferentiated macrophages. In some embodiments, the macrophages are naive macrophages. In some embodiments, the macrophages are M0 macrophages. In some embodiments, the macrophages are M2 macrophages. In some embodiments, the macrophages are mildly polarized towards M2. The skilled person is aware of surface markers expressed by MO or M2 macrophages or macrophages that are mildly polarized towards M2.
  • the macrophages and/ or monocytes are human macrophages and/or monocytes.
  • the population of macrophages and/or monocytes characterized by a high expression of MSR-1 is obtained from a population of macrophages and/or monocytes with low expression of MSR-1 via cell sorting, such as FACS or MACS.
  • the population of macrophages and/or monocytes characterized by a high expression of MSR-1 is obtained from a donor identified via the methods of donor selection described below.
  • the population of macrophages and/or monocytes is genetically modified to overexpress MSR-1.
  • the overexpression is transient or stable, preferably transient. Exemplary methods of overexpressing MSR-1 in macrophages and/or monocytes are described in the examples section.
  • the invention provides an isolated targeted delivery system obtained by or obtainable by the method of the invention described above.
  • the invention provides a method of selecting a monocyte donor, comprising a) generating a population of macrophages from a population of monocytes obtained from a donor; b) determining the percentage of
  • the percentage of MSR-1 -expressing monocytes is high, preferably at least 2%, at least 3%, or at least 4%,
  • the percentage of MSR-1 -expressing macrophages is high, preferably at least 5%, at least 10%, at least 15%, or at least 20%, and/or
  • the invention provides a method of selecting a monocyte donor, comprising a) providing a population of macrophages generated from a population of monocytes obtained from a donor; b) determining the percentage of
  • the percentage of MSR-1 -expressing monocytes is high, preferably at least 2%, at least 3%, or at least 4%,
  • the percentage of MSR-1 -expressing macrophages is high, preferably at least 5%, at least 10%, at least 15%, or at least 20%, and/or
  • Determining the percentage of MSR-1 -expressing monocytes in the population of monocytes can be carried out prior to generating a population of macrophages from the population of monocytes or in a second population of monocytes obtained from the same donor.
  • the percentage of MSR-1 -expressing macrophages in the population of macrophages is determined after 5-10 days, preferably 6-8 days, more preferably 7 days of culture.
  • the macrophage donor is selected to obtain monocytes that are then differentiated into macrophages.
  • the macrophages are used for the generation of a targeted delivery system according to the method of the invention.
  • the donor is a patient
  • the method is a method of patient stratification for autologous therapy.
  • the autologous therapy comprises administering to the patient the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical composition of the invention.
  • the donor is selected for heterologous or allogeneic therapy using the isolated targeted delivery system according to the first aspect of the invention or the pharmaceutical composition of the invention.
  • the invention provides a method of providing or generating macrophages and/or monocytes with high expression of MSR-1, comprising the steps of
  • the method comprises overexpressing MSR-1 in a population of macrophages and/or monocytes.
  • the overexpression can be transient or stable, preferably transient.
  • MSR-1 is transiently overexpressed in a population of monocytes.
  • the method comprises selecting macrophages and/or monocytes with high MSR-1 expression from a population of macrophages and/or monocytes.
  • the selection comprises cell sorting.
  • the selection comprises FACS or MACS.
  • Fig. 1 Comparison ofHFt uptake by human monocytes and macrophages.
  • the level of HFt uptake by human monocytes and macrophages derived from (A) peripheral blood or (C) THP-1 cell line and their viability (B and D, respectively) were measured using flow cytometry.
  • Data are presented as geometric mean fluorescence intensity (MFI, A and C) of HFt-AF488 or % of live cells (B and D). Values represent mean ⁇ SEM from three separate donors (blood-derived cells) or three independent experiments (THP-1).
  • Statistical difference was calculated using two-way ANOVA with Sidak’s multiple comparison test (****p ⁇ 0.0001, ***p ⁇ 0.001, **p ⁇ 0.01, ns > 0.5).
  • Fig. 2 Comparison of HFt uptake by differently polarised hMDM.
  • A, B MSR-1 expression on variously polarised hMDM was evaluated using western blot. The level of expression of target protein was normalised to the expression of P-actin. A representative blot (A) is shown. Values obtained in quantitative western blot analysis represent mean ⁇ SEM from four independent experiments (B). Statistical difference was calculated using one-way ANOVA with Dunnett’s multiple comparison test (****p ⁇ 0.1, ns > 0.5).
  • C - E Differently polarised hMDM were incubated with HF1-AF488 at concentration of (C) 25 or (D) 100 pg/ml for 20 or 60 mm.
  • HF1-AF488 uptake by macrophages and their viability were measured using flow cytometry. Data are presented as geometric mean fluorescence intensity (MFI, C and D) of Alexa Fluor 488 or % of live cells (E). Values represent mean ⁇ SEM from four separate donors. Statistical difference was calculated using two-way ANOVA with Dunnett’s multiple comparison test (****p ⁇ 0.0001, ***p ⁇ 0.001, **p ⁇ 0.01, ns > 0.5).
  • Fig. 3 Comparison of HFt uptake by differently polarised THP-1 macrophages. (A, B) MSR-1 expression in variously polarised THP-1 macrophages was evaluated using western blot.
  • THP-1 macrophages type 1 and 2 were incubated with HFt-AF488 at concentration of (C) 25 or (D) 100 pg/ml for 20 or 60 min. The level of HFt-AF488 uptake by variously polarised THP-1 macrophages and their viability were measured using flow cytometry.
  • Fig. 4 Time-dependent internalisation of MSR-1 upon binding with HFt in human macrophages.
  • the level of expression of MSR-1 on (A) hMDM and (B) THP-1 was evaluated after incubation with unlabelled HFt for different time periods by surface immunostaining using flow cytometry.
  • Data are presented as geometric mean fluorescence intensity (MFI) of anti-MSR-l-PE antibody. Values represent mean ⁇ SEM from three separate donors (hMDM) or three independent experiments (THP-1). Statistical difference was calculated using one-way ANOVA with Dunnet’s multiple comparison test (***p ⁇ 0.001, **p ⁇ 0.01, *p ⁇ 0.1).
  • Fig. 5 The effect of siRNA silencing on expression of MSR-1 and on HFt-AF488 uptake by human macrophages.
  • siRNA targeting chosen receptors scrambled siRNA (siScrl) was used as control.
  • siScrl scrmbled siRNA
  • A, B The effect of RNA interference on MSR-1 expression was assessed using western blot. Protein lysates isolated from transfected cells were examined for (A) MSR-1 expression by immunobloting. The level of expression of target proteins was normalised to the expression of GAPDH. A representative blot is shown. Values obtained in quantitative western blot analysis represent mean ⁇ SEM from three independent experiments.
  • Fig. 6 The effect of overexpression of MSR-1 on HFt uptake in CHO-K1 cells.
  • CHO-K1 cells were transfected with the plasmid encoding either MSR-1 (pMSRl) or SCARA5 (pSCARA5, positive control) or with the empty plasmid (pCMV, negative control).
  • A The effect of plasmid transfection was assessed using western blot. Protein lysate isolated from transfected cells was examined for MSR-1 and SCARA5 receptor expression by immunoblotting. A representative blot from one of the experiment repetitions is shown.
  • B, C The level of expression of target proteins was evaluated by surface immunostainmg using flow cytometry.
  • Fig. 7 Kinetic analysis of HFt binding with MSR-1 using biolayer interferometry.
  • Fig. 8 Kinetic analysis of HFt interaction with MSR-1 using microscale thermophoresis. Doseresponse curves between unlabelled (A) MSR-1 and (B) SCARA5 receptor. Data were analysed using software provided by the manufacturer. Ligand-dependent changes in temperature-related fluorescence intensity are plotted on a graph as Fnorm values (normalised fluorescence, %o) vs. ligand concentration in a dose-response curve. Kd value was derived by fitting data with a 1 : 1 binding model.
  • Fig. 9 Kinetic analysis of HFt binding with MSR-1 using spectral shift technology.
  • A Spectral shift dose-response curve between unlabelled HFt and MSR-1 conjugated with the RED-tris- NTA dye.
  • B Spectral shift dose-response curve between unlabelled MSR-1 and HFt conjugated with Alexa Fluor 488. Data was analysed using software provided by the manufacturer and are presented as the emission fluorescence ratio 670/650 vs. ligand concentration. Kd value was derived by fitting data with a 1: 1 binding model.
  • Ligand concentrations indicated on the x-axis are: IxlO' 11 , IxlO' 10 , IxlO' 9 , IxlO' 8 , IxlO' 7 , IxlO' 6 , IxlO' 5 .
  • Fig. 10 Tfn and HFt uptake by hMDM and THP1 with siRNA for TfRl.
  • a - D Four days after transfection with siRNA against TfR-1, hMDM (A, B) and THP-1 macrophages (C, D, E, F) were incubated with HF1-AF488 or Tfn-AF488.
  • the influence of TfR-l knockdown on ligand uptake by human macrophages was assessed using flow cytometry.
  • the level of (A, C) HFt or (B, D) Tfh internalisation by transfected human macrophages was normalised to ligand uptake by non-transfected cells.
  • Fig. ll Ferritin uptake by human monocyte derived macrophages is positively correlated with MSR-1 expression on macrophages surface and with MSR-1 positive cells.
  • Fig. 12 Correlation between expression of MSR-1 receptor and ferritin uptake by human macrophages obtained from monocytes from 6 different donors. For each bar: top: HFt uptake, bottom: MSR-1 surface staining.
  • the human monocytic cell line THP-1 (ATCC® TIB-202TM) and Chinese hamster ovary cell line CHO-K1 (ATCC® CCL-61TM) were purchased from American Type Culture Collection - ATCC (USA). Primary human monocytes were obtained from buffy coats of healthy individuals from Regional Centre for Blood Donation and Haemotherapy in Warsaw.
  • THP-1 monocytes were cultured in RPMI-1640 medium (Sigma Aldrich, #R8758) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Hyclone, #SV30160.03), 100 U/ml of penicillin and 100 pg/mL of streptomycin (Gibco, #15140122) (called from then on complete medium) at 37°C in a humidified 5% CO2 atmosphere. Cells were split every 2-4 days to assure that a density was kept between 3 x 10 5 and 1 x 10 6 cells/ml.
  • FBS heat-inactivated fetal bovine serum
  • streptomycin Gibco, #15140122
  • cells were harvested by 5 min centrifugation at 350xg, resuspended in fresh, complete medium and seeded at 3-4 x 10 5 cells/ml into a new cell culture flasks.
  • the cell count and viability was determined by trypan blue dye exclusion. Equal volume of cells and trypan blue were mixed and 10 pl was transferred to one of the chambers of the cell counting slide (Countess® Cell Counting Chamber Slides, Thermo Fisher Scientific, #C10228). Cell number and viability was determined using the automated cell counting device Countess® II Automated Cell Counter (Thermo Fisher Scientific).
  • THP-1 monocytes were differentiated into macrophages by incubation with phorbol 12-myristate 13-acetate (PMA, Sigma Aldrich, #P8139) at concentration of 100 ng/ml for 72 h at 37°C in a humidified 5% CO2 atmosphere followed by 24 h of rest in complete medium only.
  • PMA phorbol 12-myristate 13-acetate
  • macrophages were further polarised into Ml subtype by incubation with 1 or 10 ng/ml LPS (InvivoGen, #TLRL-PEKLPS) and 50 ng/ml IFN-y (Peprotech, #300-02) or into M2 subtype by incubation with 20 ng/ml of IL-4 (Peprotech, #200-1104) and 20 ng/ml IL-13 (Peprotech, #200-13) for additional 48 h.
  • LPS InvivoGen, #TLRL-PEKLPS
  • IFN-y IFN-y
  • CHO-K1 cells were cultured in Ham's F-12K medium (Gibco, #21127022) with 10% heat- inactivated bovine calf serum (BCS) (Hyclone, #SH30072.04), lOOU/ml of penicillin and 100 pg/ml of streptomycin (called later complete medium) at 37°C in a humidified 5% CO2 atmosphere.
  • BCS heat- inactivated bovine calf serum
  • lOOU/ml of penicillin 100 pg/ml of streptomycin (called later complete medium) at 37°C in a humidified 5% CO2 atmosphere.
  • CHO-K1 were passaged every 2-3 days (or if they reach 90% confluence).
  • cells were wash with PBS (Biowest, #L0615-1000), detached by incubation with trypsin-EDTA (0.25%) (Gibco, #25200072) for 3-5 min at 37°C, followed by trypsin neutralisation with fresh, complete medium, and split at the ratio of 1:6 to 1: 12 (depending on the demand for further experiments) into new culture flasks. Determination of cell count and viability was performed as for THP-1.
  • hMDM Human monocyte-derived macrophages
  • a buffy coat was mixed with equal volume of PBS (without Ca2+ and Mg2+) and was carefully layered on top of Histopaque®-1077 (Sigma Aldrich, #10771), a density gradient cell separation medium. Tubes were then centrifuged for 30 min at 400xg with acceleration and break switched off. PBMC were harvested from the interphase, transferred to a fresh tube and washed twice with ice-cold PBS by centrifugation at 300xg for 10 min at 4°C. A cell pellet was then resuspended in 10 ml of PEA buffer and the number and viability of PBMC was determined by trypan blue dye exclusion as described before for THP-1 cells.
  • PBMC peripheral blood mononuclear cells
  • PEA buffer by centrifugation for 10 min at 300xg at 4°C.
  • Supernatant was discarded and cells were resuspended with 800 pl PEA buffer followed by addition of 200 pl of CD14 MicroBeads (Miltenyi, #130-050-201).
  • Suspension was mixed well and incubated for 20 min in the refrigerator (2-8°C).
  • CD 14 a monocyte marker
  • LS columns (Miltenyi, #130-042-401) were placed in the magnetic field of a QuadroMACSTM Separator (Miltenyi, #130-090-976) and rinsed with 3 ml of PEA buffer. After centrifugation supernatant was discarded, PBMC were resuspended in 3 ml of fresh PEA buffer and transferred on previously prepared LS columns. Unlabelled cells that passed through were discarded and columns were washed three times with 3 ml PEA buffer (new portion was added only when reservoir was empty). Columns were then removed from the separator, placed on a fresh collection tube and CD 14+ cells were eluted by flushing them out with 5 ml of PEA buffer using a provided plunger.
  • monocytes Positively selected monocytes were centrifuged for 10 min at 300xg to remove PEA buffer, resuspended in 10 ml of complete RPMI-1640 medium and counted using trypan blue dye as described previously.
  • monocytes cells were seeded on non-adherent plates in complete RPMI 1640 medium and left overnight to rest before further treatment.
  • monocytes were seeded on 9-cm or 15 -cm bacteriological Petri dishes at the density of 1 x 106 cells/ml in 10 or 25 ml, respectively, of complete RPMI-1640 medium supplemented with 50 ng/ml of human recombinant M-CSF (Biolegend, #574808) and subsequently cultured for 7 days at 37°C in a humidified 5% CO2 atmosphere. On day 5 an extra volume of fresh culture medium was added (5 or 12 ml, depending on a plate size).
  • hMDM were harvested by incubation for 30 min at RT with Accutase (Sigma Aldrich, #A6964) followed by gentle scraping and re-plated on non-adherent plates for further experiments. If needed, macrophages were further polarised into Ml subtype by incubation with 1 or 10 ng/ml LPS and 50 ng/ml IFN-y or into M2 subtype by incubation with 20 ng/ml of IL-4 and 20 ng/ml IL-13 for additional 48 h.
  • WT HFt human heavy chain ferritin
  • cells were resuspended at 20 mM HEPES pH 7.5 with 300 mM NaCl and disrupted in a homogenizer (PandaPLUS 2000, GEA Niro Soavi) on ice. After centrifugation at 15 OOOxg for 30 min at 4°C, the supernatant was subjected to following purification steps: 2 h incubation with 30% ammonium sulfate, dialysis for 48 h against deionized water and heating at 60°C for 10 min. Afterwards, the protein was precipitated by incubation with 50% ammonium sulfate overnight at 4°C, followed by centrifugation at 15 OOOxg for 1 h at 4°C.
  • a homogenizer PandaPLUS 2000, GEA Niro Soavi
  • the protein was loaded onto a 60/600 Superdex 200 column and purified using NGC chromatography system (Bio-Rad). The main fraction of ferritin was collected and the purity of the protein was check by SDS-PAGE electrophoresis. The concentration of purified HFt was determined by UV-Vis measurement using an extinction coefficient of 18 600 M-lcm-1. The purification process was carried out by the Core Facility for Crystallography and Biophysics, University of Warsaw (Poland).
  • WT HFt was conjugated with following fluorescent dyes: Alexa FluorTM 488 NHS Ester (Thermo Fisher Scientific, A20000) and Alexa FluorTM 647 NHS Ester (Thermo Fisher Scientific, A20006). Labelling of protein was based on reaction between succinimidyl ester groups present in the dye and primary amines on ferritin.
  • Cells were transfected with isolated plasmids using lipofection method with commercially available kit LipofectamineTM LTX Reagent with PLUSTM Reagent (Thermo Fisher Scientific, #15338100) according to the transfection protocol optimised for CHO-K1 provided by the manufacturer.
  • siRNA small-interfering RNA
  • siRNA was delivered into cells by electroporation using the NeonTM Transfection System (Thermo Fisher Scientific) and the NeonTM Transfection System 100 pl Kit (Thermo Fisher Scientific, #MPK10096), instead of the liposome-based method.
  • Flow cytometry is a useful tool for a rapid analysis of thousands of individual cells, well suited to collect multiple information about every cell at the same time. In this study it was used to check the ligand-induced internalisation of a scavenger receptor and to assess the efficiency of overexpression and knock-down of selected genes.
  • cells were harvested either with Accutase as described before, washed with PBS, counted and transferred to a 96 V-bottom plate (3-4 x 105 cells/well). They were then resuspended in 80 pl of FACS buffer (Table 3.7), mixed with 20 pl of the human FcR Blocking Reagent (Miltenyi, #130-059-901) and incubated for 10 min on ice to block non-specific antibody binding to Fc receptors.
  • the cells were then incubated either with a specific fluorochrome-conjugated antibody or with its isotype control (Table 3.8) for 1 h on ice in the dark. After immunostaining, cells were washed twice with ice-cold PBS to remove excess of an antibody, resuspended in 100 pl of FACS buffer and acquired on FACSCantoTM II flow cytometer (BD Biosciences) . Data were analysed using the FlowJoTM Software (version 7.6.1, Tree Star, Inc).
  • monocytes were used one day after isolation, then 6 days later macrophages from the same donors were harvested and seeded on a 24-well plate and left overnight to attach,
  • cells were transferred on ice, washed twice with ice- cold PBS and harvested by incubation with trypsin-EDTA (0.25%) for 5 min at 37°C (CHO-K1) or with Accutase for 30 min on ice (macrophages). They were then transferred to FACS tubes, centrifuged for 5 min at 350xg at 4°C and resuspended in 100 pl of FACS buffer mixed with Draq7, a non-fixable dead cell dye (Biolegend, #424001) diluted 1:500. Immediately after, samples were acquired with FACSCantoTM II flow cytometer. Data were analysed using the FlowJoTM Software.
  • Permeabilised cells were incubated overnight at 4°C with the primary antibody against Early Endosome Antigen 1 (EEA1, Invitrogen, #MA514794) diluted 1:500 in 0,5 ml of solution II (Table 3.12). On the next day, cells were washed 2x5 min with solution II and incubated with the fluorochrome- conjugated secondary antibody (Invitrogen, #A-21244) diluted 1:500 in 0,3 ml of solution II with addition of Hoechst 33342 at concentration 2pg/ml (Invitrogen, #H3570) for 30 min at RT.
  • EAA1 Early Endosome Antigen 1
  • Biolayer interferometry is a label-free method for measuring interactions between molecules. It is an optical analytical technique that analyses the interference pattern of white light reflected from two surfaces: a layer of immobilized protein and an internal reference layer.
  • a bite molecule ligand
  • the binding between the immobilized ligand and analyte generates a change in optical thickness at the tip of biosensor which results in a shifts in an interference pattern of light proportional to the number of molecules bound to the surface of the biosensor.
  • BLI was employed to confirm the interaction between HFt and two scavenger receptors - MSR-1 and SCARA5. Measurements were conducted using BLItz system from Sartorius (previously ForteBio) with nickel nitriloacetic acid (Ni-NTA) biosensors, as both recombinant proteins used in this assay were fused with His-tag at N terminus.
  • Pre-hydrated biosensor was put on the mount (without touching the tip) and plunged into tube containing 250 pl of assay buffer for equilibration (1) for 30-60s (depending on assay). In order to reach the maximum binding capacity in every experiment, the time of following steps was optimized and each assay was designed accordingly.
  • biosensor was moved into 4 pl of ligand solution on drop holder (2) and incubated for an appropriate length of time. Subsequently, biosensor was placed in the assay buffer (in tube) to wash away an excess of the ligand and establish new stable baseline signal (3).
  • association step (4) biosensor was plunged into 4 pl of ferritin solution on a drop holder and incubated for an appropriate period of time to allow binding to the ligand.
  • step (5) in which biosensor was transferred into a tube containing fresh portion of assay buffer. These steps were repeated for all the concentrations of the ligand, every time using a fresh biosensor. After each step a drop holder and a tube were washed thoroughly and rinsed with the assay buffer.
  • Nonspecific binding of the ligand to the sensor surface can be a problem for label-free methods such as BLI. Even though this issue is usually minimized by addition of BSA and Tween 20 to the assay buffer (both present in the kinetics buffer from the supplier), every assay included one control in which ligand loading step was performed with assay buffer instead of ligand solution followed by association step with the highest concentration of analyte (HFt).
  • thermophoresis Spectral shift technology and microscale thermophoresis
  • MST MicroScale Thermophoresis
  • MST is an immobilization-free, biophysical assay used to analyse the interaction between molecules in solution.
  • MST technology is based on two effects: thermophoresis (the directed movement of molecules in microscopic temperature gradients) and temperature-related intensity-change of fluorescent molecules.
  • the assay detects the interaction between two molecules by quantifying the thermophoretic movement of fluorescently-labelled molecules caused by a temperature change induced by an infrared laser.
  • the fluorescently-labelled target at fixed concentration
  • the binding between two molecules changes their molecular properties such as size or charge, which influences thermophoretic mobility or fluorescently-labelled molecule.
  • Isothermal spectral shift technique is based on the phenomenon that both peak intensity and wavelength maxima of fluorophore attached to the target can change when their chemical microenvironment is altered.
  • a free fluorophore-labelled target generates a particular emission spectrum, and if unlabelled ligand binds to this target, the fluorophore’s chemical environment is changed, which leads to a shift in its fluorescence spectra.
  • In-solution spectral shift measurements enable precise characterization of molecular interactions for a variety of biomolecules.
  • Monolith X allows characterization of interaction for very challenging molecules such as membrane proteins and since both molecules are in solution (in contrast to BLI method when receptor is bound to biosensor), there are no lost activities due to immobilization.
  • a working buffer (used for sample dilution and as a binding buffer), was prepared by addition of Tween 20 to PBS to a final concentration 0,05%.
  • the His-tagged MSR-1 served as a target and it was conjugated with a red dye using His-Tag Labelling Kit RED-tris-NTA (Nanotemper, MO-L018) according to the manufacturer's protocol.
  • MSR-l-NTA- RED was then diluted to yield concentration of 50 pM and mixed with a set of dilutions of unlabelled HFt (0,61-2500nM). Well-mixed solutions were then transferred to capillaries (provided in kit) and the measurement was performed at 25 or 37°C.
  • HF1-AF488, used as labelled target was diluted to a final concentration of 20 nM and mixed with a set of dilutions of unlabelled SCARA5 (0,082-2700 nM) or MSR-1 (0,09-2950 nM) receptors, which were used as ligands. Well-mixed solutions were then transferred to capillaries and the measurement was performed at 25 °C. Data were analysed with the provided software.
  • the goal is selection of human monocyte derived macrophages with high expression of SR-A1 antigen to optimize ferritin uptake.
  • Macrophages with high expression of SR-A1 significantly more efficient uptake the ferritin in comparison to macrophages with low expression or without SR-A1 antigen.
  • Cells are stained with antibody for SR-A1 and with live/dead marker.
  • Flow cytometry analysis is then performed to evaluate SR-A1 relative expression (MFI) and SR- A1 positive cells on live single cells.
  • MFI SR-A1 relative expression
  • SR-A1 positive cells live single cells.
  • the level of SR-A1 expression is compared to unstained or FMO or stained with isotype antibody cells.
  • SR-A1 mRNA can be synthesized in vitro using a commercial kit such as the mMESSAGE mMACHINE T7 Ultra Kit (Thermo Fisher Scientific). For the transfection, commercial transfection reagent like Lipofectamine MessengerMAX (Thermo Fisher Scientific) can be used followed the manufacturer's protocol for transfection. After 24-48 hours, SR-A1 overexpression using techniques such as qPCR or Western Blot will be assessed, which will verify the overexpression at the mRNA and protein levels respectively.
  • Method 2 SR-A1 gene cloning into a lentiviral transfer plasmid.
  • This plasmid should also contain necessary elements for virus production (like viral long terminal repeats) and a reporter gene (like GFP) for tracking the infection.
  • HFt-AF488 uptake by human monocytes and macrophages was compared to check whether these two cell populations differ in the amount of internalized HFt.
  • the uptake of the protein was examined using both primary cells and the established cell line (THP-1). Monocytes and macrophages were incubated at 37°C with two different concentrations of HFt-AF488 (20 and 200 pg/ml) for 3 different time periods (30, 90 and 180 min). Uptake of HFt and cell viability were measured using flow cytometry.
  • both primary macrophages and macrophages derived from THP-1 monocytes took up significantly more HFt- AF488 than corresponding monocytes, regardless of the incubation time and HFt concentration
  • the lower uptake of HFt by human monocytes was not due to their low viability as both monocytes and macrophages were almost equally viable.
  • MSR-1 is one of the markers of alternatively polarised macrophages (M2), therefore if HFt is taken up by cells via this receptor, classically polarised macrophages (Ml) should internalise less HFt in comparison to M2 cells.
  • M2 alternatively polarised macrophages
  • Ml classically polarised macrophages
  • variously polarised macrophages were either lysed with RIPA buffer for western blot analysis or incubated at 37°C with two different concentrations of HF1-AF488 (25 and 100 pg/ml) for 2 different time periods (20 and 60 min). Uptake of HFt and cell viability were measured using flow cytometry and the difference in MSR-1 expression between variously polarised macrophages was assessed using immunoblotting.
  • Results from western blot analysis confirmed higher expression of MSR-1 in M2 macrophages in comparison to Ml subtype (especially ones generated using higher concentration of LPS) for both hMDM ( Figure 2) and THP-1 ( Figure 3).
  • HFt-AF488 internalised by M2 macrophages derived from primary monocytes was significantly higher in comparison to the Ml subtype, regardless of the LPS concentration, incubation time and HFt concentration.
  • THP-derived M2 macrophages significantly higher HFt-AF488 uptake was observed for cells incubated with HFt for 60 min at both concentrations.
  • polarisation of THP-1 macrophages with 10 ng/ml LPS affected their viability, therefore the influence of cell death on the lower HFt uptake by Ml macrophages stimulated with higher LPS concentration cannot be excluded for this cell type (LPS stimulation did not affect viability of hMDM).
  • hMDM and THP-1 macrophages were incubated with unlabelled ferritin at concentration of 100 pg/ml for different time intervals (5-180 min) at 37°C.
  • the expression of MSR-1 on the cell membrane in human macrophages was measured by cell surface immunostaining with a fluorophore-conjugated antibody against MSR-1 followed by analysis using flow cytometry ( Figure 4).
  • Example 4 The influence of TfR-1 knockdown on HFt uptake by human macrophages
  • Example 5 The influence of MSR-1 knockdown on HFt uptake by human macrophages
  • MSR-1 plays a significant role in HFt uptake in human macrophages.
  • Example 6 The effect of overexpression of MSR-1 on HFt uptake by CHO-K1 cells
  • CHO-K1 cells which normally do not express MSR-1, were transiently transfected with a plasmid encoding MSR-1 (pMSRl) using lipofection.
  • the empty plasmid (pCMV) was used as a negative control and plasmid encoding SCARA5 (pSCARA5) served as a positive control, as, according to the literature, this scavenger receptor can bind HFt and mediate its internalisation, however this receptor is not expressed by macrophages.
  • pCMV empty plasmid
  • SCARA5 SCARA5
  • results from both western blot and flow cytometry confirmed MSR-1 and SCARA5 overexpression in CHO-K1 cells ( Figure 6), however transfection with the plasmid encoding SCARA5 was much less efficient (lower signal form surface expression and lower % of successfully transfected cells).
  • the overexpression of MSR-1 led to a significant increase in HFt-AF488 uptake ( ⁇ 3.7 times higher MFI in comparison to the control cells) by CHO-K1.
  • the amount of acLDL-AF488 taken up by transfected cells was also significantly higher ( ⁇ 4.8 times in comparison to the control cells), which confirms that the overexpression of MSR-1 had an actual functional effect.
  • CHO-K1 expressing SCARA5 the internalisation of HFt-AF488 was also significantly increased (2.5 times higher MFI in comparison to control cells), however, the presence of this receptor on cell surface did not significantly influence acLDL uptake.
  • HFt conjugated with Alexa Fluor 488 at the concentration of 20 nM was mixed with a serial dilution of MSR-1 or SCARA5 in a working buffer, transferred to capillaries provided in the kit and subjected to temperature gradient. Temperature-related fluorescence intensity change caused by interaction between fluorochrome-conjugated target and the ligand was measured using Monolith Pico. Kd was obtained by fitting the data with a 1 : 1 binding model. SCARA5 was used as a positive control for this interaction analysis as it is known to bind ferritin (according to literature).
  • the dose-response curves presented in Figures 8 and 9 indicate a specific interaction between HFt and both MSR-1 and SCARA5 receptors with Kd values of 195 nM and 168 nM, respectively.
  • SEQ ID NO. 5 alternative mammalian consensus ferritin MTTASXSQVRQNYXQXSEAAXXRQINLELXASYVYLSMSXYFDRDDVALKNFAKY FLHQSHEEREHAE
  • An isolated targeted delivery system comprising a population of macrophages and/or monocytes comprising a complex of ferritin and a pharmaceutically active substance, label or pharmaceutically active substance and label, wherein the population of macrophages and/or monocytes is characterized by a high expression of MSR-1.
  • the isolated targeted delivery system according to item 1 wherein the population of macrophages and/or monocytes and/or monocytes is characterized by a high endogenous expression of MSR- 1.
  • the isolated targeted delivery system according to item 1 or 2 wherein the population of macrophages and/or monocytes is genetically modified to overexpress MSR-1.
  • the isolated targeted delivery system according to item 3 wherein the overexpression is transient or stable, preferably transient.
  • the isolated targeted delivery system according to any one of items 1 to 4, wherein at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes express MSR-1.
  • the macrophage is an activated macrophage, preferably a CD1 lb+ macrophage, more preferably a CD1 lb+ CD 16+ macrophage, CDl lb+ CD32+ macrophage, CDl lb+ CD64+ macrophage, CDl lb+ CD68+ macrophage, preferably a CDl lb+ CD86+ Ml macrophage, preferably producing iNOS and/or secreting interleukin 12 (IL-12) or preferably CD1 lb+ CCR2+ M2 macrophage, CD1 lb+ CD204+ M2 macrophage, CD1 lb+ CD206+ M2 macrophage, CD1 lb+ CD204+ CD206+ M2 macrophage, CDl lb+ Mayor Histocompatibility Complex 11+ (MHCII+) (low or hi expression) M2 macrophage, CD1 lb+ CD200R+ M2 macrophage,
  • the isolated targeted delivery system according to any one of items 1 to 6, wherein the macrophage expresses at least one chemokine receptor, preferably selected from the group consisting of CCR1, CCR2 + , CXCR4 + , and CXCR6 + , or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony stimulating factor Receptor (CD115), granulocyte colony stimulating factor Receptor (CD 114), and granulocyte-macrophage colony stimulating factor Receptor (consisting of CD 116 and CD 131).
  • the activated macrophage is preferably selected from the group consisting of CCR1, CCR2 + , CXCR4 + , and CXCR6 + , or at least one growth factor receptor, preferably selected from the group consisting of macrophage colony stimulating factor Receptor (CD115), granulocyte colony stimulating factor Receptor (CD 114), and granulocyte-macrophage colony stimulating factor Receptor (consisting of
  • cytokines preferably IL-10 and IL-12, chemokines and/or to produce iNOS, arginase or other immunomodulating enzymes
  • (ii) is characterized by expression of at least one of following antigens: CD64, CD86, CD 16, CD32 HLA-DR, and/or production of iNOS and/or IL-12;
  • (iv) is characterized by expression of at least one of following antigens: CD204, CD206, CD200R; CCR2, transferrin receptor (TfR), CXC-motive chemokine receptor 4 (CXCR4), CD163, and/or show low expression of HLA-DR;
  • cytokine secretion preferably of IL-12, or IL-10, or production of inducible nitric oxide synthetase (iNOS), pro-inflammatory compounds, arginase immunosuppressive compounds or anti-inflammatory compounds, preferably wherein
  • iNOS inducible nitric oxide synthetase
  • the Ml inducer is selected from the group consisting of LPS, INF-y, GM-CSF, and viral or bacterial proteins or products; or
  • the M2 inducer is selected from the group consisting of IL-4, IL-10, IL-13, immune complex of an antigen and antibody, IgG, heat activated gamma-globulin, glucocorticosteroid, TGF-p, IL-1R, CCL-2, IL-6, M-CSF, PPARy agonist, leukocyte inhibitory factor, adenosine, helminth or fungal proteins or products.
  • ferritin and the pharmaceutically active substance or label are covalently and/or non- covalently linked, and/or (ii) the pharmaceutically active substance or label is encapsulated by the ferritin or multimers thereof.
  • cleavable linker is a peptide-based linker that is cleavable by a lysosomal protease, preferably a lysosomal cysteine protease, more preferably cathepsin B, most preferably the linker is a maleimidocaproyl-valine-citrulline-para-aminobenzoyloxycarbonyl(mc-vc-PAB) linker.
  • ferritin comprises an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to any one of SEQ ID NO: 1-4 and has at least 70%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the ability of wild-type ferritin, in particular human wild type ferritin, to bind iron ion(s) and/or form ferritin 24-mers.
  • the pharmaceutically active substance is an anticancer drug selected from the group consisting of a protein, a peptide, a nucleic acid, a non-protein non-nucleic acid compound with a molecular weight of less than 1 ,5kD, a photosensitizing compound, a virus, and pharmaceutically active radioactive isotope.
  • - is selected from the group consisting of an apoptosis-inducing drug, an alkylating substance, anti-metabolites, antibiotics, an antimitotic agent, a DNA-modifying drug, a DNA minor groove interstrand crosslinking drug, an inhibitor of DNA synthesis, an inhibitor of RNA synthesis, epothilones, nuclear receptor agonists and antagonists, an anti-androgene, an antiestrogen, a platinum compound, a hormone, a antihormone, an interferon, an inhibitor of cell cycle-dependent protein kinases (CDKs), an inhibitor of cyclooxygenases and/or lipoxygenases, a biogenic fatty acid, a biogenic fatty acid derivative, including prostanoids and leukotrienes, an inhibitor of protein kinases, an inhibitor of protein phosphatases, an inhibitor of lipid kinases, a platinum coordination complex, an ethyleneimine, a methylmelamine, a triazine, a vinca alka
  • - is selected from the group consisting of acediasulfone, aclarubicine, ambazone, aminoglutethimide, L-asparaginase, auristatin, azathioprine, banoxantrone, bendamustine, bleomycin, busulfan, calcium folinate, carboplatin, carpecitabine, carmustine, celecoxib, chaliceamycin, chlorambucil, cis-platin, cladribine, cyclophosphamide, cytarabine, dacarbazine, dactinomycindapsone, daunorubicin, deruxtecan, dibrompropamidine, diethylstilbestrole, docetaxel, doxorubicin, dolastatm 10, dolastatin 15, dynemycinA, enediynes, epirubicm, epothilone B, epothilone D, estramucm
  • the immunomodulatory drug is a ligand or antagonist of Pattern Recognition Receptors, particularly Toll-like Receptors, NOD-like receptors (NLR), RIG-I-like receptors (RLR)or Stimulator of interferon genes (STING) protein; and/or
  • - is a proliferation inhibiting protein or peptide, preferably a cell cycle inhibitor or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a nucleic acid, preferably encoding a proliferation inhibiting protein or an antibody or antibody like binding protein that specifically binds to a proliferation promoting protein or a siRNA, oligonucleotide, LNA, or DNAzyme.
  • hypoxia-activated prodrug preferably selected from the group consisting of benzotriazine N-oxides, apaziquone (EO9), tirapazamine (TPN), SN30000, PR-104A, TH- 302, TH-4000 and AQ4N, or
  • the isolated targeted delivery system for use according to any of items 1 to 19, wherein the label is selected from the group consisting of a fluorescent dye, a fluorescence emitting isotope, a radioisotope, a detectable polypeptide or nucleic acid encoding a detectable polypeptide and a contrast agent or wherein the label comprises a chelating agent which forms a complex with divalent or trivalent metal cations, preferably wherein
  • the chelating agent is selected from the group consisting of 1,4,7,10- tctraazacvclododccanc-N.N'.N.
  • A"-tctraacctic acid (DOTA), ethylenediaminetetraacetic acid(EDTA), l,4,7-triazacyclononane-l,4,7-triacetic acid (NOTA), triethylenetetramine (TETA), iminodiacetic acid, diethylenetriamine-N,N,N',N',N"-pentaacetic acid (DTPA) and 6-hydrazinopyridine-3-carboxylic acid (HYNIC);
  • the contrast agent comprises a paramagnetic agent, preferably selected from Gd, Eu, W and Mn, or ferrihydride;
  • the radioisotope/fluorescence emitting isotope is selected from the group consisting of alpha radiation emitting isotopes, gamma radiation emitting isotopes, Auger electron emitting isotopes, X-ray emitting isotopes, fluorescent isotopes, such as 65 Tb, fluorescence emitting isotopes, such as 18 F, 51 Cr, 67 Ga, 68 Ga, 89 Zr, ul In, " m Tc, 140 La, 175 Yb, 153 Sm, 1 66 Ho, 88 Y, 90 Y, 149 Pm, 177 LU, 47 SC, 142 Pr, 159 Gd, 212 BI, 72 As, 72 Se, 97 Ru, 109 Pd, 105 Rh, 101ml5 Rh, 1 19 Sb, 128 Ba, 123 1, 124 I, 131 I, 197 Hg, 211 At, 169 Eu, 203 Pb, 212 Pb, 64 Cu,
  • the fluorescence dye is selected from the group consisting of the following classes of fluorescent dyes: xanthens, acridines, oxazines, cynines, styryl dyes, coumarines, porphines, metal-ligand-complexes, fluorescent proteins, nanocrystals, perylenes and phtalocyanines as well as conjugates and combinations of these classes of dyes; and/or
  • the detectable polypeptide is an auto fluorescent protein, preferably green fluorescent protein or any structural variant thereof with an altered adsorption and/or emission spectrum.
  • a pharmaceutical composition comprising the isolated targeted delivery system according to any of items 1 to 20.
  • An isolated targeted delivery system according to any of items 1 to 19 or the pharmaceutical composition of item 21 for use in medicine, preferably in the treatment of cancer.
  • a method of preparing an isolated targeted delivery system comprising the steps of a) providing purified ferritin; b) covalently or non-covalently linking a pharmaceutically active substance, label or pharmaceutically active substance and label to and/or encapsulating a pharmaceutically active substance, label or pharmaceutically active substance and label in ferritin; c) providing a population of macrophages and/or monocytes characterized by a high expression of MSR-1, wherein preferably at least 1.5% , at least 2%, at least 2.5%, at least 5%, at least 10%, at least 15%, at least 20% of the macrophages and/or monocytes comprised in the population of macrophages and/or monocytes express MSR-1; and d) incubating the macrophage in the presence of the ferritin produced in step b) until the macrophage is at least partially loaded with the complex of the ferritin and the a pharmaceutically active substance, label or pharmaceutically active
  • a method of selecting a monocyte donor comprising a) generating a population of macrophages from a population of monocytes obtained from a donor; b) determining the percentage of
  • the percentage of MSR-1 -expressing monocytes is high, preferably at least 2%, at least 3%, or at least 4%,
  • the percentage of MSR-1 -expressing macrophages is high, preferably at least 5%, at least 10%, at least 15%, or at least 20%, and/or

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Virology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un système d'administration ciblée isolé qui comprend un macrophage comportant un complexe de ferritine et une substance et/ou un marqueur pharmaceutiquement actif, le macrophage étant caractérisé par une expression élevée de MSR-1. L'invention concerne en outre l'utilisation du système d'administration ciblée isolé en médecine, en particulier dans le traitement du cancer, un procédé de production d'un système d'administration ciblée isolé, un procédé de sélection d'un donneur de monocytes et un procédé de fourniture d'un macrophage ou d'un monocyte ayant une expression de MSR-1 accrue.
PCT/EP2024/079642 2023-10-19 2024-10-21 Système d'administration ciblée isolé amélioré Pending WO2025083276A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP23204741.5 2023-10-19
EP23204741 2023-10-19

Publications (1)

Publication Number Publication Date
WO2025083276A1 true WO2025083276A1 (fr) 2025-04-24

Family

ID=88510988

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/079642 Pending WO2025083276A1 (fr) 2023-10-19 2024-10-21 Système d'administration ciblée isolé amélioré

Country Status (1)

Country Link
WO (1) WO2025083276A1 (fr)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5582981A (en) 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
WO2007002931A2 (fr) 2005-06-29 2007-01-04 Threshold Pharmaceuticals, Inc. Prodrogues d'alkylateur de phosphoramidate
WO2008083101A1 (fr) 2006-12-26 2008-07-10 Threshold Pharmaceuticals, Inc. Promédicaments d'alkylation de phosphoramidate pour le traitement du cancer
WO2016207256A1 (fr) 2015-06-22 2016-12-29 Cellis Sp. Z O.O. [Ltd.] Système de distribution d'étiquettes ciblée cellulaire
WO2017222398A1 (fr) 2016-06-22 2017-12-28 Cellis Sp. Z O.O. Système de distribution d'étiquettes ou de substances pharmaceutiquement actives à ciblage cellulaire
WO2022195092A1 (fr) 2021-03-18 2022-09-22 Cellis Ag Variants de ferritine ayant une stabilité et une capacité de complexation accrues

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5582981A (en) 1991-08-14 1996-12-10 Gilead Sciences, Inc. Method for identifying an oligonucleotide aptamer specific for a target
WO2007002931A2 (fr) 2005-06-29 2007-01-04 Threshold Pharmaceuticals, Inc. Prodrogues d'alkylateur de phosphoramidate
WO2008083101A1 (fr) 2006-12-26 2008-07-10 Threshold Pharmaceuticals, Inc. Promédicaments d'alkylation de phosphoramidate pour le traitement du cancer
WO2016207256A1 (fr) 2015-06-22 2016-12-29 Cellis Sp. Z O.O. [Ltd.] Système de distribution d'étiquettes ciblée cellulaire
WO2016207257A1 (fr) 2015-06-22 2016-12-29 Cellis Sp. Z O.O. [Ltd.] Système cellulaire d'administration ciblée de principe actif
WO2017222398A1 (fr) 2016-06-22 2017-12-28 Cellis Sp. Z O.O. Système de distribution d'étiquettes ou de substances pharmaceutiquement actives à ciblage cellulaire
WO2022195092A1 (fr) 2021-03-18 2022-09-22 Cellis Ag Variants de ferritine ayant une stabilité et une capacité de complexation accrues

Non-Patent Citations (28)

* Cited by examiner, † Cited by third party
Title
"Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
ADAM S. VENABLE, METHODS IN MOLECULAR BIOLOGY, 2015
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
BAOS SELENE ET AL: "Expression of Macrophage Scavenger Receptor (MSR1) in Peripheral Blood Cells from Patients with Different Respiratory Diseases: Beyond Monocytes", vol. 11, no. 5, 5 March 2022 (2022-03-05), CH, pages 1439, XP093122176, ISSN: 2077-0383, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8910889/pdf/jcm-11-01439.pdf> DOI: 10.3390/jcm11051439 *
BECKER ET AL., EUR. J. IMMUNOL., vol. 36, 2006, pages 950 - 960
BEHRENS CRLIU B: "Methods for site-specific drug conjugation to antibodies", MABS, vol. 6, no. 1, January 2014 (2014-01-01), pages 46 - 53, XP055293357, DOI: 10.4161/mabs.26632
BRUDNO M., BIOINFORMATICS, vol. 19, 2003, pages 154 - 162
CRAWFORD, R. ET AL., BIOPHYS J., vol. 105, no. 11, 2013, pages 2439
DENG GUODONG ET AL: "Ferritin in cancer therapy: A pleiotropic tumoraffin nanocage-based transport", CANCER MEDICINE, vol. 12, no. 10, 31 March 2023 (2023-03-31), GB, pages 11570 - 11588, XP093098031, ISSN: 2045-7634, DOI: 10.1002/cam4.5778 *
GUDGEON JACK ET AL: "The role of macrophage scavenger receptor 1 (MSR1) in inflammatory disorders and cancer", vol. 13, 17 October 2022 (2022-10-17), Lausanne, CH, XP093122184, ISSN: 1664-3224, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9618966/pdf/fimmu-13-1012002.pdf> DOI: 10.3389/fimmu.2022.1012002 *
HAUSMAN RECOOPER GM: "The cell: a molecular approach", 2004, ASM PRESS, pages: 51
JAYASENA, CLIN. CHEM., vol. 45, 1999, pages 1628 - 50
KARLINALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877
KLUGFAMULOK, M. MOL. BIOL. REP., vol. 20, 1994, pages 97 - 107
KLUSSMANN ET AL., NAT. BIOTECHNOL., vol. 14, 1996, pages 1112 - 5
LI L ET AL., PROC. NATL. ACAD. SCI. USA, vol. 107, no. 8, 2010, pages 3505 - 3510
MEITAL LARA T ET AL: "A simple and effective method for the isolation and culture of human monocytes from small volumes of peripheral blood", JOURNAL OF IMMUNOLOGICAL METHODS, ELSEVIER SCIENCE PUBLISHERS B.V.,AMSTERDAM, NL, vol. 472, 20 June 2019 (2019-06-20), pages 75 - 78, XP085736313, ISSN: 0022-1759, [retrieved on 20190620], DOI: 10.1016/J.JIM.2019.04.005 *
MURRAYWYNN, J LEUKOCBIOL, vol. 89, no. 4, 2011, pages 557 - 63
NOGRADY: "Medicinal Chemistry A Biochemical Approach", 1985, OXFORD UNIVERSITY PRESS, pages: 388 - 392
PEI JGRISHIN NV: "PROMALS: towards accurate multiple sequence alignments of distantly related proteins", BIOINFORMATICS, vol. 23, 2007, pages 802 - 808
POIROT OSUHRE KABERGEL CO'TOOLE ENOTREDAME C, NUCLEIC ACIDS RES., vol. 32, 1 July 2004 (2004-07-01), pages 37 - 40
SVENSSON P-A ET AL: "Identification of genes predominantly expressed in human macrophages", ATHEROSCLEROSIS, ELSEVIER, AMSTERDAM, NL, vol. 177, no. 2, 1 December 2004 (2004-12-01), pages 287 - 290, XP004628484, ISSN: 0021-9150, DOI: 10.1016/J.ATHEROSCLEROSIS.2004.07.031 *
TADA YOICHIRO ET AL: "Infiltration of CD204-overexpressing Macrophages Contributes to the Progression of Stage II and III Colorectal Cancer", vol. 41, no. 10, 1 October 2021 (2021-10-01), GR, pages 4857 - 4865, XP093122190, ISSN: 0250-7005, Retrieved from the Internet <URL:https://ar.iiarjournals.org/content/anticanres/41/10/4857.full.pdf> DOI: 10.21873/anticanres.15299 *
THEIL EC, ANNUAL REVIEW OF BIOCHEMISTRY, vol. 56, no. 1, 1987, pages 289 - 315
THOMPSON, J. D.HIGGINS, D. G.GIBSON, T. J., NUCLEIC ACIDS RES., vol. 22, 1994, pages 4673 - 80
UHLMANN, E.PEYMAN, A., CHEMICAL REVIEWS, vol. 90, 1990, pages 543 - 584
YANG ET AL., BIOMARK RES, vol. 2, no. 1, 2014
YU BOWEN ET AL: "Interactions of ferritin with scavenger receptor class A members", vol. 295, no. 46, 9 September 2020 (2020-09-09), US, pages 15727 - 15741, XP093122157, ISSN: 0021-9258, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667961/pdf/zbc15727.pdf> DOI: 10.1074/jbc.RA120.014690 *

Similar Documents

Publication Publication Date Title
KR101216008B1 (ko) 바이포달 펩타이드 바인더
KR102447396B1 (ko) 세포 표적화된 활성 성분 전달 시스템
IL286804B (en) Protease-cleavage resistant, shiga toxin a subunit effector polypeptides and cell-targeted molecules comprising the same
US20150132367A1 (en) Yopm as delivery vehicle for cargo molecules and as biological therapeutic for immunomodulation of the inflammatory reactions
WO2011071279A2 (fr) Système de transfert de charge à base de liant peptidique bipode
WO2011071280A2 (fr) Liant peptidique bipode à ciblage intracellulaire
JP2013535954A (ja) ペプチド、構造体およびその使用
JP6218175B2 (ja) ヘルパーｔ細胞誘導性ポリペプチドの改変
JP7436710B2 (ja) 新規のヌクレオリン－結合ペプチド及びその用途
WO2025083276A1 (fr) Système d&#39;administration ciblée isolé amélioré
AU2021412432B2 (en) Chlorotoxin derivatives and use thereof
EP4587062A1 (fr) Système isolé d&#39;administration ciblée pour le traitement du gliome
WO2024133541A1 (fr) Système isolé d&#39;administration ciblée pour le traitement du cancer de l&#39;ovaire
EP3650046A1 (fr) Système d&#39;administration d&#39;ingrédients actifs ciblés à base de cellules souches mésenchymateuses
HK40011495A (en) Protease-cleavage resistant cytotoxic cell-targeting molecules
HK40011495B (en) Protease-cleavage resistant cytotoxic cell-targeting molecules
HK1253919B (en) Cellular targeted active ingredient delivery system

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24790595

Country of ref document: EP

Kind code of ref document: A1