TW202214857A - New conjugated nucleic acid molecules and their uses - Google Patents

New conjugated nucleic acid molecules and their uses Download PDF

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TW202214857A
TW202214857A TW110122234A TW110122234A TW202214857A TW 202214857 A TW202214857 A TW 202214857A TW 110122234 A TW110122234 A TW 110122234A TW 110122234 A TW110122234 A TW 110122234A TW 202214857 A TW202214857 A TW 202214857A
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克麗斯特爾 贊達內爾
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法商昂席歐公司
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Abstract

The present invention relates to new nucleic acid molecules of therapeutic interest, in particular for use in the treatment of cancer.

Description

新型結合核酸分子及其用途Novel binding nucleic acid molecules and their uses

本發明係關於醫學領域，尤其腫瘤學領域。The present invention relates to the field of medicine, in particular the field of oncology.

DNA損傷反應(DDR)偵測DNA損傷且促進其修復。DNA損傷類型之廣泛多樣性需要多種基本不同的DNA修復機制，諸如錯配修復(MMR)、鹼基切除修復(BER)、核苷酸切除修復(NER)、單股斷裂修復(SSB)及雙股斷裂修復(DSB)。舉例而言，聚腺苷酸核糖聚合酶(PARP)主要參與修復SSB，而兩種主要機制用於修復DNA中之DSB：非同源末端連接(NHEJ)及同源重組(HR)。PARP-1充當第一反應者，其偵測DNA損傷且隨後促進修復路徑之選擇。在NHEJ中，DSB由Ku蛋白識別，隨後結合且活化蛋白激酶DNA-PKcs，引起末端處理酶之募集及活化。已證明，癌細胞修復治療性誘導之DNA損傷的能力會影響治療功效。DNA damage response (DDR) detects DNA damage and promotes its repair. The broad diversity of DNA damage types requires a number of fundamentally different DNA repair mechanisms, such as mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), single-strand break repair (SSB), and double Strand Break Repair (DSB). For example, polyadenylation ribose polymerase (PARP) is primarily involved in repairing SSBs, while two major mechanisms are used to repair DSBs in DNA: non-homologous end joining (NHEJ) and homologous recombination (HR). PARP-1 acts as a first responder that detects DNA damage and subsequently facilitates the selection of repair pathways. In NHEJ, DSBs are recognized by the Ku protein, which subsequently binds and activates the protein kinase DNA-PKcs, resulting in the recruitment and activation of end-processing enzymes. The ability of cancer cells to repair therapeutically induced DNA damage has been shown to affect therapeutic efficacy.

此已致使靶向DNA修復路徑及蛋白質來開發抗癌劑，從而增加對傳統遺傳毒性治療(化學治療劑、放射線療法)之敏感性。癌症療法之合成致死方法提供新穎機制來特異性靶向癌細胞，同時避開非癌細胞，從而減少與治療相關之毒性。This has led to the development of anticancer agents targeting DNA repair pathways and proteins, thereby increasing susceptibility to traditional genotoxic treatments (chemotherapeutic agents, radiation therapy). Synthetic lethal approaches to cancer therapy provide novel mechanisms to specifically target cancer cells while avoiding non-cancer cells, thereby reducing therapy-related toxicity.

在此等合成致死方法中，Dbait分子為模擬雙股DNA損傷之核酸分子。其充當DNA損傷信號傳導酶PARP及DNA-PK之誘鉺，誘導「假」DNA損傷信號，且最終抑制參與DSB及SSB路徑之許多蛋白質在損傷部位之募集。In these synthetic lethal methods, Dbait molecules are nucleic acid molecules that mimic double-stranded DNA damage. It acts as an inducer for the DNA damage signaling enzymes PARP and DNA-PK, induces "pseudo" DNA damage signaling, and ultimately inhibits the recruitment of many proteins involved in the DSB and SSB pathways at the site of damage.

Dbait分子已廣泛描述於PCT專利申請案WO2005/040378、WO2008/034866、WO2008/084087、WO2011/161075、WO2017/013237、WO2017/148976及WO2019/175132中。Dbait分子可由其治療活性所必需之許多特徵來定義，諸如其最小長度可為可變的，只要其足以允許包含Ku及DNA-PKcs蛋白之Ku蛋白複合物的適當結合即可。因此已表明，Dbait分子之長度必須大於20 bp，較佳為約32 bp，以確保與此類Ku複合物結合且實現DNA-PKcs活化。Dbait molecules have been extensively described in PCT patent applications WO2005/040378, WO2008/034866, WO2008/084087, WO2011/161075, WO2017/013237, WO2017/148976 and WO2019/175132. A Dbait molecule can be defined by a number of characteristics necessary for its therapeutic activity, such as its minimum length can be variable, so long as it is sufficient to allow proper binding of the Ku protein complex comprising Ku and DNA-PKcs protein. It has thus been shown that the length of the Dbait molecule must be greater than 20 bp, preferably about 32 bp, to ensure binding to such Ku complexes and enable DNA-PKcs activation.

已表徵用此類Dbait分子治療之潛在預測性生物標記。如PCT專利申請案WO2018/162439中所述，對Dbait分子之敏感性確實與具有微核(MN)之細胞的高自發頻率相關。連續在43個來自不同組織之實體腫瘤細胞株及16個細胞及患者來源之異種移植物模型中進行驗證，提出將高基礎水準之MN作為用Dbait分子治療之預測性生物標記。Potential predictive biomarkers for treatment with such Dbait molecules have been characterized. As described in PCT patent application WO2018/162439, sensitivity to Dbait molecules does correlate with a high spontaneous frequency of cells with micronuclei (MN). Serial validation in 43 solid tumor cell lines from different tissues and 16 cell and patient-derived xenograft models proposes a high basal level of MN as a predictive biomarker for treatment with Dbait molecules.

此外，最近已提出，微核(MN)將提供關鍵平台作為DNA損傷誘導之免疫反應的一部分(Gekara J Cell Biol. 2017年10月2日;216(10):2999-3001)。近期研究證明MN形成在DNA損傷誘導之免疫活化中的作用。有趣的是，胞質DNA感測路徑確實已成為DNA損傷與先天性免疫之間的主要連接。DNA正常駐存於細胞核及粒線體中；因此，其在細胞質中之存在充當危險相關分子模式(DAMP)來觸發免疫反應。環單磷酸鳥苷(GMP)-單磷酸腺苷(AMP)合成酶(cGAS)為將DNA偵測為DAMP且誘導I型IFN及其他細胞介素之感測器。DNA以序列非依賴性方式與cGAS結合；此結合誘導cGAS之催化中心的構形變化，使得此酶可將三磷酸鳥苷(GTP)及ATP轉化為第二信使環狀GMP-AMP (cGAMP)。此cGAMP分子為轉接蛋白IFN基因刺激因子STING之內源性高親和力配體。STING路徑之活化可隨後包括例如刺激炎性細胞介素IP-10 (亦稱為CXCL10)及CCL5或受體NGK2及PD-L1。Furthermore, it has recently been suggested that micronuclei (MN) will provide a key platform as part of DNA damage-induced immune responses (Gekara J Cell Biol. 2017 Oct 2;216(10):2999-3001). Recent studies demonstrate the role of MN formation in DNA damage-induced immune activation. Interestingly, the cytoplasmic DNA sensing pathway has indeed emerged as a major link between DNA damage and innate immunity. DNA normally resides in the nucleus and mitochondria; thus, its presence in the cytoplasm acts as a danger-associated molecular pattern (DAMP) to trigger an immune response. Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) is a sensor that detects DNA as DAMP and induces type I IFN and other cytokines. DNA binds to cGAS in a sequence-independent manner; this binding induces a conformational change in the catalytic center of cGAS, allowing the enzyme to convert guanosine triphosphate (GTP) and ATP to the second messenger cyclic GMP-AMP (cGAMP) . This cGAMP molecule is an endogenous high-affinity ligand for the adaptor protein IFN gene stimulator STING. Activation of the STING pathway can then include, for example, stimulation of the inflammatory cytokines IP-10 (also known as CXCL10) and CCL5 or the receptors NGK2 and PD-L1.

近期證據表明STING (干擾素基因刺激因子)路徑參與抗腫瘤免疫反應之誘導。因此，現在正廣泛開發STING促效劑作為一類新型癌症治療劑。已表明，癌細胞中STING依賴性路徑之活化可導致腫瘤經免疫細胞浸潤且調節抗癌免疫反應。Recent evidence suggests that the STING (stimulator of interferon gene) pathway is involved in the induction of antitumor immune responses. Therefore, STING agonists are now being widely developed as a new class of cancer therapeutics. It has been shown that activation of STING-dependent pathways in cancer cells can lead to tumor infiltration by immune cells and modulate anticancer immune responses.

STING為一種內質網轉接蛋白，其促進先天性免疫信號傳導(一種快速的非特異性免疫反應，可對抗環境侵害，包括但不限於病原體，諸如細菌或病毒)。據報導，STING能夠活化NF-kB、STAT6及IRF3轉錄路徑以誘導I型干擾素(例如，IFN-α及IFN-β)之表現且在表現後發揮強效抗病毒狀態。然而，迄今為止開發的STING促效劑能夠活化所有細胞類型中之STING路徑，且可觸發與其在樹突狀細胞中之活化有關的顯著副作用。因此，局部投與STING促效劑。STING is an endoplasmic reticulum transfer protein that promotes innate immune signaling (a rapid non-specific immune response against environmental aggressions, including but not limited to pathogens such as bacteria or viruses). It has been reported that STING is able to activate the NF-kB, STAT6 and IRF3 transcriptional pathways to induce the expression of type I interferons (eg, IFN-α and IFN-β) and exert a potent antiviral state upon expression. However, the STING agonists developed to date are capable of activating the STING pathway in all cell types and can trigger significant side effects associated with their activation in dendritic cells. Therefore, the STING agonist is administered topically.

因此，確實需要尋找一種特異性活化腫瘤細胞中之STING路徑的方式。Therefore, there is a real need to find a way to specifically activate the STING pathway in tumor cells.

因此，仍需要治療癌症之療法，尤其依賴於數種機制之藥物，尤其DNA修復路徑及STING路徑活化劑，及可幫助檢查點抑制劑在更多患者及更廣泛的癌症中起作用的藥物。Therefore, there remains a need for therapeutics to treat cancer, especially drugs that rely on several mechanisms, especially DNA repair pathway and STING pathway activators, and drugs that can help checkpoint inhibitors work in more patients and a wider range of cancers.

癌細胞具有獨特的能量代謝以維持快速增殖。在正常氧氣條件下對厭氧糖酵解之偏好為癌症代謝之獨特性狀，且命名為瓦氏效應(Warburg effect)。增強之糖酵解亦支持產生核苷酸、胺基酸、脂質及葉酸作為癌細胞分裂之構建塊。菸鹼醯胺腺嘌呤二核苷酸(NAD)為一種輔酶，其在包括糖酵解之許多代謝途徑中介導氧化還原反應。NAD水準升高會增強糖酵解且為癌細胞提供能量。在此情形下，NAD水準耗盡隨後經由抑制能量產生路徑，諸如糖酵解、三羧酸(TCA)循環及氧化磷酸化來抑制癌細胞增殖。NAD亦充當數種酶之受質，從而經由此等酶調節DNA修復、基因表現及壓力反應。因此，NAD代謝與能量代謝以外的癌症發病機制有關，且視為用於癌症治療之有前景的治療目標，特別是對於因DNA修復基因缺陷(例如ERCC1及ATM缺陷)或IDH (異檸檬酸去氫酶)突變而呈現NAD缺陷之癌細胞。Cancer cells have a unique energy metabolism to sustain rapid proliferation. The preference for anaerobic glycolysis under normoxic conditions is a unique trait of cancer metabolism and is named the Warburg effect. Enhanced glycolysis also supports the production of nucleotides, amino acids, lipids and folate as building blocks for cancer cell division. Nicotinamide adenine dinucleotide (NAD) is a coenzyme that mediates redox reactions in many metabolic pathways including glycolysis. Elevated levels of NAD enhance glycolysis and provide energy to cancer cells. In this case, NAD level depletion subsequently inhibits cancer cell proliferation by inhibiting energy-producing pathways such as glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. NAD also acts as a substrate for several enzymes through which DNA repair, gene expression, and stress responses are regulated. Therefore, NAD metabolism is implicated in cancer pathogenesis other than energy metabolism and is considered a promising therapeutic target for cancer therapy, especially for genes caused by DNA repair defects (eg, ERCC1 and ATM defects) or IDH (isocitrate depletion). NAD-deficient cancer cells mutated.

亦需要新型治療方法來成功解決癌細胞群，而不出現對療法具有抗性之癌細胞。There is also a need for novel therapeutic approaches to successfully address cancer cell populations without the emergence of cancer cells that are resistant to therapy.

本發明提供新型結合核酸分子，其靶向DNA修復路徑且特定性刺激癌細胞中之STING路徑。更特定言之，該核酸分子能夠活化PARP但不會活化DNA-PK。The present invention provides novel binding nucleic acid molecules that target the DNA repair pathway and specifically stimulate the STING pathway in cancer cells. More specifically, the nucleic acid molecule is capable of activating PARP but not DNA-PK.

本發明係關於一種結合核酸分子，其包含16個鹼基對(bp)雙股核酸部分，第一股之5'端及互補股之3'端藉由環連接在一起，及視情況存在之促進胞吞作用的分子，該分子與該環連接， 其中： -   該16個鹼基對(bp)雙股核酸具有以下序列

Figure 02_image004
SEQ ID NO: 1 其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵；且其中加下劃線之核苷酸為2'修飾之核苷酸， -   該環具有選自下式中之一者的結構： -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中r及s獨立地為整數0或1；g及h獨立地為1至7之整數且g + h之和為4至7； 其中K為
Figure 02_image006
其中i、j、k及l獨立地為0至6、較佳1至3之整數； 或 -O-P(X)OH-O-[(CH 2) d-C(O)-NH] b-CHR-[C(O)-NH-(CH 2) e] c-O-P(X)OH-O-    (II) 其中b及c獨立地為0至4之整數，且b + c之和為3至7； d及e獨立地為1至3、較佳1至2之整數；且 其中R為-L f-J， X為O或S，L為連接子且f為整數0或1，且J為促進胞吞作用之分子或為H。 促進胞吞作用之分子可選自由以下組成之群：膽固醇、單鏈或雙鏈脂肪酸、靶向細胞受體實現受體介導之胞吞作用的配體或運鐵蛋白。更特定言之，促進胞吞作用之分子為膽固醇。 The present invention relates to a binding nucleic acid molecule comprising a 16 base pair (bp) double-stranded nucleic acid moiety, the 5' end of the first strand and the 3' end of the complementary strand are linked together by loops, and optionally A molecule that promotes endocytosis, the molecule is linked to the loop, wherein: - the 16 base pair (bp) double-stranded nucleic acid has the following sequence
Figure 02_image004
SEQ ID NO: 1 wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; and wherein the underlined nucleotide is a 2' modified nucleotide, - the loop has a The structure of one of the formulas: -OP(X)OH-O-{[(CH 2 ) 2 -O] g -P(X)OH-O} r -KOP(X)OH-O-{[( CH 2 ) 2 -O] h -P(X)OH-O-} s (I) wherein r and s are independently an integer of 0 or 1; g and h are independently an integer from 1 to 7 and g + h is an integer and from 4 to 7; where K is
Figure 02_image006
wherein i, j, k and l are independently an integer from 0 to 6, preferably 1 to 3; or -OP(X)OH-O-[( CH2 ) d -C(O)-NH] b -CHR -[C(O)-NH-(CH 2 ) e ] c -OP(X)OH-O- (II) wherein b and c are independently an integer from 0 to 4, and the sum of b + c is 3 to 4 7; d and e are independently an integer from 1 to 3, preferably 1 to 2; and wherein R is -L f -J, X is O or S, L is a linker and f is an integer 0 or 1, and J Molecules that promote endocytosis or H. Molecules that promote endocytosis can be selected from the group consisting of cholesterol, single or double chain fatty acids, ligands targeting cellular receptors for receptor-mediated endocytosis, or transferrin. More specifically, the molecule that promotes endocytosis is cholesterol.

視情況，環可具有式(I)且r為1，s為0且g為5至7之整數，較佳為6。Optionally, the ring may have formula (I) and r is 1, s is 0 and g is an integer from 5 to 7, preferably 6.

環可具有式(I)，且當i及j為1且k及l均為1或2時，K為

Figure 02_image007
或
Figure 02_image008
。 The ring may be of formula (I), and when i and j are 1 and k and l are both 1 or 2, K is
Figure 02_image007
or
Figure 02_image008
.

視情況，f為1且L-J為-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J或-C(O)-(CH 2) m-NH-[C(O)-CH 2-O] t-[(CH 2) 2-O] n-(CH 2) p-[C(O)] v-J，其中m為0至10之整數；n為0至6之整數；且p為0至2之整數；t及v為整數0或1，其中t及v中之至少一者為1。 f is 1 and LJ is -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O)-J or -C( as appropriate O)-(CH 2 ) m -NH-[C(O)-CH 2 -O] t -[(CH 2 ) 2 -O] n -(CH 2 ) p -[C(O)] v -J , wherein m is an integer from 0 to 10; n is an integer from 0 to 6; and p is an integer from 0 to 2; t and v are integers 0 or 1, wherein at least one of t and v is 1.

視情況，f為1且L-J選自由以下組成之群：-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-J、C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-C(O)-J及-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中m為0至10之整數；n為0至15之整數；且p為0至3之整數。 Optionally, f is 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O) -J, -C(O)-(CH 2 ) m -NH-C(O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, -C(O)-(CH 2 ) m -NH-C(O )-[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J and -C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O- [( CH2 ) 2 -O] n- ( CH2 ) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3.

視情況，m為4與6之間的整數，較佳為5。Optionally, m is an integer between 4 and 6, preferably 5.

視情況，環具有式(I) -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中X為S，r為1，g為6，s為0，且當i及j為1且k及l為2時，K為

Figure 02_image008
其中f為1且L為C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-(CH 2) 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-(CH 2) 3-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 5-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 9-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 Optionally, the ring has the formula (I)-OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X)OH-O} r -KOP(X)OH-O-{[ ( CH2 ) 2 -O] h -P(X)OH-O-} s (I) where X is S, r is 1, g is 6, s is 0, and when i and j are 1 and k and When l is 2, K is
Figure 02_image008
where f is 1 and L is C(O)-(CH 2 ) 5 -NH-[(CH 2 ) 2 -O] 3 -(CH 2 ) 2 -C(O)-J, -C(O)- (CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3 -(CH 2 ) 3 -J, -C(O)-(CH 2 ) 5 -NH-C(O) -CH 2 -O-[(CH 2 ) 2 -O] 5 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O -[(CH 2 ) 2 -O] 9 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 13 -CH 2 -C(O)-J or -C(O)-(CH 2 ) 5 -NH-C(O)-J.

視情況，f為1且L-J為-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J或-C(O)-(CH 2) m-NH-[C(O)-CH 2-O] t-[(CH 2) 2-O] n-(CH 2) p-[C(O)] v-J，其中m為0至10之整數；n為0至6之整數；且p為0至2之整數；t及v為整數0或1，其中t及v中之至少一者為1。在一態樣中，m為4與6之間的整數，較佳為5。 f is 1 and LJ is -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O)-J or -C( as appropriate O)-(CH 2 ) m -NH-[C(O)-CH 2 -O] t -[(CH 2 ) 2 -O] n -(CH 2 ) p -[C(O)] v -J , wherein m is an integer from 0 to 10; n is an integer from 0 to 6; and p is an integer from 0 to 2; t and v are integers 0 or 1, wherein at least one of t and v is 1. In one aspect, m is an integer between 4 and 6, preferably 5.

視情況，f為1且L-J選自由以下組成之群：-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-C(O)-J及-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中m為0至10之整數；n為0至15之整數；且p為0至3之整數。在一態樣中，m為4與6之間的整數，較佳為5。 Optionally, f is 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O) -J, -C(O)-(CH 2 ) m -NH-C(O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, -C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, -C(O)-(CH 2 ) m -NH-C( O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J and -C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O -[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J, where m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3 . In one aspect, m is an integer between 4 and 6, preferably 5.

在一極特定態樣中，L可選自由以下組成之群：-C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-(CH 2) 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-(CH 2) 3-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 5-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 9-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 In a very specific aspect, L can be selected from the group consisting of -C(O)-( CH2 ) 5 -NH-[( CH2 ) 2 -O] 3- ( CH2 ) 2 -C( O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3 -(CH 2 ) 3 -J, -C(O)-( CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 5 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 - NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 9 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O ) -CH2 -O-[( CH2 ) 2 -O] 13 - CH2 -C(O)-J or -C(O)-( CH2 ) 5 -NH-C(O)-J.

在另一特定態樣中，環具有式(I)-O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中X為S，r為1，g為6，s為0，i及j為1且k及l為2， 其中f為1且L為C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-(CH 2) 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-(CH 2) 3-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 5-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 9-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 In another specific aspect, the ring has the formula (I)-OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X)OH-O} r -KOP(X)OH -O-{[(CH 2 ) 2 -O] h -P(X)OH-O-} s (I) where X is S, r is 1, g is 6, s is 0, and i and j are 1 and k and 1 are 2, where f is 1 and L is C(O)-( CH2 ) 5 -NH-[( CH2 ) 2 -O] 3- ( CH2 ) 2 -C(O)-J , -C(O)-(CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3 -(CH 2 ) 3 -J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 5 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C( O)-CH 2 -O-[(CH 2 ) 2 -O] 9 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[( CH2 ) 2 -O] 13 - CH2 -C(O)-J or -C(O)-( CH2 ) 5 -NH-C(O)-J.

視情況，2'修飾之核苷酸獨立地選自由以下組成之群：2'-去氧-2'-氟、2'-O-甲基、2'-O-甲氧基乙基(2'-O-MOE)、2'-O-胺基丙基(2'-O-AP)、2'-O-二甲胺基乙基(2'-O-DMAE)、2'-O-二甲胺基丙基(2'-O-DMAP)、2'-O-二甲胺基乙氧基乙基(2'-O-DMAEOE)、2'-O-N-甲基乙醯胺基(2'-O-NMA)修飾、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸(LNA)，較佳為2'-去氧-2'-氟核苷酸、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸(LNA)。Optionally, the 2'-modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-methoxyethyl) '-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAE), 2'-O- Dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido ( 2'-O-NMA) modification, 2'-deoxy-2'-fluoroarabinonucleotide (FANA) and 2' bridged nucleotide (LNA), preferably 2'-deoxy-2'- Fluoronucleotides, 2'-deoxy-2'-fluoroarabinose nucleotides (FANA) and 2' bridging nucleotides (LNA).

在一特定態樣中，2'修飾之核苷酸為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)。在另一態樣中，2'修飾之核苷酸為2'橋接核苷酸(LNA)。在另一態樣中，2'修飾之核苷酸為2'-去氧-2'-氟核苷酸。In a specific aspect, the 2' modified nucleotide is 2'-deoxy-2'-fluoroarabinonucleotide (FANA). In another aspect, the 2' modified nucleotide is a 2' bridging nucleotide (LNA). In another aspect, the 2' modified nucleotide is a 2'-deoxy-2'-fluoronucleotide.

在一極特定態樣中，結合核酸分子為：

Figure 02_image010
其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵；且 其中加下劃線之核苷酸為2'修飾之核苷酸。 或其醫藥學上可接受之鹽。 In a very specific aspect, the binding nucleic acid molecule is:
Figure 02_image010
wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; and wherein the underlined nucleotide is a 2' modified nucleotide. or its pharmaceutically acceptable salt.

在一個態樣中，2'修飾之核苷酸為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)且該分子為OX413。在另一態樣中，2'修飾之核苷酸為2'橋接核苷酸(LNA)。在另一態樣中，2'修飾之核苷酸為2'-去氧-2'-氟核苷酸。In one aspect, the 2' modified nucleotide is 2'-deoxy-2'-fluoroarabinonucleotide (FANA) and the molecule is OX413. In another aspect, the 2' modified nucleotide is a 2' bridging nucleotide (LNA). In another aspect, the 2' modified nucleotide is a 2'-deoxy-2'-fluoronucleotide.

本發明亦關於一種醫藥組合物，其包含根據本揭示內容之結合核酸分子。視情況，該醫藥組合物進一步包含額外治療劑，其較佳選自免疫調節劑，諸如免疫檢查點抑制劑(ICI)；基於T細胞之癌症免疫療法，諸如過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)；或習知化學治療劑、放射治療劑或抗血管生成劑；或靶向免疫毒素。The present invention also relates to a pharmaceutical composition comprising a binding nucleic acid molecule according to the present disclosure. Optionally, the pharmaceutical composition further comprises additional therapeutic agents, preferably selected from immunomodulatory agents, such as immune checkpoint inhibitors (ICI); T cell-based cancer immunotherapy, such as adoptive cell transfer (ACT), gene transfer Modified T cells or engineered T cells, such as chimeric antigen receptor cells (CAR-T cells); or conventional chemotherapeutic, radiotherapeutic, or anti-angiogenic agents; or targeted immunotoxins.

本發明亦關於根據本揭示內容之結合核酸分子或醫藥組合物或獸用組合物，其用作藥物，尤其用於治療癌症。其進一步關於一種治療有需要之個體之癌症的方法，其包含反覆或長期投與治療有效量之根據本發明之結合核酸分子或醫藥組合物。視情況，該方法包含投與重複治療週期，較佳至少兩個投與週期，甚至更佳至少三個或四個投與週期。The present invention also relates to binding nucleic acid molecules or pharmaceutical or veterinary compositions according to the present disclosure for use as a medicament, especially for the treatment of cancer. It further relates to a method of treating cancer in an individual in need thereof, comprising repeated or chronic administration of a therapeutically effective amount of a binding nucleic acid molecule or a pharmaceutical composition according to the invention. Optionally, the method comprises administering repeated treatment cycles, preferably at least two administration cycles, and even more preferably at least three or four administration cycles.

重複或長期投與根據本發明之結合核酸分子不會使癌細胞對療法產生抗性。其可與免疫調節劑組合使用，諸如免疫檢查點抑制劑(ICI)，或與基於T細胞之癌症免疫療法組合使用，包括過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)。Repeated or chronic administration of a binding nucleic acid molecule according to the present invention does not render cancer cells resistant to therapy. It can be used in combination with immunomodulatory agents, such as immune checkpoint inhibitors (ICIs), or in combination with T cell-based cancer immunotherapy, including adoptive cell transfer (ACT), genetically modified T cells, or engineered T cells, such as chimeric antigen receptor cells (CAR-T cells).

因此，結合核酸分子或醫藥組合物與較佳選自以下之額外治療劑組合用於治療癌症：免疫調節劑，諸如免疫檢查點抑制劑(ICI)；基於T細胞之癌症免疫療法，諸如過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)；或習知化學治療劑、放射治療劑或抗血管生成劑；或靶向免疫毒素。Thus, the binding nucleic acid molecule or pharmaceutical composition is used in the treatment of cancer in combination with additional therapeutic agents preferably selected from the group consisting of: immunomodulatory agents, such as immune checkpoint inhibitors (ICIs); T cell based cancer immunotherapy, such as adoptive cells Transfer (ACT), genetically modified T cells, or engineered T cells, such as chimeric antigen receptor cells (CAR-T cells); or conventional chemotherapeutic, radiotherapeutic, or anti-angiogenic agents; or Targeted immunotoxins.

在一特定態樣中，本發明亦關於一種針對具有攜帶NAD +合成缺陷之腫瘤之患者之可能選擇策略或臨床分層策略的方式。此等患者對於根據本發明之藥物治療可為更好的反應者，特別是具有攜帶DNA修復路徑缺陷(例如ERCC1及ATM缺陷)或IDH突變之腫瘤的患者。 In a particular aspect, the invention also relates to a possible means of selection strategy or clinical stratification strategy for patients with tumors harboring defects in NAD + synthesis. Such patients may be better responders to drug therapy according to the present invention, particularly patients with tumors harboring DNA repair pathway defects (eg, ERCC1 and ATM defects) or IDH mutations.

在一特定態樣中，結合核酸分子或醫藥組合物用於癌症治療中針對攜帶NAD +合成缺陷之腫瘤細胞的靶向作用。更特定言之，腫瘤細胞進一步攜帶選自ERCC1或ATM缺陷之DNA修復路徑缺陷或IDH突變。 In a particular aspect, the binding nucleic acid molecule or pharmaceutical composition is used in the targeting of tumor cells carrying defects in NAD + synthesis in cancer therapy. More specifically, the tumor cells further carry a DNA repair pathway defect selected from ERCC1 or ATM deficiency or an IDH mutation.

本發明係關於與促進胞吞作用之分子結合的新核酸分子，諸如膽固醇-核酸結合物，其特異性靶向且活化PARP，誘導細胞NAD之深度下調，且因此特別專用於癌症治療，特別是對於因DNA修復基因缺陷(例如ERCC1及ATM缺陷)或IDH (異檸檬酸去氫酶)突變而呈現NAD缺陷之癌細胞。The present invention relates to novel nucleic acid molecules in combination with molecules that promote endocytosis, such as cholesterol-nucleic acid conjugates, which specifically target and activate PARP, induce deep downregulation of cellular NAD, and are therefore particularly specific for cancer therapy, in particular For cancer cells that exhibit NAD deficiency due to DNA repair gene defects (eg, ERCC1 and ATM deficiency) or IDH (isocitrate dehydrogenase) mutations.

本發明係關於與促進胞吞作用之分子結合的新核酸分子，諸如膽固醇-核酸結合物，其靶向DDR機制且亦為STING促效劑，允許其與免疫檢查點療法(ICT)組合用於癌症之最佳治療。The present invention relates to novel nucleic acid molecules that bind to molecules that promote endocytosis, such as cholesterol-nucleic acid conjugates, which target the DDR mechanism and are also STING agonists, allowing their use in combination with immune checkpoint therapy (ICT) The best treatment for cancer.

根據本發明之新型結合核酸分子提供： 1) 與Dbait分子相比，本發明之結合核酸分子活化PARP但不會活化DNA-PK，獨立使用會使得具有微核、細胞質染色質片段(CCF)及細胞毒性之癌細胞增加。 2) 癌細胞中微核(MN)及細胞質染色質片段(CCF)之特異性增加引起STING路徑活化之早期增加，如藉由炎性細胞介素(CCL5)釋放以及PD-L1及NKG2D配體(MIC-A)在癌細胞上之表現增加所示。此等效應對癌細胞具有特異性。此類癌細胞特異性排除普遍及廣泛的炎症以及隨後可能出現的有害副作用。 3) 經由抑制DNA修復路徑活化STING路徑及產生微核及CCF代表一種特異性活化腫瘤細胞中之STING路徑的非常有吸引力的方式，特別是藉由先天性免疫活化。 The novel binding nucleic acid molecules according to the present invention provide: 1) Compared with Dbait molecule, the binding nucleic acid molecule of the present invention activates PARP but does not activate DNA-PK, and independent use can increase cancer cells with micronuclei, cytoplasmic chromatin fragment (CCF) and cytotoxicity. 2) Specific increases in micronuclei (MN) and cytoplasmic chromatin fragments (CCF) in cancer cells lead to an early increase in STING pathway activation, such as by inflammatory cytokine (CCL5) release and PD-L1 and NKG2D ligands (MIC-A) shown by increased expression on cancer cells. This equivalence should be specific for cancer cells. Such cancer cells specifically preclude generalized and widespread inflammation and possible subsequent deleterious side effects. 3) Activation of the STING pathway and generation of micronuclei and CCF via inhibition of the DNA repair pathway represents a very attractive way to specifically activate the STING pathway in tumor cells, especially by innate immune activation.

基於此等觀察結果，本發明係關於： -   一種如下文所述之結合核酸分子； -   一種醫藥組合物，其包含如下文所述之結合核酸分子及醫藥學上可接受之載劑，尤其用於治療癌症； -   一種如下文所述之結合核酸分子，其用作藥物，尤其用於治療癌症； -   一種如下文所述之結合核酸分子之用途，其用於製造藥物，尤其用於治療癌症； -   一種用於治療有需要之患者之癌症的方法，其包含投與有效量之如本文所揭示之結合核酸分子； -   一種醫藥組合物，其包含如下文所述之結合核酸分子、額外治療劑及醫藥學上可接受之載劑，尤其用於治療癌症； -   一種產品或套組，其含有(a)如下文所揭示之結合核酸分子及視情況選用之b)額外治療劑，作為組合製劑同時、單獨或依序使用，尤其用於治療癌症； -   一種組合製劑，其包含(a)如下文所揭示之髮夾核酸分子，b)如下文所述之額外治療劑，用於同時、單獨或依序使用，尤其用於治療癌症； -   一種醫藥組合物，其包含如下文所揭示之結合核酸分子，用於與額外治療劑組合治療癌症； -   一種包含如下文所揭示之結合核酸分子之醫藥組合物的用途，其用於製造與額外治療劑組合治療癌症之藥物； -   一種用於治療有需要之患者之癌症的方法，其包含投與有效量之a)如下文所揭示之結合核酸分子及b)有效量之額外治療劑； -   一種用於治療有需要之患者之癌症的方法，其包含投與有效量之包含如本文所揭示之結合核酸分子之醫藥組合物及有效量之額外治療劑； -   一種用於有需要之患者中提高用治療性抗腫瘤劑治療癌症之效率，或增強腫瘤對用治療性抗腫瘤劑治療之敏感性的方法，其包含投與有效量之如下文所揭示之結合核酸分子； -   一種用於治療癌症之方法，其包含藉由重複治療週期，較佳至少兩個投與週期，甚至更佳至少三個或四個投與週期，反覆或長期投與如本文所揭示之結合核酸分子； -   一種治療患者之癌症的方法，該等患者之腫瘤細胞攜帶NAD+合成缺陷及視情況存在之選自ERCC1或ATM缺陷之DNA修復路徑缺陷或IDH突變。 定義 Based on these observations, the present invention relates to: - a binding nucleic acid molecule as described below; - a pharmaceutical composition comprising a binding nucleic acid molecule as described below and a pharmaceutically acceptable carrier, especially for the treatment of cancer; - a binding nucleic acid molecule as described below for use as a medicament, especially for the treatment of cancer; - a use of a nucleic acid molecule as described below for the manufacture of a medicament, in particular for the treatment of cancer; - a method for treating cancer in a patient in need thereof, comprising administering an effective amount of a binding nucleic acid molecule as disclosed herein; - a pharmaceutical composition comprising a binding nucleic acid molecule as described below, an additional therapeutic agent and a pharmaceutically acceptable carrier, especially for the treatment of cancer; - a product or kit comprising (a) a binding nucleic acid molecule as disclosed below and optionally b) additional therapeutic agents for simultaneous, separate or sequential use as a combined preparation, especially for the treatment of cancer; - a combined preparation comprising (a) a hairpin nucleic acid molecule as disclosed below, b) additional therapeutic agents as described below for simultaneous, separate or sequential use, especially for the treatment of cancer; - a pharmaceutical composition comprising a binding nucleic acid molecule as disclosed below for the treatment of cancer in combination with additional therapeutic agents; - a use of a pharmaceutical composition comprising a binding nucleic acid molecule as disclosed below for the manufacture of a medicament for the treatment of cancer in combination with additional therapeutic agents; - a method for treating cancer in a patient in need thereof, comprising administering an effective amount of a) a binding nucleic acid molecule as disclosed below and b) an effective amount of an additional therapeutic agent; - a method for treating cancer in a patient in need thereof, comprising administering an effective amount of a pharmaceutical composition comprising a binding nucleic acid molecule as disclosed herein and an effective amount of an additional therapeutic agent; - A method for increasing the efficiency of treating cancer with a therapeutic antineoplastic agent, or enhancing the sensitivity of a tumor to treatment with a therapeutic antineoplastic agent in a patient in need thereof, comprising administering an effective amount of as disclosed below binding nucleic acid molecules; - A method for treating cancer comprising repeated or chronic administration of a combination as disclosed herein by repeating treatment cycles, preferably at least two administration cycles, even more preferably at least three or four administration cycles nucleic acid molecules; - A method of treating cancer in patients whose tumor cells carry a defect in NAD+ synthesis and optionally a defect in the DNA repair pathway selected from ERCC1 or ATM deficiency, or an IDH mutation. definition

每當在整個本說明書中提及本發明之醫藥組合物、套組、產品及組合製劑時，提到「癌症治療」或其類似物意謂：a)用於治療癌症之方法，該方法包含向需要此類治療之患者投與本發明之醫藥組合物、套組、產品及組合製劑；b)用於治療癌症之本發明之醫藥組合物、套組、產品及組合製劑；c)本發明之醫藥組合物、套組、產品及組合製劑用於製造供治療癌症用之藥物的用途；及/或d)用於治療癌症之本發明之醫藥組合物、套組、產品及組合製劑。Whenever reference is made to the pharmaceutical compositions, kits, products and combined preparations of the present invention throughout this specification, reference to "cancer treatment" or the like means: a) a method for treating cancer, the method comprising Administration of pharmaceutical compositions, kits, products and combined preparations of the present invention to patients in need of such treatment; b) pharmaceutical compositions, kits, products and combined preparations of the present invention for the treatment of cancer; c) the present invention Use of the pharmaceutical composition, kit, product and combined preparation of the present invention for the manufacture of a medicament for the treatment of cancer; and/or d) the pharmaceutical composition, kit, product and combined preparation of the present invention for the treatment of cancer.

在本發明之上下文內，術語「治療」表示治癒性、對症性及預防性治療。本發明之醫藥組合物、套組、產品及組合製劑可用於患有現有癌症或腫瘤之人類，包括在癌症進展之早期或晚期。本發明之醫藥組合物、套組、產品及組合製劑不一定能治癒患有癌症之患者，但將延緩或減緩疾病之進展或防止疾病之進一步進展，從而改善患者之病況。特定言之，本發明之醫藥組合物、套組、產品及組合製劑減少腫瘤之發展、減少腫瘤負荷、在哺乳動物宿主中產生腫瘤消退及/或防止轉移發生及癌症復發。在治療癌症時，本發明之醫藥組合物、套組、產品及組合製劑以治療有效量投與。In the context of the present invention, the term "treatment" refers to curative, symptomatic and prophylactic treatment. The pharmaceutical compositions, kits, products and combined preparations of the present invention can be used in humans with existing cancers or tumors, including in early or late stages of cancer progression. The pharmaceutical compositions, kits, products and combined preparations of the present invention may not necessarily cure a patient suffering from cancer, but will delay or slow the progression of the disease or prevent further progression of the disease, thereby improving the patient's condition. In particular, the pharmaceutical compositions, kits, products and combined formulations of the present invention reduce tumor development, reduce tumor burden, produce tumor regression in mammalian hosts and/or prevent the development of metastases and cancer recurrence. In the treatment of cancer, the pharmaceutical compositions, kits, products and combined preparations of the present invention are administered in a therapeutically effective amount.

如本文所用，術語「套組」、「產品」或「組合製劑」特別定義「分裝部分之套組」，意義在於如上文所定義之組合搭配物(a)及(b)可獨立地或藉由使用具有不同量之組合搭配物(a)及(b)的不同固定組合給藥，亦即同時或在不同時間點給藥。分裝部分之套組的組分可隨後例如同時或按時間順序交錯投與，亦即在不同時間點且對於分裝部分之套組的任何部分具有相等或不同的時間間隔。在組合製劑中待投與之組合搭配物(a)與組合搭配物(b)之總量的比率可變化。組合搭配物(a)及(b)可藉由相同途徑或藉由不同途徑投與。As used herein, the terms "kit", "product" or "combination formulation" specifically define a "kit of parts" in the sense that the combination partners (a) and (b) as defined above may be independently or Administration is by using different fixed combinations with different amounts of combination partners (a) and (b), ie at the same time or at different time points. The components of the kit of dispensed portions may then be administered, eg, simultaneously or chronologically staggered, ie, at different points in time and with equal or different time intervals for any portion of the kit of dispensed portions. The ratio of the total amount of combination partner (a) to combination partner (b) to be administered in the combination formulation may vary. Combination partners (a) and (b) can be administered by the same route or by different routes.

「有效量」意謂本發明之醫藥組合物、套組、產品及組合製劑單獨或與醫藥組合物、套組、產品或組合製劑之其他活性成分組合預防、移除或減少哺乳動物(包括人類)之癌症之不利影響的量。應理解，投與劑量可由熟習此項技術者根據患者、病理、投與模式等進行調適。"Effective amount" means that the pharmaceutical compositions, kits, products and combined formulations of the present invention, alone or in combination with other active ingredients of the pharmaceutical compositions, kits, products or combined formulations, prevent, remove or reduce mammalian (including human) ) of the adverse effects of cancer. It will be appreciated that the dose administered can be tailored by one skilled in the art depending on the patient, pathology, mode of administration, and the like.

術語「STING」係指干擾素基因刺激因子受體，亦稱為TMEM173、ERIS、MITA、MPYS、SAVI或NET23)。如本文所用，術語「STING」及「STING受體」可互換使用，且包括STING之不同同功異型物及變異體。人類STING同功異構物1 (最長同功異構物)之mRNA及蛋白質序列具有NCBI參考序列[NM_198282.3]及[NP_938023.1]。人類STING同功異構物2 (較短同功異構物)之mRNA及蛋白質序列具有NCBI參考序列[NM_001301738.1]及[NP_001288667.1]。The term "STING" refers to the receptor for interferon gene stimulator, also known as TMEM173, ERIS, MITA, MPYS, SAVI or NET23). As used herein, the terms "STING" and "STING receptor" are used interchangeably and include different isoforms and variants of STING. The mRNA and protein sequences of human STING isoform 1 (the longest isoform) have NCBI reference sequences [NM_198282.3] and [NP_938023.1]. The mRNA and protein sequences of human STING isoform 2 (the shorter isoform) have NCBI reference sequences [NM_001301738.1] and [NP_001288667.1].

如本文所用，術語「STING活化劑」係指能夠活化STING路徑之分子。STING路徑之活化可包括例如刺激炎性細胞介素，包括干擾素，諸如1型干擾素，包括IFN-α、IFN-β，3型干擾素，例如IFN-λ、IP-10 (干擾素-γ誘導蛋白，亦稱為CXCL10)、PD-L1、TNF、IL-6、CXCL9、CCL4、CXCL11、NKG2D配體(MICA/B)、CCL5、CCL3或CCL8。STING路徑之活化亦可包括刺激TANK結合激酶(TBK) 1磷酸化、干擾素調節因子(IRF)活化(例如IRF3活化)、分泌IP-10或其他炎性蛋白質及細胞介素。STING路徑之活化可例如藉由化合物刺激STING路徑之活化的能力來確定，如使用干擾素刺激分析、報導基因分析(例如hSTING wt分析或THP-1 Dual分析)、TBK1活化分析、IP-10分析或熟習此項技術者已知的其他分析所偵測。STING路徑之活化亦可藉由化合物增加編碼由STING或STING路徑活化之蛋白質之基因的轉錄水準的能力來確定。此類活化可例如使用RNAseq分析來偵測。As used herein, the term "STING activator" refers to a molecule capable of activating the STING pathway. Activation of the STING pathway can include, for example, stimulation of inflammatory interferons, including interferons, such as type 1 interferons, including IFN-α, IFN-β, type 3 interferons, such as IFN-λ, IP-10 (interferon- gamma-inducing protein, also known as CXCL10), PD-L1, TNF, IL-6, CXCL9, CCL4, CXCL11, NKG2D ligand (MICA/B), CCL5, CCL3 or CCL8. Activation of the STING pathway may also include stimulation of TANK-binding kinase (TBK) 1 phosphorylation, interferon regulatory factor (IRF) activation (eg, IRF3 activation), secretion of IP-10 or other inflammatory proteins and interferons. Activation of the STING pathway can be determined, for example, by the ability of a compound to stimulate activation of the STING pathway, such as using interferon stimulation assays, reporter gene assays (eg hSTING wt assay or THP-1 Dual assay), TBK1 activation assay, IP-10 assay or detected by other assays known to those skilled in the art. Activation of the STING pathway can also be determined by the ability of a compound to increase the level of transcription of a gene encoding a protein that is activated by STING or the STING pathway. Such activation can be detected, for example, using RNAseq analysis.

STING路徑之活化可藉由選自以下之一或多種「STING分析」來確定：干擾素刺激分析、hSTING wt分析、THP1-Dual分析、TANK結合激酶1 (TBK1)分析、干擾素-γ誘導蛋白10 (IP-10)分泌分析或PD-L1分析。Activation of the STING pathway can be determined by one or more "STING assays" selected from the group consisting of: interferon stimulation assay, hSTING wt assay, THP1-Dual assay, TANK-binding kinase 1 (TBK1) assay, interferon-gamma-inducible protein 10 (IP-10) secretion assay or PD-L1 assay.

更特定言之，若分子能夠在STING表現細胞中刺激一或多種STING依賴性細胞介素的產生為未處理之STING表現細胞的至少1.1倍、1.2倍、1.3倍、1.4倍、1.5倍、1.6倍、1.7倍、1.8倍、1.9倍、2倍或更多，則該分子為STING活化劑。較佳地，STING依賴性細胞介素係選自干擾素、1型干擾素、IFN-α、IFN-β、3型干擾素、IFN-λ、CXCL10 (IP-10)、PD-L1 TNF、IL-6、CXCL9、CCL4、CXCL11、NKG2D配體(MICA/B)、CCL5、CCL3或CCL8，更佳CCL5或CXCL10。 結合核酸分子 More specifically, if the molecule is capable of stimulating the production of one or more STING-dependent cytokines in STING-expressing cells at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold compared to untreated STING-expressing cells times, 1.7 times, 1.8 times, 1.9 times, 2 times or more, the molecule is a STING activator. Preferably, the STING-dependent interferon is selected from interferon, type 1 interferon, IFN-α, IFN-β, type 3 interferon, IFN-λ, CXCL10 (IP-10), PD-L1 TNF, IL-6, CXCL9, CCL4, CXCL11, NKG2D ligand (MICA/B), CCL5, CCL3 or CCL8, more preferably CCL5 or CXCL10. bind nucleic acid molecules

根據本發明之一些結合核酸分子的另一優點係基於以下事實：其可藉由僅使用寡核苷酸固相合成而作為一個分子合成，從而允許低成本及高製造規模。Another advantage of some binding nucleic acid molecules according to the invention is based on the fact that they can be synthesized as one molecule by using only oligonucleotide solid phase synthesis, allowing low cost and high scale of manufacture.

本發明之結合核酸分子包含16個鹼基對(bp)雙股核酸部分，第一股之5'端及互補股之3'端藉由環連接在一起，及視情況存在之促進胞吞作用的分子，該分子與該環連接。雙股核酸部分之另一端為自由的。The binding nucleic acid molecule of the present invention comprises a double-stranded nucleic acid portion of 16 base pairs (bp), the 5' end of the first strand and the 3' end of the complementary strand are linked together by a loop, and optionally promote endocytosis , which is attached to the ring. The other end of the double-stranded nucleic acid portion is free.

根據本發明之結合核酸分子可由其治療活性所必需之許多特徵來定義，諸如其16-bp長度、存在至少一個自由端及存在雙股部分，較佳存在硫代磷酸酯核苷酸間鍵及對應於核苷酸之核糖2'位之核苷酸修飾的雙股DNA部分。硫代磷酸酯核苷酸間鍵與2'修飾之核苷酸之特定組合令人驚訝地與改良之活性及藥物動力學相關。A binding nucleic acid molecule according to the present invention can be defined by a number of characteristics necessary for its therapeutic activity, such as its 16-bp length, the presence of at least one free end and the presence of a double-stranded moiety, preferably the presence of phosphorothioate internucleotide linkages and A portion of double-stranded DNA corresponding to a nucleotide modification at the 2' position of the ribose sugar of the nucleotide. The specific combination of phosphorothioate internucleotide linkages and 2' modified nucleotides was surprisingly associated with improved activity and pharmacokinetics.

結合核酸分子能夠活化PARP-1蛋白。另一方面，結合核酸分子不活化DNA-PK。Binding nucleic acid molecules can activate the PARP-1 protein. On the other hand, binding nucleic acid molecules do not activate DNA-PK.

本發明亦關於本發明之結合核酸分子的醫藥學上可接受之鹽。 核酸分子 The present invention also relates to pharmaceutically acceptable salts of the binding nucleic acid molecules of the present invention. nucleic acid molecule

本發明之核酸分子包含雙股核酸部分，第一股之5'端及互補股之3'端藉由環連接在一起，結合核酸分子之長度為16個鹼基對(bp)，允許適當結合及活化PARP (PARP-1)蛋白，且不足以允許適當結合包含Ku及DNA-PKcs蛋白之Ku蛋白複合物。「bp」意謂分子包含指定長度之雙股部分。The nucleic acid molecule of the present invention comprises a double-stranded nucleic acid portion, the 5' end of the first strand and the 3' end of the complementary strand are connected together by a loop, and the length of the binding nucleic acid molecule is 16 base pairs (bp), allowing for proper binding And activate PARP (PARP-1) protein, and not enough to allow proper binding of Ku protein complex containing Ku and DNA-PKcs protein. "bp" means that the molecule contains a double-stranded portion of the specified length.

在嚴格條件下，結合核酸分子不與人類基因體DNA雜交。Under stringent conditions, the binding nucleic acid molecule does not hybridize to human genomic DNA.

為了有利於與PARP-1之相互作用，在PARP-1與核苷酸間鍵之間具有確定相互作用之核苷酸經2'修飾之核苷酸諸如F-ANA置換，但保留硫代磷酸酯之分子3'端之核苷酸除外。舉例而言，胸腺嘧啶可經2'-去氧-2'-氟阿拉伯糖胸腺嘧啶置換，鳥苷可經2'-去氧-2'-氟阿拉伯糖鳥苷置換；胞苷可經2'-去氧-2'-氟阿拉伯糖胞苷置換；或腺嘌呤可經2'-去氧-2'-氟阿拉伯糖腺嘌呤置換。To facilitate interaction with PARP-1, nucleotides with defined interactions between PARP-1 and the internucleotide bond are replaced with 2' modified nucleotides such as F-ANA, but the phosphorothioate is retained The nucleotide at the 3' end of the ester molecule is excluded. For example, thymine can be replaced by 2'-deoxy-2'-fluoroarabinosethymine, guanosine can be replaced by 2'-deoxy-2'-fluoroarabinoguanosine; cytidine can be replaced by 2'-deoxy-2'-fluoroarabinoguanosine - Deoxy-2'-fluoroarabinocytidine replacement; or adenine can be replaced by 2'-deoxy-2'-fluoroarabinose adenine.

當確定核苷酸之2'位與PARP-1之間具有相互作用時，使核苷酸在2'位無任何修飾。當確定核苷酸之2'位與PARP-1之間具有相互作用時，或當核苷酸與PARP-1無任何已知相互作用時，此等核苷酸之核苷酸間鍵藉由引入硫代磷酸酯(「s」)進行化學修飾，以保護其免受降解。由於雙股核酸分子具有16個鹼基對，故已進行對稱的化學修飾，亦即在各股之5'端存在6個2'修飾之核苷酸及在各股之3'端存在3個2'修飾之核苷酸，且此等2'修飾之核苷酸鏈段之間的大部分核苷酸具有硫代磷酸酯鍵。When an interaction between the 2' position of the nucleotide and PARP-1 was determined, the nucleotide was left without any modification at the 2' position. When the 2' position of the nucleotides is determined to have an interaction with PARP-1, or when the nucleotides do not have any known interaction with PARP-1, the internucleotide linkages between these nucleotides are determined by A phosphorothioate ("s") was introduced for chemical modification to protect it from degradation. Since the double-stranded nucleic acid molecule has 16 base pairs, it has been chemically modified symmetrically, that is, there are 6 2' modified nucleotides at the 5' end of each strand and 3 nucleotides at the 3' end of each strand 2' modified nucleotides, and most of the nucleotides between these 2' modified nucleotide stretches have phosphorothioate linkages.

根據一個實施例，結合核酸分子包含對應於核糖2'位之修飾。舉例而言，結合核酸分子可包含至少一個2'修飾之核苷酸，例如具有2'-去氧、2'-去氧-2'-氟、2'-O-甲基、2'-O-甲氧基乙基(2'-O-MOE)、2'-O-胺基丙基(2'-O-AP)、2'-O-二甲胺基乙基(2'-O-DMAE)、2'-O-二甲胺基丙基(2'-O-DMAP)、2'-O-二甲胺基乙氧基乙基(2'-O-DMAEOE)或2'-O-N-甲基乙醯胺基(2'-O-NMA)修飾或例如2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)。在另一個實施例中，結合核酸分子包含對應於2'-去氧-2'-氟核苷酸、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸(LNA)之2'位之修飾。According to one embodiment, the binding nucleic acid molecule comprises a modification corresponding to the 2' position of the ribose sugar. For example, a binding nucleic acid molecule can comprise at least one 2' modified nucleotide, eg, with 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O -Methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O- DMAE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-O-N - Methylacetamido (2'-O-NMA) modification or eg 2'-deoxy-2'-fluoroarabinonucleotide (FANA). In another embodiment, the binding nucleic acid molecule comprises nucleotides corresponding to 2'-deoxy-2'-fluoronucleotides, 2'-deoxy-2'-fluoroarabinonucleotides (FANA) and 2' bridging cores Modification of the 2' position of the nucleotide (LNA).

在一特定態樣中，結合核酸分子具有2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)。在另一態樣中，2'修飾之核苷酸為2'橋接核苷酸(LNA)。在另一態樣中，2'修飾之核苷酸為2'-去氧-2'-氟核苷酸。In a specific aspect, the binding nucleic acid molecule has 2'-deoxy-2'-fluoroarabinonucleotide (FANA). In another aspect, the 2' modified nucleotide is a 2' bridging nucleotide (LNA). In another aspect, the 2' modified nucleotide is a 2'-deoxy-2'-fluoronucleotide.

在一特定態樣中，雙股核酸部分具有以下序列

Figure 02_image012
SEQ ID NO: 1 其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵；且其中加下劃線之核苷酸為2'修飾之核苷酸。 In a specific aspect, the double-stranded nucleic acid portion has the following sequence
Figure 02_image012
SEQ ID NO: 1 wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; and wherein the underlined nucleotide is a 2' modified nucleotide.

視情況，2'修飾之核苷酸獨立地選自由以下組成之群：2'-去氧-2'-氟、2'-O-甲基、2'-O-甲氧基乙基(2'-O-MOE)、2'-O-胺基丙基(2'-O-AP)、2'-O-二甲胺基乙基(2'-O-DMAE)、2'-O-二甲胺基丙基(2'-O-DMAP)、2'-O-二甲基胺基乙氧基乙基(2'-O-DMAEOE)、2'-O-N-甲基乙醯胺基(2'-O-NMA)修飾、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸，較佳為2'-去氧-2'-氟核苷酸、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸(LNA)。Optionally, the 2'-modified nucleotides are independently selected from the group consisting of 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-methoxyethyl) '-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAE), 2'-O- Dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamido (2'-O-NMA) modified, 2'-deoxy-2'-fluoroarabinonucleotide (FANA) and 2' bridging nucleotide, preferably 2'-deoxy-2'-fluoro core nucleotides, 2'-deoxy-2'-fluoroarabinonucleotides (FANA) and 2' bridging nucleotides (LNA).

在一特定態樣中，2'修飾之核苷酸為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)。FANA採用DNA樣結構，使得所關注蛋白質對結合核酸分子之識別未改變。FANA包括以下嘧啶2'-氟阿拉伯糖核苷及嘌呤2'-氟阿拉伯糖核苷： 9-(2-去氧-2-氟-ß-D-阿拉伯呋喃糖基)腺嘌呤(2'-FANA-A)； 9-(2-去氧-2-氟-ß-D-阿拉伯呋喃糖基)鳥嘌呤(2'-FANA-G)； 1-(2-去氧-2-氟-ß-D-阿拉伯呋喃糖基)胞嘧啶(2'-FANA-C)； 1-(2-去氧-2-氟-ß-D-阿拉伯呋喃糖基)尿嘧啶(2'-FANA-U)；及 1-(2-去氧-2-氟-ß-D-阿拉伯呋喃糖基)胸苷(2'-FANA-T)。 環 In a specific aspect, the 2' modified nucleotide is 2'-deoxy-2'-fluoroarabinonucleotide (FANA). FANA adopts a DNA-like structure such that recognition of the binding nucleic acid molecule by the protein of interest is unchanged. FANA includes the following pyrimidine 2'-fluoroarabinoside and purine 2'-fluoroarabinoside: 9-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)adenine (2'-FANA-A); 9-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)guanine (2'-FANA-G); 1-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)cytosine (2'-FANA-C); 1-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)uracil (2'-FANA-U); and 1-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl)thymidine (2'-FANA-T). ring

環與雙股部分之第一股之5'端及互補股之3'端連接，且視情況與促進胞吞作用之分子連接。The loop is attached to the 5' end of the first strand of the double-stranded portion and the 3' end of the complementary strand, and optionally to a molecule that promotes endocytosis.

環較佳包含10至100個原子、較佳15至25個原子之鏈。The ring preferably contains a chain of 10 to 100 atoms, preferably 15 to 25 atoms.

環可包括2至10個核苷酸，較佳3、4或5個核苷酸。非核苷酸環非窮盡地包括無鹼基核苷酸、聚醚、聚胺、聚醯胺、肽、碳水化合物、脂質、聚烴或其他聚合化合物(例如，寡聚乙二醇，諸如具有2至10個乙二醇單元，較佳4、5、6、7或8個乙二醇單元之寡聚乙二醇)。在一個實施例中，環可選自由以下組成之群：N-(5-羥甲基-6-磷酸己基)-11-(3-(6-磷酸己硫基)琥珀醯亞胺基))十一醯胺、1,3-雙-[5-羥基戊醯胺基]丙基-2-(6-磷酸己基)、六乙二醇、四去氧胸苷酸(T4)、1,19-雙(磷酸基)-8-氫氮雜-2-羥基-4-氧雜-9-側氧基-十九烷及2,19-雙(磷酸基)-8-氫氮雜-1-羥基-4-氧雜-9-側氧基-十九烷。The loop may comprise 2 to 10 nucleotides, preferably 3, 4 or 5 nucleotides. Non-nucleotide rings include non-exhaustively abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, oligoethylene glycols, such as those with 2 to 10 ethylene glycol units, preferably oligoethylene glycols of 4, 5, 6, 7 or 8 ethylene glycol units). In one embodiment, the ring can be selected from the group consisting of: N-(5-hydroxymethyl-6-phosphate hexyl)-11-(3-(6-phosphate hexylthio)succinimidyl)) Undecylamide, 1,3-bis-[5-hydroxypentamido]propyl-2-(hexyl phosphate), hexaethylene glycol, tetradeoxythymidylic acid (T4), 1,19 -Bis(phosphoryl)-8-hydroaza-2-hydroxy-4-oxa-9-oxo-nonadecane and 2,19-bis(phosphoryl)-8-hydroaza-1- Hydroxy-4-oxa-9-pendoxyl-nonadecane.

促進胞吞作用之分子視情況經由連接子與環結合。此項技術中已知的任何連接子可用於將促進胞吞作用之分子共價連接至環。舉例而言，WO09/126933在第38-45頁提供對方便連接子之廣泛綜述。連接子可非窮盡地為脂族鏈、聚醚、聚胺、聚醯胺、肽、碳水化合物、脂質、聚烴或其他聚合化合物(例如寡聚乙二醇，諸如具有2至10個乙二醇單元，較佳3、4、5、6、7或8個乙二醇單元，更佳6個乙二醇單元之寡聚乙二醇)，以及併入任何可藉由化學或酶促方式分解之鍵，諸如二硫鍵、受保護之二硫鍵、酸不穩定鍵(例如腙鍵)、酯鍵、原酸酯鍵、膦醯胺鍵、生物可裂解肽鍵、偶氮鍵或醛鍵。此類可裂解連接子詳述於WO2007/040469第12-14頁、WO2008/022309第22-28頁中。Molecules that promote endocytosis are optionally bound to the loop via a linker. Any linker known in the art can be used to covalently attach a molecule that promotes endocytosis to the loop. For example, WO09/126933 provides an extensive review of convenient linkers on pages 38-45. Linkers can be non-exhaustive aliphatic chains, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (eg, oligoethylene glycols, such as those having 2 to 10 ethylene glycols) alcohol units, preferably 3, 4, 5, 6, 7 or 8 ethylene glycol units, more preferably 6 ethylene glycol units (oligoethylene glycols), and incorporating any chemical or enzymatic means Cleaved bonds, such as disulfide bonds, protected disulfide bonds, acid labile bonds (eg, hydrazone bonds), ester bonds, orthoester bonds, phosphinamide bonds, biocleavable peptide bonds, azo bonds, or aldehydes key. Such cleavable linkers are detailed in WO2007/040469 pages 12-14, WO2008/022309 pages 22-28.

促進胞吞作用之分子藉由熟習此項技術者已知的任何方式與環結合，視情況經由寡聚乙二醇間隔基。Molecules that promote endocytosis are bound to the loop by any means known to those skilled in the art, optionally via an oligoethylene glycol spacer.

在一特定實施例中，促進胞吞作用之分子與環之間的連接子包含C(O)-NH-(CH 2-CH 2-O) n或NH-C(O)-(CH 2-CH 2-O) n，其中n為1至10之整數，較佳n選自由3、4、5及6組成之群。在一極特定實施例中，連接子為CO-NH-(CH 2-CH 2-O) 4(甲醯胺基三乙二醇)。 In a specific embodiment, the linker between the endocytosis-promoting molecule and the loop comprises C(O)-NH-( CH2 - CH2 -O) n or NH-C(O) - (CH2- CH 2 -O) n , wherein n is an integer from 1 to 10, preferably n is selected from the group consisting of 3, 4, 5 and 6. In a very specific embodiment, the linker is CO-NH-( CH2 - CH2 -O) 4 (formamidotriethylene glycol).

在另一特定實施例中，促進胞吞作用之分子與環分子之間的連接子為二烷基-二硫化物{例如(CH 2) p-S-S-(CH 2) q，其中p及q為1至10、較佳3至8之整數，例如6}。 In another specific embodiment, the linker between the endocytosis promoting molecule and the ring molecule is a dialkyl-disulfide {eg ( CH2 ) p -SS-( CH2 ) q , where p and q is an integer from 1 to 10, preferably from 3 to 8, such as 6}.

在另一特定實施例中，環已經開發以與寡核苷酸固相合成相容。因此，有可能在核酸分子之合成期間併入環，從而促進合成且降低其成本。In another specific embodiment, the loops have been developed to be compatible with oligonucleotide solid phase synthesis. Thus, it is possible to incorporate loops during the synthesis of nucleic acid molecules, thereby facilitating synthesis and reducing its cost.

環可具有選自下式中之一者的結構： -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中r及s獨立地為整數0或1；g及h獨立地為1至7之整數且g + h之和為4至7； 其中K為

Figure 02_image013
其中i、j、k及l獨立地為0至6、較佳1至3之整數； 或 -O-P(X)OH-O-[(CH 2) d-C(O)-NH] b-CHR-[C(O)-NH-(CH 2) e] c-O-P(X)OH-O- (II) 其中b及c獨立地為0至4之整數，且b + c之和為3至7； d及e獨立地為1至3、較佳1至2之整數； 其中R為-L f-J， 其中X為O或S，L為連接子，較佳為直鏈伸烷基及/或寡聚乙二醇，視情況雜有一個或若干個選自胺基、醯胺及側氧基之基團，f為整數0或1，且J為促進胞吞作用之分子或為H。 The ring may have a structure selected from one of the following formulae: -OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X)OH-O} r -KOP(X)OH -O-{[(CH 2 ) 2 -O] h -P(X)OH-O-} s (I) wherein r and s are independently integers 0 or 1; g and h are independently one of 1 to 7 Integer and the sum of g + h is 4 to 7; where K is
Figure 02_image013
wherein i, j, k and l are independently an integer from 0 to 6, preferably 1 to 3; or -OP(X)OH-O-[( CH2 ) d -C(O)-NH] b -CHR -[C(O)-NH-(CH 2 ) e ] c -OP(X)OH-O- (II) wherein b and c are independently an integer from 0 to 4, and the sum of b + c is 3 to 4 7; d and e are independently an integer of 1 to 3, preferably 1 to 2; wherein R is -L f -J, wherein X is O or S, L is a linker, preferably a straight-chain alkylene extension and / or oligoethylene glycol, optionally mixed with one or more groups selected from amine group, amide group and pendant oxygen group, f is an integer of 0 or 1, and J is a molecule that promotes endocytosis or is H .

當J為H時，該分子可用作合成子，以製備與促進胞吞作用之分子結合的分子。或者，該分子亦可用作藥物，無需與促進胞吞作用之分子進行任何結合。When J is H, the molecule can be used as a synthon to make molecules that bind to molecules that promote endocytosis. Alternatively, the molecule can also be used as a drug without any binding to molecules that promote endocytosis.

在第一態樣中，環具有根據式(I)之結構： -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) X為O或S。X在式(I)中每次出現-O-P(X)OH-O-時可在O及S之間變化。較佳地，X為S。 In a first aspect, the ring has the structure according to formula (I): -OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X)OH-O} r -KOP( X)OH-O-{[( CH2 ) 2 -O] h -P(X)OH-O-} s (I) X is O or S. X can vary between O and S for each occurrence of -OP(X)OH-O- in formula (I). Preferably, X is S.

g + h之和較佳為5至7，尤其為6。因此，若r為0，則h可為5至7 (其中s為1)；若g為1，則h可為4至6 (其中r及s為1)；若g為2，則h可為3至5 (其中r及s為1)；若g為3，則h可為2至4 (其中r及s為1)；若g為4，則h可為1至3 (其中r及s為1)；若g為5，則h可為1至2 (其中r為1且s為0或1)；或若g為6或7，則s為0 (其中r為1)。The sum of g+h is preferably 5 to 7, especially 6. Thus, if r is 0, h can be from 5 to 7 (where s is 1); if g is 1, then h can be from 4 to 6 (where r and s are 1); if g is 2, then h can be is 3 to 5 (where r and s are 1); if g is 3, then h may be 2 to 4 (where r and s are 1); if g is 4, then h may be 1 to 3 (where r and s is 1); if g is 5, then h can be 1 to 2 (where r is 1 and s is 0 or 1); or if g is 6 or 7, then s is 0 (where r is 1).

較佳地，i及j可為相同整數或可不同。i及j可選自整數1、2、3、4、5或6，較佳1、2或3，更特定言之1或2，尤其為1。Preferably, i and j may be the same integer or may be different. i and j may be selected from the integers 1, 2, 3, 4, 5 or 6, preferably 1, 2 or 3, more particularly 1 or 2, especially 1.

較佳地，k及l為相同整數。在一個態樣中，k及l為選自1、2或3之整數，較佳為1或2，更佳為2。Preferably, k and l are the same integer. In one aspect, k and 1 are integers selected from 1, 2 or 3, preferably 1 or 2, more preferably 2.

因此，K可為

Figure 02_image007
或
Figure 02_image008
。 Therefore, K can be
Figure 02_image007
or
Figure 02_image008
.

在一較佳態樣中，K為

Figure 02_image008
。 In a preferred form, K is
Figure 02_image008
.

在一個特定態樣中，環具有式(I) -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中X為S，r為1，g為6，s為0，且K為

Figure 02_image008
。 In a particular aspect, the ring has the formula (I)-OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X)OH-O} r -KOP(X)OH- O-{[(CH 2 ) 2 -O] h -P(X)OH-O-} s (I) where X is S, r is 1, g is 6, s is 0, and K is
Figure 02_image008
.

在一特定態樣中，f為1且L-J為-C(O)-(CH 2) m-NH-[C(O)] t-[(CH 2) 2-O] n-(CH 2) p-[C(O)] v-J或-C(O)-(CH 2) m-NH-[C(O)-CH 2-O] t-[(CH 2) 2-O] n-(CH 2) p-[C(O)] v-J，其中m為0至10之整數；n為0至15之整數；p為0至4之整數；t及v為整數0或1，其中t及v中之至少一者為1。 In a particular aspect, f is 1 and LJ is -C(O)-( CH2 ) m -NH-[C(O)] t -[( CH2 ) 2 -O] n- ( CH2 ) p -[C(O)] v -J or -C(O)-(CH 2 ) m -NH-[C(O)-CH 2 -O] t -[(CH 2 ) 2 -O] n - (CH 2 ) p -[C(O)] v -J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; p is an integer from 0 to 4; t and v are integers 0 or 1, wherein at least one of t and v is 1.

更特定言之，f為1且L-J選自由以下組成之群：-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-J、C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-C(O)-J及-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中m為0至10之整數；n為0至15之整數；且p為0至3之整數。 More specifically, f is 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C( O)-J, -C(O)-(CH 2 ) m -NH-C(O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, -C(O)-(CH 2 ) m -NH-C (O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J and -C(O)-(CH 2 ) m -NH-C(O)-CH 2 - O-[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3 Integer.

視情況，f為1且L-J選自由以下組成之群：-C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-13-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-13-CH 2-J、C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 3-13-CH 2-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-13-CH 2-C(O)-J及-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 3-13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 Optionally, f is 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) 5 -NH-[( CH2 ) 2 -O] 3-13 - CH2 -C(O)- J, -C(O)-(CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3-13 -CH 2 -J, C(O)-(CH 2 ) 5 - NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 3-13 -CH 2 -J, -C(O)-(CH 2 ) 5 -NH-C(O)-[ (CH 2 ) 2 -O] 3-13 -CH 2 -C(O)-J and -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 3-13 -CH 2 -C(O)-J or -C(O)-(CH 2 ) 5 -NH-C(O)-J.

舉例而言，f可為1且L-J選自由以下組成之群：-C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-(CH 2) 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-(CH 2) 3-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 5-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 9-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 For example, f can be 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) 5 -NH-[( CH2 ) 2 -O] 3- ( CH2 ) 2 -C( O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3 -(CH 2 ) 3 -J, -C(O)-( CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 5 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 - NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 9 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O ) -CH2 -O-[( CH2 ) 2 -O] 13 - CH2 -C(O)-J or -C(O)-( CH2 ) 5 -NH-C(O)-J.

在一極特定態樣中，f為1且L-J為-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中m為0至10、較佳4至6之整數，尤其為5；n為0至6之整數；且p為0至2之整數。在一特定態樣中，m為5，且n及p為0。在另一特定態樣中，m為5，n為3且p為2。 In a very specific aspect, f is 1 and LJ is -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O)- J, wherein m is an integer from 0 to 10, preferably 4 to 6, especially 5; n is an integer from 0 to 6; and p is an integer from 0 to 2. In a particular aspect, m is 5, and n and p are 0. In another specific aspect, m is 5, n is 3 and p is 2.

在本揭示內容之第二態樣中，環具有根據式(II)之結構： -O-P(X)OH-O-[(CH 2) d-C(O)-NH] b-CHR-[C(O)-NH-(CH 2) e] c-O-P(X)OH-O- (II) 其中X為O或S； b及c獨立地為0至4之整數，且b + c之和為3至7； d及e獨立地為1至3、較佳1至2之整數； 其中R為-(CH 2) 1-5-C(O)-NH-L f-J或-(CH 2) 1-5-NH-C(O)-L f-J，且 其中L為連接子，較佳為直鏈伸烷基或寡聚乙二醇，f為整數0或1，且J為促進胞吞作用之分子。 In a second aspect of the present disclosure, the ring has the structure according to formula (II): -OP(X)OH-O-[( CH2 ) d -C(O)-NH] b -CHR-[C (O)-NH-( CH2 ) e ] c -OP(X)OH-O- (II) wherein X is O or S; b and c are independently an integer from 0 to 4, and the sum of b + c is 3 to 7; d and e are independently an integer of 1 to 3, preferably 1 to 2; wherein R is -(CH 2 ) 1-5 -C(O)-NH-L f -J or -(CH 2 ) 1-5 -NH-C(O)-L f -J, and wherein L is a linker, preferably a straight-chain alkyl extension or oligoethylene glycol, f is an integer 0 or 1, and J is Molecules that promote endocytosis.

若b及/或c為2或更大，則d及e在[(CH 2) d-C(O)-NH]或-[C(O)-NH-(CH 2) e]每次出現時可不同。 If b and/or c are 2 or greater, then each occurrence of d and e in [( CH2 ) d -C(O)-NH] or -[C(O)-NH-( CH2 ) e ] can be different.

在一個態樣中，當d及e為2時，b + c之和為3至5，尤其為4。舉例而言，b可為0且c為3至5；b可為1且c為2至4；b可為2且c為1至3；或b可為3至5且c為0。In one aspect, when d and e are 2, the sum of b + c is 3 to 5, especially 4. For example, b can be 0 and c is 3-5; b can be 1 and c is 2-4; b can be 2 and c is 1-3; or b can be 3-5 and c is 0.

在一個態樣中，當d及e為1時，b + c之和為4至7，尤其為5或6。舉例而言，b可為0且c為3至6；b可為1且c為2至5；b可為2且c為1至4；或b可為3至6且c為0。In one aspect, when d and e are 1, the sum of b+c is 4 to 7, especially 5 or 6. For example, b can be 0 and c is 3-6; b can be 1 and c is 2-5; b can be 2 and c is 1-4; or b can be 3-6 and c is 0.

在一個態樣中，b、c、d及e經選擇以便環包含10至100個原子、較佳15至25個原子之鏈。In one aspect, b, c, d and e are selected such that the ring comprises a chain of 10 to 100 atoms, preferably 15 to 25 atoms.

在實例之非詳盡清單中，環可為以下中之一者： -O-P(X)OH-O-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-CHR-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-O-P(X)OH-O- -O-P(X)OH-O-(CH 2) 2-C(O)-NH-CHR-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-O-P(X)OH-O- -O-P(X)OH-O-CHR-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-O-P(X)OH-O- -O-P(X)OH-O-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-CHR-C(O)-NH-(CH 2) 2-O-P(X)OH-O- -O-P(X)OH-O-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-CHR-O-P(X)OH-O- -O-P(X)OH-O-(CH 2) 2-C(O)-NH-(CH 2)-C(O)-NH-CHR-C(O)-NH-(CH 2)-C(O)-NH-(CH 2) 2-O-P(X)OH-O- -O-P(X)OH-O-(CH 2)-C(O)-NH-(CH 2) 2-C(O)-NH-CHR-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2)-O-P(X)OH-O-或 -O-P(X)OH-O-(CH 2)-C(O)-NH-(CH 2)-C(O)-NH-CHR-C(O)-NH-(CH 2)-C(O)-NH-(CH 2)-O-P(X)OH-O- In a non-exhaustive list of examples, the ring can be one of the following: -OP(X)OH-O-( CH2 ) 2 -C(O)-NH-( CH2 ) 2 -C(O)- NH-CHR-C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -OP(X)OH-O- -OP(X)OH-O-(CH 2 ) 2 -C(O)-NH-CHR-C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -OP(X)OH-O- -OP(X)OH-O-CHR-C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -C(O) -NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -OP(X)OH-O- -OP(X)OH-O-(CH 2 ) 2 -C(O)- NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -C(O)-NH-CHR-C(O)-NH-(CH 2 ) 2 -OP(X)OH-O - -OP(X)OH-O-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -C(O)-NH-( CH 2 ) 2 -C(O)-NH-CHR-OP(X)OH-O- -OP(X)OH-O-(CH 2 ) 2 -C(O)-NH-(CH 2 )-C (O)-NH-CHR-C(O)-NH-(CH 2 )-C(O)-NH-(CH 2 ) 2 -OP(X)OH-O- -OP(X)OH-O- (CH 2 )-C(O)-NH-(CH 2 ) 2 -C(O)-NH-CHR-C(O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 )-OP(X)OH-O- or -OP(X)OH-O-(CH 2 )-C(O)-NH-(CH 2 )-C(O)-NH-CHR-C(O) -NH-(CH 2 )-C(O)-NH-(CH 2 )-OP(X)OH-O-

在一特定態樣中，環可為以下： -O-P(X)OH-O-(CH 2) 2-C(O)-NH-(CH 2) 2-C(O)-NH-CHR-C(O)-NH-(CH 2) 2-C(O)-NH-(CH 2) 2-O-P(X)OH-O- 其中R為-L f-J；且 其中L為連接子，較佳為直鏈伸烷基及/或寡聚乙二醇，其視情況雜有一個或若干個選自胺基、醯胺及側氧基之基團，且f為整數0或1。 In a particular aspect, the ring can be the following: -OP(X)OH-O-( CH2 ) 2 -C(O)-NH-( CH2 ) 2 -C(O)-NH-CHR-C (O)-NH-(CH 2 ) 2 -C(O)-NH-(CH 2 ) 2 -OP(X)OH-O- wherein R is -L f -J; and wherein L is a linker, more It is preferably a straight-chain alkyl group and/or oligoethylene glycol, which is optionally mixed with one or more groups selected from amine group, amide group and pendant oxygen group, and f is an integer of 0 or 1.

較佳地，X為S。Preferably, X is S.

L可為-(CH 2) 1-5-C(O)-J，較佳為-CH 2-C(O)-J或-(CH 2) 2-C(O)-J。 L can be -(CH 2 ) 1-5 -C(O)-J, preferably -CH 2 -C(O)-J or -(CH 2 ) 2 -C(O)-J.

或者，L-J可為-(CH2) 4-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中n為0至6之整數；且p為0至2之整數。在一特定態樣中，n為3且p為2。 促進胞吞作用之分子 Alternatively, LJ can be -(CH2) 4 -NH-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O)-J, where n is an integer from 0 to 6; and p is 0 an integer up to 2. In a particular aspect, n is 3 and p is 2. Molecules that promote endocytosis

本發明之核酸分子視情況與促進胞吞作用之分子(在上式中稱為J)結合。因此，在第一態樣中，J為促進胞吞作用之分子。在一替代態樣中，J為氫。The nucleic acid molecules of the present invention are optionally combined with a molecule that promotes endocytosis (referred to as J in the above formula). Thus, in the first aspect, J is a molecule that promotes endocytosis. In an alternative aspect, J is hydrogen.

促進胞吞作用之分子可為親脂性分子，諸如膽固醇、單鏈或雙鏈脂肪酸，或靶向細胞受體實現受體介導之胞吞作用的配體，諸如葉酸及葉酸衍生物或運鐵蛋白(Goldstein等人 Ann. Rev. Cell Biol. 1985 1:1-39；Leamon及Lowe, Proc Natl Acad Sci USA. 1991, 88: 5572-5576.)。脂肪酸可為飽和或不飽和的，且為C 4-C 28，較佳C 14-C 22，更佳C 18，諸如油酸或硬脂酸。特定言之，脂肪酸可為十八烷基或二油醯基。脂肪酸可發現以雙鏈形式與適當連接子(諸如甘油、磷脂醯膽鹼或乙醇胺及其類似物)連接，或藉由用於連接在結合核酸分子上之連接子連接在一起。如本文所用，術語「葉酸」意欲指葉酸及葉酸衍生物，包括喋酸衍生物及類似物。適用於本發明之葉酸類似物及衍生物包括但不限於抗葉酸劑、二氫葉酸、四氫葉酸、醛葉酸、喋醯聚麩胺酸、1-去氮、3-去氮、5-去氮、8-去氮、10-去氮、1,5-去氮、5,10-二去氮、8,10-二去氮及5,8二去氮葉酸、抗葉酸劑及喋酸衍生物。額外葉酸類似物描述於US2004/242582中。 Molecules that promote endocytosis can be lipophilic molecules, such as cholesterol, single- or double-chain fatty acids, or ligands that target cellular receptors for receptor-mediated endocytosis, such as folic acid and folic acid derivatives or transferrin Proteins (Goldstein et al. Ann. Rev. Cell Biol. 1985 1:1-39; Leamon and Lowe, Proc Natl Acad Sci USA. 1991, 88:5572-5576.). Fatty acids may be saturated or unsaturated, and are C4 - C28 , preferably C14 - C22 , more preferably C18 , such as oleic acid or stearic acid. In particular, the fatty acid can be octadecyl or dioleyl. Fatty acids can be found in double-stranded form attached to appropriate linkers such as glycerol, phosphatidylcholine or ethanolamine and the like, or linked together by linkers for attachment to binding nucleic acid molecules. As used herein, the term "folate" is intended to refer to folic acid and folic acid derivatives, including pteroic acid derivatives and analogs. Folic acid analogs and derivatives suitable for use in the present invention include, but are not limited to, antifolates, dihydrofolate, tetrahydrofolate, aldofolic acid, pteropolyglutamic acid, 1-deaza, 3-deaza, 5-deaza Nitrogen, 8-deaza, 10-deaza, 1,5-deaza, 5,10-dideza, 8,10-dideza and 5,8-didezafolate, antifolates and pteroic acid derivatives thing. Additional folic acid analogs are described in US2004/242582.

因此，促進胞吞作用之分子可選自由單鏈或雙鏈脂肪酸、葉酸及膽固醇組成之群。更佳地，促進胞吞作用之分子選自由二油醯基、十八烷基、葉酸及膽固醇組成之群。在一最佳實施例中，促進胞吞作用之分子為膽固醇。Thus, molecules that promote endocytosis can be selected from the group consisting of single or double chain fatty acids, folic acid and cholesterol. More preferably, the molecule that promotes endocytosis is selected from the group consisting of dioleyl, octadecyl, folic acid and cholesterol. In a preferred embodiment, the molecule that promotes endocytosis is cholesterol.

因此，在一個較佳實施例中，結合核酸分子具有下式：

Figure 02_image014
其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵；且其中加下劃線之核苷酸為2'修飾之核苷酸。在一個態樣中，2'修飾之核苷酸為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)且該分子稱為OX413。在另一態樣中，2'修飾之核苷酸為2'橋接核苷酸(LNA)。在另一態樣中，2'修飾之核苷酸為2'-去氧-2'-氟核苷酸。 Therefore, in a preferred embodiment, the binding nucleic acid molecule has the following formula:
Figure 02_image014
wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; and wherein the underlined nucleotide is a 2' modified nucleotide. In one aspect, the 2' modified nucleotide is 2'-deoxy-2'-fluoroarabinonucleotide (FANA) and the molecule is designated OX413. In another aspect, the 2' modified nucleotide is a 2' bridging nucleotide (LNA). In another aspect, the 2' modified nucleotide is a 2'-deoxy-2'-fluoronucleotide.

或者，促進胞吞作用之分子亦可為生育酚，糖諸如半乳糖及甘露糖及其寡醣，肽諸如RGD及鈴蟾素，及蛋白質諸如整合素。 核酸分子之治療用途 Alternatively, molecules that promote endocytosis may also be tocopherols, sugars such as galactose and mannose and their oligosaccharides, peptides such as RGD and bombesin, and proteins such as integrins. Therapeutic uses of nucleic acid molecules

根據本發明之結合核酸分子能夠活化PARP。其導致癌細胞中微核及細胞毒性之增加。其顯示出對癌細胞之特異性，由此可排除或限制副作用。另外，癌細胞中微核之特異性增加導致STING路徑之早期活化。The binding nucleic acid molecules according to the present invention are capable of activating PARP. It results in increased micronuclei and cytotoxicity in cancer cells. It shows specificity for cancer cells, whereby side effects can be excluded or limited. In addition, increased specificity of micronuclei in cancer cells leads to early activation of the STING pathway.

因此，根據本發明之結合核酸分子可用作藥物，尤其用於治療癌症。Thus, the binding nucleic acid molecules according to the present invention can be used as medicaments, especially for the treatment of cancer.

因此，本發明係關於根據本發明之結合核酸分子，其用作藥物。其進一步關於包含根據本發明之結合核酸分子的醫藥組合物，尤其用於治療癌症。Accordingly, the present invention relates to a binding nucleic acid molecule according to the present invention for use as a medicament. It further relates to pharmaceutical compositions comprising the binding nucleic acid molecules according to the invention, especially for the treatment of cancer.

除活性成分以外，本文考慮之醫藥組合物可包括醫藥學上可接受之載劑。術語「醫藥學上可接受之載劑」意謂涵蓋不干擾活性成分之生物活性之有效性且對其所投與之宿主無毒的任何載劑(例如支持物、物質、溶劑等)。舉例而言，對於非經腸投與，活性化合物可調配成用於在媒劑(諸如生理食鹽水、右旋糖溶液、血清白蛋白及林格氏溶液(Ringer's solution))中注射之單位劑型。In addition to the active ingredient, the pharmaceutical compositions contemplated herein may include pharmaceutically acceptable carriers. The term "pharmaceutically acceptable carrier" is meant to encompass any carrier (eg, supports, substances, solvents, etc.) that does not interfere with the effectiveness of the active ingredient's biological activity and is non-toxic to the host to which it is administered. For example, for parenteral administration, the active compound may be formulated in unit dosage form for injection in vehicles such as physiological saline, dextrose solution, serum albumin, and Ringer's solution .

醫藥組合物可調配為醫藥學相容性溶劑中之溶液或調配為適合之醫藥溶劑或媒劑中之乳液、懸浮液或分散液，或以此項技術中已知的方式調配為含有固體媒劑之丸劑、錠劑或膠囊。適用於經口投與之本發明調配物可呈離散單元形式，如膠囊、藥囊、錠劑或口含錠，各含有預定量之活性成分；呈粉末或顆粒形式；呈於水性液體或非水性液體中之溶液或懸浮液形式；或呈水包油乳液或油包水乳液形式。適於非經腸投與之調配物宜包含活性成分之無菌油性或水性製劑，其較佳與接受者之血液等張。每一此類調配物亦可含有其他醫藥學上相容且無毒性之輔助劑，諸如穩定劑、抗氧化劑、黏合劑、染料、乳化劑或調味物質。本發明之調配物因此包含活性成分以及醫藥學上可接受之載劑及視情況選用之其他治療成分。在與調配物之其他成分相容且對其接受者無害的意義上，載劑必須為「可接受的」。醫藥組合物有利地藉由注射或靜脈內輸注適合之無菌溶液或作為經消化道之口服劑量施加。安全且有效投與大部分此等化學治療劑之方法為熟習此項技術者已知的。另外，其投與描述於標準文獻中。Pharmaceutical compositions can be formulated as solutions in pharmaceutically compatible solvents or as emulsions, suspensions or dispersions in suitable pharmaceutical solvents or vehicles, or as a solid vehicle in a manner known in the art. pills, lozenges or capsules. Suitable for oral administration The formulations of the present invention may be in discrete unit form such as capsules, sachets, lozenges or lozenges, each containing a predetermined amount of the active ingredient; in powder or granule form; in aqueous liquid or non-aqueous form as a solution or suspension in an aqueous liquid; or as an oil-in-water emulsion or a water-in-oil emulsion. Formulations suitable for parenteral administration suitably contain sterile oily or aqueous preparations of the active ingredient, which are preferably isotonic with the blood of the recipient. Each such formulation may also contain other pharmaceutically compatible and non-toxic adjuvants such as stabilizers, antioxidants, binders, dyes, emulsifiers or flavoring substances. The formulations of the present invention thus comprise the active ingredient together with a pharmaceutically acceptable carrier and optionally other therapeutic ingredients. A carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to its recipient. The pharmaceutical compositions are advantageously administered by injection or intravenous infusion of a suitable sterile solution or as an oral dose via the digestive tract. Methods of safely and effectively administering most of these chemotherapeutic agents are known to those skilled in the art. Additionally, its administration is described in standard literature.

本發明中所述之醫藥組合物及產品、套組或組合製劑可用於治療個體之癌症。The pharmaceutical compositions and products, kits or combined formulations described in the present invention can be used to treat cancer in an individual.

術語「癌症」及「癌性」係指或描述哺乳動物中通常以不受調控之細胞生長為特徵的生理病況。癌症之實例包括但不限於實體腫瘤及血液癌，包括癌瘤、淋巴瘤、母細胞瘤(包括髓母細胞瘤及視網膜母細胞瘤)、肉瘤(包括脂肪肉瘤及滑膜細胞肉瘤)、神經內分泌腫瘤(包括類癌腫瘤、胃泌素瘤及胰島細胞癌)、間皮瘤、神經鞘瘤(包括聽神經瘤)、腦膜瘤、腺癌、黑素瘤及白血病或淋巴惡性疾病。此類癌症之更特定實例包括鱗狀細胞癌(例如上皮鱗狀細胞癌)；肺癌，包括小細胞肺癌、廣泛期小細胞肺癌(ES-SCLC)、非小細胞肺癌(NSCLC)、肺腺癌及肺鱗狀癌；腹膜癌；肝細胞癌；胃癌，包括胃腸癌；胰臟癌；神經膠質母細胞瘤；神經母細胞瘤；子宮頸癌；卵巢癌；肝癌；膀胱癌；泌尿道癌；肝癌；乳癌；大腸癌；直腸癌；結腸直腸癌；子宮內膜或子宮癌；唾液腺癌；腎癌；前列腺癌；外陰癌；甲狀腺癌；腎細胞癌(RCC)；肝癌；肛門癌；陰莖癌；睪丸癌；食道癌；膽道腫瘤以及頭頸癌。本文揭示額外癌症適應症。The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancers include, but are not limited to, solid tumors and blood cancers, including carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine Tumors (including carcinoid tumors, gastrinomas, and pancreatic islet cell carcinomas), mesothelioma, schwannomas (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, extensive stage small cell lung cancer (ES-SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma and squamous cell carcinoma of the lung; peritoneal carcinoma; hepatocellular carcinoma; gastric cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; neuroblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urinary tract cancer; Liver cancer; Breast cancer; Colorectal cancer; Rectal cancer; Colorectal cancer; Endometrial or uterine cancer; Salivary gland cancer; Kidney cancer; Prostate cancer; Vulvar cancer; Thyroid cancer; Renal cell carcinoma (RCC); ; testicular cancer; esophageal cancer; biliary tract tumors and head and neck cancer. Additional cancer indications are disclosed herein.

在一特定實施例中，「癌症」係指攜帶NAD +耗盡(例如選自ERCC1或ATM缺陷)之腫瘤細胞或攜帶IDH突變之癌細胞。 In a specific embodiment, "cancer" refers to tumor cells carrying NAD + depletion (eg, selected from ERCC1 or ATM deficiency) or cancer cells carrying IDH mutations.

在極特定實施例中，對於具有顯示NAD +合成缺陷之腫瘤的患者，尤其對於具有攜帶NAD +耗盡之腫瘤的患者，臨床分層或選擇較佳反應者為可能的。 In very specific embodiments, clinical stratification or selection of better responders is possible for patients with tumors that display defects in NAD + synthesis, especially for patients with tumors that carry NAD + depletion.

確定最佳劑量一般將涉及平衡治療益處水準與本發明之治療的任何風險或有害副作用。所選劑量水準將視多種因素而定，包括但不限於結合核酸分子之活性、投與途徑、投與時間、化合物之排泄率、治療持續時間、組合使用之其他藥物、化合物及/或材料，以及患者之年齡、性別、體重、狀況、一般健康狀況及先前病史。結合核酸分子之量及投與途徑將最終由醫師決定，但一般而言，劑量將在作用部位達到局部濃度，從而達到所需效果。Determining the optimal dose will generally involve balancing the level of therapeutic benefit with any risks or deleterious side effects of the treatments of the present invention. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the binding nucleic acid molecule, the route of administration, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds and/or materials used in combination, As well as the patient's age, gender, weight, condition, general health and prior medical history. The amount of binding nucleic acid molecule and the route of administration will ultimately be determined by the physician, but in general, the dosage will achieve a local concentration at the site of action to achieve the desired effect.

如本文所揭示之結合核酸分子的投與途徑可為經口、非經腸、靜脈內、瘤內、皮下、顱內、動脈內、局部、經直腸、經皮、皮內、經鼻、肌肉內、腹膜內、骨內及其類似途徑。在一較佳實施例中，結合核酸分子將在待治療之腫瘤部位附近投與或注射。Routes of administration of the binding nucleic acid molecules as disclosed herein can be oral, parenteral, intravenous, intratumoral, subcutaneous, intracranial, intraarterial, topical, rectal, transdermal, intradermal, nasal, intramuscular intraperitoneal, intraosseous and similar routes. In a preferred embodiment, the binding nucleic acid molecule will be administered or injected in the vicinity of the tumor site to be treated.

舉例而言，結合核酸分子之有效量為0.01至1000 mg，例如較佳0.1至100 mg。當然，熟習此項技術者可考慮化學療法及/或放射療法方案來調適劑量及方案。For example, the effective amount of binding nucleic acid molecule is 0.01 to 1000 mg, such as preferably 0.1 to 100 mg. Of course, those skilled in the art can consider chemotherapy and/or radiation therapy regimens to adjust doses and regimens.

根據本發明之結合核酸分子可與額外治療劑組合使用。額外治療劑可為例如免疫調節劑，諸如免疫檢查點抑制劑；基於T細胞之癌症免疫療法，包括過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)；習知化學治療劑、放射治療劑或抗血管生成劑；或靶向免疫毒素。 與免疫調節劑/免疫檢查點抑制劑(ICI)之組合 Binding nucleic acid molecules according to the present invention can be used in combination with additional therapeutic agents. Additional therapeutic agents can be, for example, immunomodulatory agents, such as immune checkpoint inhibitors; T cell-based cancer immunotherapy, including adoptive cell transfer (ACT), genetically modified T cells, or engineered T cells, such as chimeric Antigen receptor cells (CAR-T cells); conventional chemotherapeutic, radiotherapeutic, or anti-angiogenic agents; or targeted immunotoxins. Combination with immunomodulators/immune checkpoint inhibitors (ICIs)

本發明人證明結合核酸分子與免疫調節劑諸如免疫檢查點抑制劑(ICI)，較佳PD-1/PD-L1路徑之抑制劑之組合具有高抗腫瘤治療潛力，如由STING路徑之活化及PD-L1表現之增加所表明。因此，本發明提供組合療法，其中本發明之結合核酸分子與免疫調節劑諸如免疫檢查點抑制劑(ICI)一起、在其之前或之後投與患者。The present inventors demonstrate that the combination of binding nucleic acid molecules with immunomodulatory agents such as immune checkpoint inhibitors (ICIs), preferably inhibitors of the PD-1/PD-L1 pathway, has high antitumor therapeutic potential, such as by activation of the STING pathway and As indicated by an increase in PD-L1 expression. Accordingly, the present invention provides a combination therapy wherein a binding nucleic acid molecule of the present invention is administered to a patient together with, before or after an immunomodulatory agent such as an immune checkpoint inhibitor (ICI).

因此，本發明係關於一種醫藥組合物，其包含本發明之結合核酸分子及免疫調節劑，更特定言之，用於治療癌症。本發明亦關於一種包含本發明之結合核酸分子及免疫調節劑之產品，作為組合製劑同時、單獨或依序使用，更特定言之，用於治療癌症。在一較佳實施例中，免疫調節劑為PD-1/PD-L1路徑之抑制劑。Accordingly, the present invention relates to a pharmaceutical composition comprising the binding nucleic acid molecule of the present invention and an immunomodulatory agent, more particularly, for the treatment of cancer. The present invention also relates to a product comprising the binding nucleic acid molecule of the present invention and an immunomodulatory agent for simultaneous, separate or sequential use as a combined preparation, more particularly, for the treatment of cancer. In a preferred embodiment, the immunomodulatory agent is an inhibitor of the PD-1/PD-L1 pathway.

本發明亦提供一種治療癌症之方法，其係藉由向有需要之患者投與本發明之結合核酸分子與一或多種免疫調節劑(例如，共刺激分子之活化劑或免疫檢查點分子之抑制劑中之一或多者)之組合。在一較佳實施例中，免疫調節劑為PD-1/PD-L1路徑之抑制劑。 共刺激分子之活化劑： The present invention also provides a method of treating cancer by administering to a patient in need thereof a binding nucleic acid molecule of the present invention and one or more immunomodulatory agents (eg, an activator of a costimulatory molecule or an inhibitor of an immune checkpoint molecule) one or more of the agents) in combination. In a preferred embodiment, the immunomodulatory agent is an inhibitor of the PD-1/PD-L1 pathway. Activators of costimulatory molecules:

在某些實施例中，免疫調節劑為共刺激分子之活化劑。在一個實施例中，共刺激分子之促效劑係選自以下之促效劑(例如促效抗體或其抗原結合片段，或可溶性融合物)：OX40、CD2、CD27、CDS、ICAM-1、LFA-1 (CD11a/CD18)、ICOS (CD278)、4-1 BB (CD137)、GITR、CD30、CD40、BAFFR、HVEM、CD7、LIGHT、NKG2C、SLAMF7、NKp80、CD160、B7-H3或CD83配體。 免疫檢查點分子之抑制劑： In certain embodiments, the immunomodulatory agent is an activator of a costimulatory molecule. In one embodiment, the agonist of the costimulatory molecule is selected from the following agonists (eg, agonist antibodies or antigen-binding fragments thereof, or soluble fusions): OX40, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 body. Inhibitors of immune checkpoint molecules:

在某些實施例中，免疫調節劑為免疫檢查點分子之抑制劑。在一個實施例中，免疫調節劑為PD-1、PD-L1、PD-L2、CTLA-4、TIM-3、LAG-3、NKG2D、NKG2L、KIR、VISTA、BTLA、TIGIT、LAIR1、CD160、2B4及/或TGFRβ之抑制劑。在一個實施例中，免疫檢查點分子之抑制劑抑制PD-1、PD-L1、LAG-3、TIM-3、TIGIT或CTLA-4，或其任何組合。術語「抑制」或「抑制劑」包括給定分子(例如免疫檢查點抑制劑)之某一參數(例如活性)降低。舉例而言，此術語包括對活性(例如PD-1或PD-L1活性)之至少5%、10%、20%、30%、40%、50%或更大的抑制。因此，抑制不必為100%。In certain embodiments, the immunomodulatory agent is an inhibitor of an immune checkpoint molecule. In one embodiment, the immunomodulatory agent is PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, NKG2D, NKG2L, KIR, VISTA, BTLA, TIGIT, LAIR1, CD160, Inhibitors of 2B4 and/or TGFRβ. In one embodiment, the inhibitor of the immune checkpoint molecule inhibits PD-1, PD-L1, LAG-3, TIM-3, TIGIT or CTLA-4, or any combination thereof. The terms "inhibit" or "inhibitor" include a decrease in a certain parameter (eg, activity) of a given molecule (eg, an immune checkpoint inhibitor). For example, this term includes at least 5%, 10%, 20%, 30%, 40%, 50% or greater inhibition of activity (eg, PD-1 or PD-L1 activity). Therefore, the suppression need not be 100%.

抑制性分子之抑制可以DNA、RNA或蛋白質水準進行。在一些實施例中，抑制性核酸(例如dsRNA、siRNA或shRNA)可用於抑制抑制性分子之表現。在其他實施例中，抑制性信號之抑制劑為與抑制性分子結合之多肽，例如可溶性配體(例如PD-1 Ig或CTLA-4 Ig)或抗體或其抗原結合片段；例如與PD-1、PD-L1、PD-L2、CTLA-4、TIM-3、LAG-3、NKG2D、NKG2L、KIR VISTA、BTLA、TIGIT、LAIR1、CD160、2B4及/或TGFRβ或其組合結合之抗體或其片段(在本文中亦稱為「抗體分子」)。Inhibition by inhibitory molecules can be performed at the DNA, RNA or protein level. In some embodiments, inhibitory nucleic acids (eg, dsRNA, siRNA, or shRNA) can be used to inhibit the expression of inhibitory molecules. In other embodiments, the inhibitor of the inhibitory signal is a polypeptide that binds to an inhibitory molecule, such as a soluble ligand (eg, PD-1 Ig or CTLA-4 Ig) or an antibody or antigen-binding fragment thereof; , PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, NKG2D, NKG2L, KIR VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and/or TGFRβ or combinations thereof, antibodies or fragments thereof (Also referred to herein as an "antibody molecule").

在一個實施例中，抗體分子為完整抗體或其片段(例如Fab、F(ab')2、Fv或單鏈Fv片段(scFv))。在其他實施例中，抗體分子具有重鏈恆定區(Fc)，其選自例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD及IgE之重鏈恆定區；特定言之，選自例如IgG1、IgG2、IgG3及IgG4之重鏈恆定區，更特定言之，IgG1或IgG4 (例如人類IgG1或IgG4)之重鏈恆定區。在一個實施例中，重鏈恆定區為人類IgG1或人類IgG4。在一個實施例中，恆定區經改變，例如經突變以調節抗體分子之特性(例如增加或減少Fc受體結合、抗體糖基化、半胱胺酸殘基數、效應細胞功能或補體功能中之一或多者)。在某些實施例中，抗體分子呈雙特異性或多特異性抗體分子形式。 PD-1抑制劑 In one embodiment, the antibody molecule is an intact antibody or a fragment thereof (eg, Fab, F(ab')2, Fv, or single-chain Fv fragment (scFv)). In other embodiments, the antibody molecule has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgGl, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; in particular, selected from Examples are heavy chain constant regions of IgGl, IgG2, IgG3, and IgG4, more specifically, heavy chain constant regions of IgGl or IgG4 (eg, human IgGl or IgG4). In one embodiment, the heavy chain constant region is human IgGl or human IgG4. In one embodiment, the constant region is altered, eg, mutated, to modulate the properties of the antibody molecule (eg, increasing or decreasing Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function, or complement function) one or more). In certain embodiments, the antibody molecule is in the form of a bispecific or multispecific antibody molecule. PD-1 inhibitors

在一些實施例中，本發明之結合核酸分子與PD-1抑制劑組合投與。在一些實施例中，PD-1抑制劑係選自PDR001 (Novartis)、納武單抗(Nivolumab) (Bristol-Myers Squibb)、派姆單抗(Pembrolizumab) (Merck & Co)、皮立珠單抗(Pidilizumab) (CureTech)、MEDI0680 (Medimmune)、REGN2810 (Regeneron)、TSR-042 (Tesaro)、PF-06801591 (Pfizer)、BGB-A317 (Beigene)、BGB-108 (Beigene)、INCSHR1210 (Incyte)、AMP-224 (Amplimmune)、IBI308 (Innovent及Eli Lilly)、JS001、JTX-4014 (Jounce Therapeutics)、PDR001 (Novartis)或MGA012 (Incyte及MacroGenics)。 例示性PD-1抑制劑 In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is selected from the group consisting of PDR001 (Novartis), Nivolumab (Bristol-Myers Squibb), Pembrolizumab (Merck & Co), pilizumab Anti-(Pidilizumab) (CureTech), MEDI0680 (Medimmune), REGN2810 (Regeneron), TSR-042 (Tesaro), PF-06801591 (Pfizer), BGB-A317 (Beigene), BGB-108 (Beigene), INCSHR1210 (Incyte) , AMP-224 (Amplimmune), IBI308 (Innovent and Eli Lilly), JS001, JTX-4014 (Jounce Therapeutics), PDR001 (Novartis) or MGA012 (Incyte and MacroGenics). Exemplary PD-1 Inhibitors

在一些實施例中，抗PD-1抗體為納武單抗(CAS登記號：946414-94-4)。納武單抗之替代名稱包括MDX-1106、MDX-1106-04、ONO-4538、BMS-936558或OPDIVO®。納武單抗為完全人類lgG4單株抗體，其特異性阻斷PD1。納武單抗(純系5C4)及其他與PD1特異性結合之人類單株抗體揭示於美國專利第8,008,449號及PCT公開案第WO 2006/121168號，其以全文引用之方式併入本文中。In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4). Alternative names for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, BMS-936558 or OPDIVO®. Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (pure line 5C4) and other human monoclonal antibodies that specifically bind PDl are disclosed in US Patent No. 8,008,449 and PCT Publication No. WO 2006/121168, which are incorporated herein by reference in their entirety.

在其他實施例中，抗PD-1抗體為派姆單抗。派姆單抗(商品名KEYTRUDA，以前稱為蘭利珠單抗(Lambrolizumab)，亦稱為Merck 3745、MK-3475或SCH-900475)為與PD1結合之人類化lgG4單株抗體。派姆單抗揭示於例如Hamid, O.等人 (2013) New England Journal of Medicine 369 (2): 134-44、PCT公開案第WO 2009/114335號及美國專利案第8,354,509號中，其以全文引用之方式併入本文中。In other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (trade name KEYTRUDA, formerly known as Lambrolizumab, also known as Merck 3745, MK-3475 or SCH-900475) is a humanized IgG4 monoclonal antibody that binds to PD1. Pembrolizumab is disclosed, for example, in Hamid, O. et al. (2013) New England Journal of Medicine 369(2): 134-44, PCT Publication No. WO 2009/114335, and U.S. Patent No. 8,354,509, which are described in Incorporated herein by reference in its entirety.

在一些實施例中，抗PD-1抗體為皮立珠單抗。皮立珠單抗(CT-011；CureTech)為與PD1結合之人類化lgG1 k單株抗體。皮立珠單抗及其他人類化抗PD-1單株抗體揭示於PCT公開案第WO 2009/101611號中，其以全文引用之方式併入本文中。In some embodiments, the anti-PD-1 antibody is pilizumab. Pilibizumab (CT-011; CureTech) is a humanized IgG1 k monoclonal antibody that binds to PD1. Pirizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in PCT Publication No. WO 2009/101611, which is incorporated herein by reference in its entirety.

其他抗PD1抗體揭示於美國專利案第8,609,089號、美國公開案第2010028330號及/或美國公開案第20120114649號中，其以全文引用之方式併入本文中。其他抗PD1抗體包括AMP514 (Amplimmune)。Additional anti-PDl antibodies are disclosed in US Patent No. 8,609,089, US Publication No. 2010028330, and/or US Publication No. 20120114649, which are incorporated herein by reference in their entirety. Other anti-PD1 antibodies include AMP514 (Amplimmune).

在一個實施例中，抗PD-1抗體分子為MEDI0680 (Medimmune)，亦稱為AMP-514。MEDI0680及其他抗PD-1抗體揭示於US 9,205,148及WO 2012/145493中，其以全文引用之方式併入本文中。In one embodiment, the anti-PD-1 antibody molecule is MEDI0680 (Medimmune), also known as AMP-514. MEDI0680 and other anti-PD-1 antibodies are disclosed in US 9,205,148 and WO 2012/145493, which are incorporated herein by reference in their entirety.

在一個實施例中，抗PD-1抗體分子為REGN2810 (Regeneron)，亦稱為西米普利單抗(Cemiplimab)。In one embodiment, the anti-PD-1 antibody molecule is REGN2810 (Regeneron), also known as Cemiplimab.

在一個實施例中，抗PD-1抗體分子為PF-06801591 (Pfizer)。In one embodiment, the anti-PD-1 antibody molecule is PF-06801591 (Pfizer).

在一個實施例中，抗PD-1抗體分子為BGB-A317 (Beigene)，亦稱為BGB-108或替雷利珠單抗(Tislelizumab)。In one embodiment, the anti-PD-1 antibody molecule is BGB-A317 (Beigene), also known as BGB-108 or Tislelizumab.

在一個實施例中，抗PD-1抗體分子為INCSHR1210 (Incyte)，亦稱為INCSHR01210或SHR-1210或卡瑞利珠單抗(Camrelizumab)。In one embodiment, the anti-PD-1 antibody molecule is INCSHR1210 (Incyte), also known as INCSHR01210 or SHR-1210 or Camrelizumab.

在一個實施例中，抗PD-1抗體分子為TSR-042 (Tesaro)，亦稱為ANB011或多塔利單抗(Dostarlimab)。In one embodiment, the anti-PD-1 antibody molecule is TSR-042 (Tesaro), also known as ANB011 or Dostarlimab.

在一個實施例中，抗PD-1抗體分子為IBI308 (Innovent及Eli Lilly)，亦稱為信迪利單抗(Sintilimab)。In one embodiment, the anti-PD-1 antibody molecule is IBI308 (Innovent and Eli Lilly), also known as Sintilimab.

在一個實施例中，抗PD-1人類化IgG4單株抗體分子為JS 001，亦稱為特瑞普利單抗(Toripalimab)。In one embodiment, the anti-PD-1 humanized IgG4 monoclonal antibody molecule is JS 001, also known as Toripalimab.

在一個實施例中，抗PD-1抗體分子為JTX-4014 (Jounce Therapeutics)。In one embodiment, the anti-PD-1 antibody molecule is JTX-4014 (Jounce Therapeutics).

在一個實施例中，抗PD-1單株抗體分子為PDR001 (Novartis)，亦稱為斯帕塔利單抗(Spartalizumab)。In one embodiment, the anti-PD-1 monoclonal antibody molecule is PDR001 (Novartis), also known as Spartalizumab.

在一個實施例中，抗PD-1人類化IgG4單株抗體分子MGA012 (Incyte及MacroGenics)亦稱為INCMGA00012或瑞弗利單抗(Retifanlimab)。In one embodiment, the anti-PD-1 humanized IgG4 monoclonal antibody molecule MGA012 (Incyte and MacroGenics) is also known as INCMGA00012 or Retifanlimab.

其他已知抗PD-1抗體包括例如WO 2015/1 12800、WO 2016/092419、WO 2015/085847、WO 2014/179664、WO 2014/194302、WO 2014/209804、WO 2015/2001 19、US 8,735,553、US 7,488,802、US 8,927,697、US 8,993,731及US 9, 102,727中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。Other known anti-PD-1 antibodies include, for example, WO 2015/1 12800, WO 2016/092419, WO 2015/085847, WO 2014/179664, WO 2014/194302, WO 2014/209804, WO 2015/2001 19, US 8,735,553, Such antibodies are described in US 7,488,802, US 8,927,697, US 8,993,731 and US 9,102,727, which are incorporated herein by reference in their entirety.

在一個實施例中，抗PD-1抗體為與本文所述之抗PD-1抗體中之一者競爭結合及/或結合於PD-1上之相同抗原決定基的抗體。In one embodiment, the anti-PD-1 antibody is an antibody that competes for binding to and/or binds to the same epitope on PD-1 as one of the anti-PD-1 antibodies described herein.

在一個實施例中，PD-1抑制劑為抑制PD-1信號傳遞路徑之肽，如US 8,907,053中所述，其以全文引用之方式併入本文中。在一些實施例中，PD-1抑制劑為免疫黏附素{例如包含與恆定區(例如免疫球蛋白序列之Fc區)融合之PD-L1或PD-L2之胞外或PD-1結合部分的免疫黏附素}。在一些實施例中，PD-1抑制劑為AMP-224 (B7-DCIg (Amplimmune)，例如揭示於WO 2010/027827及WO 2011/066342中，其以全文引用之方式併入本文中。 PD-L1抑制劑 In one embodiment, the PD-1 inhibitor is a peptide that inhibits the PD-1 signaling pathway, as described in US 8,907,053, which is incorporated herein by reference in its entirety. In some embodiments, the PD-1 inhibitor is an immunoadhesin {eg, one comprising the extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (eg, the Fc region of an immunoglobulin sequence) Immunoadhesin}. In some embodiments, the PD-1 inhibitor is AMP-224 (B7-DCIg (Amplimmune), eg, disclosed in WO 2010/027827 and WO 2011/066342, which are incorporated herein by reference in their entirety. PD-L1 inhibitors

在某些實施例中，免疫檢查點分子之抑制劑為PD-L1之抑制劑。在一些實施例中，本發明之結合核酸分子與PD-L1抑制劑組合投與。在一些實施例中，PD-L1抑制劑係選自FAZ053 (Novartis)、阿特珠單抗(Atezolizumab) (Genentech/Roche)、阿維魯單抗(Avelumab) (Merck Serono及Pfizer)、度伐利尤單抗(Durvalumab) (Medlmmune/AstraZeneca)或BMS-936559 (Bristol-Myers Squibb)。 例示性PD-L1抑制劑 In certain embodiments, the inhibitor of the immune checkpoint molecule is an inhibitor of PD-L1. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is selected from the group consisting of FAZ053 (Novartis), Atezolizumab (Genentech/Roche), Avelumab (Merck Serono and Pfizer), durval Durvalumab (Medlmmune/AstraZeneca) or BMS-936559 (Bristol-Myers Squibb). Exemplary PD-L1 Inhibitors

在一個實施例中，PD-L1抑制劑為抗PD-L1抗體分子。在一個實施例中，抗PD-L1抗體分子為阿維魯單抗(Merck Serono及Pfizer)，亦稱為MSB0010718C。阿維魯單抗及其他抗PD-L1抗體揭示於WO 2013/079174中，其以全文引用之方式併入本文中。In one embodiment, the PD-L1 inhibitor is an anti-PD-L1 antibody molecule. In one embodiment, the anti-PD-L1 antibody molecule is avelumab (Merck Serono and Pfizer), also known as MSB0010718C. Avelumab and other anti-PD-L1 antibodies are disclosed in WO 2013/079174, which is incorporated herein by reference in its entirety.

在一個實施例中，抗PD-L1抗體分子為度伐利尤單抗(Medlmmune/AstraZeneca)，亦稱為MEDI4736。度伐利尤單抗及其他抗PD-L1抗體揭示於US 8,779,108中，其以全文引用之方式併入本文中。In one embodiment, the anti-PD-L1 antibody molecule is durvalumab (Medlmmune/AstraZeneca), also known as MEDI4736. Durvalumab and other anti-PD-L1 antibodies are disclosed in US 8,779,108, which is incorporated herein by reference in its entirety.

在一個實施例中，抗PD-L1抗體分子為BMS-936559 (Bristol-Myers Squibb)，亦稱為MDX-1105或12A4。BMS-936559及其他抗PD-L1抗體揭示於US 7,943,743及WO 2015/081 158中，其以全文引用之方式併入本文中。In one embodiment, the anti-PD-L1 antibody molecule is BMS-936559 (Bristol-Myers Squibb), also known as MDX-1105 or 12A4. BMS-936559 and other anti-PD-L1 antibodies are disclosed in US 7,943,743 and WO 2015/081 158, which are incorporated herein by reference in their entirety.

其他已知抗PD-L1抗體包括例如WO 2015/181342、WO 2014/100079、WO 2016/000619、WO 2014/022758、WO 2014/055897、WO 2015/061668、WO 2013/079174、WO 2012/145493、WO 2015/112805、WO 2015/109124、WO 2015/195163、US 8,168,179、US 8,552,154、US 8,460,927及US 9,175,082中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。Other known anti-PD-L1 antibodies include, for example, WO 2015/181342, WO 2014/100079, WO 2016/000619, WO 2014/022758, WO 2014/055897, WO 2015/061668, WO 2013/079174, WO 2012/145493, Such antibodies are described in WO 2015/112805, WO 2015/109124, WO 2015/195163, US 8,168,179, US 8,552,154, US 8,460,927 and US 9,175,082, which are incorporated herein by reference in their entirety.

在一個實施例中，抗PD-L1抗體為與本文所述之抗PD-L1抗體中之一者競爭結合及/或結合於PD-L1上之相同抗原決定基的抗體。 CTLA-4 (細胞毒性T淋巴球相關蛋白4)抑制劑 In one embodiment, the anti-PD-L1 antibody is an antibody that competes for binding to and/or binds to the same epitope on PD-L1 as one of the anti-PD-L1 antibodies described herein. CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitor

在某些實施例中，免疫檢查點分子之抑制劑為CLTA-4之抑制劑。在一些實施例中，本發明之結合核酸分子與CLTA-4抑制劑組合投與。在一些實施例中，CLTA-4抑制劑為伊匹單抗(Ipilimumab)。 例示性CTLA-4抑制劑 In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of CLTA-4. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a CLTA-4 inhibitor. In some embodiments, the CLTA-4 inhibitor is Ipilimumab. Exemplary CTLA-4 Inhibitors

在一個實施例中，CLTA-4抑制劑為抗CLTA-4抗體分子。在一個實施例中，抗CLTA-4抗體分子為伊匹單抗(Bristol-Myers Squibb)，亦稱為MDX-010。 LAG-3抑制劑 In one embodiment, the CLTA-4 inhibitor is an anti-CLTA-4 antibody molecule. In one embodiment, the anti-CLTA-4 antibody molecule is ipilimumab (Bristol-Myers Squibb), also known as MDX-010. LAG-3 inhibitors

在某些實施例中，免疫檢查點分子之抑制劑為LAG-3之抑制劑。在一些實施例中，本發明之結合核酸分子與LAG-3抑制劑組合投與。在一些實施例中，LAG-3抑制劑係選自LAG525 (Novartis)、BMS-986016 (Bristol-Myers Squibb)、TSR-033 (Tesaro)、MK-4280 (Merck)、REGN3767 (Regeneron)、BI-754111 (Boehringer Ingelheim)、SYM-022 (Symphogen)、FS118 (F-star)或MGD013 (MacroGenics)。 例示性LAG-3抑制劑 In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of LAG-3. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a LAG-3 inhibitor. In some embodiments, the LAG-3 inhibitor is selected from LAG525 (Novartis), BMS-986016 (Bristol-Myers Squibb), TSR-033 (Tesaro), MK-4280 (Merck), REGN3767 (Regeneron), BI- 754111 (Boehringer Ingelheim), SYM-022 (Symphogen), FS118 (F-star) or MGD013 (MacroGenics). Exemplary LAG-3 Inhibitors

在一個實施例中，LAG-3抑制劑為抗LAG-3抗體分子。在一個實施例中，LAG-3抑制劑為BMS-986016 (Bristol-Myers Squibb)，亦稱為BMS986016或瑞拉單抗(Relatlimab)。BMS-986016及其他抗LAG-3抗體揭示於WO 2015/116539及US 9,505,839中，其以全文引用之方式併入本文中。In one embodiment, the LAG-3 inhibitor is an anti-LAG-3 antibody molecule. In one embodiment, the LAG-3 inhibitor is BMS-986016 (Bristol-Myers Squibb), also known as BMS986016 or Relatlimab. BMS-986016 and other anti-LAG-3 antibodies are disclosed in WO 2015/116539 and US 9,505,839, which are incorporated herein by reference in their entirety.

在一個實施例中，抗LAG-3抗體分子為TSR-033 (Tesaro)。In one embodiment, the anti-LAG-3 antibody molecule is TSR-033 (Tesaro).

在一個實施例中，抗LAG-3抗體分子為IMP731或GSK2831781 (GSK及Prima BioMed)。IMP731及其他抗LAG-3抗體揭示於WO2008/132601及US 9,244,059中，其以全文引用之方式併入本文中。In one embodiment, the anti-LAG-3 antibody molecule is IMP731 or GSK2831781 (GSK and Prima BioMed). IMP731 and other anti-LAG-3 antibodies are disclosed in WO2008/132601 and US 9,244,059, which are incorporated herein by reference in their entirety.

在一個實施例中，抗LAG-3抗體分子為LAG525 (Novartis)，亦稱為埃拉利單抗(Ieramilimab)。In one embodiment, the anti-LAG-3 antibody molecule is LAG525 (Novartis), also known as Ieramilimab.

在一個實施例中，抗LAG-3抗體分子為MK-4280 (Merck)，亦稱為瑪維澤利單抗(Mavezelimab)。In one embodiment, the anti-LAG-3 antibody molecule is MK-4280 (Merck), also known as Mavezelimab.

在一個實施例中，抗LAG-3抗體分子為REGN3767 (Regeneron)，亦稱為弗安利單抗(Fianlimab)。In one embodiment, the anti-LAG-3 antibody molecule is REGN3767 (Regeneron), also known as Fianlimab.

在一個實施例中，抗LAG-3抗體分子為BI-754111 (Boehringer Ingelheim)，亦稱為邁納利單抗(Miptenalimab)。In one embodiment, the anti-LAG-3 antibody molecule is BI-754111 (Boehringer Ingelheim), also known as Mipenalimab.

在一個實施例中，抗LAG-3抗體分子為SYM-022 (Symphogen)。In one embodiment, the anti-LAG-3 antibody molecule is SYM-022 (Symphogen).

在一個實施例中，抗LAG-3抗體分子為FS118 (F-star)。In one embodiment, the anti-LAG-3 antibody molecule is FS118 (F-star).

在一個實施例中，抗LAG-3抗體分子為MGD013 (MacroGenics)，亦稱為特泊利單抗(Tebotelimab)。In one embodiment, the anti-LAG-3 antibody molecule is MGD013 (MacroGenics), also known as Tebotelimab.

其他已知抗LAG-3抗體包括例如WO 2008/132601、WO 2010/019570、WO 2014/140180、WO 2015/116539、WO 2015/200119、WO 2016/028672、US 9,244,059、US 9,505,839中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。 TIM-3抑制劑 Other known anti-LAG-3 antibodies include, for example, those described in WO 2008/132601, WO 2010/019570, WO 2014/140180, WO 2015/116539, WO 2015/200119, WO 2016/028672, US 9,244,059, US 9,505,839 and other antibodies, which are incorporated herein by reference in their entirety. TIM-3 inhibitors

在某些實施例中，免疫檢查點分子之抑制劑為TIM-3之抑制劑。在一些實施例中，本發明之結合核酸分子與TIM-3抑制劑組合投與。在一些實施例中，TIM-3抑制劑為MGB453 (Novartis)、TSR-022 (Tesaro)、BMS-986258 (Bristol-Myers Squibb)、SHR-1702、RO7121661 (La Roche)、MBG453 (Novartis)、Sym023 (Symphogen)、INCAGN2390 (Agenus)或LY3321367 (Eli Lilly)。 例示性TIM-3抑制劑 In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIM-3. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a TIM-3 inhibitor. In some embodiments, the TIM-3 inhibitor is MGB453 (Novartis), TSR-022 (Tesaro), BMS-986258 (Bristol-Myers Squibb), SHR-1702, RO7121661 (La Roche), MBG453 (Novartis), Sym023 (Symphogen), INCAGN2390 (Agenus) or LY3321367 (Eli Lilly). Exemplary TIM-3 Inhibitors

在一個實施例中，抗TIM-3抗體分子為TSR-022 (AnaptysBio/Tesaro)。In one embodiment, the anti-TIM-3 antibody molecule is TSR-022 (AnaptysBio/Tesaro).

在一個實施例中，抗TIM-3抗體為APE5137或APE5121。APE5137、APE512及其他抗TIM-3抗體揭示於WO 2016/161270中，其以全文引用之方式併入本文中。In one embodiment, the anti-TIM-3 antibody is APE5137 or APE5121. APE5137, APE512 and other anti-TIM-3 antibodies are disclosed in WO 2016/161270, which is incorporated herein by reference in its entirety.

在一個實施例中，抗TIM-3抗體分子為BMS-986258 (Bristol-Myers Squibb)，亦稱為ONO 7807。In one embodiment, the anti-TIM-3 antibody molecule is BMS-986258 (Bristol-Myers Squibb), also known as ONO 7807.

在一個實施例中，抗TIM-3抗體分子為SHR-1702。In one embodiment, the anti-TIM-3 antibody molecule is SHR-1702.

在一個實施例中，抗TIM-3抗體分子為RO7121661 (La Roche)。In one embodiment, the anti-TIM-3 antibody molecule is RO7121661 (La Roche).

在一個實施例中，抗TIM-3抗體分子為MBG453 (Novartis)，亦稱為薩巴托利單抗(Sabatolimab)。In one embodiment, the anti-TIM-3 antibody molecule is MBG453 (Novartis), also known as Sabatolimab.

在一個實施例中，抗TIM-3抗體分子為Sym023 (Symphogen)。In one embodiment, the anti-TIM-3 antibody molecule is Sym023 (Symphogen).

在一個實施例中，抗TIM-3抗體分子為INCAGN2390 (Agenus)。In one embodiment, the anti-TIM-3 antibody molecule is INCAGN2390 (Agenus).

在一個實施例中，抗TIM-3抗體分子為LY3321367 (Eli Lilly)。In one embodiment, the anti-TIM-3 antibody molecule is LY3321367 (Eli Lilly).

其他已知抗TIM-3抗體包括例如WO 2016/1 1 1947、WO 2016/071448、WO 2016/144803、US 8,552,156、US 8,841,418及US 9,163,087中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。 NKG2D抑制劑 Other known anti-TIM-3 antibodies include, for example, those described in WO 2016/1 1 1947, WO 2016/071448, WO 2016/144803, US 8,552,156, US 8,841,418 and US 9,163,087, which are incorporated by reference in their entirety manner is incorporated herein. NKG2D inhibitors

在某些實施例中，NKG2D/NKG2DL路徑之抑制劑為NKG2D之抑制劑。在一些實施例中，本發明之結合核酸分子與NKG2D抑制劑組合投與。在一些實施例中，NKG2D抑制劑為抗NKG2D抗體分子，諸如抗NKG2D抗體NNC0142-0002 (亦稱為NN 8555、IPH2301或JNJ-4500)。 例示性NKG2D抑制劑 In certain embodiments, the inhibitor of the NKG2D/NKG2DL pathway is an inhibitor of NKG2D. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with an NKG2D inhibitor. In some embodiments, the NKG2D inhibitor is an anti-NKG2D antibody molecule, such as the anti-NKG2D antibody NNC0142-0002 (also known as NN 8555, IPH2301 or JNJ-4500). Exemplary NKG2D Inhibitors

在一個實施例中，抗NKG2D抗體分子為NNC0142-0002 (Novo Nordisk)，如WO 2009/077483及US 7,879,985中所揭示，其以全文引用之方式併入本文中。In one embodiment, the anti-NKG2D antibody molecule is NNC0142-0002 (Novo Nordisk), as disclosed in WO 2009/077483 and US 7,879,985, which are incorporated herein by reference in their entirety.

在另一個實施例中，抗NKG2D抗體分子為JNJ-64304500 (Janssen)，如WO 2018/035330中所揭示，其以全文引用之方式併入本文中。In another embodiment, the anti-NKG2D antibody molecule is JNJ-64304500 (Janssen), as disclosed in WO 2018/035330, which is incorporated herein by reference in its entirety.

在一些實施例中，抗NKG2D抗體為如US 7,879,985中所述產生、分離且在結構及功能上表徵之人類單株抗體16F16、16F31、MS及21F2。其他已知抗NKG2D抗體包括例如WO 2009/077483、WO 2010/017103、WO 2017/081190、WO 2018/035330及WO 2018/148447中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。In some embodiments, the anti-NKG2D antibodies are human monoclonal antibodies 16F16, 16F31, MS, and 21F2, produced, isolated, and structurally and functionally characterized as described in US 7,879,985. Other known anti-NKG2D antibodies include, for example, those described in WO 2009/077483, WO 2010/017103, WO 2017/081190, WO 2018/035330, and WO 2018/148447, which are incorporated by reference in their entirety in this article.

在一些其他實施例中，NKG2D抑制劑為免疫黏附素{例如包含與恆定區(例如，免疫球蛋白序列之Fc區，如WO 2010/080124、WO 2017/083545及WO 2017/083612中所揭示，其以全文引用之方式併入本文中)融合之NKG2DL之胞外或NKG2D結合部分的免疫黏附素。 NKG2DL抑制劑 In some other embodiments, the NKG2D inhibitor is an immunoadhesin {eg, comprising an Fc region with a constant region (eg, an immunoglobulin sequence, as disclosed in WO 2010/080124, WO 2017/083545, and WO 2017/083612, The immunoadhesin of the extracellular or NKG2D binding portion of the fused NKG2DL, which is incorporated herein by reference in its entirety. NKG2DL inhibitors

在一些實施例中，NKG2D/NKG2DL路徑之抑制劑為NKG2DL之抑制劑，諸如MICA、MICB、ULBP1、ULBP2、ULBP3、ULBP4或RAET1家族之成員。在一些實施例中，本發明之結合核酸分子與NKG2DL抑制劑組合投與。在一些實施例中，NKG2DL抑制劑為抗NKG2DL抗體分子，諸如抗MICA/B抗體。 例示性MICA/MICB抑制劑 In some embodiments, the inhibitor of the NKG2D/NKG2DL pathway is an inhibitor of NKG2DL, such as a member of the MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, or RAET1 family. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with an NKG2DL inhibitor. In some embodiments, the NKG2DL inhibitor is an anti-NKG2DL antibody molecule, such as an anti-MICA/B antibody. Exemplary MICA/MICB Inhibitors

在一個實施例中，抗MICA/B抗體分子為IPH4301 (Innate Pharma)，如WO 2017/157895中所揭示，其以全文引用之方式併入本文中。In one embodiment, the anti-MICA/B antibody molecule is IPH4301 (Innate Pharma) as disclosed in WO 2017/157895, which is incorporated herein by reference in its entirety.

其他已知抗MICA/B抗體包括例如WO 2014/140904及WO 2018/073648中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。 KIR抑制劑 Other known anti-MICA/B antibodies include, for example, those described in WO 2014/140904 and WO 2018/073648, which are incorporated herein by reference in their entirety. KIR inhibitors

在某些實施例中，免疫檢查點分子之抑制劑為KIR之抑制劑。在一些實施例中，本發明之結合核酸分子與KIR抑制劑組合投與。在一些實施例中，KIR抑制劑為利瑞路單抗(Lirilumab) (先前亦稱為BMS-986015或IPH2102)。 例示性KIR抑制劑 In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of KIR. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a KIR inhibitor. In some embodiments, the KIR inhibitor is Lirilumab (also previously known as BMS-986015 or IPH2102). Exemplary KIR Inhibitors

在一個實施例中，抗KIR抗體分子為利瑞路單抗(Innate Pharma/AstraZeneca)，如WO 2008/084106及WO 2014/055648中所揭示，其以全文引用之方式併入本文中。In one embodiment, the anti-KIR antibody molecule is rilumab (Innate Pharma/AstraZeneca), as disclosed in WO 2008/084106 and WO 2014/055648, which are incorporated herein by reference in their entirety.

其他已知抗KIR抗體包括例如WO 2005/003168、WO 2005/009465、WO 2006/072625、WO 2006/072626、WO 2007/042573、WO 2008/084106、WO 2010/065939、WO 2012/071411及WO/2012/160448中所述之彼等抗體，該等文獻以全文引用之方式併入本文中。 TIGIT抑制劑 Other known anti-KIR antibodies include, for example, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO 2012/071411 and WO/ Those antibodies described in 2012/160448, which are incorporated herein by reference in their entirety. TIGIT inhibitors

在某些實施例中，免疫檢查點分子之抑制劑為TIGIT之抑制劑。在一些實施例中，本發明之結合核酸分子與TIGIT抑制劑組合投與。在一些實施例中，TIGIT抑制劑為MK-7684、厄提吉利單抗(Etigilimab)、替拉格魯單抗(Tiragolumab)或BMS-986207。 例示性TIGIT抑制劑 In certain embodiments, the inhibitor of an immune checkpoint molecule is an inhibitor of TIGIT. In some embodiments, the binding nucleic acid molecules of the invention are administered in combination with a TIGIT inhibitor. In some embodiments, the TIGIT inhibitor is MK-7684, Etigilimab, Tiragolumab, or BMS-986207. Exemplary TIGIT Inhibitors

在一個實施例中，TIGIT抑制劑為抗TIGIT抗體分子。在一個實施例中，抗TIGIT抗體分子係選自MK-7684 (Merck Sharp & Dohme)、厄提吉利單抗(OncoMed Pharmaceuticals, Mereo BioPharma)、替拉格魯單抗(Genentech, Roche)或BMS-986207 (Bristol-Myers Squibb)。In one embodiment, the TIGIT inhibitor is an anti-TIGIT antibody molecule. In one embodiment, the anti-TIGIT antibody molecule is selected from MK-7684 (Merck Sharp & Dohme), ertigilimab (OncoMed Pharmaceuticals, Mereo BioPharma), tilagrumab (Genentech, Roche) or BMS- 986207 (Bristol-Myers Squibb).

在一個實施例中，抗TIGIT抗體分子為MK-7684 (Merck Sharp & Dohme)，亦稱為維博利單抗(Vibostolimab)。In one embodiment, the anti-TIGIT antibody molecule is MK-7684 (Merck Sharp & Dohme), also known as Vibostolimab.

在一個實施例中，抗TIGIT抗體分子為厄提吉利單抗(OncoMed Pharmaceuticals, Mereo BioPharma)。In one embodiment, the anti-TIGIT antibody molecule is ertigilimab (OncoMed Pharmaceuticals, Mereo BioPharma).

在一個實施例中，抗TIGIT抗體分子為替拉格魯單抗(Genentech, Roche)，亦稱為RO7092284。In one embodiment, the anti-TIGIT antibody molecule is tilagrumab (Genentech, Roche), also known as RO7092284.

在一個實施例中，抗TIGIT抗體分子為BMS-986207 (Bristol-Myers Squibb)。 與習知化學治療劑、放射治療劑、抗血管生成劑之組合 In one embodiment, the anti-TIGIT antibody molecule is BMS-986207 (Bristol-Myers Squibb). Combinations with conventional chemotherapeutic, radiotherapeutic, anti-angiogenic agents

本發明亦提供組合療法，其中本發明之結合核酸分子與手術或放射治療同時、在其之前或之後使用；或與習知化學治療劑、放射治療劑或抗血管生成劑或靶向免疫毒素一起、在其之前或之後投與患者。The invention also provides combination therapy wherein the binding nucleic acid molecules of the invention are used concurrently with, before or after surgery or radiation therapy; or with conventional chemotherapeutic, radiotherapeutic or anti-angiogenic or targeted immunotoxins , administered to a patient before or after it.

本發明亦提供一種治療癌症之方法，其係藉由向有需要之患者投與本發明之結合核酸分子與習知化學治療劑、放射治療劑或抗血管生成劑或靶向免疫毒素之組合。本發明亦關於一種醫藥組合物，其包含本發明之結合核酸分子及習知化學治療劑、放射治療劑或抗血管生成劑或靶向免疫毒素，更特定言之，用於治療癌症。本發明亦關於一種產品，其包含本發明之結合核酸分子及習知化學治療劑、放射治療劑或抗血管生成劑或靶向免疫毒素，作為組合製劑同時、單獨或依序使用，更特定言之，用於治療癌症。The present invention also provides a method of treating cancer by administering to a patient in need thereof a binding nucleic acid molecule of the present invention in combination with conventional chemotherapeutic, radiotherapeutic or anti-angiogenic or targeted immunotoxins. The present invention also relates to a pharmaceutical composition comprising a binding nucleic acid molecule of the present invention and a conventional chemotherapeutic, radiotherapeutic or anti-angiogenic agent or targeted immunotoxin, more particularly, for the treatment of cancer. The present invention also relates to a product comprising a binding nucleic acid molecule of the present invention and a conventional chemotherapeutic, radiotherapeutic or anti-angiogenic agent or targeted immunotoxin for simultaneous, separate or sequential use as a combined preparation, more particularly One, for the treatment of cancer.

本發明之其他態樣及優點將在以下實驗部分中揭示，該實驗部分應被視為說明性的且不限制本申請案之範疇。在本說明書中引用許多參考文獻；此等所引用參考文獻各自以引用之方式併入本文中。 實例 實例1：核酸分子之合成 材料及方法 OX401 Other aspects and advantages of the present invention will be disclosed in the following experimental section, which should be considered illustrative and not limiting of the scope of this application. A number of references are cited in this specification; each of these cited references is incorporated herein by reference. example Example 1: Synthesis of Nucleic Acid Molecules Materials and Methods OX401

在實例中，OX401用作對照分子。OX401為一種合成的膽固醇結合16個鹼基對雙螺旋DNA，其具有經修飾之磷酸二酯主鏈，更特定言之在各股上具有3個硫代磷酸酯鍵。In the examples, OX401 was used as a control molecule. OX401 is a synthetic cholesterol-binding 16 base pair duplex DNA with a modified phosphodiester backbone, more specifically 3 phosphorothioate linkages on each strand.

OX401之合成係基於使用固體胺基亞磷酸酯化學試劑(dA(Bz)；dC(Bz)；dG(Ibu)；dT (-))、HEG及Chol6胺基磷酸酯之標準固相DNA合成。

Figure 02_image016
The synthesis of OX401 is based on standard solid phase DNA synthesis using solid phosphoramidate chemistries (dA(Bz); dC(Bz); dG(Ibu); dT(-)), HEG and Chol6 phosphoramidate.
Figure 02_image016

去三苯甲基化步驟係用含3% DCA之甲苯進行，氧化係用含50 mM碘之吡啶/水9/1進行，且硫化係用含50 mM DDTT之吡啶//ACN 1/1進行。封端係用含20% NMI之ACN以及含20% Ac2O之2,6-二甲基吡啶/ACN (40/60)進行。裂解及去保護分別用含20%二乙胺之ACN移除磷酸酯/硫代磷酸酯上之氰乙基保護基25分鐘及用濃氨水在45℃下18小時來進行。The detritylation step was performed with 3% DCA in toluene, the oxidation was performed with 50 mM iodine in pyridine/water 9/1, and the sulfurization was performed with 50 mM DDTT in pyridine//ACN 1/1 . Endcapping was performed with 20% NMI in ACN and 20% Ac2O in 2,6-lutidine/ACN (40/60). Cleavage and deprotection were performed with 20% diethylamine in ACN to remove the cyanoethyl protecting group on the phosphate/phosphorothioate for 25 minutes and concentrated ammonia at 45°C for 18 hours, respectively.

將粗制溶液裝載於製備型AEX-HPLC管柱(TSK gel SuperQ 5PW20)上。隨後用含有20體積%乙腈之pH 12之溴化鈉鹽梯度溶離來進行純化。在彙集溶離份後，藉由TFF在再生纖維素上進行去鹽。The crude solution was loaded on a preparative AEX-HPLC column (TSK gel SuperQ 5PW20). Purification was followed by gradient elution with sodium bromide salt pH 12 containing 20 vol% acetonitrile. After pooling the fractions, desalting was performed on regenerated cellulose by TFF.

OX401之純度：藉由AEX-HPLC為91.8%；藉由ESI-MS之分子量：11046.5 Da。 HEG胺基亞磷酸酯(六乙二醇胺基亞磷酸酯)

Figure 02_image018
(No CLP-9765, ChemGenes Corp) Chol6胺基亞磷酸酯
Figure 02_image020
(N° 51230, AM Chemicals) OX413 Purity of OX401: 91.8% by AEX-HPLC; molecular weight by ESI-MS: 11046.5 Da. HEG amino phosphite (hexaethylene glycol amino phosphite)
Figure 02_image018
(No CLP-9765, ChemGenes Corp) Chol6 aminophosphite
Figure 02_image020
(N° 51230, AM Chemicals) OX413

來自Axolabs (德國)之OX413之合成係基於使用胺基亞磷酸酯化學試劑、HEG及Chol6胺基磷酸酯之標準固相DNA合成，隨後為去三苯甲基化、硫化、封端及純化步驟。The synthesis of OX413 from Axolabs (Germany) is based on standard solid phase DNA synthesis using aminophosphite chemistry, HEG and Chol6 phosphoramidate, followed by detritylation, sulfurization, capping and purification steps .

OX413之純度：藉由AEX-HPLC為93.8%；藉由ESI-MS之分子量：11434.9 Da。 實例2：OX413活化PARP 材料及方法 細胞培養 Purity of OX413: 93.8% by AEX-HPLC; molecular weight by ESI-MS: 11434.9 Da. Example 2: OX413 activates PARP Materials and Methods cell culture

使用來自ATCC之三陰性乳癌細胞株MDA-MB-231作為細胞模型。細胞根據供應商之說明書在補充有10%胎牛血清(FBS)之L15 Leibovitz培養基中生長，且在37℃及0% CO2下之潮濕氛圍中維持。 藉由免疫螢光分析對PAR化進行定量 A triple-negative breast cancer cell line MDA-MB-231 from ATCC was used as a cell model. Cells were grown in L15 Leibovitz medium supplemented with 10% fetal bovine serum (FBS) according to the supplier's instructions and maintained in a humidified atmosphere at 37°C and 0% CO2. Quantification of PARylation by immunofluorescence analysis

細胞以2×10 4個細胞之濃度接種於LabTek室(Fischer scientific)且在24小時期間在37℃下培育。隨後用5 µM OX401或OX413處理細胞。在處理後六小時、二十四小時及四十八小時，細胞在4%多聚甲醛/PBS 1x中固定20分鐘，在0.5% Triton X-100中透化10分鐘，用10% FBS阻斷15分鐘，且在室溫下與初級抗體(抗泛ADP-核糖結合試劑，1/300，Millipore)培育1小時。與Alexa-488 (Molecular Probes)結合之二級山羊抗兔IgG以1/200之稀釋度在室溫下使用45分鐘，且用6-二甲脒基-2-苯基吲哚(DAPI)對DNA進行染色。陽性細胞(顯示聚ADP-核糖聚合物，PAR化)之頻率估計為相對於細胞總數之陽性細胞數。分析各樣品之至少100個細胞。 結果 Cells were seeded in LabTek chambers (Fischer scientific) at a concentration of 2 x 104 cells and incubated at 37°C during 24 hours. Cells were then treated with 5 µM OX401 or OX413. At six, twenty-four, and forty-eight hours after treatment, cells were fixed in 4% paraformaldehyde/PBS 1x for 20 minutes, permeabilized in 0.5% Triton X-100 for 10 minutes, and blocked with 10% FBS for 15 minutes , and incubated with primary antibody (anti-pan ADP-ribose binding reagent, 1/300, Millipore) for 1 hour at room temperature. Secondary goat anti-rabbit IgG conjugated to Alexa-488 (Molecular Probes) was used at a dilution of 1/200 for 45 minutes at room temperature and quenched with 6-dimethylamidino-2-phenylindole (DAPI). DNA was stained. The frequency of positive cells (indicating poly ADP-ribose polymer, PARylation) was estimated as the number of positive cells relative to the total number of cells. At least 100 cells from each sample were analyzed. result

本發明人分析在結合OX413或模擬雙股斷裂之OX401寡核苷酸部分後MDA-MB-231細胞中聚(ADP-核糖)聚合酶(PARP)之活化。用OX401處理之MDA-MB-231細胞在處理後24小時開始顯示聚(ADP-核糖) (PAR)聚合物累積(PAR化，PARP活化之結果)，處理後24小時後具有大致10%之PAR化細胞且48小時後為20% (圖1A、B)。與用OX401處理之細胞相比，用OX413處理之細胞顯示更高的PARP活化，尤其在處理後48小時，具有超過40%之PAR化細胞(圖1A、B)。因此，發明人觀察到與OX401相比，OX413在MDA-MB-231細胞中之目標接合更高，由假DNA損傷信號傳導(PAR化)顯示。 實例3：OX413呈現高抗腫瘤活性 材料及方法 細胞培養 The inventors analyzed the activation of poly(ADP-ribose) polymerase (PARP) in MDA-MB-231 cells following binding of OX413 or a double-strand break-mimicking oligonucleotide moiety of OX401. MDA-MB-231 cells treated with OX401 began to show poly(ADP-ribose) (PAR) polymer accumulation (PARylation, a result of PARP activation) 24 hours after treatment, with approximately 10% PAR after 24 hours of treatment cells and 20% after 48 hours (Fig. 1A,B). Compared to cells treated with OX401, cells treated with OX413 showed higher PARP activation, especially at 48 hours post-treatment, with more than 40% of PARylated cells (Fig. 1A,B). Therefore, the inventors observed that OX413 has higher target engagement in MDA-MB-231 cells compared to OX401, as indicated by pseudo DNA damage signaling (PARylation). Example 3: OX413 exhibits high antitumor activity Materials and Methods cell culture

使用來自ATCC之三陰性乳癌細胞株MDA-MB-231作為細胞模型。細胞根據供應商之說明書在補充有10%胎牛血清(FBS)之L15 Leibovitz培養基中生長，且在37℃及0% CO2下之潮濕氛圍中維持。 藥物處理及量測細胞存活率 A triple-negative breast cancer cell line MDA-MB-231 from ATCC was used as a cell model. Cells were grown in L15 Leibovitz medium supplemented with 10% fetal bovine serum (FBS) according to the supplier's instructions and maintained in a humidified atmosphere at 37°C and 0% CO2. Drug treatment and measurement of cell viability

將MDA-MB-231 (5.103個細胞/孔)接種於96孔盤中，且在+37℃下培育24小時，隨後添加遞增濃度之藥物7天。在藥物暴露後，使用XTT分析(Sigma Aldrich)量測細胞存活率。簡言之，將XTT溶液直接添加至含有細胞培養物之各孔中，且在37℃下培育細胞5小時，隨後使用微量盤讀取器(BMG Fluostar, Galaxy)在490 nm及690 nm下讀取吸光度。細胞存活率以活的經處理細胞與活的模擬處理細胞之比率計算。藉由使用GraphPad Prism軟體(版本5.04)之非線性回歸模型，藉由繪製各細胞株之活力百分比對藥物濃度之對數來計算IC50 (其表示50%之細胞存活的劑量)。 結果 MDA-MB-231 (5.103 cells/well) were seeded in 96-well plates and incubated at +37°C for 24 hours, followed by the addition of increasing concentrations of drug for 7 days. After drug exposure, cell viability was measured using XTT assay (Sigma Aldrich). Briefly, the XTT solution was added directly to each well containing the cell culture, and the cells were incubated at 37°C for 5 hours before reading at 490 nm and 690 nm using a microplate reader (BMG Fluostar, Galaxy). Take the absorbance. Cell viability was calculated as the ratio of live treated cells to live mock-treated cells. IC50 (which represents the dose at which 50% of the cells survive) was calculated by plotting the logarithm of the percent viability of each cell line against the drug concentration by a nonlinear regression model using GraphPad Prism software (version 5.04). result

為了評定OX413之抗腫瘤功效，在1週期間用OX413 (黑色)或OX401 (深灰色)處理MDA-MB-231腫瘤細胞以估計IC 50(中位抑制濃度)，且在處理後7天使用XTT分析量測存活率(圖2)。有趣的是，與OX401相比，OX413呈現更高的抗腫瘤活性，IC 50值比OX401低30倍(圖2)。 實例4：醫藥特性/PK實驗 材料及方法 To assess the antitumor efficacy of OX413, MDA-MB-231 tumor cells were treated with OX413 (black) or OX401 (dark gray) during 1 week to estimate IC50 (median inhibitory concentration), and XTT was used 7 days after treatment The analysis measures survival (Figure 2). Interestingly, compared to OX401, OX413 exhibited higher antitumor activity with a 30-fold lower IC50 value than OX401 (Figure 2). Example 4: Pharmaceutical Properties/PK Experimental Materials and Methods

雌性Nu/NU NMRI小鼠係購自Janvier-labs。藉由腹膜內(i.p.)途徑將兩毫克OX401或OX413注射至三隻小鼠，且在不同時間採集血液：15分鐘、30分鐘、1小時、2小時、4小時及24小時。前五個時間點藉由下頜靜脈穿刺進行血液採集，且24小時時間點藉由在深度氣體麻醉下之心內終末穿刺進行血液採集。將血液採集至具有抗凝血劑(K2-EDTA)之管中，且在+4℃下以1,200 g離心15分鐘以回收血漿。將血漿樣品在80℃下儲存於丙烯管中。The female Nu/NU NMRI mouse line was purchased from Janvier-labs. Three mice were injected with two mg of OX401 or OX413 by the intraperitoneal (i.p.) route, and blood was collected at different times: 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 24 hours. Blood collection was performed by mandibular vein puncture for the first five time points and by terminal intracardiac puncture under deep gas anesthesia at the 24-hour time point. Blood was collected into tubes with anticoagulant (K2-EDTA) and centrifuged at 1,200 g for 15 minutes at +4°C to recover plasma. Plasma samples were stored in propylene tubes at 80°C.

藉由用20 µL緩衝液(400 µL CaCl2 0.5M、100 µl HEPES 1M、500 µl.eau)及75 µL MilliQ水稀釋5 µL之＞ 20 mg/mL蛋白酶k溶液來製備蛋白酶K溶液(solK)。固定體積之樣品(15至20 µL)用相同體積之蛋白酶K溶液(solK)稀釋，且在55℃下升溫1小時，隨後直接注入配備有DNA-Pac管柱之高壓液相層析中。藉由隨時間推移增加Tris-乙腈-過氯酸鈉相之百分比來執行梯度。 結果 Proteinase K solution (solK) was prepared by diluting 5 µL of a >20 mg/mL proteinase k solution with 20 µL buffer (400 µL CaCl2 0.5M, 100 µl HEPES 1M, 500 µl.eau) and 75 µL MilliQ water. A fixed volume of sample (15 to 20 µL) was diluted with the same volume of proteinase K solution (solK) and warmed at 55°C for 1 hour before being injected directly into a high pressure liquid chromatography equipped with a DNA-Pac column. Gradients were performed by increasing the percentage of Tris-acetonitrile-sodium perchlorate phase over time. result

如藉由HPLC方法所量測，在小鼠中藉由i.p.途徑注射2 mg劑量之OX413產生80 μg/mL之血漿濃度最大值(Cmax)，達到與在相同實驗條件下用OX401獲得之血漿濃度最大值(76 μg/mL)等效的水準。Injection of a 2 mg dose of OX413 in mice by the i.p. route resulted in a plasma concentration maximum (Cmax) of 80 μg/mL as measured by the HPLC method, reaching the plasma concentration obtained with OX401 under the same experimental conditions Maximum (76 μg/mL) equivalent level.

與OX401 (245 µg.h/mL)相比，投與OX413時量測之血漿藥物濃度-時間曲線下面積(AUC)略高(261 µg.h/mL) (圖3)。當與OX401相比時，OX413之Cmax及AUC參數保持在良好水準(圖3)。Compared to OX401 (245 µg.h/mL), the plasma area under the drug concentration-time curve (AUC) measured when OX413 was administered was slightly higher (261 µg.h/mL) (Figure 3). The Cmax and AUC parameters of OX413 remained at good levels when compared to OX401 (Figure 3).

在OX413之i.p.投與或OX401之i.p.投與後之Cmax及AUC值展示於下表1中：   達到Cmax之時間(h) Cmax (µg/mL) AUC (µg.h/mL) OX401 2 76 245 OX413 2 162 625 實例5：締合/解離之動力學及相互作用之強度(KD) 材料及方法 The Cmax and AUC values following ip administration of OX413 or ip administration of OX401 are shown in Table 1 below: Time to reach Cmax (h) Cmax (µg/mL) AUC (µg.h/mL) OX401 2 76 245 OX413 2 162 625 Example 5: Kinetics of Association/Dissociation and Strength of Interaction (KD) Materials and Methods

OX413與人類聚[ADP-核糖聚合酶1蛋白(PARP-1) (115kDa)之相互作用已藉由SPR技術，使用來自GE Healthcare Life Sciences之Biacore T100儀器，且使用購自Thermofisher之人類His標記之PARP-1蛋白進行表徵。為了評估PARP1/髮夾相互作用，PARP1-His已捕捉於固定在羧甲基化晶片表面上之抗His抗體上。 結果 The interaction of OX413 with human poly[ADP-ribose polymerase 1 protein (PARP-1) (115 kDa) has been analyzed by SPR technique using a Biacore T100 instrument from GE Healthcare Life Sciences and using human His-tagged from Thermofisher. PARP-1 protein was characterized. To assess the PARP1/hairpin interaction, PARP1-His has been captured on an anti-His antibody immobilized on the surface of a carboxymethylated wafer. result

OX413及OX401之締合動力學(kon)以及相互作用強度(KD)報導於下表2中：   K D(nM) k on(M -1s -1) k off(s -1) OX401 7 1.0 10 5 6.9 10 -4 OX413 4 2.6 10 5 9.0 10 -4 The association kinetics (kon) and interaction strength (KD) of OX413 and OX401 are reported in Table 2 below: K D (nM) k on (M -1 s -1 ) k off (s -1 ) OX401 7 1.0 10 5 6.9 10-4 OX413 4 2.6 10 5 9.0 10-4

與OX401相比，OX413與PARP-1蛋白具有更好的相互作用，亦突出表現在實驗期間使用之晶片難以回收，意味著OX413與PARP-1強有力地連接。Compared with OX401, OX413 has a better interaction with PARP-1 protein, and it is also highlighted that the wafers used during the experiment are difficult to recover, which means that OX413 is strongly linked to PARP-1.

總體而言，OX413在兩股上之化學修飾，亦即各股在特定位置攜帶9個FANA及在不同位置增加硫代磷酸酯數目，對其活性(提高IC 50)及對其與PARP-1蛋白之相互作用強度(K D值降低及解離動力學(k off)增加)具有顯著影響，顯示強PARP相互作用，幾乎不可逆。此等修飾沒有對OX413之藥物暴露產生負面影響，因為Cmax及AUC參數保持在良好水準。 實例6：OX413誘導細胞質DNA累積且觸發先天性免疫反應 材料及方法 細胞培養 Overall, chemical modification of OX413 on both strands, ie, each strand carrying 9 FANAs at specific positions and increasing the number of phosphorothioates at different positions, was associated with its activity (increased IC50 ) and its interaction with PARP-1 protein. The strength of the interaction (decreased K D value and increased dissociation kinetics (k off )) has a significant effect, indicating a strong PARP interaction, which is almost irreversible. These modifications did not negatively affect the drug exposure of OX413, as the Cmax and AUC parameters remained at good levels. Example 6: OX413 Induces Cytoplasmic DNA Accumulation and Triggers Innate Immune Response Materials and Methods Cell Culture

使用來自ATCC之三陰性乳癌細胞株MDA-MB-231作為細胞模型。細胞根據供應商之說明書在補充有10%胎牛血清(FBS)之L15 Leibovitz培養基中生長，且在37℃及0% CO2下之潮濕氛圍中維持。 藉由免疫螢光分析對PAR化進行定量 A triple-negative breast cancer cell line MDA-MB-231 from ATCC was used as a cell model. Cells were grown in L15 Leibovitz medium supplemented with 10% fetal bovine serum (FBS) according to the supplier's instructions and maintained in a humidified atmosphere at 37°C and 0% CO2. Quantification of PARylation by immunofluorescence analysis

細胞以2×10 4個細胞之濃度接種於LabTek室(Fischer scientific)且在37℃下培育24小時。隨後用OX413 (200 nM)處理細胞。在處理後四十八小時，細胞在4%多聚甲醛/PBS 1x中固定20分鐘，在0.5% Triton X-100中透化10分鐘，用10% FBS阻斷15分鐘，且在室溫下與初級抗體(抗泛ADP-核糖結合試劑，1/300，Millipore)培育1小時。與Alexa-488 (Molecular Probes)結合之二級山羊抗兔IgG以1/200之稀釋度在室溫下使用45分鐘，且用6-二甲脒基-2-苯基吲哚(DAPI)對DNA進行染色。 微核(MN)及細胞質染色質片段(CCF)偵測 Cells were seeded in LabTek chambers (Fischer scientific) at a concentration of 2 x 104 cells and incubated at 37°C for 24 hours. Cells were then treated with OX413 (200 nM). Forty-eight hours after treatment, cells were fixed in 4% paraformaldehyde/PBS 1x for 20 min, permeabilized in 0.5% Triton X-100 for 10 min, blocked with 10% FBS for 15 min, and incubated with primary Antibody (anti-pan ADP-ribose binding reagent, 1/300, Millipore) was incubated for 1 hour. Secondary goat anti-rabbit IgG conjugated to Alexa-488 (Molecular Probes) was used at a dilution of 1/200 for 45 minutes at room temperature and quenched with 6-dimethylamidino-2-phenylindole (DAPI). DNA was stained. Micronucleus (MN) and cytoplasmic chromatin fragment (CCF) detection

將MDA-MB-231細胞自適當密度之6孔盤中以5E4個細胞接種於蓋玻片(Menzel, Braunschweig, Germany)上，且隨後在具有或不具有OX413之情況下處理48小時。處理後，細胞藉由4%多聚甲醛/PBS 1X固定20分鐘，在0.5% Triton X-100中透化10分鐘且用10% FBS阻斷15分鐘。隨後，細胞用PBS洗滌，藉由picogreen (Invitrogen，用於CCF偵測)及/或DAPI (用於MN分析)染色5分鐘。MN之百分比估計為總細胞數中呈現MN結構之細胞的數目。針對各條件分析約150個細胞。 流動式細胞測量術分析 MDA-MB-231 cells were seeded on coverslips (Menzel, Braunschweig, Germany) at 5E4 cells from 6-well plates of appropriate density and then treated with or without OX413 for 48 hours. After treatment, cells were fixed by 4% paraformaldehyde/PBS 1X for 20 minutes, permeabilized in 0.5% Triton X-100 for 10 minutes and blocked with 10% FBS for 15 minutes. Subsequently, cells were washed with PBS and stained by picogreen (Invitrogen, for CCF detection) and/or DAPI (for MN analysis) for 5 minutes. The percentage of MN was estimated as the number of cells presenting the MN structure out of the total number of cells. About 150 cells were analyzed for each condition. Flow cytometry analysis

將MDA-MB-231細胞以2E5個細胞/毫升接種於T25燒瓶中，且隨後在具有或不具有200 nM OX413之情況下處理48小時。對於細胞內染色(pSTING分析)，洗滌細胞，隨後在至少1小時期間在4℃下在PBS/70%乙醇中固定。隨後洗滌細胞，在室溫下用PBS/0.2% TritonX-100溶液透化10分鐘，且在室溫下用PBS/2%牛血清白蛋白(BSA)溶液飽和10分鐘。隨後，在流動式細胞測量術分析(Guava EasyCyte 12H, Luminex, Germany)前，細胞用PBS洗滌且用Alexa488結合之抗pSTING抗體(cell signaling, Netherlands, 1/200)培育1小時。對於細胞表面受體染色，在處理結束後直接收穫及洗滌細胞，且隨後在4℃下用Alexa-488偶合之抗MIC-A抗體(R&D System, 1/200)及APE偶合之抗PD-L1抗體(Abcam, 1/200)培育1小時。隨後用PBS洗滌染色細胞，且用Guava EasyCyte 12H流式細胞儀(Luminex, Germany)獲取螢光強度。使用FlowJo軟體(Tree Star, CA)分析資料。 ELISA偵測CCL5 MDA-MB-231 cells were seeded in T25 flasks at 2E5 cells/ml and then treated with or without 200 nM OX413 for 48 hours. For intracellular staining (pSTING analysis), cells were washed and then fixed in PBS/70% ethanol at 4°C for at least 1 hour. Cells were then washed, permeabilized with PBS/0.2% TritonX-100 solution for 10 minutes at room temperature, and saturated with PBS/2% bovine serum albumin (BSA) solution for 10 minutes at room temperature. Subsequently, cells were washed with PBS and incubated with Alexa488-conjugated anti-pSTING antibody (cell signaling, Netherlands, 1/200) for 1 hour before flow cytometry analysis (Guava EasyCyte 12H, Luminex, Germany). For cell surface receptor staining, cells were harvested and washed directly after treatment, and then with Alexa-488-conjugated anti-MIC-A antibody (R&D System, 1/200) and APE-conjugated anti-PD-L1 at 4°C Antibody (Abeam, 1/200) was incubated for 1 hour. Stained cells were then washed with PBS and fluorescence intensities were acquired with a Guava EasyCyte 12H flow cytometer (Luminex, Germany). Data were analyzed using FlowJo software (Tree Star, CA). Detection of CCL5 by ELISA

在具有或不具有T淋巴球之情況下，用OX401 (5 µM)或OX413 (500 nM)處理MDA-MB-231腫瘤細胞24及48小時。隨後收穫細胞培養上清液，且以1,500×g離心10分鐘以移除殘渣。套組(Human SimpleStep CCL5 ELISA Kit - Abcam - ab174446)包括之96孔盤條可隨時使用。將50 µl各上清液與50 µl抗體混合液一式兩份地添加至各孔中，且隨後在室溫下在設置為400 rpm之盤震盪器上培育1小時，之後用1X洗滌緩衝液PT洗滌。隨後，向各孔中添加100 µl TMB受質且在設置為400 rpm之盤震盪器上在黑暗中培育10分鐘。隨後將100 µl終止溶液添加至各孔中，在盤震盪器上1分鐘，且在微量盤讀取器(EnspireTM Perkin-Almer)上量測所得發光信號。 結果 MDA-MB-231 tumor cells were treated with OX401 (5 μM) or OX413 (500 nM) for 24 and 48 hours with or without T lymphocytes. Cell culture supernatants were then harvested and centrifuged at 1,500 xg for 10 minutes to remove debris. The kit (Human SimpleStep CCL5 ELISA Kit - Abcam - ab174446) includes 96-well coils ready to use. 50 µl of each supernatant and 50 µl of antibody mix were added to each well in duplicate and then incubated for 1 hour at room temperature on a disk shaker set to 400 rpm, followed by 1X wash buffer PT washing. Subsequently, 100 μl of TMB substrate was added to each well and incubated for 10 minutes in the dark on a pan shaker set to 400 rpm. 100 μl of stop solution was then added to each well, 1 minute on a disk shaker, and the resulting luminescent signal was measured on a microplate reader (Enspire™ Perkin-Almer). result

為了證實經最佳化之OX413分子的效率，本發明人測試化學修飾是否不影響其固定PARP1蛋白之能力。OX413處理之MDA-MB-231細胞中PARP蛋白之過度活化係藉由對PAR化之免疫螢光分析來評定(圖4A)。OX413處理之細胞對「假」核PAR化信號傳導呈陽性，證實目標接合(圖4A)。To confirm the efficiency of the optimized OX413 molecule, the inventors tested whether chemical modification did not affect its ability to immobilize PARP1 protein. Hyperactivation of PARP protein in OX413-treated MDA-MB-231 cells was assessed by immunofluorescence analysis of PARylation (FIG. 4A). OX413-treated cells were positive for "pseudo" nuclear PARylation signaling, confirming target engagement (Figure 4A).

為了檢查此較高抗腫瘤功效是否係由於對細胞應激及DNA修復之更強力作用，本發明人藉由免疫染色經由定量微核(MN)及細胞質染色質片段(CCF)研究由OX413處理(以50及100 nM之劑量)誘導之細胞質未修復DNA的量。OX413誘導具有MN (圖4B)及CCF (圖4C)之細胞顯著增加。為了檢查OX413誘導之細胞質DNA累積是否可活化STING路徑，本發明人藉由流動式細胞測量術分析STING之磷酸化及活化形式(pSTING)。OX413在處理後48小時誘導STING之活化(平均螢光為57.4，相較於未處理之細胞為35) (圖4D)。為了證實STING路徑活化，本發明人亦分析OX413處理之細胞上清液中CCL5趨化介素的分泌。OX413在處理後48小時誘導CCL5分泌增加(圖4E)。腫瘤細胞中STING路徑活化的後果之一為PD-L1 (計劃性死亡配體1)上調，其可能為一種保護免疫系統之反應。本發明人分析OX413處理之細胞中細胞表面相關PD-L1的水準。與未處理之細胞相比，OX413誘導膜相關PD-L1增加2倍(圖4F)。一些報導亦已顯示，腫瘤STING活化可增加腫瘤細胞上之NK細胞配體，諸如NKG2D配體(MIC-A、MIC-B、ULBP1/6)。因此，分析MIC-A在OX413處理之細胞表面的表現。有趣的是，用OX413處理之細胞顯示細胞表面MIC-A表現增加超過2倍(圖4G)。To examine whether this higher anti-tumor efficacy was due to a more robust effect on cellular stress and DNA repair, the inventors investigated treatment with OX413 ( Amount of cytoplasmic unrepaired DNA induced at doses of 50 and 100 nM). OX413 induced a significant increase in cells with MN (FIG. 4B) and CCF (FIG. 4C). To examine whether OX413-induced cytoplasmic DNA accumulation can activate the STING pathway, the inventors analyzed the phosphorylated and activated form of STING (pSTING) by flow cytometry. OX413 induced activation of STING 48 hours after treatment (average fluorescence was 57.4, compared to 35 in untreated cells) (Fig. 4D). To confirm STING pathway activation, the inventors also analyzed the secretion of CCL5 chemokine in the supernatants of OX413-treated cells. OX413 induced an increase in CCL5 secretion 48 hours after treatment (Fig. 4E). One of the consequences of STING pathway activation in tumor cells is the upregulation of PD-L1 (programmed death ligand 1), which may be a protective immune system response. The inventors analyzed the levels of cell surface-associated PD-L1 in OX413-treated cells. OX413 induced a 2-fold increase in membrane-associated PD-L1 compared to untreated cells (FIG. 4F). Several reports have also shown that tumor STING activation can increase NK cell ligands, such as NKG2D ligands (MIC-A, MIC-B, ULBP1/6) on tumor cells. Therefore, the expression of MIC-A on the surface of OX413-treated cells was analyzed. Interestingly, cells treated with OX413 showed a more than 2-fold increase in cell surface MIC-A expression (Fig. 4G).

此等結果證實，藉由增加OX401之穩定性最佳化其結構可增加到達目標之分子的量，從而提高抗腫瘤功效及抗腫瘤免疫反應。 實例7：OX413誘導活體內腫瘤內PARP及STING路徑活化，導致腫瘤浸潤性白血球之水準增加 材料及方法 ELISA偵測CCL5 These results demonstrate that optimizing the structure of OX401 by increasing its stability increases the amount of molecules reaching the target, thereby enhancing anti-tumor efficacy and anti-tumor immune responses. Example 7: OX413 induces activation of PARP and STING pathways in tumors in vivo, resulting in increased levels of tumor-infiltrating leukocytes Materials and Methods Detection of CCL5 by ELISA

為了定量腫瘤微環境(TME)中CCL之水準，收穫腫瘤解離後之上清液，且以1,500×g離心10分鐘以移除殘渣。套組(Human SimpleStep CCL5 ELISA Kit - Abcam - ab174446)包括之96孔盤條可隨時使用。將50 µl各上清液與50 µl抗體混合液一式兩份地添加至各孔中，且隨後在室溫下在設置為400 rpm之盤震盪器上培育1小時，之後用1X洗滌緩衝液PT洗滌。隨後，向各孔中添加100 µl TMB受質且在設置為400 rpm之盤震盪器上在黑暗中培育10分鐘。隨後將100 µl終止溶液添加至各孔中，在盤震盪器上1分鐘，且在微量盤讀取器(EnspireTM Perkin-Almer)上量測所得發光信號。 活體內實驗、腫瘤消化、細胞分選及流動式細胞測量術 To quantify the levels of CCL in the tumor microenvironment (TME), post-tumor dissociation supernatants were harvested and centrifuged at 1,500 xg for 10 minutes to remove debris. The kit (Human SimpleStep CCL5 ELISA Kit - Abcam - ab174446) includes 96-well coils ready to use. 50 µl of each supernatant and 50 µl of antibody mix were added to each well in duplicate and then incubated for 1 hour at room temperature on a disk shaker set to 400 rpm, followed by 1X wash buffer PT washing. Subsequently, 100 μl of TMB substrate was added to each well and incubated for 10 minutes in the dark on a pan shaker set to 400 rpm. 100 μl of stop solution was then added to each well, 1 minute on a disk shaker, and the resulting luminescent signal was measured on a microplate reader (Enspire™ Perkin-Almer). In vivo experiments, tumor digestion, cell sorting and flow cytometry

藉由將4.10 5個細胞注射至6至8週齡成年Balb/c雌性(Janvier)之右側腹中獲得EMT6細胞衍生之異種移植物(CDX)。動物在腫瘤移植前至少1週圈養在明暗(12小時/12小時)、相對濕度(55%)及溫度(21℃)之受控條件下。使用卡尺一週評估腫瘤生長三次，且使用下式計算腫瘤體積：(長度×寬度×寬度)/2。當地動物實驗倫理委員會批准所有實驗。 EMT6 cell-derived xenografts (CDX) were obtained by injecting 4.10 5 cells into the right flank of 6- to 8-week-old adult Balb/c females (Janvier). Animals were housed under controlled conditions of light and dark (12h/12h), relative humidity (55%) and temperature (21°C) for at least 1 week prior to tumor implantation. Tumor growth was assessed three times a week using calipers, and tumor volume was calculated using the formula: (length x width x width)/2. The local animal experimentation ethics committee approved all experiments.

當移植腫瘤達到150至300 mm 3時，將小鼠隨機分組。腹膜內投與OX413 (10 mg/kg)。在指定時間(處理後6、24或72小時)處死小鼠，且根據製造商說明書，使用小鼠腫瘤解離套組(Miltenyi Biotec, 130-096-730)提取EMT6 CDX，精細絞碎且與gentleMACS octo解離劑(Miltenyi Biotec)摻合。解離的腫瘤細胞用DMEM培養基洗滌，且紅血球用RBC溶解溶液(Miltenyi Biotec, 130-094-183)溶解。隨後使用CD45微珠(Miltenyi Biotec, 130-110-618)及MultiMACS Cell24 Separator plus (Miltenyi Biotec)富集腫瘤浸潤性白血球(TIL)。 Mice were randomized when transplanted tumors reached 150 to 300 mm. OX413 (10 mg/kg) was administered intraperitoneally. Mice were sacrificed at the indicated times (6, 24 or 72 hours after treatment) and EMT6 CDX was extracted, finely minced and mixed with gentleMACS using a mouse tumor dissociation kit (Miltenyi Biotec, 130-096-730) according to the manufacturer's instructions octo dissociation agent (Miltenyi Biotec) was blended. Dissociated tumor cells were washed with DMEM medium, and red blood cells were lysed with RBC lysis solution (Miltenyi Biotec, 130-094-183). Tumor-infiltrating leukocytes (TILs) were then enriched using CD45 microbeads (Miltenyi Biotec, 130-110-618) and MultiMACS Cell24 Separator plus (Miltenyi Biotec).

將CD45+細胞再懸浮於FACS緩衝液(含有2% BSA及2 mM EDTA之PBS)中，且在4℃下在30分鐘期間用抗體組(抗CD45-VioBlue、CD3-FITC、CD8a-PE-Vio770、CD4-APC-Vio770、CD49b-PE、CD335-APC、CD11c-PerCP-Vio700)或相應同型染色。基本上含有EMT6腫瘤細胞之CD45-細胞用抗PD-L1-PE抗體染色(30分鐘，4℃)，且隨後使用FoxP3/轉錄因子染色緩衝液組(ThermoFisher, 00-5523-00)根據製造商指南固定及透化，且在4℃下與抗聚(ADP)-核糖抗體(純系10H；MERCK, MABC547)培育30分鐘。隨後洗滌細胞，再懸浮於PBS中且使用Guava EasyCyte 12HT流式細胞儀(Luminex)進行分析。使用單色及同型對照手動進行補償。使用全染色、未染色及同型對照定義信號臨限值定義。在FlowJo軟體上進行分析。 結果 CD45+ cells were resuspended in FACS buffer (PBS containing 2% BSA and 2 mM EDTA) and treated with antibody groups (anti-CD45-VioBlue, CD3-FITC, CD8a-PE-Vio770 during 30 min at 4°C). , CD4-APC-Vio770, CD49b-PE, CD335-APC, CD11c-PerCP-Vio700) or the corresponding isotype staining. CD45-cells essentially containing EMT6 tumor cells were stained with anti-PD-L1-PE antibody (30 min, 4°C) and subsequently using FoxP3/transcription factor staining buffer set (ThermoFisher, 00-5523-00) according to the manufacturer Guides were fixed and permeabilized, and incubated with anti-poly(ADP)-ribose antibody (clone 10H; MERCK, MABC547) for 30 minutes at 4°C. Cells were then washed, resuspended in PBS and analyzed using a Guava EasyCyte 12HT flow cytometer (Luminex). Compensation was performed manually using monochromatic and isotype controls. Signal threshold definitions were defined using whole-stained, unstained, and isotype controls. Analysis was performed on FlowJo software. result

本發明人評估OX413在來源於異體同質乳房腫瘤模型EMT6之異種移植物中的功效。用媒劑或OX413處理EMT6細胞衍生之異種移植物，且在處理後6、24或72小時收穫腫瘤(圖5A)。為了確認OX413吸收至腫瘤中及目標接合，本發明人在自解離腫瘤分選之EMT6細胞中分析PARP活化及細胞表面之PD-L1水準。OX413處理自處理後6小時開始誘導顯著PARP活化，表明腫瘤吸收及目標接合(圖5B)。此OX413誘導之PAR化在處理後72小時恢復至基礎水準，表明一週重複處理兩次為維持高目標接合所必需的(圖5B)。為了檢查此PAR化是否與STING路徑活化相關，如活體外實驗所觀測，本發明人定量腫瘤微環境(TME)中之腫瘤CCL5釋放。有趣的是，TME-CCL5以類似於PAR化之方式增加，處理後24小時達到峰值且在72小時下降(圖5C)。腫瘤細胞表面PD-L1在處理後亦增加，證實本發明人先前的活體外結果及PARP活性消除與PD-L1增加之間的聯繫，其可能有助於免疫抑制(圖5D)。The inventors evaluated the efficacy of OX413 in xenografts derived from the allogeneic homogenous breast tumor model EMT6. EMT6 cell-derived xenografts were treated with vehicle or OX413, and tumors were harvested 6, 24, or 72 hours after treatment (FIG. 5A). To confirm uptake of OX413 into tumors and target engagement, the inventors analyzed PARP activation and cell surface PD-L1 levels in EMT6 cells sorted from dissociated tumors. OX413 treatment induced significant PARP activation starting 6 hours after treatment, indicating tumor uptake and target engagement (Figure 5B). This OX413-induced PARylation returned to basal levels 72 hours after treatment, indicating that repeated treatments twice a week were necessary to maintain high target engagement (FIG. 5B). To examine whether this PARylation correlates with STING pathway activation, the inventors quantified tumor CCL5 release in the tumor microenvironment (TME) as observed in in vitro experiments. Interestingly, TME-CCL5 increased in a manner similar to PARylation, peaking at 24 hours post treatment and decreasing at 72 hours (Fig. 5C). Tumor cell surface PD-L1 was also increased after treatment, confirming the inventors' previous in vitro results and the link between ablation of PARP activity and increased PD-L1, which may contribute to immunosuppression (Fig. 5D).

本發明人接下來想定義OX413對免疫微環境之影響。腫瘤浸潤性白血球(TIL；CD45+細胞)之流動式細胞測量術分析顯示，OX413早在處理後3天就顯著增加總TIL，如藉由CD45染色所量測(圖5E)。響應於OX413處理，CD45+細胞中T細胞(CD3+)之比例顯著增加(圖5E)。OX413不僅增強T細胞腫瘤浸潤(CD45+、CD3+)，且亦增強自然殺手(NK)細胞(總浸潤性NK細胞：CD3-、CD49b+；活化浸潤性NK細胞：CD3-、CD49b+、CD335+，圖5E)，表明先天性及適應性免疫反應之活化。此外，在OX413處理後，TME填入樹突狀細胞(DC)中(CD45+、CD11c+)。The inventors next wanted to define the effect of OX413 on the immune microenvironment. Flow cytometry analysis of tumor-infiltrating leukocytes (TIL; CD45+ cells) showed that OX413 significantly increased total TIL as early as 3 days after treatment, as measured by CD45 staining (Figure 5E). The proportion of T cells (CD3+) in CD45+ cells was significantly increased in response to OX413 treatment (Fig. 5E). OX413 not only enhanced T cell tumor infiltration (CD45+, CD3+) but also natural killer (NK) cells (total infiltrating NK cells: CD3-, CD49b+; activated infiltrating NK cells: CD3-, CD49b+, CD335+, Figure 5E) , indicating activation of innate and adaptive immune responses. Furthermore, after OX413 treatment, TME was populated in dendritic cells (DC) (CD45+, CD11c+).

總之，此等結果證明OX413誘導之PARP及STING路徑活化在腫瘤中有效，其增加先天性及適應性免疫細胞募集且促進富有成效的抗腫瘤免疫反應。Taken together, these results demonstrate that OX413-induced activation of the PARP and STING pathways is effective in tumors, increasing innate and adaptive immune cell recruitment and promoting a productive anti-tumor immune response.

無。none.

圖1. OX413誘導之目標接合。用OX413 (5 µM)或OX401 (5 µM)處理細胞，且藉由量測細胞PAR化來評定PARP活化。(A)在OX401或OX413處理後48小時PAR化之代表性影像；(B)具有PAR化信號之陽性細胞(PAR+細胞)%的定量。Figure 1. OX413-induced target engagement. Cells were treated with OX413 (5 µM) or OX401 (5 µM) and PARP activation was assessed by measuring cellular PARylation. (A) Representative images of PARylation 48 hours after OX401 or OX413 treatment; (B) Quantification of % positive cells with PARylation signal (PAR+ cells).

圖2. OX413呈現高抗腫瘤細胞毒性。用遞增劑量之OX401或OX413處理MDA-MB-231細胞，且使用XTT分析評定細胞存活率。細胞存活率以活的經處理細胞與活的未處理細胞之比率計算。使用GraphPadPrism軟體根據劑量反應曲線計算IC 50。 Figure 2. OX413 exhibits high antitumor cytotoxicity. MDA-MB-231 cells were treated with increasing doses of OX401 or OX413, and cell viability was assessed using XTT analysis. Cell viability was calculated as the ratio of viable treated cells to viable untreated cells. IC50s were calculated from dose-response curves using GraphPad Prism software.

圖3. OX413之藥物動力學。在腹膜內藥物(OX413或OX401)投與後，小鼠血液中OX413 (粗線)及OX401 (虛線)隨時間推移之平均濃度。Figure 3. Pharmacokinetics of OX413. Mean concentrations of OX413 (thick line) and OX401 (dashed line) in mouse blood over time following intraperitoneal drug (OX413 or OX401) administration.

圖4. OX413誘導細胞質DNA累積且觸發先天性免疫反應。(A)在OX413處理(200 nM)後48小時PAR化之代表性影像。(B, C)在OX413 (50及100 nM)處理後48小時，藉由免疫螢光分析具有微核(MN) (B)或細胞質染色質片段(CCF) (C)之細胞的水準。(D, F, G) (D) pSTING、(F) PD-L1及(G) MIC-A生物標記之流動式細胞測量術分析。(E)在OX413 (200 nM)處理後48小時，藉由ELISA分析細胞上清液中分泌之CCL5。Figure 4. OX413 induces cytoplasmic DNA accumulation and triggers innate immune responses. (A) Representative images of PARylation 48 hours after OX413 treatment (200 nM). (B, C) Levels of cells with micronuclei (MN) (B) or cytoplasmic chromatin fragments (CCF) (C) were analyzed by immunofluorescence 48 hours after OX413 (50 and 100 nM) treatment. (D, F, G) Flow cytometry analysis of (D) pSTING, (F) PD-L1 and (G) MIC-A biomarkers. (E) Secreted CCL5 in cell supernatants was analyzed by ELISA 48 hours after OX413 (200 nM) treatment.

圖5. OX413活體內誘導PARP及STING活化。提取在6、24或72小時期間用OX413 (10 mg/kg)處理之EMT6細胞衍生之異種移植腫瘤，解離且分選CD45+或Cd45- (主要為EMT6細胞) (A)。在EMT6細胞上，針對各條件分析(B) PARP、(D) PD-L1及(C) CCL5表現。(E)藉由流動式細胞測量術分析測定腫瘤浸潤性白血球(TIL)百分比(CD45+、CD3+、DC細胞、NK細胞)。Figure 5. OX413 induces PARP and STING activation in vivo. Xenograft tumors derived from EMT6 cells treated with OX413 (10 mg/kg) during 6, 24 or 72 hours were extracted, dissociated and sorted for CD45+ or Cd45- (mainly EMT6 cells) (A). On EMT6 cells, (B) PARP, (D) PD-L1 and (C) CCL5 expression were analyzed for each condition. (E) Percent tumor-infiltrating leukocytes (TIL) (CD45+, CD3+, DC cells, NK cells) were determined by flow cytometry analysis.

Figure 12_A0101_SEQ_0001
Figure 12_A0101_SEQ_0001

無。none.

Claims (18)

一種結合核酸分子，其包含16個鹼基對雙股核酸部分，第一股之5'端及互補股之3'端藉由環連接在一起； 該雙股核酸部分具有以下序列
Figure 03_image022
SEQ ID NO: 1 其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵，且 其中加下劃線之核苷酸為2'修飾之核苷酸； 該環為 -O-P(X)OH-O-{[(CH2)2-O]g-P(X)OH-O}r-K-O-P(X)OH-O-{[(CH2)2-O]h-P(X)OH-O-}s- (I) 其中r及s獨立地為整數0或1；g及h獨立地為1至7之整數且g + h之和為4至7； X為O或S； 其中K為
Figure 03_image023
其中i、j、k及l獨立地為0至6、較佳1至3之整數； 或 -O-P(X)OH-O-[(CH 2) d-C(O)-NH] b-CHR-[C(O)-NH-(CH 2) e] c-O-P(X)OH-O-    (II) 其中b及c獨立地為0至4之整數，且b + c之和為3至7； d及e獨立地為1至3、較佳1至2之整數；且 其中R為-L f-J， X為O或S，L為連接子且f為整數0或1，且J為促進胞吞作用之分子或為H。 A binding nucleic acid molecule comprising a 16-base pair double-stranded nucleic acid portion, the 5' end of the first strand and the 3' end of the complementary strand are connected together by a loop; the double-stranded nucleic acid portion has the following sequence
Figure 03_image022
SEQ ID NO: 1 wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond, and wherein the underlined nucleotide is a 2' modified nucleotide; the loop is -OP(X )OH-O-{[(CH2)2-O]gP(X)OH-O}rKOP(X)OH-O-{[(CH2)2-O]hP(X)OH-O-}s- (I) wherein r and s are independently the integers 0 or 1; g and h are independently an integer from 1 to 7 and the sum of g + h is 4 to 7; X is O or S; wherein K is
Figure 03_image023
wherein i, j, k and l are independently an integer from 0 to 6, preferably 1 to 3; or -OP(X)OH-O-[( CH2 ) d -C(O)-NH] b -CHR -[C(O)-NH-(CH 2 ) e ] c -OP(X)OH-O- (II) wherein b and c are independently an integer from 0 to 4, and the sum of b + c is 3 to 4 7; d and e are independently an integer from 1 to 3, preferably 1 to 2; and wherein R is -L f -J, X is O or S, L is a linker and f is an integer 0 or 1, and J Molecules that promote endocytosis or H. 如請求項1所述之結合核酸分子，其中該促進胞吞作用之分子係選自由以下組成之群：膽固醇、單鏈或雙鏈脂肪酸、靶向細胞受體實現受體介導之胞吞作用的配體或運鐵蛋白。The binding nucleic acid molecule of claim 1, wherein the endocytosis-promoting molecule is selected from the group consisting of cholesterol, single-chain or double-chain fatty acids, targeting cellular receptors for receptor-mediated endocytosis ligand or transferrin. 如請求項1或2所述之結合核酸分子，其中該促進胞吞作用之分子為膽固醇。The binding nucleic acid molecule according to claim 1 or 2, wherein the molecule that promotes endocytosis is cholesterol. 如請求項1至3所述之結合核酸分子，其中r為1，s為0且g為5至7之整數，較佳為6。The binding nucleic acid molecule according to claims 1 to 3, wherein r is 1, s is 0 and g is an integer from 5 to 7, preferably 6. 如請求項1至4中任一項所述之結合核酸分子，其中i及j為1且k及l均為1或2。The binding nucleic acid molecule of any one of claims 1 to 4, wherein i and j are 1 and k and 1 are both 1 or 2. 如請求項1至5中任一項所述之結合核酸分子，其中f為1且L-J選自由以下組成之群：-C(O)-(CH 2) m-NH-[(CH 2) 2-O] n-(CH 2) p-C(O)-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-J、-C(O)-(CH 2) m-NH-C(O)-[(CH 2) 2-O] n-(CH 2) p-C(O)-J及-C(O)-(CH 2) m-NH-C(O)-CH 2-O-[(CH 2) 2-O] n-(CH 2) p-C(O)-J，其中m為0至10之整數；n為0至15之整數；且p為0至3之整數。 The binding nucleic acid molecule of any one of claims 1 to 5, wherein f is 1 and LJ is selected from the group consisting of: -C(O)-( CH2 ) m -NH-[( CH2 ) 2 -O] n -(CH 2 ) p -C(O)-J, -C(O)-(CH 2 ) m -NH-C(O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, -C(O)-(CH 2 ) m -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] n -(CH 2 ) p -J, - C(O)-(CH 2 ) m -NH-C(O)-[(CH 2 ) 2 -O] n -(CH 2 ) p -C(O)-J and -C(O)-(CH 2 ) m -NH-C(O) -CH2 -O-[( CH2 ) 2 -O] n- ( CH2 ) p -C(O)-J, wherein m is an integer from 0 to 10; n is an integer from 0 to 15; and p is an integer from 0 to 3. 如請求項6所述之結合核酸分子，其中m為4與6之間的整數，較佳為5。The binding nucleic acid molecule according to claim 6, wherein m is an integer between 4 and 6, preferably 5. 如請求項1至7中任一項所述之結合核酸分子，其中該環具有式(I) -O-P(X)OH-O-{[(CH 2) 2-O] g-P(X)OH-O} r-K-O-P(X)OH-O-{[(CH 2) 2-O] h-P(X)OH-O-} s(I) 其中X為S，r為1，g為6，s為0，i及j為1且k及l為2， 其中f為1且L為C(O)-(CH 2) 5-NH-[(CH 2) 2-O] 3-(CH 2) 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-[(CH 2) 2-O] 3-(CH 2) 3-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 5-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 9-CH 2-C(O)-J、-C(O)-(CH 2) 5-NH-C(O)-CH 2-O-[(CH 2) 2-O] 13-CH 2-C(O)-J或-C(O)-(CH 2) 5-NH-C(O)-J。 The binding nucleic acid molecule of any one of claims 1 to 7, wherein the loop has the formula (I)-OP(X)OH-O-{[( CH2 ) 2 -O] g -P(X) OH-O} r -KOP(X)OH-O-{[(CH 2 ) 2 -O] h -P(X)OH-O-} s (I) where X is S, r is 1, and g is 6, s is 0, i and j are 1 and k and 1 are 2, where f is 1 and L is C(O)-( CH2 ) 5 -NH-[( CH2 ) 2 -O] 3- ( CH 2 ) 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-[(CH 2 ) 2 -O] 3 -(CH 2 ) 3 -J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 5 -CH 2 -C(O)-J, -C(O) -(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 9 -CH 2 -C(O)-J, -C(O)-(CH 2 ) 5 -NH-C(O)-CH 2 -O-[(CH 2 ) 2 -O] 13 -CH 2 -C(O)-J or -C(O)-(CH 2 ) 5 -NH-C (O)-J. 如請求項1至8中任一項所述之結合核酸分子，其中該2'修飾之核苷酸獨立地選自由以下組成之群：2'-去氧-2'-氟、2'-O-甲基、2'-O-甲氧基乙基(2'-O-MOE)、2'-O-胺基丙基(2'-O-AP)、2'-O-二甲胺基乙基(2'-O-DMAE)、2'-O-二甲胺基丙基(2'-O-DMAP)、2'-O-二甲胺基乙氧基乙基(2'-O-DMAEOE)、2'-O-N-甲基乙醯胺基(2'-O-NMA)修飾、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸，較佳為2'-去氧-2'-氟核苷酸、2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)及2'橋接核苷酸。The binding nucleic acid molecule of any one of claims 1 to 8, wherein the 2'-modified nucleotides are independently selected from the group consisting of: 2'-deoxy-2'-fluoro, 2'-O -Methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylamino Ethyl (2'-O-DMAE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O -DMAEOE), 2'-O-N-methylacetamido (2'-O-NMA) modification, 2'-deoxy-2'-fluoroarabinonucleotide (FANA) and 2' bridging nucleotide , preferably 2'-deoxy-2'-fluoronucleotides, 2'-deoxy-2'-fluoroarabinose nucleotides (FANA) and 2' bridging nucleotides. 如請求項9所述之結合核酸分子，其中該2'修飾之核苷酸為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)。The binding nucleic acid molecule of claim 9, wherein the 2'-modified nucleotide is 2'-deoxy-2'-fluoroarabinonucleotide (FANA). 如請求項1至10中任一項所述之結合核酸分子，其中該核酸分子為：
Figure 03_image024
其中核苷酸間鍵「s」係指硫代磷酸酯核苷酸間鍵；且 其中加下劃線之核苷酸為2'修飾之核苷酸，較佳為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)、2'-去氧-2'-氟核苷酸或2'橋接核苷酸(LNA)，更佳為2'-去氧-2'-氟阿拉伯糖核苷酸(FANA)，或其醫藥學上可接受之鹽。 The binding nucleic acid molecule of any one of claims 1 to 10, wherein the nucleic acid molecule is:
Figure 03_image024
wherein the internucleotide bond "s" refers to a phosphorothioate internucleotide bond; and wherein the underlined nucleotide is a 2' modified nucleotide, preferably 2'-deoxy-2'- Fluoroarabinonucleotide (FANA), 2'-deoxy-2'-fluoronucleotide or 2' bridged nucleotide (LNA), more preferably 2'-deoxy-2'-fluoroarabinose nucleus glycoside (FANA), or a pharmaceutically acceptable salt thereof. 一種醫藥組合物或獸用組合物，其包含如請求項1至11中任一項之結合核酸分子。A pharmaceutical or veterinary composition comprising the binding nucleic acid molecule of any one of claims 1 to 11. 如請求項12所述之醫藥組合物或獸用組合物，其中該醫藥組合物進一步包含額外治療劑，其較佳選自免疫調節劑，諸如免疫檢查點抑制劑(ICI)；基於T細胞之癌症免疫療法，諸如過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)；或習知化學治療劑、放射治療劑或抗血管生成劑；或靶向免疫毒素。The pharmaceutical composition or veterinary composition of claim 12, wherein the pharmaceutical composition further comprises an additional therapeutic agent, preferably selected from immunomodulatory agents, such as immune checkpoint inhibitors (ICIs); T cell-based Cancer immunotherapy, such as adoptive cell transfer (ACT), genetically modified T cells or engineered T cells, such as chimeric antigen receptor cells (CAR-T cells); or conventional chemotherapeutics, radiotherapeutics or anti-angiogenic agents; or targeted immunotoxins. 如請求項1至11中任一項所述之結合核酸分子或如請求項12或13所述之醫藥組合物或獸用組合物，其用作藥物。The binding nucleic acid molecule according to any one of claims 1 to 11 or the pharmaceutical composition or veterinary composition according to claim 12 or 13 for use as a medicament. 如請求項1至11中任一項所述之結合核酸分子或如請求項12或13所述之醫藥組合物或獸用組合物，其用於治療癌症。The binding nucleic acid molecule according to any one of claims 1 to 11 or the pharmaceutical composition or veterinary composition according to claim 12 or 13, for use in the treatment of cancer. 如請求項15所用之結合核酸分子或醫藥組合物或獸用組合物，其中該癌症係選自白血病、淋巴瘤、肉瘤、黑素瘤及頭頸癌、腎癌、卵巢癌、胰臟癌、前列腺癌、甲狀腺癌、肺癌、食道癌、乳癌、膀胱癌、腦癌、結腸直腸癌、肝癌及子宮頸癌。The binding nucleic acid molecule or pharmaceutical composition or veterinary composition as used in claim 15, wherein the cancer is selected from leukemia, lymphoma, sarcoma, melanoma and head and neck cancer, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer cancer, thyroid cancer, lung cancer, esophageal cancer, breast cancer, bladder cancer, brain cancer, colorectal cancer, liver cancer and cervical cancer. 如請求項15或16所用之結合核酸分子，其與較佳選自以下之額外治療劑組合：免疫調節劑，諸如免疫檢查點抑制劑(ICI)；基於T細胞之癌症免疫療法，諸如過繼細胞轉移(ACT)、經基因修飾之T細胞或經工程改造之T細胞，諸如嵌合抗原受體細胞(CAR-T細胞)；或習知化學治療劑、放射治療劑或抗血管生成劑；或靶向免疫毒素。A binding nucleic acid molecule as used in claim 15 or 16 in combination with an additional therapeutic agent preferably selected from the group consisting of: immunomodulatory agents, such as immune checkpoint inhibitors (ICIs); T cell based cancer immunotherapy, such as adoptive cells Transfer (ACT), genetically modified T cells, or engineered T cells, such as chimeric antigen receptor cells (CAR-T cells); or conventional chemotherapeutic, radiotherapeutic, or anti-angiogenic agents; or Targeted immunotoxins. 如請求項15至17中任一項所用之結合核酸分子或醫藥組合物或獸用組合物，其用於癌症治療中針對攜帶NAD +合成缺陷之腫瘤細胞的靶向作用，視情況腫瘤細胞進一步攜帶選自ERCC1或ATM缺陷之DNA修復路徑缺陷或IDH突變。 A binding nucleic acid molecule or a pharmaceutical composition or a veterinary composition as used in any one of claims 15 to 17, for targeting tumor cells with NAD + synthesis defects in cancer therapy, optionally the tumor cells further Carry a DNA repair pathway defect or IDH mutation selected from ERCC1 or ATM deficiency.
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