(43) International Publication Date
`4 December 2014 (04.12.2014)
`
`(51)
`
`International Patent Classification:
`A61K 39/395 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US2014/039821
`
`(22)
`
`International Filing Date:
`
`(25)
`
`(26)
`
`(30)
`
`(71)
`
`(72)
`
`Filing Language:
`
`Publication Language:
`
`28 May 2014 (28.05.2014)
`
`English
`
`English
`
`Priority Data:
`61/827,763
`
`28 May 2013 (28.05.2013)
`
`US
`
`Applicants: DCB-USA LLC [US/US]; 1007 North Or-
`ange Street, Ninth floor, New Castle County, Wilmington,
`Delaware 19801 (US). KAOHSIUNG MEDICAL UNI-
`VERSITY [CN/CN]; No. 100, Shih-Chuan lst Road, Ka-
`ohsiung, 80708 (TW).
`
`Inventors: CHENG, 'l'ian—Lu; No. 100, Shih—Chuan 1st
`Road, Kaohsiung, 80708 (TW). CHUANG, Chih-Hung;
`No. 100, Shih—Chuan 1st Road, Kaohsiung, 80708 (TW).
`KO, Hsiu-Fen; No. 100, Shih-Chuan 1st Road, Ka-
`ohsiung, 80708 (TW). LU, Yun—Chi; No. 100, Shih-Chuan
`1st Road, Kaohsiung, 80708 (TW).
`
`(74)
`
`Agent: MUNCY, GEISSLER, OLDS & LOWE, P.C.;
`4000 Legato Rd., Suite 310, Fairfax, Virginia 22033 (US).
`
`\9
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`WIPOI PCT
`
`(81)
`
`(10) International Publication Number
`
`WO 2014/193973 A2
`
`Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ,
`UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV,
`MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`KM, ML, MR, NE, SN, TD, TG).
`Published:
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`without international search report and to be republished
`upon receipt ofthat report (Rule 48.2(g))
`
`with sequence listing part ofdescription (Rule 5.2(a))
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`
`
`W02014/193973A2|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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`(54) Title: ANTIBODY LOCKER FOR THE INACTIVATION OF PROTEIN DRUG
`
`1,09
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`a
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`
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`or ’r
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`Disease site
`
`FIG. 2
`
`(57) Abstract: Disclosed herein is a hinge antibody capable of being selectively activated in a target cell or tissue to treat a condition
`therein. The hinge antibody includes a functional antibody, two inhibitory domains and four cleavable linkers. The functional anti—
`body is capable of treating the condition in an activated state, and has two light chains and two heavy chains. Each inhibitory domain
`includes a hinge domain of an immunoglobulin and consists of two peptide arms. Each cleavable linker includes a peptide substrate
`cleavable by an enzyme specifically or highly expressed in the target cell or tissue, and connects one of the peptide arms of the inhib -
`itory domains to the N—terminal of one of the light chains and heavy chains of the functional antibody. Also disclosed herein are
`methods for preparing and using this hinge antibody.
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`

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`WO 2014/193973
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`PCT/U82014/039821
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`ANTIBODY LOCKER FOR THE INACTIVATION OF PROTEIN DRUG
`
`BACKGROUND OF THE INVENTION
`
`[0001] 1.
`
`FIELD OF THE INVENTION
`
`[0002] The
`
`present disclosure
`
`relates generally to
`
`antibody-based
`
`molecules useful as therapeutics for treating various medical conditions. More
`
`particularly, the disclosed invention relates to hinge antibodies that are selectively
`
`activated in a target cell or tissue so as to treat the medical conditions therein.
`
`[0003] 2. DESCRIPTION OF RELATED ART
`
`[0004] Antibody-based therapeutic agents, including monoclonal antibodies,
`
`are emerging as one of the major classes of drugs effective in the treatment of
`
`various diseases.
`
`Of the top 10 drugs by global sales in 2012,
`
`five are
`
`therapeutic
`
`antibodies,
`
`including, HUMIRATM, REMICADETM, RITUXANTM,
`
`HERCEPTINT'V', and AVASTINTM.
`
`Said five drugs grossed about $45 billion
`
`around the globe, approximating 60% of the global antibody-based therapeutic
`
`agent market in that year. The global market is expected to grow continuously as
`
`existing products expand their approved usage and new entrants launch into the
`
`marketplace.
`
`[0005] Although the field continues to advance, many challenges remain in
`
`order to bring more efficacious and affordable antibody-based candidates to the
`
`market. One problem associated with current antibody-based therapeutic agents
`
`is the poor selectivity of site of action. Monoclonal antibodies and soluble fusion
`
`proteins are specific for binding to and neutralizing their intended target molecules
`
`(such as antigens and cell surface receptors). However, most target molecules
`
`are not specific to the disease site; rather, they may be present in cells or tissues
`
`other than the disease site. Accordingly, the therapeutic agent may act in these
`
`non—disease normal cells or tissues. This off—target action may result in unwanted
`
`side
`
`effects.
`
`Consequently,
`
`developing
`
`highly
`
`targeted
`
`antibody—based
`
`therapeutic agents is desirable.
`
`[0006] One possible scheme of avoiding off-target action and increasing
`
`selectivity is to provide a pro-antibody activatable in the target site.
`
`For example,
`
`U.S. Patent No. 8,399,219 and U.S. Patent Application Publication No.
`
`2010/0189651 disclose protease activatable antibodies that are modified by a
`
`peptide mask or masking moiety.
`
`In these documents,
`
`the phage display
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`technique is used to screen peptides or moieties capable of inhibiting/reducing the
`
`binding of the functional antibody to its binding target. However, the masking
`
`moieties obtained by such methods could not be universally applied to all
`
`antibodies for they are identified based on their inhibitory ability toward a specific
`
`target. Therefore, it is necessary in their approach to develop a masking moiety
`
`for each antibody-based therapeutic agent, which is time consuming, expensive,
`
`and complicated. Additionally, the introduction of masking moieties runs the risk
`
`of inducing unnecessary immuno response to the subject.
`
`[0007] A similar approach is described in U.S. Patent Application
`
`Publication No. 2010/0189727, which proposed a masking ligand non—covalently
`
`bound to an antigen binding site of an antibody so as to inactivate the antibody.
`
`In particular, the masking ligand comprises two copies of the epitope of the antigen
`
`to which the antibody specifically binds and a cleavable polypeptide cleavable
`
`linkerjoined to each copy of the epitope. Similar to the phage display technique
`
`described above, the masking ligand also needs to be specifically designed with
`
`respect to each antibody, and hence the development of such inactivated antibody
`
`is also time-consuming and with high cost.
`
`Further, since the masking ligand has
`
`a high affinity toward the therapeutic antibody, there might be certain masking
`
`ligands attached to the antibody after the cleavage of the cleavable polypeptide
`
`cleavable linker. These residual masking ligands may hinder the therapeutic
`
`action of the antibody.
`
`[0008] In View of the foregoing, there exists a need in the art for providing
`
`next generation therapeutics that are carefully designed and engineered to
`
`possess features such as improved selectivity of site of action as well as enhanced
`
`efficacy. Further, such design and engineering schemes shall be applicable to a
`
`wide variety of antibody—based therapeutic agents, and would not incur unwanted
`
`immuno response.
`
`SUMMARY
`
`[0009] 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
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`purpose is to present some concepts disclosed herein in a simplified form as a
`
`prelude to the more detailed description that is presented later.
`
`[0010] In one aspect, the present disclosure is directed to a hinge antibody.
`
`This antibody—based therapeutic agent is capable of being selectively activated in a
`
`target cell or tissue to treat a condition in the target cell or tissue.
`
`[0011]According to various embodiments of the present disclosure,
`
`the
`
`hinge antibody comprises a functional antibody, two inhibitory domains, and four
`
`cleavable linkers. The functional antibody is capable of treating the condition in
`
`an activated state and comprises two light chains and two heavy chains. Each of
`
`the two inhibitory domains consists of two peptide arms interconnected by disulfide
`
`bonds.
`
`Each inhibitory domain consists of
`
`two peptide arms
`
`that are
`
`interconnected by disulfide bonds. Each of the four cleavable linkers comprises a
`
`peptide substrate cleavable by an enzyme that is specifically or highly expressed in
`
`the target cell or tissue. Each cleavable linker connects one of the two peptide
`
`arms of the two inhibitory domains to the N—terminals of one of the two light chains
`
`and two heavy chains of the functional antibody.
`
`[0012] According to certain embodiments of the present disclosure, each of
`
`the two inhibitory domains is a hinge domain of an immunoglobulin A (lgA), an
`
`immunoglobulin D or an immunoglobulin G (IgG), or a fragment of the hinge
`
`domain. For example,
`
`the inhibitory domain may comprise any of
`
`fowling
`
`sequences, SEQ ID Nos. 10, 11, 12 and 13 of I96, 14 and 15 of IgA, and 54 and
`
`55 of lgD.
`
`[0013] In optional embodiments, the functional antibody is an anti-TNF-q
`
`antibody,
`
`anti-RANKL antibody,
`
`anti-CTLA-4 antibody,
`
`anti-HER2 antibody,
`
`anti-EG FR antibody, anti-VEGF antibody, anti-VEGFRZ) antibody, anti-IL6R
`
`antibody,
`
`anti—IL12/23
`
`antibody,
`
`anti—CD3
`
`antibody,
`
`anti—CD1 1a
`
`antibody,
`
`anti-CD20 antibody, anti-CD25 antibody, anti-CD30 antibody, anti-CD33 antibody
`
`or anti-CD52 antibody.
`
`For example, the amino acid sequence of the light chain
`
`of the functional antibody is any of the amino acid sequences of SEQ ID Nos. 1, 2,
`
`3, 4, 5, 6, 7, 8 and 9; while the amino acid sequence of the heavy chain of the
`
`functional antibody is any of the amino acid sequences of SEQ ID Nos. 58, 59, 60,
`
`61, 62, 63, 64, 65 and 66.
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`[0014] In certain embodiments, the peptide substrate is cleavable by any of
`
`the following enzyme: a matrix metalloproteinase (MMP), a cathepsin (CTS), a
`
`caspase (CASP), or a disintegrin and metalloproteinase (ADAM).
`
`For example,
`
`according to some embodiments,
`
`the enzyme is MMP—2 or MMP—9 and each
`
`cleavable linker comprises the amino acid sequence of SEQ ID No. 16.
`
`[0015] According to some embodiments of the present disclosure,
`
`the
`
`functional antibody is an anti-TNF-or antibody, which has a light chain having the
`
`amino acid sequence of SEQ ID No.
`
`1 and a heavy chain having the amino acid
`
`sequence of SEQ ID No. 58, each of the cleavable linkers comprises the amino
`
`acid sequence ofSEQ ID No. 16; and each of the inhibitory domain comprises the
`
`amino acid sequence of SEQ ID No. 10.
`
`[0016] In another aspect, the present disclosure is directed to an expression
`
`system for
`
`producing
`
`the
`
`hinge
`
`antibodies
`
`according
`
`to
`
`the
`
`above
`
`aspect/embodiments of the present disclosure.
`
`[0017] According to various embodiments of the present disclosure,
`
`the
`
`expression system for producing comprises a first nucleic acid sequence and a
`
`second nucleic acid sequence. The first nucleic acid sequence, comprising, from
`
`5’ to 3’, a first inhibitory domain-encoding region, a first cleavable linker-encoding
`
`region and a light chain-encoding region. The first
`
`inhibitory domain-encoding
`
`region encodes a first peptide arm of an inhibitory domain of any of the
`
`above—described hinge antibodies. The first cleavable linker—encoding region
`
`encodes a cleavable linker of the above-mentioned hinge antibody, and the
`
`cleavable linker is a peptide substrate cleavable by an enzyme that is specifically
`
`or highly expressed in the target cell or tissue. The light chain-encoding region
`
`encodes a light chain of a functional antibody of the above-mentioned hinge
`
`antibody, in which the functional antibody is capable of treating the condition in an
`
`activated state. The second nucleic acid sequence, comprising, from 5’ to 3’, a
`
`second inhibitory domain inhibitory domain-encoding region, a second cleavable
`
`linker-encoding region and a heavy chain-encoding region. The second inhibitory
`
`domain-encoding region encodes a second peptide arm of the inhibitory domain of
`
`the hinge antibody. The second cleavable linker—encoding region encodes the
`
`cleavable linker of the hinge antibody. The heavy chain—encoding region encodes
`
`a heavy chain of the functional antibody of the hinge antibody.
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`[0018] In some optional embodiments of the present disclosure, the first and
`
`second nucleic acid sequences can be constructed in a single expression vector.
`
`For example, the expression system may further comprise a connecting nucleic
`
`acid sequence that connects the first nucleic acid sequence and the second
`
`nucleic acid sequence. Non-limiting examples of the connecting nucleic acid
`
`sequence include a sequence encoding a Furin-2A polypeptide or an internal
`
`ribosome entry site (IRES) sequence.
`
`[0019] In the case where the first and second nucleic acid sequences are
`
`constructed in a single expression vector,
`
`the expression system may further
`
`optionally comprise a regulatory sequence operably linked to the first nucleic acid
`
`sequence and the second nucleic acid sequence, so as to regulate the translation
`
`of the first nucleic acid sequence,
`
`the second nucleic acid sequence, and,
`
`optionally, the connecting nucleic acid sequence in a host cell. Alternatively, the
`
`expression system may comprise at least two separate regulatory sequences
`
`operably linked to the first and the second nucleic acid sequences, respectively, to
`
`allow the individual regulation of the expression of the first and second nucleic acid
`
`sequences.
`
`[0020] In some other embodiments,
`
`the first and second nucleic acid
`
`sequences may be constructed in two separate expression vectors.
`
`For instance,
`
`the first nucleic acid sequence, together with an operably-linked first regulatory
`
`sequence is constructed in a first expression vector, while the second nucleic acid
`
`sequence,
`
`along with
`
`an operably-linked second regulatory sequence is
`
`constructed in a second expression vector. The first and second expression
`
`vectors may then be delivered into and expressed in a same host cell or different
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`host cells.
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`[0021]According to certain embodiments of the present disclosure,
`
`the
`
`inhibitory domain is a hinge domain of an immunoglobulin A (IgA), an
`
`immunoglobulin D or an immunoglobulin G (IgG), or a fragment of the hinge
`
`domain.
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`
`[0022] According to various embodiments of the present disclosure,
`
`the
`
`expression system encodes any of the above—mentioned hinge antibodies.
`
`For
`
`example, when the expression system is embodied by a single construct, the
`
`nucleic acid sequence of the construct can be any of SEQ ID Nos. 17, 18, 19, 20,
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`PCT/US2014/039821
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`21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
`
`43, 44, 45, 46, 47, 48, 49 and 50.
`
`In the case where expression system is
`
`embodied as a two-vector (or two-plasmid) system, the first nucleic acid sequence
`
`is any of SEQ ID Nos. 67, 69, 71, 73, 75, and 77; whereas the second nucleic acid
`
`sequence is any of SEQ ID Nos. 68, 70, 72, 74, 76 and 78.
`
`[0023] In yet another aspect,
`
`the present disclosure is directed to a
`
`recombinant vector suitable for use in manufacturing the hinge antibodies
`
`according to the above aspect/embodiments of the present disclosure.
`
`[0024] According to certain embodiments of the present disclosure,
`
`the
`
`recombinant vector comprises the synthetic nucleic acid molecule according to the
`
`above-mentioned aspect/embodiments of the present disclosure, and one or more
`
`regulatory sequences operatively linked to the synthetic nucleic acid molecule, so
`
`that the vector, under suitable conditions and in an appropriate host cell, is capable
`
`of
`
`expressing
`
`the
`
`hinge
`
`antibody
`
`according
`
`to
`
`the
`
`above-mentioned
`
`aspect/embodiments of the present disclosure.
`
`[0025] In still another aspect, the present invention is directed to a method
`
`for treating a subject;
`
`in particular, a subject with cancer or an autoimmune
`
`disease.
`
`[0026] According to some embodiments of
`
`the present
`
`invention,
`
`the
`
`method comprises administering to the subject a therapeutically effective amount
`
`of the hinge antibodies according to the above aspect/embodiments of the present
`
`disclosure.
`
`For example,
`
`the hinge antibody may be administered orally,
`
`subcutaneously, intravenously, intrathecally or intramuscularly to the subject.
`
`[0027] Many of the attendant features and advantages of the present
`
`disclosure will becomes better understood with reference to the following detailed
`
`description considered in connection with the accompanying drawings.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`[0028] The present description will be better understood from the following
`
`detailed description read in light of the accompanying drawings, where:
`
`[0029] Figure 1
`
`is a schematic diagram illustrating the structure of a hinge
`
`antibody according to certain embodiments of the present disclosure;
`
`[0030] Figure 2 is a schematic diagram illustrating the design scheme of the
`
`hinge antibody according to embodiments of the present disclosure;
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`[0031] Figure 3 is a schematic diagram illustrating a nucleic acid molecule
`
`encoding a hinge antibody according to certain embodiments of the present
`
`disclosure;
`
`[0032] Figure 4 is a schematic diagram illustrating the overall structure of a
`
`hinge antibody according to one embodiment of the present disclosure;
`
`[0033] Figure 5 is a photograph of an SDS PAGE gel according to one
`
`working example of the present disclosure;
`
`[0034] Figure 6 is a photograph of two SDS PAGE gels according to one
`
`working example of the present disclosure;
`
`[0035] Figure 7 is a bar graph illustrating the binding capacity of various
`
`antibodies according to one working example of the present disclosure;
`
`[0036] Figure 8 is a bar graph illustrating the TNF-or signal according to one
`
`working example of the present disclosure;
`
`[0037] Figure 9 is a bar graph illustrating the binding capacity of various
`
`antibodies according to another working example of the present disclosure;
`
`[0038] Figure 10 is a bar graph illustrating the binding capacity of various
`
`antibodies according to yet another working example of the present disclosure;
`
`[0039] Figure 11 provides photographs illustrating the in vivo localization
`
`and activation of hinge-dEG FR antibody at the tumor site of mice, according to one
`
`Example of the present disclosure; and
`
`[0040] Figure 12 is line graph indicating the in vivo anti—inflammatory effects
`
`of hinge-TNFo antibody against collagen-induced arthritis.
`
`[0041] 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.
`
`DESCRIPTION
`
`[0042] 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,
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`the same or equivalent functions and sequences may be accomplished by different
`
`examples.
`
`[0043] For convenience, certain terms employed in
`
`the specification,
`
`examples and appended claims are collected here. Unless defined otherwise, all
`
`technical and scientific terms used herein have the same meaning as commonly
`
`understood by one of the ordinary skill in the art to which this invention belongs.
`
`[0044] Unless
`
`othen/vise
`
`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
`
`othenNise 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 indicates othenNise. 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.
`
`[0045] Notwithstanding that the numerical ranges and parameters setting
`
`forth the broad scope of the invention are approximations, the numerical values set
`
`forth in the specific examples are reported as precisely as possible. Any
`
`numerical value, however, inherently contains certain errors necessarily resulting
`
`from the standard deviation found in the respective testing measurements. Also,
`
`as used herein, the term “about” generally means within 10%, 5%, 1%, or 0.5% of
`
`a given value or
`
`range.
`
`Alternatively,
`
`the term “about” means within an
`
`acceptable standard error of the mean when considered by one of ordinary skill in
`
`the art.
`
`[0046] The term “antibody-based therapeutic agent” is intended to mean a
`
`therapeutic agent that inhibits the pharmacological actions of endogenous human
`
`proteins or pathogens.
`
`Said
`
`"therapeutic
`
`agent," when present
`
`in
`
`a
`
`therapeutically effective amount, produces a desired therapeutic effect on a
`
`subject.
`
`For the purpose of the present disclosure, antibody-based therapeutic
`
`agents encompass antibodies and fusion proteins that are highly specific for
`
`binding to and neutralizing their intended target molecules.
`
`[0047] The term "antibody" as used herein includes full—length antibodies
`
`and any antigen binding fragment or single chains thereof. The basic functional
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`unit of each antibody is an immunoglobulin monomer which is a Y-shaped
`
`molecule consisting of two heavy chains and two light chains interconnected by
`
`disulfide bonds. A “functional antibody” encompasses a full-length antibody or
`
`one or more fragments of the antibody that maintain the specific binding ability
`
`thereof; example of such functional fragments including Fab (antigen-binding
`
`fragment), Fv (variable fragment), and F(ab')2, Fab', scFv (single chain fragment
`
`variable), and the like. An antibody may be monoclonal or polyclonal and may be
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`of human or non-human origin or a chimeric protein.
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`[0048] Here, a “cleavable linker“ is a peptide substrate cleavable by an
`
`enzyme. Operatively, the cleaveable linker, upon being cleaved by the enzyme,
`
`allows for activation of the present hinge antibody.
`
`Preferably, the cleaveable
`
`linker is selected so that activation occurs at the desired site of action, which can
`
`be a site in or near the target cells (e.g., carcinoma cells) or tissues. For example,
`
`the cleaveable linker
`
`is a peptide substrate specific for an enzyme that
`
`is
`
`specifically or highly expressed in the site of action, such that the cleavage rate of
`
`the cleavable linker in the target site is greater than that in sites other than the
`
`target site.
`
`[0049] The term "ligand" means any molecule that specifically binds or
`
`reactively associates or complexes with a
`
`receptor,
`
`substrate,
`
`antigenic
`
`determinant, or other binding site on a target cell or tissue. Examples of ligands
`
`include antibodies and fragments thereof (e.g., a monoclonal antibody or fragment
`
`thereof), enzymes (e.g., fibrinolytic enzymes), biologic response modifiers (e.g.,
`
`interleukins,
`
`interferons, erythropeoitin, or colony stimulating factors), peptide
`
`hormones, and antigen-binding fragments thereof.
`
`[0050] As used herein,
`
`the term “nucleic acid” designates single- or
`
`double—stranded RNA, mRNA, and DNA including cDNA and genomic DNA.
`
`Unless otherwise indicated, a particular nucleic acid sequence also implicitly
`
`encompasses conservatively modified variants thereof (e.g., degenerate codon
`
`substitutions) and complementary sequences, as well as the sequence explicitly
`
`indicated. Also, the left-hand end of single-stranded polynucleotide sequences is
`
`the 5' end; the left—hand direction of double—stranded polynucleotide sequences is
`
`referred to as the 5' direction, unless specified othenNise.
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`[0051] The
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`terms
`
`"polypeptide,"
`
`"peptide"
`
`and
`
`"protein"
`
`are used
`
`interchangeably herein to refer to a polymer of amino acid residues. These terms
`
`also encompass the term "antibody." The term "amino acid" refers to naturally
`
`occurring and synthetic amino acids, as well as amino acid analogs and amino
`
`acid mimetics that function in a manner similar to the naturally occurring amino
`
`acids.
`
`In the polypeptide notation used herein, the left-hand direction is the amino
`
`(N)— terminal direction and the right-hand direction is the carboxyl
`
`(C)—terminal
`
`direction, in accordance with standard usage and convention.
`
`[0052] Throughout the present disclosure, the term "synthetic" nucleic acid
`
`or amino acid means a nucleic acid or amino acid sequence that is not found in
`
`nature.
`
`It
`
`is intended that synthetic sequences designed by the method be
`
`included in the invention in any form, e.g., paper or computer readable, and
`
`physically created nucleic acids or polypeptides. Physically created nucleic acids
`
`and polypeptides of the invention are part of the invention, whether derived directly
`
`from the designed sequence, or copies of such sequences (e.g., made by PCR,
`
`plasmid replication, chemical synthesis, and the like). The term "synthetic nucleic
`
`acid" can include, for example, nucleic acid sequences derived or designed from
`
`wholly artificial amino acid sequences, or nucleic acid sequences with single or
`
`multiple nucleotide changes as compared to the naturally occurring sequence,
`
`those created by random or directed mutagenesis, chemical synthesis, DNA
`
`shuffling methods, DNA reassembly methods, or by any means known to one of
`
`skill
`
`in the art. Such alterations can be done without changing the amino acid
`
`sequence encoded by the nucleic acid sequence, or can modify the amino acid
`
`sequence to leave a desired function of the encoded protein unaltered or
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`enhanced.
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`[0053] As used herein, the term “vector” refers to composition of matter (e.g.,
`
`phage, plasmid, viral vectors as well as artificial chromosomes, such as bacterial
`
`or yeast artificial chromosomes) used to transmit genetic material into a host cell.
`
`A vector may be composed of either DNA or RNA. The vector may be introduced
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`into a host cell by various techniques well known in the art. The regulatory
`
`sequence of a vector is a nucleic acid sequence required for expression of a target
`
`gene product operably linked thereto. The term "operatively linked" as used
`
`herein means that the regulatory nucleic acid and the nucleic acid of interest are
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`linked so that the expression of the said nucleic acid of interest can be governed by
`
`the said regulatory nucleic acid, i.e. the regulatory nucleic acid sequence shall be
`
`functionally linked to the
`
`said nucleic acid sequence to be expressed.
`
`Accordingly, the regulatory nucleic acid sequence and, the nucleic acid sequence
`
`to be expressed may be physically linked to each other, e.g., by inserting the
`
`regulatory nucleic acid sequence at the 5'end of the nucleic acid sequence to be
`
`expressed. Alternatively, the regulatory nucleic acid sequence and the nucleic
`
`acid to be expressed may be merely in physical proximity so that the regulatory
`
`nucleic acid sequence is capable of governing the expression of at least one
`
`nucleic acid sequence of interest. The regulatory nucleic acid sequence and the
`
`nucleic acid to be expressed are, preferably, separated by not more than 500 bp,
`
`300 bp, 100 bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 bp or 5 bp.
`
`[0054] The term “treating” as used herein refers to the application or
`
`administration of the present hinge antibody to a subject, who has a medical
`
`condition, a symptom of the condition, a disease or disorder secondary to the
`
`condition, or a predisposition toward the condition, with the purpose to partially or
`
`completely alleviate, ameliorate, relieve, delay onset of,
`
`inhibit progression of,
`
`reduce severity of, and/or reduce incidence of one or more symptoms or features
`
`of a particular disease, disorder, and/or condition. Generally, a “treatment”
`
`includes not just the improvement of symptoms or decrease of markers of the
`
`disease, but also a cessation or slowing of progress or worsening of a symptom
`
`that would be expected in absence of treatment. Beneficial or desired clinical
`
`results include, but are not
`
`limited to, alleviation of one or more symptom(s),
`
`diminishment of extent of disease, stabilized (i.e., not worsening) state of disease,
`
`delay or slowing of disease progression, amelioration or palliation of the disease
`
`state, and remission (whether partial or total), whether detectable or undetectable.
`
`[0055] The term “effective amount” as used herein refers to the quantity of a
`
`component which is sufficient
`
`to yield a desired therapeutic response.
`
`A
`
`therapeutically effective amount is also one in which any toxic or detrimental
`
`effects of the compound or composition are outweighed by the therapeutically
`
`beneficial effects. The specific effective or sufficient amount will vary with such
`
`factors as the particular condition being treated,
`
`the physical condition of the
`
`patient (e.g., the patient's body mass, age, or gender), the type of mammal or
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`animal being treated,
`
`the duration of the treatment,
`
`the nature of concurrent
`
`therapy (if any), and the specific formulations employed and the structure of the
`
`compounds or its derivatives. Effective amount may be expressed, for example,
`
`in grams, milligrams or micrograms or as milligrams per kilogram of body weight
`
`(mg/kg)-
`
`[0056] The term “subject” refers to a mammal including the human species
`
`that is treatable with the hinge antibody and/or methods of the present invention.
`
`The term “subject” is intended to refer to both the male and female gender unless
`
`one gender is specifically indicated.
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`[0057] The present
`
`invention is directed to hinge antibodies that are
`
`selectively activatable in a target cell or tissue. Methods and composition of
`
`matters (e.g., nucleic acid sequences and vectors) for preparing the present hinge
`
`antibodies, the pharmaceutical compositions comprising the hinge antibodies, as
`
`well treating methods using the same, also fall within the scope of the present
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`invenfion.
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`[0058] Figure 1
`
`is a schematic diagram illustrating the general structure of
`
`the hinge antibody 100 according to certain embodiments of the present invention,
`
`and Figure 2 is a schematic diagram illustrating the design scheme and action
`
`mechanism of the hinge antibody 100. As illustrated in Figure 1,
`
`the hinge
`
`antibody 100 comprises a functional antibody 110, two inhibitory domains 120, and
`
`four cleavable linkers 130 connecting the inhibitory domains 120 to the functional
`
`antibody 110. Referring to Figure 2,
`
`in the original, uncleaved form, the binding
`
`ability of said hinge antibody 100 toward its target ligand (L)
`
`is substantially
`
`inhibited (inactivated). Once the hinge antibody 100 is administered to a subject
`
`and reaches the target site, an enzyme (E) that is specifically or highly expressed
`
`in the target site would cleave the hinge antibo