Patent Application
20160340435
1. Field of the Invention
The present disclosure relates to the field of pharmaceuticals; more particularly, to multi-functional molecular constructs, e.g., those having targeting and effector elements for delivering the effector (e.g., therapeutic drug) to targeted sites.
2. Description of the Related Art
The continual advancement of a broad array of methodologies for screening and selecting monoclonal antibodies (mAbs) for targeted antigens has helped the development of a good number of therapeutic antibodies for many diseases that were regarded as untreatable just a few years ago. According to Therapeutic Antibody Database, approximately 2,800 antibodies have been studied or are being planned for studies in human clinical trials, and approximately 80 antibodies have been approved by governmental drug regulatory agencies for clinical uses. The large amount of data on the therapeutic effects of antibodies has provided information concerning the pharmacological mechanisms how antibodies act as therapeutics.
One major pharmacologic mechanism for antibodies acting as therapeutics is that, antibodies can neutralize or trap disease-causing mediators, which may be cytokines or immune components present in the blood circulation, interstitial space, or in the lymph nodes. The neutralizing activity inhibits the interaction of the disease-causing mediators with their receptors. It should be noted that fusion proteins of the soluble receptors or the extracellular portions of receptors of cytokines and the Fc portion of IgG, which act by neutralizing the cytokines or immune factors in a similar fashion as neutralizing antibodies, have also been developed as therapeutic agents.
Several therapeutic antibodies that have been approved for clinical applications or subjected to clinical developments mediate their pharmacologic effects by binding to receptors, thereby blocking the interaction of the receptors with their ligands. For those antibody drugs, Fc-mediated mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), are not the intended mechanisms for the antibodies.
Some therapeutic antibodies bind to certain surface antigens on target cells and render Fc-mediated functions and other mechanisms on the target cells. The most important Fc-mediated mechanisms are antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytolysis (CMC), which both will cause the lysis of the antibody-bound target cells. Some antibodies binding to certain cell surface antigens can induce apoptosis of the bound target cells.
The concept and methodology for preparing antibodies with dual specificities germinated more than three decades ago. In recent year, the advancement in recombinant antibody engineering methodologies and the drive to develop improved medicine has stimulated the development bi-specific antibodies adopting a large variety of structural configurations.
For example, the bi-valent or multivalent antibodies may contain two or more antigen-binding sites. A number of methods have been reported for preparing multivalent antibodies by covalently linking three or four Fab fragments via a connecting structure. For example, antibodies have been engineered to express tandem three or four Fab repeats.
Several methods for producing multivalent antibodies by employing synthetic crosslinkers to associate, chemically, different antibodies or binding fragments have been disclosed. One approach involves chemically cross-linking three, four, and more separately Fab fragments using different linkers. Another method to produce a construct with multiple Fabs that are assembled to one-dimensional DNA scaffold was provided. Those various multivalent Ab constructs designed for binding to target molecules differ among one another in size, half-lives, flexibility in conformation, and ability to modulate the immune system. In view of the foregoing, several reports have been made for preparing molecular constructs with a fixed number of effector elements or with two or more different kinds of functional elements (e.g., at least one targeting element and at least one effector element). However, it is often difficult to build a molecular construct with a particular combination of the targeting and effector elements either using chemical synthesis or recombinant technology. Accordingly, there exists a need in the related art to provide novel molecular platforms to build a more versatile molecule suitable for covering applications in a wide range of diseases.
The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the present invention or delineate the scope of the present invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.
<I> Molecular Construct for Treating Central Nervous System Diseases and Uses Thereof
In this aspect, the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin. Targeting and effector elements of the present Fc-based molecular constructs are specifically selected such that these Fc-based molecular constructs are suitable for use in the treatment of central nervous system (CNS) diseases, or for use in the manufacture of a medicament for treating CNS diseases. As could be appreciated, methods for treating CNS diseases using such Fc-based molecular constructs also fall within the aspect of the present disclosure.
According to certain embodiments of the present disclosure, the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a pair of effector elements, and a pair of targeting elements. The pair of effector element is interferon β1a (INF-β1a) or INF-β1b, or an antibody fragment specific for integrin α4 or β-amyloid, while the pair targeting elements is an antibody fragment specific for human transferrin receptor or human insulin receptor.
In the case where the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa. Alternatively, when the pair of effectors elements and the pair of targeting elements is both in the form of single-chain variable fragments (scFvs), then the pair of targeting elements is linked to the N-termini of the pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
In certain embodiments, the pair of CH2-CH3 segments is derived from human IgG heavy chain γ4 or human IgG heavy chain γ1.
In some examples, the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the VH-CH1 domain and the VL-Cκ domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of elements that is not in the Fab configuration is linked to the C-termini of the pair of CH2-CH3 segments.
According to certain optional embodiments, the effector element is INF-β1a, INF-β1b, or an scFv specific for integrin α4, while the targeting element is an scFv specific for human transferrin receptor. In particular, this molecular construct is suitable for treating multiple sclerosis.
According to other optional embodiments, the effector element is an scFv specific for β-amyloid, while the targeting element is an scFv specific for human transferrin receptor. In particular, this molecular construct is suitable for treating Alzheimer's disease.
Methods for treating CNS diseases in a subject in need thereof comprise the step of administering to the subject an effective amount of the molecular construct of this aspect. CNS diseases treatable by this method include multiple sclerosis and Alzheimer's disease.
<II> Molecular Construct for Treating Infectious Diseases
In this aspect, the present disclosure is directed to a fragment crystallizable (Fc)-based molecular construct that has at least one targeting element and at least one effector element linked, directly or indirectly, to a CH2-CH3 domain of an immunoglobulin. Targeting and effector elements of the present Fc-based molecular constructs are specifically selected such that these Fc-based molecular constructs are suitable for use in the treatment of diseases/conditions associated with viral or bacterial infection, or for use in the manufacture of a medicament for treating such diseases/conditions. As could be appreciated, methods for treating diseases/conditions associated with viral or bacterial infection using such Fc-based molecular constructs also fall within the aspect of the present disclosure.
According to certain embodiments of the present disclosure, the Fc-based molecular construct comprises a pair of CH2-CH3 segments of an IgG.Fc, a pair of effector elements, and a pair of targeting elements. The pair of effector element is an antibody fragment specific for CD32 or CD16b, while the pair targeting elements is an antibody fragment specific for a viral protein or a bacterial protein.
In the case where the pair of effector elements is linked to the N-termini of the pair of CH2-CH3 segments, the pair of targeting elements is linked to the C-termini of the pair of CH2-CH3 segments, and vice versa. Alternatively, when the pair of effectors elements and the pair of targeting elements is both in the form of single-chain variable fragments (scFvs), then the pair of targeting elements is linked to the N-termini of the pair of effector elements in a tandem or diabody configuration, thereby forming a pair of bispecific scFvs that are linked to the N-termini of the pair of CH2-CH3 segments.
In certain embodiments, the pair of CH2-CH3 segments is derived from human IgG heavy chain γ4 or human IgG heavy chain γ1.
In some examples, the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the VH-CH1 domain and the VL-Cκ domain); this Fab fragment is linked to the N-termini of the first and second heavy chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of elements that is not in the Fab configuration is linked to the C-termini of the pair of CH2-CH3 segments.
According to certain optional embodiments, the effector element is an scFv specific for CD32 or CD16b, while the targeting element is an scFv specific for a viral protein. For example, the viral protein can be F protein of respiratory syncytia virus (RSV), gp120 protein of human immunodeficiency virus type 1 (HIV-1), hemagglutinin A (HA) protein of influenza A virus, or glycoprotein of cytomegalovirus. In particular, this molecular construct is suitable for treating viral infections.
According to other optional embodiments, the effector element is an scFv specific for Cd35 or C16b, while the targeting element is an scFv specific for a bacterial protein. Examples of the bacterial protein include, but are not limited to, the endotoxin of Gram(−) bacteria, the surface antigen of Clostridium difficile, the lipoteichoic acid of Staphylococcus aureus, the anthrax toxin of Bacillus anthracis, or the Shiga-like toxin type I or II of Escherichia coli. In particular, such molecular construct is suitable for treating bacterial infections.
Methods for treating diseases/conditions associated with infections (e.g., viral or bacterial infections) in a subject in need thereof comprise the step of administering to the subject an effective amount of the molecular construct of this aspect.
The present description will be better understood from the following detailed description read in light of the accompanying drawings briefly discussed below.
In accordance with common practice, the various described features/elements are not drawn to scale but instead are drawn to best illustrate specific features/elements relevant to the present invention. Also, like reference numerals and designations in the various drawings are used to indicate like elements/parts, where possible.
The detailed description provided below in connection with the appended drawings is intended as a description of the present examples and is not intended to represent the only forms in which the present example may be constructed or utilized. The description sets forth the functions of the example and the sequence of steps for constructing and operating the example. However, the same or equivalent functions and sequences may be accomplished by different examples.
For convenience, certain terms employed in the specification, examples and appended claims are collected here. Unless otherwise defined herein, scientific and technical terminologies employed in the present disclosure shall have the meanings that are commonly understood and used by one of ordinary skill in the art.
Unless otherwise required by context, it will be understood that singular terms shall include plural forms of the same and plural terms shall include the singular. Specifically, as used herein and in the claims, the singular forms “a” and “an” include the plural reference unless the context clearly indicated otherwise. Also, as used herein and in the claims, the terms “at least one” and “one or more” have the same meaning and include one, two, three, or more. Furthermore, the phrases “at least one of A, B, and C”, “at least one of A, B, or C” and “at least one of A, B and/or C,” as use throughout this specification and the appended claims, are intended to cover A alone, B alone, C alone, A and B together, B and C together, A and C together, as well as A, B, and C together.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in the respective testing measurements. Also, as used herein, the term “about” generally means within 10%, 5%, 1% or 0.5% of a given value or range. Alternatively, the term “about” means within an acceptable standard error of the mean when considered by one of ordinary skill in the art. Other than in the operating/working examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for quantities of materials, durations of times, temperatures, operating conditions, ratios of amounts, and the likes thereof disclosed herein should be understood as modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present disclosure and attached claims are approximations that can vary as desired. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Ranges can be expressed herein as from one endpoint to another endpoint or between two endpoints. All ranges disclosed herein are inclusive of the endpoints, unless specified otherwise.
This present disclosure pertains generally to molecular constructs, in which each molecular construct comprises a targeting element (T) and an effector element (E), and these molecular constructs are sometimes referred to as “T-E molecules”, “T-E pharmaceuticals” or “T-E drugs” in this document.
As used herein, the term “targeting element” refers to the portion of a molecular construct that directly or indirectly binds to a target of interest (e.g., a receptor on a cell surface or a protein in a tissue) thereby facilitates the transportation of the present molecular construct into the interested target. In some example, the targeting element may direct the molecular construct to the proximity of the target cell. In other cases, the targeting element specifically binds to a molecule present on the target cell surface or to a second molecule that specifically binds a molecule present on the cell surface. In some cases, the targeting element may be internalized along with the present molecular construct once it is bound to the interested target, hence is relocated into the cytosol of the target cell. A targeting element may be an antibody or a ligand for a cell surface receptor, or it may be a molecule that binds such antibody or ligand, thereby indirectly targeting the present molecular construct to the target site (e.g., the surface of the cell of choice). The localization of the effector (therapeutic agent) in the diseased site will be enhanced or favored with the present molecular constructs as compared to the therapeutic without a targeting function. The localization is a matter of degree or relative proportion; it is not meant for absolute or total localization of the effector to the diseased site.
According to the present invention, the term “effector element” refers to the portion of a molecular construct that elicits a biological activity (e.g., inducing immune responses, exerting cytotoxic effects and the like) or other functional activity (e.g., recruiting other hapten tagged therapeutic molecules), once the molecular construct is directed to its target site. The “effect” can be therapeutic or diagnostic. The effector elements encompass those that bind to cells and/or extracellular immunoregulatory factors. The effector element comprises agents such as proteins, nucleic acids, lipids, carbohydrates, glycopeptides, drug moieties (both small molecule drug and biologics), compounds, elements, and isotopes, and fragments thereof.
Although the terms, first, second, third, etc., may be used herein to describe various elements, components, regions, and/or sections, these elements (as well as components, regions, and/or sections) are not to be limited by these terms. Also, the use of such ordinal numbers does not imply a sequence or order unless clearly indicated by the context. Rather, these terms are simply used to distinguish one element from another. Thus, a first element, discussed below, could be termed a second element without departing from the teachings of the exemplary embodiments.
Here, the terms “link,” “couple,” and “conjugates” are used interchangeably to refer to any means of connecting two components either via direct linkage or via indirect linkage between two components.
The term “polypeptide” as used herein refers to a polymer having at least two amino acid residues. Typically, the polypeptide comprises amino acid residues ranging in length from 2 to about 200 residues; preferably, 2 to 50 residues. Where an amino acid sequence is provided herein, L-, D-, or beta amino acid versions of the sequence are also contemplated. Polypeptides also include amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. In addition, the term applies to amino acids joined by a peptide linkage or by other, “modified linkages,” e.g., where the peptide bond is replaced by an α-ester, a β-ester, a thioamide, phosphoramide, carbomate, hydroxylate, and the like.
In certain embodiments, conservative substitutions of the amino acids comprising any of the sequences described herein are contemplated. In various embodiments, one, two, three, four, or five different residues are substituted. The term “conservative substitution” is used to reflect amino acid substitutions that do not substantially alter the activity (e.g., biological or functional activity and/or specificity) of the molecule. Typically, conservative amino acid substitutions involve substitution one amino acid for another amino acid with similar chemical properties (e.g., charge or hydrophobicity). Certain conservative substitutions include “analog substitutions” where a standard amino acid is replaced by a non-standard (e.g., rare, synthetic, etc.) amino acid differing minimally from the parental residue. Amino acid analogs are considered to be derived synthetically from the standard amino acids without sufficient change to the structure of the parent, are isomers, or are metabolite precursors.
In certain embodiments, polypeptides comprising at least 80%, preferably at least 85% or 90%, and more preferably at least 95% or 98% sequence identity with any of the sequences described herein are also contemplated.
“Percentage (%) amino acid sequence identity” with respect to the polypeptide sequences identified herein is defined as the percentage of polypeptide residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percentage sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, sequence comparison between two polypeptide sequences was carried out by computer program Blastp (protein-protein BLAST) provided online by Nation Center for Biotechnology Information (NCBI). The percentage amino acid sequence identity of a given polypeptide sequence A to a given polypeptide sequence B (which can alternatively be phrased as a given polypeptide sequence A that has a certain % amino acid sequence identity to a given polypeptide sequence B) is calculated by the formula as follows:
where X is the number of amino acid residues scored as identical matches by the sequence alignment program BLAST in that program's alignment of A and B, and where Y is the total number of amino acid residues in A or B, whichever is shorter.
The term “PEGylated amino acid” as used herein refers to a polyethylene glycol (PEG) chain with one amino group and one carboxyl group. Generally, the PEGylated amino acid has the formula of NH2—(CH2CH2O)n—COOH. In the present disclosure, the value of n ranges from 1 to 20; preferably, ranging from 2 to 12.
As used herein, the term “terminus” with respect to a polypeptide refers to an amino acid residue at the N- or C-end of the polypeptide. With regard to a polymer, the term “terminus” refers to a constitutional unit of the polymer (e.g., the polyethylene glycol of the present disclosure) that is positioned at the end of the polymeric backbone. In the present specification and claims, the term “free terminus” is used to mean the terminal amino acid residue or constitutional unit is not chemically bound to any other molecular.
The term “antigen” or “Ag” as used herein is defined as a molecule that elicits an immune response. This immune response may involve a secretory, humoral and/or cellular antigen-specific response. In the present disclosure, the term “antigen” can be any of a protein, a polypeptide (including mutants or biologically active fragments thereof), a polysaccharide, a glycoprotein, a glycolipid, a nucleic acid, or a combination thereof.
In the present specification and claims, the term “antibody” is used in the broadest sense and covers fully assembled antibodies, antibody fragments that bind with antigens, such as antigen-binding fragment (Fab/Fab′), F(ab′)2 fragment (having two antigen-binding Fab portions linked together by disulfide bonds), variable fragment (Fv), single chain variable fragment (scFv), bi-specific single-chain variable fragment (bi-scFv), nanobodies, unibodies and diabodies. “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen-binding region or variable region of the intact antibody. Typically, an “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The well-known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. A typical immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, with each pair having one “light” chain (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains, respectively. According to embodiments of the present disclosure, the antibody fragment can be produced by modifying the nature antibody or by de novo synthesis using recombinant DNA methodologies. In certain embodiments of the present disclosure, the antibody and/or antibody fragment can be bispecific, and can be in various configurations. For example, bispecific antibodies may comprise two different antigen binding sites (variable regions). In various embodiments, bispecific antibodies can be produced by hybridoma technique or recombinant DNA technique. In certain embodiments, bispecific antibodies have binding specificities for at least two different epitopes.
The term “specifically binds” as used herein, refers to the ability of an antibody or an antigen-binding fragment thereof, to bind to an antigen with a dissociation constant (Kd) of no more than about 1×10−6 M, 1×10−7 M, 1×10−8 M, 1×10−9 M, 1×10−10 M, 1×10−11 M, 1×10−12 M, and/or to bind to an antigen with an affinity that is at least two-folds greater than its affinity to a nonspecific antigen.
The term “treatment” as used herein includes preventative (e.g., prophylactic), curative or palliative treatment; and “treating” as used herein also includes preventative (e.g., prophylactic), curative or palliative treatment. In particular, the term “treating” as used herein refers to the application or administration of the present molecular construct or a pharmaceutical composition comprising the same to a subject, who has a medical condition a symptom associated with the medical condition, a disease or disorder secondary to the medical condition, or a predisposition toward the medical condition, with the purpose to partially or completely alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of said particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition, and/or to a subject who exhibits only early signs of a disease, disorder and/or condition, for the purpose of decreasing the risk of developing pathology associated with the disease, disorder and/or condition.
The term “effective amount” as used herein refers to the quantity of the present molecular construct that is sufficient to yield a desired therapeutic response. An effective amount of an agent is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered or prevented, or the disease or condition symptoms are ameliorated. The effective amount may be divided into one, two, or more doses in a suitable form to be administered at one, two or more times throughout a designated time period. The specific effective or sufficient amount will vary with such factors as particular condition being treated, the physical condition of the patient (e.g., the patient's body mass, age, or gender), the type of subject being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. Effective amount may be expressed, for example, as the total mass of active component (e.g., in grams, milligrams or micrograms) or a ratio of mass of active component to body mass, e.g., as milligrams per kilogram (mg/kg).
The terms “application” and “administration” are used interchangeably herein to mean the application of a molecular construct or a pharmaceutical composition of the present invention to a subject in need of a treatment thereof.
The terms “subject” and “patient” are used interchangeably herein and are intended to mean an animal including the human species that is treatable by the molecular construct, pharmaceutical composition, and/or method of the present invention. The term “subject” or “patient” intended to refer to both the male and female gender unless one gender is specifically indicated. Accordingly, the term “subject” or “patient” comprises any mammal, which may benefit from the treatment method of the present disclosure. Examples of a “subject” or “patient” include, but are not limited to, a human, rat; mouse; guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human. The term “mammal” refers to all members of the class Mammalia, including humans, primates, domestic and farm animals, such as rabbit, pig, sheep, and cattle; as well as zoo, sports or pet animals; and rodents, such as mouse and rat. The term “non-human mammal” refers to all members of the class Mammalis except human.
The present disclosure is based, at least on the construction of the T-E pharmaceuticals that can be delivered to target cells, target tissues or organs at increased proportions relative to the blood circulation, lymphoid system, and other cells, tissues or organs. When this is achieved, the therapeutic effect of the pharmaceuticals is increased, while the scope and severity of the side effects and toxicity is decreased. It is also possible that a therapeutic effector is administered at a lower dosage in the form of a T-E molecule, than in a form without a targeting component. Therefore, the therapeutic effector can be administered at lower dosages without losing potency, while lowering side effects and toxicity.
Diseases that can Benefit from Better Drug Targeting
Drugs used for many diseases can be improved for better efficacy and safety, if they can be targeted to the disease sites, i.e., if they can be localized or partitioned to the disease sites more favorably than the normal tissues or organs. Certain antibody drugs, which target infectious microorganisms or their toxic products, can be improved, if they are empowered with the ability to recruit immunocytes, which phagocytose and clear the antibody-bound particles. Following are primary examples of diseases, in which drugs can be improved if they can be preferentially distributed to the disease sites or cells or if they can recruit phagocytic immunocytes.
I Central Nervous System Diseases
For treating diseases of the central nervous system (CNS), the therapeutic agents are often required to pass through the blood-brain barrier (BBB) to get into the CNS. Some therapeutic agents do not get into the CNS; they regulate certain activities, such as immune activities, in the peripheral, which then modulates the diseased conditions in the CNS. The BBB is formed by the endothelial cells lining the capillaries of blood vessels in the CNS. Unlike the capillaries in the peripheral tissues and organs, the capillary endothelial cells in the BBB are connected by tight junctions formed by occludin, claudins, and junctional adhesion molecules.
At least six antibodies, namely, aducanumab, bapinerumab, crenezumab, gantenerumab, ponezumab, and solanezumab, specific for β-amyloid, which is responsible for causing Alzheimer's disease, have been developed and placed in clinical development. These antibodies generally fall short of satisfactory therapeutic efficacy in improving Alzheimer's disease. A general belief is that if those antibodies are to achieve therapeutic efficacy, a significant portion must get across the BBB to enter the injured sites in the CNS. However, only very minute portions of those antibodies get across the BBB.
Interferon-β-1a (IFN-β-1a) and interferon-β-1b (IFN-β-1b) have been used for the treatment of multiple sclerosis (MS). The pharmaceuticals, IFN-β-1a produced by mammalian cells and IFN-β-1b produced in E. coli, are one-chain protein of 166 amino acid residues containing one disulfide bond. It has been claimed that those therapeutic agents reduce relapse of MS in 18-38% of treated patients. The mechanisms of action of IFN-β-1a and IFN-β-1b are very complex and not completely understood, involving the increased generation of anti-inflammatory immune cells and factors and the down-regulation of pro-inflammatory cells and factors. IFN-β treatment in MS patients also reduces the trafficking of pro-inflammatory T cells across the BBB. It is yet unanswered whether IFN-β-1a and IFN-β-1b mediate their pharmacologic effects in part by getting into the injured sites in the CNS.
When an antibody or protein therapeutic is administered in the body's peripheral, only a very minute amount (about 0.1%) reaches to the CNS, because only a very minute portion of the protein therapeutic gets across the BBB. However, it has also been found that in many diseases of the CNS, including Alzheimer's disease and multiple sclerosis, the inflammation at the diseased sites renders the BBB to breakdown, leading to increased permeability. Therefore, we rationalize that if a larger proportion of an administered antibody specific for β-amyloid or IFN-β-1a and IFN-β-1b is channeled to the BBB, a higher percentage of the therapeutic agents can pass through the BBB and better therapeutic effects can be achieved.
Furthermore, some therapeutic agents have been developed to inhibit the entry of inflammatory immunocytes to across the BBB. A notable example is natalizumab specific for the cell adhesion molecule integrin α4. The antibody functions by inhibiting inflammatory immune cells to attach to and pass through the epithelial layer lining the BBB. While natalizumab has been shown to be therapeutic efficacious, it has serious immunosuppressive side effect. In particular, it causes progressive multifocal leukoencephalopathy, an opportunistic infection caused by John Cunningham virus (JC virus). We therefor rationalize that if a larger proportion of an antibody specific for integrin α4 is recruited to the BBB, a smaller dose will be required, better therapeutic effects can be achieved, and fewer side effects will occur.
The endothelial cells in the capillaries forming the BBB express transferrin receptors and insulin receptors, which mediate the transcytosis of transferrin and insulin molecules, respectively, to the cerebral parenchyma. For using the transferrin receptor as a ferry, only a small proportion gets through while the reaming bulk are trapped or degraded. Because the endothelial cells lining the capillaries in other parts of the vasculature do not express transferrin receptors, the transferrin receptors on the endothelial cells in the BBB can serve as site-specific antigen for recruiting administered therapeutics. Once the therapeutic is concentrated in the BBB, an increased proportion of it will pass through the capillaries.
We also rationalize that when the mechanisms for channeling pharmaceuticals to the BBB is established, anti-inflammatory drugs, such as anti-TNF-α, anti-IL12/IL-23, anti-IL17, and anti-CD3, should be investigated for their therapeutic effects on many types of diseases of the CNS.
For the antibody therapeutic specific for integrin α4, the transferrin receptor is used as a target site recruiter. For Alzheimer disease, the effector moiety can be a few copies of scFv specific for β-amyloid; for treating multiple sclerosis, the effector moiety can be a few copies of IFN-β-1a or IFN-β-1b, or a few copies of scFv specific for integrin α4.
Embodiments of the present disclosure disclose several T-E molecules respectively exist in single multi-arm linker-units or joint-linker configurations, each contains scFv specific for transferrin receptor as the targeting element and IFN-β-1a or IFN-β-1b or scFv specific for integrin α-4 as the effector element. Alternative embodiments disclose T-E molecules respectively exist in single linker-units or joint-linker configurations, each contains scFv specific for transferrin receptor as the targeting element and scFv specific for β-amyloid as the effector element.
Fingolimod is an immunosuppressive drug that is derived from a natural product myriocin originally isolated from certain fungi. Fingolimod has been approved for reducing the relapse of relapsing-remitting multiple sclerosis. Fingolimod is phosphorylated in vivo to form fingolimod-phosphate, which resembles naturally occurring sphingosine-1-phosphate (S1P), an extracellular lipid mediator, and can bind to 4 of the 5 S1P receptors. The S1P receptors are expressed on lymphocytes and involved in lymphocyte migration. A generally pharmacologic mechanism of fingolimod is that it inhibits lymphocytes egress from the lymphoid tissues to the circulation and hence to the CNS. Fingolimod can cross BBB to enter CNS and many cell types in the CNS express S1P receptors, which play roles in cell proliferation, morphology, and migration. It is believed that fingolimod can have direct on the CNS. The administration of fingolimod causes common side effects of headache and fatigue, and severe side effects of skin cancer, macular edema, and fatal infections, such as hemorrhaging focal encephalitis.
A fingolimod molecule has an NH2 group and thus provides a functional group to couple with a bi-functional linker with an NHS group. One preferred embodiment of the present invention is to prepare a T-E construct, which contains a targeting element for delivery to the BBB and a drug bundle of fingolimod as an effector element. For a bundle of fingolimod, 5-10 molecules are incorporated to a linker unit, using either a cleavable linker or non-cleavable linker to conjugate fingolimod molecules to the linking arms of a linker unit. Since fingolimod, after uptake in a patient, is modified to fingolimod phosphate to resemble sphingosine1-phosphate and become active, the drug bundle is alternatively prepared with fingolimod phosphate. A linker unit with fingolimod or fingolimod phosphate bundle is conjugated with 1 or 2 scFv specific for a transferrin receptor I. Upon administration of the molecular construct, a portion of it is carried to the BBB. The fingolimod molecules released from the cleavable linkers pass through the BBB and enter the CNS. Or, a portion of the entire construct enters the CNS. Cleavable linkers can be designed by employing a number of cleaving mechanisms. An installment of S—S bond is often used, since S—S disulfide bond can be cleaved by a reduction reaction at the target tissue site. A peptide bond between amino acids, which is sensitive to proteases, such as matrix metalloproteinases in many tissues and cathepsins in endosomes in target cells, is also commonly used as a cleavable bond in many linker designs.
II Infectious Disease
Although large numbers of monoclonal antibodies have been made against components of a various viruses, bacteria, and fungi, which cause serious infectious in humans and animals, few monoclonal antibodies have been developed into preventive treatments or therapeutic agents to counter infections. These shortcomings can be attributed to a few major factors. One major factor is the infectious microorganisms and their products have different serotypes and variable reactivity toward a particular antibody. Another reason is that the targeted microorganisms undergo mutations and escape the targeting of a particular antibody.
The T-E molecular design of the present invention can also be applied for the prevention and treatment of infectious diseases. The plurality of the linking arms can enhance the avidity and specificity of binding to target infectious microorganisms or their products and elicit immune functions to facilitate the clearance of the microorganisms and their products. We reason that the avidity enhancement and the recruitment of immune clearance function can somehow overcome the serotypic difference and mutational problems. Such improvements should increase the efficacy of the candidate antibodies for the prevention and therapy of infectious diseases. Many antibodies, which have failed to meet expectation in clinical trials, may be configured with the present invention and re-investigated.
A preferred set of embodiment of the present invention is to employ joint-linkers configuration with one linker-unit for targeting and one linker-unit for recruiting effector function. An alternative set of preferred embodiment is to employ single linker-units with multiple linking arms for targeting elements and a coupling arm for an effector element. The targeting elements may be one of the two categories: (1) scFv or sdAb specific for a surface component of a microorganism or its product, e.g., envelope protein gp120 of human immunodeficiency virus type 1 (HIV-1), F protein of respiratory syncytia virus (RSV), a surface antigen of Clostridium difficile or Staphylococcus aureus, or endotoxin of Gram-negative bacteria or Shiga-like toxin of Escherichia coli, or (2) the extracellular portions of cell surface receptors of viruses, such as the HIV-1 gp120-binding CD4 domain.
The effector elements are 1 or 2 scFv or sdAb specific for one Fc receptor of IgG, e.g. FcγRIIA (CD32), FcγRIIIB (CD16b), or FcγRI (CD64). Those receptors are expressed on neutrophils, macrophages, and eosinophils and are the key molecules mediating phagocytosis of antibody-bound microorganisms. FcγRIIA and FcγRIIIB bind to IgG with low affinity (Kd in the range of 10−6 to 10−7), and FcγRI binds to IgG1 and IgG3 with high affinity (Kd 10−9). It is advantageous to employ scFv or sdAb specific for FcγRIIA or FcγRIIIB, because they can compete favorably with IgG in binding to the receptors.
The antibodies specific for carbohydrate antigens on bacterial surface are usually weak in binding affinity and are expressed in IgM rather than IgG. An IgM molecule has 10 Fv's (antigen-binding sites). However, an IgM molecule, which has a molecular weight of about 1000 kd, cannot cross capillaries and reach to extravascular space. With the configuration of the present invention, a molecular construct carrying 6 scFv or 10 sdAb will have a molecular weight of about 150 kd.
In employing antibody-based therapeutics for clearing viruses, it is important that the therapeutic does not lead to FcR-mediated enhancement of viral infection. In those cases, the bound viral particles are not phagocytosed and digested. Some viruses, such as Dengue virus can multiply in phagocytes. Thus, if the viral particles gain access to a cell and enter the bound cells without being destroyed, the virus can multiply in the infected cells. Therefore, a set of preferred embodiments of this invention is that the molecular construct contains 2 or more scFv specific for an Fcγ receptor and can bind to multiple Fcγ receptor molecules on phagocyte cell surface, so that the bound viral particles are destined to phagocytosis pathway.
Among the many antibodies specific for viruses, bacteria, or their products, which have been in clinical trials, only antibodies specific for RSV have been approved for clinical uses. Even for antibodies against RSV, they are only approved for prevention, and not for treatment of on-going infection. It is desirable that an anti-RSV antibody can be developed for treating already-infected subjects. The other antibodies are still in clinical development or have failed in clinical trials. With the molecular construct platforms of this invention, all of these antibodies can be employed for improved efficacy. A partial list of those antibodies are:
Fc-Based Molecular Constructs for Treating Central Nervous System Diseases or Infectious Diseases
In the broad sense of the Fc-based configuration, immunoglobulin antibody can serve as the base of a targeting or effector element, and its corresponding effector or targeting element can be incorporated at the C-terminal of its two heavy γ chains in the form of scFv domains. For a typical “Fc-based” configuration, two-chain IgG.Fc is used as the base of the molecular platform. Each of the polypeptide chain is fused with one or two targeting and one or two effector elements, for a total of two to three elements on each chain. The T-E molecule with an Fc-based configuration will have a total of four to six elements (e.g., scFv or any other antibody fragments). Optionally, the Fc portion of the molecular constructs also carries Fc-mediated effector functions, ADCC, and/or complement-mediated activation. While in certain other applications, such Fc-mediated effector functions are avoided.
In designing the Fc-based molecular constructs, targeting elements are positioned at the N- or C-terminus. If the effector elements function by binding to a cell surface component, they should also be positioned at the terminus. If the effector elements function by binding to and neutralizing soluble factors, they can be positioned between a terminal targeting or effector element and CH2-CH3.
By selecting the T-E elements of the present Fc-based molecular construct, the molecular construct can be used to treat central nervous system (CNS) diseases or infectious diseases. The present disclosure is also advantageous in that, in some embodiments, it utilizes the linker unit according to the first aspect of the present disclosure, which provides a facile means for controlling the number of the targeting and effector elements of the present Fc-based molecular constructs. Depending on the targeting and/or effector elements selected, the present Fc-based molecular construct may take different configurations, which are discussed below, respectively.
In the present Fc-based molecular constructs, both the targeting element and effector element are antibodies or fragments thereof.
Referring to
The Fc-based molecular construct 800B illustrated in
According to certain embodiments, both the effector elements and targeting elements are linked to the N-termini of the CH2-CH3 chains. For example, when both the effector element and the targeting element are in the form of single-chain variable fragments (scFvs), the effector element and the targeting element may be linked in a tandem or diabody configuration, thereby forming a bispecific scFv that is linked to the N-terminus of the CH2-CH3 chain.
The Fc-based molecular construct 800C (
In some examples, the pair of effector elements or the pair of the targeting elements takes a Fab configuration (i.e., consisting of the VH-CH1 domain and the VL-Cκ domain); this Fab fragment is linked to the N-termini of the CH2-CH3 chains, so that the Fc-based molecular construct adopts an IgG configuration. In these cases, the pair of elements that is not in the Fab configuration may be linked to the C-termini of the pair of CH2-CH3 segments.
For example, in the Fc-based molecular construct 900 of
According to some embodiments, the present Fc-based molecular construct has an effector element which is a peptide.
For example, according to certain embodiments of the present disclosure, the effector element can be a peptide with certain therapeutic effect, while the targeting element is an antibody or a fragment thereof (see,
Similarly, in the Fc-based molecular construct 1000B of
As could be appreciated, for Fc-base molecular constructs that use a peptide as the effector element, the targeting element can be constructed into a Fab fragment, so that the molecular constructs take the IgG configuration.
In the configuration illustrated in
Functional Elements Suitable for Use with Fc-Based Molecular Construct
Now that the basic structural arrangements of the Fc-based molecular constructs have been discussed above, certain combinations of particular effector element(s) and targeting element(s) are provided below for the illustration purpose.
To treat central nervous system (CNS) diseases, an antibody (or a fragment thereof) specific for transferrin receptor can be used as the targeting element, in connection with effector elements suitable for the particular CNS disease. For example, Fc-based molecular constructs for the treatment of multiple sclerosis may use an scFv specific for integrin-α4 as the effector element. In the case of Alzheimer's disease, illustrative Fc-based molecular constructs can use an scFv specific for β-amyloid as the effector element. The above-mentioned Fc-based molecular constructs for treating CNS diseases may take the configuration described in connection with any of
Fc-based molecular constructs for treating multiple sclerosis may also use INF-β1a or INF-β1b as the effector elements. In this case, the Fc-based molecular constructs may take the configuration described in connection with
In constructing Fc-based molecular constructs for treating diseases/conditions associated with infection (such as viral infections or bacterial infections), one may use an antibody (or a fragment thereof) specific for a viral protein or bacterial protein as the targeting element. As to the effector elements for treating infections, an antibody (or a fragment thereof) specific for CD32 or CD16b can be used. These Fc-based molecular constructs may take the configuration described in connection with any of
The essence of this invention is the rationalization and conception of the specific combination or pairing of the targeting and effector elements. The adoption of Fc-fusion configuration in the molecular constructs is a preferred embodiment. It is conceivable for those skilled in the arts to link the pairs of targeting and effector elements of this invention employing other molecular platforms, such as peptides, proteins (e.g., albumin), polysaccharides, polyethylene glycol, and other types of polymers, which serve as a structural base for attaching multiple molecular elements.
III-(iii) Use of Fc-Based Molecular Construct
The present disclosure also pertains to method for treating CNS diseases using the suitable Fc-based molecular construct. Generally, the method comprises the step of administering to a subject in need of such treatment an effective amount of the Fc-based molecular construct according to embodiments of the present disclosure.
The present disclosure further pertains to method for treating infections using the suitable Fc-based molecular construct. Generally, the method comprises the step of administering to a subject in need of such treatment an effective amount of the Fc-based molecular construct according to embodiments of the present disclosure.
The scFv1-CH2-CH3-scFv2 (human γ1) recombinant chain was configured by fusing two scFvs, in which the first one specific for Protein F of RSV fused to the N-terminal of CH2 domain of IgG1.Fc through a flexible hinge region, while the second one specific for ectodomain of CD32a was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)3.
Both of the scFvs had an orientation of VL-linker-VH. The VL and VH in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the IgG1.Fc fusion protein molecular construct is shown as SEQ ID NO: 1.
Illustrated below is the configuration of the prepared 2-chain (scFv α RSV)-(scFv α CD32a)-hIgG1.Fc molecular construct.
In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. Expi293F cells were seeded at a density of 2.0×106 viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, 7.5×108 cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ 293 transfection reagent. The transfected cells were incubated at 37° C. for 16 to 18 hours post-transfection in an orbital shaker (125 rpm) and the cells were added ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 to the shaker flask, and incubated for 7 days. Culture supernatants were harvested and recombinant 2-chain (scFv α RSV)-hIgG1.Fc-(scFv α CD32a) fusion protein in the media was purified using Protein A chromatography. Following buffer exchange to PBS, the concentration of (scFv α RSV)-hIgG1.Fc-(scFv α CD32a) protein was determined and analyzed by 12% SDS-PAGE shown in
To examine the binding ability of recombinant 2-chain (scFv α RSV)-hIgG1.Fc-(scFv α CD32a) fusion protein to both Protein F of RSV and ectodomain of CD32a, ELISA assay was performed. ELISA plates were coated with 2 μg/mL of Protein F of RSV (Sino biological Inc.). Recombinant 2-chain (scFv α RSV)-hIgG1.Fc-(scFv α CD32a) fusion protein and (scFv α RSV)-hIgG1.Fc were detected by HRP-conjugated goat anti-human IgG1.Fc. The ELISA results in
The scFv1-CH2-CH3-scFv2 (human γ1) recombinant chain was configured by fusing two scFvs, in which the first one specific for endotoxin was fused to the N-terminal of CH2 domain of IgG1.Fc through a flexible hinge region, and the second one specific for ectodomain was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)3.
Both of the scFvs had an orientation of VL-linker-VH. The VL and VH in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG.
The sequence of the recombinant chain in the IgG1.Fc fusion protein molecular construct is shown as SEQ ID NO: 2. The expression of the constructed gene in Expi293F cells and the purification of the expressed fusion protein were performed as in preceding Examples. Characterization of the new construct was performed with SDS-PAGE and ELISA. The SDA-PAGE results in
Illustrated below is the configuration of the thus-prepared 2-chain (scFv α endotoxin)-(scFv α CD32)-hIgG1.Fc molecular construct.
The 2-chain IgG.Fc fusion protein was prepared by configuring (interferon-β-1a)-CH2-CH3-(scFv α TfR1) (human γ4) in a recombinant chain. The C-terminal of the interferon-β-1a was fused to the N-terminal of CH2 via a linker, GGGGSGGGASGGS. The scFv specific for ectodomain of TfR1 was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)3.
The scFv (specific for ectodomain of TfR1) had an orientation of VL-linker-VH. The VL and VH in the scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 3.
Illustrated herein is the configuration of the prepared 2-chain (interferon-β-1a)-IgG4.Fc-(scFv α TfR1) molecular construct.
In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. Expi293F cells were seeded at a density of 2.0×106 viable cells/ml in Expi293F expression medium and maintained for 18 to 24 hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, 7.5×108 cells in 255-ml medium in a 2-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ 293 transfection reagent. The transfected cells were incubated at 37° C. for 16 to 18 hours post-transfection in an orbital shaker (125 rpm) and the cells were added ExpiFectamine™ 293 transfection enhancer 1 and enhancer 2 to the shaker flask, and incubated for 7 days. Culture supernatants were harvested and recombinant 2-chain (Interferon-β-1a)-hIgG1.Fc-(scFv α TfR1) fusion protein in the media was purified using Protein A chromatography. Following buffer exchange to PBS, the concentration of (Interferon-β-1a)-hIgG1.Fc-(scFv α TfR1) protein was determined and analyzed by 8% SDS-PAGE shown in
Binding activity of recombinant (Interferon-β-1a)-hIgG1.Fc-(scFv α TfR1) was assayed by ELISA using a 96-well plate coated with recombinant (Interferon-β-1a)-hIgG1.Fc-(scFv α TfR1) protein in 5μg/ml concentration, 100 μl per well. The scFv specific for ectodomain of TfR1 is as a negative control. Recombinant 2-chain (Interferon-β-1a)-hIgG1.Fc-(scFv α TfR1) was detected by HRP-conjugated rabbit anti-human interferon-β polyclonal antibody (Santa Cruz Biotechnology, Dallas, USA). Next, 50 μl of TMB substrate was added for color development. The reaction was stopped by 50 μl of 1M HCl. Absorbance at 450 nm was measured with a plate reader, Each bar represents the mean OD450 value of duplicate samples.
The VL and VH of the scFv specific for integrin α4 were from monoclonal antibody natalizumab. The 2-chain IgG.Fc fusion protein was prepared by configuring (scFv α integrin α4)-CH2-CH3-(scFv α TfR1) (human γ4) in a recombinant chain. The C-terminal of the scFv specific for integrin α4 was fused to the N-terminal of CH2 via a linker, GGGGSGGGASGGS. The scFv specific for ectodomain of TfR1 was fused to the C-terminal of CH3 domain through a flexible linker, (GGGGS)3. The result of 8% SDA-PAGE in
The two scFv had the orientation of VL-linker-VH. The VL and VH in each of the two scFv were connected by a hydrophilic linker, GSTSGSGKPGSGEGSTKG. The sequence of the recombinant chain in the IgG4.Fc fusion protein molecular construct is shown as SEQ ID NO: 4.
Illustrated herein is the configuration of the prepared 2-chain (scFv α integrin α4)-IgG4.Fc-(scFv α TfR1) molecular construct.
To examine the binding ability of recombinant 2-chain (scFv α integrin α4)-IgG4.Fc-(scFv α TfR1) protein to integrin α4-expressing Jurkat T cells, cell-binding assay was performed by flow cytometry.
1×106 Jurkat T cells was maintained in the RPMI1640 medium supplemented with 10% FBS at a density of 1×105. The cells were kept in 37° C. with 5% CO2 in a humidified chamber. 1×106 Jurkat T cells were washed with the binding buffer (phosphate-buffered saline with 0.1% FBS, 2 mM EDTA and 20 ng/ml NaN3) twice. 10 μg/ml of Human BD Fc Block™ (BD Biosciences, San Jose, US) was added to the washed Jurkat T cells to block Fc receptor mediated. Cells were washed and incubated with 10 μg/ml of recombinant (scFv α integrin α4)-IgG4.Fc-(scFv α TfR1) protein on ice for 15 minutes, using recombinant 2-chain (interferon-β-1a)-IgG4.Fc-(scFv α TfR1) as a negative control. Cells were washed again and incubated with FITC-conjugated goat anti-human IgG.Fc (Caltag, Buckingham, UK), diluted 1:200 in blocking buffer, at on ice for 15 min in the dark. The stained cells were analyzed on a FACSCanto II flow cytometer (BD Biosciences).
To test the effects of recombinant 2-chain (scFv α endotoxin)-hIgG1.Fc-(scFv α CD32a) fusion protein on inhibiting TNF-α secretion, ELISA was to determine the amount of secreted TNF-α in the supernatant by macrophage-like U937 cells.
U937 cells were maintained in RPMI1640 supplemented with 10% fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin (Gibco), at the density between 3×105 and 2×106 cells/ml. The cells were kept in 37° C. with 5% CO2 in a humidified chamber. To differentiate U937 into macrophage-like cells, 1×106 cells/ml of U937 were incubated with 10 ng/ml of phorbol 12-myristate β-acetate (PMA, Sigma Aldrich). After 48 hours, non-adherent cells were removed, and adherent cells were washed and seeded into 96-well plates.
5×104 cells/well of differentiated 0937 were seeded into 96-well plates the day before assay. Cells were stimulated with 1 μg/ml E. coli LPS 0111:B4 (Sigma Aldrich) alone, or premixes of LPS and 10 μg/ml of (scFv α endotoxin)-hIgG1Fc, 15 μg/ml of (scFv α endotoxin)-hIgG1 Fc-(scFv α CD32a) or 2.5 μg/ml of anti-CD32a scFv. The stimulation proceeded for 2 hours before the supernatant was collected. TNF-α production was measured by commercially available ELISA kit (Biolegend).
TNF-α levels in U937 supernatant were measured using an ELISA kit from R&D Systems. The wells of ELISA plates (Greiner Bio-One) were coated with 4 μg/mL of capture antibody in PBS at 4° C. overnight. Wells were subsequently blocked by 0.5% in PBS for 1 hour and incubated with diluted culture supernatant for 2 hours. 400 ng/mL of biotin-labeled detection antibody was used followed by Streptavidin-HRP to detect bound TNF-α. Chromogenic reaction was carried out using TMB substrate (Clinical Science Products), and stopped by adding 1N HCl. Plates were read at 450 nm absorbance. Concentrations of TNF-α were determined by extrapolation from four-parameter logistic fit standard curves generated from dilutions of standard protein supplied by the manufacturer.
It will be understood that the above description of embodiments is given by way of example only and that various modifications may be made by those with ordinary skill in the art. The above specification, examples and data provide a complete description of the structure and use of exemplary embodiments of the invention. Although various embodiments of the invention have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those with ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of this invention.
| Number | Date | Country | |
|---|---|---|---|
| 62308349 | Mar 2016 | US | |
| 62213012 | Sep 2015 | US | |
| 62164400 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14997764 | Jan 2016 | US |
| Child | 15159862 | US | |
| Parent | 14997827 | Jan 2016 | US |
| Child | 14997764 | US | |
| Parent | 14997849 | Jan 2016 | US |
| Child | 14997827 | US | |
| Parent | 14997874 | Jan 2016 | US |
| Child | 14997849 | US |
