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WO2025049967A2 - Inhibition de pcbp2 pour améliorer l'efficacité d'une immunothérapie - Google Patents

Inhibition de pcbp2 pour améliorer l'efficacité d'une immunothérapie Download PDF

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WO2025049967A2
WO2025049967A2 PCT/US2024/044755 US2024044755W WO2025049967A2 WO 2025049967 A2 WO2025049967 A2 WO 2025049967A2 US 2024044755 W US2024044755 W US 2024044755W WO 2025049967 A2 WO2025049967 A2 WO 2025049967A2
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cancer
pcbp2
cells
antibody
inhibitor
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WO2025049967A3 (fr
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Christopher A. KLEBANOFF
Korbinian Nepomuk KROPP
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4268—MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4269—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
    • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • T cell-based immunotherapies including immune checkpoint inhibitors, have curative potential for the treatment of cancer and have shown clinical success in multiple malignancies. Nevertheless, a significant proportion of patients fail to respond to current immunotherapies or relapse after an initial response either due to defects in the immune cell compartment or due to tumor cell-intrinsic immune escape mechanisms.
  • the present disclosure provides a method for sensitizing a cancer patient to immunotherapy comprising administering to the cancer patient an effective amount of a PCBP2 inhibitor separately, sequentially or simultaneously with the -1- 4856-7271-5486.1 Atty. Dkt.
  • the immunotherapy may comprise immune checkpoint blockade therapy and/or an adoptive cell therapeutic composition comprising T cells.
  • the adoptive cell therapeutic composition comprises one or more of tumor infiltrating T cells, CD8+ T cells, CD4+ T cells, delta-gamma T-cells, and alpha-beta T-cells.
  • the T cells may comprise a native TCR or a heterologous TCR.
  • the native TCR or the heterologous TCR is HLA-I restricted or HLA- II restricted.
  • the adoptive cell therapeutic composition is obtained from an autologous donor or allogeneic donor.
  • the cancer patient has previously received immunotherapy.
  • the cancer patient is resistant or non-responsive to immunotherapy.
  • the immune checkpoint blockade therapy comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB antibody, an anti-CD73 antibody, an anti-GITR antibody, and an anti-LAG-3 antibody.
  • immune checkpoint blockade therapy examples include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, ticlimumab, JTX-4014, Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), Dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, AMP-514, KN035, CK-301, AUNP12, CA-170, or BMS-986189.
  • PDR001 Spartalizumab
  • SHR1210 Camrelizumab
  • Sintilimab IBI308
  • Tislelizumab B
  • the PCBP2 inhibitor reduces the expression and/or activity of PCBP2 mRNA or PCBP2 polypeptides including exon 8 of PCBP2.
  • the PCBP2 inhibitor is a small molecule, a PCBP2-specific inhibitory nucleic acid, or a PROTAC that specifically targets PCBP2.
  • the PCBP2-specific inhibitory nucleic acid may be a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.
  • the PROTAC that specifically targets PCBP2 comprises an E3 ubiquitin ligase binding moiety (“ULM”) selected from among an IAP E3 ubiquitin ligase binding moiety (an “ILM”), a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM), and a mouse double minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM).
  • EMLM E3 ubiquitin ligase binding moiety
  • PROTAC that specifically targets PCBP2 comprises a PCBP2 binding moiety (PBM) selected from among HNRPK, PTBP1, and HNRNPL.
  • PBM PCBP2 binding moiety
  • the PBM is coupled to a ULM directly or via a chemical linker.
  • the PCBP2 inhibitor is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.
  • the immune checkpoint blockade therapy and/or the adoptive cell therapeutic composition is administered pleurally, parenterally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.
  • the cancer is selected from among an HLA class-II expressing cancer, melanoma, breast cancer, cervical cancer, adrenal cancer, bladder cancer, bone cancer, brain cancer, carcinoma, colon cancer, colorectal cancer, corpus uterine cancer, ear, nose and throat (ENT) cancer, endometrial cancer, esophageal cancer, gastrointestinal cancer, glioblastoma, head and neck cancer, intestinal cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, mesothelioma, nasopharynx cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, penile cancer, pharynx cancer, prostate cancer, rectal cancer, sarcoma, seminoma, stomach cancer, teratoma, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vascular tumor, and metastases thereof.
  • HLA class-II expressing cancer melanoma, breast cancer, cervical cancer, adrenal cancer, bladder cancer, bone cancer, brain
  • the methods of the present technology further comprise administering a cytokine to the cancer patient.
  • the cytokine may be administered prior to, during, or subsequent to administration of the adoptive cell therapeutic composition or the immune checkpoint blockade therapy.
  • the cytokine is selected from a group consisting of interferon a, interferon ⁇ , interferon ⁇ , complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCRIO, CCR2, CCR5, CCR6, CCR7, CCR8, CCRLl, CCRL2, CX3CL1, CX3CR,
  • FIGs.1A-1B Whole genome CRISPR knockout screen identifies knockout of PCBP2 as a sensitizer to killing mediated by CD4+ T cells expressing an HLA-II restricted TCR.
  • FIG.1A Schematic overview of the whole genome CRISPR knockout screen.
  • the melanoma cell line A375 was modified to stably express Cas9.
  • A375/Cas9 was infected with the whole genome Brunello CRISPR knockout library and subsequently challenged with either control CD4+ T cells or CD4+ T-cells expressing the HLA-II restricted TCR 6F9 recognizing the tumor- associated antigen MAGE-A3/A6.
  • FIG.1B Volcano plot depicting depleted and enriched sgRNAs conferring sensitization or resistance to CD4+ T-cell killing, respectively.
  • FIG.2. Focused CRISPR knockout screen confirms PCBP2 as a sensitizer to killing mediated by CD4+ T cells expressing an HLA-II restricted TCRs.
  • a custom made CRISPR knockout library (4 sgRNAs per gene) was generated targeting the 200 most significantly depleted and enriched genes discovered in the whole genome screen shown in FIG.1.
  • the melanoma cell lines A375 and SK-MEL-130, the cervical cancer cell line HeLa and the breast cancer cell line MB-468 were modified to stably express Cas9.
  • Cas9- expressing tumor cells were infected with the focused library and subsequently challenged with either control CD4+ T cells or CD4+ T cells expressing the HLA-II restricted TCR 6F9 recognizing the tumor-associated antigen MAGE-A3/A6 (A375 and SK-MEL-130) or expressing the HLA-II restricted TCR TA10 recognizing the tumor-antigen KK-LC-1 (HeLa and MB-468). Volcano plots depict depleted and enriched sgRNAs conferring sensitization or resistance to CD4+ T-cell killing, respectively. [0015] FIGs.3A-3B.
  • Genomic deletion of PCBP2 sensitizes tumor cells to killing mediated by CD4+ T cells expressing an HLA-II restricted TCR in in vitro cytotoxicity assays.
  • the indicated melanoma cell lines stably expressing a nuclear localized mCherry reporter were electroporated with CRISPR/Cas9 RNPs targeting either the safer harbor locus AAVS1 or PCBP2.
  • Knockout of PCBP2 was confirmed by ICE analysis (inference of CRISPR edits) and the indel rate is indicated.
  • FIG.3A Edited tumor cells were exposed to either control CD4+ T cells or CD4+ T cells expressing the HLA-II restricted TCR 6F9 and -4- 4856-7271-5486.1 Atty.
  • FIG.3B Fold sensitization relative to control CD4+ T cell is shown. Data points represent technical replicates of three different T-cell donors. Error bars indicate SD. Statistical analysis was performed by an unpaired t- test. [0016] FIGs.4A-4B. Disruption of the HLA class I antigen presentation pathway impairs HLA class I but not HLA class II expression.
  • FIG.4A The indicated sgRNA guide sequences (represented as SEQ ID NOs: 1-12 in order of appearance) targeting components of the HLA class I antigen presentation pathway (B2M, TAP1, TAP2, TAPBP) or targeting the HLA class II master transcription factor CIITA were cloned into the lentiviral plasmid lentiCRISPR v2 (Addgene: #52961).
  • FIG.4B A375 melanoma knockout cell lines were generated by lentiviral transduction and were selected with puromycin for at least 7 days prior to experiments. Cell lines were stained for HLA class I and HLA class II expression using the monoclonal antibodies W6/32-FITC or Tu39-APC, respectively.
  • FIGs.5A-5B Disruption of the HLA class I antigen presentation pathway impairs cytolysis by tumor specific HLA class I-restricted CD8 + T cell but not by HLA class II-restricted CD4 + T cells.
  • mCherry + A375 melanoma cells were generated by retroviral transduction of a nuclear localized mCherry reporter.
  • CD8 + T cells were transduced with the HLA-A*02:01-restricted TCR 1G4 recognizing a peptide processed from NY-ESO-1 and CD4 + T cells were transduced with the HLA-DP*04:01-restricted TCR 6F9 recognizing a peptide processed from MAGE-A3/A6.
  • FIG.5A Representative Incucyte data after 48 h of co-culture.
  • FIG.5B Cytolysis after 48 h averaged across two sgRNAs. Data points represent technical replicates. Error bars indicate SD. Statistical analysis was performed by a one-way ANOVA test.
  • FIGs.6A-6D Multiple PCBP2 splice variants are expressed in cancer cells.
  • FIG.6A Exon/Intron structure of PCBP2 full length (PCBP2 fl) and PCBP2 lacking coding Exon 8 (PCBP2 ⁇ E8).
  • FIG.6B A375 melanoma cells were edited by electroporation with CRISPR/Cas9 RNPs targeting either the safe harbor locus AAVS1 or -5- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 PCBP2 coding Exon 2. Western Blot showing presence of multiple PCBP2 isoforms in control tumor cells and deletion of both PCBP2 fl and PCBP2 ⁇ E8 in PCBP2 knockout tumor cells.
  • FIG.6C Domain structure of PCBP2 fl and PCBP2 ⁇ E8.
  • FIG.6D Protein structure prediction of PCBP2 fl and PCBP2 ⁇ E8 using AlphaFold (Jumper J. et al., Nature, 2021).
  • FIGs.7A-7B Genomic deletion of PCBP2 primes tumor cells for apoptosis induced by T cell-derived inflammatory cytokines. Induction of apoptosis was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive for active Caspase 3/7) over time.
  • FIG.7A Conditioned media (CM) was generated by culturing parental A375 melanoma cells with control CD4+ T cells or CD4+ T cells expressing the HLA-II restricted TCR 6F9. CM was added to A375 melanoma cells edited at the AAVS1 or PCBP2 locus in the presence or absence of TNF- and IFN ⁇ - neutralizing antibodies, and induction of apoptosis was assessed. Left, experimental setup. Right, exemplary Incucyte images.
  • FIG.7B A375 melanoma cells edited at the AAVS1 or PCBP2 locus were exposed to recombinant human TNF (100 ng/ml) and IFN ⁇ (100 ng/ml), and induction of apoptosis was assessed. Left, experimental setup. Right, exemplary Incucyte images.
  • a clonal A375 melanoma cell line lacking expression of endogenous PCBP2 was generated by electroporation with a CRISPR/Cas9 RNP targeting PCBP2 followed by single cell-cloning.
  • N-terminally FLAG- tagged full length (fl) PCBP2 cDNA, PCBP2 cDNA lacking coding Exon 8 ( ⁇ E8), or an empty control vector was retrovirally expressed in A375 PCBP2 null cells.
  • FIG.8A Expression of the FLAG tag was confirmed by intracellular flow cytometry.
  • FIG.8B Expression of PCBP2 fl and PCBP2 ⁇ E8, and lack of PCBP2 expression in A375 null cells was confirmed by Western Blot.
  • FIGs.8C-8D Induction of apoptosis was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive -6- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 for active Caspase 3/7) over time. A375 PCBP2 null cells modified as indicated were exposed to recombinant human TNF (100 ng/ml) and IFN ⁇ (100 ng/ml), and induction of apoptosis was assessed.
  • FIG.8C exemplary Incucyte images.
  • FIG.8D Incucyte data over time. Data points show mean of technical replicates. Error bars represent SD.
  • FIGs.9A-9D Selective deletion of PCBP2 full length using Cas9 phenocopies re-expression of PCBP2 ⁇ E8 cDNA in A375 PCBP2 null cells. A375 melanoma cells were edited by electroporation with CRISPR/Cas9 RNPs targeting either the safe harbor locus AAVS1, PCBP2 coding Exon 2 or PCBP2 coding Exon 8.
  • FIG.9A Schematic overview of the CRISPR/Cas9 knockout strategy.
  • RNA was -7- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 isolated from edited-tumor cells and transcribed into cDNA.
  • Graphs show fold expression of PCBP2 fl (left) or PCBP2 ⁇ E8 (right) in PCBP2 ⁇ E8 and CD71 knockdown tumor cells relative to control tumor cells (NTC RfxCas13d guide) normalized to HPRT1 expression using the 2 – ⁇ Ct method. Data points show mean of technical replicates. Error bars represent SD.
  • Statistical analysis was performed by a one-way ANOVA test using background-editing (CD71 guide) as control. One exemplary experiment of two with similar results is shown.
  • FIG.10C Induction of apoptosis in RfxCas13d-transduced tumor cells upon exposure to recombinant human TNF (1 ng/ml) and IFN ⁇ (1 ng/ml) was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive for active Caspase 3/7) over time. Data points show mean of technical replicates. Error bars represent SD. Statistical analysis was performed on the 72 h data points by a one-way ANOVA test using background-editing (CD71 guide) as control. One exemplary experiment of two with similar results is shown. [0023] FIGs.11A-11B.
  • Cytokine responsiveness is selectively regulated by the RNA-binding domain KH3 of PCBP2 ⁇ E8.
  • A375 melanoma cells stably expressing a nuclear localized mCherry reporter were electroporated with a CRISPR/Cas9 RNP targeting PCBP2 followed by single cell-cloning to generate A375/mCherry PCBP2 null cells.
  • N- terminally FLAG-tagged PCBP2 cDNA lacking coding Exon 8 ( ⁇ E8) harboring the GXXG -> GDDG mutation in the indicated KH domain(s), or an empty control vector was retrovirally expressed in A375/mCherry PCBP2 null cells.
  • FIG.11A Expression of the FLAG tag was confirmed by intracellular flow cytometry.
  • FIG.11B Induction of apoptosis upon exposure to recombinant human TNF (100 ng/ml) and IFN ⁇ (100 ng/ml) was assessed after 48 h in an Incucyte instrument by measuring green object count per field (corresponding to tumor cells positive for active Caspase 3/7) relative to red object count per field (corresponding to the number of tumor cells per field). Left, relative apoptosis as measured for individual KH-domain mutants.
  • HLA class-II expression is found in diverse cancer types, including ⁇ 30% of melanomas (Johnson et al.2016), and its expression is differently regulated than HLA class- I.
  • the presently disclosed subject matter provides methods for enhancing the efficacy of immunotherapy in cancers, wherein inhibition of the RNA-binding protein PCBP2 is combined with immunotherapy (e.g., with immune checkpoint blockade or adoptive cell therapeutic composition) to treat HLA expressing cancers, including but not limited to melanoma, breast cancer, and cervical cancer.
  • immunotherapy e.g., with immune checkpoint blockade or adoptive cell therapeutic composition
  • HLA expressing cancers including but not limited to melanoma, breast cancer, and cervical cancer.
  • a cell includes a combination of two or more cells, and the like.
  • nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.
  • the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).
  • the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, or topically. Administration includes self- administration and the administration by another.
  • “adoptive cell therapeutic composition” refers to any composition comprising cells suitable for adoptive cell transfer. In exemplary embodiments, the adoptive cell therapeutic composition comprises a cell type selected from a group -10- 4856-7271-5486.1 Atty. Dkt.
  • the adoptive cell therapeutic composition comprises a cell type selected from a group consisting of T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T-cells, regulatory T-cells and peripheral blood mononuclear cells.
  • TILs, T-cells, CD8+ cells, CD4+ cells, NK-cells, delta-gamma T- cells, regulatory T-cells or peripheral blood mononuclear cells form the adoptive cell therapeutic composition.
  • the adoptive cell therapeutic composition comprises T cells.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.”
  • sequence “5'-A-G-T-3'” is complementary to the sequence “3'-T-C-A-5.”
  • Certain bases not commonly found in naturally-occurring nucleic acids may be included in the nucleic acids described herein. These include, for example, inosine, 7- deazaguanine, Locked Nucleic Acids (LNA), and Peptide Nucleic Acids (PNA).
  • Complementarity need not be perfect; stable duplexes may contain mismatched base pairs, degenerative, or unmatched bases.
  • Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs.
  • a complementary sequence can also be an RNA sequence complementary to the DNA sequence or its complementary sequence, and can also be a cDNA.
  • a “control” is an alternative sample used in an experiment for comparison purpose.
  • a control can be “positive” or “negative.”
  • a positive control a compound or composition known to exhibit the desired therapeutic effect
  • a negative control a subject or a sample that does not receive the therapy or receives a placebo
  • the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and/or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein.
  • the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein.
  • a “therapeutically effective amount” of a composition refers to composition levels in which the physiological effects of a disease or condition are ameliorated or eliminated.
  • a therapeutically effective amount can be given in one or more administrations.
  • “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and/or other modifications of the translation product, if required for proper expression and function.
  • the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.
  • “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleobase or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.
  • a polynucleotide or polynucleotide region has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, -12- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.
  • This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment.
  • One alignment program is BLAST, using default parameters.
  • Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity.
  • hybridize refers to a process where two substantially complementary nucleic acid strands (at least about 65% complementary over a stretch of at least 14 to 25 nucleotides, at least about 75%, or at least about 90% complementary) anneal to each other under appropriately stringent conditions to form a duplex or heteroduplex through formation of hydrogen bonds between complementary base pairs. Nucleic acid hybridization techniques are well known in the art.
  • Hybridization and the strength of hybridization is influenced by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, and the thermal melting point (Tm) of the formed hybrid.
  • Tm thermal melting point
  • the bases are arranged on the backbone in such a way that they can bind with a nucleic acid having a sequence of bases that are complementary to the bases of the oligonucleotide.
  • the most common oligonucleotides have a backbone of sugar phosphate units.
  • Oligonucleotides may also include derivatives, in which the hydrogen of the hydroxyl group is replaced with organic groups, e.g., an allyl group.
  • the oligonucleotide may be modified e.g., by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides.
  • pharmaceutically-acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20 th edition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).
  • polynucleotide or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA.
  • Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions.
  • polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
  • polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins.
  • Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
  • Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature.
  • “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to the untreated control sample.
  • sample refers to clinical samples obtained from a subject.
  • Biological samples may include tissues, cells, protein or membrane extracts of cells, mucus, sputum, bone marrow, bronchial alveolar lavage (BAL), bronchial wash (BW), and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids (blood, plasma, saliva, urine, serum etc.) present within a subject. -15- 4856-7271-5486.1 Atty. Dkt.
  • BAL bronchial alveolar lavage
  • BW bronchial wash
  • biological fluids e.g., ascites fluid or cerebrospinal fluid (CSF)
  • oligonucleotide sequence of the oligonucleotide has at least 12 bases of sequence identity with a portion of a target nucleic acid when the oligonucleotide and the target nucleic acid are aligned.
  • An oligonucleotide that is specific for a target nucleic acid is one that, under the stringent hybridization or washing conditions, is capable of hybridizing to the target nucleic acid of interest and not substantially hybridizing to nucleic acids which are not of interest.
  • sequence identity is desirable and include at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 98% sequence identity.
  • stringent hybridization conditions refers to hybridization conditions at least as stringent as the following: hybridization in 50% formamide, 5xSSC, 50 mM NaH 2 PO 4 , pH 6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5x Denhart's solution at 42 o C. overnight; washing with 2x SSC, 0.1% SDS at 45 o C; and washing with 0.2x SSC, 0.1% SDS at 45 o C.
  • target sequence and “target nucleic acid sequence” refer to a specific nucleic acid sequence to be modulated (e.g., inhibited or downregulated).
  • therapeutic agent is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.
  • Treating”, “treat”, or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.
  • the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved.
  • the treatment may be a continuous prolonged -16- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.
  • ubiquitin ligase refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein, targeting the substrate protein for degradation.
  • an E3 ubiquitin ligase protein that alone or in combination with an E2 ubiquitin-conjugating enzyme causes the attachment of ubiquitin to a lysine on a target protein, and subsequently targets the specific protein substrates for degradation by the proteasome.
  • E3 ubiquitin ligase alone or in complex with an E2 ubiquitin conjugating enzyme is responsible for the transfer of ubiquitin to targeted proteins.
  • the ubiquitin ligase is involved in polyubiquitination such that a second ubiquitin is attached to the first; a third is attached to the second, and so forth. Polyubiquitination marks proteins for degradation by the proteasome.
  • ubiquitination events that are limited to mono-ubiquitination, in which only a single ubiquitin is added by the ubiquitin ligase to a substrate molecule.
  • Mono-ubiquitinated proteins are not targeted to the proteasome for degradation, but may instead be altered in their cellular location or function, for example, via binding other proteins that have domains capable of binding ubiquitin.
  • different lysines on ubiquitin can be targeted by an E3 to make chains. The most common lysine is Lys48 on the ubiquitin chain. This is the lysine used to make polyubiquitin, which is recognized by the proteasome.
  • PCBP2 Inhibitors of the Present Technology provides therapeutic agents that inhibit the activity or expression of PCBP2.
  • the PCBP2 inhibitor is a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), an antibody (e.g., a neutralizing antibody) or a PROTAC that specifically targets PCBP2.
  • an inhibitory nucleic acid e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes
  • an antibody e.g., a neutralizing antibody
  • PROTAC e.g., a PROTAC that specifically targets PCBP2.
  • Exemplary mRNA sequences of PCBP2 are provided below, represented by SEQ ID NOs: 13-19.
  • NM_001098620.3 Homo sapiens poly(rC) binding protein 2 (PCBP2), transcript variant 3, mRNA (SEQ ID NO: 13) CCCAGACCAGCAGAGGCAGCAGCCGGAGCAGCCGCAGCCTGCGCCCTCTCCCGCCCGCCCTCCGCC CGCCCGCCCTCCACCCGCCCCGGGGTCTCTTTCCCCCTTCCTCCTCCTCCTCCTCC ACCCCCCCTTCCTCCTCCGCCCGCCCGCGGGGCCCCCCTCGCCTTCCCGCCCGCCCCTATTGTTCCGCCC CCGGCCTCCCGCCCTTCCTTCCCGCCCGCTCCCCTTTTCCCCTCAGTCGCCTCGCGCCTGCAGTTTTT GGCTTTCACCCCCAACCAGTGACCAAAGACTTGACCACTCAAAGTCCAGCTCCCCAGAACACTGCTCGAC -17- 4856-7271-5486.1 Atty.
  • PCBP2 poly(rC) binding protein 2
  • transcript variant 3 mRNA (SEQ ID NO: 13) CCCAGACCAG
  • the present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 13-19 (PCBP2 mRNA), thereby reducing or inhibiting expression of PCBP2.
  • the antisense nucleic acid may be antisense RNA, or antisense DNA.
  • Antisense nucleic acids based on the known PCBP2 gene sequence can be readily designed and engineered using methods known in the art.
  • the antisense nucleic acid comprises a nucleic acid sequence that binds to exon 8 of any one of SEQ ID NOs: 13-19 or a complement thereof.
  • Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire PCBP2 coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame).
  • the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the mRNA coding region of PCBP2.
  • an antisense nucleic acid molecule can be complementary to a noncoding region of the PCBP2 coding strand.
  • the noncoding region refers to the 5′ and 3′ untranslated regions that flank the coding region and are not translated into amino acids.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of PCBP2.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • an antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-2-thouridine, 5-carboxymethylaminometh-yluracil, -24- 4856-7271-5486.1 Atty. Dkt.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • the antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule.
  • nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res.15:6625- 6641(1987)).
  • the antisense nucleic acid molecule can also comprise a 2′-O - methylribonucleotide (Inoue et al., Nucleic Acids Res.15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).
  • the present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 13-19 (mRNA of -25- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 PCBP2), thereby reducing or inhibiting expression of PCBP2.
  • shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length.
  • Double-stranded RNA can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos.
  • RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA.
  • a double-stranded siRNA or shRNA molecule is engineered to complement and hybridize to a mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA-induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as mRNA of PCBP2).
  • RISC RNA-induced silencing complex
  • the shRNA or siRNA comprises a nucleic acid sequence that binds to exon 8 of any one of SEQ ID NOs: 13-19 or a complement thereof.
  • the present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 13-19 (PCBP2 mRNA), thereby reducing or inhibiting expression of PCBP2.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single-stranded nucleic acid, such as an mRNA.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))
  • a ribozyme having specificity for a nucleic acid encoding PCBP2 can be designed based upon a nucleic acid sequence of PCBP2.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA encoding PCBP2.
  • mRNA of a PCBP2 can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418, incorporated herein by reference.
  • the present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 13-19 (mRNA of PCBP2).
  • Guide RNAs for use in CRISPR-Cas systems are typically generated as a single guide RNA comprising a crRNA -26- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 segment and a tracrRNA segment.
  • the crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules.
  • the crRNA segment comprises the targeting sequence that binds to a portion of any one of SEQ ID NOs: 13-19, and a stem portion that hybridizes to a tracrRNA.
  • the tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme.
  • the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, the crRNA segment and the tracrRNA segment are provided as separate RNAs.
  • the combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO2015/089465.
  • a synthetic guide RNA is a single RNA represented as comprising the following elements: 5 ⁇ -X1-X2-Y-Z-3 ⁇ [0072] where X1 and X2 represent the crRNA segment, where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 13-19, X2 is a stem sequence the hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence.
  • the linker sequence comprises two or more nucleotides and links the crRNA and tracrRNA segments.
  • the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA.
  • a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5 ⁇ -X1-X2-3 ⁇ where X1 is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 13-19, X2 is a stem sequence the hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.
  • exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO/2015/089465, which is incorporated by reference herein.
  • a -27- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to the tracrRNA.
  • degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the stem sequence or the complementary sequence in the tracrRNA.
  • the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the tracrRNA has additional complementary sequences that form hairpins.
  • the tracrRNA has at least two or more hairpins.
  • the tracrRNA has two, three, four or five hairpins.
  • the tracrRNA has at most five hairpins. [0075] In a hairpin structure, the portion of the sequence 5 ⁇ of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3 ⁇ of the loop corresponds to the tracrRNA sequence.
  • single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5 ⁇ to 3 ⁇ ), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator: (a) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTTTT (SEQ ID NO: 20); (b) NNNNNNNNNNNNNNNNNNNN
  • the sgRNA comprises a nucleic acid sequence that binds to exon 8 of any one of SEQ ID NOs: 13-19 or a complement thereof.
  • Selection of suitable oligonucleotides for use in as a targeting sequence in a CRISPR Cas system depends on several factors including the particular CRISPR enzyme to be used and the presence of corresponding proto-spacer adjacent motifs (PAMs) downstream of the target sequence in the target nucleic acid.
  • PAMs proto-spacer adjacent motifs
  • a suitable PAM is 5'- NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
  • the PAM sequences should be present between about 1 to about 10 nucleotides of the target sequence to generate efficient cleavage of the target nucleic acid.
  • the guide RNA forms a complex with the CRISPR enzyme, the complex locates the target and PAM sequence, unwinds the DNA duplex, and the guide RNA anneals to the complementary sequence on the opposite strand. This enables the Cas9 nuclease to create a double-strand break.
  • CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure.
  • the CRISPR enzyme is a Type II CRISPR enzyme.
  • the CRISPR enzyme catalyzes DNA cleavage.
  • the CRISPR enzyme catalyzes RNA cleavage.
  • the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, -29- 4856-7271-5486.1 Atty. Dkt.
  • the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site.
  • the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.
  • the present disclosure provides PCBP2 inhibitors comprising an E3 ubiquitin ligase binding moiety (“ULM”) that is an IAP E3 ubiquitin ligase binding moiety (an “ILM”), a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM), and/or a mouse double minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM).
  • ULM E3 ubiquitin ligase binding moiety
  • ILM IAP E3 ubiquitin ligase binding moiety
  • CLM Cerblon E3 ubiquitin ligase binding moiety
  • VHL Von Hippel-Lindae E3 ubiquitin ligase binding moiety
  • MDM2 mouse double minute 2 homologue
  • ULMs useful in the bifunctional or multifunctional PCBP2 inhibitors (e.g., PROTACs) of the present technology are disclosed in US Patent No.11707452, the contents of which are incorporated herein in their entirety.
  • the ULM is coupled to a target protein binding moiety via a chemical linker (L) according to the structure: [0081] PBM-L-ULM (A), [0082] wherein L is a bond or a chemical linker group, ULM is a E3 ubiquitin ligase binding moiety, and PBM is a PCBP2 binding moiety.
  • ULM is inclusive of all ULMs, including those that bind IAP (i.e., ILMs), MDM2 (i.e., MLM), cereblon (i.e., CLM), and VHL (i.e., VLM).
  • the term ILM is inclusive of all possible IAP E3 ubiquitin ligase binding moieties
  • the term MLM is inclusive of all possible MDM2 E3 ubiquitin ligase binding moieties
  • the term VLM is inclusive of all possible VHL binding moieties
  • the term CLM is inclusive of all cereblon binding moieties.
  • the present disclosure provides bifunctional or multifunctional PCBP2 inhibitors (e.g., PROTACs) useful for regulating protein activity by inducing the degradation of PCBP2 protein.
  • the PCBP2 inhibitor comprises an -30- 4856-7271-5486.1 Atty. Dkt.
  • a PCBP2 protein i.e., a PCBP2 targeting moiety or a “PBM”.
  • the ILM/VLM/CLM/MLM and PBM are joined or coupled via a chemical linker (L).
  • L chemical linker
  • the ILM binds the IAP E3 ubiquitin ligase
  • the VLM binds VHL.
  • CLM binds the cereblon E3 ubiquitin ligase
  • MLM binds the MDM2 E3 ubiquitin ligase
  • the PBM recognizes PCBP2 protein and the interaction of the respective moieties with their targets facilitates the degradation of the PCBP2 protein by placing the PCBP2 protein in proximity to the ubiquitin ligase protein.
  • An exemplary bifunctional PCBP2 inhibitor can be depicted as: [0085] PBM-ILM (B) [0086] PBM-CLM (C) [0087] PBM-VLM (D) [0088] PBM-MLM (E) [0089]
  • the bifunctional PCBP2 inhibitor further comprises a chemical linker (“L”).
  • the bifunctional PCBP2 inhibitor can be depicted as: [0090] PBM-L-ILM (F) [0091] PBM-L-CLM (G) [0092] PBM-L-VLM (H) [0093] PBM-L-MLM (I), [0094] wherein the PBM is a PCBP2 protein/polypeptide targeting moiety, the L is a chemical linker, the ILM is a IAP E3 ubiquitin ligase binding moiety, the CLM is a cereblon E3 ubiquitin ligase binding moiety, the VLM is a VHL binding moiety, and the MLM is a MDM2 E3 ubiquitin ligase binding moiety.
  • the ULM (e.g., a ILM, a CLM, a VLM, or a MLM) shows activity or binds to the E3 ubiquitin ligase (e.g., IAP E3 ubiquitin ligase, cereblon E3 ubiquitin ligase. VHL, or MDM2 E3 ubiquitin ligase) with an IC50 of less than about 200 ⁇ M.
  • the IC 50 can be determined according to any method known in the art, e.g., a fluorescent polarization assay. -31- 4856-7271-5486.1 Atty. Dkt.
  • the bifunctional PCBP2 inhibitors described herein demonstrate an activity with an IC50 of less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 mM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 ⁇ M, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 nM, or less than about 100, 50, 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001 ⁇ M.
  • the PCBP2 inhibitors as described herein comprise multiple PBMs, one or more ULMs (i.e., moieties that bind specifically to multiple/different E3 ubiquitin ligase, e.g., VHL, IAP, cereblon, and/or MDM2) or a combination thereof.
  • the PBMs and ULMs e.g., ILM. VLM, CLM, and/or MLM
  • the PBMs and ULMs can be coupled directly or via one or more chemical linkers or a combination thereof.
  • the ULMs can be for the same E3 ubiquintin ligase or each respective ULM can bind specifically to a different E3 ubiquitin ligase.
  • a PCBP2 inhibitor has multiple PBMs.
  • PBMs useful in the bifunctional or multifunctional PCBP2 inhibitors (e.g., PROTACs) of the present technology include HNRPK, PTBP1, and HNRNPL.
  • the PCBP2 inhibitor comprises multiple ULMs, the ULMs are identical.
  • the PCBP2 inhibitor comprising a plurality of ULMs (e.g., ULM, ULM′, etc.), at least one PBM coupled to a ULM directly or via a chemical linker (L) or both.
  • the PCBP2 inhibitor comprising a plurality of ULMs further comprises multiple PBMs.
  • the PBMs are the same or, optionally, different.
  • the PCBP2 inhibitor may comprise a plurality of ULMs.
  • the PCBP2 inhibitor comprising at least two different ULMs, and/or a plurality of ULMs further comprises at least one PBM coupled to a ULM directly or via a chemical linker or both.
  • the T cell is a CD8 + T cell.
  • the presently disclosed adoptive cell therapeutic compositions of the present technology may further include at least one recombinant or exogenous co-stimulatory ligand.
  • the presently disclosed adoptive cell therapeutic compositions can be further transduced with at least one co- stimulatory ligand, such that the immune cells co- express or are induced to co-express the at least one co-stimulatory ligand.
  • ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands.
  • TNF tumor necrosis factor
  • Ig immunoglobulin
  • TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells.
  • TNF superfamily share a number of common features. The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region.
  • immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins — they possess an immunoglobulin domain (fold).
  • Immunoglobulin superfamily ligands include, but are not limited to, CD80 and CD86, both ligands for CD28, PD-L1/(B7-H1) that ligands for PD-1.
  • the at least one co-stimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.
  • the presently disclosed adoptive cell therapeutic compositions can further comprise at least one exogenous cytokine.
  • a presently disclosed adoptive cell therapeutic composition can be further transduced with at least one cytokine, such that the adoptive cell therapeutic compositions secrete the at least one cytokine.
  • the at least one cytokine is selected from the group consisting of IL-2, IL- 3, IL-6, IL-7, IL-11, IL-12, IL-15, IL-17, and IL-21.
  • the cytokine is IL-12.
  • Monoclonal antibodies are particularly useful for identifying markers associated with particular cell lineages and/or stages of differentiation for both positive and negative selections.
  • a large proportion of terminally differentiated cells can be initially removed by a relatively crude separation.
  • magnetic bead separations can be used initially to remove large numbers of irrelevant cells.
  • at least about 80%, usually at least 70% of the total hematopoietic cells will be removed prior to cell isolation.
  • Procedures for separation include, but are not limited to, density gradient centrifugation; resetting; coupling to particles that modify cell density; magnetic separation with antibody-coated magnetic beads; affinity chromatography; cytotoxic agents joined to or used in conjunction with a mAb, including, but not limited to, complement and cytotoxins; and panning with antibody attached to a solid matrix, e.g., plate, chip, elutriation or any other convenient technique.
  • Techniques for separation and analysis include, but are not limited to, flow cytometry, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, impedance channels.
  • the cells can be selected against dead cells, by employing dyes associated with dead cells such as propidium iodide (PI).
  • the cells are collected in a medium comprising 2% fetal calf serum (FCS) or 0.2% bovine serum albumin (BSA) or any other suitable, preferably sterile, isotonic medium.
  • the adoptive cell therapeutic compositions comprise one or more additional modifications.
  • the adoptive cell therapeutic compositions comprise and express (is transduced to express) a chimeric co- stimulatory receptor (CCR).
  • CCR is described in Krause et al. (1998) J. Exp. Med. -35- 4856-7271-5486.1 Atty.
  • CCRs mimic co-stimulatory signals, but unlike, engineered receptors, do not provide a T-cell activation signal, e.g., CCRs lack a CD3 ⁇ polypeptide.
  • CCRs provide co-stimulation, e.g., a CD28-like signal, in the absence of the natural co-stimulatory ligand on the antigen-presenting cell.
  • a combinatorial antigen recognition i.e., use of a CCR in combination with an engineered receptor, can augment T-cell reactivity against the dual- antigen expressing T cells, thereby improving selective tumor targeting.
  • These methods often involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick in a target DNA sequence such that repair of the break by an error born process such as non-homologous end joining (NHEJ) or repair using a repair template (homology directed repair or HDR) can result in the knock out of a gene or the insertion of a sequence of interest (targeted integration).
  • DSB double strand break
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • the adoptive cell therapeutic compositions are modified to disrupt or reduce expression of an endogenous T-cell receptor gene (see, e.g. WO 2014153470, which is incorporated by reference in its entirety).
  • the adoptive cell therapeutic compositions are modified to result in disruption or inhibition of PD1, PDL-1 or CTLA-4 (see, e.g. U.S.
  • the adoptive cell therapeutic compositions provided herein express a T-cell receptor (TCR) or other cell-surface ligand that binds to a target antigen, such as a tumor antigen.
  • T cell receptor is a wild-type or native T-cell receptor.
  • the TCR is an engineered receptor or a non-native receptor.
  • the engineered receptor is an engineered TCR (eTCR). In some embodiments, the engineered receptor is a chimeric antibody TCR (caTCR). In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR).
  • the adoptive cell therapeutic compositions provided herein express a native receptor, a non-native receptor, or an engineered receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a tumor antigen.
  • the adoptive cell therapeutic compositions provided herein express a native receptor, a non-native receptor, or an engineered receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a tumor antigen presented in the context of an MHC molecule.
  • the adoptive cell therapeutic compositions provided herein express a native receptor, a non-native receptor or an engineered receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a tumor antigen presented in the context of an HLA-A2 molecule.
  • the adoptive cell therapeutic compositions provided herein express a native receptor, a non-native receptor or an engineered receptor (e.g., a CAR, caTCR, or eTCR) or other cell-surface ligand that binds to a tumor antigen.
  • a native receptor e.g., a CAR, caTCR, or eTCR
  • an engineered receptor e.g., a CAR, caTCR, or eTCR
  • other cell-surface ligand that binds to a tumor antigen.
  • compositions comprising a PCBP2 inhibitor as described herein.
  • the pharmaceutical compositions of the present disclosure may be prepared by any of the methods known in the pharmaceutical arts.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated and the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.
  • compositions of the present technology may contain one or more pharmaceutically-acceptable carriers, which as used herein, generally refers to a pharmaceutically-acceptable composition, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.
  • pharmaceutically-acceptable carriers such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, useful for introducing the active agent into the body.
  • aqueous and non-aqueous carriers examples include, for example, water, -38- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate), and suitable mixtures thereof.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • auxiliary agents such as wetting agents, emulsifiers, lubricants (e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, release agents, coating agents, sweetening agents, flavoring agents, preservative agents, and antioxidants can also be included in the pharmaceutical composition of the present technology.
  • antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecit
  • the pharmaceutical formulation includes an excipient selected from, for example, celluloses, liposomes, micelle-forming agents (e.g., bile acids), and polymeric carriers, e.g., polyesters and polyanhydrides.
  • Suspensions in addition to the active compounds, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • any method known to those in the art for contacting a cell, organ or tissue with one or more PCBP2 inhibitors and/or immunotherapeutic agents (e.g., immune checkpoint blockade or adoptive cell therapeutic compositions) disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more PCBP2 inhibitors and/or immunotherapeutic agents (e.g., immune checkpoint blockade or adoptive cell therapeutic compositions) to a mammal, suitably a human.
  • An effective amount of one or more PCBP2 inhibitors and/or immunotherapeutic agents (e.g., immune checkpoint blockade or adoptive cell therapeutic compositions) useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the one or more PCBP2 inhibitors and/or immunotherapeutic agents (e.g., immune checkpoint blockade or adoptive cell therapeutic compositions) described herein can be incorporated into pharmaceutical compositions for administration, singly or in -40- 4856-7271-5486.1 Atty. Dkt.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).
  • a treatment course e.g. 7 days of treatment.
  • Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. -41- 4856-7271-5486.1 Atty. Dkt.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier.
  • the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules.
  • Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.
  • Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, -42- 4856-7271-5486.1 Atty. Dkt.
  • a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • transmucosal or transdermal administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • transdermal administration may be performed by iontophoresis.
  • a therapeutic agent can be formulated in a carrier system.
  • the carrier can be a colloidal system.
  • the colloidal system can be a liposome, a phospholipid bilayer vehicle.
  • the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity.
  • One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)).
  • An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or liposomes.
  • Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems. -43- 4856-7271-5486.1 Atty. Dkt.
  • the carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix.
  • the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity.
  • the polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly ⁇ -hydroxy acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof.
  • the polymer is poly-lactic acid (PLA) or copoly lactic/glycolic acid (PGLA).
  • the polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)). [00133] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy, et al.), U.S. Pat.
  • U.S. Pat. Nos.5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.
  • the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No.4,522,811. [00135] The therapeutic compounds can also be formulated to enhance intracellular delivery.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day.
  • dosages can be 1 mg/kg body weight or 10 mg/kg body weight every day, every two days or every three days or within the range of 1-10 mg/kg every week, every two weeks or every three weeks.
  • a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight.
  • PCBP2 inhibitor and/or immunotherapeutic agent concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter.
  • An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
  • a therapeutically effective amount of one or more PCBP2 inhibitors and/or immunotherapeutic agents may be defined as a concentration of inhibitor at the target tissue of 10 -32 to 10 -6 molar, e.g., approximately 10 -7 molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg/kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).
  • a composition comprising a PCBP2 inhibitor and/or immunotherapeutic agent (e.g., immune checkpoint blockade or adoptive cell therapeutic composition) disclosed herein, is administered to the subject. -46- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 [00143]
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered more than five times per day.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered for six weeks or more.
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered for twelve weeks or more.
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered for a period of less than one year.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered throughout the subject’s life.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent -47- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 (e.g., immune checkpoint blockade or adoptive cell therapeutic composition) is administered daily for 3 weeks or more.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered daily for 4 weeks or more.
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered daily for 6 weeks or more.
  • the PCBP2 inhibitor and/or immunotherapeutic agent e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and/or immunotherapeutic agent is administered daily throughout the subject’s life.
  • Therapeutic Methods of the Present Technology provides a method for sensitizing a cancer patient to immunotherapy comprising administering to the cancer patient an effective amount of a PCBP2 inhibitor separately, sequentially or simultaneously with the immunotherapy.
  • the immunotherapy may comprise immune checkpoint blockade therapy and/or an adoptive cell therapeutic composition comprising T cells.
  • the adoptive cell therapeutic composition comprises one or more of tumor infiltrating T cells, CD8+ T cells, CD4+ T cells, delta-gamma T-cells, and alpha-beta T-cells.
  • the T cells may comprise a native TCR or a heterologous TCR.
  • the native TCR or the heterologous TCR is HLA-I restricted or HLA- II restricted.
  • the adoptive cell therapeutic composition is obtained from an autologous donor or allogeneic donor.
  • the cancer patient has previously received immunotherapy. In some embodiments, the cancer patient is resistant or non-responsive to immunotherapy.
  • the immune checkpoint blockade therapy comprises one or more of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB antibody, an anti-CD73 antibody, an anti-GITR antibody, and an anti-LAG-3 antibody.
  • immune checkpoint blockade therapy -48- 4856-7271-5486.1 Atty. Dkt.
  • the PROTAC that specifically targets PCBP2 comprises an E3 ubiquitin ligase binding moiety (“ULM”) selected from among an IAP E3 ubiquitin ligase binding moiety (an “ILM”), a cereblon E3 ubiquitin ligase binding moiety (a “CLM”), a Von Hippel-Lindae E3 ubiquitin ligase (VHL) binding moiety (VLM), and a mouse double minute 2 homologue (MDM2) E3 ubiquitin ligase binding moiety (MLM).
  • EMLM E3 ubiquitin ligase binding moiety
  • the immune checkpoint blockade therapy and/or the adoptive cell therapeutic composition is administered pleurally, parenterally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intrapleurally or intraperitoneally.
  • the methods of the present technology further comprise administering a cytokine to the cancer patient.
  • the cytokine may be administered prior to, during, or subsequent to administration of the adoptive cell therapeutic composition or the immune checkpoint blockade therapy.
  • the cytokine is selected from a group consisting of interferon a, interferon ⁇ , interferon ⁇ , complement C5a, IL-2, TNF alpha, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, CCL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCRIO, C
  • the PCBP2 inhibitor and immunotherapy are administered to a patient, for example, a mammal, such as a human, in a sequence and within a time interval such that the inhibitor that is administered first acts together with the inhibitor that is administered second to provide greater benefit than if each inhibitor were administered alone.
  • a patient for example, a mammal, such as a human
  • the PCBP2 inhibitor and immunotherapy can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, the PCBP2 inhibitor and immunotherapy (e.g., immune checkpoint blockade or adoptive cell therapeutic composition) are administered sufficiently close in time so as to provide the desired therapeutic or prophylactic effect of the combination of the at least two therapeutic agents.
  • the PCBP2 inhibitor and immunotherapy e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the PCBP2 inhibitor and immunotherapy are each administered as separate dosage forms, in any appropriate form and by any suitable route.
  • the PCBP2 inhibitor and immunotherapy e.g., immune checkpoint blockade or adoptive cell therapeutic composition
  • the frequency with which any of these therapeutic agents can be administered can be once or more than once over a period of about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 20 days, about 28 days, about a week, about 2 weeks, about 3 weeks, about 4 weeks, about a month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, about every 6 months, about every 7 months, about every 8 months, about every 9 months, about every 10 months, about every 11 months, about every year, about every 2 years, about every 3 years, about every 4 years, or about every 5 years.
  • kits for the treatment of an HLA expressing cancer comprises a therapeutic composition containing an effective amount of any and all embodiments of the PCBP2 inhibitor disclosed herein.
  • the kits further comprise at least one of an immune checkpoint inhibitor, and/or reagents for an adoptive cell therapeutic composition.
  • reagents for an adoptive cell therapeutic composition include polynucleotides and vectors comprising TCR (i.e. heterologous T-cell receptor) constructs or CAR (i.e. chimeric antigen receptor) constructs.
  • the kit comprises a sterile -51- 4856-7271-5486.1 Atty.
  • Examples of immune checkpoint inhibitor may include an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4-1BB antibody, an anti-CD73 antibody, an anti-GITR antibody, and an anti-LAG-3 antibody.
  • the immune checkpoint inhibitor comprises one or more of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, tremelimumab, ticlimumab, JTX-4014, Spartalizumab (PDR001), Camrelizumab (SHR1210), Sintilimab (IBI308), Tislelizumab (BGB-A317), Toripalimab (JS 001), Dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, AMP-514, KN035, CK-301, AUNP12, CA-170, or BMS-986189.
  • pembrolizumab nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimum
  • the PCBP2 inhibitor, the immune checkpoint inhibitor, and/or the adoptive cell therapeutic composition is formulated for intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intradermal, intraperitoneal, transtracheal, subcutaneous, intracerebroventricular, oral or intranasal administration.
  • the above described components of the kits of the present technology are packed in suitable containers and labeled for treatment of an HLA expressing cancer in a subject.
  • the above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for reconstitution.
  • the kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition.
  • the kit may comprise instructions for diluting the pharmaceutical composition and/or instructions for administering the pharmaceutical composition, whether diluted or not.
  • the containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle).
  • the kit may further comprise more containers -52- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, etc.
  • the instructions will generally include information about the use of the composition for the treatment of cancer.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter-indications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Identification of PCBP2 as a Sensitizer to CD4+ T cell-mediated Killing in Cancer Cells [00161] The melanoma cell line A375 was modified to stably express Cas9.
  • FIG.1A shows a volcano plot depicting the depleted -53- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 and enriched sgRNAs conferring sensitization or resistance to CD4+ T-cell killing, respectively.
  • FIG.1B PCBP2 depletion in HLA-II expressing A375 cells were significantly sensitized to killing mediated by CD4+ T cells expressing the HLA-II restricted TCR.
  • a custom made CRISPR knockout library (4 sgRNAs per gene) was generated targeting the 200 most significantly depleted and enriched genes discovered in the whole genome screen shown in FIG.1.
  • the melanoma cell lines A375 and SK-MEL-130, the cervical cancer cell line HeLa, and the breast cancer cell line MB-468 were modified to stably express Cas9.
  • Cas9-expressing tumor cells were infected with the focused library and subsequently challenged with either control CD4+ T cells or CD4+ T cells expressing the HLA-II restricted TCR 6F9 recognizing the tumor-associated antigen MAGE-A3/A6 (A375 and SK-MEL-130) or expressing the HLA-II restricted TCR TA10 recognizing the tumor-antigen KK-LC-1 (HeLa and MB-468).
  • PCBP2 depletion in HLA-II expressing cancers (melanoma cells, cervical cancer cells, and breast cancer cells) were significantly sensitized to killing mediated by CD4+ T cells expressing the HLA-II restricted TCR. See FIG.2.
  • FIG.4A The indicated sgRNA guide sequences (FIG.4A) targeting components of the HLA class I antigen presentation pathway (B2M, TAP1, TAP2, TAPBP) or targeting the HLA class II master transcription factor CIITA were cloned into the lentiviral plasmid lentiCRISPR v2 (Addgene: #52961).
  • A375 melanoma knockout cell lines were generated by lentiviral transduction and were selected with puromycin for at least 7 days prior to experiments. Cell lines were stained for HLA class I and HLA class II expression using the monoclonal antibodies W6/32-FITC or Tu39-APC, respectively.
  • mCherry + A375 melanoma cells were generated by retroviral transduction of a nuclear localized mCherry reporter.
  • CD8 + T cells were transduced with the HLA-A*02:01- restricted TCR 1G4 recognizing a peptide processed from NY-ESO-1 and CD4 + T cells were transduced with the HLA-DP*04:01-restricted TCR 6F9 recognizing a peptide processed from MAGE-A3/A6.
  • mCherry + A375 HLA-A*02:01 + , HLA-DP*04:01 + ) were co-cultured with the indicated T cell populations or without T cells and tumor cell growth was followed over time with Incucyte.
  • disruption of the HLA class I antigen presentation pathway impaired cytolysis by tumor specific HLA class I- restricted CD8+ T cells but not by HLA class II-restricted CD4+ T cells.
  • ablation of HLA class II antigen presentation pathway inhibited cytolysis by HLA class II-restricted CD4+ T cells but not by HLA class I-restricted CD8+ T cells.
  • Cancer cells express multiple PCBP2 splice variants (FIGs.6A-6D).
  • Conditioned media was generated by culturing parental A375 melanoma cells with control CD4+ T cells or CD4+ T cells expressing the HLA-II restricted TCR 6F9.
  • CM was added to A375 melanoma cells edited at the AAVS1 or PCBP2 locus in the presence or absence of TNF- and IFN ⁇ -neutralizing antibodies, and induction of apoptosis was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive for active Caspase 3/7) over time.
  • genomic deletion of PCBP2 primes tumor cells for apoptosis induced by T cell-derived inflammatory cytokines.
  • a clonal A375 melanoma cell line lacking expression of endogenous PCBP2 was generated by electroporation with a CRISPR/Cas9 RNP targeting PCBP2 followed by single cell-cloning.
  • A375 PCBP2 null cells modified as indicated were exposed to recombinant human TNF (100 ng/ml) and IFN ⁇ (100 ng/ml), and induction of apoptosis was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive for active Caspase 3/7) over time. Cytokine responsiveness was selectively regulated by PCBP2 lacking coding Exon 8 (FIGs.8A-8D.) [00168] A375 melanoma cells were edited by electroporation with CRISPR/Cas9 RNPs targeting either the safe harbor locus AAVS1, PCBP2 coding Exon 2 or PCBP2 coding Exon 8.
  • qRT-PCR primer were designed that specifically amplify PCBP2 fl or PCBP2 ⁇ E8.
  • RNA was isolated from edited-tumor cells and transcribed into cDNA. Induction of -55- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 apoptosis in edited tumor cells upon exposure to recombinant human TNF (1 ng/ml) and IFN ⁇ (1 ng/ml) was assessed in an Incucyte instrument by measuring green object count (corresponding to tumor cells positive for active Caspase 3/7) over time.
  • A375 melanoma cells were transduced with lentiviral constructs expressing RfxCas13d and a non-targeting (NTC) Cas13d guide, Cas13d guides targeting the PCBP2 coding exon 7/9 splice junction specific to PCBP2 ⁇ E8 mRNA or Cas13d guides targeting CD71 as a specificity control.
  • qRT-PCR primer were designed that specifically amplify PCBP2 fl or PCBP2 ⁇ E8.
  • A375 melanoma cells stably expressing a nuclear localized mCherry reporter were electroporated with a CRISPR/Cas9 RNP targeting PCBP2 followed by single cell- cloning to generate A375/mCherry PCBP2 null cells.
  • N-terminally FLAG-tagged PCBP2 cDNA lacking coding Exon 8 ( ⁇ E8) harboring the GXXG -> GDDG mutation in the indicated KH domain(s), or an empty control vector was retrovirally expressed in A375/mCherry PCBP2 null cells.
  • the GXXG -> GDDG mutation impairs the RNA- binding capacity of the KH-domain (Hollingworth D.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups -57- 4856-7271-5486.1 Atty. Dkt. No.: 115872-3074 having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

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Abstract

La présente invention concerne des méthodes permettant d'améliorer l'efficacité d'une immunothérapie (p. ex., un blocage de point de contrôle immunitaire ou une composition thérapeutique cellulaire adoptive) chez un patient cancéreux, consistant à administrer une quantité efficace d'au moins un agent qui inhibe l'expression et/ou l'activité de PCBP2.
PCT/US2024/044755 2023-09-01 2024-08-30 Inhibition de pcbp2 pour améliorer l'efficacité d'une immunothérapie Pending WO2025049967A2 (fr)

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