WO2023179370A1 - Anticorps bispécifique peptidique, ses procédés de préparation et ses utilisations - Google Patents

Anticorps bispécifique peptidique, ses procédés de préparation et ses utilisations Download PDF

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WO2023179370A1
WO2023179370A1 PCT/CN2023/080473 CN2023080473W WO2023179370A1 WO 2023179370 A1 WO2023179370 A1 WO 2023179370A1 CN 2023080473 W CN2023080473 W CN 2023080473W WO 2023179370 A1 WO2023179370 A1 WO 2023179370A1
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biomarker
antibody
bispecific antibody
binding peptides
cells
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Clarence Tsun Ting Wong
Chihao SHAO
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The Hong Kong Polytechnic University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • a sequence listing file with a file name “P24144PCT00_sequence_listing. xml” in ST.26 XML file format having a file size of 8KB created on March 7 th , 2023 is incorporated herein by reference in its entirety
  • Blinatumomab the first T-cell engaging bispecific antibody, was approved by FDA in 2015, which contains bifunctionality that binds to both B-lineage leukemia CD19 and CD3 of T-cell. Such engaging mechanism redirects the immune cells to engage the target cells for the cytotoxic killing of cancer cells.
  • Another T-cell engager binds to both PD-1 and CTLA4, is currently in phase I and II clinical trials. Up to date, there are more than 90 bispecific antibodies at different stages of clinical and pre-clinical trials. Thus far, the bispecific antibodies have been shown to enhance the drug response and therapeutic index compared to monoclonal antibodies.
  • a first aspect of the present invention provides a bispecific antibody chemically or enzymatically conjugated with one or more biomarker-binding peptides capable of conjugating with multiple biomolecules of one or more targets simultaneously.
  • the monoclonal antibody can be an IgG antibody from human or any other animal origin with any specificity.
  • biomarker-binding peptides may be selected from one or more amino acid sequences of SEQ ID NOs: 1 to 8.
  • the one or more biomolecules or biomarkers which the biomarker-binding peptides capable of targeting include, but not limited to, epidermal growth factor receptor (EGFR) , epithelial cellular adhesion molecule (EpCAM) , programmed death-ligand 1 (PD-L1) , integrin, human epidermal growth factor receptor 2 (HER-2) and glypican-3 (GPC3) .
  • EGFR epidermal growth factor receptor
  • EpCAM epithelial cellular adhesion molecule
  • PD-L1 programmed death-ligand 1
  • HER-2 human epidermal growth factor receptor 2
  • GPC3 glypican-3
  • each bispecific antibody can conjugate with two or more different biomolecules on a surface of the same target.
  • the target of the bispecific antibody includes, but not limited to, non-living materials and living cells such as NK cells, T-cells, macrophages, dendritic cells, red blood cells, B-cells, cancer cells, viruses, bacteria, fungi, yeasts, and parasites.
  • non-living materials and living cells such as NK cells, T-cells, macrophages, dendritic cells, red blood cells, B-cells, cancer cells, viruses, bacteria, fungi, yeasts, and parasites.
  • the one or more biomarker-binding peptides can be linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
  • the chemical conjugation is performed by a bifunctional linker having a first functional domain capable of cyclizing the biomarker-binding peptide and a second functional domain chemically conjugating to the region of the monoclonal antibody.
  • the immunoglobulins or any fragments thereof include, but not limited to, IgG, IgA, IgM, nanobody, Fab, scFv, or peptibody.
  • the monoclonal antibody can be an IgG antibody from human or any other animal origin with any specificity.
  • the monoclonal antibody and the one or more biomarker-binding peptides are conjugated with each other through a chemical linker.
  • biomarker-binding peptides may be selected from one or more amino acid sequences of SEQ ID NOs: 1 to 8.
  • each bispecific antibody can conjugate with two or more different biomolecules on a surface of the same target.
  • the target of the bispecific antibody includes, but not limited to, non-living materials and living materials such as NK cells, T-cells, macrophages, dendritic cells, red blood cells, B-cells, cancer cells, viruses, bacteria, fungi, yeasts, and parasites.
  • non-living materials and living materials such as NK cells, T-cells, macrophages, dendritic cells, red blood cells, B-cells, cancer cells, viruses, bacteria, fungi, yeasts, and parasites.
  • the one or more biomolecules (or biomarkers) which the biomarker-binding peptides capable of targeting include, but not limited to, epidermal growth factor receptor (EGFR) , epithelial cellular adhesion molecule (EpCAM) , programmed death-ligand 1 (PD-L1) , integrin, human epidermal growth factor receptor 2 (HER-2) and glypican-3.
  • EGFR epidermal growth factor receptor
  • EpCAM epithelial cellular adhesion molecule
  • PD-L1 programmed death-ligand 1
  • HER-2 human epidermal growth factor receptor 2
  • glypican-3 glypican-3
  • the one or more biomarker-binding peptides can be linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
  • cyclic biomarker-binding peptides are formed by cyclizing the biomarker-binding peptide through a chemical bond between N-terminus and C-terminus, between one side chain and the other side chain, or between one of the N-and C-termini and a side chain, such that a cyclic peptide backbone is formed.
  • the chemical bond between N-terminus and C-terminus, between a side chain and the other side chain, or between one of the N-and C-termini and a side chain, for forming the cyclic peptide backbone can be a covalent bond, supramolecular interaction or disulfide bond.
  • the biomarker-binding peptide is cyclized by one of the functional domains of the bifunctional linker, while the other functional domain of the bifunctional linker will form a chemical conjugation with the region of the monoclonal antibody.
  • a third aspect of the present invention provides a method for treating a disease in a subject in need of an immunotherapy, where the method includes administering a composition comprising a therapeutically effective amount of bispecific antibodies described herein to the subject, or a use of the bispecific antibodies in preparation of a composition for treating a disease in said subject as an immunotherapy.
  • the composition is capable of triggering an antibody-dependent cellular phagocytosis (ADCP) .
  • ADCP antibody-dependent cellular phagocytosis
  • the receptor on the macrophages initially inactivated by an antigen of the cancer cells is activated by the administration of the composition comprising the therapeutically effective amount of the bispecific antibodies to the subject.
  • the one or more biomolecules (or biomarkers) which the biomarker-binding peptides capable of targeting include, but not limited to, epidermal growth factor receptor (EGFR) , epithelial cellular adhesion molecule (EpCAM) , programmed death-ligand 1 (PD-L1) , integrin, human epidermal growth factor receptor 2 (HER-2) and glypican-3.
  • EGFR epidermal growth factor receptor
  • EpCAM epithelial cellular adhesion molecule
  • PD-L1 programmed death-ligand 1
  • HER-2 human epidermal growth factor receptor 2
  • glypican-3 glypican-3
  • the composition can be administered via intravenous, intratumoral, subcutaneous, intraperitoneal, or intramuscular injection.
  • composition can be administered in conjunction with, prior to, or after other cancer therapies including, but not limited to, radiotherapy and chemotherapy.
  • the disease to be treated includes cancers such as head and neck, ovarian, cervical, bladder, oesophageal, gastric, breast, endometrial, colorectal, lung, pancreatic, skin, and non-small cell lung cancers.
  • cancers such as head and neck, ovarian, cervical, bladder, oesophageal, gastric, breast, endometrial, colorectal, lung, pancreatic, skin, and non-small cell lung cancers.
  • the bispecific antibodies may be further conjugated with an indicator to determine a presence of the target cell types, tissues or biomolecules in a sample in vitro or in vivo when the bispecific antibody specifically conjugates with the corresponding biomarker (s) expressed by the target cell types, tissues or biomolecules, thereby activating the indicator.
  • the indicator of the present kit includes, but not limited to, fluorescent and colorimetric dyes.
  • the kit or the bispecific antibodies according to certain embodiments are able to detect the presence of corresponding cell type (s) or tissues overexpressing certain biomarker (s) in vitro and in vivo.
  • Signals generated by the corresponding indicator associated with the conjugation between the bispecific antibodies and the target cell type/tissue can also be used to quantify cell viability of the target cells/tissues with respect to certain treatment regime thereby evaluating an efficacy thereof on certain cell type/tissues.
  • the kit may also be used for disease prognosis by indication of the number or extent of abnormal cells or tissues in the sample with overexpression of certain biomarker (s) detectable by the bispecific antibodies and the indicator, indicating the presence of the disease or a likelihood to progress into certain stages of a disease.
  • FIG. 2A schematically depicts a general synthesis scheme of the one-pot peptide cyclization and antibody conjugation according to certain embodiments of the present invention.
  • FIG. 2B schematically depicts a more detailed synthesis scheme of the one-pot peptide cyclization and antibody conjugation according to certain embodiments of the present invention.
  • FIG. 2C schematically depicts an embodiment of the present pBsAb derived from an anti-signal regulatory protein ⁇ (SIRP ⁇ ) conjugated with ortho-phthalaaldehyde-functionalized cyclic epidermal growth factor receptor (EGFR) -targeting peptide (hereinafter as “cEBP-OPA” ) and how it relates to an activation of a macrophage-mediated cancer cell phagocytosis.
  • SIRP ⁇ anti-signal regulatory protein ⁇
  • cEBP-OPA ortho-phthalaaldehyde-functionalized cyclic epidermal growth factor receptor
  • FIG. 3 shows confocal images of HT29 and HeLa cells after incubation for 1 h with native (non-specific) IgG (20 nM) (left) and the cyclic peptide-modified IgG (cEBP-IgG) (20 nM) according to certain embodiments of the present invention.
  • FIG. 4 shows results of ELISA binding assay of the present pBsAb against (A) EGFR and (B) SIRP- ⁇ . Data are expressed as the mean value ⁇ standard error of the mean (SEM) of three independent experiments, each performed in triplicate.
  • FIG. 5 shows results of cellular binding test on anti-SIRP mAb and the present pBsAb against RAW264.7, HT29, A549, and HeLa cells by (A) confocal microscopic images; (B) flow cytometric data after treated with anti-SIRP mAb and the present pBsAb for 30 mins. Data are expressed as the mean value ⁇ SEM of three independent experiments.
  • FIG. 6A shows confocal microscopic images of co-culture binding experiment.
  • RAW264.7 macrophage was incubated with either EGFR-overexpressing cell HT29 (upper panel) and low-EGFR expressing cell HeLa (lower panel) .
  • the data show that higher number of cell clusters (white circles) were formed between HT29 and RAW264.7 in the presence of pBsAb compared to HeLa.
  • FIG. 6B shows results of a macrophage-cancer cell binding assay on unlabeled A549 EGFR-overexpressing cells in the presence of the anti-SIRP mAb and the present pBsAb compared with a control (without any antibody) by confocal microscopic images (upper panel) and a chart (lower panel) illustrating the number of macrophages bounded to the surface of A549 in different treatment groups.
  • FIGs. 7A-7B show results of an antibody-dependent cellular phagocytosis (ADCP) assay in different cell lines, in which: FIG. 7A shows confocal microscopic images of the co-cultured RAW264.7 macrophages (CFSE) and A549, HT29 and HeLa cells (CellTracker Red) treated with the present pBsAb according to certain embodiments and the anti-SIRP- ⁇ monoclonal antibody (20 nM) for 24 h. The arrows show the phagocytic macrophages; FIG.
  • ADCP antibody-dependent cellular phagocytosis
  • FIG. 7B shows flow cytometry quadrant analysis of the co-cultured RAW264.7 macrophages (CFSE) with A549, HT29, and HeLa cells (CellTracker Red) treated with pBsAb (20 nM) or anti-SIRP- ⁇ mAb (20 nM) or without any treatment control for 2 h.
  • the gray squares show the percentage of phagocytotic macrophages at different conditions.
  • FIG. 8 shows a quantitative analysis of the ADCP assay of RAW264.7 macrophages against EGFR overexpressing cells (A549 and HT29) and EGFR-low expressing cells (HeLa and HepG2) treated with different concentrations of the present pBsAb according to certain embodiments and the anti-SIRP- ⁇ mAb.
  • FIG. 9A shows confocal Z-stack maximum projection microscopic images of HT29 cancer cell spheroids co-cultured with RAW264.7 macrophages (second left column) treated with the present pBsAb according to certain embodiments and the anti-SIRP- ⁇ mAb (50 nM) for 24 h.
  • the death cells were stained with PI (second right column) .
  • FIG. 9B shows a confocal microscopic image of 3D spheroids of HT29 cells with infiltration of green fluorescent-labeled macrophages in the presence of the present pBsAb according to certain embodiments.
  • FIG. 11 shows MALDI-TOF mass spectrum of EBP peptide as shown in FIG. 2B.
  • pBsAb peptidic bispecific antibody
  • FIG. 1 a novel type of bispecific antibody
  • pBsAb peptidic bispecific antibody
  • the key molecule for this reaction is a bifunctional linker that contains a dibromomethyl benzene unit for peptide cyclization and a phthalaldehyde for protein conjugation.
  • the pBsAb is provided by conjugating EGFR-targeting cyclic peptides onto an anti-SIRP- ⁇ monoclonal antibody, forming the EGFR x SIRP- ⁇ pBsAb.
  • the preparation of pBsAb starts from a monoclonal antibody, a bifunctional linker, and a linear tumor-targeting peptide.
  • the bifunctional linker is provided to generate serum-stable cyclic peptide-dye conjugates via a one-pot peptide cyclization and dye conjugation reaction.
  • One end of the bifunctional linker contains a dibromomethyl benzene (DBMB) unit for site-selective alkylation of the sulfhydryl (SH) side chains of two cysteine residues of a fully deprotected peptide (an amino acid sequence as shown in (ii) of FIG. 2B) to form a monocyclic structure.
  • DBMB dibromomethyl benzene
  • a proof-of-concept experiment was performed by conjugating the EGFR-targeting cyclic peptide to a non-specific IgG that does not have any selectivity against tumor cells but has an ability to target macrophage.
  • a schematic diagram is provided in FIG. 2C illustrating how an EGFR ⁇ SIRP- ⁇ pBsAb synthesized by the one-pot orthogonal manner as described in FIGs. 2A-2B is used in targeting and initiating a macrophage-mediated phagocytosis of certain cancer cell types.
  • the EGFR-positive HT29 colorectal carcinoma as the target cell and low EGFR-expressing HeLa cervix adenocarcinoma as the control cell line were chosen.
  • PBS phosphate buffered saline
  • the data show that the peptide-modified IgG was able to bind to the HT29 cell surface but not the HeLa cell. In comparison, the native IgG did not bind to any cell surfaces (FIG. 3) .
  • pBsAb (20 nM) with different ratios of cyclic peptides (1: 1.10: 1, 20: 1, 50: 1, and 100: 1) were synthesized and incubated with either SIRP- ⁇ (0.5 ⁇ g/ml) or EGFR (1 ⁇ g/ml) on ELISA plate for 2 h at room temperature. After washing, anti-rabbit-HRP secondary antibody was added and incubated for 1 h at room temperature in dark. Plate reader was used to measure the absorbance and the readout were recorded in plots as shown in FIG. 4. The results show that pBsAb conjugated with 100-fold excess of cyclic peptide gave the best binding affinity against EGFR among five different ratios.
  • FIG. 6A Cellular adhesion assay also demonstrated that in the presence of pBsAb, the addition of fluorescent labeled macrophages on top of the seeded A549 cells led to the enhanced binding between cancer cell and immune cell.
  • FIG. 6B Cellular adhesion assay also demonstrated that in the presence of pBsAb, the addition of fluorescent labeled macrophages on top of the seeded A549 cells led to the enhanced binding between cancer cell and immune cell.
  • FIGs. 7A shows that in the presence of 20 nM pBsAb, the cytoplasm of green-labelled RAW264.7 macrophages had intense red fluorescence originated from the EGFR-overexpressed A549 and HT29 cells. While the EGFR-low expressing cells HeLa did not show the same phenomenon. This further indicates that the phagocytosis occurred to engulf the red-labelled cancer cells.
  • the ADCP activity of RAW264.7 macrophage against HT29 and A549 cells were determined by using a CellTiter-Glo Luminescent Cell Viability Kit (Promega) in terms of cell viability and the relative antibody dependent cellular phagocytotic activity was calculated as an EC 50 .
  • pBsAb of various concentrations could enhance the phagocytotic activity that leads to anticancer effect, compared to the unmodified anti-SIRP- ⁇ mAb at 50 nM. Therefore, it is suggested that the present pBsAb induces a significant enhancement of ADCP against target EGFR-high expression cell lines compared to native anti-SIRP antibody.
  • the pBsAb also significantly enhances the ADCP against EGFR-high expression cell lines compared to EGFR-low expressing HeLa cell line. Overall, the present one-pot chemical conjugation strategy is proven to turn a single target monoclonal antibody into a bispecific antibody.
  • the HT29 cells (5x10 4 cells) were seeded at a low-attachment 96-well plate for 3 days to form the spheroids, which were further co-cultured with the carboxyfluorescein succinimideyl ester (CFSE) -stained RAW264.7 cells (1x10 3 cells) together with the pBsAb or anti-SIRP- ⁇ mAb (50 nM) , respectively, for 24 h at 37°C in a complete cell culture medium. The spheroids were also stained with propidium iodide (PI) after 24 h incubation for the detection of dead cells. Finally, the spheroids were transferred to confocal dishes for imaging. As shown in FIG.
  • the Z-stack maximum projection confocal images clearly reveal that the number of macrophages (fluorescent signal in second left column) inside the core of the spheroid treated with the pBsAb was significantly higher than that the spheroid treated with the anti-SIRP- ⁇ mAb.
  • a higher intensity of red-fluorescent PI was observed in the spheroid when it was treated with pBsAb, including those at the inner core of the spheroid (fluorescent signal in the second right column) .
  • FIG. 9B shows that some CFSE-stained macrophages successfully entered into the spheroids (indicated by solid arrows) .
  • the present invention also includes any structures of peptides such as, but not limited to, linear, cyclic, stapled, branched, dendrimeric, and scaffold peptides.
  • molecules other than peptides which can target tumor, or have tumor-targeting ability should also be considered as potential candidates to be chemically conjugated on the antibody to generate the bispecific antibodies of the present invention.
  • N, N-Dimethylformamide (DMF) , tetrahydrofuran (THF) , and CH 2 Cl 2 were dried using an INERT solvent drying system prior to use.
  • Acetonitrile was of HPLC grade. All other solvents were of analytical grade and used as received without further purification. All the reactions were performed under an atmosphere of nitrogen and monitored by thin layer chromatography (TLC; Merck pre-coated silica gel 60 F254 plates) . Chromatographic purification was performed on a silica gel (Macherey-Nagel, 230–400 mesh) column with the indicated eluent.
  • Compounds 1, 2, cEBP-OPA, and pBsAB were prepared according to the literature procedure.
  • Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer or a Bruker SolariX 9.4 Tesla FTICR mass spectrometer.
  • Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectra were recorded on a Bruker Daltonics Autoflex III spectrometer.
  • UV-Vis and steady-state fluorescence spectra were taken on a Shimazu UV-1800 UV-Vis spectrophotometer and a Horiba FluoroMax-4 spectrofluorometer, respectively.
  • Reverse-phase HPLC separation was performed on a XBridge BEH300 C18 column (5 ⁇ m, 4.6 mm ⁇ 150 mm) at a flow rate of 1 mL min -1 for analytical purpose or on a XBridge BEH300 Prep C18 column (5 ⁇ m, 10 mm ⁇ 250 mm) at a flow rate of 3 mL min -1 for preparative purpose using a Waters system equipped with a Waters 1525 binary pump and a Waters 2998 photodiode array detector.
  • the solvents used for HPLC analysis were of HPLC grade.
  • solvent A 0.1%trifluoroacetic acid (TFA) in acetonitrile
  • solvent B 0.1%TFA in deionized water
  • EBP AcNH-CMYIEALDRYAC-COHN 2 ) peptide (SEQ ID NO: 1) was synthesized manually using a modified 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol with the commercially available N- ⁇ -Fmoc-protected amino acids.
  • the rink amide resin was used as the solid support.
  • a solution of 20%piperidine in DMF was used to remove the Fmoc protecting group, and 1- [bis (dimethylamino) methylene] -1H-1, 2, 3-triazolo- [4, 5-b] pyridinium 3-oxide hexafluorophosphate (HATU) was used as the carboxyl group activating agent.
  • the resin was treated with a solution containing 95%TFA, 2.5%triisopropylsilane (TIS) , and 2.5%CH 2 Cl 2 for 1 h to cleave the peptide from the resin and remove the protecting groups.
  • the resin was removed by filtration and the filtrate was precipitated by the addition of diethyl ether. After centrifugation, the supernatant was removed. The solid was redissolved in DMSO and then the solution was precipitated again using diethyl ether. Lyophilization of the precipitated peptide afforded the crude peptide, which was purified by reverse-phase HPLC, followed by lyophilization.
  • RGD peptide could be used to substitute EBP peptide or in conjunction therewith.
  • RGD peptide could be synthesized in-situ according to the standard protocol or acquired from the corresponding manufacturers (e.g., SIGMA Cas No.: 99896-85-2) .
  • HRMS (MALDI-TOF) m/z calcd for C 64 H 99 N 16 O 19 S 3 [M+H] + , 1491.6429; found, 1491.6347.
  • HRMS (MALDI-TOF) m/z calcd for C 29 H 45 N 10 O 9 S 2 [M+H] + , 741.2807; found, 741.2610.
  • Monoclonal antibody was first dissolved in phosphate-buffered saline (PBS) (pH 7.4) to afford a 5 ⁇ M stock solution.
  • PBS phosphate-buffered saline
  • cEBP-OPA was then added to the protein solution in PBS with a 20: 1 mol ratio.
  • the mixture was stirred at room temperature for 30 min, and then filtrated through a molecular membrane filter (cut-off at 3 kDa) to remove the excess unconjugated peptide to form the cEBPxSIRP-alpha pBsAb.
  • the pBsAb was retained on the molecular was membrane filter and re-dissolved in PBS for further use.
  • NUNC Maxisorp plates (Thermo Scientific) were coated with equimolar of EGFR or SIRP-alpha protein at 4C overnight. Plates were washed three times with PBS containing 0.05%Tween-20 and blocked with 2%bovine serum albumin in PBS containing 0.1%Tween-20 at room temperature for 2 hours. Five-fold serial dilutions of pBsAb starting at 80nM were added and plates were incubated for 2 hrs at room temperature. Plates were washed three times and incubated with horseradish peroxidase (HRP) -conjugated goat anti-rabbit secondary antibody (ITK Southern Biotech) diluted 1: 2000 in blocking buffer for 1 hour at room temperature.
  • HRP horseradish peroxidase
  • ITK Southern Biotech horseradish peroxidase
  • HTB-22 were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, cat. no. 23400-021) supplemented with FBS (10%) and penicillin ⁇ streptomycin (100 unit mL -1 and 100 ⁇ g mL -1 , respectively) .
  • RPMI Roswell Park Memorial Institute
  • U87-MG human glioblastoma cells ATCC, no. HTB-14
  • MEM Minimum Essential Medium
  • FBS penicillin ⁇ streptomycin
  • the cells after being rinsed with PBS, were incubated with phycoerythrin labelled pBsAb, in a serum-free medium at 37 °C for 30 min.
  • the cells were rinsed with PBS twice, followed by post-incubation in a serum-free medium for further 3 h.
  • the solution was then removed, and the cells were rinsed with PBS twice before being examined using a Leica TCS SP8 high speed confocal microscope equipped with solid-state lasers.
  • the pBsAb was excited at 488 nm and its fluorescence was monitored at 500-530nm.
  • the images were digitized and analyzed using a Leica Application Suite X software.
  • the activity of trypsin was quenched with a serum-containing medium (0.5 mL) , and the mixture was centrifuged at 1500 rpm for 3 min at room temperature. The pellet was washed with PBS (1.0 mL) and then subjected to centrifugation. The cells were then suspended in PBS (1.0 mL) and the intracellular fluorescence intensities were measured using a BD FACSVerse flow cytometer (Becton Dickinson) with 10 4 cells counted in each sample. The data collected were analyzed using the BD FACSuite. All experiments were performed in triplicate.
  • RAW264.7 macrophages were harvested by washing the adherent differentiated cells in 10 mL of cold PBS, incubating for 10 min, and then gently scraping to detach. Macrophages were counted using an automated cell counter that also calculated viability at 3x10 6 (Vi-CELL XR Cell Viability Analyzer, Beckman Coulter, Brea, CA) . Macrophages were loaded with 10 ⁇ M CFSE (Thermo Fisher Scientific, Eugene OR) as per the manufacturer's instructions. The other cells, HT29 or HeLa were stained by Cell Tracker Red per manufacture instruction. Two cells were then mixed together in a round bottom plate in the presence of pBsAb at 20 nM for 2 hr at 37°C.
  • biomarker-binding peptide of the present invention is not limited to the EBP peptide described in Part (B) of Examples section for targeting EGFR, but also includes any possible peptide (s) that conjugate with the immunoglobin or its fragments for targeting multiple biomolecules simultaneously.
  • the potential candidates should also include, but not limited to, a peptide according to any amino acid sequence of SEQ ID NOs: 2-8, which targets EpCAM, PD-L1, integrin, HER2, and GPC3, respectively.
  • conjugation chemistry to conjugate tumor-targeting peptides on antibody allow flexibility in bispecificity

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Abstract

L'invention concerne des anticorps bispécifiques synthétisés par une réaction chimique monotope comprenant la cyclisation de peptides ciblant une tumeur et la conjugaison des peptides ciblant une tumeur cyclisés sur la surface d'un anticorps monoclonal pour créer une nouvelle forme d'anticorps bispécifiques, nommés anticorps bispécifiques peptidiques (pBsAb), qui combine les avantages de l'anticorps monoclonal et des peptides cycliques ciblant une tumeur stable au sérum, donnant à l'anticorps monoclonal une capacité de ciblage supplémentaire pour former un anticorps bispécifique. Le pBsAb selon l'invention présente des activités pour initier une interaction cellule-cellule entre une cellule cancéreuse et un macrophage, suivie d'une phagocytose cellulaire dépendante des anticorps. La synthèse monotope peut également être appliquée pour conjuguer d'autres molécules ciblant une tumeur avec diverses formes d'immunoglobulines afin de produire des anticorps bispécifiques destinés à être utilisés en immunothérapie, un médicament pour différentes infections pathogènes, et un diagnostic médical/clinique.
PCT/CN2023/080473 2022-03-21 2023-03-09 Anticorps bispécifique peptidique, ses procédés de préparation et ses utilisations WO2023179370A1 (fr)

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