Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-6, 8-9, 11-14, 17, 21-26, 54 and 55 are pending.
Claims 1-6, 8-9, 11-14, 17, 21-26, 54 and 55, drawn to a therapeutic combination that read on (A) SO1861 as the species of saponin, (B) EGF as the first binding molecule, (C) an antibody as the second binding molecule, (D) a ligand for binding to the second binding site of the cell-surface molecule are being acted upon in this Office Action.
Priority
Receipt is acknowledged of papers submitted under 35 U.S.C. 119(a)-(d), which papers have been placed of record in the file.
Objection and Rejection Withdrawn
The objection to claim 2, 17 and 22 is withdrawn in light of the claim amendment.
The rejection of claims 4, 5, 8 and 26 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph is withdrawn in view of the claim amendment.
The provisional rejection of claims 1-6, 8, 9, 11-14, 17, 21-26 and 54 on the ground of nonstatutory obviousness-type double patenting as being unpatentable over 28-29 of copending Application No. 17/312,104 (reference).
Claim rejections under - 35 U.S.C. 112
The following is a quotation of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), first paragraph:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Written Description Guidelines for examination of patent applications indicates, “the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical characteristics and/or other chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show applicant was in possession of the claimed genus.” (see MPEP 2163).
Claim 1 encompasses a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second binding molecule different from the first binding molecule, the second binding molecule comprising any second binding region different from the first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from the first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule,
wherein the first pharmaceutical composition and the second pharmaceutical composition optionally further comprise a pharmaceutically acceptable excipient and optionally further comprise a pharmaceutically acceptable diluent.
Claim 2 encompasses a pharmaceutical composition comprising:
any conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any triterpenoid saponin of the monodesmosidic type or the bidesmosidic type; and
any conjugate comprising any second binding molecule different from said first binding molecule, the second binding molecule comprising any second binding region different from said first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from said first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule, and optionally further comprising a pharmaceutically acceptable excipient and optionally further comprising a pharmaceutically acceptable diluent.
Claim 3 encompasses the therapeutic combination of claim 1, wherein the first binding molecule is a first proteinaceous binding molecule or a first non-proteinaceous ligand comprising the first binding region for binding to the first binding site of the cell-surface molecule, and/or wherein the second binding molecule is a second proteinaceous binding molecule or a second non- proteinaceous ligand comprising the second binding region for binding to the second binding site of the cell-surface molecule.
Claim 4 encompasses the therapeutic combination of claim 1, wherein the first binding molecule is a first proteinaceous binding molecule and wherein the saponin is covalently bound to an amino acid residue of the first binding molecule, or via a linker.
Claim 5 encompasses the therapeutic combination of claim 1, wherein the first binding site is a first epitope of said cell-surface molecule such as any cell-surface receptor and wherein the second binding site is a second epitope of said, same, cell-surface molecule, wherein the second epitope is different from the first epitope.
Claim 6 encompasses the therapeutic combination of claim 1, wherein the saponin is a bidesmosidic triterpene saponin.
Claim 8 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule comprises any ligand for binding to the first binding site of the cell-surface molecule such as EGF, or
wherein the first binding region of the first binding molecule comprises or consists of an immunoglobulin or at least one binding fragment or binding domain of said immunoglobulin comprising the first binding region for binding to the first binding site of the cell-surface molecule, and/or
wherein the second binding region of the second binding molecule comprises or consists of a ligand for binding to the second binding site of the cell-surface molecule, or
wherein the second binding region of the second binding molecule comprises or consists of an immunoglobulin or at least one binding fragment or binding domain of said immunoglobulin comprising the second binding region for binding to the second binding site of the cell-surface molecule.
Claim 9 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule comprises any monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment, and/or
wherein the second binding region of the second binding molecule comprises or consists of a monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment.
Claim 11 encompasses the therapeutic combination of claim 1, wherein the first binding region and the second binding region are selected to simultaneously bind the same cell-surface molecule at the first binding site and at the second binding site.
Claim 12 encompasses the therapeutic combination of claim 1, wherein the first binding region is selected to bind to the first binding site of the cell-surface molecule without competing for the binding of the second binding region to the second binding site of the same cell-surface molecule, and wherein the second binding region is selected to bind to the second binding site of the cell-surface molecule without competing for the binding of the first binding region to the first binding site of the same cell-surface molecule.
Claim 13 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23, the saponin comprising a first saccharide chain at the C3beta-OH group of the saponin, the first saccharide chain optionally comprising a glucuronic acid moiety, and the saponin comprising a second saccharide chain linked to C28 of the saponin and comprising or consisting of a monosaccharide or a linear or branched oligosaccharide wherein optionally at least one saccharide moiety of the second saccharide chain comprises at least one acetyl group, for example 1, 2, 3 or 4 acetyl groups.
Claim 14 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a saponin isolated from any one or more of a Gypsophila species, a Saponaria species, an Agrostemma species and a Quillaja species.
Claim 17 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is Quillaja bark saponin, or a saponin derivative, or any of their stereoisomers or any combination thereof.
Claim 21 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene glycoside belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 of the aglycone core structure of the saponin, wherein the saponin is covalently bound to the first binding molecule, via an aldehyde function in the saponin via at least one linker.
Claim 22 encompasses the therapeutic combination of claim 21, wherein the aldehyde function in position C23 of the aglycone core structure of the at least one saponin is covalently bound to linker ε-maleimidocaproic acid hydrazide (EMCH), which linker is covalently bound via a thio-ether bond to a sulfhydryl group in the first binding molecule.
Claim 23 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene glycoside belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 of the aglycone core structure of the saponin and comprising a glucuronic acid unit in a first saccharide chain at the C3beta- OH group of the aglycone core structure of the saponin, wherein the saponin is covalently bound to an amino-acid residue of the first binding molecule via the carboxyl group of the glucuronic acid unit in the first saccharide chain.
Claim 24 encompasses the therapeutic combination of claim 23, wherein the at least one saponin comprises a glucuronic acid unit in its first saccharide chain at the C3beta-OH group of the aglycone core structure of the at least one saponin, wherein the glucuronic acid unit is covalently bound to linker 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), wherein the linker is covalently bound via an amide bond to an amine group in the first binding molecule.
Claim 25 encompasses the therapeutic combination of claim 1, wherein the cell-surface molecule is any cell-surface receptor.
Claim 26 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule and the second binding region of the second binding molecule comprise any antibody or any cell-surface molecule binding fragment thereof or cell-surface molecule binding domain(s) thereof and/or comprise or consist of any ligand for binding to the undisclosed cell-surface molecule,
with the proviso that the first binding region and the second binding region are different and with the proviso that the first binding site and the second binding site are different.
Claim 54 encompasses a kit comprising the therapeutic combination of claim 1, and instructions for use.
Claim 55 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is any one of: dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate, AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R, or any saponin derivative, a stereoisomer or a combination thereof.
Regarding “binding molecule”, the specification define as follow:
[0039] The term “binding molecule” has its regular scientific meaning and here refers to a molecule capable of specifically binding to another molecule such as a cell-surface molecule, e.g. a cell-surface receptor. Typical binding molecules are peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to e.g. a protein, a lipid, a (poly)saccharide, such as a cell-surface receptor or a cell-surface molecule. “Specifically binding” here refers to specific and selective binding with higher affinity than non-specific background binding.
Regarding “binding region”, the specification define as follow:
[0020] The term “binding region” has its regular scientific meaning and here refers to a part of a molecule or (a) chemical group(s) of a molecule or a(n) (linear or non-linear) amino-acid sequence of a protein or peptide and the like, that has the capacity to bind to a binding partner molecule. A typical binding region are the CDR loops of an immunoglobulin. A typical binding region of a protein is or are loop(s) of amino-acid residues comprised by said protein and capable of specifically binding to the binding site on a binding partner molecule such as a protein, cell-surface receptor, etc.
Regarding “binding site”, the specification define as follow:
[0021] The term “binding site” has its regular scientific meaning and here refers to a region on a macromolecule such as a protein, for example a cell-surface molecule such as a cell-surface receptor, that binds to another molecule such as a protein, for example a ligand, with specificity.
Regarding “cell-surface molecule”, the specification define as follow:
[0022] The term “cell-surface molecule” has its regular scientific meaning and here refers to a molecule that is present and exposed at the outside surface of a cell such as a blood cell or an organ cell, such as a mammalian cell, such as a human cell. Typically, a cell-surface molecule is a protein such as a receptor, or a lipid molecule or a polysaccharide.
Regarding “mono-desmosidic saponin”, the specification define as follow:
[0026] The term “mono-desmosidic saponin” has its regular scientific meaning and here refers to a triterpenoid saponin containing a single saccharide chain bound to the aglycone core, wherein the saccharide chain consists of one or more saccharide moieties.
Regarding “bi-desmosidic saponin”, the specification define as follow:
[0027] The term “bi-desmosidic saponin” has its regular scientific meaning and here refers to a triterpenoid saponin containing two saccharide chains bound to the aglycone core, wherein each of the two saccharide chains consists of one or more saccharide moieties.
Regarding “triterpenoid saponin”, the specification define as follow:
[0028] The term “triterpenoid saponin” has its regular scientific meaning and here refers to a saponin having a triterpenoid-type of aglycone core structure. The triterpenoid saponin differs from a saponin based on a steroid glycoside such as sapogenol in that such saponin comprising steroid glycoside has a steroid core structure, and the triterpenoid saponin differs from a saponin based on an alkaloid glycoside such as tomatidine in that such saponin comprising alkaloid glycoside has a alkaloid core structure.
Regarding “saponin”, the specification define as follow:
[0023] The term “saponin” has its regular scientific meaning and here refers to a group of amphipathic glycosides which comprise one or more hydrophilic glycone moieties combined with a lipophilic aglycone core which is a sapogenin. The saponin may be naturally occurring or synthetic (i.e. non-naturally occurring). The term “saponin” includes naturally-occurring saponins, derivatives of naturally-occurring saponins as well as saponins synthesized de novo through chemical and/or biotechnological synthesis routes. Saponin has a triterpene backbone, which is a pentacyclic C30 terpene skeleton, also referred to as sapogenin or aglycone. Within the context of the invention saponin is not considered an effector molecule nor an effector moiety in the conjugates according to the invention. Thus, in conjugates comprising a saponin and an effector moiety, the effector moiety is a different molecule than the conjugated saponin.
Regarding “effector molecule”, the specification define as follow:
[0033] The term “effector molecule”, or “effector moiety” when referring to the effector molecule as part of e.g. a covalent conjugate, has its regular scientific meaning and here refers to a molecule that can selectively bind to for example any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and regulates the biological activity of such one or more target molecule(s). In the conjugate of the invention the effector moiety for example exerts its effect in the cytosol, in the cell nucleus, is delivered intracellularly in the endosome and/or lysosome, and/or is active after exiting or escaping the endosomal-lysosomal pathway. The effector molecule is for example a molecule selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof. Thus, for example, an effector molecule or an effector moiety is a molecule or moiety selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or any combination thereof, that can selectively bind to any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and that upon binding to the target molecule regulates the biological activity of such one or more target molecule(s). For example, an effector moiety is a toxin or an active toxic fragment thereof or an active toxic derivative or an active toxic domain thereof. Typically, an effector molecule can exert a biological effect inside a cell such as a mammalian cell such as a human cell, such as in the cytosol of said cell. An effector molecule or moiety of the invention is thus any substance that affects the metabolism of a cell by interaction with an intracellular effector molecule target, wherein this effector molecule target is any molecule or structure inside cells excluding the lumen of compartments and vesicles of the endocytic and recycling pathway but including the membranes of these compartments and vesicles. Said structures inside cells thus include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the inner part of the plasma membrane and the cytosol. Typical effector molecules are thus drug molecules, an enzyme, plasmid DNA, toxins such as toxins comprised by antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, nucleic acids comprised by an antibody-oligonucleotide conjugate (AOC). For example, an effector molecule is a molecule which can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling. In the context of the invention, an effector molecule or effector moiety when the effector molecule is part of a conjugate, is not a saponin, and is not a cell-surface molecule binding molecule such as an antibody such as an sdAb. Typically, an effector moiety comprised by the conjugate exerts its therapeutic (for example toxic, enzymatic, inhibitory, gene silencing, etc.) effect in the cytosol and/or in the cell nucleus. Typically, the effector moiety is delivered intracellularly in the endosome and/or in the lysosome, and typically the effector moiety is active after exiting or escaping the endosomal-lysosomal pathway.
Thus, the claim encompasses 1) a broad genus of first and second cell-surface binding molecules that are different; 2) a broad genus of effector moieties; and 3) a broad genus of saponins. Given BRI, cell-surface molecule binding molecules or effector moieties could be small molecules, peptides, proteins, antibodies (or fragment thereof), ligand, engineered receptors, and various types of nucleic acids etc. These compounds vary significantly, have different structures, physical, and chemical properties, function through different mechanisms. In addition a cell-surface molecule encompasses any possible molecule occurs in any cell-surface which may be cell-type specific or not. The combinations of a first cell-surface molecule targeting molecule conjugated to any possible saponin, e.g., monodesmosidic triterpene glycoside or bidesmosidic triterpene glycoside from any one or more Gypsophila species, Saponaria species, Agrostemma species and Quillaja species and a second cell-surface binding molecule different from the first cell-surface binding molecule conjugated to any one or more effector moiety are even more complex. These claims lack written description because the specification lacks a representative number of species that satisfies the entirety of the genus.
The MPEP states that the purpose of the written description requirement is to ensure that the inventor had possession, as of the filing date of the application, of the specific subject matter later claimed. The MPEP lists factors that can be used to determine if sufficient evidence of possession has been furnished in the disclosure of the application. These include "level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention."
The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, disclosure of drawings, or by disclosure of relevant identifying characteristics, for example, structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the
Applicants were in possession of the claimed genus.
Vas-Gath, Inc. v. Mahurkar, 935 F.2d 1555, 1562-63, 19 USPQ2d 1111 (Fed. Cir. 1991), makes clear that: "applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the 'written description' inquiry, whatever is now claimed." (See page 1117.) The specification does not "clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed." (See Vas-Gath at page 1116.).
Given that the structures and properties of the encompassed combination of first cell-surface binding molecules conjugated to any saponin and second cell-surface binding molecules conjugated to any effector moieties vary significantly, one of ordinary skilled in the art would not be able to readily visualize or recognize the combination of conjugates with all possible combinations of cell-surface binding molecules, saponins and effector moieties. One of ordinary skilled in the art would not be able to predict the function and activity of all possible combinations of first conjugate comprising any cell-surface binding molecules covalently linked to saponins and second conjugate comprising any cell surface molecules covalently linked to any effector moieties.
The specification discloses just saponin SO1861, a triterpenoid saponin from the common soapwort Saponaria officinalis, which is a member of the Caryophyllaceae family, See para. [0157].
However, the synthesis methods described in the specification would not be able to make all possible conjugates encompassed by the claim.
The state of the prior art teaches that saponin represents a heterogenous phytochemicals with different structures and properties.
For example, Fuchs (of record, Fuchs et al., Biomedicines, 5(14): 1-25, 2017; PTO 1449) teaches that saponins represent a wide spectrum in the field of secondary plant compounds and are subdivided into two groups, the steroid saponins and triterpenoid saponins, even including steroid alkaloid saponins (page 2, para. 3). The wide range of structural variation options, both in the aglycone and sugar moieties explains the variety of different saponins with diverse effects (page 2, para. 5). For example, only saponins of a certain electrophoretic mobility are able to enhance the endosomal escape (page 8, para. 2). In addition, the enhancing effect of saponin (SA1641) on a targeted toxin is clathrin- and actin-dependent. Six inhibitory agents that are known to inhibit either clathrin-mediated endocytosis, GTPase activity of dynamin-2, actin-polymerization, endosomal acidification, or caveolae-dependent endocytosis were tested. Inhibition of clathrin-mediated endocytosis, actin-polymerization, and endosomal acidification blocked the enhancer effect of Saponinum album (page 12, para. 2).
Likewise, Barr (of record, Advanced Drug Delivery Reviews 32: 247-271, 1998; PTO 892) teaches that saponins have different molecular weight, different adjuvant activity, different toxicity, and different structures (Table1, and § 3. Structural and functional analysis of purified Quillaias saponins).
Bhargava et al (of record, Molecular Oncology 11: 1527-1543, 2017; PTO 1449) teaches that effect by endosomal escape enhancers saponin depends on the cell line, the targeting moiety, target receptor expression, and the structure of the enhancer, see p. 1539, right col.
In view of above, however, the specification exemplifies conjugates comprising only pertuzumab and trastuzumab that bind to human HER2, cetuximab and matuzumab that bind to human EGFR, EGF ligand (elected species), VHH 7D12 comprises the amino acid sequence of SEQ ID NO: 1 and VHH 9G8 comprising the amino acid sequence of SEQ ID NO: 2. These two single domain antibodies bind to domain III, compete for EGF binding to just human EGFR. In addition 7D12 compete for binding of either cetuximab or matuzumab (9G8); only one type of saponin (SO1861, the elected species); and two effector moieties (saporin and dianthin toxin). The specification discloses:
Example 1. 1T2C, Non-Competing HER2 Targeting
[0147] SO1861-EMCH was conjugated via cysteine residues (Cys) to pertuzumab, with a DAR 4, (pertuzumab-(Cys-L-SO1861).sup.4. Pertuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin, saporin, with a DAR4). Pertuzumab and Trastuzumab recognize and bind human HER2 at different epitopes (non-competing). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50=0.5 nM; FIG. 2A) whereas equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM trastuzumab-saporin could not induce any cell killing activity in HER2 expressing cells. When we compare these data with the combination of Trastuzumab-(Cys-L-SO1861).sup.4+50 pM trastuzumab-saporin we observe that at high concentrations Trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity is reduced due to competition of both trastuzumab antibody conjugates for binding the HER2 receptor. In MDA-MB-468 cells (no HER2 expression) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 2B) for any of the treatments.
[0148] All this shows that the use of two different antibodies recognizing the same receptor but binding at different epitopes (different binding sites), effectively induce cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of trastuzumab-saporin in high HER2 expressing cells, but not in cells that do not express HER2. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals activity at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
[0149] Next, trastuzumab-saporin was titrated on a fixed concentration of 2.5 nM and 75 nM pertuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and HER2 non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing at low concentrations trastuzumab-saporin in combination with 2.5 nM, or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 in SK-BR-3 (HER2.sup.++; IC50=0.5 pM; FIG. 3A), whereas Trastuzumab-saporin or Trastuzumab-saporin+2.5 nM or 75 nM pertuzumab showed only at high concentrations cell killing in SK-BR-3 cells (IC50>1000 pM; FIG. 3A). When these data is compared with the combination of trastuzumab-saporin+2.5 nM or 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity was strongly reduced when combined with 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9. In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 3B).
[0150] All this shows that the combination of low concentrations of trastuzumab-saporin+2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 induce effective cell killing in high HER2 expressing cells. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals effective cell killing at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
Example 2. 1T2C, Non-Competing HER2 Targeting
[0151] Next, pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM pertuzumab-dianthin (pertuzumab conjugated to the protein toxin, dianthin, with a DAR4). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low concentrations of trastuzumab-(Cys-L-SO1861).sup.4 or pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50<0.1 nM; FIG. 4A). At higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4 cell killing activity was reduced whereas higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 were still effective. Equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM pertuzumab-dianthin were not effective in HER2 expressing cells (SK-BR-3: IC50>1000 nM; FIG. 4A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 4B) for any of the treatments.
[0152] All this shows that the use of two different antibodies recognizing the same receptor but bind at a different epitopes, effectively induce cell killing at low and higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of pertuzumab-dianthin in high HER2 expressing cells.
[0153] Next, pertuzumab-dianthin was titrated on a fixed concentration of 2.5 nM and 25 nM pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing of SK-BR-3 cells (HER2.sup.++) at low concentrations pertuzumab-dianthin in combination with 2.5 nM, trastuzumab-(Cys-L-SO1861).sup.4 or 2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 (IC50=0.5 pM; IC50=0.5 pM, resp. FIG. 5A), whereas pertuzumab-dianthin+25 nM trastuzumab-(Cys-L-SO1861).sup.4 showed more efficient cell killing compared to the combination of pertuzumab-dianthin+25 nM pertuzumab-(Cys-L-SO1861).sup.4. Equivalent concentrations of pertuzumab-dianthin or pertuzumab-dianthin+25 nM pertuzumab showed only at high concentrations some slight cell killing activity in SK-BR-3 cells (IC50>10.000 pM; FIG. 5A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 5B).
[0154] All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
Example 3. 1T2C, Non-Competing EGFR Targeting
[0155] SO1861-EMCH was conjugated via cysteine residues (Cys) to matuzumab, with a DAR 3,3, (matuzumab-SO1861). Matuzumab-SO1861 was titrated on a fixed concentration of 10 pM cetuximab-saporin (cetuximab conjugated to the protein toxin, saporin, with a DAR4) or 10 pM EGFdianthin (recombinant toxin fusion protein). Matuzumab recognizes and binds human EGFR at a different epitope compared to cetuximab and EGF, whereas Cetuximab and EGF compete for binding the EGFR receptor. Targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) and non-expressing cells (A2058, EGFR.sup.−) was determined. This revealed strong cell killing at low and higher concentrations of matuzumab-(SO1861)+10 pM cetuximab-saporin or 10 pM EGFdianthin in A431 cells (IC50=2 nM; FIG. 6A) whereas equivalent concentrations matuzumab, matuzumab-SO1861, matuzumab+10 pM cetuximab-saporin or matuzumab+10 pM EGFdianthin could not induce any cell killing activity in A431 cells (IC50>1000 nM; FIG. 6A). When we compare these data with the combination of cetuximab-(Cys-L-SO1861).sup.4+10 pM cetuximab-saporin or cetuximab-SO1861+10 pM EGF-dianthin we observe that at higher concentrations Cetuximab-SO1861, cell killing activity is reduced due to competition of both cetuximab conjugate and EGF for binding the EGFR receptor. In A2058 cells (EGFR.sup.−) no cell killing was observed (IC50>1000 nM; FIG. 6B) for any of the treatments. All this shows that the use of two different antibodies or antibody/ligand combinations recognizing the same receptor but bind at different epitopes, effectively induces cell killing in EGFR.sup.++ expressing cells at low and high concentrations of matuzumab-SO1861 in combination with a fixed low concentration (10 pM) of cetuximab-saporin or EGFdianthin. Thus the use of the combination according to the invention omits competition for receptor binding and reveals very effective endosomal escape and cytoplasmic toxin delivery resulting in efficient and selective tumor cell killing.
[0156] Next, cetuximab-saporin was titrated on a fixed concentration of 10 nM and 75 nM matuzumab-SO1861 and targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) was determined. This revealed that, 10 nM and 75 nM matuzumab-SO1861 in combination with low concentrations cetuximab-saporin induced efficient cell killing in EGFR expressing cells (A431: 1050=0.5 pM; FIG. 7A), whereas cetuximab-saporin or cetuximab-saporin+10 nM or 75 nM matuzumab showed only at high concentrations cell killing (1050=1000 pM FIG. 3A). When we compared these data with the combination of cetuximab-saporin+10 nM and 75 nM cetuximab-SO1861, cell killing activity was reduced with increased concentrations of cetuximab-SO1861. In A2058 cells (EGFR.sup.−) no cell killing was observed (A2058: IC50 >1000 pM; FIG. 7B). All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
SO1861-EMCH Synthesis
[0159] To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture stirred at room temperature. After 1.5 hours the reaction mixture was subjected to preparative MP-LC..sup.1 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS 96%.
[0160] LRMS (m/z): 2069 [M-1].sup.1−
[0161] LC-MS r.t. (min): 1.08.sup.4
mAb-SO1861 Synthesis
To Matuzumab freshly prepared TCEP solution (1.00 mg/ml, 1.971 mole equivalents, 2.80×10.sup.−5 mmol) was added. The reaction mixture was vortexed briefly then incubated for 90 minutes at 20° C. with roller-mixing. After incubation (prior to addition of SO1861-EMCH), a 0.5 mg (0.101 ml) aliquot of Matuzumab-SH was removed and purified by gel filtration using zeba spin desalting column eluting into TBS pH 7.5. This aliquot was characterised by UV-vis analysis and Ellman's assay. To the bulk Matuzumab-SH was added an aliquot of freshly prepared SO1861-EMCH solution (2.00 mg/ml, 8 mole equivalents, 8.54×10.sup.−5 mmol, 0.089 ml), the mixture vortexed briefly then incubated for 120 minutes at 20° C. Besides the conjugation reaction, two aliquots of desalted Matuzumab-SH (0.10 mg, 0.022 ml, 6.70×10.sup.−7 mmol) were reacted with NEM (8.00 equivalents, 5.36×10.sup.−6 mmol, 0.67 pg, 2.7 μl of a 0.25 mg/ml solution) or TBS pH 7.5 buffer (2.7 μl) for 120 minutes at 20° C., as positive and negative controls, respectively. After incubation (prior to addition of NEM), a ca. 60 pg (0.020 ml) aliquot of Matuzumab-SO1861 mixture was removed and characterised by Ellman's assay alongside positive and negative controls to obtain SO1861 incorporation. To the bulk Matuzumab-SO1861 mixture was added an aliquot of freshly prepared NEM solution (0.25 mg/ml, 5 mole equivalents, 5.34×10.sup.−5 mmol, 0.007 mg) to quench the reaction. The conjugate was purified by zeba 40K MWCO spin column eluting with DPBS pH 7.5 to give purified Matuzumab-SO1861 conjugate. The product was normalized to 2.0 mg/ml and filtered to 0.2 μm, to afford Matuzumab-SO1861 (total yield=1.10 mg, 52%, Matuzumab:SO1861-EMCH=3.3).
[0162] Similar procedures were followed to produce pertuzumab-SO1861 (DAR4), cetuximab-SO1861 (DAR4), trastuzumab-SO1861 (DAR4)
Pertuzumab-Dianthin Synthesis
[0163] Dianthin-Cys (17.0 ml, ˜9.6 mg) was concentrated by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 10 minutes). The resulting 3.25 ml aliquot was gel filtered using zeba 10 ml spin columns eluting with TBS pH 7.5.
[0164] Pertuzumab (0.30 ml, ˜10 mg) was diluted to 10 mg/ml with DPBS pH 7.5, desalted via zeba 5 ml spin column eluting with DPBS pH 7.5 and normalised to 2.50 mg/ml. To an aliquot of Pert (5.00 mg, 3.30×10.sup.−5 mmol, 2.593 mg/ml) was added an aliquot of freshly prepared SMCC solution (1.00 mg/ml, 4.20 mole equivalents, 13.9×10.sup.−5 mmol) in DMSO, the mixture vortexed briefly then incubated for 60 minutes at 20° C. with roller-mixing. After, the reaction was quenched by the addition of an aliquot of a freshly prepared glycine solution (2.0 mg/ml, 5.0 mole equivalents, 69.5×10.sup.−5 mmol) in DPBS pH 7.5. Pert-SMCC (4.27 mg, 2.80×10.sup.−5 mmol, 1.514 mg/ml) was obtained after gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0165] To Dianthin-Cys (7.54 mg, 25.3×10.sup.−5 mmol, 2.258 mg/ml) was added an aliquot of freshly prepared TCEP solution (1.00 mg/ml, 0.5 mole equivalents, 12.6×10.sup.−5 mmol) in TBS pH 7.5, the mixture briefly vortexed then incubated for 60 minutes at 20° C. with roller-mixing. After, Dianthin-SH (6.0 mg, 20.2×10.sup.−5 mmol, 1.722 mg/ml, Dianthin:SH=1.1) was obtained by gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0166] To the bulk Pert-SMCC was added the aliquot of Dianthin-SH (7.20 mole equivalents), the mixture vortexed briefly then incubated overnight at 20° C. After ca. 16 hours, the reaction was quenched by the addition of an aliquot of freshly prepared NEM solution (2.50 mg/ml, 5.0 mole equivalents, 101×10.sup.−5 mmol) in TBS pH 7.5. The reaction mixture was filtered to 0.45 μm and then concentrated to <2 ml by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 15 minutes). The conjugate was purified by gel filtration using a 1.6×35 cm Superdex 200PG column eluting with DPBS pH 7.5.
Example 4. SO1861+EGFR/HER2/CD71 Targeted mAb
[0169] SO1861 was titrated on a fixed concentration of 10 pM CD71-saporin (DAR4), 10 pM cetuximab-saporin (DAR4), 10 pM matuzumab-dianthin (DAR4), 10 pM pertuzumab-saporin (DAR4), 10 pM or 50 pM pertuzumab-saporin (DAR4) and 50 pM trastuzumab-saporin (DAR4) and targeted protein toxin-mediated cell killing on A431 (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) and A2058 (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) was determined. In A431 cells (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) this revealed cell killing activity for all EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) as well as 10 pM CD71-saporin and 50 pM pertuzumab-saporin at SO1861: IC50=200 nM, whereas 50 pM trastuzumab or 10 pM pertuzumab-saporin showed activity at IC50=250 nM and IC50=300 nM, respectively (FIG. 8A), In A2058 cells (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) the EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) were not active but 10 pM CD71mab-saporin, 10 or 50 pM Pertuzumab-saporin and 50 pM trastuzumab-saporin all showed activity at SO1861: IC50=200 nM (FIG. 8B).
However, the specification does not describe the structure, e.g., amino acid sequences of the first binding molecules that encompassed any and all peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands (claims 1, 54), any proteinaceous binding molecule or any non-proteinaceous ligand (claim 2) or any ligand, any ligand such as EGF (claim 8) wherein the undisclosed first binding molecule can bind to any and all cell-surface molecule, any cell-surface molecule is any cell-surface receptor or different epitope of the same undisclosed cell-surface receptor (claim 5) conjugated to any saponin from any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside via a linker (claim 3) in combination with any second binding molecule that different from the first binding molecule, wherein the second binding molecule comprising any and all peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to any cell-surface molecule, e.g., any cell-surface receptor of the same but different epitope (claim 5) conjugated to any effector molecule, e.g., a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, toxin and regulates the biological activity of such one or more target molecule(s). One of skill in the art cannot visualize or recognize member of the genus of first and second conjugates encompassed by claims.
Even assuming the first binding molecule and second binding molecule comprise an immunoglobulin (claim 8) or antibody such as any monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment (claim 9), the specification discloses VHH 7D12 comprises the amino acid sequence of SEQ ID NO: 1 and VHH 9G8 comprising the amino acid sequence of SEQ ID NO: 2. These two single domain antibodies bind to domain III of human EGFR, and compete with EGF (elected species) for binding to human EGFR. In addition, VHH 7D12 competes for binding of either cetuximab (anti-EGFR) or matuzumab (9G8). The specification also discloses pertuzumab and trastuzumab that bind to human HER2.
However, specification does not describe the structure, e.g., amino acid sequences of the heavy chain variable region and the light chain variable regions of all possible antibodies that correlated with binding to any and all cell surface molecules (claims 1, 2, 8, 9, 11, 26, 54), any region of any cell surface receptor (claims 5, 25), or any different first and second binding sites (aka different epitope) of the cell-surface molecules simultaneously (claim 11), wherein the first binding region is selected to bind to the undisclosed first binding site of the cell-surface molecule without competing for binding of the second binding region to the second binding site (epitope) of the same-surface molecule (claim 12) or different epitopes (different first and second binding site of the same surface molecule (claim 26). There are no limitation on the structure of the first and second antibodies or ligand, and the binding sites (epitope) to which the first and second antibodies or ligands bind.
The specification does not describe a representative number of species of the first and second conjugates falling within the scope of the genus or structure common to the members of the genus so the one of skill in the art can visualize or recognize the member of the genus of the actual claimed conjugates themselves in combination.
An adequate written description must contain enough information about the actual makeup of the claimed products – “a precise definition, such as structure, formula, chemic name, physical properties of other properties, of species falling with the genus sufficient to distinguish the gene from other materials”, which may be present in “functional terminology when the art has established a correlation between structure and function” (Amgen page 1361).
It is known in the art that antibodies have a large repertoire of distinct structures and that a huge variety of antibodies can be made to bind to a single epitope.
For example, Lloyd et al. taught that hundreds of functional antibody fragments can be isolated from an antibody library that bind to the same antigen wherein these antibodies have distinct heavy and light chain sequences (Lloyd et al. Protein Engineering, Design & Selection 22:159-168, 2009; PTO 892; see, e.g., Discussion).
Similarly, Edwards et al., (J Mol Biol. 334(1): 103-118, 2003; PTO 892), found that over 1000 antibodies, all different in amino acid sequence, were generated to a single protein; 568 different amino acid sequences identified for the V(H) CDR3 domains of these antibodies (Abstract).
Poosarla et al (Biotechn. Bioeng. 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.)
Regarding antibody drug conjugate, the state of the prior art is such that the location or site of conjugation on the drug and the antibody affect conjugate stability, and pharmacokinetics of antibody drug conjugates.
For example, Strop et al (Chemistry and Biology 20: 161-167, 2013; PTO 892) teach drug position can have a significant effect on linker stability and antibody pharmacokinetics. The site of conjugation on the drug and antibody can influence ADC properties differently in mice and rats, highlighting potential pitfalls of examining efficacy in mouse xenograft models and toxicity in rats or nonhuman primates, see abstract, p 166, p. 168 right col, in particular.
Nejadmoghaddam (Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Regarding derivatives based on any one or more saponin (claim 17), the specification does not describe the chemical structures of any such derivatives, including derivative of SO1861 (elected species). One of ordinary skilled would not be able to readily visualize or recognize any of the undisclosed derivatives conjugated to any first cell-surface binding molecule in combination with any second cell-surface binding molecules conjugated to any effector molecule. One of ordinary skilled in the art would not expect that all conjugates encompassed by claims will function in a given or predictable manner as the examples shown in the specification, e.g. enhance killing of targeted cells.
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method for isolating it. See Fiers v. Revel, 25 USPQ2d 1601, 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016.
One cannot describe what one has not conceived. See Fiddles v. Baird, 30 USPQ2d 1481, 1483. In Fiddles v. Baird, claims directed to mammalian FGF’s were found unpatentable due to lack of written description for the broad class. The specification provided only the bovine sequence.
Therefore, only (1) a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising a first antibody that binds to domain III of human EGFR wherein the first antibody is an anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 1 or matuzumab, wherein the first antibody is covalently linked to at least one saponin SO1861 and wherein the first antibody does not compete for binding with the second antibody matuzumab or cetuximab or EGF that binds to human EGFR; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second antibody that binds to the extracellular domain of human EGFR wherein the second antibody comprises the amino acid sequence of SEQ ID NO: 2 or pertuzumab or cetuximab, wherein the second antibody is conjugated to an effector, wherein the second antibody does not compete for binding to the EGFR with the first anti-EGFR antibody and wherein the effector is saporin or dianthin,
(2) a first pharmaceutical composition comprising a conjugate comprising (a) a first antibody that binds to human HER2 wherein the antibody is covalently bound to at least one saponin SO1861; and (b) a second pharmaceutical composition comprising a conjugate comprising a second antibody that binds to human HER2, wherein the second antibody is conjugated to an effector molecule, wherein the first or second antibody is pertuzumab or trastuzumab, wherein the effector is saporin or dianthin and wherein the first antibody and second antibody binds to different epitope of the same human HER2 for saponin mediated endosomal escape of targeted toxin, and
(3) a kit comprising the therapeutic combination above and instructions for use, but not the full breadth of the claims meets the written description provision of 35 U.S.C. § 112, first paragraph.
Applicant is reminded that Vas-Cath makes clear that the written description provision of 35 U.S.C. § 112 is severable from its enablement provision (see page 1115).
Applicant's arguments filed July 23, 2026 have been fully considered but they are not persuasive.
Applicant respectfully submits that the written description requirement for a claimed genus may be satisfied by disclosure of a representative number of species, relevant identifying structural or functional characteristics, or a combination thereof. The specification satisfies this standard for at least the following reasons:
The specification provides working examples spanning multiple receptor targets (HER2, EGFR, and CD71), multiple binding-molecule formats (monoclonal antibodies, single-domain antibodies, and ligands), and multiple effector molecules (saporin and dianthin). Representative species include: pertuzumab and trastuzumab (HER2); matuzumab, cetuximab, EGF, VHH 7D12 (SEQ ID NO: 1), and VHH 9G8 (SEQ ID NO: 2) (EGFR); and CD71mab-saporin (CD71). See As-Filed Specification, [0147]-[0156] and [0169].
The specification does not merely list species in isolation but provides detailed structural definitions for all key genus components. These include "binding molecule" ([0039]), "binding region" ( [0020]), "binding site" ( [0021]), "saponin" ([0023]), "mono-desmosidic saponin" ([0026]), "bi-desmosidic saponin" ( [0027]), and "effector molecule" ([0033]). The specification further identifies the structural feature correlating with function: the aldehyde function in the aglycone core of the saponin mediates endosomal escape, which is released under acidic endosomal conditions. See As-Filed Specification, p. 22, lines 1-7. Table Al provides an extensive list of named saponins with their aglycone core structure, first saccharide chain, and second saccharide chain, further establishing the structure-function relationship across the saponin genus.
The specification discloses conjugation via the C23 aldehyde function using an EMCH linker (covalently bound via a thio-ether bond to a cysteine sulfhydryl group) and via the glucuronic acid unit using a HATU linker (covalently bound via an amide bond to a lysine amine group). See As-Filed Specification, [0022] (as-filed claims 22 and 24). Complete synthesis protocols are provided for SO1861-EMCH and mAb-SO 1861 conjugation, expressly confirmed as applicable to pertuzumab, cetuximab, and trastuzumab conjugates. See As-Filed Specification, [0162]. Written description does not require a separate synthesis protocol for every species within a claimed genus; these well-established bioconjugation techniques are routine in the art and confirmed as generally applicable. See As-Filed Specification, [0159]-[0166].
Bhargava itself constitutes an additional working example within the scope of claim 1: a combination of HisDianthin-EGF (effector molecule dianthin with EGF as binding molecule) and saponin SO1861 to treat EGFR-overexpressing cells, confirming the claimed principle that a saponin potentiates the intracellular activity of a co-delivered effector molecule. See Bhargava, p. 1527. The cited passage concerns variability in the degree of enhancement across targeting
constructs (including panitumumab, trastuzumab, cetuximab, and EGF), not whether enhancement occurs, it occurs in each case. Because claims 1 and 2 recite a structural therapeutic combination rather than a minimum enhancement threshold, this variability is legally irrelevant to possession.
The inventive contribution is not a newly-discovered binding molecule, but rather a therapeutic combination architecture: a first binding molecule conjugated to a saponin and a second, different binding molecule conjugated to an effector molecule, wherein the two binding molecules bind different, non-competing sites on the same cell-surface molecule, enabling co- delivery of the saponin and effector molecule into the same target cell. The concern underlying the written description standard for antibody genera , a claim to all antibodies capable of binding a newly-identified target defined only by function, is therefore absent here.
The binding molecules recited in the claims (antibodies, antibody fragments, and ligands such as EGF) and their target receptors (HER2, EGFR, CD71) were all conventional and readily available to a POSA at the time of filing. The art has an established structure-function correlation between specific binding to these known receptors and the readily-available structures that accomplish it. Claims 8 and 9 recite binding molecules by well-established structural categories, including monoclonal antibodies, single-domain antibodies, VHH domains, VH domains, VNAR domains, Fab, scFv, Fv, dAb, F(ab)2, and Fcab fragments, and the specification discloses representative species spanning these classes (full-length mAbs: pertuzumab, trastuzumab, matuzumab, cetuximab; single-domain antibodies: VHH 7D 12, VHH 9G8; non-proteinaceous ligand: EGF). Disclosure of amino acid sequences for conventional binding molecules directed to known receptors is not required.
For at least the foregoing reasons, Applicant respectfully requests withdrawal of the rejection of claims 1-6, 8, 9, 11-14, 17, 21-26, and 54 under 35 U.S.C. § 112(a) for failure to comply with the written description requirement.
In response, Claim 1 encompasses a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second binding molecule different from the first binding molecule, the second binding molecule comprising any second binding region different from the first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from the first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule,
wherein the first pharmaceutical composition and the second pharmaceutical composition optionally further comprise a pharmaceutically acceptable excipient and optionally further comprise a pharmaceutically acceptable diluent.
Claim 2 encompasses a pharmaceutical composition comprising:
any conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any triterpenoid saponin of the monodesmosidic type or the bidesmosidic type; and
any conjugate comprising any second binding molecule different from said first binding molecule, the second binding molecule comprising any second binding region different from said first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from said first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule, and optionally further comprising a pharmaceutically acceptable excipient and optionally further comprising a pharmaceutically acceptable diluent.
Regarding “binding molecule”, the specification define as follow:
[0039] The term “binding molecule” has its regular scientific meaning and here refers to a molecule capable of specifically binding to another molecule such as a cell-surface molecule, e.g. a cell-surface receptor. Typical binding molecules are peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to e.g. a protein, a lipid, a (poly)saccharide, such as a cell-surface receptor or a cell-surface molecule. “Specifically binding” here refers to specific and selective binding with higher affinity than non-specific background binding.
Regarding “binding region”, the specification define as follow:
[0020] The term “binding region” has its regular scientific meaning and here refers to a part of a molecule or (a) chemical group(s) of a molecule or a(n) (linear or non-linear) amino-acid sequence of a protein or peptide and the like, that has the capacity to bind to a binding partner molecule. A typical binding region are the CDR loops of an immunoglobulin. A typical binding region of a protein is or are loop(s) of amino-acid residues comprised by said protein and capable of specifically binding to the binding site on a binding partner molecule such as a protein, cell-surface receptor, etc.
Regarding “binding site”, the specification define as follow:
[0021] The term “binding site” has its regular scientific meaning and here refers to a region on a macromolecule such as a protein, for example a cell-surface molecule such as a cell-surface receptor, that binds to another molecule such as a protein, for example a ligand, with specificity.
Regarding “cell-surface molecule”, the specification define as follow:
[0022] The term “cell-surface molecule” has its regular scientific meaning and here refers to a molecule that is present and exposed at the outside surface of a cell such as a blood cell or an organ cell, such as a mammalian cell, such as a human cell. Typically, a cell-surface molecule is a protein such as a receptor, or a lipid molecule or a polysaccharide.
Other than the particular antibodies pertuzumab and trastuzumab that bind to different epitope on HER2, matuzumab and cetuximab that bind to different epitope on human EGFR; single-domain antibodies VHH 7D 12, VHH 9G8 that bind to EGFR and EGF ligand, the specification fails to describe the (i) Complete structure, e.g. amino acid sequence of the heavy and light chain variable domains, ii. Partial structure, e.g., the six CDRs of all potential first or second binding region of all first or second binding molecules, e.g., antibody or antigen binding fragment thereof that correlated with binding to which binding site, e.g., amino acid sequence (epitope) of any and all cell-surface molecule, e.g., any receptor, or any lipid molecule or any polysaccharide covalently conjugated to any one or more saponin from any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside or effector molecule encompassed by the claimed therapeutic combination to reasonably convey to the skilled artisan that Applicant had possession of the claimed invention at the time the application was filed.
Even assuming the first and second binding molecule bind to EGFR, the specification discloses just two antibodies matuzumab and cetuximab that bind to different epitope on human EGFR.
Considering an antibody will have six CDRs, each CDR comprising approximately ten amino acid residues. With twenty naturally occurring amino acids, the potential size of the CDR variation, of just human antibodies within this indeterminate genus, is 2060.
The instant disclosure, including the claims fail to disclose a representative number of species falling with the scope of the genus or structural common to the members of the genus so the one of skill in the art can visualize or recognize the member of the genus first and second binding molecules (claims 1-2), proteinaceous binding molecule, e.g., antibodies or non-proteinaceous ligand (claim 3) that bind to different epitope on same undisclosed cell-surface molecule (claim 5), any first and second binding molecule such as any immunoglobulin (claim 8), any monoclonal antibody, any single domain antibody, any VHH, any VNAR domain, any Fab, any scFv, any Fv, any dAb, any F(ab)2, any Fab that bind to different epitope of any and all potential cell surface molecule (claims 9, 11-12).
The specification does not describe the structure-identifying information about the claimed first and second binding molecules that bind to different binding site (epitope) of any and all cell-surface molecule, nor describe a representative number of species falling with the scope of the genus or structural common to the members of the genus so the one of skill in the art can visualize or recognize the member of the genus of the actual claimed binding molecules themselves conjugated to any and all saponin and effector molecule.
It is known in the art that antibodies have a large repertoire of distinct structures and that a huge variety of antibodies can be made to bind to a single epitope.
For example, Lloyd et al. taught that hundreds of functional antibody fragments can be isolated from an antibody library that bind to the same antigen wherein these antibodies have distinct heavy and light chain sequences (of record, Lloyd et al. Protein Engineering, Design & Selection 22:159-168, 2009; PTO 892; see, e.g., Discussion).
Similarly, Edwards et al., (of record, J Mol Biol. 334(1): 103-118, 2003; PTO 892), found that over 1000 antibodies, all different in amino acid sequence, were generated to a single protein; 568 different amino acid sequences identified for the V(H) CDR3 domains of these antibodies (Abstract).
Poosarla et al (of record, Biotechn. Bioeng. 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.) Given that hundreds of unique antibody structures may bind a single epitope on a single antigen, the structure of an antibody cannot be predicted from the structure of the antigen (as held in Amgen), and a single species, or small group of species, cannot define a structure-function relationship so as to be representative of all the antibodies that bind to that antigen (as held in Abbvie).
Regarding effector molecule conjugated to the undisclosed second binding molecule, the specification define effector as follow:
[0033] The term “effector molecule”, or “effector moiety” when referring to the effector molecule as part of e.g. a covalent conjugate, has its regular scientific meaning and here refers to a molecule that can selectively bind to for example any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and regulates the biological activity of such one or more target molecule(s). In the conjugate of the invention the effector moiety for example exerts its effect in the cytosol, in the cell nucleus, is delivered intracellularly in the endosome and/or lysosome, and/or is active after exiting or escaping the endosomal-lysosomal pathway. The effector molecule is for example a molecule selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof. Thus, for example, an effector molecule or an effector moiety is a molecule or moiety selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or any combination thereof, that can selectively bind to any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and that upon binding to the target molecule regulates the biological activity of such one or more target molecule(s). For example, an effector moiety is a toxin or an active toxic fragment thereof or an active toxic derivative or an active toxic domain thereof. Typically, an effector molecule can exert a biological effect inside a cell such as a mammalian cell such as a human cell, such as in the cytosol of said cell. An effector molecule or moiety of the invention is thus any substance that affects the metabolism of a cell by interaction with an intracellular effector molecule target, wherein this effector molecule target is any molecule or structure inside cells excluding the lumen of compartments and vesicles of the endocytic and recycling pathway but including the membranes of these compartments and vesicles. Said structures inside cells thus include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the inner part of the plasma membrane and the cytosol. Typical effector molecules are thus drug molecules, an enzyme, plasmid DNA, toxins such as toxins comprised by antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, nucleic acids comprised by an antibody-oligonucleotide conjugate (AOC). For example, an effector molecule is a molecule which can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling. In the context of the invention, an effector molecule or effector moiety when the effector molecule is part of a conjugate, is not a saponin, and is not a cell-surface molecule binding molecule such as an antibody such as an sdAb. Typically, an effector moiety comprised by the conjugate exerts its therapeutic (for example toxic, enzymatic, inhibitory, gene silencing, etc.) effect in the cytosol and/or in the cell nucleus. Typically, the effector moiety is delivered intracellularly in the endosome and/or in the lysosome, and typically the effector moiety is active after exiting or escaping the endosomal-lysosomal pathway.
The specification exemplifies just Dianthin or saporin conjugated to cetuximab (anti-EGFR) or matuzumab (anti-HER2), see working examples. However, two species are not representative of the genus of effector molecule, e.g., small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof to allow one of ordinary skill in the art would recognize applicant as being in possession of the extremely diverse conjugates.
The state of the prior art is such that the location or site of conjugation on the drug and the antibody affect conjugate stability, and pharmacokinetics of antibody drug conjugates.
For example, Strop et al (of record, Chemistry and Biology 20: 161-167, 2013; PTO 892) teach drug position can have a significant effect on linker stability and antibody pharmacokinetics. The site of conjugation on the drug and antibody can influence ADC properties differently in mice and rats, highlighting potential pitfalls of examining efficacy in mouse xenograft models and toxicity in rats or nonhuman primates, see abstract, p 166, p. 168 right col, in particular.
Nejadmoghaddam (of record, Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Regarding “saponin”, the specification define as follow:
[0023] The term “saponin” has its regular scientific meaning and here refers to a group of amphipathic glycosides which comprise one or more hydrophilic glycone moieties combined with a lipophilic aglycone core which is a sapogenin. The saponin may be naturally occurring or synthetic (i.e. non-naturally occurring). The term “saponin” includes naturally-occurring saponins, derivatives of naturally-occurring saponins as well as saponins synthesized de novo through chemical and/or biotechnological synthesis routes. Saponin has a triterpene backbone, which is a pentacyclic C30 terpene skeleton, also referred to as sapogenin or aglycone. Within the context of the invention saponin is not considered an effector molecule nor an effector moiety in the conjugates according to the invention. Thus, in conjugates comprising a saponin and an effector moiety, the effector moiety is a different molecule than the conjugated saponin.
The specification exemplifies just saponin SO1861 conjugate via the C23 aldehyde function using an EMCH linker (covalently bound via a thio-ether bond to a cysteine sulfhydryl group) or via the glucuronic acid unit using a HATU linker (covalently bound via an amide bond to a lysine amine group) to pertuzumab, cetuximab, or trastuzumab.
However, the specification does not describe the chemical structures of any such derivatives (claim 17 and 55), or stereoisomers (claim 17 and 55) of any Quillaja bark saponin or any one or more of dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate, AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861 (elected species), QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R. One of ordinary skilled would not be able to readily visualize or recognize any of the undisclosed derivatives conjugated to any first cell-surface binding molecule encompassed by the claim therapeutic combination to demonstrate possession at the time of filing.
The state of the prior art teaches that saponin represents a heterogenous phytochemicals with different structures and properties.
For example, Fuchs (of record, Fuchs et al., Biomedicines, 5(14): 1-25, 2017; PTO 1449) teaches that saponins represent a wide spectrum in the field of secondary plant compounds and are subdivided into two groups, the steroid saponins and triterpenoid saponins, even including steroid alkaloid saponins (page 2, para. 3). The wide range of structural variation options, both in the aglycone and sugar moieties explains the variety of different saponins with diverse effects (page 2, para. 5). For example, only saponins of a certain electrophoretic mobility are able to enhance the endosomal escape (page 8, para. 2). In addition, the enhancing effect of saponin (SA1641) on a targeted toxin is clathrin- and actin-dependent. Six inhibitory agents that are known to inhibit either clathrin-mediated endocytosis, GTPase activity of dynamin-2, actin-polymerization, endosomal acidification, or caveolae-dependent endocytosis were tested. Inhibition of clathrin-mediated endocytosis, actin-polymerization, and endosomal acidification blocked the enhancer effect of Saponinum album (page 12, para. 2).
Likewise, Barr (of record, Advanced Drug Delivery Reviews 32: 247-271, 1998; PTO 892) teaches that saponins have different molecular weight, different adjuvant activity, different toxicity, and different structures (Table1, and § 3. Structural and functional analysis of purified Quillaias saponins).
Bhargava et al (of record, Molecular Oncology 11: 1527-1543, 2017; PTO 1449) teaches that effect by endosomal escape enhancers saponin depends on the cell line, the targeting moiety, target receptor expression, and the structure of the enhancer, see p. 1539, right col.
A "representative number of species" means that those species that are adequately described are representative of the entire genus. AbbVie Deutschland GMBH v. Janssen Biotech. III USPQ2d 1780, 1790 (Fed. Cir. 2014).
Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of isolating it. The compound itself is required. See Fiers v. Revel, 25 USPQ2d 1601 at 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016 (Fed. Cir. 1991). One cannot describe what one has not conceived. See Fiddes v. Baird, 30 USPQ2d 1481 at 1483 (BPAI 1993). In Fiddes, claims directed to mammalian FGFs were found to be unpatentable due to lack of written description for that broad class. The specification provided only the bovine sequence.
Vas-Cath Inc, v. Mahurkar. 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the written description inquiry, whatever is now claimed.” (See page 1117.) The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116.). Consequently, Applicant was not in possession of the instant claimed invention. See University of California v. Eli Lilly and Co. 43 USPQ2d 1398.
As such, of skill in the art would reasonably conclude that applicant was not in possession of the claimed therapeutic combination of claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 at the time of filing.
For these reasons, the rejection is maintained.
Claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for only (1) a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising a first antibody that binds to domain III of human EGFR wherein the first antibody is an anti-EGFR antibody comprising the amino acid sequence of SEQ ID NO: 1 or matuzumab, wherein the first antibody is covalently linked to at least one saponin SO1861 and wherein the first antibody does not compete for binding with the second antibody matuzumab or cetuximab or EGF that binds to human EGFR; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second antibody that binds to the extracellular domain of human EGFR wherein the second antibody comprises the amino acid sequence of SEQ ID NO: 2 or pertuzumab or cetuximab, wherein the second antibody is conjugated to an effector, wherein the second antibody does not compete for binding to the EGFR with the first anti-EGFR antibody and wherein the effector is saporin or dianthin, (2) a first pharmaceutical composition comprising a conjugate comprising (a) a first antibody that binds to human HER2 wherein the antibody is covalently bound to at least one saponin SO1861; and (b) a second pharmaceutical composition comprising a conjugate comprising a second antibody that binds to human HER2, wherein the second antibody is conjugated to an effector molecule, wherein the first or second antibody is pertuzumab or trastuzumab, wherein the effector is saporin or dianthin and wherein the first antibody and second antibody binds to different epitope of the same human HER2 for saponin mediated endosomal escape of targeted toxin, and (3) a kit comprising the therapeutic combination above and instructions for use, does not reasonably provide enablement for any therapeutic combination as set forth in claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Enablement is considered in view of the Wands factors (MPEP 2164.01(a)). These factors include, but are not limited to: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. . In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988).
Claim 1 encompasses a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second binding molecule different from the first binding molecule, the second binding molecule comprising any second binding region different from the first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from the first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule,
wherein the first pharmaceutical composition and the second pharmaceutical composition optionally further comprise a pharmaceutically acceptable excipient and optionally further comprise a pharmaceutically acceptable diluent.
Claim 2 encompasses a pharmaceutical composition comprising:
any conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any triterpenoid saponin of the monodesmosidic type or the bidesmosidic type; and
any conjugate comprising any second binding molecule different from said first binding molecule, the second binding molecule comprising any second binding region different from said first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from said first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule, and optionally further comprising a pharmaceutically acceptable excipient and optionally further comprising a pharmaceutically acceptable diluent.
Claim 3 encompasses the therapeutic combination of claim 1, wherein the first binding molecule is a first proteinaceous binding molecule or a first non-proteinaceous ligand comprising the first binding region for binding to the first binding site of the cell-surface molecule, and/or wherein the second binding molecule is a second proteinaceous binding molecule or a second non- proteinaceous ligand comprising the second binding region for binding to the second binding site of the cell-surface molecule.
Claim 4 encompasses the therapeutic combination of claim 1, wherein the first binding molecule is a first proteinaceous binding molecule and wherein the saponin is covalently bound to an amino acid residue of the first binding molecule, or via a linker.
Claim 5 encompasses the therapeutic combination of claim 1, wherein the first binding site is a first epitope of said cell-surface molecule such as any cell-surface receptor and wherein the second binding site is a second epitope of said, same, cell-surface molecule, wherein the second epitope is different from the first epitope.
Claim 6 encompasses the therapeutic combination of claim 1, wherein the saponin is a bidesmosidic triterpene saponin.
Claim 8 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule comprises any ligand for binding to the first binding site of the cell-surface molecule such as EGF, or
wherein the first binding region of the first binding molecule comprises or consists of an immunoglobulin or at least one binding fragment or binding domain of said immunoglobulin comprising the first binding region for binding to the first binding site of the cell-surface molecule, and/or
wherein the second binding region of the second binding molecule comprises or consists of a ligand for binding to the second binding site of the cell-surface molecule, or
wherein the second binding region of the second binding molecule comprises or consists of an immunoglobulin or at least one binding fragment or binding domain of said immunoglobulin comprising the second binding region for binding to the second binding site of the cell-surface molecule.
Claim 9 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule comprises any monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment, and/or
wherein the second binding region of the second binding molecule comprises or consists of a monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment.
Claim 11 encompasses the therapeutic combination of claim 1, wherein the first binding region and the second binding region are selected to simultaneously bind the same cell-surface molecule at the first binding site and at the second binding site.
Claim 12 encompasses the therapeutic combination of claim 1, wherein the first binding region is selected to bind to the first binding site of the cell-surface molecule without competing for the binding of the second binding region to the second binding site of the same cell-surface molecule, and wherein the second binding region is selected to bind to the second binding site of the cell-surface molecule without competing for the binding of the first binding region to the first binding site of the same cell-surface molecule.
Claim 13 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23, the saponin comprising a first saccharide chain at the C3beta-OH group of the saponin, the first saccharide chain optionally comprising a glucuronic acid moiety, and the saponin comprising a second saccharide chain linked to C28 of the saponin and comprising or consisting of a monosaccharide or a linear or branched oligosaccharide wherein optionally at least one saccharide moiety of the second saccharide chain comprises at least one acetyl group, for example 1, 2, 3 or 4 acetyl groups.
Claim 14 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a saponin isolated from any one or more of a Gypsophila species, a Saponaria species, an Agrostemma species and a Quillaja species.
Claim 17 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is Quillaja bark saponin, or a saponin derivative, or any of their stereoisomers or any combination thereof.
Claim 21 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene glycoside belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 of the aglycone core structure of the saponin, wherein the saponin is covalently bound to the first binding molecule, via an aldehyde function in the saponin via at least one linker.
Claim 22 encompasses the therapeutic combination of claim 21, wherein the aldehyde function in position C23 of the aglycone core structure of the at least one saponin is covalently bound to linker ε-maleimidocaproic acid hydrazide (EMCH), which linker is covalently bound via a thio-ether bond to a sulfhydryl group in the first binding molecule.
Claim 23 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is a bidesmosidic triterpene glycoside belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 of the aglycone core structure of the saponin and comprising a glucuronic acid unit in a first saccharide chain at the C3beta- OH group of the aglycone core structure of the saponin, wherein the saponin is covalently bound to an amino-acid residue of the first binding molecule via the carboxyl group of the glucuronic acid unit in the first saccharide chain.
Claim 24 encompasses the therapeutic combination of claim 23, wherein the at least one saponin comprises a glucuronic acid unit in its first saccharide chain at the C3beta-OH group of the aglycone core structure of the at least one saponin, wherein the glucuronic acid unit is covalently bound to linker 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), wherein the linker is covalently bound via an amide bond to an amine group in the first binding molecule.
Claim 25 encompasses the therapeutic combination of claim 1, wherein the cell-surface molecule is any cell-surface receptor.
Claim 26 encompasses the therapeutic combination of claim 1, wherein the first binding region of the first binding molecule and the second binding region of the second binding molecule comprise any antibody or any cell-surface molecule binding fragment thereof or cell-surface molecule binding domain(s) thereof and/or comprise or consist of any ligand for binding to the undisclosed cell-surface molecule,
with the proviso that the first binding region and the second binding region are different and with the proviso that the first binding site and the second binding site are different.
Claim 54 encompasses a kit comprising the therapeutic combination of claim 1, and instructions for use.
Claim 55 encompasses the therapeutic combination of claim 1, wherein the at least one saponin is any one of: dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate, AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R, or any saponin derivative, a stereoisomer or a combination thereof.
Regarding “binding molecule”, the specification define as follow:
[0039] The term “binding molecule” has its regular scientific meaning and here refers to a molecule capable of specifically binding to another molecule such as a cell-surface molecule, e.g. a cell-surface receptor. Typical binding molecules are peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to e.g. a protein, a lipid, a (poly)saccharide, such as a cell-surface receptor or a cell-surface molecule. “Specifically binding” here refers to specific and selective binding with higher affinity than non-specific background binding.
Regarding “binding region”, the specification define as follow:
[0020] The term “binding region” has its regular scientific meaning and here refers to a part of a molecule or (a) chemical group(s) of a molecule or a(n) (linear or non-linear) amino-acid sequence of a protein or peptide and the like, that has the capacity to bind to a binding partner molecule. A typical binding region are the CDR loops of an immunoglobulin. A typical binding region of a protein is or are loop(s) of amino-acid residues comprised by said protein and capable of specifically binding to the binding site on a binding partner molecule such as a protein, cell-surface receptor, etc.
Regarding “binding site”, the specification define as follow:
[0021] The term “binding site” has its regular scientific meaning and here refers to a region on a macromolecule such as a protein, for example a cell-surface molecule such as a cell-surface receptor, that binds to another molecule such as a protein, for example a ligand, with specificity.
Regarding “cell-surface molecule”, the specification define as follow:
[0022] The term “cell-surface molecule” has its regular scientific meaning and here refers to a molecule that is present and exposed at the outside surface of a cell such as a blood cell or an organ cell, such as a mammalian cell, such as a human cell. Typically, a cell-surface molecule is a protein such as a receptor, or a lipid molecule or a polysaccharide.
Regarding “mono-desmosidic saponin”, the specification define as follow:
[0026] The term “mono-desmosidic saponin” has its regular scientific meaning and here refers to a triterpenoid saponin containing a single saccharide chain bound to the aglycone core, wherein the saccharide chain consists of one or more saccharide moieties.
Regarding “bi-desmosidic saponin”, the specification define as follow:
[0027] The term “bi-desmosidic saponin” has its regular scientific meaning and here refers to a triterpenoid saponin containing two saccharide chains bound to the aglycone core, wherein each of the two saccharide chains consists of one or more saccharide moieties.
Regarding “triterpenoid saponin”, the specification define as follow:
[0028] The term “triterpenoid saponin” has its regular scientific meaning and here refers to a saponin having a triterpenoid-type of aglycone core structure. The triterpenoid saponin differs from a saponin based on a steroid glycoside such as sapogenol in that such saponin comprising steroid glycoside has a steroid core structure, and the triterpenoid saponin differs from a saponin based on an alkaloid glycoside such as tomatidine in that such saponin comprising alkaloid glycoside has a alkaloid core structure.
Regarding “saponin”, the specification define as follow:
[0023] The term “saponin” has its regular scientific meaning and here refers to a group of amphipathic glycosides which comprise one or more hydrophilic glycone moieties combined with a lipophilic aglycone core which is a sapogenin. The saponin may be naturally occurring or synthetic (i.e. non-naturally occurring). The term “saponin” includes naturally-occurring saponins, derivatives of naturally-occurring saponins as well as saponins synthesized de novo through chemical and/or biotechnological synthesis routes. Saponin has a triterpene backbone, which is a pentacyclic C30 terpene skeleton, also referred to as sapogenin or aglycone. Within the context of the invention saponin is not considered an effector molecule nor an effector moiety in the conjugates according to the invention. Thus, in conjugates comprising a saponin and an effector moiety, the effector moiety is a different molecule than the conjugated saponin.
Regarding “effector molecule”, the specification define as follow:
[0033] The term “effector molecule”, or “effector moiety” when referring to the effector molecule as part of e.g. a covalent conjugate, has its regular scientific meaning and here refers to a molecule that can selectively bind to for example any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and regulates the biological activity of such one or more target molecule(s). In the conjugate of the invention the effector moiety for example exerts its effect in the cytosol, in the cell nucleus, is delivered intracellularly in the endosome and/or lysosome, and/or is active after exiting or escaping the endosomal-lysosomal pathway. The effector molecule is for example a molecule selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof. Thus, for example, an effector molecule or an effector moiety is a molecule or moiety selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or any combination thereof, that can selectively bind to any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and that upon binding to the target molecule regulates the biological activity of such one or more target molecule(s). For example, an effector moiety is a toxin or an active toxic fragment thereof or an active toxic derivative or an active toxic domain thereof. Typically, an effector molecule can exert a biological effect inside a cell such as a mammalian cell such as a human cell, such as in the cytosol of said cell. An effector molecule or moiety of the invention is thus any substance that affects the metabolism of a cell by interaction with an intracellular effector molecule target, wherein this effector molecule target is any molecule or structure inside cells excluding the lumen of compartments and vesicles of the endocytic and recycling pathway but including the membranes of these compartments and vesicles. Said structures inside cells thus include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the inner part of the plasma membrane and the cytosol. Typical effector molecules are thus drug molecules, an enzyme, plasmid DNA, toxins such as toxins comprised by antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, nucleic acids comprised by an antibody-oligonucleotide conjugate (AOC). For example, an effector molecule is a molecule which can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling. In the context of the invention, an effector molecule or effector moiety when the effector molecule is part of a conjugate, is not a saponin, and is not a cell-surface molecule binding molecule such as an antibody such as an sdAb. Typically, an effector moiety comprised by the conjugate exerts its therapeutic (for example toxic, enzymatic, inhibitory, gene silencing, etc.) effect in the cytosol and/or in the cell nucleus. Typically, the effector moiety is delivered intracellularly in the endosome and/or in the lysosome, and typically the effector moiety is active after exiting or escaping the endosomal-lysosomal pathway.
The specification exemplifies just saponin SO1861 purified by Analyticon Discovery GmbH from raw plant extract obtained from Saponaria officinalis. See para. [0157].
Example 1. 1T2C, Non-Competing HER2 Targeting
[0147] SO1861-EMCH was conjugated via cysteine residues (Cys) to pertuzumab, with a DAR 4, (pertuzumab-(Cys-L-SO1861).sup.4. Pertuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin, saporin, with a DAR4). Pertuzumab and Trastuzumab recognize and bind human HER2 at different epitopes (non-competing). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50=0.5 nM; FIG. 2A) whereas equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM trastuzumab-saporin could not induce any cell killing activity in HER2 expressing cells. When we compare these data with the combination of Trastuzumab-(Cys-L-SO1861).sup.4+50 pM trastuzumab-saporin we observe that at high concentrations Trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity is reduced due to competition of both trastuzumab antibody conjugates for binding the HER2 receptor. In MDA-MB-468 cells (no HER2 expression) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 2B) for any of the treatments.
[0148] All this shows that the use of two different antibodies recognizing the same receptor but binding at different epitopes (different binding sites), effectively induce cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of trastuzumab-saporin in high HER2 expressing cells, but not in cells that do not express HER2. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals activity at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
[0149] Next, trastuzumab-saporin was titrated on a fixed concentration of 2.5 nM and 75 nM pertuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and HER2 non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing at low concentrations trastuzumab-saporin in combination with 2.5 nM, or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 in SK-BR-3 (HER2.sup.++; IC50=0.5 pM; FIG. 3A), whereas Trastuzumab-saporin or Trastuzumab-saporin+2.5 nM or 75 nM pertuzumab showed only at high concentrations cell killing in SK-BR-3 cells (IC50>1000 pM; FIG. 3A). When these data is compared with the combination of trastuzumab-saporin+2.5 nM or 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity was strongly reduced when combined with 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9. In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 3B).
[0150] All this shows that the combination of low concentrations of trastuzumab-saporin+2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 induce effective cell killing in high HER2 expressing cells. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals effective cell killing at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
Example 2. 1T2C, Non-Competing HER2 Targeting
[0151] Next, pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM pertuzumab-dianthin (pertuzumab conjugated to the protein toxin, dianthin, with a DAR4). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low concentrations of trastuzumab-(Cys-L-SO1861).sup.4 or pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50<0.1 nM; FIG. 4A). At higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4 cell killing activity was reduced whereas higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 were still effective. Equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM pertuzumab-dianthin were not effective in HER2 expressing cells (SK-BR-3: IC50>1000 nM; FIG. 4A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 4B) for any of the treatments.
[0152] All this shows that the use of two different antibodies recognizing the same receptor but bind at a different epitopes, effectively induce cell killing at low and higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of pertuzumab-dianthin in high HER2 expressing cells.
[0153] Next, pertuzumab-dianthin was titrated on a fixed concentration of 2.5 nM and 25 nM pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing of SK-BR-3 cells (HER2.sup.++) at low concentrations pertuzumab-dianthin in combination with 2.5 nM, trastuzumab-(Cys-L-SO1861).sup.4 or 2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 (IC50=0.5 pM; IC50=0.5 pM, resp. FIG. 5A), whereas pertuzumab-dianthin+25 nM trastuzumab-(Cys-L-SO1861).sup.4 showed more efficient cell killing compared to the combination of pertuzumab-dianthin+25 nM pertuzumab-(Cys-L-SO1861).sup.4. Equivalent concentrations of pertuzumab-dianthin or pertuzumab-dianthin+25 nM pertuzumab showed only at high concentrations some slight cell killing activity in SK-BR-3 cells (IC50>10.000 pM; FIG. 5A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 5B).
[0154] All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
Example 3. 1T2C, Non-Competing EGFR Targeting
[0155] SO1861-EMCH was conjugated via cysteine residues (Cys) to matuzumab, with a DAR 3,3, (matuzumab-SO1861). Matuzumab-SO1861 was titrated on a fixed concentration of 10 pM cetuximab-saporin (cetuximab conjugated to the protein toxin, saporin, with a DAR4) or 10 pM EGFdianthin (recombinant toxin fusion protein). Matuzumab recognizes and binds human EGFR at a different epitope compared to cetuximab and EGF, whereas Cetuximab and EGF compete for binding the EGFR receptor. Targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) and non-expressing cells (A2058, EGFR.sup.−) was determined. This revealed strong cell killing at low and higher concentrations of matuzumab-(SO1861)+10 pM cetuximab-saporin or 10 pM EGFdianthin in A431 cells (IC50=2 nM; FIG. 6A) whereas equivalent concentrations matuzumab, matuzumab-SO1861, matuzumab+10 pM cetuximab-saporin or matuzumab+10 pM EGFdianthin could not induce any cell killing activity in A431 cells (IC50>1000 nM; FIG. 6A). When we compare these data with the combination of cetuximab-(Cys-L-SO1861).sup.4+10 pM cetuximab-saporin or cetuximab-SO1861+10 pM EGF-dianthin we observe that at higher concentrations Cetuximab-SO1861, cell killing activity is reduced due to competition of both cetuximab conjugate and EGF for binding the EGFR receptor. In A2058 cells (EGFR.sup.−) no cell killing was observed (IC50>1000 nM; FIG. 6B) for any of the treatments. All this shows that the use of two different antibodies or antibody/ligand combinations recognizing the same receptor but bind at different epitopes, effectively induces cell killing in EGFR.sup.++ expressing cells at low and high concentrations of matuzumab-SO1861 in combination with a fixed low concentration (10 pM) of cetuximab-saporin or EGFdianthin. Thus the use of the combination according to the invention omits competition for receptor binding and reveals very effective endosomal escape and cytoplasmic toxin delivery resulting in efficient and selective tumor cell killing.
[0156] Next, cetuximab-saporin was titrated on a fixed concentration of 10 nM and 75 nM matuzumab-SO1861 and targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) was determined. This revealed that, 10 nM and 75 nM matuzumab-SO1861 in combination with low concentrations cetuximab-saporin induced efficient cell killing in EGFR expressing cells (A431: 1050=0.5 pM; FIG. 7A), whereas cetuximab-saporin or cetuximab-saporin+10 nM or 75 nM matuzumab showed only at high concentrations cell killing (1050=1000 pM FIG. 3A). When we compared these data with the combination of cetuximab-saporin+10 nM and 75 nM cetuximab-SO1861, cell killing activity was reduced with increased concentrations of cetuximab-SO1861. In A2058 cells (EGFR.sup.−) no cell killing was observed (A2058: IC50 >1000 pM; FIG. 7B). All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
SO1861-EMCH Synthesis
[0159] To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture stirred at room temperature. After 1.5 hours the reaction mixture was subjected to preparative MP-LC..sup.1 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS 96%.
[0160] LRMS (m/z): 2069 [M-1].sup.1−
[0161] LC-MS r.t. (min): 1.08.sup.4
mAb-SO1861 Synthesis
To Matuzumab freshly prepared TCEP solution (1.00 mg/ml, 1.971 mole equivalents, 2.80×10.sup.−5 mmol) was added. The reaction mixture was vortexed briefly then incubated for 90 minutes at 20° C. with roller-mixing. After incubation (prior to addition of SO1861-EMCH), a 0.5 mg (0.101 ml) aliquot of Matuzumab-SH was removed and purified by gel filtration using zeba spin desalting column eluting into TBS pH 7.5. This aliquot was characterised by UV-vis analysis and Ellman's assay. To the bulk Matuzumab-SH was added an aliquot of freshly prepared SO1861-EMCH solution (2.00 mg/ml, 8 mole equivalents, 8.54×10.sup.−5 mmol, 0.089 ml), the mixture vortexed briefly then incubated for 120 minutes at 20° C. Besides the conjugation reaction, two aliquots of desalted Matuzumab-SH (0.10 mg, 0.022 ml, 6.70×10.sup.−7 mmol) were reacted with NEM (8.00 equivalents, 5.36×10.sup.−6 mmol, 0.67 pg, 2.7 μl of a 0.25 mg/ml solution) or TBS pH 7.5 buffer (2.7 μl) for 120 minutes at 20° C., as positive and negative controls, respectively. After incubation (prior to addition of NEM), a ca. 60 pg (0.020 ml) aliquot of Matuzumab-SO1861 mixture was removed and characterised by Ellman's assay alongside positive and negative controls to obtain SO1861 incorporation. To the bulk Matuzumab-SO1861 mixture was added an aliquot of freshly prepared NEM solution (0.25 mg/ml, 5 mole equivalents, 5.34×10.sup.−5 mmol, 0.007 mg) to quench the reaction. The conjugate was purified by zeba 40K MWCO spin column eluting with DPBS pH 7.5 to give purified Matuzumab-SO1861 conjugate. The product was normalised to 2.0 mg/ml and filtered to 0.2 μm, to afford Matuzumab-SO1861 (total yield=1.10 mg, 52%, Matuzumab:SO1861-EMCH=3.3).
[0162] Similar procedures were followed to produce pertuzumab-SO1861 (DAR4), cetuximab-SO1861 (DAR4), trastuzumab-SO1861 (DAR4)
Pertuzumab-Dianthin Synthesis
[0163] Dianthin-Cys (17.0 ml, ˜9.6 mg) was concentrated by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 10 minutes). The resulting 3.25 ml aliquot was gel filtered using zeba 10 ml spin columns eluting with TBS pH 7.5.
[0164] Pertuzumab (0.30 ml, ˜10 mg) was diluted to 10 mg/ml with DPBS pH 7.5, desalted via zeba 5 ml spin column eluting with DPBS pH 7.5 and normalised to 2.50 mg/ml. To an aliquot of Pert (5.00 mg, 3.30×10.sup.−5 mmol, 2.593 mg/ml) was added an aliquot of freshly prepared SMCC solution (1.00 mg/ml, 4.20 mole equivalents, 13.9×10.sup.−5 mmol) in DMSO, the mixture vortexed briefly then incubated for 60 minutes at 20° C. with roller-mixing. After, the reaction was quenched by the addition of an aliquot of a freshly prepared glycine solution (2.0 mg/ml, 5.0 mole equivalents, 69.5×10.sup.−5 mmol) in DPBS pH 7.5. Pert-SMCC (4.27 mg, 2.80×10.sup.−5 mmol, 1.514 mg/ml) was obtained after gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0165] To Dianthin-Cys (7.54 mg, 25.3×10.sup.−5 mmol, 2.258 mg/ml) was added an aliquot of freshly prepared TCEP solution (1.00 mg/ml, 0.5 mole equivalents, 12.6×10.sup.−5 mmol) in TBS pH 7.5, the mixture briefly vortexed then incubated for 60 minutes at 20° C. with roller-mixing. After, Dianthin-SH (6.0 mg, 20.2×10.sup.−5 mmol, 1.722 mg/ml, Dianthin:SH=1.1) was obtained by gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0166] To the bulk Pert-SMCC was added the aliquot of Dianthin-SH (7.20 mole equivalents), the mixture vortexed briefly then incubated overnight at 20° C. After ca. 16 hours, the reaction was quenched by the addition of an aliquot of freshly prepared NEM solution (2.50 mg/ml, 5.0 mole equivalents, 101×10.sup.−5 mmol) in TBS pH 7.5. The reaction mixture was filtered to 0.45 μm and then concentrated to <2 ml by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 15 minutes). The conjugate was purified by gel filtration using a 1.6×35 cm Superdex 200PG column eluting with DPBS pH 7.5.
Example 4. SO1861+EGFR/HER2/CD71 Targeted mAb
[0169] SO1861 was titrated on a fixed concentration of 10 pM CD71-saporin (DAR4), 10 pM cetuximab-saporin (DAR4), 10 pM matuzumab-dianthin (DAR4), 10 pM pertuzumab-saporin (DAR4), 10 pM or 50 pM pertuzumab-saporin (DAR4) and 50 pM trastuzumab-saporin (DAR4) and targeted protein toxin-mediated cell killing on A431 (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) and A2058 (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) was determined. In A431 cells (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) this revealed cell killing activity for all EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) as well as 10 pM CD71-saporin and 50 pM pertuzumab-saporin at SO1861: IC50=200 nM, whereas 50 pM trastuzumab or 10 pM pertuzumab-saporin showed activity at IC50=250 nM and IC50=300 nM, respectively (FIG. 8A), In A2058 cells (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) the EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) were not active but 10 pM CD71mab-saporin, 10 or 50 pM Pertuzumab-saporin and 50 pM trastuzumab-saporin all showed activity at SO1861: IC50=200 nM (FIG. 8B).
However, enablement is not commensurate in scope with how to make and use the claim invention as broadly as claim. The specification discloses:
Example 1. 1T2C, Non-Competing HER2 Targeting
[0147] SO1861-EMCH was conjugated via cysteine residues (Cys) to pertuzumab, with a DAR 4, (pertuzumab-(Cys-L-SO1861).sup.4. Pertuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM trastuzumab-saporin (trastuzumab conjugated to the protein toxin, saporin, with a DAR4). Pertuzumab and Trastuzumab recognize and bind human HER2 at different epitopes (non-competing). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50=0.5 nM; FIG. 2A) whereas equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM trastuzumab-saporin could not induce any cell killing activity in HER2 expressing cells. When we compare these data with the combination of Trastuzumab-(Cys-L-SO1861).sup.4+50 pM trastuzumab-saporin we observe that at high concentrations Trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity is reduced due to competition of both trastuzumab antibody conjugates for binding the HER2 receptor. In MDA-MB-468 cells (no HER2 expression) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 2B) for any of the treatments.
[0148] All this shows that the use of two different antibodies recognizing the same receptor but binding at different epitopes (different binding sites), effectively induce cell killing at low and high concentrations of pertuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of trastuzumab-saporin in high HER2 expressing cells, but not in cells that do not express HER2. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals activity at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
[0149] Next, trastuzumab-saporin was titrated on a fixed concentration of 2.5 nM and 75 nM pertuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and HER2 non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing at low concentrations trastuzumab-saporin in combination with 2.5 nM, or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 in SK-BR-3 (HER2.sup.++; IC50=0.5 pM; FIG. 3A), whereas Trastuzumab-saporin or Trastuzumab-saporin+2.5 nM or 75 nM pertuzumab showed only at high concentrations cell killing in SK-BR-3 cells (IC50>1000 pM; FIG. 3A). When these data is compared with the combination of trastuzumab-saporin+2.5 nM or 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9, cell killing activity was strongly reduced when combined with 75 nM trastuzumab-(Cys-L-SO1861).sup.3,9. In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 3B).
[0150] All this shows that the combination of low concentrations of trastuzumab-saporin+2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 or 75 nM pertuzumab-(Cys-L-SO1861).sup.4 induce effective cell killing in high HER2 expressing cells. Thus, the use of the combination of both conjugates according to the invention omits competition for receptor binding and reveals effective cell killing at low and higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4.
Example 2. 1T2C, Non-Competing HER2 Targeting
[0151] Next, pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 was titrated on a fixed concentration of 50 pM pertuzumab-dianthin (pertuzumab conjugated to the protein toxin, dianthin, with a DAR4). Targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed strong cell killing at low concentrations of trastuzumab-(Cys-L-SO1861).sup.4 or pertuzumab-(Cys-L-SO1861).sup.4 (SK-BR-3: IC50<0.1 nM; FIG. 4A). At higher concentrations of pertuzumab-(Cys-L-SO1861).sup.4 cell killing activity was reduced whereas higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 were still effective. Equivalent concentrations pertuzumab, pertuzumab-(Cys-L-SO1861).sup.3,9 or pertuzumab+50 pM pertuzumab-dianthin were not effective in HER2 expressing cells (SK-BR-3: IC50>1000 nM; FIG. 4A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed (MDA-MB-468: IC50>1000 nM; FIG. 4B) for any of the treatments.
[0152] All this shows that the use of two different antibodies recognizing the same receptor but bind at a different epitopes, effectively induce cell killing at low and higher concentrations of trastuzumab-(Cys-L-SO1861).sup.4 in combination with a fixed low concentration (50 pM) of pertuzumab-dianthin in high HER2 expressing cells.
[0153] Next, pertuzumab-dianthin was titrated on a fixed concentration of 2.5 nM and 25 nM pertuzumab-(Cys-L-SO1861).sup.4 or trastuzumab-(Cys-L-SO1861).sup.4 and targeted protein toxin mediated cell killing on HER2 expressing cells (SK-BR-3, HER2.sup.++) and non-expressing cells (MDA-MB-468, HER2.sup.−) was determined. This revealed efficient cell killing of SK-BR-3 cells (HER2.sup.++) at low concentrations pertuzumab-dianthin in combination with 2.5 nM, trastuzumab-(Cys-L-SO1861).sup.4 or 2.5 nM pertuzumab-(Cys-L-SO1861).sup.4 (IC50=0.5 pM; IC50=0.5 pM, resp. FIG. 5A), whereas pertuzumab-dianthin+25 nM trastuzumab-(Cys-L-SO1861).sup.4 showed more efficient cell killing compared to the combination of pertuzumab-dianthin+25 nM pertuzumab-(Cys-L-SO1861).sup.4. Equivalent concentrations of pertuzumab-dianthin or pertuzumab-dianthin+25 nM pertuzumab showed only at high concentrations some slight cell killing activity in SK-BR-3 cells (IC50>10.000 pM; FIG. 5A). In MDA-MB-468 cells (HER2.sup.−) no cell killing was observed for any of the treatments (MDA-MB-468: IC50>10.000 pM; FIG. 5B).
[0154] All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
Example 3. 1T2C, Non-Competing EGFR Targeting
[0155] SO1861-EMCH was conjugated via cysteine residues (Cys) to matuzumab, with a DAR 3,3, (matuzumab-SO1861). Matuzumab-SO1861 was titrated on a fixed concentration of 10 pM cetuximab-saporin (cetuximab conjugated to the protein toxin, saporin, with a DAR4) or 10 pM EGFdianthin (recombinant toxin fusion protein). Matuzumab recognizes and binds human EGFR at a different epitope compared to cetuximab and EGF, whereas Cetuximab and EGF compete for binding the EGFR receptor. Targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) and non-expressing cells (A2058, EGFR.sup.−) was determined. This revealed strong cell killing at low and higher concentrations of matuzumab-(SO1861)+10 pM cetuximab-saporin or 10 pM EGFdianthin in A431 cells (IC50=2 nM; FIG. 6A) whereas equivalent concentrations matuzumab, matuzumab-SO1861, matuzumab+10 pM cetuximab-saporin or matuzumab+10 pM EGFdianthin could not induce any cell killing activity in A431 cells (IC50>1000 nM; FIG. 6A). When we compare these data with the combination of cetuximab-(Cys-L-SO1861).sup.4+10 pM cetuximab-saporin or cetuximab-SO1861+10 pM EGF-dianthin we observe that at higher concentrations Cetuximab-SO1861, cell killing activity is reduced due to competition of both cetuximab conjugate and EGF for binding the EGFR receptor. In A2058 cells (EGFR.sup.−) no cell killing was observed (IC50>1000 nM; FIG. 6B) for any of the treatments. All this shows that the use of two different antibodies or antibody/ligand combinations recognizing the same receptor but bind at different epitopes, effectively induces cell killing in EGFR.sup.++ expressing cells at low and high concentrations of matuzumab-SO1861 in combination with a fixed low concentration (10 pM) of cetuximab-saporin or EGFdianthin. Thus the use of the combination according to the invention omits competition for receptor binding and reveals very effective endosomal escape and cytoplasmic toxin delivery resulting in efficient and selective tumor cell killing.
[0156] Next, cetuximab-saporin was titrated on a fixed concentration of 10 nM and 75 nM matuzumab-SO1861 and targeted protein toxin mediated cell killing on EGFR expressing cells (A431, EGFR.sup.++) was determined. This revealed that, 10 nM and 75 nM matuzumab-SO1861 in combination with low concentrations cetuximab-saporin induced efficient cell killing in EGFR expressing cells (A431: 1050=0.5 pM; FIG. 7A), whereas cetuximab-saporin or cetuximab-saporin+10 nM or 75 nM matuzumab showed only at high concentrations cell killing (1050=1000 pM FIG. 3A). When we compared these data with the combination of cetuximab-saporin+10 nM and 75 nM cetuximab-SO1861, cell killing activity was reduced with increased concentrations of cetuximab-SO1861. In A2058 cells (EGFR.sup.−) no cell killing was observed (A2058: IC50 >1000 pM; FIG. 7B). All this shows that the combination according to the invention omits receptor competition, revealing very effective endosomal escape and cytoplasmic toxin delivery resulting in very efficient and selective tumor cell killing.
SO1861-EMCH Synthesis
[0159] To SO1861 (121 mg, 0.065 mmol) and EMCH.TFA (110 mg, 0.325 mmol) was added methanol (extra dry, 3.00 mL) and TFA (0.020 mL, 0.260 mmol). The reaction mixture stirred at room temperature. After 1.5 hours the reaction mixture was subjected to preparative MP-LC..sup.1 Fractions corresponding to the product were immediately pooled together, frozen and lyophilized overnight to give the title compound (120 mg, 90%) as a white fluffy solid. Purity based on LC-MS 96%.
[0160] LRMS (m/z): 2069 [M-1].sup.1−
[0161] LC-MS r.t. (min): 1.08.sup.4
mAb-SO1861 Synthesis
To Matuzumab freshly prepared TCEP solution (1.00 mg/ml, 1.971 mole equivalents, 2.80×10.sup.−5 mmol) was added. The reaction mixture was vortexed briefly then incubated for 90 minutes at 20° C. with roller-mixing. After incubation (prior to addition of SO1861-EMCH), a 0.5 mg (0.101 ml) aliquot of Matuzumab-SH was removed and purified by gel filtration using zeba spin desalting column eluting into TBS pH 7.5. This aliquot was characterised by UV-vis analysis and Ellman's assay. To the bulk Matuzumab-SH was added an aliquot of freshly prepared SO1861-EMCH solution (2.00 mg/ml, 8 mole equivalents, 8.54×10.sup.−5 mmol, 0.089 ml), the mixture vortexed briefly then incubated for 120 minutes at 20° C. Besides the conjugation reaction, two aliquots of desalted Matuzumab-SH (0.10 mg, 0.022 ml, 6.70×10.sup.−7 mmol) were reacted with NEM (8.00 equivalents, 5.36×10.sup.−6 mmol, 0.67 pg, 2.7 μl of a 0.25 mg/ml solution) or TBS pH 7.5 buffer (2.7 μl) for 120 minutes at 20° C., as positive and negative controls, respectively. After incubation (prior to addition of NEM), a ca. 60 pg (0.020 ml) aliquot of Matuzumab-SO1861 mixture was removed and characterised by Ellman's assay alongside positive and negative controls to obtain SO1861 incorporation. To the bulk Matuzumab-SO1861 mixture was added an aliquot of freshly prepared NEM solution (0.25 mg/ml, 5 mole equivalents, 5.34×10.sup.−5 mmol, 0.007 mg) to quench the reaction. The conjugate was purified by zeba 40K MWCO spin column eluting with DPBS pH 7.5 to give purified Matuzumab-SO1861 conjugate. The product was normalised to 2.0 mg/ml and filtered to 0.2 μm, to afford Matuzumab-SO1861 (total yield=1.10 mg, 52%, Matuzumab:SO1861-EMCH=3.3).
[0162] Similar procedures were followed to produce pertuzumab-SO1861 (DAR4), cetuximab-SO1861 (DAR4), trastuzumab-SO1861 (DAR4)
Pertuzumab-Dianthin Synthesis
[0163] Dianthin-Cys (17.0 ml, ˜9.6 mg) was concentrated by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 10 minutes). The resulting 3.25 ml aliquot was gel filtered using zeba 10 ml spin columns eluting with TBS pH 7.5.
[0164] Pertuzumab (0.30 ml, ˜10 mg) was diluted to 10 mg/ml with DPBS pH 7.5, desalted via zeba 5 ml spin column eluting with DPBS pH 7.5 and normalised to 2.50 mg/ml. To an aliquot of Pert (5.00 mg, 3.30×10.sup.−5 mmol, 2.593 mg/ml) was added an aliquot of freshly prepared SMCC solution (1.00 mg/ml, 4.20 mole equivalents, 13.9×10.sup.−5 mmol) in DMSO, the mixture vortexed briefly then incubated for 60 minutes at 20° C. with roller-mixing. After, the reaction was quenched by the addition of an aliquot of a freshly prepared glycine solution (2.0 mg/ml, 5.0 mole equivalents, 69.5×10.sup.−5 mmol) in DPBS pH 7.5. Pert-SMCC (4.27 mg, 2.80×10.sup.−5 mmol, 1.514 mg/ml) was obtained after gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0165] To Dianthin-Cys (7.54 mg, 25.3×10.sup.−5 mmol, 2.258 mg/ml) was added an aliquot of freshly prepared TCEP solution (1.00 mg/ml, 0.5 mole equivalents, 12.6×10.sup.−5 mmol) in TBS pH 7.5, the mixture briefly vortexed then incubated for 60 minutes at 20° C. with roller-mixing. After, Dianthin-SH (6.0 mg, 20.2×10.sup.−5 mmol, 1.722 mg/ml, Dianthin:SH=1.1) was obtained by gel filtration using a zeba 10 ml spin column eluting with TBS pH 7.5.
[0166] To the bulk Pert-SMCC was added the aliquot of Dianthin-SH (7.20 mole equivalents), the mixture vortexed briefly then incubated overnight at 20° C. After ca. 16 hours, the reaction was quenched by the addition of an aliquot of freshly prepared NEM solution (2.50 mg/ml, 5.0 mole equivalents, 101×10.sup.−5 mmol) in TBS pH 7.5. The reaction mixture was filtered to 0.45 μm and then concentrated to <2 ml by ultrafiltration using a vivaspin T15 filter tube (3,000 g, 20° C., 15 minutes). The conjugate was purified by gel filtration using a 1.6×35 cm Superdex 200PG column eluting with DPBS pH 7.5.
Example 4. SO1861+EGFR/HER2/CD71 Targeted mAb
[0169] SO1861 was titrated on a fixed concentration of 10 pM CD71-saporin (DAR4), 10 pM cetuximab-saporin (DAR4), 10 pM matuzumab-dianthin (DAR4), 10 pM pertuzumab-saporin (DAR4), 10 pM or 50 pM pertuzumab-saporin (DAR4) and 50 pM trastuzumab-saporin (DAR4) and targeted protein toxin-mediated cell killing on A431 (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) and A2058 (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) was determined. In A431 cells (EGFR.sup.++/HER2.sup.+/−/CD71.sup.+) this revealed cell killing activity for all EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) as well as 10 pM CD71-saporin and 50 pM pertuzumab-saporin at SO1861: IC50=200 nM, whereas 50 pM trastuzumab or 10 pM pertuzumab-saporin showed activity at IC50=250 nM and IC50=300 nM, respectively (FIG. 8A), In A2058 cells (EGFR.sup.−/HER2.sup.+/−/CD71.sup.+) the EGFR targeted antibody-toxins (10 pM cetuximab-saporin, and 10 pM matuzumab-dianthin) were not active but 10 pM CD71mab-saporin, 10 or 50 pM Pertuzumab-saporin and 50 pM trastuzumab-saporin all showed activity at SO1861: IC50=200 nM (FIG. 8B).
However, the specification does not teach the structure, e.g., amino acid sequences of the first binding molecules that encompassed any and all peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands (claims 1, 54), any proteinaceous binding molecule or any non-proteinaceous ligand (claim 2) or any ligand (claim 8) wherein the undisclosed first binding molecule can bind to any and all cell-surface molecule, any cell-surface molecule is any cell-surface receptor or different epitope of the same undisclosed cell-surface receptor (claim 5) conjugated to any saponin from any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside via a linker (claim 3) in combination with a second binding molecule that different from the first binding molecule, wherein the second binding molecule comprising any possible peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to any cell-surface molecule, e.g., any cell-surface receptor of the same but different epitope (claim 5) conjugated to any effector molecule, e.g., a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, toxin and regulates the biological activity of such one or more target molecule(s).
The state of the art is such that saponin represents a heterogenous phytochemicals with different structures and properties.
For example, Fuchs (of record, Fuchs et al., Biomedicines, 5(14): 1-25, 2017; PTO 1449) teaches that saponins represent a wide spectrum in the field of secondary plant compounds and are subdivided into two groups, the steroid saponins and triterpenoid saponins, even including steroid alkaloid saponins (page 2, para. 3). The wide range of structural variation options, both in the aglycone and sugar moieties explains the variety of different saponins with diverse effects (page 2, para. 5). For example, only saponins of a certain electrophoretic mobility are able to enhance the endosomal escape (page 8, para. 2). In addition, the enhancing effect of saponin (SA1641) on a targeted toxin is clathrin- and actin-dependent. Six inhibitory agents that are known to inhibit either clathrin-mediated endocytosis, GTPase activity of dynamin-2, actin-polymerization, endosomal acidification, or caveolae-dependent endocytosis were tested. Inhibition of clathrin-mediated endocytosis, actin-polymerization, and endosomal acidification blocked the enhancer effect of Saponinum album (page 12, para. 2).
Likewise, Barr (of record, Advanced Drug Delivery Reviews 32: 247-271, 1998; PTO 892) teaches that saponins have different molecular weight, different adjuvant activity, different toxicity, and different structures (Table1, and § 3. Structural and functional analysis of purified Quillaias saponins).
Bhargava et al (of record, Molecular Oncology 11: 1527-1543, 2017; PTO 1449) teaches that effect of endosomal escape enhancer saponin depends on the cell line, the targeting moiety, target receptor expression, and the structure of the enhancer, see p. 1539, right col. There are insufficient working examples. One of skill in the art cannot predict the structures correlated with function share by members of the genus of first and second conjugates in combination encompassed by claims.
Even assuming the first binding molecule and second binding molecule comprise an immunoglobulin (claim 8) or antibody such as any monoclonal antibody, a single-domain antibody, at least one VHH domain, at least one VH domain, a variable heavy chain new antigen receptor (VNAR) domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, or a Fcab fragment (claim 9), the specification discloses VHH 7D12 comprises the amino acid sequence of SEQ ID NO: 1 and VHH 9G8 comprising the amino acid sequence of SEQ ID NO: 2. These two single domain antibodies bind to domain III of human EGFR, and compete with EGF (elected species) for binding to human EGFR. In addition 7D12 compete for binding of either cetuximab (anti-EGFR) or matuzumab (9G8). The specification also discloses pertuzumab and trastuzumab that bind to human HER2.
However, specification does not teach the structure, e.g., amino acid sequences of the heavy chain variable domain and the light chain variable domain of all possible antibodies that correlated with binding to any and all cell surface molecules (claims 1, 2, 8, 9, 11, 26, 54), or any region of any cell surface receptor (claims 5, 25), or any different first and second binding sites (aka different epitope) of the undisclosed cell-surface molecules simultaneously (claim 11) wherein the first binding region is selected to bind to the undisclosed first binding site of the cell-surface molecule without competing for binding of the second binding region to the second binding site (epitope) of the same-surface molecule (claim 12) or different epitopes (different first and second binding site of the same surface molecule (claim 26). There is no limitation on the structure of the first and second antibodies or ligand, and the binding sites (epitope) to which the first and second antibodies or ligands bind. The specification does not disclose a representative number of species of first and second binding molecules or antibodies comprising different binding region, e.g., heavy and light chains region for binding to different epitopes of all cell surface molecules or receptor wherein the first binding molecule is conjugated to any saponin and wherein the second binding molecule is conjugated to any effector falling within the scope of the genus or structure common to the members of the genus so the one of skill in the art can make and use the claimed invention without undue experimentation.
It is known in the art that antibodies have a large repertoire of distinct structures and that a huge variety of antibodies can be made to bind to a single epitope.
For example, Lloyd et al. taught that hundreds of functional antibody fragments can be isolated from an antibody library that bind to the same antigen wherein these antibodies have distinct heavy and light chain sequences (of record, Lloyd et al. Protein Engineering, Design & Selection 22:159-168, 2009; PTO 892; see, e.g., Discussion).
Similarly, Edwards et al., (of record, J Mol Biol. 334(1): 103-118, 2003; PTO 892), found that over 1000 antibodies, all different in amino acid sequence, were generated to a single protein; 568 different amino acid sequences identified for the V(H) CDR3 domains of these antibodies (Abstract).
Poosarla et al (of record, Biotechn. Bioeng. 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.)
Regarding the first or second binding molecule comprising at least one VH domain (claim 9), it is unlikely that the first binding or the second binding molecule as defined by the claims, which may contain less than the full complement of CDRs from the heavy chain variable region (VH) and light chain variable region (VL) have the required binding function.
Regarding antibody drug conjugate, the state of the prior art is such that the location or site of conjugation on the drug and the antibody affect conjugate stability, and pharmacokinetics of antibody drug conjugates.
For example, Strop et al (of record, Chemistry and Biology 20: 161-167, 2013; PTO 892) teach drug position can have a significant effect on linker stability and antibody pharmacokinetics. The site of conjugation on the drug and antibody can influence ADC properties differently in mice and rats, highlighting potential pitfalls of examining efficacy in mouse xenograft models and toxicity in rats or nonhuman primates, see abstract, p 166, p. 168 right col, in particular.
Nejadmoghaddam (of record, Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Regarding derivatives based on any one or more saponin (claims 17, 55), the specification does not teach the chemical structures of any such derivatives, including derivatives of SO1861 (elected species) and what effects that they may have without specific guidance. One of ordinary skilled cannot make and use the undisclosed derivatives conjugated to any first cell-surface binding molecule in combination with any second cell-surface binding molecules conjugated to any effector molecule. It is unpredictable which combination of all conjugates encompassed by claim will function in a given or predictable manner as the examples shown in the specification, e.g. enhance killing of targeted cells. There are no in vivo working examples.
Also, when combining two antibody conjugates it is not known whether the toxicities are additive, which would lead to an unacceptable level of toxicity. For example, Tol et al (of record, N Engl J Med. 5;360(6):563-72, February 2009; PTO 892) teaches that it is not always predictable that a combination of antibodies, each of which has had positive results, will produce a positive effect. Thus, a skilled person would not have been able to predict whether a combination of antibodies/conjugates would have an additive or synergistic effect or whether the antibodies/conjugates would interfere with each other so that the combination would have a reduced effect. It is also a possibility that since both antibodies/conjugates affect normal cells, they could act in concert to be more toxic than either alone. There are no in vivo working examples.
As such, it would require undue experimentation of one skilled in the art to practice the claimed invention. See page 1338, footnote 7 of Ex parte Aggarwal, 23 USPQ2d 1334 (PTO Bd. Pat App. & Inter. 1992).
Applicant's arguments filed July 23, 2026 have been fully considered but they are not persuasive.
Applicant respectfully traverses this rejection. As discussed above, the relevant binding molecules and receptor targets were well known in the art at the time of filing. The references cited by the Examiner themselves demonstrate the mature state of the field and the routine availability of suitable targeting molecules which indicates enablement already.
Regarding the amount of direction provided by the inventor and the existence of working examples, the specification provides detailed synthesis protocols and employs well-established conjugation methodologies that were routine in the art, including EMCH-mediated conjugation to cysteine residues, SMCC-mediated conjugation of effector molecules, and HATU-mediated conjugation via amine groups. See As-Filed Specification, paragraphs [0159]-[0166]. As discussed above, the specification further demonstrates the applicability of these methods across multiple antibodies, confirming that a POSA could readily prepare the claimed conjugates without undue experimentation.
The specification provides multiple working examples across different receptor targets,
binding-molecule formats, and effector molecules, demonstrating practical implementation of the claimed invention and providing substantial guidance to a POSA.
Regarding predictability, Applicant incorporates by reference the discussion of Bhargava above. Bhargava reports substantial enhancement of intracellular activity by SO1861 across multiple targeting molecules, demonstrating that the claimed approach is broadly applicable and predictable. Although the magnitude of enhancement varies depending on biological context, Bhargava consistently reports the presence of the enhancement effect. Because claims 1 and 2 do not require any particular magnitude of enhancement, such variability does not create undue experimentation or undermine enablement.
For at least the foregoing reasons, Applicant respectfully requests withdrawal of the rejection of claims 1-6, 8, 9, 11-14, 17, 21-26 and 54 under 35 U.S.C. § 112(a) for failure to comply with the enablement requirement.
In response, Claim 1 encompasses a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside; and
(b) a second pharmaceutical composition comprising a conjugate comprising a second binding molecule different from the first binding molecule, the second binding molecule comprising any second binding region different from the first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from the first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule,
wherein the first pharmaceutical composition and the second pharmaceutical composition optionally further comprise a pharmaceutically acceptable excipient and optionally further comprise a pharmaceutically acceptable diluent.
Claim 2 encompasses a pharmaceutical composition comprising:
any conjugate comprising any first binding molecule comprising any first binding region for binding to any first binding site of any cell-surface molecule and the conjugate comprising at least one saponin covalently bound to said first binding molecule, wherein the saponin is any triterpenoid saponin of the monodesmosidic type or the bidesmosidic type; and
any conjugate comprising any second binding molecule different from said first binding molecule, the second binding molecule comprising any second binding region different from said first binding region, the second binding region for binding to any second binding site of said cell-surface molecule different from said first binding site of said cell-surface molecule, and the conjugate comprising any effector molecule covalently bound to said second binding molecule, and optionally further comprising a pharmaceutically acceptable excipient and optionally further comprising a pharmaceutically acceptable diluent.
Regarding “binding molecule”, the specification define as follow:
[0039] The term “binding molecule” has its regular scientific meaning and here refers to a molecule capable of specifically binding to another molecule such as a cell-surface molecule, e.g. a cell-surface receptor. Typical binding molecules are peptides, proteins, non-protein molecules, cell-surface receptor ligands, protein ligands, that can bind to e.g. a protein, a lipid, a (poly)saccharide, such as a cell-surface receptor or a cell-surface molecule. “Specifically binding” here refers to specific and selective binding with higher affinity than non-specific background binding.
Regarding “binding region”, the specification define as follow:
[0020] The term “binding region” has its regular scientific meaning and here refers to a part of a molecule or (a) chemical group(s) of a molecule or a(n) (linear or non-linear) amino-acid sequence of a protein or peptide and the like, that has the capacity to bind to a binding partner molecule. A typical binding region are the CDR loops of an immunoglobulin. A typical binding region of a protein is or are loop(s) of amino-acid residues comprised by said protein and capable of specifically binding to the binding site on a binding partner molecule such as a protein, cell-surface receptor, etc.
Regarding “binding site”, the specification define as follow:
[0021] The term “binding site” has its regular scientific meaning and here refers to a region on a macromolecule such as a protein, for example a cell-surface molecule such as a cell-surface receptor, that binds to another molecule such as a protein, for example a ligand, with specificity.
Regarding “cell-surface molecule”, the specification define as follow:
[0022] The term “cell-surface molecule” has its regular scientific meaning and here refers to a molecule that is present and exposed at the outside surface of a cell such as a blood cell or an organ cell, such as a mammalian cell, such as a human cell. Typically, a cell-surface molecule is a protein such as a receptor, or a lipid molecule or a polysaccharide.
Other than the particular antibodies pertuzumab and trastuzumab that bind to different epitope on HER2, matuzumab and cetuximab that bind to different epitope on human EGFR; single-domain antibodies VHH 7D 12, VHH 9G8 that bind to EGFR and EGF ligand, the specification does not teach the (i) Complete structure, e.g. amino acid sequence of the heavy and light chain variable domains, ii. Partial structure, e.g., the six CDRs of all potential first or second binding region of all first or second binding molecules, e.g., antibody or antigen binding fragment thereof that correlated with binding to which binding site, e.g., amino acid sequence (epitope) of any and all cell-surface molecule, e.g., any a receptor, or a lipid molecule or a polysaccharide covalently conjugated to any one or more saponin from any monodesmosidic triterpene glycoside or any bidesmosidic triterpene glycoside or effector molecule encompassed by the claimed therapeutic combination.
Considering an antibody will have six CDRs, each CDR comprising approximately ten amino acid residues. With twenty naturally occurring amino acids, the potential size of the CDR variation, of just human antibodies within this indeterminate genus, is 2060.
The specification does not teach the genus first and second binding molecules (claims 1-2), proteinaceous binding molecule, e.g., antibodies or non-proteinaceous ligand (claim 3) that bind to different epitope on same undisclosed cell-surface molecule (claim 5), any first and second binding molecule such as any immunoglobulin (claim 8), any monoclonal antibody, any single domain antibody, any VHH, any VNAR domain, any Fab, any scFv, any Fv, any dAb, any F(ab)2, any Fab that bind to different epitope of any and all potential cell surface molecule (claims 9, 11-12).
The specification does not teach the structure-identifying information about the claimed first and second binding molecules that bind to different binding site (epitope) of any and all cell-surface molecule, or structure common to the members of the genus so the one of skill in the art can make and use without undue experimentation.
It is known in the art that antibodies have a large repertoire of distinct structures and that a huge variety of antibodies can be made to bind to a single epitope.
For example, Lloyd et al. taught that hundreds of functional antibody fragments can be isolated from an antibody library that bind to the same antigen wherein these antibodies have distinct heavy and light chain sequences (of record, Lloyd et al. Protein Engineering, Design & Selection 22:159-168, 2009; PTO 892; see, e.g., Discussion).
Similarly, Edwards et al., (of record, J Mol Biol. 334(1): 103-118, 2003; PTO 892), found that over 1000 antibodies, all different in amino acid sequence, were generated to a single protein; 568 different amino acid sequences identified for the V(H) CDR3 domains of these antibodies (Abstract).
Poosarla et al (of record, Biotechn. Bioeng. 114(6): 1331-1342, 2017; PTO 892) teach substantial diversity in designed mAbs (sharing less than 75% sequence similarity to all existing natural antibody sequences) that bind to the same 12-mer peptide, binding to different epitopes on the same peptide. Said reference further teaches “most B-cell epitopes... in nature consist of residues from different regions of the sequence and are discontinuous...de novo antibody designs against discontinuous epitopes present additional challenges...". (See entire reference.) Given that hundreds of unique antibody structures may bind a single epitope on a single antigen, the structure of an antibody cannot be predicted from the structure of the antigen, and a single species, or small group of species, cannot define a structure-function relationship so as to be representative of all the antibodies that bind to that antigen.
Regarding effector molecule conjugated to the undisclosed second binding molecule, the specification define effector as follow:
[0033] The term “effector molecule”, or “effector moiety” when referring to the effector molecule as part of e.g. a covalent conjugate, has its regular scientific meaning and here refers to a molecule that can selectively bind to for example any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and regulates the biological activity of such one or more target molecule(s). In the conjugate of the invention the effector moiety for example exerts its effect in the cytosol, in the cell nucleus, is delivered intracellularly in the endosome and/or lysosome, and/or is active after exiting or escaping the endosomal-lysosomal pathway. The effector molecule is for example a molecule selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof. Thus, for example, an effector molecule or an effector moiety is a molecule or moiety selected from any one or more of a small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or any combination thereof, that can selectively bind to any one or more of the target molecules: a protein, a peptide, a carbohydrate, a saccharide such as a glycan, a (phospho)lipid, a nucleic acid such as DNA, RNA, an enzyme, and that upon binding to the target molecule regulates the biological activity of such one or more target molecule(s). For example, an effector moiety is a toxin or an active toxic fragment thereof or an active toxic derivative or an active toxic domain thereof. Typically, an effector molecule can exert a biological effect inside a cell such as a mammalian cell such as a human cell, such as in the cytosol of said cell. An effector molecule or moiety of the invention is thus any substance that affects the metabolism of a cell by interaction with an intracellular effector molecule target, wherein this effector molecule target is any molecule or structure inside cells excluding the lumen of compartments and vesicles of the endocytic and recycling pathway but including the membranes of these compartments and vesicles. Said structures inside cells thus include the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, other transport vesicles, the inner part of the plasma membrane and the cytosol. Typical effector molecules are thus drug molecules, an enzyme, plasmid DNA, toxins such as toxins comprised by antibody-drug conjugates (ADCs), oligonucleotides such as siRNA, BNA, nucleic acids comprised by an antibody-oligonucleotide conjugate (AOC). For example, an effector molecule is a molecule which can act as a ligand that can increase or decrease (intracellular) enzyme activity, gene expression, or cell signaling. In the context of the invention, an effector molecule or effector moiety when the effector molecule is part of a conjugate, is not a saponin, and is not a cell-surface molecule binding molecule such as an antibody such as an sdAb. Typically, an effector moiety comprised by the conjugate exerts its therapeutic (for example toxic, enzymatic, inhibitory, gene silencing, etc.) effect in the cytosol and/or in the cell nucleus. Typically, the effector moiety is delivered intracellularly in the endosome and/or in the lysosome, and typically the effector moiety is active after exiting or escaping the endosomal-lysosomal pathway.
The specification exemplifies just Dianthin or saporin as effector molecules conjugated to just cetuximab (anti-EGFR) or matuzumab (anti-HER2), see working examples.
However, two species are not representative of the genus of effector molecule, e.g., small molecule such as a drug molecule, a toxin such as a protein toxin, an oligonucleotide such as a BNA, a Xeno nucleic acid or an siRNA, an enzyme, a peptide, a protein, or an active fragment or an active domain thereof, or any combination thereof to enable one of skilled in the art to make and use without undue experimentation.
The state of the prior art is such that the location or site of conjugation on the drug (aka effector molecule) and the antibody affect conjugate stability, and pharmacokinetics of antibody drug conjugates.
For example, Strop et al (of record, Chemistry and Biology 20: 161-167, 2013; PTO 892) teach drug position can have a significant effect on linker stability and antibody pharmacokinetics. The site of conjugation on the drug and antibody can influence ADC properties differently in mice and rats, highlighting potential pitfalls of examining efficacy in mouse xenograft models and toxicity in rats or nonhuman primates, see abstract, p 166, p. 168 right col, in particular.
Nejadmoghaddam (of record, Avicenna Journal of Medical Biotechnology 2(1): 3-23, 2019; PTO 892) discusses major obstacles of antibody-drug conjugates include off-target toxicity, tumor marker selection, antibody specificity, adequately affinity and receptor-mediated internalization are major aspects of choice, cytotoxic payload (e.g., up to 7 drugs per antibody), cytotoxic payload linkage strategy, aqueous solubility, non-immunogenic and stability in storage and bloodstream, see entire document, abstract, p. 15, in particular.
Regarding “saponin”, the specification define as follow:
[0023] The term “saponin” has its regular scientific meaning and here refers to a group of amphipathic glycosides which comprise one or more hydrophilic glycone moieties combined with a lipophilic aglycone core which is a sapogenin. The saponin may be naturally occurring or synthetic (i.e. non-naturally occurring). The term “saponin” includes naturally-occurring saponins, derivatives of naturally-occurring saponins as well as saponins synthesized de novo through chemical and/or biotechnological synthesis routes. Saponin has a triterpene backbone, which is a pentacyclic C30 terpene skeleton, also referred to as sapogenin or aglycone. Within the context of the invention saponin is not considered an effector molecule nor an effector moiety in the conjugates according to the invention. Thus, in conjugates comprising a saponin and an effector moiety, the effector moiety is a different molecule than the conjugated saponin.
The specification exemplifies just saponin SO1861 conjugated via the C23 aldehyde function using an EMCH linker (covalently bound via a thio-ether bond to a cysteine sulfhydryl group) or via the glucuronic acid unit using a HATU linker (covalently bound via an amide bond to a lysine amine group) to pertuzumab, cetuximab, or trastuzumab.
However, the specification does not teach the chemical structures of any and all derivatives (claim 17 and 55), or stereoisomers (claim 17 and 55) of any Quillaja bark saponin or any one or more of dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate, AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861 (elected species), QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R. One of ordinary skilled cannot predict which undisclosed derivative or stereoisomer conjugated to any and all potential first binding molecule different from the second binding molecule is effective as a therapeutic combination.
The state of the prior art teaches that saponin represents a heterogenous phytochemicals with different structures and properties.
For example, Fuchs (of record, Fuchs et al., Biomedicines, 5(14): 1-25, 2017; PTO 1449) teaches that saponins represent a wide spectrum in the field of secondary plant compounds and are subdivided into two groups, the steroid saponins and triterpenoid saponins, even including steroid alkaloid saponins (page 2, para. 3). The wide range of structural variation options, both in the aglycone and sugar moieties explains the variety of different saponins with diverse effects (page 2, para. 5). For example, only saponins of a certain electrophoretic mobility are able to enhance the endosomal escape (page 8, para. 2). In addition, the enhancing effect of saponin (SA1641) on a targeted toxin is clathrin- and actin-dependent. Six inhibitory agents that are known to inhibit either clathrin-mediated endocytosis, GTPase activity of dynamin-2, actin-polymerization, endosomal acidification, or caveolae-dependent endocytosis were tested. Inhibition of clathrin-mediated endocytosis, actin-polymerization, and endosomal acidification blocked the enhancer effect of Saponinum album (page 12, para. 2).
Likewise, Barr (of record, Advanced Drug Delivery Reviews 32: 247-271, 1998; PTO 892) teaches that saponins have different molecular weight, different adjuvant activity, different toxicity, and different structures (Table1, and § 3. Structural and functional analysis of purified Quillaias saponins).
Bhargava et al (of record, Molecular Oncology 11: 1527-1543, 2017; PTO 1449) teaches that effect by endosomal escape enhancers saponin depends on the cell line, the targeting moiety, target receptor expression, and the structure of the enhancer, see p. 1539, right col. Bhargava teaches EGF conjugated to dianthin (toxin) alone or in combination with saponin SO1861 isolated from Saponaria officinalis L for enhance the effect of EGF conjugated to dianthin in various cell lines. Bhargava does not teach the saponin SO1861 is conjugated to any antibody that binds to EGFR.
There are insufficient in vivo working examples. It is unpredictable with combination of conjugates set forth in claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 is effective as a “pharmaceutical” combination for treating disease such as cancer given the lack of guidance as to the binding specificity of the first and second binding molecules to deliver the effector and saponin in the same cell in vivo.
Therefore, in view of the state of the art, the quantity of experimentation necessary, the breadth of the claims, lack of guidance in the specification, and the absence of in vivo working examples, it would require undue experimentation for one skilled in the art to practice the invention as claimed.
In response to the argument that Bhargava reports substantial enhancement of intracellular activity by SO1861 across multiple targeting molecules, demonstrating that the claimed approach is broadly applicable and predictable, Bhargava et al (of record, Molecular Oncology 11: 1527-1543, 2017; PTO 1449) teaches EGF conjugated to dianthin (toxin) alone or in combination with saponin SO1861 isolated from Saponaria officinalis L for enhance the effect of EGF conjugated to dianthin in various cell lines.
Bhargava does not teach the saponin SO1861 is conjugated to any second binding molecule, much less antibody that binds to EGFR.
For these reasons, the rejection is maintained.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived by the manner in which the invention was made.
The factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1, 2, 3, 5, 6, 8-9, 11-14, 17, 25, 26 and 54 are rejected under 35 U.S.C. 103 as being unpatentable over Gilabert-Oriol et al (Biochemical Pharmacology 97: 247-255, 2015; PTO 1449) as evidenced by Voigt (Neoplasia 14(11): 1023-1031, 2012; PTO 892) in view of Gao et al (WO2015157595 publication, published Oct 15, 2015; PTO 1449).
Claim 1 is drawn to a therapeutic combination comprising:
(a) a first pharmaceutical composition comprising a conjugate comprising a first binding molecule comprising a first binding region for binding to a first binding site (epitope) of a cell-surface molecule and the conjugate comprising at least one of any saponin covalently bound to said first binding molecule, wherein the saponin is a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside; and
(b) a second pharmaceutical composition comprising any conjugate comprising a second binding molecule different from the first binding molecule, the second binding molecule comprising a second binding region different from the first binding region, the second binding region for binding to a second binding site of said cell-surface molecule different from the first binding site of said cell-surface molecule, and the conjugate comprising an effector molecule covalently bound to said second binding molecule, wherein the first pharmaceutical composition and the second pharmaceutical composition optionally further comprises a pharmaceutically acceptable excipient and optionally further comprising a pharmaceutically acceptable diluent.
Regarding claims 1(b), 2, 5, 6, 9, 11, 17, 25, 26, Gilabert-Oriol teaches a pharmaceutical composition comprising a combination of immunotoxin conjugate, e.g., dianthin-cetuximab or dianthin-panitumumab or dianthin-trastuzumab (anti-HER2) conjugate and a bidesmosidic triterpene saponin, e.g., SO1861 isolated from Saponaria officinalis, see entire document, abstract, p. 248, right col., p. 249, right col. SO1861 is a plant saponin with a triterpenoidal skeleton of oleanane type and two sugar side chains (bidesmosidic) attached to it at positions C-3 and C-28, see p. 248, left col. in particular.
Evidentiary reference Voigt teaches that chimeric cetuximab (anti-EGFR monoclonal antibody) and panitumumab (a fully human monoclonal antibody specific for human EGFR) that bind to different conformational epitope in domain III on the same human EGFR (aka cell surface molecule) wherein the binding region (aka CDRs of cetuximab and panitumumab) are different from each other, see p. 1024, left col, p. 1025, in particular. Gilabert-Oriol teaches each anti-EGFR antibody is covalently linked (conjugated) to an effector molecule, e.g., dianthin (see p. 248, right col) via a cleavable linker SPDP to the cysteine amino acid on the antibody (see p. 248, right col). The dianthin-cetuximab or dianthin-panitumumab or dianthin-trastuzumab is in combination with a second composition comprising a bidesmosidic triterpene saponin, e.g., SO1861 isolated from Saponaria officinalis, see entire document, abstract, p. 248, right col., p. 249, right col. Gilabert-Oriol teaches that saponin-mediated the endo/lysosomal escape of targeted dianthin toxin (see p. 251) without disrupting the plasma membrane, enhance the efficacy of the therapeutic antibodies, see abstract, in particular.
Regarding claim 3, the reference chimeric cetuximab (anti-EGFR monoclonal antibody) and panitumumab (a fully human monoclonal antibody specific for human EGFR) are proteinaceous ligands.
Regarding claims 8, 9, Gilabert-Oriol teaches that the first or second binding molecule is a monoclonal antibody, e.g., cetuximab (anti-EGFR) or panitumumab (anti-EGFR), or trastuzumab (anti-HER2), see p. 248, right col., Cross-linking and purification of conjugates, in particular.
Claims 5 and 12 are included because it is within the purview of one of ordinary skill in the art to combine different antibodies, e.g., cetuximab conjugated to dianthin and panitumumab conjugated at least one saponins SO1816 would bind to the same cell-surface receptor, e.g., EGFR without competing for binding as the two antibodies bind to different epitopes on the same EGFR as taught by Voigt.
Regarding claim 13, Gilabert-Oriol teaches that the SO1861 is a plant saponin with a triterpenoidal skeleton of oleanane type and two sugar side chains (bidesmosidic) attached to it at positions C-3 and C-28, see p. 248, left col, in particular.
Regarding claim 14, Gilabert-Oriol teaches that the SO1861 saponin is from a Saponaria species, see p. 254, left col.
Gilabert-Oriol does not teach the saponin is covalently linked to an antibody as per claims 1, and 2, wherein the first antibody that binds to the first epitope without competing for the binding of the second antibody (second binding region) that binds to the second epitope (aka second binding site) of the same cell-surface receptor as per claim 12 and wherein the first binding region and the second binding region are different and that the first binding site and the second binding site are different as per claim 26.
However, Gao teaches a conjugate comprising a first binding molecule, e.g., a cysteine engineered antibody that binds to a cell-surface receptor, e.g., EGFR wherein the antibody is covalently linked to at least one heterologous moieties, e.g., saponin, see para. [0130], [0140]. Examples of heterologous moieties (aka effector) attached to a thiol group of an engineered cysteine antibody include toxin, drug, cytokine, enzyme, oligonucleotide, DNA, peptide, lipid, carbohydrate, scaffolding molecule, see page 39, para. [0131] in particular. Examples of cysteine-engineered antibody for binding to EGFR (ErbB1), HER2 (ErbB0 or p185neu, see para. [0170]) include cetuximab as per claims 13 and 26 (which is an anti-EGFR antibody, also known as ERBITUX®, p. 57, line 5), panitumumab (also known as ABX-EGF, see para. [0171]), trastuzumab (also known as HERCEPTIN® for binding to HER2, see para. [0171], [0188]). The cetuximab and panitumumab are monoclonal antibodies (mAbs, para. [0002],[0037], [0039], [0040]) that bind to a cell-surface molecule, e.g., human EGFR. Since the two antibodies bind to the same EGFR but on different epitope, the two antibodies intrinsically comprise different six CDRs and the cetuximab antibody does not competing for the binding to the EGFR with the second antibody panitumumab or vice versa.
Regarding claim 54, Gao et al teaches a kit comprising the claimed conjugate compound and all the components necessary for use, including directions, see p. 76, para. [0231].
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of Gilabert-Oriol in view of Gao by covalently linked Gilabert-Oriol’s bidesmosidic triterpenoid saponin SO1861 to any one of Gao’s antibody cetuximab or panitumumab that binds to different epitope on the same human cell surface EGFR with a reasonable expectation of success, e.g., targeting saponin to the same cell surface EGFR for saponin-mediated the endo/lysosomal escape of targeted dianthin toxin without competing for binding to EGFR (see p. 251) and enhance the efficacy of the therapeutics, see abstract, in particular.
One of ordinary skill in the art would have had an expectation of success at the time the invention was made to conjugate saponins SO1816 to any one of the antibody that binds to a different epitope on the same human EGFR or different receptor, e.g., HER2 of Gilabert-Oriol in order to avoid competition for binding of the conjugated antibodies to the same EGFR, and the conjugated saponin is expected to enhance the endo/lysosomal escape of targeted dianthin (see p. 251) without disrupting the plasma membrane, see abstract, in particular.
In addition, the claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Applicant's arguments filed July 23, 2026 have been fully considered but they are not persuasive.
Applicant’s position is that independent claim 1 recites a therapeutic combination comprising (a) a first conjugate in which "at least one saponin" that is "a monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside" is "covalently bound to said first binding molecule," the first binding molecule comprising a first binding region for binding to a first binding site of a cell-surface molecule, together with (b) a second conjugate in which "an effector molecule" is "covalently bound to said second binding molecule," the second binding molecule being different from the first binding molecule and comprising a second binding region for binding to a second, different binding site of the same cell-surface molecule. Claim 2 recites the same two-conjugate architecture as a pharmaceutical composition. The claims thus require two distinct conjugates, a saponin- conjugate and an effector-conjugate, each targeting a different, non-competing binding site on the same cell-surface molecule.
The Examiner has alleged that it would have been obvious to covalently link Gilabert- Oriol's bidesmosidic triterpenoid saponin S01861 to one of Gao's cysteine-engineered antibodies (e.g. cetuximab or panitumumab) that binds a different epitope on EGFR, in combination with a second antibody-toxin conjugate, with a reasonable expectation of success. Applicant respectfully submits that the cited references, taken individually or in combination, fail to teach or suggest the claimed two-conjugate architecture, and that there would have been no motivation to make, and no reasonable expectation of success in making, the combination recited by claim 1.
Applicant respectfully submits that the role of the saponin in the claimed combination is to provide an endosomal escape enhancing effect, that is, to facilitate the release of the co-delivered effector molecule from the endosome into the cytosol of the target cell. Gilabert-Oriol itself confirms this function, teaching that "S01861 specifically mediated the endo/lysosomal escape of dianthin without disrupting the plasma membrane." See Gilabert-Oriol, p. 247. Gilabert-Oriol further proposes "the use of dianthin and SO1861 as a new platform technology to enhance the efficacy of therapeutic antibodies." See Gilabert-Oriol, p. 247. Critically, however, in Gilabert- Oriol the saponin SO1861 is administered as a free, unconjugated molecule alongside a single immunotoxin conjugate (dianthin-cetuximab, dianthin-panitumumab, or dianthin-trastuzumab). Gilabert-Oriol does not teach or suggest covalently conjugating the saponin to a binding molecule, let alone combining such a saponin-conjugate with a separate effector-conjugate that binds a different, non-competing binding site on the same cell-surface molecule. The Examiner concedes that Gilabert-Oriol does not teach the saponin covalently linked to an antibody.
Claim 1 requires that the endosomal-escape-enhancing saponin be covalently bound to the first binding molecule (i.e., a saponin-conjugate) and that this saponin-conjugate be combined with a separate second conjugate bearing an effector molecule and targeting a different, non-competing binding site on the same cell-surface molecule. This two-conjugate architecture is premised on the recognition that saponins preserve their endosomal escape enhancing activity even when covalently conjugated to a binding molecule, and that non-competing co-delivery of the saponin- conjugate and the effector-conjugate to the same target cell avoids receptor competition and thereby enables effective intracellular delivery of the effector molecule. None of the cited references teaches, suggests, or provides any reason to expect that a saponin would retain its endosomal escape enhancing activity after covalent conjugation to a targeting molecule. Nor do the references teach or suggest co-administration of a saponin-conjugate and a separate effector- conjugate directed to different, non-competing binding sites on the same cell-surface molecule.
The Examiner has relied on Gao to cure the deficiency of Gilabert-Oriol. However, Gao merely lists "saponin" as one item in a long enumeration of toxins that may be conjugated to an engineered-cysteine antibody, alongside abrin, brucine, cicutoxin, diphtheria toxin, botulinum toxin, shiga toxin, ricin, strychnine, tetrodotoxin, and numerous others. See Gao, paragraph [0140]. Gao thus treats saponin as a cytotoxic payload, not as an endosomal escape enhancer. Gao does not teach or suggest that a saponin conjugated to an antibody would retain endosomal escape enhancing activity, nor does Gao teach or suggest combining a saponin-conjugate with a separate effector-conjugate binding a different, non-competing epitope of the same cell-surface molecule. Accordingly, even if one were to conjugate a saponin to an antibody as generically suggested by Gao, there would have been no motivation to combine such a saponin-conjugate with a separate effector-conjugate targeting a different binding site on the same receptor. Neither Gilabert-Oriol nor Gao recognizes that a conjugated saponin retains its endosomal escape enhancing function, let alone provides any reasonable expectation that such a construct would successfully promote intracellular delivery of a separately targeted effector molecule.
In response to applicant's argument that the references fail to show certain features of applicant’s invention, it is noted that the features upon which applicant relies (i.e., endosomal escape enhancing effect) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
In response to the argument that Gilabert- Oriol the saponin SO1861 is administered as a free, unconjugated molecule alongside a single immunotoxin conjugate (dianthin-cetuximab, dianthin-panitumumab, or dianthin-trastuzumab), it is noted that Gao teaches antibody conjugated to saponin, see para. [0140]. Gao further teaches that the cysteine-engineered antibody binds to two different epitopes (aka non-competing binding sites) on the same molecule, see para. [0164]. Examples of antibody that bind to EGFR include panitumumab (also known as ABX-EGF, VECTIBIX®), cetuximab (also known as ERBITUX®), matuzumab (also known as EMD72000), see para. [0171]. Examples of antibody that bind to HER2 include trastuzumab (also known as HERCEPTIN®), and pertuzumab (also known as 2C4, OMNITARG®) see para. [0171]. Gao further teaches that pharmaceutical composition can comprise a combination of conjugate compounds that bind to different epitopes, or that have complementary activities (see para. [0204]) would have led one of ordinary skill in the art to combine such a saponin-conjugate with a separate effector-conjugate targeting a different epitope on the same EGFR or HER2 receptor in order to target the saponin SO1861 to the same target cells for saponin-mediated the endo/lysosomal escape of targeted dianthin toxin (see p. 251) without disrupting the plasma membrane, and enhance the efficacy of the therapeutic antibodies as taught by Gilabert-Oriol, see abstract, in particular.
For these reasons, the rejection is maintained.
Claims 4, 13, 21 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Gilabert-Oriol et al (Biochemical Pharmacology 97: 247-255, 2015; PTO 892) as evidenced by Voigt (Neoplasia 14(11): 1023-1031, 2012; PTO 892) in view of Gao et al (WO2015157595 publication, published Oct 15, 2015; PTO 1449) as applied to claims 1, 2, 3, 5, 6, 8-9, 11, 13-14, 17, 25, 26 mentioned above and further in view of Fuchs et al (US20080064762, published March 13, 2008; PTO 892) and Doronina et al (US20040157782, published August 12, 2004; PTO 892).
The combine teachings of Gilabert-Oriol, Voigt and Gao have been discussed supra.
The references do not teach the at least one saponin is a bidesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 wherein the saponin is covalently bound to the first antibody via an aldehyde function in the saponin as per claim 21, wherein the at least one saponin is covalently bound to a linker EMCH, which linker is covalently bound via a thioether bond to a sulfhydryl group in the first antibody as per claim 22, and wherein the saponin comprising a glucuronic acid unit in a first saccharide chain at the C3beta-OH group of the saponin, wherein the saponin is covalently bound to an amino acid of the first antibody as per claim 23.
However, Fuchs teaches a composition comprising at least one pharmacologically active agent, e.g., toxin (see para. [0045]) coupled to a target cell specific component, e.g., antibody or antigen binding fragment or natural EGF ligand (elected species) that binds to a cell surface receptor, e.g., EGFR or HER2 (see para. [0002], [0010], [0014]), and saponins, see entire document, reference claims 1 and 11. Typical target antigens are the epidermal growth factor (EGF) receptor, the proto-oncogene receptor ErbB2 (also known as HER-2 in humans), the interleukin-2 receptor or cancer-associated carbohydrates, see paragraph[0002], in particular. Examples of conjugate include EGF ligand directly coupled to Sap-3 (SE), see para. [0077]. Examples of saponins are triterpenoic saponins, preferably having an aldehyde function at the aglycone and two sugar residues glycosidically bound to the aglycone. The triterpenoic saponins with basic structures belonging to the type of 12,13-dehydrooleanane with an aldehyde function in position 23 and a glucuronic acid unit at the C3beta-OH group of the aglycone core structure, see reference claim 11; the saponins is isolated from Saponinum album of Gypsophila paniculata, see para. [0020]. The triterpenoic saponins with basic aglycone structure
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belonging to the type of 12,13-dehydrooleanane with an aldehyde function in position 23 of the aglycone core, and a glucuronic acid unit at the C3beta-OH group of the aglycone core structure, see reference claim 11.
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Fuchs further teaches by combining a pharmacologically active agent with a saponin, it is possible to enhance the function of the pharmacological active agent, e.g., cytotoxicity of immunotoxins, e.g., Sap-3 (saporin-3 ribosome inactivating protein), see para. [0051].
Gilabert-Oriol, Voigt and Gao and Fuchs I do not teach the saponins are covalently bound to an amino acid residue of the antibody via a linker as per claim 4, wherein the linker is EMCH, which covalently bound via a thio-ether bond to a sulfhydryl group in the antibody as per claim 22.
However, Doronina teaches heterobifunctional linker includes, on one end, a thiol acceptor i.e., a chemical moiety that will react with a thiol group so that the sulfur atom of the thiol group becomes covalently bonded to the thiol acceptor. Thiol acceptors are well known in the art, where exemplary thio acceptor group are maleimide and haloacetamide groups. The thiol acceptor group provides a convenient site for ligand attachment. At the other end, in a preferred aspect the heterobifunctional linker has a hydrazide group. Hydrazide groups are useful end groups for a heterobifunctional linker because they readily react with carbonyl groups, i.e., aldehyde and ketone groups, where carbonyl groups can be readily incorporated into a pentapeptide compound as disclosed herein, to provide a hydrazone linkage. Incorporation of a hydrazone into the conjugate is particularly preferred in order to impart desired pH-sensitivity to the conjugate. Thus, under low pH conditions, as in acidic intracellular compartments (e.g., lysosomes), a hydrazone group will cleave and release the drug free from the ligand, see para. [0274]. Commercially available various linkers include maleimido hydrazides (e.g., .beta.-maleimido propionic acid hydrazide, Epsilon-maleimidocaproic acid hydrazide (aka EMCH), and SMCC hydrazide, available from Molecular Biosciences, Inc. Boulder Colo.), see para. [0298], thio-ether modified linker for linking any agent of interest on the mAb via the sulfhydryl group of cysteines, see Examples 22-23, in particular. Doronina teaches targeting ligand is an antibody, e.g., chimeric or humanized or a fragment thereof, see para. [0327]. Examples of antibody for treatment of cancer include, but not limited to, Herceptin™ (trastuzumab), see para. [0343], anti-transferrin receptor (also known as CD71 as per claims 11) for carcinomas, see para. [0344], in particular). Doronina teaches that the mAb-pentapeptide conjugates can be used to deliver the cytotoxic drug to tumor cells. Once the antibody has bound to tumor associated antigens, e.g., EGFR or CD71 (transferrin receptor), it is taken up inside cells through receptor-mediated endocytosis into endosomes and lysosomes. These intracellular vesicles are acidic and can induce the hydrolysis of an acid-sensitive hydrazone bond, between the drugs and the mAbs. In addition, ester bonds can be cleaved by proteases and esterase, which are in abundance within lysosomes wherein the pH is < 6.5. The released drug is then free to migrate in the cytosol and induce cytotoxic activities.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of Gilabert-Oriol, Voigt and Gao in view of Fuchs I and Doronina by linking any one of Gilabert-Oriol or Fuchs’ saponins to the cetuximab or panitumumab anti-EGFR antibody using any commercially available heterobifunctional linker such as Epsilon maleimidocaproic acid hydrazide (EMCH) as taught by Doronina for covalently linking sulfhydryl (cysteines) on the antibody to the aldehyde group on the saponins to arrive at the claimed invention with a reasonable expectation of success, e.g., targeting saponins and effector toxins to the same cell surface EGF receptor.
One of ordinary skill in the art would have been motivated to do so because Fuchs teaches that by combining a pharmacologically active agent with a saponin, it is possible to enhance the function of the pharmacological active agent, e.g., cytotoxicity of immunotoxins, para. [0054].
One of ordinary skill in the art would have been motivated to do so because Doronina teaches that the linker epsilon-maleimidocaproic acid hydrazide is commercial available and the acid-sensitive hydrazone bond forms between the saponin or effector and the mAbs can be hydrolyzed by the acidic environment in endosomes and lysosomes to release the saponin.
In addition, the claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Applicants’ arguments filed July 23, 2026 have been fully considered but are not found persuasive.
Applicants’ position is that the secondary references relied upon for the dependent claims do not remedy these deficiencies. Fuchs and Doronina (relied upon for claims 4, 13, 21 and 22) are cited only for linker and conjugation chemistry, specifically, the EMCH linker and thio-ether bond to a sulfhydryl group, and do not teach or suggest the claimed two-conjugate combination. Likewise, Gin and Aaronson (relied upon for claims 23-24) are cited only for the HATU linker and amide conjugation chemistry and similarly do not teach or suggest the claimed combination. Because the base Gilabert-Oriol/Voigt/Gao combination fails to render obvious the two-conjugate architecture of independent claim 1, the dependent claims are allowable at least by virtue of their dependency on claim 1.
In response, the arguments with respect to base Gilabert-Oriol/Voigt/Gao combination have been discussed supra and incorporated here by references. Given the lack of any additional arguments with respect to the secondary references, the rejection is maintained for reasons of record.
Claims 23-24 are rejected under 35 U.S.C. 103 as being unpatentable over Gilabert-Oriol et al (Biochemical Pharmacology 97: 247-255, 2015; PTO 892) as evidenced by Voigt (Neoplasia 14(11): 1023-1031, 2012; PTO 892) in view of Gao et al (WO2015157595 publication, published Oct 15, 2015; PTO 1449) as applied to claims 1, 2, 3, 5, 6, 8-9, 11, 13-14, 17, 25, 26 mentioned above and further in view of Fuchs et al (US20080064762, published March 13, 2008; PTO 892), Gin et al (US20110300177, published December 8, 2011, PTO 892) and/or Aaronson et al (Bioconjugate Chemistry 22: 1723-1728, 2011; PTO 892).
The combine teachings of Gilabert-Oriol, Voigt and Gao have been discussed supra.
The references do not teach the combination wherein the at least one saponin is a bidesmosidic triterpene glycoside belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C23 of the aglycone core structure of the saponin and comprising a glucuronic acid unit in a first saccharide chain at the C3beta-OH group of the aglycone core structure of the saponin, wherein the saponin is covalently bound to an amino-acid residue of the first binding molecule via the carboxyl group of the glucuronic acid unit in the first saccharide chain as per claim 23 and wherein the glucuronic acid unit is covalently bound to linker 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), wherein the linker is covalently bound via an amide bond to an amine group in the first binding molecule, e.g., antibody as per claim 24.
However, Fuchs teaches a composition comprising at least one pharmacologically active agent, e.g., toxin (see para. [0045]) coupled to a target cell specific component, e.g., antibody or antigen binding fragment or natural EGF ligand (elected species) that binds to a cell surface receptor, e.g., EGFR or HER2 (see para. [0002], [0010], [0014]), and saponins, see entire document, reference claims 1 and 11. Typical target antigens are the epidermal growth factor (EGF) receptor, the proto-oncogene receptor ErbB2 (also known as HER-2 in humans), the interleukin-2 receptor or cancer-associated carbohydrates, see paragraph[0002], in particular. Examples of conjugate include EGF ligand directly coupled to Sap-3 (SE), see para. [0077]. Examples of saponins are triterpenoic saponins, preferably having an aldehyde function at the aglycone and two sugar residues glycosidically bound to the aglycone. The triterpenoic saponins with basic structures belonging to the type of 12,13-dehydrooleanane with an aldehyde function in position 23 and a glucuronic acid unit at the C3beta-OH group of the aglycone core structure, see reference claim 11; the saponins is isolated from Saponinum album of Gypsophila paniculata, see para. [0020]. The triterpenoic saponins with basic aglycone structure
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belonging to the type of 12,13-dehydrooleanane with an aldehyde function in position 23 of the aglycone core, and a glucuronic acid unit at the C3beta-OH group of the aglycone core structure, see reference claim 11.
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Fuchs further teaches by combining a pharmacologically active agent with a saponin, it is possible to enhance the function of the pharmacological active agent, e.g., cytotoxicity of immunotoxins, e.g., Sap-3 (saporin-3 ribosome inactivating protein), see para. [0051].
Gilabert-Oriol, Voigt, Gao and Fuchs do not teach the glucuronic acid unit is covalently bound to linker 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), wherein the linker is covalently bound via an amide bond to an amine group in the first binding molecule, e.g., antibody as per claim 24.
However, Gin teaches various triterpene saponins such as QS-21 (Figure 6), QS-7 Api (Figure 7) or QS-7 (Figure 8) from Quillaia bark saponins as per claim 17, see para. [0004]) wherein the C-3OH comprises a glucuronic acid (see para. [0194]) coupled to any suitable peptide using linker such as 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU), see para. [0353]. The major components of Quil-A showed that QS-21 had low toxicity and QS-7 showed no lethality at the doses tested in CD-1 mice intradermally. QS-7 showed no hemolytic activity at levels up to 200 .mu.g/ml of saponin, see para. [0358]. Gin further teaches Saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in cell culture, even at nonpermeabilizing concentrations, see para. [0361]. Gin further teaches that the reference compounds may be used to enhance the uptake of other cytotoxic agents, see para. [0362].
Likewise, Aaronson teaches that the amidation strategy was selected primarily because of the commercial and synthetic availability of carboxylated substrates, allowing for the preparation of a wide variety of desirable conjugates. Aaronson teaches oligonucleotide (non-proteinaceous ligand as per claim 3) conjugation using linker 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU). Activation of the carboxylic acid substrate with HATU was performed in a separate step using anhydrous DMSO as a solvent to further minimize contract of the coupling reagent with water. One-pot coupling in which HATU, carboxylic acid, and siRNA are introduced into the same reaction vessel without pre-activation, see p. 1725, in particular. The successful conjugation of various molecule, e.g., lipophilic moieties (4a h), an affinity ligand (4i), a fluorescent label (4j), a linear PEG (4k), and a protected amino acid (4l) illustrates the versatility and utility of this method, see p. 1725, Table 1, in particular. Aaronson concludes that the principal advantage of the protocol described in this technical note is the ability to rapidly access diverse conjugates without isolating a preactivated ester. This is particularly useful when carboxylic acid partners are synthetically precious or their activated ester is not commercially available. The relatively small excess of carboxylic acid required reduces waste, conserving expensive or difficult to synthesize coupling partners. In addition to eliminating the need to isolate a preactivated ester, this protocol is rapid, with a total reaction time of 30 min. Finally, these conditions are generally applicable to a diverse set of conjugates and do not require significant alterations to solvent composition or RNA counterions for different classes of substrates, see p. 1727.
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filling date of the claimed invention to combine the teachings of Gilabert-Oriol, Voigt and Gao in view of Fuchs, Gin and Aaronson by linking any one of bidesmosidic saponins belonging to the type of 12,13-dehydrooleanane having an aldehyde function in position 23 and a glucuronic acid unit at the C3beta-OH group of the aglycone core structure isolated from Saponinum album of Fuchs or any monodesmoside Quillaja saponins Q-20 or QS-7 or QS-7api of Gin using the linker HATU as taught by Gin and/or Aaronson in a one pot-reaction for covalently linking carboxyl group of the glucuronic acid at the C3beta-OH of saponins via an amide bond to the lysine residue on the first antibody in order to target the saponins to the same EGFR expressing cell when combine with a second conjugate comprising anti-EGFR covalently linked to a toxin, to enhance the saponins mediated endosomal escape of the targeted toxin in the same cell.
One of ordinary skill in the art would have had an expectation of success at the time the invention was made to use HATU linker to covalently link any saponins to any first antibody or EGF ligand because Gin teaches that coupling conditions of HATU are well known in the art, see para. [0353].
One of ordinary skill in the art would have been motivated to use HATU linker because Aaronson teaches that the one-pot coupling without pre-activation eliminating the need to isolate a preactivated ester; this protocol is rapid, with a total reaction time of 30 min. Finally, these conditions are generally applicable to a diverse set of conjugates and do not require significant alterations to solvent composition for different classes of substrates, see p. 1727.
One of ordinary skill in the art would have been motivated to do so because Fuchs teaches saponin enhance the specific cytotoxicity of chimeric toxin or toxin conjugate or immunotoxins and the combined application of a saponin and a pharmacologically active agent coupled to a target cell specific component is a promising tool for enhancing the function of the pharmacologically active agent, as exemplified by the improved specific cytotoxicity of immunotoxins (ITs) on target cells and reduced side-effects during tumor therapy, see para. [0080].
One of ordinary skill in the art would have had an expectation of success at the time the invention was made to combine the conjugate dianthin-cetuximab or dianthin-panitumumab of Gilabert-Oriol, Voigt and Gao with the conjugate comprising bidesmosidic triterpene saponin belonging to the type of 12,13-dehydrooleanane with a glucuronic acid function in position C-3OH of any saponins to the panitumumab or EGF ligand using HATU linker of Gin or Aaronson in order to targeted toxin and saporin to same EGFR expressing cancer cells and thereby avoid off-targeted effects.
In this case, “The simple substitution of a known element, e.g., linker for another, e.g., HATU is likely to be obvious when it does no more than yield predictable results.”
In addition, the claims would have been obvious because "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense". See KSR International Co. v. Teleflex Inc., 82 USPQ2d 1385 (U.S. 2007).
“The test of obviousness is not express suggestion of the cl aimed invention in any or all of the references but rather what the references taken collectively would suggest to those of ordinary skill in the art presumed to be familiar with them.” See In re Rosselet 146 USPQ 183, 186 (CCPA 1965).
“There is no requirement (under 35 USC 103(a)) that the prior art contain an express suggestion to combine known elements to achieve the claimed invention. Rather, the suggestion to combine may come from the prior art, as filtered through the knowledge of one skilled in the art.,” Motorola, Inc, v. Interdigital Tech. Corn., 43 USPQ2d 1481, 1489 (Fed. Cir. 1997).
Accordingly, the claimed invention as a whole was prima facie obvious to one of ordinary skill in the art before the effective filling date of the claimed invention especially in the absence of evidence to the contrary.
Applicants’ arguments filed July 23, 2026 have been fully considered but are not found persuasive.
Applicants’ position is that the secondary references relied upon for the dependent claims do not remedy these deficiencies. Fuchs and Doronina (relied upon for claims 4, 13, 21 and 22) are cited only for linker and conjugation chemistry, specifically, the EMCH linker and thio-ether bond to a sulfhydryl group, and do not teach or suggest the claimed two-conjugate combination. Likewise, Gin and Aaronson (relied upon for claims 23-24) are cited only for the HATU linker and amide conjugation chemistry and similarly do not teach or suggest the claimed combination. Because the base Gilabert-Oriol/Voigt/Gao combination fails to render obvious the two-conjugate architecture of independent claim 1, the dependent claims are allowable at least by virtue of their dependency on claim 1.
In response, the arguments with respect to base Gilabert-Oriol/Voigt/Gao combination have been discussed supra and incorporated here by references. Given the lack of any additional arguments with respect to the secondary references, the rejection is maintained for reasons of record.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the claims at issue are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); and In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on a nonstatutory double patenting ground provided the reference application or patent either is shown to be commonly owned with this application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The USPTO internet Web site contains terminal disclaimer forms which may be used. Please visit http://www.uspto.gov/forms/. The filing date of the application will determine what form should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to http://www.uspto.gov/patents/process/file/efs/guidance/eTD-info-I.jsp.
Claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 are provisionally rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 14, 16, 19, 27, 29-30 and 50 of copending Application No. 17/312,193 (reference). Although the conflicting claims are not identical, they are not patentably distinct from each other because the reference claims recite:
Claim 14 recites a therapeutic combination, wherein the therapeutic combination comprises:(a) a first pharmaceutical composition comprising a first proteinaceous molecule comprising a first binding site for binding to a first epitope of a first target cell-surface molecule on a target cell, the first proteinaceous molecule provided with at least one endosomal escape enhancer saponin covalently bound via at least one linker or via an oligomeric or polymeric scaffold to an amino-acid residue of said first proteinaceous molecule or via at least one linker and an oligomeric or polymeric scaffold to an amino-acid residue of said first proteinaceous molecule, or covalently bound directly to an amino-acid residue of said first proteinaceous molecule, wherein the first binding site comprises an antibody, or at least one antigen-binding domain thereof or at least one antigen-binding fragment thereof, wherein binding of the first proteinaceous molecule to the first epitope on the target cell is followed by endocytosis of the complexes of the first proteinaceous molecule and the first target cell-surface molecule, wherein the at least one saponin is a bisdesmosidic triterpene saponin belonging to the type of a 12,13-dehydrooleanane with an aldehyde function in position C-23 and comprising a glucuronic acid function in a carbohydrate substituent at the C-3beta-OH group of the saponin and optionally a pharmaceutically acceptable excipient; and (b) a second pharmaceutical composition comprising a second proteinaceous molecule different from the first proteinaceous molecule, the second proteinaceous molecule comprising a second binding site for binding to a second epitope of a second cell-surface molecule different from the first cell-surface molecule, and comprising an effector moiety, the second pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, wherein the second epitope is different from the first epitope.
16. (Previously Presented) A therapeutic combination, wherein the therapeutic combination comprises:(a) the first pharmaceutical composition of claim 14 and comprising the first binding site for binding to the first epitope on the first cell-surface molecule, the first pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient; and (b) a third pharmaceutical composition comprising a third proteinaceous molecule, the third proteinaceous molecule comprising the first binding site for binding to the first epitope on the cell-surface molecule of (a) and an effector moiety, the third pharmaceutical composition optionally further comprising a pharmaceutically acceptable excipient, wherein the first binding site of the first proteinaceous molecule and the first binding site of the third proteinaceous molecule are the same, and wherein the first cell-surface molecule and the first epitope on the first cell-surface molecule, to which the first proteinaceous molecule can bind, and the first cell-surface molecule and the first epitope on the first cell-surface molecule, to which the third proteinaceous molecule can bind, are the same.
19. (Currently amended) The therapeutic combination of claim 14, wherein the second binding site of the second proteinaceous molecule and/or the first binding site of a third proteinaceous molecule comprises an immunoglobulin, at least one binding domain of an immunoglobulin and/or at least one binding fragment of an immunoglobulin, or an antibody-.
26. (Currently Amended) The first proteinaceous molecule of claim 1, wherein the first binding site comprises a monoclonal antibody or at least one cell-surface molecule binding fragment and/or domain thereof, and optionally comprise cetuximab.
27. (Currently Amended) The therapeutic combination of claim 16, wherein the first binding site of the first proteinaceous molecule and the third proteinaceous molecule comprise a monoclonal antibody or at least one of a cell-surface molecule binding domain and/or fragment thereof, and optionally comprise cetuximab, with the proviso that the first binding site of the first proteinaceous molecule is the same as the first binding site of the third proteinaceous molecule.
28. (Currently amended) The therapeutic combination of claim 14, wherein the second binding site of the second proteinaceous molecule and/or the first binding site of a third proteinaceous molecule comprises a monoclonal antibody.
29. (Currently amended) The therapeutic combination of claim 14, wherein the effector moiety that is comprised by the second proteinaceous molecule and/or by a third proteinaceous molecule comprises any one or more of an oligonucleotide, a nucleic acid, a xeno nucleic acid.
30. (Currently amended) The therapeutic combination of claim 14, wherein the effector moiety that is comprised by the second proteinaceous molecule and/or by a third proteinaceous molecule comprises at least one proteinaceous molecule.
50. (Currently amended) The therapeutic combination of claim 14, wherein the second binding site of the second proteinaceous molecule and/or the first binding site of a third proteinaceous molecule comprises at least one ligand for binding to a cell-surface molecule or EGF or a cytokine.
Applicants’ arguments filed July 23, 2026 have been fully considered but are not found persuasive.
Applicants’ position is that the reference application contains claim 1 as amended in May 2026, which requires that the at least one saponin further comprises a glucuronic acid moiety in a carbohydrate substituent attached to the C-3β-OH group of the saponin. Additionally, US '193 discloses, among other alternatives, covalent conjugation through an oligomeric or polymeric scaffold, a feature that is not encompassed by the claims presently under examination. Accordingly, the pending claims are patentably distinct from the cited claims, and the provisional ODP rejection should be withdrawn.
In response, the amendment to copending claims is acknowledged.
The copending claims limit the first proteinaceous molecule which requires that the at least one saponin further comprises a glucuronic acid moiety in a carbohydrate substituent attached to the C-3β-OH group of the saponin (species) whereas instant claims are generic with respect to the site of attachment in the saponin (genus). Further, instant specification discloses saponin comprising a glucuronic acid function in a carbohydrate substituent at the C-3beta-OH group, see p. 12, lines 3-4. Thus, the claims are not patently distinct. For these reasons, the rejection is maintained.
Claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 are provisionally rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 57-65, 67-76 of copending Application No. 18/012,741 (reference). Although the conflicting claims are not identical, they are not patentably distinct from each other because the reference claims recite:
57. (Currently Amended) A pharmaceutical combination comprising:- a first conjugate comprising an effector molecule and a single-domain antibody (sdAb) for binding to a first cell-surface molecule, wherein the effector molecule and the sdAb are covalently linked to each other, wherein the effector molecule comprises
57. (Currently Amended) A pharmaceutical combination comprising:- a first conjugate comprising an effector molecule and a single-domain antibody (sdAb) for binding to a first cell-surface molecule, wherein the effector molecule and the sdAb are covalently linked to each other, wherein the effector molecule comprises or consists of an oligonucleotide;- a second conjugate comprising a saponin and a binding molecule for binding to a second cell-surface molecule, wherein o the saponin and the binding molecule are covalently linked to each other,
o the saponin is a monodesmosidic triterpene glycoside or
a bidesmosidic triterpene glycoside comprising
an aglycone core structure which is quillaic acid or gypsogenin and
(ii) an aldehyde at position C23 of the aglycone core structure or an acid-sensitive bond at position C23 of the aglycone core structure,
wherein the acid-sensitive bond breaks under acidic conditions such that it forms the aldehyde at position C23 of the aglycone core structure, and
o the second cell-surface molecule and the first cell-surface molecule are present on the surface of the same target cell; and optionally - a pharmaceutically acceptable excipient and/or pharmaceutically acceptable diluent.
58. (Previously Presented) The pharmaceutical combination of claim 57 comprising two pharmaceutical compositions wherein:- the first pharmaceutical composition comprises the first conjugate; and- the second pharmaceutical composition comprises the second conjugate.
59. (Previously Presented) The pharmaceutical combination of claim 57, wherein the first conjugate and the second conjugate are provided in a single pharmaceutical composition.
60. (Currently Amended) The pharmaceutical combination of claim 57, wherein the sdAb is a VHH domain.
61. (Previously Presented) The pharmaceutical combination of claim 57, wherein the first conjugate comprises at least two sdAbs that are the same sdAbs or are capable of binding to the same binding site on the first cell-surface molecule.
62. (Currently Amended) The pharmaceutical combination of claim 57, wherein the first cell- surface molecule is a tumor-cell surface receptor and/or a tumor-cell specific receptor and/or a receptor selected from: CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin receptor, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146, CD56, CD19, CD27L receptor, integrin-alphaV, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, CD25, ephrinA4, MUC-1, Trop2, CEACAMS, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2,C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA-4, CD52, PDGFRA, VEGFR1, VEGFR2, c-Met (HGFR), EGFR1, RANKL, ADAMTS5,CD16, CXCR7 (ACKR3), and glucocorticoid-induced TNFR-related protein (GITR).
63. (Previously Presented) The pharmaceutical combination of claim 57, wherein the first conjugate comprises an sdAb that can bind to HER2 or CD71.
64. (Previously Presented) The pharmaceutical combination of claim 57, wherein the oligonucleotide is a natural, synthetic, or modified oligonucleotide that is selected from any one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA (siRNA), anti-microRNA (anti-miRNA), DNA aptamer, RNA aptamer, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA), 2'-deoxy-2'-fluoroarabino nucleic acid (FANA), 2'-O-methoxyethyl-RNA (MOE), 3'-fluoro hexitol nucleic acid (FHNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA). Attorney Docket No.: 40306-54337/US 65. (Previously Presented) The pharmaceutical combination of claim 57, wherein the saponin comprises an aglycone core structure selected from: 2alpha-hydroxy oleanolic acid; 16alpha-hydroxy oleanolic acid; hederagenin (23-hydroxy oleanolic acid); 16alpha,23-dihydroxy oleanolic acid; gypsogenin; quillaic acid; protoaescigenin-21(2-methylbut-2-enoate)-22-acetate; 23-oxo-barringtogenol C-21,22-bis(2-methylbut-2-enoate); 23-oxo-barringtogenol C-21(2-methylbut-2-enoate)-16,22-diacetate; 3,16,28-trihydroxy oleanan-12-en; and gypsogenic acid.
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68. (Previously Presented) The pharmaceutical combination of claim 57, wherein the saponin is selected from any one or more of: Quillaja bark saponin, dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate, AS6.2, NP- 005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP-017777, NP-017778, NP-017774, NP- 018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP- 017674, NP-017810, AG1, NP-003881, NP-017676, NP-017677, NP-017706, NP-017705, NP- 017773, NP-017775, SA1657, AG2, S01861, GE1741, S01542, S01584, S01658, S01674, S01832, S01904, S01862, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1, and AS64R.
69. (Previously Presented) The pharmaceutical combination of claim 57, wherein the saponin is selected from any one or more of QS-21, S01861, SA1641, and GE1741.
70. (Previously Presented) The pharmaceutical combination of claim 57, wherein the binding molecule of the second conjugate is selected from any one or more of: a ligand, an immunoglobulin, and a binding fragment or domain of an immunoglobulin.
71. (Previously Presented) The pharmaceutical combination of claim 57, wherein the binding molecule of the second conjugate binds to the second cell-surface molecule, wherein the second cell-surface molecule is selected from a second cell-surface receptor and/or a second tumor-cell specific receptor.
72. (Previously Presented) The pharmaceutical combination of claim 57, wherein the saponin is covalently linked to the binding molecule via a linker.
73. (Previously Presented) The pharmaceutical combination of claim 72, wherein linker is an acid-sensitive linker.
74. (Previously Presented) The pharmaceutical combination of claim 73, wherein the acid- sensitive linker is covalently bound to the saponin at position C23 of the aglycone core structure via an acid-sensitive bond.
75. (Currently Amended) The pharmaceutical combination of claim 72, wherein the saponin comprises a first saccharide chain at the C3beta-OH group position of the aglycone core structure, the first saccharide chain comprising a glucuronic acid unit that is covalently bound to the linker.
76. (Previously Presented) The pharmaceutical combination of claim 57, wherein the second conjugate comprises more than one molecule of the saponin and/or of the saponin derivative, and further comprises an oligomeric molecule or polymeric molecule to which the more than one molecule of the saponin and/or of the saponin derivative is covalently bound.
Applicants’ arguments filed July 23, 2026 have been fully considered but are not found persuasive.
Applicants’ position is that claim 57 of the reference 3 application was amended on 20 January 2026 to recite that "the acid-sensitive bond breaks under acidic conditions such that it forms the aldehyde at position C23 of the aglycone core structure." This limitation is not present in the claims of the instant application and narrows the scope of the reference claims to a specific mechanism and product resulting from cleavage of the acid-sensitive bond.
In response, the amendment to copending 57 is acknowledged. It should be noted that instant specification discloses quillaic acid is the aglycone core structure for SO1861, QS-7, QS21, see para. [0024]. The instant specification also discloses quillaic acid or gypsogenin, having an aldehyde group at position C.sub.23 of the aglycone, see para. [0057]. Thus, the claims are not patently distinct. For these reasons, the rejection is maintained.
New Ground of objection and rejection necessitated by claim amendment filed July 23, 2026
Claim objection
Claim 55 is objected to because of the following informalities:
“AG2,SO1861” should have been “AG2, SO1861”.
the claim uses the abbreviations AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, and AS64R without first defining it. To clarify the claim, applicant should first spell out the full term before using an abbreviation. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5, 8, 26, 55 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which applicant regards as the invention.
Regarding claims 5 and 8, the phrase “such as” renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 55 is rejected on the judicially-created basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). The improper Markush grouping includes species of the claimed invention that do not share both a substantial structural feature and a common use that flows from the substantial structural feature. The members of the improper Markush grouping do not share a substantial feature and/or a common use that flows from the substantial structural feature for the following reasons: AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R do not share the same structure obtained from the same family and a common use, e.g., enhance endosome escape that flows from the substantial structural feature. In response to this rejection, Applicant should either amend the claim(s) to recite only individual species or grouping of species that share a substantial structural feature as well as a common use that flows from the substantial structural feature, or present a sufficient showing that the species recited in the alternative of the claims(s) in fact share a substantial structural feature as well as a common use that flows from the substantial structural feature. This is a rejection on the merits and may be appealed to the Board of Patent Appeals and Interferences in accordance with 35 U.S.C. §134 and 37 CFR 41.31(a)(1) (emphasis provided).
Claims 8, 13, 26 and 55 recite a broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c).
In this case, claim 8 recites the broad limitation “comprises” and the claim also recites “consists of”.
Claim 13 recites the broad limitation “comprising” and the claim also recites “consisting of”.
Claim 26 recites the broad limitation “comprise” and the claim also recites “consists of”.
Claim 55 recites the broad limitation “dipsacoside B, saikosaponin A, saikosaponin D, macranthoidin A, esculentoside A, phytolaccagenin, aescinate” and the claim also recites AS6.2, NP-005236, AMA-1, AMR, alpha-Hederin, NP-012672, NP- 017777, NP-017778, NP-017774, NP-018110, NP-017772, NP-018109, NP-017888, NP-017889, NP-018108, SA1641, AE X55, NP-017674, NP-017810, AG1, NP-003881, NP-017676, NP- 017677, NP-017706, NP-017705, NP-017773, NP-017775, SA1657, AG2,S01861, GE1741, SO1542, S01584, S01658, 501674, S01832, 501862, S01904, QS-7, QS1861, QS-7 api, QS1862, QS-17, QS-18, QS-21 A-apio, QS-21 A-xylo, QS-21 B-apio, QS-21 B-xylo, beta-Aescin, Aescin Ia, Teaseed saponin I, Teaseedsaponin J, Assamsaponin F, Digitonin, Primula acid 1 and AS64R, which are narrow statement of the range/limitation.
Claims 1-6, 8, 9, 11-14, 17, 21-26, 54 and 55 are provisionally rejected on the ground of nonstatutory obviousness-type double patenting as being unpatentable over claims 57-59, 61-65, 67-68-74 of copending Application No. 18/012,754 (reference). Although the conflicting claims are not identical, they are not patentably distinct from each other because copending claims 57 limit one of the two conjugates to single-domain antibody (sdAb) wherein the single-domain antibody is VHH that bind to CD71 wherein the VHH is conjugated to effector, e.g., nucleic acid or a xeno nucleic acid and a second conjugate which comprises a binding molecule for binding to a second cell-surface molecule and the modified saponin, which binding molecule and the modified saponin are covalently linked to each other, either directly or via a linker, and wherein the modified saponin is covalently bound to the binding molecule via the hydrazone bond at C-23 of said quillaic acid or of said gypsogenin aglycone core structure (species) whereas in instant claims are generic with respect to the first binding molecule covalently coupled to any monodesmosidic triterpene glycoside or a bidesmosidic triterpene glycoside and the second binding molecule conjugated to any receptor.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to PHUONG HUYNH whose telephone number is (571)272-0846. The examiner can normally be reached on 9:00 a.m. to 6:30 p.m. The examiner can also be reached on alternate alternative Friday from 9:00 a.m. to 5:30 p.m.
If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Misook Yu, can be reached at 571-272-0839. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/PHUONG HUYNH/ Primary Examiner, Art Unit 1641
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