EP4504772A1

EP4504772A1 - Peptidic bispecific antibody, methods for preparation and uses thereof

Info

Publication number: EP4504772A1
Application number: EP23773619.4A
Authority: EP
Inventors: Clarence Tsun Ting Wong; Chihao SHAO
Original assignee: Hong Kong Polytechnic University HKPU
Current assignee: Hong Kong Polytechnic University HKPU
Priority date: 2022-03-21
Filing date: 2023-03-09
Publication date: 2025-02-12
Also published as: WO2023179370A1; CN119095870A

Abstract

Provided herein are bispecific antibodies synthesized by a one-pot chemical reaction including cyclization of tumor-targeting peptides and conjugation of the cyclized tumor-targeting peptides on the surface of monoclonal antibody to create a new form of bispecifics, named the peptidic bispecific antibodies (pBsAb), which combines the merits of monoclonal antibody and serum stable tumor-targeting cyclic peptides, giving the monoclonal antibody an additional targeting ability to form a bispecific antibody. The proposed pBsAb exhibits activities to initiate cell-cell interaction between cancer cell and macrophage, followed by antibody-dependent cellular phagocytosis. The one-pot synthesis can also be applied to conjugate other tumor-targeting molecules with various forms of immunoglobulins to generate bispecifics for use in immunotherapy, medicament for different pathogenic infections, and medical/clinical diagnosis.

Description

PEPTIDIC BISPECIFIC ANTIBODY, METHODS FOR PREPARATION AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION
The present application claims priority from the U.S. provisional patent application number 63/269,638 filed March 21, 2022, the disclosure of which is incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE DISCLOSURE
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

TECHNICAL FIELD

The present invention relates to a peptidic bispecific antibody, methods for preparation and uses thereof.

BACKGROUND

The bispecific antibody is a next-generation of antibody-based immunotherapy with the potential to improve clinical efficacy and safety. The first concept of the bispecific antibody was introduced in 1960 by Nisonoff and co-workers. The bispecific antibodies were designed to recognize two or more different epitopes or antigens on single or multiple target cells. Such multiple targeting functions allow the antibody to enhance the heterogeneity targeting ability against tumors and create interaction and linkage between immune cells/effector cells and cancer cells to enhance the immunotherapeutic activities. The bispecific antibodies can be grouped into IgG-like and non-IgG-like structures, which are differentiated by the presence of Fc-mediated effector functions. (FIG. 1) . Over 100 different types of constructs have been reported, and five FDA approved bispecific antibodies are now in the market. 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. The fragment crystallizable (Fc) region of the bispecific antibodies additionally binds to a cell that expresses Fc receptors, such as macrophage, natural killer cell or dendritic cell. It is able to trigger common immune responses when recognized by an Fc receptor, such as antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity. Despite the aforementioned advantages and potential applications, bispecific antibody production remains challenging, tedious, and expensive with limited design flexibility. For example, Blinatumomab can cost about US$ 178,000 per treatment, which is one of the most expensive oncology drugs on the market. Chemical approach to develop bispecific antibodies has emerged recently, but the methodologies remained rare.
To summarize, major technical problems of the existing technologies include:
· Tedious production of bispecific antibody using traditional methods;
· Limited flexibility in the production of traditional bispecific antibody;
· Expensive to produce;
· Existing bispecific antibodies usually having a two binding site ratio in 1: 1;
· Slow to produce new bispecific antibody.
· Challenging characterization and quality control
There is a need for a robust platform that can rapidly and effectively generate novel bispecifics for therapeutics and diagnosis of disease.

SUMMARY OF INVENTION

Accordingly, 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.
In certain embodiments, the bispecific antibody is derived from native, denatured, or synthetic immunoglobulins, or any fragments thereof, including, but not limited to, IgG, IgA, IgM, nanobody, fragment antigen-binding region (Fab) , single-chain variable fragment (scFv) , or peptibody.
In certain embodiments, the bispecific antibody is derived from polyclonal or a monoclonal antibody.
In certain embodiments, the monoclonal antibody can be an IgG antibody from human or any other animal origin with any specificity.
In certain embodiments, the monoclonal antibody and the one or more biomarker-binding peptides are conjugated through a chemical or enzymatic reaction.
In certain embodiments, the one or more biomarker-binding peptides include 1 to 100 biomarker-binding peptides per monoclonal antibody.
In certain embodiments, each of the biomarker-binding peptides includes at least one sequence capable of targeting one or more biomolecules including, but not limited to, proteins, peptides, cell surface, organelles, and/or any soluble substances of the same or different targets.
In certain embodiments, the biomarker-binding peptides may be selected from one or more amino acid sequences of SEQ ID NOs: 1 to 8.
In certain embodiments, 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) .
In certain embodiments, each bispecific antibody can conjugate with two or more different biomolecules on a surface of the same target.
In certain embodiments, each bispecific antibody can conjugate with two or more different molecules on surfaces of different targets. In those embodiments, the different targets can be linked to each other through the conjugated bispecific antibody when they are in sufficient proximity.
In certain embodiments, 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.
In certain embodiments, the one or more biomarker-binding peptides can be linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
In certain embodiments, linear biomarker-binding peptides may be conjugated to the N-terminus, C-terminus, disulfide, or any amino acids’ side chain of the monoclonal antibody.
In certain embodiments, 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.
In certain embodiments, the chemical bond between N-terminus and C-terminus, between a side chain and the other 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.
In certain embodiments, the cyclic peptide backbone of the cyclic biomarker-binding peptides chemically conjugate with a region of the monoclonal antibody through a chemical conjugation whereas the at least one sequence capable of targeting the one or more biomolecules of the target is disposed away from the region of the monoclonal antibody chemically conjugated with the cyclic peptide backbone.
In certain embodiments, 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.
In an embodiment, the bifunctional linker is selected from a phthalaldehyde linker and the biomarker-binding peptide is cyclized by one of the functional domains of the phthalaldehyde linker through site-specific dialkylation of sulfhydryl side chains of two cysteine residues in the biomarker-binding peptide, while the other functional domain of the phthalaldehyde linker will form a chemical conjugation with the region of the monoclonal antibody.
A second aspect of the present invention provides a method for preparing a bispecific antibody chemically or enzymatically conjugated with one or more biomarker-binding peptides, where the method includes:
providing immunoglobulins or any fragments thereof;
providing the one or more biomarker-binding peptides each comprising a sequence capable of targeting one or more biomolecules of a target; and
conjugating the one or more biomarker-binding peptides to a region of the immunoglobulins or any fragments thereof through a chemical or enzymatic reaction.
In certain embodiments, the immunoglobulins or any fragments thereof include, but not limited to, IgG, IgA, IgM, nanobody, Fab, scFv, or peptibody.
In certain embodiments, the bispecific antibody is derived from a monoclonal antibody.
In certain embodiments, the monoclonal antibody can be an IgG antibody from human or any other animal origin with any specificity.
In certain embodiments, the monoclonal antibody and the one or more biomarker-binding peptides are conjugated with each other through a chemical linker.
In certain embodiments, the one or more biomarker-binding peptides include 1 to 100 biomarker-binding peptides per monoclonal antibody.
In certain embodiments, each of the biomarker-binding peptides includes at least one sequence capable of targeting one or more biomolecules including, but not limited to, proteins, peptides, cell surface, organelles, and/or any soluble substances of a target.
In certain embodiments, the biomarker-binding peptides may be selected from one or more amino acid sequences of SEQ ID NOs: 1 to 8.
In certain embodiments, each bispecific antibody can conjugate with two or more different biomolecules on a surface of the same target.
In certain embodiments, each bispecific antibody can conjugate with two or more different molecules on surfaces of different targets. In those embodiments, the different targets can be linked to each other through the conjugated bispecific antibody when they are in proximity.
In certain embodiments, 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.
In certain embodiments, 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.
In certain embodiments, the one or more biomarker-binding peptides can be linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
In certain embodiments, linear biomarker-binding peptides may be conjugated to the N-terminus, C-terminus, disulfide, or any amino acids’ side chain of the monoclonal antibody.
In certain embodiments, 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.
In certain embodiments, 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.
In certain embodiments, the cyclic peptide backbone of the cyclic biomarker-binding peptides chemically conjugates with a region of the monoclonal antibody through the chemical linker whereas the at least one sequence capable of targeting the one or more biomolecules of the target is disposed away from the region of the monoclonal antibody chemically conjugated with the cyclic peptide backbone.
In certain embodiments, the chemical linker is 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.
In an embodiment, 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.
In certain embodiments, the composition is capable of triggering an antibody-dependent cellular phagocytosis (ADCP) .
In certain embodiments, each of the bispecific antibodies has one or more chemically conjugated biomarker-binding peptides each comprising a sequence capable of conjugating with one or more corresponding biomolecules of target cells while the bispecific antibody is capable of targeting a receptor on macrophages of the subject to trigger a macrophage-mediated phagocytosis on the target cells.
In certain embodiments, the target cells include, but not limited to, cancer cells or tumor-initiating cells.
In certain embodiments, 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.
In certain embodiments, 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.
In certain embodiments, the composition can be administered via intravenous, intratumoral, subcutaneous, intraperitoneal, or intramuscular injection.
In certain embodiments, the composition can be administered in conjunction with, prior to, or after other cancer therapies including, but not limited to, radiotherapy and chemotherapy.
In certain embodiments, 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.
Other aspects of the present invention include a method of using the bispecific antibody described herein for preparing a medicament or pharmaceutical composition for immunotherapy or treating pathogenic infection or diseases. Depending on the nature of the target or biomolecules to be conjugated, the configuration of one or more biomarker-binding peptides and/or the conjugation strategy between the immunoglobulins and the biomarker-binding peptides can vary. The present invention also includes a kit for targeting certain cell types, tissues or biomolecules comprising the bispecific antibodies described herein which are chemically conjugated with the one or more biomarker-binding peptides having a sequence capable of specifically conjugating with one or more biomarkers of certain cell types, tissues or biomolecules. In certain embodiments, 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 according to certain embodiments 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.
This summary introduces a selection of concepts in a simplified form that is further described below in the Detailed Description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Other aspects of the present invention are disclosed as illustrated by the embodiments hereinafter.

BRIEF DESCRIPTION OF DRAWINGS

The appended drawings, where like reference numerals refer to identical or functionally similar elements, contain figures of certain embodiments to illustrate further and clarify the above and other aspects, advantages, and features of the present invention. It will be appreciated that these drawings depict embodiments of the invention and are not intended to limit its scope. The invention will be described and explained with additional specificity and detail through the use of the accompanying drawings, in which:
FIG. 1 schematically depicts structures of different conventional monoclonal and bispecific antibodies (left two diagrams) and the present peptidic bispecific antibody (pBsAb) (right diagram) .
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 α (SIRPα) 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.
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-α. 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-α monoclonal antibody (20 nM) for 24 h. The arrows show the phagocytic macrophages; 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-α 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-α 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-α 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. 10 shows (a) HPLC chromatogram and (b) MALDI-TOF mass spectrum of cEBP-OPA as shown in FIG. 2B.
FIG. 11 shows MALDI-TOF mass spectrum of EBP peptide as shown in FIG. 2B.
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been depicted to scale.

DETAILED DESCRIPTION OF THE INVENTION

It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation.
Provided herein is a novel type of bispecific antibody named the peptidic bispecific antibody (abbreviated as “pBsAb” ) (right diagram in FIG. 1) . It utilizes a one-pot peptide cyclization and protein conjugation reaction strategy to efficiently cyclize and conjugate tumor-targeting peptide on a monoclonal antibody to create the bispecificity. 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. In certain embodiments, the pBsAb is provided by conjugating EGFR-targeting cyclic peptides onto an anti-SIRP-α monoclonal antibody, forming the EGFR x SIRP-α pBsAb.
The preparation of pBsAb according to certain embodiments 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. The other end incorporated with a phthalaldehyde moiety for protein conjugation via a phthalaldehyde-amine capture (PAC) reaction. The two reactions can be performed in a one-pot orthogonal manner. (FIGs. 2A and 2B) .
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-α 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. In that experiment, the EGFR-positive HT29 colorectal carcinoma as the target cell and low EGFR-expressing HeLa cervix adenocarcinoma as the control cell line were chosen. To this end, a bifunctional linker was first synthesized. In brief, methyl-ester acetal-protected phthalaldehyde 1 prepared according to the previous literature was reduced with LiAlH₄ to give hydroxy 2. Then 2 was reacted with 1, 3, 5-tris (bromomethyl) -benzene in the presence of NaH to yield linker 3 via nucleophilic mono-substitution. Details of synthesis of this bifunctional linker can be referred to scheme S1 in the following Examples section.
To prepare the phthalaldehyde-functionalized cyclic EBP, compound 3 was treated with trifluoroacetic acid (TFA) for the in situ deprotection. Then, it was reacted with a linear peptide, EBP, in a 1: 1 molar ratio (1 mM) to a mixture of N, N-dimethylformamide (DMF) and borate buffer (pH 9.0) (100 μL, 1: 1 v/v) for 2 h at room temperature to afford EBP-OPA. RP-HPLC and mass spectrometry were used to characterize the cyclization process (FIGs. 10 and 11) . Without further purification, the cyclized product EBP-OPA was mixed with non-specific human IgG in phosphate buffered saline (PBS) (pH = 7.4) (1 mL) with 10: 1 mol ratio of peptide to protein and the excess peptides was removed by dialysis. 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) . These data show that an additional tumor-targeting function has been successfully incorporated into an antibody following the proposed one-pot synthesis method.
With the plausible data, the same approach was used to conjugate EGFR-binding cyclic peptide to commercially available rabbit anti-SIRP-α monoclonal antibody to form the novel EGFR x SIRP-α pBsAb. To optimize the pBsAb, the best ratio of cyclic peptide to the antibody needs to be determined that could generate the highest binding affinity against both SIRP-α and EGFR. To this end, 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-α (0.5 μg/ml) or EGFR (1 μg/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. While at 50: 1 of cyclic peptide to antibody, the binding affinity against SIRP-α was not changed significantly when compared to the native anti-SIRP-α antibody. Thus, the results show that 50: 1 of cyclic peptide: antibody is an ideal candidate for binding both EGFR and SIRP-α, and this construct was used in subsequent tests.
After optimization of the EGFR x SIRP-α pBsAb, in vitro binding assay was performed to demonstrate the binding ability of pBsAb against EGFR positive cell lines and macrophages. Confocal microscopy reveals that when PE-tagged pBsAb was incubated with EGFR overexpressing cell lines A549 and HT29, fluorescence signal was observed on the cell surface. When the pBsAb incubated with SIRP-α positive RAW264.7 murine macrophages, strong fluorescent signal was also observed. However, fluorescent signal was not observed when the pBsAb was incubated with HeLa cell, which is a low EGFR-expressing cell line (FIG. 5A) . Flow cytometry was used to further quantify the binding of pBsAb against different cell line (FIG. 5B) . The results show that comparing between the anti-SIRP-α native antibody and the present EGFR x SIRP-α pBsAb, the binding ability of pBsAb against EGFR-positive cell lines HT29 is highly significant, proving its bispecificity against EGFR and SIRP-α in cellular level.
Cell culture formation assay showed that when two cells added together at the same dish, cell clusters formation between macrophages and cancer cells were observed in the presence of pBsAB. (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)
With the high binding specificities against EGFR and SIRP-α positives cell lines, the antibody-dependent cellular phagocytosis (ADCP) activity of the macrophages was further tested. To this end, different cell lines were co-cultured in the presence and absence of the proposed pBsAb. 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. On the other hand, when the co-cultured cells were incubated with the anti-SIRP-α mAb, the phagocytosis occurred in a much smaller extent. This series of imaging data clearly demonstrates that the antibody-dependent cellular phagocytosis activity was enhanced by the proposed EGFR x SIRP-α pBsAb. The confocal microscopic data were further quantified by flow cytometry and analyzed by quadrant analysis. In brief, the RAW264.7 was stained with CFSE and the HT29 or HeLa was stained with Cell Tracker Red before the assay. The macrophage and cancer cells were then added into round bottom 96-well plate at about 1: 1 ratio in the presence or absence of 20 nM pBsAb for 2 hours at 37℃ with 5%CO₂ in full medium culture environment. After the incubation, flow cytometry was performed to determine the percentage of phagocytosis by the RAW264.7 macrophage. From the quadrant analysis, the data clearly shows that the percentage of cells at the double positive (top right-hand corner) had increased significantly in the presence of pBsAb (FIG. 7B, upper two panels) compared to those in the absence of pBsAb (FIG. 7B, lower two panels) . The increase of cells in the double positive area indicates that an increased number of macrophages have performed phagocytotic activity. In contrast, no enhancement of phagocytotic activity was observed on the EGFR-low expressing HeLa cells. Thus, these quantitative data suggest the specificity of the present bispecific antibody. It also demonstrates that the enhanced macrophage-mediated phagocytosis is antibody-dependent.
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 EC₅₀. As shown in FIG. 8, pBsAb of various concentrations could enhance the phagocytotic activity that leads to anticancer effect, compared to the unmodified anti-SIRP-α 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.
Lastly, the macrophage infiltration and ADCP antitumor effect in a three-dimensional HT29 spheroid model were demonstrated in the presence of pBsAb or unmodified anti-SIRP-α mAb. A three-dimensional tumor spheroid model was used to resemble the tumor situation in vivo more closely than the monolayer setting. In brief, the HT29 cells (5x10⁴ 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 (1x10³ cells) together with the pBsAb or anti-SIRP-α mAb (50 nM) , respectively, for 24 h at 37℃ 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. 9A, 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-α mAb. In addition, 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) .
In summary, a one-pot chemical conjugation method provided herein successfully converts monospecific antibody, e.g., monoclonal antibody, into a new type of bispecific antibody, peptidic bispecific antibody (pBsAb) , with proven bispecificity and enhanced antibody-dependent cellular phagocytosis (ADCP) . The EGFR x SIRP-alpha pBsAb prepared according to certain embodiments has been demonstrated to be able to bind to the EGFR-overexpressing cells as well as the SIRP-alpha of the macrophage, linking together to form a new cell-cell interaction between the target cell and the macrophage. Such design enhances the antibody-dependent cellular phagocytosis. The establishment of the present platform allows a low-cost and rapid production of bispecifics for immunotherapy.
In certain embodiments, the present invention also includes any structures of peptides such as, but not limited to, linear, cyclic, stapled, branched, dendrimeric, and scaffold peptides.
In certain embodiments, 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.
It should be understood that the above embodiments and examples are intended to assist the illustration of the present invention, but should not be considered to limit the scope of the present invention, e.g., to limit to certain mechanism of linking peptides on antibody. Any chemical reactions within the knowledge of an ordinary skilled artisan in the relevant field that are able to link the peptides on the antibody without departing the objectives and spirit of the present invention should also fall within the scope of the present invention and be used to enable the present invention.
The following descriptions further characterize the structure, properties and the method of making the novel bispecific antibodies of the present invention as various examples and be supported by corresponding experiments, which should not be considered to limit the scope of the present invention thereto.
EXAMPLES
(A) General Materials and Characterization Methods:
N, N-Dimethylformamide (DMF) , tetrahydrofuran (THF) , and CH₂Cl₂ 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.
¹H and ¹³C {¹H} NMR spectra were recorded on a Bruker Avance III 500 spectrometer (¹H, 500 MHz; ¹³C, 125.7 MHz) in deuterated solvents. Spectra were referenced internally by using the residual solvent {¹H, δ = 7.26 (for CDCl₃) , δ = 3.31 (for CD₃OD) , δ = 2.50 [for dimethylsulfoxide (DMSO) -d₆] } or solvent [¹³C, δ = 77.2 (for CDCl₃) , δ = 49.0 (for CD₃OD) , δ = 39.5 (for DMSO-d₆) ] resonances relative to SiMe₄. 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 μm, 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 μm, 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. The condition used for the analysis was set as follows: solvent A = 0.1%trifluoroacetic acid (TFA) in acetonitrile and solvent B = 0.1%TFA in deionized water; gradient: 5%A + 95%B in the first 5 min, then changed to 15%A + 85%B in 10 min, further changed to 100%A + 0%B in 30 min, maintained under this condition for 5 min, changed to 0%A + 100%B in 5 min, maintained under this condition for further 5 min.
(B) Preparation of EBP and Arginine-glycine-aspartate (RGD) peptides:
EBP (AcNH-CMYIEALDRYAC-COHN₂) peptide (SEQ ID NO: 1) was synthesized manually using a modified 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis protocol with the commercially available N-α-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. An excess of the Fmoc-protected amino acid (4 equiv. ) , HATU (4 equiv. ) , and N, N-diisopropylethylamine (DIPEA) (8 equiv. ) in DMF were used for each coupling at room temperature. For the N-terminal acetylation, a mixture of CH₂Cl₂/pyridine/acetic anhydride (2/1/1 v/v/v) was added and the mixture was stirred at room temperature for 30 min. After washing with DMF and CH₂Cl₂, the resin was treated with a solution containing 95%TFA, 2.5%triisopropylsilane (TIS) , and 2.5%CH₂Cl₂ 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. Alternatively, 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) . For EBP: HRMS (MALDI-TOF) : m/z calcd for C₆₄H₉₉N₁₆O₁₉S₃ [M+H] ⁺, 1491.6429; found, 1491.6347. For RGD: HRMS (MALDI-TOF) : m/z calcd for C₂₉H₄₅N₁₀O₉S₂ [M+H] ⁺, 741.2807; found, 741.2610.
Scheme S1. Scheme of preparing the phthalaldehyde:
(C) Preparation of compound 2:
Methyl-ester acetal-protected phthalaldehyde 1 (0.5 g, 1.9 mmol) was treated with LiAlH₄ (75 mg, 2.0 mmol) in THF (20 mL) at 0℃. The reaction mixture was kept stirring for 3 h and then quenched by the addition of methanol. The mixture was filtered through celite and the filtrate was evaporated under reduced pressure. Water (20 mL) was then added to the residue, and the crude product was extracted with ethyl acetate (20 mL × 3) . The combined organic phase was dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with hexane/ethyl acetate (1: 1 v/v) as eluent to afford 2 (0.42 g, 94 %) . ¹H NMR (400 MHz, CDCl₃) : δ 7.27-7.32 (m, 2 H, ArH) , 7.23 (s, 1 H, ArH) , 6.29-6.31 (m, 1 H, CH) , 6.04 (d, J = 8.4 Hz, 1 H, CH) , 3.67 (q, J = 5.6 Hz, 2 H, OCH₂) , 3.42-3.46 (m, 6 H, CH₃) , 2.77 (t, J = 7.6 Hz, 2 H, CH₂) , 1.89 (quintet, J = 6.4 Hz, 2 H, CH₂) , 1.31 (t, J = 4.8 Hz, 1 H, OH) . ¹³C {¹H} NMR (100.6 MHz, CDCl₃) : δ 144.0, 143.9, 138.8, 138.7, 136.1, 136.0, 130.2, 130.1, 122.8, 122.7, 106.5, 105.4, 61.9, 54.3, 54.1, 34.1, 31.9. HRMS (ESI) : m/z calcd for C₁₃H₁₈NaO₄ [M+Na] ⁺, 261.1097; found, 261.1096.
(D) Preparation of compound 3:
A mixture of 2 (0.4 g, 1.7 mmol) and Et₃N (0.5 mL, 3.6 mmol) in CH₂Cl₂ (20 mL) was stirred at 0℃. p-Toluenesulfonyl chloride (0.4 g, 2.1 mmol) was then slowly added to the reaction mixture. After stirring overnight, the mixture was evaporated under reduced pressure. Water (20 mL) was then added to the residue, and the crude product was extracted with ethyl acetate (20 mL × 3) . The combined organic phase was dried over anhydrous Na₂SO₄, and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel with hexane/ethyl acetate (3: 1 v/v) as eluent to afford 3 (0.56 g, 85 %) . ¹H NMR (400 MHz, CDCl₃) : δ 7.79 (d, J = 8.4 Hz, 2 H, ArH) , 7.35 (d, J = 8.4 Hz, 2 H, ArH) , 7.28 (s, 1 H, ArH) , 7.13-7.15 (m, 2 H, ArH) , 6.25-6.28 (m, 1 H, CH) , 6.01 (d, J = 13.6 Hz, 1 H, CH) , 4.02 (t, J = 6.4 Hz, 2 H, OCH₂) , 3.42-3.44 (m, 6 H, CH₃) , 2.71 (t, J = 7.6 Hz, 2 H, CH₂) , 2.46 (s, 3 H, CH₃) , 1.96 (quintet, J = 7.2 Hz , 2 H, CH₂) . ¹³C {¹H} NMR (100.6 MHz, CDCl₃) : δ 144.9, 142.7, 142.5, 139.1, 139.0, 136.7, 136.6, 133.2, 130.3, 130.2, 130.0, 128.0, 123.1, 122.8, 106.6, 105.5, 69.5, 54.5, 54.4, 54.3, 31.5, 30.6, 21.8. HRMS (ESI) : m/z calcd for C₂₀H₂₄NaO₆S [M+Na] ⁺, 415.1186; found, 415.1186.
(E) Preparation of compound 4:
A mixture of 1, 3, 5-tris (bromomethyl) benzene (0.28 g, 0.8 mmol) and sodium hydride (60%suspension in mineral oil, 25.2 mg, 0.6 mmol) in THF (5 mL) was stirred at 0℃. A solution of compound 3 (75.0 mg, 0.3 mmol) in THF (5 mL) was added to the suspension in dropwise. The reaction mixture was kept stirring at 0℃ for 1 h and then slowly warmed to room temperature for overnight. The solvent was evaporated under reduced pressure. Water (20 mL) was then added to the residue, and the crude product was extracted with CH₂Cl₂ (20 mL × 3) . The combined organic phase was dried over anhydrous Na₂SO₄. After evaporating the solvent under reduced pressure, the residue was purified by column chromatography on silica gel with hexane/ethyl acetate (3: 1 v/v) as eluent to afford 4 (56.0 mg, 35 %) . ¹H NMR (400 MHz, CDCl₃) : δ 7.22-7.34 (m, 6 H, ArH) , 6.29-6.30 (m, 1 H, CH) , 6.04 (d, J = 6.8 Hz, 1 H, CH) , 4.48 (s, 6 H, CH₂) , 3.49 (t, J = 6.4 Hz, 2 H, OCH₂) , 3.43-3.45 (m, 6 H, CH₃) , 2.78 (t, J = 7.6 Hz, 2 H, CH₂) , 1.92-1.99 (m, 2 H, CH₂) . ¹³C {¹H} NMR (125.8 MHz, CDCl₃) : δ 144.0, 143.9, 140.1, 138.9, 138.8, 138.6, 136.2, 136.1, 130.3, 130.2, 128.8, 128.1, 122.9, 122.8, 122.8, 122.7, 106.6, 105.5, 72.2, 69.7, 54.3, 54.2, 32.8, 32.3, 31.3, 31.0. HRMS (ESI) : m/z calcd for C₂₂H₂₆Br₂NaO₄ [M+Na] ⁺, 537.0071; found, 537.0081.
(F) Preparation of cEBP-OPA:
Compound 4 was treated with a mixture of TFA and water (1: 1 v/v) for 30 min at room temperature. After the in situ deprotection of ortho-phthalaldehyde, the intermediate product was treated with EBP (1 mM) in borate buffer (pH 8.5) for 1 h at room temperature. Based on the HPLC analysis, the conversion for cEBP-OPA was found to be 92%and 89%, respectively. For cEBP-OPA: HRMS (MALDI-TOF) : m/z calcd for C₈₄H₁₁₇N₁₆O₂₂S₃ [M+H] ⁺, 1797.7685; found, 1797.7608.
(G) Preparation of Cyclic Peptide-antibody Conjugates:
Monoclonal antibody was first dissolved in phosphate-buffered saline (PBS) (pH 7.4) to afford a 5 μM stock solution. 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.
(H) ELISA analysis of pBsAb binding against EGFR and SIRP-alpha:
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. 100 ul of TMB substrate (Biogene, China) was added into each well for 6 min. The HRP activity was measured at 450 nm by ELISA plat reader. Half-maximum effective concentration (EC₅₀) binding values were calculated by non-linear regression analysis on the binding curves using GraphPad Prism.
(I) Cell Lines and Culture Conditions:
All cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher Scientific, cat. no. 12100-046) supplemented with fetal bovine serum (FBS) (10%) and penicillin‐streptomycin (100 unit mL^-1 and 100 μg mL^-1, respectively) . HT29 human colorectal adenocarcinoma cells (ATCC, no. HTB-38) , HeLa human cervical carcinoma cells (ATCC, no. CCL-2) , MDA-MB-231 human breast adenocarcinoma cells (ATCC, no. HTB-26) , and MCF-7 human breast adenocarcinoma cells (ATCC, no. 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 μg mL^-1, respectively) . U87-MG human glioblastoma cells (ATCC, no. HTB-14) were maintained in Minimum Essential Medium (MEM) (Sigma-Aldrich, no. M5650) supplemented with FBS (10%) and penicillin‐streptomycin (100 unit mL^-1 and 100 μg mL^-1, respectively) . All the cells were grown at 37 ℃ in a humidified 5%CO₂ atmosphere.
(J) Confocal Fluorescence Microscopic Study:
Approximately 2 × 10⁵ cells in DMEM, RPMI 1640 medium, or MEM (2 mL) were seeded on a glass-bottom confocal dish and incubated overnight at 37 ℃ in a humidified 5%CO₂ atmosphere. For the study of cell binding, the cells, after being rinsed with PBS, were incubated with phycoerythrin labelled pBsAb, in a serum-free medium at 37 ℃ 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.
(K) Flow Cytometric Analysis:
Approximately 2 × 10⁵ cells per well in DMEM, RPMI 1640 medium, or MEM (2 mL) were seeded on a 6-multiwell plate and incubated overnight at 37℃ in a humidified 5%CO₂ atmosphere. The cells, after being rinsed with PBS, were incubated with different concentrations of pBsAb or the native mAb in a serum-free medium at 37℃ for 1 h. The cells were then rinsed with PBS twice, and harvested by 0.25%trypsin-ethylenediaminetetraacetic acid (Invitrogen, 0.2 mL) for 5 min. 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 10⁴ cells counted in each sample. The data collected were analyzed using the BD FACSuite. All experiments were performed in triplicate.
(L) Double-staining phagocytosis experiment:
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 3x10⁶ (Vi-CELL XR Cell Viability Analyzer, Beckman Coulter, Brea, CA) . Macrophages were loaded with 10 μM 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℃. After incubation, the cells were fixed with 4%paraformaldehyde and three times washing with PBS. The intracellular fluorescence intensities were measured using a BD FACSVerse flow cytometer (Becton Dickinson) with 10⁴ cells counted in each sample. The data collected were analyzed using the BD FACSuite. All experiments were performed in triplicate.
It will be apparent to those skilled in the art that modifications, including additions and/or substitutions, may be made without departing from the scope and spirit of the invention. Specific details may be omitted so as not to obscure the invention; however, the disclosure is written to enable one skilled in the art to practice the teachings herein without undue experimentation. For example, the 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.
Although the invention has been described in terms of certain embodiments, other embodiments apparent to those of ordinary skill in the art are also within the scope of this invention.

INDUSTRIAL APPLICABILITY

The proposed bispecific antibody is not just a potential therapeutic agent for use in immunotherapy of various cancers or tumors, the proposed one-pot chemical method in the present disclosure which is able to conjugate tumor-targeting molecules with various forms of immunoglobulins to generate bispecifics has potentials to be applied in developing next generation immuno-therapeutics, medicaments for treating different pathogenic infections or diseases, and medical/clinical diagnostic tools.
Some advantages of the present disclosure include:
- Using chemical approach to produce bispecific antibody is generally cheaper and faster;
- Using conjugation chemistry to conjugate tumor-targeting peptides on antibody allow flexibility in bispecificity;
- Allowing construction and adjusting the avidity and affinity of the two targets on one bispecific; and
- Being a novel platform technology that can produce unlimited combination of bispecific antibody against two different targets.

Claims

A bispecific antibody comprising an immunoglobulin or any fragment thereof chemically or enzymatically conjugated with one or more biomarker-binding peptides capable of conjugating with multiple biomolecules on one or more targets simultaneously.
The bispecific antibody of claim 1, wherein the immunoglobulin or any fragment thereof is a native, denatured, or synthetic immunoglobulin comprising IgG, IgA, IgM, nanobody, fragment antigen-binding region (Fab) , single-chain variable fragment (scFv) , or peptibody.
The bispecific antibody of claim 1, wherein the immunoglobulin is a monoclonal antibody.
The bispecific antibody of claim 3, wherein the monoclonal antibody is an IgG antibody from human or any other animal origin with any specificity.
The bispecific antibody of claim 3, wherein the one or more biomarker-binding peptides comprises 1 to 100 biomarker-binding peptides per monoclonal antibody.
The bispecific antibody of claim 1, wherein each of the biomarker-binding peptides comprises at least one sequence capable of targeting at least two biomolecules on the same or different targets simultaneously, said biomolecules comprising proteins, peptides, cell surface, organelles, and/or any soluble substances of the same or different targets.
The bispecific antibody of claim 6, wherein the biomolecules which said biomarker-binding peptides are capable of targeting comprise 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) .
The bispecific antibody of claim 1, wherein two or more different targets conjugated with the bispecific antibody are linked together through the conjugated bispecific antibody when the two or more different targets are in sufficient proximity.
The bispecific antibody of claim 1, wherein the target of the bispecific antibody comprises non-living and living materials including NK cells, T-cells, macrophages, dendritic cells, red blood cells, B-cells, cancer cells, viruses, bacteria, fungi, yeasts, and parasites.
The bispecific antibody of claim 1, wherein the one or more biomarker-binding peptides are linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
The bispecific antibody of claim 10, wherein the linear biomarker-binding peptides are conjugated to N-terminus, C-terminus, disulfide, or any amino acids’ side chain of the immunoglobin, wherein the immunoglobin is a monoclonal antibody.
The bispecific antibody of claim 10, wherein the cyclic biomarker-binding peptides are formed by cyclizing the biomarker-binding peptides 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 bispecific antibody of claim 12, wherein the chemical bond between the N-terminus and C-terminus, between the side chain and the other chain, or between one of the N-and C-termini and the side chain, for forming the cyclic peptide backbone is a covalent bond, supramolecular interaction or disulfide bond.
The bispecific antibody of claim 12, wherein the cyclic peptide backbone of the cyclic biomarker-binding peptides chemically conjugates with a region of a monoclonal antibody through a chemical conjugation whereas at least one sequence capable of targeting the one or more biomolecules of the target is disposed away from the region of the monoclonal antibody chemically conjugated with the cyclic peptide backbone.
The bispecific antibody of claim 14, wherein 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 bispecific antibody of claim 15, wherein the bifunctional linker is selected from a phthalaldehyde linker and the linear biomarker-binding peptide is cyclized by one of the functional domains of the phthalaldehyde linker through site-specific dialkylation of sulfhydryl side chains of two cysteine residues in the biomarker-binding peptide, while the other functional domain of the phthalaldehyde linker forms the chemical conjugation with the region of the monoclonal antibody.
A method for preparing a bispecific antibody chemically conjugated with one or more biomarker-binding peptides according to any one of the preceding claims, the method comprising:

providing immunoglobulins or any fragments thereof;

providing the one or more biomarker-binding peptides each comprising a sequence capable of targeting one or more biomolecules of a target; and

conjugating the one or more biomarker-binding peptides to a region of the immunoglobulins or any fragments thereof through a chemical or enzymatic reaction.
The method of claim 17, wherein the immunoglobulins or any fragments thereof comprise IgG, IgA, IgM, nanobody, Fab, scFv, or peptibody.
The method of claim 17, wherein the immunoglobulins are selected from a monoclonal antibody.
The method of claim 19, wherein the monoclonal antibody is an IgG antibody from human or any other animal origin with any specificity.
The method of claim 19, wherein the one or more biomarker-binding peptides comprise 1 to 100 biomarker-binding peptides per monoclonal antibody.
The method of claim 17, wherein the one or more biomarker-binding peptides are linear, cyclic, stapled, branched, dendrimeric, or scaffold peptides.
The method of claim 22, wherein the linear biomarker-binding peptides are conjugated to N-terminus, C-terminus, disulfide, or any amino acids’ side chain of the immunoglobin, wherein the immunoglobin is a monoclonal antibody.
The method of claim 22, wherein the cyclic biomarker-binding peptides are formed by cyclizing the biomarker-binding peptides 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 method of claim 24, wherein 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 is a covalent bond, supramolecular interaction or disulfide bond.
The method of claim 25, wherein the cyclic peptide backbone of the cyclic biomarker-binding peptides chemically conjugates with a region of a monoclonal antibody through a chemical linker whereas at least one sequence capable of targeting the one or more biomolecules of the target is disposed away from the region of the monoclonal antibody chemically conjugated with the cyclic peptide backbone.
The method of claim 26, wherein the chemical linker is 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 method of claim 27, wherein the linear biomarker-binding peptide is cyclized by one of the functional domains of the bifunctional linker, while the other functional domain of the bifunctional linker forms a chemical conjugation with the region of the monoclonal antibody.
The method of claim 17, wherein the one or more biomolecules of the target comprise 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) .
Use of the bispecific antibody according to any one of Claims 1 to 16 in preparation of a composition for treating a disease in a subject in need of an immunotherapy, said composition comprising a therapeutically effective amount of the bispecific antibodies.
The use of claim 30, wherein the composition is capable of triggering an antibody-dependent cellular phagocytosis (ADCP) .
The use of claim 30, wherein each of the bispecific antibodies has one or more chemically conjugated biomarker-binding peptides each comprising a sequence capable of conjugating with one or more corresponding biomolecules of target cells or tissues while the bispecific antibody is capable of targeting a receptor on macrophages of the subject to trigger a macrophage-mediated phagocytosis on the target cells or tissues.
The use of claim 32, wherein the target cells comprise cancer cells or tumor-initiating cells.
The use of claim 33, wherein the receptor on the macrophages initially inactivated by an antigen of the cancer cells or tumor-initiating cells is activated by the composition comprising the therapeutically effective amount of the bispecific antibodies after administration of the composition to the subject.
The use of claim 34, wherein the composition is administered via intravenous, intratumoral, or intramuscular injection.
The use of claim 34, wherein the composition is administered in conjunction with, prior to, or after other cancer therapies comprising radiotherapy and chemotherapy.
The use of claim 30, wherein the disease comprises cancers, said cancers comprising head and neck, ovarian, cervical, bladder, oesophageal, gastric, breast, endometrial, colorectal, lung, pancreatic, skin, and non-small cell lung cancers.
A kit comprising the bispecific antibody according to any one of Claims 1 to 15 and an indicator for detecting a presence of one or more biomarkers specific to a target cell type, tissue, or biomolecule in a sample in vitro or in vivo.
The kit of claim 38, wherein the indicator comprises fluorescent dye and colorimetric dye.