Novel antibody oligonucleotide drug conjugates containing gemcitibine for pharmaceutical compositions and related methods of use and treatment

Abstract

Antibody-oligonucleotide drug conjugates (AODC) targeting CHK1 and / or WEE1 are provided for use in pharmaceutical compositions and methods of treating various cancers. AODCs comprise an antibody, a peptide linker and an oligonucleotide containing GEM moieties. In some embodiments, the oligo comprises a double stranded RNA molecule, which comprises one or more gemcitabine (GEM) moieties replacing certain cytidine nucleotides or other nucleotides. When the sense strand is separated from the antisense strand, one or both of which contain at least one GEM moiety, the sense strand releases its GEM moieties and together with the CHK1-siRNA (containing GEM), exerts a synergistic effect that exceeds the effects of either the released GEM moieties or the CHK1-siRNA-GEM construct alone to silence the CHK1 gene. In some embodiments the RNA molecule of the AODC is an mxRNA containing GEM moieties or an muRNA construct with GEM moieties in one or both antisense strands, targeting both CHK1 and WEE1.

Description

NOVEL ANTIBODY OLIGONUCLEOTIDE DRUG CONJUGATES CONTAINING GEMCITIBINE FOR PHARMACEUTICAL COMPOSITIONS AND RELATED METHODS OF USE AND TREATMENTField

[0001] Antibody-oligonucleotide drug conjugates are provided that target CHK1 or WEE1 genes. The oligonucleotide in the conjugate contains one or more gemcitabine moieties. Also provided are pharmaceutical compositions containing the conjugates, together with methods of using the conjugates for treating a variety of cancers.Background

[0002] siRNA molecules that target CHK1 mRNA are able to silence expression of the CHK1 gene. CHK 1 has been shown to be synergistic with the small molecule chemotherapeutic Gemcitabine (“GEM”) (Azorsa et al., J Transl Med 11:7:43(2009); Fredebohm et al., J Cell Sci I. (2013) and Liang et al., OncolRep 39:1322-1330 (2018)) and CHKl siRNA plus GEM (incorporated in the sense strand in place of Cytidine nucleotides) has been shown to be very effective against pancreatic cancer cells (Simonenko et al. NAR Cancer 2:zcaa016 (2020) and, when chemically modified, against pancreatic cancer, non-small cell lung cancer (“NSCLC”), triple negative breast cancer (“TNBC”) and Ovarian cancer (Halami et al., J Oncol Res Ther 9: 10218 (2024)). GEM has been used to treat cancer for many years, but must be administered by infusion in patients with large amounts being delivered intravenously in greater amounts to enable small amounts to reach the tissue where the cancer resides. Initially, this high dose of GEM shows efficacy against many solid tumors, but resistance eventually becomes an issue as the transporters that take up the drug into the cells become down regulated or desensitized, or the proteins responsible for exporting the drug become upregulated.Brief Description of Drawings

[0003] Figure 1 shows an AODC where an siRNA is directly linked to an antibody via an acid- labile hydrazone linker.

[0004] Figure 2 shows an AODC where an siRNA is directly linked to an antibody via an enzyme cleavable Val-Cit linker.

[0005] Figure 3 shows a diagram representing an muRNA construct that may be used in an AODC targeting CHK1 and WEE1 where the GEM moieties are present in place of certain cytidines. The adaptor region is shown in faded lettering (GCCUUACUA for CHK1 and UAACUAUGU on the WEE1 strand). As shown the bases in the adaptor region are all unmodified but may be modified after the first base joined to the antisense siRNA sequence. The center light shading region highlights base pairing, the darker shading shows the region of the bulge, letters in darker font are the antisense strand of the sequence targeting the gene and the locations of the GEM moieties are shown in place of cytidines in the adaptor. Small case letters are 2’F, upper case letters are unmodified bases or alternatively 2’-0Me.

[0006] Figure 4 shows a method of coupling an antibody to an siRNA via DBCO-azide click chemistry.Detailed Description

[0007] Antibody oligonucleotide drug conjugates (AODC) targeting CHK1 and / or WEE1 are provided that me be used for cancer treatments. The AODC contains an antibody, a peptide linker, which may optionally be a cleavable linker, and an RNA molecule. The RNA molecule may be double stranded such as an miRNA or siRNA, may be an mxRNA, comprising a hybridized double-stranded portion and a hairpin loop, or an muRNA construct, comprising two antisense strands, each targeting different genes or two different portions of the same gene. mxRNA molecules are described in, for example, WO2020 / 44186 and muRNA molecules are described in W02020 / 65602, the contents of each of which are incorporated herein by reference in their entireties.

[0008] In an AODC construct one, two, three, four or more GEM moieties are incorporated into the sense strand in place of cytidine nucleotides or other nucleotides. GEM moieties may also be attached to the ends of the antisense strand or in the adaptor regions of antisense strands in mx or mu RNA, z.e., the last nine nucleotides at the 3’ end of the antisense strand. GEM moieties also can be appended to the 3’ ends of the antisense and / or sense strands. Thus, the AODC construct is unique in that both strands of the RNA comprise at least one GEM moiety, and are eachchemically modified, which stabilizes the construct and allows longer half life in vivo and can be targeted to tumor cells.

[0009] Once the antibody binds to a receptor on the cancer cell and the siRNA is taken up and is released inside the cell, the strands are separated, and the GEM moieties in the sense strand are released into the cytoplasm by the action of endogenous nucleases. The antisense strand is loaded into the RISC complex and surveils for the cognate mRNA encoding the gene of interest. The mRNA is degraded and ultimately the protein level of CHK1 or WEE1, or other gene of interest, is reduced. Both the GEM action in the cytoplasm inhibiting the tumor cell replication machinery, and the effect of gene silencing combine to show synergistic effects (versus either effect alone). The AODC construct also reduces exposure to free GEM that is typically provided to patients by infusion, thus minimizing systemic toxicity. Chemical modification of the siRNA backbones does not prevent release of the GEM moieties from the sense strand once the RNA molecule is inside the tumor cell.

[0010] RNA molecules against select targets within a cancer cell can reduce expression of a protein encoded by the silenced gene target. Silencing these genes can, in turn, inhibit growth of that cell. If the cell is specifically a diseased cell (e.g., a cancer cell) that the siRNA or other RNA molecule can access, then the siRNA may act as a therapeutic. In some cases, it has been found that the use of select therapeutics (small molecule inhibitors such as GEM, monoclonal antibodies, etc.) that are currently the ‘gold standard’ for therapy can be augmented by silencing genes in select pathways.

[0011] The siRNAs can be stabilized against nuclease degradation by chemical modification, using methods that are well known in the art, e.g., by use of 2’-0Me and / or 2’-F and / or phosphorothioate modifications. The siRNA can optionally be chemically linked (via the terminus of the SS or AS or even via the terminal GEM added to the molecule) to a targeting moiety (e.g., GalNac, RGD, Folate, TFR, EGFR, peptide targeting ligands, aptamers or other carbohydrates or even small molecules or antibodies or nanobodies). The targeting moiety has affinity for a receptor or other target on the surface of the cells, which enriches uptake into thesecells. As with unmodified siRNAs, the SS and AS strands will separate in the cell, the AS strand will result in silencing of the gene of interest while the released SS-polyGEM structure will be degraded, releasing GEM which, in combination with the silenced gene exhibits a greater effect than either alone.

[0012] FIGs. 1 and 2 show two antibody-oligonucleotide drug conjugate (AODC) embodiments depicting the general structure of the AODC with two different types of peptide linkers.

[0013] AODC structure'. A cleavable peptide linker of the AODC may contain several components, including, for example, one or more degrader motifs, a cell penetrating peptide (CPP) motif, and an endosomal release peptide. Advantageously, the peptide contains a first degrader motif, a CPP and / or an endosomal release peptide and a final degrader motif. The terminus of the peptide containing the first degrader motif is coupled to the antibody using, for example, DBCO-azide click chemistry or thiol / maleimide linker chemistry. The terminus of the peptide containing the final degrader motif is coupled to the oligo using conventional linker chemistry. The oligonucleotide of interest contains one ore more GEM moieties and other modifications. FIGs. 1 and 2 show two antibody-oligonucleotide drug conjugate (AODC) embodiments depicting the general structure of the AODC with two different types of peptide linkers. If the peptide contains no degrader motif it will not be cleaved in vivo. However, the degrader motif has to be stable in circulation and function only in the endosomes of target cells - so it is advantageously cleaved by specific enzymes such as Cathepsin B, Legumain and / or other enzymes preferentially located inside the endosomes.

[0014] Suitable antibodies for the AODC including IgG (immunoglobulins) are some of the most common in circulation. In cancer immunotherapies, IgGl and IgG4 are common, but IgG2 and IgG3 may also be used.

[0015] The antibody moiety of the AODC targets a molecule on the surface of a cancer cell, typically a receptor that is upregulated on a cancer cell. Suitable cellular targets for the antibody include integrin aVp3 integrin, ICAM1, Glut5, MUC1, EGFR, and SCEL protein in target triplenegative breast cancer. Her2 and EGFR are suitable target antigens in solid tumors. Trop-2 andNectin-4 are also suitable antigens.

[0016] Degrader motifs of the cleavable peptide linker include GGFG (GGFG tetrapeptidyl- aminomethoxy linker), EGCit, VAL-CIT PABC, aryl sulfate linker, Asn-Asn, Asn-Thr and other motifs well known in the art. For the purpose of the AODC molecules herein the degrader motifs are optionally interchangeable.

[0017] Endosomal release peptides of the peptide linker include short, unbranched peptides such as HHHKHHHK, HKP(+H), Endoporter, TAT, penetratin and poly-arginine, which is a series of nine linked arginine residues. Other endosomal release peptides an endosomal release peptides are well known in the art.

[0018] A class of such peptides is the pH-dependent membrane- active peptides (PMAPs). A prototypical example is the HA2 fusion peptide, a peptide that corresponds to the 23 N-terminal residues of the hemagglutinin A2 subunit of the influenza vims X31 strain (GLFGAIAGFIENGWEGMIDGWYG) (Pharmaceuticals 2012, 5, 1177-1209; doi:10.3390 / ph5111177). The influenza virus uses endosomal acidification as a triggering mechanism to deliver its genome into host cells. The viral hemagglutinin undergoes a complex series of conformational changes to induce fusion of the viral and host membranes. In the context of hemagglutinin, the HA2 peptide serves as an anchor that inserts into the endosomal membranes of host cells. Again, any of the endosomal release peptides may be used interchangeably with any of the others.

[0019] Enzymes involved in in vivo cleavage of peptide linkers include legumain (an Asn lysopeptidase found in lysosomes), sulfatase, cathepsin, cathepsin responsive trilinkers, and B- glucuronidase.

[0020] Peptide linker CPP motifs, include RRWQW, RRWQWR, RWQWR and CHHHHHRRRRRRRRRHHHHHC, as well as HK peptide motifs (e.g., HKP based sequence KHHHKHHHKHHHKHHHK). As with degrader sequences and endosomal release peptides, CPP motifs may be mixed and matched to compare delivery efficiencies and efficacy of the targeting oligonucleotide.Double stranded siRNA molecules in AODC constructs

[0021] For certain embodiments of the AODC, the antibody can be coupled via the peptide linker with a traditional siRNA containing cancer inhibitory molecules (such as GEM or other small molecules) or with an mxRNA or with an muRNA as discussed below.

[0022] siRNAs are double stranded RNA molecules comprising a sense strand and a complementary antisense strand. These molecules may have chemically modified backbones and may be blunt ended that are, e.g.. 19-29 bases long on each strand or they may exhibit two base overhangs (typically dTdT). Each strand of the siRNA typically is made on a synthesizer by conjugating the next base in the desired sequence to the previous base attached to the growing oligonucleotide. Amidite chemistry and other synthetic approaches are well known in the field. Once synthesized, the two strands are then annealed to each other to form the duplex.

[0023] The RNA molecules in the AODC constructs may be, for example, duplex siRNA molecules in which GEM moieties have been introduced in place of a nucleotide. In several embodiments the nucleotides replaced may be cytidines.

[0024] GEM replaces the nucleotide cytidine during DNA replication and can inhibit tumor growth since new nucleotides cannot be attached to this nucleotide mimic, resulting in apoptosis of the cells. GEM is an approved therapeutic for treating various cancers including pancreatic cancer. It acts as a nucleoside metabolic inhibitor that causes DNA damage and blocks the progression of cells through the Gl / S phase boundary, however, it has poor bioavailability and, when used in patients, requires intravenous infusion in large doses, resulting in significant toxicity. CHK1 is an integral part of DNA repair and also regulates Gl / S transition. Inhibition of CHK1 may enhance sensitization to DNA-damaging agents via downregulating ribonucleotide reductase levels, shown to be important for resistance to GEM activity. Ribonucleotide reductase is composed of the homodimeric RRM1 and RRM2 subunits that catalyze the conversion of ribonucleotides to deoxyribonucleotides (dNTs). These are used in the synthesis of DNA during replication and repair. Consequently, CHK1 inhibition results in exhaustion of dNTP and enhanced DNA damage. A small molecule inhibitor of CHK1 and an siRNA targeting CHK1decreased RRM1 and 2 and increased H2AX, an established biomarker for DNA double-strand breaks (also increased by GEM treatment). CHK1 inhibition can increase the cytotoxicity of GEM by interfering with DNA damage checkpoints independent of p53 status in pancreatic cell lines. This causes cell death.

[0025] Since GEM is a cytidine analog, it may be used to replace the cytidine moieties in an RNA oligonucleotide sequence with the GEM moieties or the deoxycytidine moieties in a DNA oligonucleotide. These GEMs will still hybridize with their counterparts on a second strand of an oligonucleotide, i.e., GEM will hybridize with a “G” (guanosine / deoxyguanosine) on the opposite strand. Accordingly, the siRNA sequence targeting a gene that, when silenced, produces inhibition of cell growth and can kill tumor cells, is augmented by inclusion of GEM moieties in place of the cytosine moieties in either the sense strand or the antisense strand of an siRNA or an miRNA or even in a single stranded sequence like for an antisense oligonucleotide (ASO).Replacing two cytosine moieties with GEMs in the antisense strand of CHK1 -targeting siRNA sequence augments the inhibitory activity of the siRNA alone. GEM additions can also be on the 3’ or 5’ end of the sense and / or antisense strand. The additions independently can contain 1, 2, 3, 4, or more GEM moieties.

[0026] Other nucleoside anticanccr agents can be attached to the oligonucleotide backbone in place of GEM. Such analogs, which may either replace their analog base (ACGU / T) or other bases in the sequence, or be appended to the ends of one or both strands, include Cytarabine (ara- C), 5-FU, 5-Deoxy-5-fluorouridine, Fludarabine, Capecitabine, Cladribine, Troxacitabine or Clofarabine, Azacytidine and 5-deoxy Azacytidine to name just a few examples see Damaraju, supra). The toxic or resistant effects of these small molecules alone can be reduced when the AODC construct is used and the small molecules are incorporated into the RNA molecule as part of the AODC construct.

[0027] Additional siRNAs may be administered with the AODS constructs. Examples of such siRNA sequences are siRNAs that silence genes encoding proteins that are counterproductive to the inhibitory mechanism of the nucleoside analogs. For example, an siRNA can be used tosilence the enzymes responsible for deamination and inactivation of GEM (Cytidine deaminase or 5 ’-nucleotidase) or those responsible for enhanced release of the nucleoside from the cell - e.g.. nucleoside transporters (see Damaraju supra and references therein).

[0028] Other potential targets of such siRNAs include Multidrug resistance (MDR) proteins such as P-glycoprotein (P-gp) and multidrug resistance-associated protein- 1 (MRP1), encoded by the MDR1 and MRP1 genes, respectively. Expression of these proteins confers a resistant phenotype to a broad spectrum of drugs used in cancer chemotherapy. These transporters arc broadly classified as ATP -binding cassette proteins (ABC) and constitute a superfamily of proteins. Silencing of the MDR1 and MRP1 genes using siRNA has been described. See Donmez and Giindiiz, Biomed Pharmacother. 65(2):85-9 (2011). Other examples of genes that can be silenced include methyl transferases, organic anion and cation transporters (OAT and OCT), oxidoreductase flavoproteins and Nucleoside Transporters (NTs), all of which are involved in drug transport and metabolism in cells and may impart drug resistance phenotypes to cancer cells.

[0029] siRNA gene targets other than CHK1 and WEE1 include those that have been validated to augment the action of GEM or the other nucleoside analogs when silenced themselves for treating cancers. Such gene targets include ATR and RADI?, among others. As other targets arc silenced and demonstrated to augment nucleoside activity, these can also have siRNAs against them coupled to the appropriate nucleoside analog. mxRNA in AODC

[0030] The mxRNA construct has been previously described. See WO2020 / 44186. Briefly, it comprises sense and antisense strands connected at the 3’ of one strand and the 5’ end of the other. One end of the long oligonucleotide is then capable of hybridizing in part at least with the other end, and in the inner fold of that partially hybridized oligonucleotide is a set of five to eight unpaired bases, forming a hairpin loop. The mxRNA hairpin is much shorter than conventional hairpin siRNA structures. In certain embodiments of the AODC, both the sense and antisense strands may contain GEM moieties replacing cytidine nucleotides. In the antisense strand, theGEM moieties can replace the cytidine nucleotides in an adaptor region at the 3’ end. muRNA in AODC

[0031] The muRNA construct has been previously described. See W02020 / 65602. An muRNA typically comprises a first strand containing, in 5’-3’ order, a first antisense sequence of about 18-19 residues and a first sense sequence of about 14 residues, hybridized to a second strand of a second antisense sequence of about 18-19 residues and a second sense strand of about 14 residues. The two strands hybridize together except for a “bulge,” approximately in the center of the hybridized strands, which contains about six to nine unpaired bases between the strands. See e.g., FIG. 3. The muRNA contains nuclease-resistant modified residues and a nuclease- susceptible unmodified site that is cleaved in vivo to release two short duplex siRNA molecules. In the AODC, the muRNA construct may be attached to the peptide linker from either antisense strand. In the AODC construct of certain embodiments, each of the antisense strands may contain GEM moieties replacing cytidine nucleotides (or other nucleotides) in the adaptor region. In other embodiments one or more other small molecules may replace the GEM moieties. The adaptor region of any antisense strand that is a part of an AODC construct is a nine-nucleotide section at the 3’ end of each antisense strand in which the GEM (or other small molecule) may replace the cytidine nucleotide (or other nucleotide). Each of the antisense strands may target a different gene or may target a different portion of the same gene. In the case of several embodiments disclosed herein, the muRNA antisense strands each containing GEM moieties in place of cytidine nucleotides, such as those sequences disclosed in Tables 1 and 2. In these embodiments each of the cleaved siRNA molecules may target CHK1 and WEE1. In other embodiments, one or the other of the antisense strands may contain GEM moieties in the adaptor region. The muRNA affords the opportunity to realize a synergistic effect from silencing two genes at the same time, e.g., CHK1 and WEE1.Definitions

[0032] As used herein, "a" or "an" may mean one or more. As used herein, "another" may mean at least a second or more.

[0033] As used herein, the term “nucleic acid” refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphorodithioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, 2’-Fluoro ribonucleotides, peptide-nucleic acids (PNAs) and unlocked nucleic acids (UNAs; see, e.g., Jensen et al. Nucleic Acids Symposium Series 52: 133-4), and derivatives thereof.

[0034] The term "in vivo" includes therapy based on injection, whether intravenous or local (e.g., intratumoral, intramuscular, subcutaneous, intratracheal, intravenous, or intraocular injection into organ or airway directly, injection into vessels of the organ, or aerosolized into airways). The term "in vivo" also includes therapy based on electroporation of tumor, tissue, or organ.

[0035] The term “adaptor” refers the terminal nine nucleotides at the 3’ end of an RNA antisense strand. This region contains the nucleotides of interest (such as cytidine) that may be replaced by GEM or other small molecules used in cancer treatments as part of the RNA molecule of the AODC construct.

[0036] The term "peptide" is inclusive of both straight and branched amino acid chains, as well as cyclic amino acid chains, which comprise at least 2 amino acid residues. The terms "peptide" and "polypeptide" are used interchangeably herein.

[0037] A "pharmaceutical agent" includes any therapeutic agent useful in preventing, delaying or reducing the severity of the onset of a disease, or in reducing the severity of an ongoing disease, or in enhancing normal physiological functioning, as well as diagnostic agents, for example, a marker gene (GFP, luciferase). A "pharmaceutical agent" may consist of one or more therapeutic agents, one or more diagnostic agents, or a combination of one or more therapeuticand one or more diagnostic agents.

[0038] As used herein, a "pharmaceutically acceptable" component (such as a salt, carrier, excipient or diluent) of a pharmaceutical agent delivery composition according to the present disclosed embodiments is a component which (1) is compatible with the other ingredients of the delivery composition in that it can be included in the delivery composition without eliminating the capacity of the composition to deliver the pharmaceutical agent; and (2) where the delivery composition is intended for therapeutic uses, is suitable for use with an animal (e.g., a human) without undue adverse side effects, such as toxicity, irritation, and allergic response. Side effects are "undue" when their risk outweighs the benefit provided by the pharmaceutical agent.

[0039] As used herein, the term "physiologic pH" is defined as a pH between about 7.2 and about 7.5.

[0040] As used herein, the term "recombinant" means a cell having genetically engineered DNA, which was prepared in vitro and includes DNA from the host organism or, more often, from a different species, genus, family, order or class as compared to the host organism.

[0041] The term "siRNA" is used as it is in the art, and includes a duplex of RNA (19 to 25 bases or fewer in each strand) that targets mRNA. siRNA may be chemically or enzymatically synthesized. siRNA in accordance with the present disclosed embodiments may be incorporated and then activated in RISC (RNA-induced silencing complex).

[0042] A "therapeutically effective amount" is an amount necessary to prevent, delay or reduce the severity of the onset of disease, or an amount necessary to arrest or reduce the severity of an ongoing disease, and also includes an amount necessary to enhance normal physiological functioning. The phrases "pharmacologically effective amount" and "therapeutically effective amount" or "effective amount" refer to that amount of the composition effective to produce the intended pharmacological, therapeutic or preventive result. For example, if a given clinical treatment is considered effective when there is at least a 30% reduction in a measurable parameter associated with a disease or disorder, a therapeutically effective amount of a drug for the treatment of that disease or disorder is the amount necessary to effect at least a 30% reductionin that parameter.

[0043] The word "transfect" is broadly used herein to refer to introduction of an exogenous compound, such as a polynucleotide sequence, into a prokaryotic or eukaryotic cell; the term includes, without limitation, introduction of an exogenous nucleic acid into a cell, which may result in a permanent or temporary alteration of genotype in an immortal or non-immortal cell line. siRNA synthesis with GEM

[0044] The siRNA sequence identified by Azorsa et al. 2009, supra, (CHK1_AZ) is an example of an siRNA that can be used to prepare molecules containing GEM moieties. Using an oligonucleotide synthesizer, the following siRNA strands were manufactured with and without GEM residues included in place of the natural cytidine (“C”) groups present in the sequence.

[0045] The anti-CHKl siRNAs containing GEM moieties were prepared. When modified with the inclusion of the gemcitabine moieties within the sense strand, we inserted these between the end of the sense strand and before the dTdT end groups. The dTdT ends may help stabilize the siRNA sequences against nuclease degradation and this, in turn, may affect the rate of release of the GEM moieties from the sense strand when it is separated from the antisense in the RISC complex during surveillance of the antisense strand for the mRNA sequence to be cleaved and silenced.

[0046] Analogs of siRNA molecules were synthesized where two, four or six GEM moieties are appended on the 3’ end of the sense strand, and we also designed several different siRNAs where the Cs within the sequence could be modified by inserting GEM in their place. GEMs were inserted in place of Cs within the sense strand and in the antisense strand separately or in combination to explore whether increasing GEM content improved efficacy or potency. Unmodified CHK1 siRNA, C1D-2A, C1D-2S and CID-4 were transfected into the pancreatic cancer cell line BxPC3 that has been shown to be sensitive to CHK1 +Gem (Azorsa 2009, supra). In previous work, a dose response of gemcitabine alone was carried out, together with transfection of non-silencing siRNA (as a control).

[0047] It was observed previously that appending additional GEMs to the 3’ end of the sense strand (ahead of the usual dTdT end groups) had no additive effect greater than adding two GEMs and there was no increase in potency and efficacy when four or six were added (Simonenko et al., 2020, supra). It was suspected that the nucleases present within a cell are unable to cleave the polyGEM sequences between the GEM moieties but can only cleave a GEM from its prior unmodified nucleotide - releasing just 1 GEM from within the polyGEM sequences irrespective of the number of GEMs in the sequence. Consequently, constructs with four or six GEMs at the 3 ’ end of the sense strand had equal or slightly worse potency when compared with two GEMs at this location.Improved CHK1 -targeted siRNAs with modified backbones and GEMs

[0048] The 25mer siRNAs described herein are blunt ended siRNAs whereas the siRNA sequence used by Azorsa 2009 (supra) was a 19mer siRNA with a 2 base (dTdT) overhang at the 3’ end. When two GEM moieties were added between the last nucleotide in the siRNA SS and the dTdT overhang the resulting sequence showed much higher efficacy against MiaPaca cells than BxPC3 cells. The inclusion of the dTdT on the end of the sequence may decrease the rate of release of the GEMs from the SS in this construct when the SS is unwound from the AS strand and released into the cytoplasm. This may allow the AS sequence to induce silencing of the gene before the GEMs are released from the SS and this may augment the efficacy of this combination in MiaPaca cells compared with BxPC3 cells.Table 1: sense and antisense strands of an RNA molecule, each containing GEM moieties and other modificationsTable 2; sense and antisense strands of RNA molecules targeting WEE1 or CHK1, identifying locations of GEM moieties in the sense strands and, for antisense strands, identifying the adaptor regions where the same (cytidine) nucleotides are replaced by GEMPreparation ofAODC molecules

[0049] The antibody, peptide linker, and oligonucleotide may be coupled together using conventional linker chemistry that is well-known in the art, such as maleimide / thiol coupling, DBCO-azide coupling and other well-known methods, including click chemistry methods.

[0050] Delivery of therapeutic siRNA in the Antibody-Oligonucleotide Drug Conjugate (AODC)

[0051] Table 1 shows the sequences of siRNA targeting Checkpoint kinase 1 (CHK1) and WEE1, and shows incorporation of GEM in place of cytidines in the sense strands. Delivery of CHK1 siRNA and / or WEE1 siRNA-containing GEM moictics allows the synergy between the inhibition of CHK1 and / or WEE1 and the mechanism of GEM when it is released freely in the cytoplasm of the cancer cell.Formulation of pharmaceutical compositions

[0052] In various embodiments, the AODCs may be further formulated into pharmaceuticalcompositions using methods that are well known in the ail. The composition may be formulated to be compatible with its intended route of administration, whether systemic or for local or regional effect, for example, parenteral, e.g., intramuscular, intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0053] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Crcmophor EL® (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases,it will be advantageous to include isotonic agents, for example, sugar’s, polyalcohols such as mannitol, trehalose, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0054] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above, sterile powders for the preparation of sterile injectable solutions may be prepared using methods such as vacuum drying and freeze-drying, generating a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0055] The compositions may also be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems such as HKP derivatives (Halami et al., 2024, supra). Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthocstcrs, and polylactic acid. Such formulations can be prepared using standard techniques. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.Methods of administration and treatment

[0056] Embodiments of the AODCs in pharmaceutical compositions described herein may be used to treat a variety of disorders and diseases, including, e.g., various cancers, characterized by expression, and particularly altered expression, of genes targeted by the nucleic acid(s) (RNA).

[0057] Suitably formulated pharmaceutical compositions as described herein may beadministered, as noted above, by means known in the ait such as by parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, intratumorally, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration. Advantageously, the pharmaceutical compositions are administered by intravenous or intraparenteral infusion or injection. In the disclosed method embodiments, the compositions may be administered as described above and, advantageously may be delivered systemically or intratumorally. The compositions may be administered as a monotherapy, i.e., in the absence of another treatment, or may be administered as part of a combination regimen that includes one or more additional medications. For example, the compositions are used as part of a combination regimen for treating cancers includes an effective amount of at least one additional chemotherapy drug. In many embodiments, pharmaceutical compositions comprising the AODC are administered intravenously.

[0058] The method of delivery allows delivery of other siRNAs that silence genes responsible for resistance to co-delivery of another therapeutic with anticancer benefits. If the therapeutic is given via a different administration route but demonstrates a build up of resistance by the cancer cell due to, e.g., upregulation of an efflux pump, then an siRNA can be used to reduce the expression of the pump - restoring efficacy of the therapy. Additional mechanisms of resistance can be inhibited in similar ways and targeted to the cancer cell using the AODC and tayloring administration I delivery mode.Determination of toxicity and dosage

[0059] Toxicity and therapeutic efficacy of the pharmaceutical compositions may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population) or IC50 (the concentration of the composition which achieves a half-maximal inhibition of symptoms). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Disclosed pharmaceutical compositions advantageously exhibit high therapeutic indices.

[0060] Data from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans and other mammals. The dosage of the compositions advantageously is within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For the compositions described herein, a therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography (HPLC). Advantageously, in some embodiments, the form of the pharmaceutical composition to be administered is a lyophilizate.

[0061] A therapeutically effective amount of a composition as described herein can be in the range of approximately 1 pg to 1000 mg. For example, 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 pg, or 10, 30, 100, or 1000 mg, or 1-5 g of the compositions can be administered. In general, a suitable dosage unit of the compositions described herein will be in the range of 0.001 to 0.25 milligrams per kilogram body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.001 to 5 micrograms per kilogram of body weight per day, or in the range of 1 to 500 nanograms per kilogram of body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day. The pharmaceutical composition can be administered once daily, or may be dosed in dosage units containing two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In that case, the RNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage unit. The dosage unit can also be compounded for a single dose over several days, e.g., using a conventional sustained release formulation which provides sustained and consistent release of the RNA over a several day period. Sustained releaseformulations are well known in the ail. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose. Regardless of the formulation, the pharmaceutical composition must contain RNA in a quantity sufficient to inhibit expression of the target gene in the animal or human being treated. The composition can be compounded in such a way that the sum of the multiple units of RNA together contain a sufficient dose.

[0062] The composition may be administered one or more times within a given period. The pharmaceutical compositions may be administered once, one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition as described herein may include a single treatment or, advantageously, can include a series of treatments.

[0063] Depending on the particular targeted gene(s) and the selected nucleic acid (e.g., siRNA) sequence(s) and the dose administered, a partial or complete loss of targeted gene function may be observed. A reduction of mRNA levels or target protein expression (either mRNA expression or encoded polypeptide expression) in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of the targeted cells is exemplary. Degradation of mRNA levels or mRNA expression refers to the absence (or observable decrease) in targeted protein levels. Specificity refers to the ability to inhibit the translation of mRNA into proteins without manifesting effects on other genes in the targeted cells. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS). Inhibition of target gene RNA sequence(s) by the nucleic acid molecules described in the disclosed embodiments also can be measured based upon the effect ofadministration of such molecules upon development / progression of an associated disease or disorder, e.g., tumor formation, growth, metastasis, etc., either in vivo or in vitro. Treatment and / or reductions in, e.g., tumor or cancer cell levels can include halting or reduction of growth of tumor or cancer cell levels or reductions of, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and can also be measured in logarithmic terms, e.g., a 10-fold, 100-fold, 1000-fold, 105 -fold, 106-fold, or 107-fold reduction in cancer cell levels could be achieved via administration of the pharmaceutical compositions containing them to cells, tissues, or to subjects in need. The subject may be a mammal, such as a human, a non-human primate or other animal used in a research, clinical or veterinary setting.

Claims

CLAIMSWhat is claimed is:

1. An antibody-oligonucleotide drug conjugate (AODC), comprising: an antibody that specifically binds to a cancer cell receptor covalently coupled to an siRNA molecule via a peptide linker, wherein said siRNA molecule inhibits expression of checkpoint kinase01 (CHK1) gene expression in cancer cells, wherein the sense and / or the antisense strand of the RNA molecule comprises at least one gemcitabine (GEM) moiety in place of a cytidine residue within the sequence or adaptor region.

2. The AODC of claim 1, wherein the RNA molecule comprises a plurality of modified nucleotides3. The AODC of claim 2, wherein said modified nucleotides are 2’-0Me and / or 2’-F modified nucleotides.

4. The AODC of any preceding claim, wherein the siRNA is an muRNA comprising a strand containing a second antisense sequence that targets WEE 1.

5. The AODC of any preceding claim, wherein the siRNA molecule is selected from the molecules of Table 1.

6. The AODC of any preceding claim, wherein the antibody moiety binds to a receptor expressed on the surface of cancer cells selected from the group consisting of non-small cell lung cancer cells, pancreatic cancer cells, triple-negative breast cancer cells, and ovarian cancer cells7. The AODC of any preceding claim, wherein the antibody binds to integrin aVp3, ICAM1, Glut5, MUC1, EGFR, and / or SCEL protein on target triple-negative breast cancer cells.

8. The AODC of any preceding claim, wherein the peptide linker comprises a cell penetrating peptide (CPP) and / or an endosomal releasing peptide, and at least two degrader motifs.

9. The AODC of claim 8, comprising a linker having the structure: first degrader motif - CPP (or endosomal releasing peptide)- second degrader motif - endosomal releasingpeptide - third degrader motif; wherein the first degrader motif is covalently attached to the antibody and the second / third degrader motif is covalently attached to the RNA molecule.

10. The AODC of claim 9, wherein the CPP is selected from the group consisting of RRWQW, RRWQWR, RWQWR and CHHHHHRRRRRRRRRHHHHHC and Histidine Lysine polymers.

11. The AODC of claim 9, wherein said two or more degrader motifs are independently selected from the group consisting of GGFG, EGCit, aryl sulfate linker, and Asn-Asn or Asn- Thr.

12. The AODC of claim 9, wherein the endosomal releasing peptide is selected from the group consisting of HHHKHHHK, HKP(+H), and poly-arginine or endoporter.

13. The AODC of any of claims 1 to 7, wherein said peptide linker contains no degrader motifs.

14. A pharmaceutical composition comprising: an AODC of any of claims 1 to 13 and a pharmaceutically acceptable carrier, diluent or excipient.

15. The pharmaceutical composition of claim 14, further comprising one or more cytotoxic drugs selected from the group consisting of alkylating agents, antimetabolites, antitumor antibiotics, topoisomerases and mitotic inhibitors, and corticosteroids.

16. A method of treating a subject suffering from cancer, comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition according to any of claim 14 or claim 15.

17. The method of treatment of claim 16, wherein the cancer is selected from the group consisting of triple negative breast cancer, non-small cell lung cancer, pancreatic cancer and ovarian cancer.

18. The method of treatment of claim 17, wherein the pharmaceutical composition is administered intravenously, intratumorally or subcutaneously.

19. The method of treatment of claim 18, wherein the pharmaceutical composition is administered intravenously.

20. The method of treatment of any of claims 16 to 19, wherein the subject is a mammal.

21. The method of treatment of claim 20, wherein the mammal is a human.

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