Patent Description:
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 <NUM>,<NUM> antibodies have been studied or are being planned for studies in human clinical trials, and approximately <NUM> 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). For example, document <CIT> discloses a human amylin derivative or analog comprising one or more lysine residues for linking an element (i.e., albumin binding residue or PEG) or a linker via forming an amide bond between the lysine residue and the element/linker. In the publication "<NPL>) from Chaudhary V K et al. an antibody OVB3 is disclosed, in which the light chain and heavy chain sequences are linked by a peptide linker. Document <CIT> teaches a polypeptide having a plurality of lysine residues for linking cancer-associated peptides. Document <CIT> discloses a salmon calcitonin peptide (sCT) comprising three K residues for linking bone targeting moiety (BP) via linker NHS-PEG-MAL. Document <CIT> teaches a fusion polypeptide comprising a Relaxin A chain polypeptide, a Relaxin B chain polypeptide, and a linker polypeptide linking the Relaxin A and B chain polypeptides. Document <CIT> discloses a multifunctional antibody conjugate (MAC) comprising an antibody, an effector moiety (e.g., an Ang2-binding peptide), and a linker of formula X-Y-Z linking the antibody and the effector moiety; and document <CIT> teaches a bioconjugate comprising a peptide or polypeptide and a half-life extending moiety linked or fused to the peptide or polypeptide via a linker. 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.

The invention is defined by a linker unit comprising, a center core and a plurality of linking arms, wherein the center core comprises a first polypeptide comprising a plurality of lysine (K) residues, wherein each K residue and its next K residue are separated by a filler sequence comprising glycine (G) and serine (S) residues, and the number of K residues ranges from <NUM> to <NUM>; each of the plurality of linking arms has an N-hydroxysuccinimidyl (NHS) group at one end and a functional group at the other end and is linked to one of the K residues of the center core by forming an amide bond between the amine group of the K residue and the NHS group, wherein each of the linking arms is a PEG chain having <NUM>-<NUM> repeats of EG units and the functional group is a NHS; and the amino acid residue at the N- or C-terminus of the center core has the azide or the alkyne group. In the first aspect, the present disclosure is directed to a linker unit that has at least two different functional elements linked thereto. For example, the linker unit may have linked thereto two different effector elements, one targeting element and one effector element, or one effector element and a polyethylene glycol (PEG) chain for prolonging the circulation time of the linker unit. The present linker unit is designed to have at least two different functional groups such that the functional elements can be linked thereto by reacting with the respective functional groups. Accordingly, the present linker unit can serve as a platform for preparing a molecular construct with two or more functional elements.

According to various embodiments of the present disclosure, the linker unit comprises a center core and a plurality of linking arms. The center core is a polypeptide core comprising a plurality of lysine (K) resides, in which each K residue and a next K residue are separated by a filler sequence comprising glycine (G) and serine (S) residues, and the number of K residues ranges from <NUM> to <NUM>. Optionally, the filler sequence consists of <NUM> to <NUM> amino acid residues. Each of the linking arms is linked to the K residues of the center core via forming an amide linkage between the K residue and the linking arm. The linking arm linked to the center core has a N-hydroxysuccinimidyl (NHS) group at its free-terminus. Also, the amino acid residue at the N- or C-terminus of the center core has an azide group or an alkyne group.

In some embodiments, the linking arm is a PEG chain, preferably having <NUM> to <NUM> repeats of EG units. Alternatively, the linking arm is a PEG chain having <NUM> to <NUM> repeats of EG units with a disulfide linkage at the free terminus thereof (i.e., the terminus that is not linked with the K residue of the center core). In some embodiments, the coupling linking arm is a PEG chain, preferably having <NUM> to <NUM> repeats of EG units.

Regarding amino acid residues having the azide group, non-limiting examples of said amino acid residues include L-azidohomoalanine (AHA), <NUM>-azido-L-phenylalanine, <NUM>-azido-D-phenylalanine, <NUM>-azido-L-alanine, <NUM>-azido-D-alanine, <NUM>-azido-L-homoalanine, <NUM>-azido-D-homoalanine, <NUM>-azido-L-ornithine, <NUM>-azido-d-ornithine, <NUM>-azido-L-lysine, and <NUM>-azido-D-lysine. As to the amino acid residues having the alkyne group, illustrative examples thereof include L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), and beta-homopropargylglycine (β-HPG).

According to various embodiments of the present disclosure, the linker unit further comprises a plurality of first elements. In some embodiments, each of the first elements is linked to one of the linking arms via forming an amide bound between the linking arm and the first element.

According to various optional embodiments of the present disclosure, the first element is an effector element suitable for eliciting an intended effect (e.g., a therapeutic effect) in a subject. Alternatively, the first element may be a targeting element for directing the linker unit to the site of interest. According to the embodiments of the present disclosure, the first element is fingolimod, fingolimod phosphate, interferon-β, or a single-chain variable fragment (scFv) specific for integrin-α4, β-amyloid, a viral protein, a bacterial protein.

Still optionally, the linker unit further comprises a second element that is different from the first elements. In some embodiments, the second element has an azide or alkyne group, so that it is linked to the center core by coupling with the corresponding alkyne or azide group of the center core via CuAAC reaction. Alternatively, in some embodiments, the second element having a cyclooctyne group is linked to the center core by coupling with the corresponding azide group of the center core or the coupling arm via SPAAC reaction.

In optional embodiments of the present disclosure, when the first element is an effector element, then the second element may be another effector element, which works additively or synergistically with or independently of the first element; alternatively, the second element may be a targeting element or an element for improving the pharmacokinetic property of the linker unit, such as solubility, clearance, half-life, and bioavailability. In some other optional embodiments, when the first element is the targeting element, then the second element is preferably an effector element or an element for improving the pharmacokinetic property of the linker unit.

According to some embodiments of the present disclosure, the first element of the present linker unit is fingolimod, fingolimod phosphate, interferon-β, or an scFv specific for integrin-α4 or β-amyloid, and the second element of the linker unit is an scFv specific for transferrin receptor.

According to other embodiments of the present disclosure, the linker units comprise an scFv specific for a viral protein or a bacterial protein as the first element, and an scFv specific for CD32 or CD16b as the second element. In one preferred embodiment, the viral protein is F protein of respiratory syncytia virus (RSV), gp120 protein of human deficiency virus type <NUM> (HIV-<NUM>), hemagglutinin A (HA) protein of influenza A virus, or glycoprotein of cytomegalovirus; and the bacterial protein is endotoxin of Gram(-) bacteria, surface antigen of Clostridium difficile, lipoteichoic acid of Staphylococcus aureus, anthrax toxin of Bacillus anthracis, or Shiga-like toxin type I or II of Escherichia coli.

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.

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 <NUM>%, <NUM>%, <NUM>%, or <NUM>% 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 <NUM> to about <NUM> residues; preferably, <NUM> to <NUM> 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 <NUM>%, preferably at least <NUM>% or <NUM>%, and more preferably at least <NUM>% or <NUM>% 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-<NUM>, 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: <MAT> 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 NH<NUM>-(CH<NUM>CH<NUM>O)n-COOH. In the present disclosure, the value of n ranges from <NUM> to <NUM>; preferably, ranging from <NUM> to <NUM>.

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')<NUM> 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 <NUM> kDa) and one "heavy" chain (about <NUM>-<NUM> kDa). The N-terminus of each chain defines a variable region of about <NUM> to <NUM> 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 <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> M, <NUM>×<NUM>-<NUM> 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 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.

The first aspect of the present disclosure pertains to a linker unit that comprises, (<NUM>) a center core that comprises <NUM>-<NUM> lysine (K) residues, and (<NUM>) <NUM>-<NUM> linking arms respectively linked to the K residues of the center core. The present center core is characterized in having or being linked with an azide group, or an alkyne group at its N- or C-terminus.

In the preparation of the present linker unit, a PEG chain having a N-hydroxysuccinimidyl (NHS) group at one terminus and a functional group (i.e., an NHS group) at the other terminus is linked to the K residue of the center core by forming an amide bond between the NHS group of the PEG chain and the amine group of the K residue. In the present disclosure, the PEG chain linked to the K residue is referred to as a linking arm, which has a functional group at the free-terminus thereof.

According to the embodiments of the present disclosure, the center core is a polypeptide that has <NUM>-<NUM> amino acid residues in length and comprises <NUM> to <NUM> lysine (K) residues, in which each K residue and the next K residue are separated by a filler sequence.

According to embodiments of the present disclosure, the filler sequence comprises glycine (G) and serine (S) residues; preferably, the filler sequence consists of <NUM>-<NUM> residues selected from G, S, and a combination thereof. For example, the filler sequence can be,.

The filler sequence placed between two lysine residues may be variations of glycine and serine residues in somewhat random sequences and/or lengths. Longer fillers may be used for a polypeptide with fewer lysine residues, and shorter fillers for a polypeptide with more lysine residues. Hydrophilic amino acid residues, such as aspartic acid and histidine, may be inserted into the filler sequences together with glycine and serine. As alternatives for filler sequences made up with glycine and serine residues, filler sequences may also be adopted from flexible, soluble loops in common human serum proteins, such as albumin and immunoglobulins.

According to certain preferred embodiments of the present disclosure, the center core comprises <NUM>-<NUM> units of the sequence of G<NUM>-<NUM>SK. Alternatively, the polypeptide comprises the sequence of (GSK)<NUM>-<NUM>; that is, the polypeptide comprises at least two consecutive units of the sequence of GSK. For example, the present center core may comprises the amino acid sequence of the following,.

in which Ac represents the acetyl group.

According to some embodiments of the present disclosure, the present center core comprises, at its N- or C-terminus, an amino acid residue having an azide group or an alkyne group. The amino acid residue having an azide group can be, L-azidohomoalanine (AHA), <NUM>-azido-L-phenylalanine, <NUM>-azido-D-phenylalanine, <NUM>-azido-L-alanine, <NUM>-azido-D-alanine, <NUM>-azido-L-homoalanine, <NUM>-azido-D-homoalanine, <NUM>-azido-L-ornithine, <NUM>-azido-d-ornithine, <NUM>-azido-L-lysine, or <NUM>-azido-D-lysine. For example, the present center core may have the sequence of,.

in which Ac represents the acetyl group, and AAH represents the AHA residue.

Exemplary amino acid having an alkyne group includes, but is not limited to, L-homopropargylglycine (L-HPG), D-homopropargylglycine (D-HPG), or beta-homopropargylglycine (β-HPG). In this case, the present center core may have the sequence of,.

in which Ac represents the acetyl group, and GHP represents the HPG residue.

It is noted that many of the amino acids containing an azide or alkyne group in their side chains and PEGylated amino acids are available commercially in t-boc (tert-butyloxycarbonyl)- or Fmoc (<NUM>-fluorenylmethyloxycarbonyl)-protected forms, which are readily applicable in solid-phase peptide synthesis.

According to some working examples of the present disclosure, the center core may comprise the sequence of,.

in which Xaa is a PEGylated amino acid having specified repeats of EG unit, Ac represents the acetyl group, AAH represents the AHA residue, and GHP represents the HPG residue; SEQ ID NOs: <NUM> and <NUM> are not within the scope of protection of the present granted patent.

The polypeptide may also be synthesized using recombinant technology by expressing designed gene segments in bacterial or mammalian host cells. It is preferable to prepare the polypeptide as recombinant proteins if the core has high numbers of lysine residues with considerable lengths. As the length of a polypeptide increases, the number of errors increases, while the purity and/or the yield of the product decrease, if solid-phase synthesis was adopted. To produce a polypeptide in bacterial or mammalian host cells, a filler sequence ranges from a few amino acid residues to <NUM>-<NUM> residues may be placed between two K residues. Further, since AHA and HPG are not natural amino acids encoded by the genetic codes, the N-terminal or C-terminal residue for those recombinant polypeptides is cysteine. After the recombinant proteins are expressed and purified, the terminal cysteine residue is then reacted with short bifunctional cross-linkers, which have maleimide group at one end, which reacts with SH group of cysteine residue, and alkyne, azide, tetrazine, or strained alkyne at the other end.

The synthesis of a polypeptide using PEGylated amino acids involves fewer steps than that with regular amino acids such as glycine and serine resides. In addition, PEGylated amino acids with varying lengths (i.e., numbers of repeated ethylene glycol units) may be employed, offering flexibility for solubility and spacing between adjacent amino groups of lysine residues. In addition to PEGylated amino acids, the center cores may also be constructed to comprise artificial amino acids, such as D-form amino acids, homo-amino acids, N-methyl amino acids, etc. Preferably, the PEGylated amino acids with varying lengths of polyethylene glycol (PEG) are used to construct the center core, because the PEG moieties contained in the amino acid molecules provide conformational flexibility and adequate spacing between conjugating groups, enhance aqueous solubility, and are generally weakly immunogenic. The synthesis of PEGylated amino acid-containing center core is similar to the procedures for the synthesis of regular polypeptides.

Optionally, for stability purpose, the present center core has an acetyl group to block the amino group at its N-terminus.

As could be appreciated, the number of the linking arms linked to the center core is mainly determined by the number of lysine resides comprised in the center core. Since there are at least two lysine residues comprised in the present center core, the present linker unit may comprise a plurality of linking arms.

Reference is now made to <FIG>. As illustrated, the linker unit 10A comprises a center core 11a comprising one HPG (GHP) residue and four lysine (K) residues respectively separated by filler sequences (denoted by the dots throughout the drawings). The filler sequences between the HPG residue and K residue or between any two K residues may comprise the same or different amino acid sequences. In this example, four linking arms 20a-20d are linked to the lysine residues by forming an amide linkage between the NHS group and the amine group of the lysine residue, respectively. As could be appreciated, certain features discussed above regarding the linker unit 10A or any other following linker units are common to other linker units disclosed herein, and hence some or all of these features are also applicable in the following examples, unless it is contradictory to the context of a specific embodiment. However, for the sake of brevity, these common features may not be explicitly repeated below.

When the release of effector elements at the targeted site is required, a cleavable bond can be installed in the linking arm. Such a bond is cleaved by acid/alkaline hydrolysis, reduction/oxidation, or enzymes. One embodiment of a class of cleavable PEG chains that can be used to form the coupling arm is NHS-PEG<NUM>-<NUM>-S-S-maleimide, where S-S is a disulfide bond that can be slowly reduced, while the NHS group is used for conjugating with the amine group of the center core, thereby linking the PEG chain onto the center core. The maleimide group at the free terminus of the linking arm may be substituted by an azide, alkyne, tetrazine, or strained alkyne group. According to some embodiments of the present disclosure, the linking arm is a PEG chain, which has <NUM>-<NUM> repeats of EG units with a disulfide linkage at the free terminus thereof (i.e., the terminus that is not linked with the center core).

According to the embodiments of the present disclosure, the linking arm linked to the K residue of the center core has a functional group (i.e., an NHS group) at its free terminus.

Depending on the functional group (i.e., an NHS group) present at the free terminus of the linking arm, it is feasible to design a functional element (such as, a targeting element, an effector element, or an element for improving the pharmacokinetic property) with a corresponding functional group (i.e., an amine group), so that the functional element may linked to the free terminus of the linking arm via
forming an amide bond therebetween.

For the sake of illustration, the functional elements linked to the linking arms are referred to as the first elements. As could be appreciated, the number of the first elements carried by the present linker unit depends on the number of K residues of the center core (and thus, the number of the linking arms). Accordingly, one of ordinary skill in the art may adjust the number of the first elements of the linker unit as necessary, for example, to achieve the desired targeting or therapeutic effect.

According to some preferred embodiments of the present disclosure, the first elements is fingolimod, fingolimod phosphate, interferon-β, or a single-chain variable fragment (scFv) specific for integrin-α4, β-amyloid, a viral protein, a bacterial protein.

Non-limiting viral protein includes F protein of respiratory syncytia virus (RSV), gp120 protein of human immunodeficiency virus type <NUM> (HIV-<NUM>), hemagglutinin A (HA) protein of influenza A virus, and glycoprotein of cytomegalovirus.

Example of the bacterial protein includes, but is not limited to, the endotoxin of Gram(-) bacteria, the surface antigen of Clostridium difficile, the lipoteichoic acid of Saphylococcus aureus, the anthrax toxin of Bacillus anthracis, or the Shiga-like toxin type I or II of Escherichia coli.

In order to increase the intended or desired effect (e.g., the therapeutic effect), the present linker unit may further comprise a second element in addition to the first element. For example, the second element can be either a targeting element or an effector element. In optional embodiments of the present disclosure, the first element is an effector element, while the second element may be another effector element, which works additively or synergistically with or independently of the first element. Still optionally, the first and second elements exhibit different properties; for example, the first element is a targeting element, and the second element is an effector element, and vice versa. Alternatively, the first element is an effector element, and the second element is an element capable of improving the pharmacokinetic property of the linker unit, such as solubility, clearance, half-life, and bioavailability. The choice of a particular first element and/or second element depends on the intended application in which the present linker unit (or multi-arm linker) is to be used. Examples of these functional elements are discussed below in Part I-(iii) of this specification.

Structurally, the second element is linked to the azide, or alkyne group at the N- or C-terminus of the center core. Specifically, the second element may be optionally conjugated with a short PEG chain (preferably having <NUM>-<NUM> repeats of EG units) and then linked to the N- or C-terminal amino acid residue having an azide group or an alkyne group (e.g., AHA residue or HPG residue).

According to some embodiments of the present disclosure, the center core comprises an amino acid having an azide group (e.g., the AHA residue) at its N- or C-terminus; and accordingly, a second element having an alkyne group is linked to the N- or C-terminus of the center core via the CuAAC reaction. According to other embodiments of the present disclosure, the center core comprises an amino acid having an alkyne group (e.g., the HPG residue) at its N- or C-terminus; and a second element having an azide group is thus capable of being linked to the N- or C-terminus of the center core via the CuAAC reaction.

<FIG> provides an example of the present linker unit <NUM> carrying a plurality of first elements and one second element. In this example, the center core 11c comprises one HPG (GHP) residue and five lysine (K) residues. Five linking arms 20a-20e are respectively linked to the five K residues of the center core 11c; and five first elements 30a-30e are respectively linked to said five linking arms 20a-20e via the thiol-maleimide reaction. In addition to the first elements, the linker unit <NUM> further comprises one second element <NUM> that is linked to one end of a short PEG chain <NUM>. Before being conjugated with the center core 11c, the other end of the short PEG chain <NUM> has an azide group. In this way, the azide group may reacted with the HPG residue that having an alkyne group via CuAAC reaction, so that the second element <NUM> is linked to the center core 11c. The solid dot <NUM> depicted in <FIG> represents the chemical bond resulted from the CuAAC reaction occurred between the HPG residue and the azide group.

Scheme <NUM> is an exemplary illustration of the process of preparing the present linker unit. In step <NUM>, the center core comprising the amino acid sequence of (GSK)<NUM> and a L-azidohomoalanine (AHA) residue at the C-terminus thereof is prepared. In step <NUM>, three linking arms are respectively linked to the lysine (K) residues of the center core via forming an amide bond between the NHS group and the amine group; the linking arm linked to the center core has a maleimide (Mal) group at the free-terminus thereof. In step <NUM>, three anti-A antigen scFvs (scFv α A) as the first element are respectively linked to the linking arms via the thiol-maleimide reaction. Meanwhile, in step <NUM>, one anti-B antigen scFv (scFv α B) as the second element is linked with a short PEG chain that has <NUM> repeats of EG units and a DBCO group at the free terminus. Finally, in step <NUM>, the second element is linked to the AHA residue of the center core via the SPAAC reaction.

<FIG> provides an alternative example of the present linker unit (linker unit 10J), in which five first elements <NUM> are respectively linked to the lysine residues via the linking arms <NUM>, and the HPG (GHP) residue of the center core 11e is linked with a PEG chain <NUM> via the CuAAC reaction. The solid dot <NUM> depicted in <FIG> represents the chemical bond resulted from the CuAAC reaction occurred between the HPG residue and the PEG chain <NUM>.

In the case where the linker unit (or multi-arm linker) comprises only the first element but not the second and/or third element(s), the first element is an effector element that may elicit a therapeutic effect in a subject. On the other hand, when the present linker unit comprises elements in addition to first element(s), then at least one of the elements is an effector element, while the other may be another effector element, a targeting element, or an element capable of enhancing one or more pharmacokinetic properties of the linker unit (e.g., solubility, clearance, half-life, and bioavailability). For example, the linker unit may have two different kinds of effector element, one effector element and one targeting element or one pharmacokinetic property-enhancing element, two different kinds of targeting elements and one kind of effector element, two different kinds of effector elements and one kind of targeting element, or one kind of targeting element, one kind of effector element and one element capable of improving the pharmacokinetic property of the linker unit.

According to certain embodiments of the present disclosure, the targeting element or the effector element is fingolimod, fingolimod phosphate, interferon-β, or a single-chain variable fragment (scFv) specific for integrin-α4, β-amyloid, a viral protein, or a bacterial protein.

Examples of viral proteins include, but are not limited to, F protein of respiratory syncytia virus (RSV), gp120 protein of human immunodeficiency virus type <NUM> (HIV-<NUM>), hemagglutinin A (HA) protein of influenza A virus, and glycoprotein of cytomegalovirus.

Illustrative examples of bacterial protein include endotoxin of Gram(-) bacteria, surface antigen of Clostridium difficile, lipoteichoic acid of Saphylococcus aureus, anthrax toxin of Bacillus anthracis, and Shiga-like toxin type I or II of Escherichia coli.

Elements that enhance one or more pharmacokinetic properties of the linker unit can be a long PEG chain having a molecular weight of about <NUM>,<NUM> to <NUM>,<NUM> daltons.

Compared with previously known therapeutic constructs, the present linker unit is advantageous in two points:.

In certain therapeutic applications, it is desirable to have a single copy of a targeting or effector element. For example, a single copy of a targeting element can be used to avoid unwanted effects due to overly tight binding. This consideration is relevant, when the scFv has a relatively high affinity for the targeted antigen and when the targeted antigen is a cell surface antigen on normal cells, which are not targeted diseased cells. As an example, in using scFv specific for CD3 or CD16a to recruit T cells or NK cells to kill targeted cells, such as thyroid gland cells in patients with Graves' disease, a single copy of the scFv specific for CD3 or CD16a is desirable, so that unwanted effects due to cross-linking of the CD3 or CD16a may be avoided. Similarly, in using scFv specific for CD32 or CD16b to recruit phagocytic neutrophils and macrophages to clear antibody-bound viral or bacterial particles or their products, a single copy of scFv may be desirable. Also, in using scFv specific for transferrin receptor to carry effector drug molecules to the BBB for treating CNS diseases, a single copy of scFv specific for transferrin receptor is desirable. In still another example, it is desirable to have only one copy of long-chain PEG for enhancing pharmacokinetic properties. Two or more long PEG chains may cause tangling and affect the binding properties of the targeting or effector elements.

The synthesized peptides <NUM>, <NUM> and <NUM> (Chinapeptide Inc. , Shanghai, China) were processed similarly. Each peptide was dissolved in <NUM> sodium phosphate buffer (pH <NUM>) containing <NUM> NaCl and <NUM> EDTA at a final concentration of <NUM>. The dissolved peptide was reduced by <NUM> tris(<NUM>-carboxyethyl)phosphine (TCEP) at <NUM> for <NUM> hours. For conjugating the SH group of the cysteine residue with maleimide-PEG<NUM>-TCO (Conju-probe Inc. ) to create a functional linking group TCO, the peptide and maleimide-PEG<NUM>-TCO were mixed at a <NUM>/<NUM> molar ratio and incubated at pH <NUM> and <NUM> for <NUM> hours. The TCO-conjugated peptide was purified by reverse phase HPLC on a Supelco C18 column (<NUM> X <NUM>; <NUM>), using a mobile phase of acetonitrile and <NUM>% trifluoroacetic acid, a linear gradient of <NUM>% to <NUM>% acetonitrile over <NUM> minutes, at a flow rate of <NUM>/min and a column temperature of <NUM>.

The identification of the synthesized TCO-peptides (illustrated below) was carried out by MALDI-TOF mass spectrometry. Mass spectrometry analyses were performed by the Mass Core Facility at the Institute of Molecular Biology (IMB), Academia Sinica, Taipei, Taiwan. Measurements were performed on a Bruker Autoflex III MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany).

The synthesized TCO-peptide <NUM>, as illustrated below, had a molecular weight (m. ) of <NUM>,<NUM> daltons.

The synthesized TCO-peptide <NUM>, as illustrated below, had a m. of <NUM>,<NUM> daltons.

The TCO-peptide <NUM>, as illustrated below, had a m. of <NUM>,<NUM> daltons.

The synthesized peptide <NUM> (Chinapeptide Inc. , Shanghai, China) was dissolved in <NUM> sodium phosphate buffer (pH <NUM>) containing <NUM> NaCl and <NUM> EDTA at a final concentration of <NUM>. The dissolved peptide was reduced by <NUM> tris(<NUM>-carboxyethyl)phosphine (TCEP) at <NUM> for <NUM> hours. For conjugating the SH group of the cysteine residue with maleimide-PEG<NUM>-tetrazine (Conju-probe Inc. , San Diego, USA) to create a functional linking group tetrazine, the peptide and maleimide-PEG<NUM>-tetrazine were mixed at a <NUM>/<NUM> ratio and incubated at pH <NUM> and <NUM> for <NUM> hours. The tetrazine-conjugated peptide was purified by reverse phase HPLC on a Supelco C18 column (<NUM> X <NUM>; <NUM>), using a mobile phase of acetonitrile and <NUM>% trifluoroacetic acid, a linear gradient of <NUM>% to <NUM>% acetonitrile over <NUM> minutes, at a flow rate of <NUM>/min and a column temperature of <NUM>.

The synthesized tetrazine-peptide <NUM>, as illustrated below, had a m. of <NUM>,<NUM> daltons.

Three linking arms of PEG<NUM>-maleimide were attached to the peptide core, TCO-peptide <NUM>. The crosslinker, NHS-PEG<NUM>-maleimide (succinimidyl-[(N-maleimido-propionamido)-dodecaethyleneglycol] ester, was purchased from Thermo Fisher Scientific Inc. (Waltham, USA). The conjugation procedure was performed per the manufacturer's instruction. Briefly, the peptide with lysine residues was dissolved in the conjugation buffer, phosphate buffered saline (pH <NUM>) at <NUM>. NHS-PEG<NUM>-maleimide crosslinker was then added to the dissolved peptide at a <NUM> final concentration (<NUM>-fold molar excess over <NUM> peptide solution). The reaction mixtures were incubated for <NUM> hours at room temperature. The maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> was purified by reverse phase HPLC on a Supelco C18 column (<NUM> X <NUM>; <NUM>), using a mobile phase of acetonitrile and <NUM>% trifluoroacetic acid, a linear gradient of <NUM>% to <NUM>% acetonitrile over <NUM> minutes, at a flow rate of <NUM>/min and a column temperature of <NUM>.

The identification of the maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> was carried out by mass spectrometry MALDI-TOF.

The synthesized maleimide-PEG<NUM>-conjugated TCO-peptide1 had a m. of <NUM>,<NUM> daltons. As illustrated below, the maleimide-PEG<NUM>-conjugated TCO-peptide1 is a peptide-core based linker unit carrying one TCO group and three PEG linking arms with maleimide groups.

Three linking arms of PEG<NUM>-maleimide were attached to the peptide core, tetrazine-peptide <NUM>. The crosslinker, NHS-PEG<NUM>-maleimide (succinimidyl-[(N-maleimido-propionamido)-dodecaethyleneglycol] ester, was purchased from Thermo Fisher Scientific Inc. (Waltham, USA). The conjugation procedure was performed per the manufacturer's instruction. Briefly, the peptide with lysine residues was dissolved in the conjugation buffer, phosphate buffered saline (pH <NUM>) at <NUM>. NHS-PEG<NUM>-maleimide crosslinker was then added to the dissolved peptide at a <NUM> final concentration (<NUM>-fold molar excess over <NUM> peptide solution). The reaction mixtures were incubated for <NUM> hours at room temperature. The maleimide-PEG<NUM>-conjugated tetrazine-peptide <NUM> was purified by reverse phase HPLC on a Supelco C18 column (<NUM> X <NUM>; <NUM>), using a mobile phase of acetonitrile and <NUM>% trifluoroacetic acid, a linear gradient of <NUM>% to <NUM>% acetonitrile over <NUM> minutes, at a flow rate of <NUM>/min and a column temperature of <NUM>.

The synthesized maleimide-PEG<NUM>-conjugated tetrazine-peptide1, as illustrated below, was a peptide-core based linker unit carrying one tetrazine group and three PEG linking arms with maleimide groups. <FIG> shows the MALDI-TOF result, indicating that the construct had a m. of <NUM>,<NUM> daltons.

Fingolimod was purchased from Biotang Inc. (Lexington, USA) and fingolimod phosphate from KM3 Scientific Corporation (New Taipei City, Taiwan). The NH<NUM> group of fingolimod molecule was reacted with a homo-bifunctional crosslinker, NHS-PEGs-NHS, as shown in scheme <NUM>. Fingolimod was dissolved in <NUM>% DMSO at a final concentration of <NUM>; NHS-PEG<NUM>-NHS was dissolved in <NUM>% DMSO at a <NUM> final concentration. To activate the NH<NUM> group of fingolimod, <NUM>% (v/v) of basic sodium phosphate buffer (pH12. <NUM>) was added to the fingolimod solution and then incubated for <NUM> minutes. NHS-PEGs-NHS crosslinker was added to the dissolved fingolimod solution at a final concentration of <NUM> (<NUM>-fold molar excess over <NUM> fingolimod solution). The reaction mixture was incubated for <NUM> hours at room temperature.

Fingolimod phosphate was dissolved in <NUM>% DMSO at a final concentration of <NUM>, and NHS-PEGs-NHS crosslinker was dissolved in <NUM>% DMSO at a final concentration of <NUM>. NHS-PEGs-NHS crosslinker was added to the dissolved fingolimod phosphate solution at a <NUM> final concentration (<NUM>-fold molar excess over <NUM> fingolimod phosphate solution). The reaction mixture was incubated for <NUM> hours at room temperature, then <NUM>% (v/v) acid sodium phosphate buffer (pH=<NUM>) was added to quench the reaction. The solvent was evaporated under vacuum.

NHS-PEGs-conjugated fingolimod and NHS-PEGs-conjugated fingolimod phosphate were dissolved in <NUM>% acetonitrile, and then purified using reverse phase HPLC on a Supelco C18 column (<NUM> X <NUM>; <NUM>), using a mobile phase of acetonitrile and <NUM>% trifluoroacetic acid, a linear gradient of <NUM>% to <NUM>% acetonitrile over <NUM> minutes, at a flow rate of <NUM>/min and a column temperature of <NUM>.

<FIG> shows that the synthesized NHS-PEGs-conjugated fingolimod, as illustrated in scheme <NUM>, had a m. of <NUM> daltons.

The synthesized NHS-PEGs-conjugated fingolimod phosphate, as illustrated below, had a m. of <NUM> daltons.

The NH<NUM> group of fingolimod molecule was reacted with a hetero-bifunctional cleavable linker, NHS-S-S-PEG<NUM>-azido (Conju-probe Inc. ), at a <NUM>:<NUM> molar ratio. The product, azido-PEG<NUM>-S-S-fingolimod was purified by HPLC to remove the excess, unreacted fingolimod molecules. The procedures for conjugation and purification were similar to those described in the preceding example.

The synthesized azido-PEG<NUM>-S-S-conjugated fingolimod, as illustrated below, had a m. of <NUM> daltons.

Azido-PEG<NUM>-S-S-conjugated fingolimod molecule was dissolved in <NUM>% DMSO at a final concentration of <NUM>, and NHS-PEG<NUM>-DBCO crosslinker was dissolved in <NUM>% DMSO at a final concentration of <NUM>. <NUM>µl of NHS-PEG<NUM>-DBCO crosslinker was added to <NUM>µl of the dissolved azido-PEG<NUM>-S-S-conjugated fingolimod solution to a final molar ratio of <NUM>/<NUM> (NHS-PEG<NUM>-DBCO: azido-PEG<NUM>-S-S-conjugated fingolimod) in <NUM> sodium phosphate buffer at pH <NUM>. The reaction mixture was incubated for <NUM> hours at room temperature.

The synthesized NHS-PEG<NUM>-PEG<NUM>-S-S-conjugated fingolimod, as illustrated below, had a m. of <NUM>,<NUM> daltons. The two isotopic peaks were also visible in the MS spectrum at <NUM>,<NUM> and <NUM>,<NUM>, corresponding to [M+H+<NUM>]+ and [M+H+<NUM>]+.

TCO-peptide <NUM> was dissolved in <NUM> sodium phosphate buffer at pH <NUM> to a concentration of <NUM>, and NHS-PEGs-conjugated fingolimod was dissolved in <NUM>% DMSO to a concentration of <NUM>. TCO-peptide <NUM> and NHS-PEGs-conjugated fingolimod were mixed at <NUM>/<NUM> molar ratio in <NUM>% DMSO and incubated for <NUM> hours at room temperature. Subsequently, additional TCO-peptide <NUM> was added to the reaction solution to a final molar ratio of <NUM>/<NUM> (TCO-peptide <NUM>: NHS PEGs-conjugated fingolimod) in <NUM>% DMSO. The mixture was further incubated for <NUM> hours at room temperature. <FIG> shows that the drug bundle of TCO-peptide <NUM> with fingolimod had a m. of <NUM>,<NUM> daltons.

TCO-peptide <NUM> was dissolved in <NUM> sodium phosphate buffer at pH <NUM> to a concentration of <NUM>, and NHS-PEGs-conjugated fingolimod was dissolved in <NUM>% DMSO to a concentration of <NUM>. TCO-peptide <NUM> and PEGs-NHS-conjugated fingolimod were mixed at <NUM>/<NUM> molar ratio at room temperature for overnight. <FIG> shows that the drug bundle of TCO-peptide <NUM> with fingolimod had a m. of <NUM>,<NUM> daltons, indicating that ten fingolimod molecules were conjugated to the TCO-peptide <NUM> linker unit.

The synthesized drug bundle, as illustrated below, was composed of a linker unit with a free TCO functional group and a set of five fingolimod molecules.

The second synthesized drug bundle, as illustrated below, was composed of a linker unit with a free TCO functional group and a set of ten fingolimod molecules.

TCO-peptide <NUM> and NHS-PEGs-conjugated fingolimod phosphate were mixed at <NUM>/<NUM> molar ratio in <NUM> sodium phosphate buffer at pH <NUM> at room temperature for <NUM> hours. Mass spectrometric analysis shows that the drug bundle of TCO-peptide <NUM> with fingolimod phosphate had a m. of <NUM>,<NUM> daltons (<FIG>).

The synthesized drug bundle, as illustrated below, was composed of a linker unit with a free TCO functional group and a set of five fingolimod phosphate molecules as effector elements.

Five NHS-PEG<NUM>-PEG<NUM>-S-S-conjugated fingolimod molecules were attached to TCO-peptide <NUM>. The conjugation of NHS-PEG<NUM>-PEG<NUM>-S-S-conjugated fingolimod molecules to the NH<NUM> groups of lysine residues of the TCO-peptide <NUM> was performed similarly as in the preceding example. The identification was carried out by mass spectrometry MALDI-TOF.

The synthesized drug bundle, as illustrated below, had a m. of <NUM>,<NUM> daltons; it was composed of a linker unit with a free TCO functional group and a set of five fingolimod molecules.

The gene-encoding sequence was placed in pG1K expression cassette. The amino acid sequence of the extracellular portion of human CD32a, which was expressed as a recombinant protein with a histidine-tag, is set forth in SEQ ID NO: <NUM>. Recombinant ectodomain of human CD32a was expressed in FreeStyle 293F suspension culture cell expression system and medium (Invitrogen, Carlsbad, USA). FreeStyle 293F cells were seeded at a cell density of <NUM> × <NUM><NUM> viable cells/ml in <NUM>-ml culture and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in a <NUM>-ml medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by using linear polyethylenimine with an average molecular weight of <NUM> kDa (Polysciences, Warrington, USA) as a transfection reagent. The transfected cells were incubated at <NUM> for <NUM> hours post-transfection in an orbital shaker (<NUM> rpm), and their cell density was then adjusted to <NUM>×<NUM><NUM> cells/ml with a fresh medium and incubated for <NUM> to <NUM> days. Culture supernatants were harvested and protein in the media was purified using nickel affinity chromatography. <FIG> shows SDS-PAGE analysis of purified protein of ectodomain of human CD32a.

The gene-encoding sequence was placed in pG1K expression cassette. The amino acid sequence of the ectodomain of human TfR1, which was expressed as a recombinant protein with a histidine-tag, is set forth in SEQ ID NO: <NUM>. Recombinant ectodomain of human TfR1 was expressed in FreeStyle 293F suspension culture cell expression system and medium (Invitrogen, Carlsbad, USA). FreeStyle 293F cells were seeded at a cell density of <NUM> × <NUM><NUM> viable cells/ml in <NUM>-ml culture and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in <NUM>-ml medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by using linear polyethylenimine with an average molecular weight of <NUM> kDa (Polysciences, Warrington, USA) as a transfection reagent. The transfected cells were incubated at <NUM> for <NUM> hours post-transfection in an orbital shaker (<NUM> rpm), and their cell density was then adjusted to <NUM>×<NUM><NUM> cells/ml with a fresh medium and incubated for <NUM> to <NUM> days. Culture supernatants were harvested and protein in the media was purified using nickel affinity chromatography. <FIG> shows SDS-PAGE analysis of the purified protein of ectodomain of human TfR1.

The VL and VH of the scFv specific for Protein F of RSV were from monoclonal antibody palivizumab; the VL and VH of the scFv specific for endotoxin were from monoclonal antibody WN1 <NUM>-<NUM> (Patent <CIT>); VL and VH of the scFv specific for ectodomain of CD32a were from MDE-<NUM> (US Patent Application publication <CIT>). The scFv derived from those antibodies were designed to contain a flexible linker of GGGGSGGGGS and a terminal cysteine residue at the C-terminus. The cysteine residue provides a sulfhydryl group for conjugation with maleimide group present at the free ends of linking arms in various linker units. To produce the scFv of mAb specific for Protein F of RSV, mAb specific for endotoxin, and mAb specific for extracellular component of CD32a, the VL and VH DNA sequences of the three antibodies with further codon optimization were used. DNA sequences encoding VL-GSTSGSGKPGSGEGSTKG-VH-(GGGGS)<NUM>-C were synthesized. The amino acid sequences of the scFv of mAb specific for Protein F of RSV, mAb specific for endotoxin, and mAb specific for ectodomain of CD32a prepared for the experiments of the invention are set forth in SEQ ID NO: <NUM> to <NUM>, respectively.

For preparing scFv proteins using a mammalian expression system, an overexpression system based on Expi293F™ cell line were used for experimentation. The system employed ExpiFectamine™ <NUM> transfection kit (Life Technologies, Carlsbad, USA) consisting of the Expi293F™ cell line, the cationic lipid-based ExpiFectamine™ <NUM> Reagent and ExpiFectamine™ <NUM> transfection Enhancers <NUM> and <NUM>, and the medium, which was part of the expression system (Gibco, New York, USA).

The scFv-encoding sequence was placed in pG1K expression cassette. Expi293F cells were seeded at a density of <NUM> × <NUM><NUM> viable cells/ml in Expi293F expression medium and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in <NUM> medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ <NUM> transfection reagent. The transfected cells were incubated at <NUM> for <NUM> to <NUM> hours post-transfection in an orbital shaker (<NUM> rpm) and the cells were added ExpiFectamine™ <NUM> transfection enhancer <NUM> and enhancer <NUM> to the shaker flask, and incubated for <NUM> to <NUM> days. Culture supernatants were harvested and scFv proteins in the media were purified using Protein L affinity chromatography.

<FIG> show SDS-PAGE and ELISA analyses of purified scFv of mAb specific for Protein F of RSV; <FIG> and <FIG> show SDS-PAGE and ELISA analyses of purified scFv of mAb specific for endotoxin; <FIG> show SDS-PAGE and ELISA analyses of purified scFv of mAb specific for ectodomain of CD32a. The <NUM>-well ELISA plates (Greiner Bio-one) were coated with <NUM>µg/ml of Protein F of RSV, <NUM>µg/ml of endotoxin, and <NUM>µg/ml of ectodomain of CD32a, respectively. Purified scFvs were detected by HRP-conjugated protein L at a ratio of <NUM>:<NUM>.

The ELISA results show that each purified scFv protein is bound specifically to its antigen (Protein F of RSV, endotoxin, or ectodomain of CD32a), using adalimumab scFv (anti-TNF-α scFv) as a negative control.

The VL and VH of the scFv specific for ectodomain of TfR1 were from monoclonal antibody OX26; the VL and VH of the scFv specific for β-amyloid were from monoclonal antibody bapineuzumab. The scFv derived from those antibodies were designed to contain a flexible linker of GGGGSGGGGS and a terminal cysteine residue at the C-terminus. The cysteine residue provides a sulfhydryl group for conjugation with maleimide group present at the free ends of linking arms in various linker units. To produce the scFv of mAb specific for ectodomain of TfR1 and mAb specific for -β-amyloid, the VL and VH DNA sequences of the two antibodies with further codon optimization were used. DNA sequences encoding VL-GSTSGSGKPGSGEGSTKG-VH-(GGGGS)<NUM>-C were synthesized. The amino acid sequences of the scFv of mAb specific for ectodomain of TfR1 and mAb specific for β-amyloid prepared for the experiments of the invention are set forth in SEQ ID NO: <NUM> and <NUM>, respectively.

For preparing scFv proteins using mammalian expression systems, the overexpression system based on Expi293F™ cell line were used. The system employed ExpiFectamine™ <NUM> transfection kit (Life Technologies, Carlsbad, USA) consisting of the Expi293F™ cell line, the cationic lipid-based ExpiFectamine™ <NUM> Reagent and ExpiFectamine™ <NUM> transfection Enhancers <NUM> and <NUM>, and the medium (Gibco, New York, USA).

The scFv-encoding sequence was placed in pG1K expression cassette. Expi293F cells were seeded at a density of <NUM> × <NUM><NUM> viable cells/ml in Expi293F expression medium and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in <NUM> medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ <NUM> transfection reagent. The transfected cells were incubated at <NUM> for <NUM> to <NUM> hours post-transfection in an orbital shaker (<NUM> rpm) and the cells were added ExpiFectamine™ <NUM> transfection enhancer <NUM> and enhancer <NUM> to the shaker flask, and incubated for <NUM> to <NUM> days. Culture supernatants were harvested and scFv proteins in the media were purified using Protein L affinity chromatography. <FIG> respectively show SDS-PAGE and ELISA analyses of purified scFv of mAb specific for ectodomain of TfR1. <FIG> and <FIG> respectively show SDS-PAGE and ELISA analyses of purified scFv of mAb specific for β-amyloid. The ELISA plates were coated with <NUM>µg/ml of ectodomain of TfR1 and <NUM>µg/ml of β-amyloid, respectively. Purified scFvs were detected by HRP-conjugated protein L at a ratio of <NUM>:<NUM>.

The ELISA results show that each purified scFv protein bound specifically to its antigen (ectodomain of TfR1 or β-amyloid), using HRP-conjugated protein L alone as a negative control.

The phage clones carrying the scFv specific for the ectodomain of human CD32a were obtained through a contractual arrangement with Dr. An-Suei Yang's laboratory at the Genomics Research Center, Academia Sinica, Taipei, Taiwan. The framework sequence of the GH2 scFv library was derived from G6 anti-VEGF Fab (Protein Bank Code 2FJG) and cloned into restriction sites Sfil and NotI of phagemid vector pCANTAB5E (GE Healthcare), carrying an ampicillin resistance, a lacZ promotor, a pelB leader sequence for secretion of scFv fragments into culture supernatants, a E-tag applicable for detection. The VH and VL domains of the scFv template were diversified separately based on the oligonucleotide-directed mutagenesis procedure; the three CDRs in each of the variable domains were diversified simultaneously. The scFv library of over <NUM><NUM> clones was used for selections on ectodomain of CD32a.

Maxisorp <NUM>-well plates (Nunc) coated with recombinant CD32a proteins (<NUM>µg/<NUM>µL PBS per well) were used for panning anti-CD32a antibodies. In brief, the wells were coated with human CD32a by shaking the coating solution in the wells for <NUM> hours at room temperature. The CD32a-coated wells were then treated with blocking buffer (<NUM>% skim milk in PBST (phosphate buffered saline with <NUM>% tween-<NUM>)) for <NUM> hour at room temperature. Recombinant phages in the blocking buffer diluted to 8x10<NUM> CFU/ml was added to the CD32a-coated wells for <NUM> hour with gentle shaking; CFU stands for colony-forming unit. The wells were then washed vigorously <NUM> times with PBST, followed by <NUM> times with PBS to remove nonspecific binding phages. The bound phages were eluted using <NUM> HCl/glycine buffer at pH <NUM>, and the elution solution was neutralized immediately by <NUM> Tris-base buffer at pH <NUM>. coli strain ER2738 (OD600 = ~<NUM>) was used for phage infection at <NUM> for <NUM> minutes; non-infected E. coli was eliminated by treating with ampicillin for <NUM> minutes. After ampicillin treatment, helper phage M13KO7 carrying kanamycin resistance was added for another one-hour incubation. The selected phages rescued by helper phage in the E. coli culture were amplified with vigorously shaking overnight at <NUM> in the presence of kanamycin. The amplified phages were precipitated in PEG/NaCl, and then resuspended in PBS for the next selection-amplification cycles. A total of three consecutive panning rounds was performed on ectodomain of CD32a by repeating this selection-amplification procedure.

Phage-infected ER2738 colonies of plates with dilution series were counted and phage titers were calculated, yielding the output titer/ml (CFU/ml) per panning round. A <NUM>-fold increase in phage output title from <NUM>. 6E+<NUM> CFU/well to <NUM>. 2E+<NUM> CFU/well was obtained after three rounds of panning. The phage output/input titer ratios from each round are shown in <FIG>. For each panning round, the phage output/input titer ratios are given on the y-axis. There was clear enrichment of the positive clones over the three rounds of panning. The third panning round resulted in a <NUM>-fold on the ratios of phage output/input titer over the first round, as the binding clones became the dominant population in the library.

In a typical selection procedure, after three rounds of antigen-panning on human CD32a-coated wells in ELISA plates, approximately <NUM>% of the bound phage particles bound to CD32a specifically in ELISA with coated CD32a.

coli strain ER2738 infected with single-clonal phages each harboring a selected scFv gene in its phagemid was grown in the mid-log phase in 2YT broth (<NUM>/L tryptone, <NUM>/L yeast extract, <NUM>/L NaCl, pH <NUM>) with <NUM>µg/ml ampicillin in deep well at <NUM> with shaking. After broth reaching an OD600 of <NUM>, IPTG was added to a final concentration of <NUM>µg/ml. The plates were incubated at <NUM> overnight with rigorously shaking; thereafter, the plates were centrifuged at <NUM> for <NUM> minutes at <NUM>.

For soluble scFv binding test, ELISA was carried out. In brief, Maxisorp <NUM>-well plate (Nunc) was coated with ectodomain of CD32a (<NUM>µg/<NUM>µl PBS per well) or a negative control antigen human transferrin-<NUM> receptor, for <NUM> hours with shaking at <NUM>. After treated with <NUM>µl of blocking buffer for <NUM> hour, <NUM>µl of secreted scFv in the supernatant was mixed with <NUM>µl of blocking buffer and then added to the coated plate for another <NUM> hour. Goat anti-E-tag antibody (conjugated with HRP, <NUM>:<NUM>, Cat. No. AB19400, Abcam) was added to the plate for <NUM> hour. TMB substrate (<NUM>µl per well) was added to the wells and the absorbance at <NUM> was measured after reactions were stopped by adding 1N HCl (<NUM>µl per well).

A total of <NUM> phage clones after the third round of panning were subjected to the present analysis. Among them, <NUM> scFv clones that bound to CD32a with a differential of OD450 greater than <NUM> were further characterized by sequencing genes encoding these scFvs. Six different DNA sequences were identified. <FIG> shows the ELISA result of an scFv clone 22D1. The amino acid sequence of an scFV clone 22D1, which binds to human CD32a with an OD450 of <NUM>, is shown in SEQ ID NO: <NUM>.

The phage clones carrying the scFv specific for the ectodomain of human TfR1 were obtained through a contractual arrangement with Dr. An-Suei Yang's laboratory at the Genomics Research Center, Academia Sinica, Taipei, Taiwan. The framework sequence of the GH2 scFv library was derived from G6 anti-VEGF Fab (Protein Bank Code 2FJG) and cloned into restriction sites Sfil and NotI of phagemid vector pCANTAB5E (GE Healthcare), carrying an ampicillin resistance, a lacZ promotor, a pelB leader sequence for secretion of scFv fragments into culture supernatants, an E-tag applicable for detection. The VH and VL domains of the scFv template were diversified separately based on the oligonucleotide-directed mutagenesis procedure; the three CDRs in each of the variable domains were diversified simultaneously. The scFv library of over <NUM><NUM> clones was used for selections on ectodomain of CD32a.

Maxisorp <NUM>-well plates (Nunc) coated with recombinant ectodomain of TfR1 proteins (<NUM>µg/<NUM>µL PBS per well) were used for panning anti-TfR1 antibodies. In brief, the wells were coated with human TfR1 by shaking the coating solution in the wells for <NUM> hours at room temperature. The TfR1-coated wells were then treated with blocking buffer (<NUM>% skim milk in PBST (phosphate buffered saline with <NUM>% tween-<NUM>)) for <NUM> hour at room temperature. Recombinant phages in the blocking buffer diluted to 8x10<NUM> CFU/ml was added to the TfR1-coated wells for <NUM> hour with gentle shaking; CFU stands for colony-form ing unit. The wells were then washed vigorously <NUM> times with PBST, followed by <NUM> times with PBS to remove nonspecific binding phages. The bound phages were eluted using <NUM> HCl/glycine buffer at pH <NUM>, and the elution solution was neutralized immediately by <NUM> Tris-base buffer at pH <NUM>. coli strain ER2738 (OD600 = ~<NUM>) was used for phage infection at <NUM> for <NUM> minutes; non-infected E. coli was eliminated by treating with ampicillin for <NUM> minutes. After ampicillin treatment, helper phage M13KO7 carrying kanamycin resistance was added for another one-hour incubation. The selected phages rescued by helper phage in the E. coli culture were amplified with vigorously shaking overnight at <NUM> in the presence of kanamycin. The amplified phages were precipitated in PEG/NaCl, and then resuspended in PBS for the next selection-amplification cycles. A total of three consecutive panning rounds was performed on ectodomain of TfR1 by repeating this selection-amplification procedure.

Phage-infected ER2738 colonies of plates with serial dilutions were counted and phage titers were calculated, yielding the output titer/ml (CFU/ml) per panning round. A <NUM><NUM>-fold increase in phage output title from <NUM>. 74E+<NUM> CFU/well to <NUM>. 5E+<NUM> CFU/well was obtained after three rounds of panning. The phage output/input titer ratios from each round are shown in <FIG>. For each panning round, the phage output/input titer ratios are given on the y-axis. There was clear enrichment of the positive clones over the three rounds of panning. The third panning round resulted in a <NUM><NUM>-fold on the ratios of phage output/input titer over the first round, as the binding clones became the dominant population in the library.

In a typical selection procedure, after three rounds of antigen-panning on human TfR1-coated wells in ELISA plates, approximately <NUM>% of the bound phage particles bound to TfR1 specifically in ELISA with coated TfR1.

For soluble scFv binding test, ELISA was carried out. In brief, Maxisorp <NUM>-well plate (Nunc) was coated with ectodomain of TfR1 (<NUM>µg/<NUM>µl PBS per well) or a negative control antigen CD16b, for <NUM> hours with shaking at <NUM>. After treated with <NUM>µl of blocking buffer for <NUM> hour, <NUM>µl of secreted scFv in the supernatant was mixed with <NUM>µl of blocking buffer and then added to the coated plate for another one-hour. Goat anti-E-tag antibody (conjugated with HRP, <NUM>:<NUM>, Cat. No. AB19400, Abcam) was added to the plate for <NUM> hour. TMB substrate (<NUM>µl per well) was added to the wells and the absorbance at <NUM> was measured after reactions were stopped by adding 1N HCl (<NUM>µl per well).

A total of <NUM> phage clones after the third round of panning were subjected to the present analysis. Among them, <NUM> scFv clones that bound to TfR1 with a differential of OD450 greater than <NUM> were further characterized by sequencing the genes encoding these scFvs. Sixteen different DNA sequences were identified. <FIG> shows the ELISA result of an scFv clone 12A1. The amino acid sequence of the scFV clone 12A1, which binds to human TfR1 with an OD450 of <NUM>, is shown in SEQ ID NO: <NUM>.

The DNA sequence encoding SEQ ID NO: <NUM> was synthesized and expressed as in the above Examples. For the conjugation with Mal-PEG<NUM>-TCO (Conju-probe, Inc. ), the cysteine residue at the C-terminal end of the purified scFv of anti-CD32a mAb was reduced by incubating with <NUM> dithiothreitol (DTT) at room temperature for <NUM> hours with gentle shaking. The buffer of reduced scFv proteins were exchanged to sodium phosphate buffer (<NUM> sodium phosphate, pH7. <NUM>, <NUM> NaCl, and <NUM> EDTA) by using NAP-<NUM> Sephadex G-<NUM> column. After the reduction reaction and buffer exchange, conjugation was conducted overnight at room temperature in a reaction molar ratio of <NUM>:<NUM> ([Mal-PEG<NUM>-TCO:[scFv]]. The excess crosslinker was removed by a desalting column and the TCO-conjugated scFv product was analyzed.

The results of mass spectroscopy MALDI-TOF analysis indicated that the sample of TCO-conjugated scFv specific for CD32a had a m. of <NUM>,<NUM> daltons. The purity of TCO-conjugated scFvs specific for CD32a was identified through Coomassie blue staining of <NUM>% SDS-PAGE. <FIG> and <FIG> show, respectively, the ELISA and Mass spectrometric analysis of TCO-conjugated scFv specific for CD32a, in which unmodified scFv specific for CD32a was used as a positive control. According to the ELISA results, TCO-conjugated scFv specific for CD32a bound to recombinant ectodomain of human CD32a.

The DNA sequence encoding SEQ ID NO: <NUM> was synthesized and expressed as in the above Examples. For the conjugation with Mal-PEG<NUM>-tetrazine (Conju-probe, Inc. ), the cysteine residue at the C-terminal end of the purified scFv of mAb specific for TfR1 was reduced by incubating with <NUM> DTT at room temperature for <NUM> hours with gentle shaking. The buffer of reduced scFv proteins were exchanged to sodium phosphate buffer (<NUM> sodium phosphate, pH <NUM>, <NUM> NaCl, and <NUM> EDTA) by using NAP-<NUM> Sephadex G-<NUM> column. After the reduction reaction and buffer exchange, conjugation was conducted overnight at <NUM> in a reaction molar ratio of <NUM>:<NUM> ([Mal-PEG<NUM>-tetrazine:[scFv]]. The excess crosslinker was removed by a desalting column and the tetrazine-conjugated scFv product was analyzed.

The results of mass spectroscopy MALDI-TOF analysis indicated that the sample of tetrazine-conjugated scFv specific for TfR1 had a m. of <NUM>,<NUM> daltons. The purity of tetrazine-conjugated scFv specific for TfR1 was identified through Coomassie blue staining of <NUM>% SDS-PAGE. <FIG> show, respectively, the ELISA and Mass spectrometric analysis of tetrazine-conjugated scFv specific for TfR1, in which unmodified scFv specific for TfR1 was used as a positive control. According to the ELISA results, tetrazine-conjugated scFv specific for TfR1 bound to recombinant ectodomain of TfR1.

This example demonstrates that three scFvs can be conjugated to the three PEG<NUM>-maleimide linking arms based on tetrazine-peptide <NUM>. Prior to conjugation with the tetrazine-peptide <NUM> that had three PEG<NUM>-maleimide linking arms, scFv specific for endotoxin was incubated with DTT at a molar ratio of <NUM>:<NUM> ([DTT]:[scFv]) at <NUM> for <NUM> hours with gentle shaking to keep its C-terminal cysteine in a reduced form. Subsequently, the buffer of reduced scFv specific endotoxin was exchanged to maleimide-SH coupling reaction buffer (<NUM> sodium phosphate, pH <NUM>, <NUM> NaCl and <NUM> EDTA) by using an NAP-<NUM> Sephadex G-<NUM> column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the tetrazine-peptide <NUM> having three maleimide-PEG<NUM> linking arms was conducted overnight at <NUM> at a molar ratio of <NUM>:<NUM> ([linker]:[Protein]).

The PEG<NUM>-maleimide-conjugated tetrazine-peptide <NUM> conjugated with three scFvs specific for endotoxin was separated from the free scFv, free PEG<NUM>-maleimide-conjugated tetrazine-peptide <NUM> and the PEG<NUM>-maleimide-conjugated tetrazine-peptide <NUM> conjugated with one and two scFvs specific for endotoxin by size exclusion chromatography column S75.

<FIG> is the FPLC elution profile on a synthesized targeting linker unit composed of a linker unit with a free tetrazine functional group and a set of three scFvs specific for endotoxin as targeting elements with retention volume of <NUM>. The product (i.e., the PEG<NUM>-maleimide-conjugated tetrazine-peptide <NUM> having a free tetrazine functional group and being conjugated with a set of three scFvs specific for endotoxin) was purified in the elution fraction and shown in lane <NUM> (indicated by arrow) of the <NUM>% SDS-PAGE analysis shown in <FIG>.

The sample of the targeting linker unit with three scFvs specific for endotoxin linked to the three maleimide-PEG<NUM> linking arms based on tetrazine-peptide <NUM> was analyzed by MALDI-TOF. The median of the experimental molecular weight was consistent with the median of theoretical molecular weight of three scFvs specific for endotoxin conjugated to tetrazine-peptide <NUM> with three maleimide-PEG<NUM> linking arms. According to the mass spectrometric profile in <FIG>, the present targeting linker unit had a median molecular weight of <NUM>,<NUM> daltons.

Illustrated below is the synthesized targeting linker unit that was composed of a linker unit with a free tetrazine functional group and a set of three scFvs specific for endotoxin as targeting elements.

The conjugation of scFv to the linker unit and the purification and analysis of the product were the same as in the preceding Examples.

Shown in <FIG> is the mass spectrometric analysis of the synthesized targeting linker unit that was composed of a linker unit with a free tetrazine functional group and a set of three scFv specific for Protein F of RSV as targeting elements (illustrated below). As indicated in <FIG>, this targeting linker unit had a molecular weight of <NUM>,<NUM> daltons.

This example was performed to demonstrate that three scFvs could be conjugated to the three maleimide-PEG<NUM> linking arms based on TCO-peptide <NUM>. Prior to conjugation with the TCO-peptide <NUM> that had three maleimide-PEG<NUM> linking arms, scFv specific for β-amyloid was incubated with DTT at a molar ratio of <NUM>:<NUM> ([DTT]:[scFv]) at room temperature for <NUM> hours with gentle shaking to keep its C-terminal cysteine in a reduced form. Subsequently, the buffer of reduced scFv specific for β-amyloid was exchanged to maleimide-SH coupling reaction buffer (<NUM> sodium phosphate, pH <NUM>, <NUM> NaCl and <NUM> EDTA) by using an NAP-<NUM> Sephadex G-<NUM> column (GE Healthcare). After the reduction and buffer exchange, the conjugation to the TCO-peptide <NUM> having three maleimide-PEG<NUM> linking arms was conducted overnight at room temperature at a molar ratio of <NUM>:<NUM> ([linker]:[Protein]).

The reaction mixture of the preceding examples was adjusted to pH <NUM> and then applied to pre-equilibrated (<NUM> EDTA, and <NUM> sodium acetate at pH <NUM>) cation exchange column SP Sepharose FF (GE Healthcare). The maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> conjugated with three scFvs specific for β-amyloid was eluted using a linear gradient of <NUM>-<NUM> sodium chloride in a flow rate of <NUM>/min for <NUM> minutes. The maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> conjugated with three scFvs specific for β-amyloid was separated from the free scFv, free maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> and the maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> conjugated with one and two scFvs specific for β-amyloid by cation exchange column SP Sepharose FF. The purified product, maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> conjugated with three scFvs specific for β-amyloid, was concentrated and buffer-exchange into click reaction buffer, <NUM> potassium phosphate at pH <NUM>.

<FIG> is the FPLC elution profile of cation exchange column SP Sepharose FF on a synthesized effector linker unit composed of a linker unit with a free TCO functional group and a set of three scFvs specific for β-amyloid as effector elements. Symbol #<NUM> and #<NUM> respectively represented the eluted peaks of maleimide-PEG<NUM>-conjugated TCO-peptide <NUM> conjugated with two scFvs and three scFvs specific for β-amyloid. The product, the maleimide- PEG<NUM>-conjugated TCO-peptide <NUM> bearing a free TCO functional group and three scFvs specific for β-amyloid was purified and revealed in lane <NUM> of the <NUM>% SDS-PAGE analysis shown in <FIG>.

The sample of the effector linker unit of three scFvs specific for β-amyloid linked to the three maleimide-PEG<NUM> linking arms based on TCO-peptide <NUM> was analyzed by MALDI-TOF. The median of the experimental molecular weight was consistent with the median of theoretical molecular weight of three scFvs specific for β-amyloid conjugated to TCO-peptide <NUM> with three maleimide-PEG<NUM> linking arms. According to the mass spectrometric profile in <FIG>, the synthesized effector linker unit had the median molecular weight of <NUM>,<NUM> daltons.

Illustrated herein is the synthesized effector linker unit that was composed of a linker unit with a free TCO functional group and a set of three scFvs specific for β-amyloid as targeting elements.

In this example, the targeting linker unit of the preceding examples and a TCO-scFv specific for ectodomain of CD32a were coupled via a tetrazine-TCO iEDDA reaction. Specifically, the targeting linker unit had three scFv specific for Protein F of RSV and one free tetrazine group.

The procedure for tetrazine-TCO ligation was performed per the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). Briefly, <NUM>µl of the targeting linker unit (<NUM>/ml) was added to the solution containing the effector element at a molar ratio of <NUM>:<NUM> ([tetrazine]:[TCO]). The reaction mixture was incubated for <NUM> hour at room temperature. The product was subjected to mass spectrometric analysis, and the result indicated a molecular weight of <NUM>,<NUM> daltons (<FIG>).

The product, a single linker unit molecular construct with three scFvs specific for Protein F of RSV as targeting elements and one scFv specific for ectodomain of CD32a as an effector element, is illustrated below.

The targeting linker unit prepared in an earlier Example and the TCO-scFv specific for ectodomain of CD32a were coupled via a tetrazine-TCO iEDDA reaction.

The targeting linker unit prepared in an earlier Example and the tetrazine-scFv specific for ectodomain of TfR1 were coupled via a tetrazine-TCO iEDDA reaction.

The procedure for tetrazine-TCO ligation was performed per the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). Briefly, <NUM>µl of the targeting element (<NUM>/ml) was added to the solution containing the linker unit with effector elements at a molar ratio of <NUM>:<NUM> ([tetrazine]:[TCO]). The reaction mixture was incubated for <NUM> hours at room temperature. The product was subjected to mass spectrometric analysis, and the result indicated a molecular weight of <NUM>,<NUM> daltons (<FIG>).

The product, as illustrated herein, was a single linker unit molecular construct with one scFv specific for ectodomain of TfR1 as a targeting element and three scFvs specific for β-amyloid as effector elements.

In this example, the molecular construct with one scFv specific for ectodomain of TfR1 and a drug bundle of five fingolimod molecules was constructed. The molecular construct was made by a TCO-tetrazine iEDDA reaction. The procedure for tetrazine-TCO ligation was performed per the manufacturer's instructions (Jena Bioscience GmbH, Jena, Germany). Briefly, <NUM>µl of the effector linker unit (<NUM>µmole) was added to the solution containing the targeting element of one scFv specific for ectodomain of TfR1 at a molar ratio of <NUM>:<NUM> ([TCO]:[tetrazine]). The reaction mixture was incubated for <NUM> hours at room temperature.

The product, as illustrated below, was the molecular construct with one scFv specific for ectodomain of TfR1 and one drug bundle bearing five fingolimod molecules. <FIG> respectively show the SDS-PAGE and mass spectrometric analyses of the present the molecular construct. A major band, arrow #<NUM>, is the molecular construct with one scFv specific for ectodomain of TfR1 and a drug bundle with five fingolimod molecules, and arrow #<NUM> is unconjugated scFv specific for ectodomain of TfR1.

The mass spectrometric analysis shows that the molecular construct with one scFv specific for ectodomain of TfR1 and a drug bundle of five fingolimod molecules has a molecular weight of <NUM>,<NUM> daltons.

Modified fingolimod molecules (NHS-PEGs-conjugated fingolimod and the drug bundle with a free TCO functional group and with five fingolimod molecules) were synthesized as described in the preceding examples. To examine the biological activities of the three compounds, S1P-driven Transwell migration assay was performed with human primary B cells isolated from human PBMC (peripheral blood mononuclear cells).

In the preparation of human primary B cells, human B cells were isolated from human PBMC (peripheral blood mononuclear cells) by B cell isolation kit (Myltenyi Biotech). Then, the isolated B cells were seeded and maintained in a <NUM>-cm dish in IMDM medium supplemented with <NUM>% fetal bovine serum (Gibco) and <NUM> ng/ml IL2 (Peprotech Inc.

<FIG> shows that staining analysis of the isolated S1P<NUM>-expressing human B cells, 2x10<NUM> B cells were incubated with <NUM>µg/ml of anti-S1P<NUM> receptor antibody (AbD Serotec) in PBS containg <NUM>% BSA on ice for <NUM> mininutes. Cells were washed and incubated with FITC-conjugated goat anti-mouse IgG, diluted <NUM>:<NUM> in PBS/BSA, on ice for <NUM> minutes in the dark. The cells were then analyzed by FACS (FACSCanto II, BD Biosciences).

For chemotaxis assays, <NUM>µl of the maintained human B cells (4x10<NUM> cells) were transferred into <NUM>-ml eppendorf tube and added with fingolimod, fingolimod phosphate, NHS-PEGs-conjugated fingolimod, and the drug bundle with a free TCO functional group and with five fingolimod molecules, respectively, at a final concentration of <NUM> and <NUM> at <NUM> for <NUM> hours. Subsequently, <NUM>µl of treated B cells were added to the upper chamber of a <NUM>-mm Trans-well with a <NUM> pore polyester membrane insert (Corning), and the lower chamber of the Trans-well had contained <NUM>µl of IMDM medium with sphingosine-<NUM>-Phosphate molecule at a final concentration of <NUM>. After <NUM> hours, the migrated cells in the lower chambers were collected and further stained with trypan blue and counted by hemocytometer. For each measurement, the specific migration was calculated as follows: [(Number of cells in lower chamber)/ (Number of cells in lower + upper chamber) x100]- (cell migration percentage at <NUM> attractant)]. The result of the percentage of specific migrated cells is shown in <FIG>.

<FIG> shows the assay results of the biological activity of NHS-PEGs-conjugated fingolimod and the drug bundle with a free TCO functional group and with five fingolimod molecules. The result indicates that the fingolimod molecule conjugated with a linking arm had similar biological activity to block B-cell migration as the unmodified fingolimod.

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)<NUM>.

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: <NUM>.

Illustrated below is the configuration of the prepared <NUM>-chain (scFv α RSV)-hlgG1. Fc-(scFv α CD32a) molecular construct.

In this Example, the gene-encoding sequence was placed in pcDNA3 expression cassette. Expi293F cells were seeded at a density of <NUM> × <NUM><NUM> viable cells/ml in Expi293F expression medium and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in <NUM>-ml medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ <NUM> transfection reagent. The transfected cells were incubated at <NUM> for <NUM> to <NUM> hours post-transfection in an orbital shaker (<NUM> rpm) and the cells were added ExpiFectamine™ <NUM> transfection enhancer <NUM> and enhancer <NUM> to the shaker flask, and incubated for <NUM> days. Culture supernatants were harvested and recombinant <NUM>-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 <NUM>% SDS-PAGE shown in <FIG>. The Fc-fusion molecular construct was revealed as the major band at about <NUM> kDa, consistent with the expected size.

To examine the binding ability of recombinant <NUM>-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 <NUM>µg/mL of Protein F of RSV (Sino biological Inc. Recombinant <NUM>-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. The ELISA results in <FIG> show that the recombinant <NUM>-chain (scFv α RSV)-hIgG1. Fc-(scFv α CD32a) fusion protein bind to Protein F of RSV, using adalimumab scFv as a control scFv.

<FIG> shows binding activity of the recombinant Fc-fusion protein to ectodomain of CD32a. ELISA plates were coated with <NUM>µg/mL of recombinant ectodomain of CD32a. Recombinant <NUM>-chain (scFv α RSV)-hIgG1. Fc-(scFv α CD32a) fusion protein was detected by HRP-conjugated goat anti-human IgG1. <FIG> shows that the recombinant <NUM>-chain (scFv α RSV)-hIgG1. Fc-(scFv α CD32a) Fc-fusion protein has binding activity to recombinant ectodomain of CD32a. Recombinant <NUM>-chain (scFv α endotoxin)-IgG1. Fc protein was used as a control antibody.

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)<NUM>.

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: <NUM>. 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 <FIG> shows that the recombinant chain of the new construct has a size of about <NUM> kDa, consistent with the expected size.

<FIG> shows ELISA results of the recombinant <NUM>-chain (scFv α endotoxin)-(scFv α CD32a)-hIgG1. Fc binding to E. coli LPS <NUM>:B4 (Sigma Aldrich). ELISA plates were coated with <NUM>µ g/ml poly-L-lysine. Subsequently, the poly-L-lysine-coated plates were further coated with <NUM>µg/ml E. coli LPS <NUM>:B4. The recombinant fusion protein was detected by HRP-conjugated goat anti-human IgG. The ELISA results show that the present recombinant Fc-fusion protein has binding activity to E. coli LPS <NUM>:B4 (Sigma Aldrich); <FIG> shows that the recombinant Fc-fusion protein has binding activity to ectodomain of CD32a.

Illustrated below is the configuration of the thus-prepared <NUM>-chain (scFv α endotoxin)-(scFv α CD32)-hIgG1. Fc molecular construct.

The <NUM>-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)<NUM>.

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: <NUM>.

Illustrated herein is the configuration of the prepared <NUM>-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 <NUM> × <NUM><NUM> viable cells/ml in Expi293F expression medium and maintained for <NUM> to <NUM> hours prior to transfection to ensure that the cells were actively dividing at the time of transfection. At the time of transfection, <NUM>×<NUM><NUM> cells in <NUM>-ml medium in a <NUM>-liter Erlenmeyer shaker flask were transfected by ExpiFectamine™ <NUM> transfection reagent. The transfected cells were incubated at <NUM> for <NUM> to <NUM> hours post-transfection in an orbital shaker (<NUM> rpm) and the cells were added ExpiFectamine™ <NUM> transfection enhancer <NUM> and enhancer <NUM> to the shaker flask, and incubated for <NUM> days. Culture supernatants were harvested and recombinant <NUM>-chain (Interferon-β-1a)-hIgG4. 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)-hIgG4. Fc-(scFv α TfR1) protein was determined and analyzed by <NUM>% SDS-PAGE shown in <FIG>. The Fc-fusion molecular construct was revealed as the major band at about <NUM> kDa, consistent with the expected size.

Binding activity of recombinant (Interferon-β-1a)-hIgG4. Fc-(scFv α TfR1) was assayed by ELISA using a <NUM>-well plate coated with recombinant (Interferon-β-1a)-hIgG4. Fc-(scFv α TfR1) protein in <NUM> µ g/ml concentration, <NUM>µl per well. The scFv specific for ectodomain of TfR1 is as a negative control. Recombinant <NUM>-chain (Interferon-β-1a)-hIgG4. Fc-(scFv α TfR1) was detected by HRP-conjugated rabbit anti-human interferon-β polyclonal antibody (Santa Cruz Biotechnology, Dallas, USA). Next, <NUM>µl of TMB substrate was added for color development. The reaction was stopped by <NUM>µl of <NUM> HCI. Absorbance at <NUM> was measured with a plate reader. Each bar represents the mean OD450 value of duplicate samples.

<FIG> shows ELISA analysis of the present the molecular construct. The ELISA results show that (Interferon-β-1a)-hIgG4. Fc-(scFv α TfR1) fusion protein bound specifically to recombinant ectodomain of TfR1 protein.

The VL and VH of the scFv specific for integrin α4 were from monoclonal antibody natalizumab. The <NUM>-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)<NUM>. The result of <NUM>% SDA-PAGE in <FIG> shows that the recombinant chain of the new construct has a size of about <NUM> kDa (indicated by arrow), consistent with the expected size.

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: <NUM>.

Illustrated herein is the configuration of the prepared <NUM>-chain (scFv α integrin α4)-IgG4. Fc-(scFv α TfR1) molecular construct.

To examine the binding ability of recombinant <NUM>-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.

1x10<NUM> Jurkat T cells was maintained in the RPMI1640 medium supplemented with <NUM>% FBS at a density of 1x10<NUM>. The cells were kept in <NUM> with <NUM>% CO<NUM> in a humidified chamber. 1x10<NUM> Jurkat T cells were washed with the binding buffer (phosphate-buffered saline with <NUM>% FBS, <NUM> EDTA and 20ng/ml NaN<NUM>) twice. <NUM>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 <NUM>µg/ml of recombinant (scFv α integrin α4)-IgG4. Fc-(scFv α TfR1) protein on ice for <NUM> minutes, using recombinant <NUM>-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 <NUM>:<NUM> in blocking buffer, at on ice for <NUM> in the dark. The stained cells were analyzed on a FACSCanto II flow cytometer (BD Biosciences).

<FIG> shows results of the cell staining analysis of recombinant <NUM>-chain (scFv α integrin α4)-IgG4. Fc-(scFv α TfR1) protein on integrin α4-expressing Jurkat T cells. The construct bound to Jurkat T cells substantially positively.

To test the effects of recombinant <NUM>-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 <NUM>% fetal bovine serum (Gibco) and <NUM> U/ml penicillin-streptomycin (Gibco), at the density between 3x10<NUM> and 2x10<NUM> cells/ml. The cells were kept in <NUM> with <NUM>% CO<NUM> in a humidified chamber. To differentiate U937 into macrophage-like cells, 1x10<NUM> cells/ml of U937 were incubated with <NUM> ng/ml of phorbol <NUM>-myristate <NUM>-acetate (PMA, Sigma Aldrich). After <NUM> hours, non-adherent cells were removed, and adherent cells were washed and seeded into <NUM>-well plates.

5x10<NUM> cells/well of differentiated U937 were seeded into <NUM>-well plates the day before assay. Cells were stimulated with <NUM>µg/ml E. coli LPS <NUM>:B4 (Sigma Aldrich) alone, or premixes of LPS and <NUM>µg/ml of (scFv α endotoxin)-hIgG1Fc, <NUM>µg/ml of (scFv α endotoxin)-hIgG1Fc-(scFv α CD32a) or <NUM>µg/ml of anti-CD32a scFv. The stimulation proceeded for <NUM> 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 <NUM>µg/mL of capture antibody in PBS at <NUM> overnight. Wells were subsequently blocked by <NUM>% in PBS for <NUM> hour and incubated with diluted culture supernatant for <NUM> hours. <NUM> 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 HCI. Plates were read at <NUM> 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.

Claim 1:
A linker unit comprising, a center core and a plurality of linking arms, wherein
the center core comprises a first polypeptide comprising a plurality of lysine (K) residues, wherein each K residue and its next K residue are separated by a filler sequence comprising glycine (G) and serine (S) residues, and the number of K residues ranges from <NUM> to <NUM>;
each of the plurality of linking arms has an N-hydroxysuccinimidyl (NHS) group at one end and a functional group at the other end and is linked to one of the K residues of the center core by forming an amide bond between the amine group of the K residue and the NHS group, wherein each of the linking arms is a PEG chain having <NUM>-<NUM> repeats of EG units and the functional group is a NHS; and
the amino acid residue at the N- or C-terminus of the center core has the azide or the alkyne group.