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WO2024254596A1 - Compositions et méthodes de détection d'expression aberrante de cdk5 - Google Patents

Compositions et méthodes de détection d'expression aberrante de cdk5 Download PDF

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WO2024254596A1
WO2024254596A1 PCT/US2024/033266 US2024033266W WO2024254596A1 WO 2024254596 A1 WO2024254596 A1 WO 2024254596A1 US 2024033266 W US2024033266 W US 2024033266W WO 2024254596 A1 WO2024254596 A1 WO 2024254596A1
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chain variable
nos
variable region
amino acid
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WO2024254596A9 (fr
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James BIBB
Priyanka Gupta
Angela Carter
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UAB Research Foundation
University of Arizona
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University of Arizona
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/14—Post-translational modifications [PTMs] in chemical analysis of biological material phosphorylation

Definitions

  • the field of the invention is general compositions and methods for detection of aberrant Cdk5 activity in biological samples such as tumor samples.
  • BACKGROUND OF THE INVENTION The “one-size-fits-all” therapeutic approach has been unsuccessful in treating majority of cancer patients, causing a spike in global incidence rates of neuroendocrine tumors (NETs) with the highest escalation rate in North America and Canada.
  • NETs neuroendocrine tumors
  • the incidence of NETs in the USA alone increased by 6 folds showing a current prevalence of ⁇ 170,000 patients.
  • colorectal cancer and melanoma are the third and fifth most common types of cancer worldwide. Estimations indicate 1.8 million cases of colorectal cancer and 486,000 cases of melanoma in 2020.
  • Cdk5 has emerged as an important instigator of tumorigenic signaling in several types of NETs including pancreatic, adrenal, thyroid, and gastrointestinal tumors.
  • Non- neuroendocrine malignancies such as colorectal cancer and melanomas are also afflicted with pathological activation of Cdk5 signaling.
  • Cdk5 has been shown to promote tumor growth and invasion by regulating the activity of proteins involved in metabolic reprogramming, cytoskeletal remodeling, and epithelial-mesenchymal transition.
  • medullary thyroid carcinoma is derived from calcitonin-secreting parafollicular neuroendocrine (NE) cells. These tumors occur as sporadic or hereditary forms with an incidence rate of approximately 75% and 25%, respectively.
  • MTC patients present clinically heterogeneous disease courses ranging from indolent to highly aggressive. The 1 45659820.1 survival rate of 10-years varies from 100% (stage I) to 21% (stage IV) [1].
  • MTC accounts for 5-10% of all thyroid malignancies and is frequently associated with germline mutations in the RET proto-oncogene. Patients may also harbor somatic mutations in HRAS, KRAS, or NRAS [2].
  • the 5-year survival rate is ⁇ 40% in patients with metastatic disease where tumors can spread to the cervical lymph nodes, or distant sites such as bones, lungs, liver, and brain [3-5].
  • Surgery is the only curative therapy for MTC, but resection of isolated metastases or other newer treatments have shown promise [6, 7].
  • RET mutations, serum calcitonin, and carcinoembryonic antigen (CEA) are known prognostic markers for MTCs.
  • compositions and methods thereof for diagnosing and treating Cdk5+ cancer such as MTC are provided.
  • Improved antibodies for detection of phosphorylated RBL1, H1.5, LARP6, SUV39H1, and FAM53C have been developed. Assays using them to detect the biomarkers: P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1, and diagnostic and prognostic applications stemming therefrom are also provided.
  • the antigen binding domain typically includes six complementarity-determining regions (CDRs), wherein the CDRs includes one, two, three, four, five, or six consensus CDRs of the CDRs of: 2 45659820.1 anti-RBL1 antibody RBL1-1; anti-H1.5 antibody H1-5-1; anti-LARP6 antibody LARP6-1; anti-SUV39H1 antibody SUV39H1-1; anti-FAM53C antibody FAM53C-1; or a variant thereof with at least 70% sequence identity thereto.
  • CDRs complementarity-determining regions
  • the antibodies and molecules include the heavy and/or light chain variable region(s) of anti-RBL1 antibody RBL1-1; anti-H1.5 antibody H1-5-1; anti-LARP6 antibody LARP6-1; anti-SUV39H1 antibody SUV39H1-1; anti-FAM53C antibody FAM53C-1; or a variant thereof with at least 70% sequence identity thereto.
  • the antibodies can be intact antibodies or functional antibody fragment or fusion proteins.
  • Examples of functional fragments or fusion proteins are selected from Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments such as single chain variable fragments (scFv), and single domain antibodies such as sdAb, sdFv, and nanobody fragments.
  • the antibodies can be intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, heteroconjugate antibodies, and multispecific antibodies such as bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv.
  • the antibodies can be IgM, IgE, IgA, IgD, or IgG optionally an IgG1, IgG2, IgG3, or IgG4. Any of the antibodies can further include a detectable label (substance) and/or a conjugated toxin, drug, receptor, enzyme, or receptor ligand. Methods of detecting Cdk5 are also provided.
  • An exemplary method includes detecting the level of phosphorylation of one or more of P-T143 FAM53C, P-T202 LARP6, P- S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1 by contacting a biological sample with one or more of the disclosed antibodies, and detecting binding between the antibody or antibodies and the one or more of FAM53C, LARP6, RBL1, H1.5, and SUV39H1.
  • the method can further include determining that the sample includes aberrant Cdk5 activity if the level of detected binding is higher in the biological sample than in a control.
  • Exemplary means of detecting the antibody or antibodies include, but are not limited to immunoassays, such as immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, or immuno-chromatography assays as well as immunohistochemistry, Western blotting, surface plasmon resonance, flow cytometry (FACS) analysis, and biochips.
  • immunoassays such as immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT
  • the biological sample is cells or a cell lysate or a fraction thereof.
  • the cells or cell lysate or fraction thereof can be derived from a biopsy from a subject. 3 45659820.1
  • the biopsy can contain or be suspected of containing tumor cells.
  • the biopsy can be a tumor biopsy. Diagnostic methods are also provided. For example, diagnosing a subject with a Cdk5- related disease or disorder can include detecting aberrant Cdk5 according to the disclosed methods.
  • the Cdk5-related disease or disorder is cancer, diabetes, or obesity.
  • the cancer is a neuroendocrine cancer, colorectal cancer, melanoma, lung cancer, head and neck squamous cell carcinoma, hepatocelluar carcinoma (HCC), pancreatic cancer, or breast cancer.
  • the neuroendocrine cancer is Medullary Thyroid Carcinoma (MTC), pancreatic, adrenal, thyroid, or gastrointestinal cancer.
  • MTC Medullary Thyroid Carcinoma
  • the treatment includes a therapy effective for treating diseases and disorders characterized by aberrant Cdk5 activity.
  • Exemplary treatments include dinaciclib (SCH727965), a proteosome inhibitor optionally Bortezomib (Velcade), or a functional nucleic acid that reduces expression of the Cdk5 gene, mRNA, or protein, optionally wherein the functional nucleic acid is siRNA, miRNA, RNAi, or shRNA.
  • Figures 1A-1G illustrate the generation of transgenic mouse models of MTC.
  • Figure 1A is a schematic showing tetracycline controlled bitransgenic system where a neuroendocrine cell-specific promoter linked to the tetracycline transactivator (tTA) activates Tet-Operon driving p25-GFP expression.
  • tTA tetracycline transactivator
  • FIGS 1B-1C are schematics showing the induction of p25GFP in doxycycline-controlled bitransgenic models driven by NSE and CGRP promoters.
  • Figure 1D are images showing gross anatomy of tumors in NSE-p25GFP and CGRP-p25GFP models (upper panel); histopathological neuroendocrine features characterized by H & E (bottom panel), scale; 50 ⁇ m, tr; trachea.
  • FIG. 1F and 1G are representative immunostains comparing expression of p25GFP and Chromogranin A (ChrA) in NSE (Fig.1F) and CGRP (Fig.1G) mice tissues extracted 4 45659820.1 from growing and arrested tumors.
  • a version of NSE-p25GFP MTC tumors (Fig.1D, left) was previously published [10] and shown here at a different magnification for comparison.
  • Figures 2A-2G illustrates the mutational landscape of mouse tumor models.
  • Figure 2B is a plot of variant classification by type; frequency of variant (y-axis), colors denote types of variation.
  • Figure 2C is a plot of variant type presented as SNP (single nucleotide polymorphism), INS (insertion), DNP (double nucleotide polymorphism), DEL (deletion), TNP (triple nucleotide polymorphism); frequency (y-axis).
  • Figure 2D is a UpSet plot showing counts of unique or overlapping mutated genes in NSE and CGRP models.
  • Figure 2E is a bar chart displaying enriched pathways associated with altered genes in NSE-p25OE mice.
  • Figure 2F is a bar chart displaying enriched pathways in CGRP-p25OE mice.
  • Figure 2G is a bar chart comparing enriched pathways common in –NSE and –CGRP p25OE mice. Significant functions are shown where p-value ⁇ 0.01.
  • Figures 3A-3D illustrate comparison of mouse and human tumors mutational profile.
  • Figure 3A is a Venn diagram showing overlapping genes between NSE-p25OE mice and human tumors [35].
  • Figure 3B is a GO term enrichment analysis shows biological processes and pathways associated with the altered genes shared by NSE-p25OE mice and human tumors.
  • Figure 3C is a Venn diagram showing overlapping mutated genes in CGRP-p25OE mice and human tumors [35].
  • Figure 3D is a GO analysis of overlapping genes shared by CGRP-p25OE mice and human tumors. Bars sorted by p-value ranking; p-value cut-off ⁇ 0.05; GO, Gene Ontology.
  • Figures 4A-4G illustrate transcriptomic analysis of mouse models.
  • Figure 4B is a volcano plot presenting differentially expressed genes (DEGs) in growing vs.
  • DEGs differentially expressed genes
  • FIG. 4D-4G are illustrations showing visualization of enrichment network of DEGs up and downregulated (Figs.4D-4E) in NSE-p25OE mice, and (Figs.4F- 4G) in CGRP-p25OE mice. Cluster annotation is indicated by color code, nodes with the same color are closely spaced and associated with the same cluster, clusters were labeled manually; enriched terms with a similarity score of >0.3 are connected by edges.
  • Figures 5A-5J illustrate the impact of genetic alterations on mRNA expression.
  • Figure 5A is an UpSet plot depicts the overlap between mutations and corresponding gene expression in NSE-p25OE mice.
  • Figure 5B and 5C are bar graphs showing pathway visualization of overlapping genes afflicted both with mutations and changes in mRNA expression: where mRNA was up- (Fig.5B) or down-regulated (Fig.5C) in NSE-p25OE mice.
  • Figure 5D is a series of violin plots comparing mRNA reads of mutated genes involved in spindle assembly and Notch signaling.
  • Figure 5E is a series of violin plots comparing mRNA reads of mutated genes enriched in cancer-associated proliferative signaling (G, growing; A, arrested tumors).
  • Figure 5F is a bar graph showing intersection size of mutated genes and corresponding mRNAs in CGRP-p25OE mice.
  • Figure 5G and 5H are bar graphs showing functional enrichment of overlapping mutated genes where mRNA was either up- (Fig.5G) or down- regulated (Fig.5H) in the CGRP-p25OE mice.
  • Figures 5I and 5J are each a pair of plots showing mRNA reads of mutated genes associated with metabolic pathways and cell migration (Fig.5I), or mRNA reads of mutated genes involved in mTOR signaling (Fig.5J).
  • Figures 6A-6E illustrate efficacy of monoclonal antibodies for detection of aberrant Cdk5 activity.
  • Figure 6B is a series of images showing immunocytochemical analysis of Cdk5-dependent phosphorylations in mouse MTC cells as indicated, scale; 70 ⁇ m.
  • Figure 6C is a series of images of immunohistochemical staining of Cdk5 phosphorylations in growing (p25OE) and arrested (p25OFF) tumor sections derived from NSE/CGRP models, scale; 50 ⁇ m.
  • Figures 7A-7D illustrate histological evaluation of aberrant Cdk5 activity in human tissue microarray.
  • Figure 7A and 7B are representative immunohistochemical micrographs showing Cdk5 phosphosites viz.
  • FIG.7A P-RBL1 (Fig.7A)and P-LARP6 (Fig.7B) in tissue microarray sections of MTC tumors. magnification; 4x (scale, 200 ⁇ m), 20x (scale, 50 ⁇ m).
  • Figures 7C and 7D are each a series of plots showing quantification presented as the optical density for (Fig.7C) P-RBL1 (TMA 1-3), and (Fig.7D) P-LARP6 (TMA 1-3).
  • Figures 8A-8B illustrate enrichment analysis of mutated genes in mouse tumors.
  • y-axis pathway description;
  • x-axis fold enrichment. Analysis performed in Shiny GO 0.76.2; the bubble size indicates the number of genes, and the bar color code signifies the corrected p-value as indicated.
  • GO Gene Ontology (biological process); KEGG: Kyoto Encyclopedia of Genes and Genomes.
  • Figures 9A-9C illustrate mitotic cell cycle pathways shared by NSE mouse and human tumors.
  • Figure 9A is a UpSet plot showing intersecting mutated genes in mouse and human tumors.
  • Figure 9B is a Lollipop chart and Figure 1C is a network visualization summarize significantly enriched GO terms common in NSE and human tumors.
  • Figures 10A-10B illustrate metabolic pathways shared by CGRP mouse and human tumors.
  • Figure 10A is a plot showing counts of unique and overlapping genes between mouse and human tumors.
  • Figure 10B is a pathway and process enrichment plot and
  • Figures 11A-11B illustrates enrichment of transcription factors in NSE-p25OE mice.
  • Figure 11A is a bar graph showing putative regulators of DEGs in NSE-p25OE mice determined via TRRUST database [73].
  • Figure 11B is a bar graph summarizing the enriched transcription factor in CGRP-p25OE mice. TRRUST database revealed putative regulators of DEGs in CGRP-p25OE mice. Terms with a p-value ⁇ 0.01; minimum count of 3, enrichment factor > 1.5 [74].
  • Figure 12 illustrates detection of Cdk5 activity in NSE and CGRP tumors. Immunoblots show expression levels of p25GFP, P-RBL1, and P-H1.5 in tissue lysates extracted from growing (p25OE) vs. arrested (p25OFF) tumors.
  • FIGS. 13A-13E illustrate characterization of recombinant phospho-specific mcAbs in mouse tumors. Comparative analysis of Cdk5 phosphorylation state-specific polyclonal antibodies (pcAb) vs. recombinant mcAbs. Lysates from growing (“G”) and arrested (“A”) tumors were analyzed via immunoblotting using Abs as indicated.
  • Antibodies include monoclonal and polyclonal antibodies as well as fragments and polymers containing the antigen binding domain and/or one or more complementarity determining regions of these antibodies.
  • antibody refers to any form of antibody or antigen binding fragment or recombinant protein, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they specifically bind the target antigen. Any specific antibody can be used in the methods and compositions provided herein.
  • the term “antibody” encompasses a molecule comprising at least one variable region from a light chain immunoglobulin molecule and at least one variable region from a heavy chain molecule that in combination form a specific binding site for the target antigen.
  • the term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain).
  • the variable region comprises a “hypervariable region” whose residues are responsible for antigen binding.
  • the hypervariable region comprises amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.
  • CDR Constantarity Determining Region
  • “hypervariable loop” i.e., residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia, C. et al. (1987) “Canonical Structures For The Hypervariable Regions Of Immunoglobulins,” J. Mol. Biol.196:901-917).
  • “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined.
  • antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies (See e.g., Muyldermans et al., 2001, Trends Biochem. Sci.26:230; Nuttall et al., 2000, Cur. Pharm. Biotech.1:253; Reichmann and Muyldermans, 1999, J. Immunol. Meth.231:25; International Publication Nos. 8 45659820.1 WO 94/04678 and WO 94/25591; U.S.
  • antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.
  • An “antibody fragment” or “antigen binding fragment” of an antibody is defined as at least a portion of the variable region of the immunoglobulin molecule that binds to its target, i.e., the antigen binding region (also antigen binding domain). In one embodiment it specifically covers single antibodies and clones thereof and anti- antibody compositions with polyepitopic specificity.
  • the antibody of the present methods and compositions can be monoclonal or polyclonal.
  • An antibody can be in the form of an antigen binding antibody fragment including a Fab fragment, F(ab')2 fragment, a single chain variable region, and the like. Fragments of intact molecules can be generated using methods well known in the art and include enzymatic digestion and recombinant means. Thus, the “fragment” may be a recombinant protein, e.g., a fusion protein. As used herein, any form of the “antigen” can be used to generate an antibody that is specific for the target antigen. Thus, the eliciting antigen may be a single epitope, multiple epitopes, or the entire protein alone or in combination with one or more immunogenicity enhancing agents known in the art.
  • the eliciting antigen may be an isolated full-length protein, a cell surface protein (e.g., immunizing with cells transfected with at least a portion of the antigen), or a soluble protein (e.g., immunizing with only the extracellular domain portion of the protein).
  • the antigen may be produced in a genetically modified cell.
  • the DNA encoding the antigen may genomic or non-genomic (e.g., cDNA) and encodes at least a portion of the extracellular domain.
  • portion refers to the minimal number of amino acids or nucleic acids, as appropriate, to constitute an immunogenic epitope of the antigen of interest.
  • any genetic vectors suitable for transformation of the cells of interest may be employed, including but not limited to adenoviral vectors, plasmids, and non-viral vectors, such as cationic lipids.
  • the antibody of the methods and compositions herein specifically bind at least a portion of the extracellular domain of the target antigen of interest.
  • the antibodies or antigen binding fragments thereof provided herein may be conjugated to a “bioactive agent.”
  • bioactive agent refers to any synthetic or 9 45659820.1 naturally occurring compound that binds the antigen and/or enhances or mediates a desired biological effect.
  • the binding fragments useful in the present invention are biologically active fragments.
  • the term “biologically active” refers to an antibody or antibody fragment that is capable of binding the desired the antigenic epitope and directly or indirectly exerting a biologic effect.
  • “Bispecific” antibodies are also useful in the present methods and compositions.
  • the term “bispecific antibody” refers to an antibody, typically a monoclonal antibody, having binding specificities for at least two different antigenic epitopes. In one embodiment, the epitopes are from the same antigen. In another embodiment, the epitopes are from two different antigens. Methods for making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly using the co-expression of two immunoglobulin heavy chain/light chain pairs.
  • bispecific antibodies can be prepared using chemical linkage. See, e.g., Brennan, et al., Science 229:81 (1985). Bispecific antibodies include bispecific antibody fragments. See, e.g., Hollinger, et al., Proc. Natl. Acad. Sci. U.S.A.90:6444-48 (1993), Gruber, et al., J. Immunol.152:5368 (1994).
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
  • an antibody specifically binds or “immuno-specifically binds” refers to the binding of an antibody to its cognate antigen while not significantly binding to other antigens.
  • an antibody “specifically binds” to an antigen with an affinity constant (Ka) greater than about 10 5 mol –1 (e.g., 10 6 mol –1 , 10 7 mol –1 , 10 8 mol –1 , 10 9 mol –1 , 10 10 mol –1 , 10 11 mol –1 , and 10 12 mol –1 or more) with that second molecule.
  • Ka affinity constant
  • mAb refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the 10 45659820.1 population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • isolated means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • the term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences.
  • a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • phosphorothioate phosphorothioate
  • methylphosphonate and the like.
  • a “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • the term “vector” encompasses an autonomously replicating plasmid or a virus.
  • the term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors 11 45659820.1 include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, and the like.
  • subject includes, but is not limited to, animals, plants, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
  • a patient refers to a subject afflicted with a disease or disorder.
  • the term “patient” includes human and veterinary subjects.
  • the subject can be any organism in which the disclosed method can be used to genetically modify the organism or cells of the organism.
  • Treatment or “treating” means to administer a composition to a subject or a system with an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological state, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological state, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder.
  • treatment while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention.
  • the effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms.
  • characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
  • “Prevention” or “preventing” means to administer a composition 12 45659820.1 to a subject or a system at risk for an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • the condition can also be a predisposition to the disease, pathological state, or disorder.
  • the effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or reduction of the chances that a particular event or characteristic will occur.
  • the terms “effective amount” or “therapeutically effective amount” means a quantity sufficient to alleviate or ameliorate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiological effect. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise quantity will vary according to a variety of factors such as subject- dependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • subject- dependent variables e.g., age, immune system health, weight, etc.
  • the disease or disorder being treated as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • polypeptides includes proteins and functional fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus.
  • amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).
  • the term “functional fragment” as used herein is a fragment of a full- length protein retaining one or more function properties of the full-length protein. 13 45659820.1
  • the terms “variant” or “active variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties (e.g., functional or biological activity).
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity.
  • polypeptide sequence Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological or functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties (e.g., functional or biological activity). Modifications and changes can be made in the structure of the polypeptides of in disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution). For example, certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity.
  • Those indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); 14 45659820.1 tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (- 3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity can also be made on the basis of hydrophilicity, particularly, where the biological functional equivalent polypeptide or peptide thereby created is intended for use in immunological forms.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamnine (+0.2); glycine (0); proline (-0.5 ⁇ 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (- 1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include (original residue: exemplary substitution): (Ala: Gly, Ser), (Arg: Lys), (Asn: Gln, His), (Asp: Glu, Cys, Ser), (Gln: Asn), (Glu: Asp), (Gly: Ala), (His: Asn, Gln), (Ile: Leu, Val), (Leu: Ile, Val), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (Val: Ile, Leu).
  • forms of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest. 15 45659820.1
  • “conservative” amino acid substitutions are substitutions wherein the substituted amino acid has similar structural or chemical properties.
  • “non-conservative” amino acid substitutions are those in which the charge, hydrophobicity, or bulk of the substituted amino acid is significantly altered.
  • identity is a relationship between two or more polypeptide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between polypeptide as determined by the match between strings of such sequences. “Identity” can also mean the degree of sequence relatedness of a polypeptide compared to the full-length of a reference polypeptide. “Identity” and “similarity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • polypeptide sequence may be identical to the reference sequence, that is be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the % identity is less than 100%.
  • Such alterations are selected from: at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the number of amino acid alterations for a given % identity is determined by 16 45659820.1 multiplying the total number of amino acids in the reference polypeptide by the numerical percent of the respective percent identity (divided by 100) and then subtracting that product from said total number of amino acids in the reference polypeptide.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.
  • antibodies and their antigen binding fragments and other molecules capable of immunospecifically binding P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1 are provided.
  • 18 45659820.1 can be, for example, a monoclonal antibody, a human antibody, a chimeric antibody or a humanized antibody, or a fragment thereof, and fusion proteins formed therefrom.
  • the antibodies and antigen binding fragments can be monospecific, bispecific, trispecific or multispecific.
  • RBL1 Polypeptides that selectively bind RB transcriptional corepressor like 1 (RBL1) are provided.
  • RBL1 regulates the cell cycle, and proliferation by modulating chromatin structure. See, e.g., RB transcriptional corepressor like 1 [ Homo sapiens (human) ], NCBI Gene ID: 5933.
  • the polypeptide specifically binds the epitope: CSIYI-pS-PHKN (SEQ ID NO:1).
  • the molecules and antibodies that bind to RBL1 can include an antigen binding domain that includes six CDRs, wherein the CDRs include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti-RBL1 antibody RBL1-1.
  • the molecule or antibody includes a heavy and/or light chain variable region of antibody RBL1-1.
  • the CDRs and/or the heavy and light chain variable regions are in the same orientation as antibody RBL1-1.
  • An exemplary consensus amino acid sequence for the mature heavy chain variable region of RBL1-1 is: QSLEESGGRLVTPGTPLKLTCTVSGFSLSDYNVGWVRQAPGKGLEWIGIMNIGISTWYASWAKGR FTISRTSTTVDLKMTSLTTEDTATYFCARGFSRNSYDIWGPGTLVTVSS (SEQ ID NO:2)
  • CDR-H3: GFSRNSYDI SEQ ID NO:6
  • An exemplary consensus amino acid sequence for the mature light chain variable region of RBL1-1 is: AVLTQTPSPVSAAVGGTVTIKCQSSQSVVKNNYLSWYQ
  • H1.5 Polypeptides that selectively bind H1.5 are provided.
  • H1.5 is a linker histone that facilitates chromatin compaction. See, e.g., H1.5 linker histone, cluster member [ Homo sapiens (human) ], NCBI Gene ID: 3009.
  • the polypeptide specifically binds the epitope: CAPVEK-pS- PAK (SEQ ID NO:14).
  • the molecules and antibodies that bind to H1.5 can include an antigen binding domain that includes six CDRs, wherein the CDRs include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti-H1.5 antibody H1-5-1.
  • the molecule or antibody includes a heavy and/or light chain variable region of antibody H1-5-1.
  • the CDRs and/or the heavy and light chain variable regions are in the same orientation as antibody H1-5-1.
  • An exemplary consensus amino acid sequence for the mature heavy chain variable region of H1-5-1 is: QSVEESGGRLVTPGTPLTLTCTVSGIDLSSNVMMWVRQAPGKGLEYIGIITNSGIRYYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCARGAPNTGNIWGPGTLVTVSL (SEQ ID NO:15)
  • CDR-H2 IITNSGIRYYASWAKG (SEQ ID NO:18)
  • CDR-H3 GAPNTGNI (SEQ ID NO:19)
  • An exemplary consensus amino acid sequence for the mature light chain variable region of H1-5-1 is: QVLTQTASSVSAAVGGTVTINCQSSQSVYDNNYLSWYQQKPGQPP
  • LARP6 Polypeptides that selectively bind La Ribonucleoprotein 6 (LARP6) are provided.
  • LARP6 is a translational regulator shuttles between cytoplasm and nucleus promotes nucleic acid binding. See, e.g., LARP6 La ribonucleoprotein 6, translational regulator [ Homo sapiens (human) ], NCBI Gene ID: 55323.
  • the polypeptide specifically binds the epitope: CALA-pT- PQKNGRV (SEQ ID NO:27).
  • the molecules and antibodies that bind to LARP6 can include an antigen binding domain that includes six CDRs, wherein the CDRs include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti-LARP6 antibody LARP6-1.
  • the molecule or antibody includes a heavy and/or light chain variable region of antibody LARP6-1.
  • the CDRs and/or the heavy and light chain variable regions are in the same orientation as antibody LARP6-1.
  • An exemplary consensus amino acid sequence for the mature heavy chain variable region of LARP6-1 is: QSLEESGGGLVQPEGSLTLTCKASGFSFSSGYYMCWVRQAPGKGLEWSGCISARSGRTYYATWAK GRFTISKTSSTTVTLQVTSLTAADTATYFCARGNRFVSSSGDSMWGPGTLVTVSS (SEQ ID NO:28) CDR-H1: SGYYMC (SEQ ID NO:30) CDR-H2: CISARSGRTYYATWAKG (SEQ ID NO:31) CDR-H3: GNRFVSSSGDSM (SEQ ID NO:32)
  • An exemplary consensus amino acid sequence for the mature light chain variable region of LARP6-1 is: DPVLTQTPSPVSAAVGGTVTINCQASQSVFSNNQLAWFQQKPGQPPKQLIYGASTLASGVSSRFK GSGYGTRFTLTISDVQCDDTATYYCLGEFTCSSVDCNAFGGGTEVVVE (SEQ ID NO:
  • SUV39H1 Polypeptides that selectively bind SUV39H1 are provided.
  • SUV39H1 is a histone lysine methyltransferase. See, e.g., SUV39H1 histone lysine methyltransferase [ Homo sapiens (human) ] NCBI Gene ID: 6839.
  • the polypeptide specifically binds the epitope: CLAGLPG-pS- PKKRVR (SEQ ID NO:40).
  • the molecules and antibodies that bind to SUV39H1 can include an antigen binding domain that includes six CDRs, wherein the CDRs include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti-SUV39H1 antibody SUV39H1-1.
  • the molecule or antibody includes a heavy and/or light chain variable region of antibody SUV39H1-1.
  • the CDRs and/or the heavy and light chain variable regions are in the same orientation as antibody SUV39H1-1.
  • An exemplary consensus amino acid sequence for the mature heavy chain variable region of SUV39H1-1 is: QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYHMCWVRQAPGKGLEYIGMINRRAITSYASWAKGR FTISKTSTTVDLKITSPTTEDTATYFCARYSSGNDFDADIWGPGTLVTVSL (SEQ ID NO:41) CDR-H1: TYHMC (SEQ ID NO:43) CDR-H2: MINRRAITSYASWAKG (SEQ ID NO:44) CDR-H3: YSSGNDFDADI (SEQ ID NO:45)
  • An exemplary consensus amino acid sequence for the mature light chain variable region of SUV39H1-1 is: AAVLTQTPSPVSAAVGGTVTISCQSSKSVYDRNLLSWFQQKPGQPPKLLIYKASTLASGVPSRFK GSGSGTQFTLTISDVQCDDAATYYCAGGYSGTSDAYPFGGGTEVVVK (SEQ ID NO:42), CDR-L1
  • FAM53C Polypeptides that selectively bind family with sequence similarity 53 member C (FAM53C) are provided. FAM53C bind to a transcriptional regulator that modulates cell 22 45659820.1 proliferation. See, e.g., FAM53C family with sequence similarity 53 member C [ Homo sapiens (human) ] NCBI Gene ID: 51307. In some embodiments, the polypeptide specifically binds the epitope: CAPSKLW-pT- PIKH (SEQ ID NO:53).
  • the molecules and antibodies that bind to FAM53C can include an antigen binding domain that includes six CDRs, wherein the CDRs include at least one, two, three, four, five, or six consensus CDRs of the CDRs of anti-FAM53C antibody FAM53C-1.
  • the molecule or antibody includes a heavy and/or light chain variable region of antibody FAM53C-1.
  • the CDRs and/or the heavy and light chain variable regions are in the same orientation as antibody FAM53C-1.
  • An exemplary consensus amino acid sequence for the mature heavy chain variable region of the FAM53C-1 is: QSLEESGGDLVKPGASLTLTCKGSGFSFTIRYNICWVRQAPGKGLEWIACVNSAYASWAKGRFTI SKTSSTTVTLQMTSLTAADTATYFCVRYVDSRYYGVDLWGPGTLVTVSS (SEQ ID NO:54)
  • CDR-H1 IRYNIC (SEQ ID NO:56)
  • CDR-H2 CVNSAYASWAKG (SEQ ID NO:57)
  • CDR-H3 YVDSRYYGVDL (SEQ ID NO:58)
  • An exemplary consensus amino acid sequence for the mature light chain variable region of FAM53C-1 is: QVLTQTPSSVSAALGGTVTISCQSSESVYKNNYLSWYQQKPGQPPKLLIYGASTLASGVPSRFKG SGSGTQFTLTISSVQCDDAATYYCQGGYRGDYSSGDGILFGGGTEVVVK (SEQ ID NO
  • the disclosed antibodies and molecule include at least a portion of an immunoglobulin constant region (Fc), for example, that of a human immunoglobulin.
  • the constant domains can be selected with respect to the proposed function of the antibody.
  • the constant domains of the antibodies are (or include) human IgA, IgD, 23 45659820.1 IgE, IgG or IgM domains.
  • human IgG constant domains, especially of the IgG1 and IgG3 isotypes are used.
  • the antibodies and other molecules contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody or other molecule further includes one or more of the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4.
  • the heavy chain constant region of the antibodies utilized in the examples below is: GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSL SSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPMCPPPELLGGPSVFIFPPKPKDTLMIS RTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEF KCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKN GKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK (SEQ ID NO:65).
  • the light chain constant regions of the antibodies utilized in the examples below are: GDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTY NLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:66), or GDPVAPTVLIFPPSADLVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTY NLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC (SEQ ID NO:67).
  • derivatives refers to an antibody or antigen-binding fragment thereof that immunospecifically binds to an antigen but which includes, one, two, three, four, five or more amino acid substitutions, additions, deletions or modifications relative to a “parental” (or wild-type) molecule.
  • derivative is or includes a variant amino acid sequence.
  • SEQ ID NOS:1-67 with at least 60%, 70%, 80%, 85%, 90%, or 95% sequence identity to the reference sequence.
  • the CDRs have or include variation relative to reference sequence. In other embodiments, the CDRs do not have or include variation relative to reference sequence. Thus, in some embodiments, the variation is limited to the heavy and light chain variable region framework residues. 24 45659820.1
  • Such amino acid substitutions or additions may be introduce naturally occurring (i.e., DNA-encoded) or non-naturally occurring amino acid residues. Such amino acids may be glycosylated (e.g., have altered mannose, 2-N-acetylglucosamine, galactose, fucose, glucose, sialic acid, 5-N-acetylneuraminic acid, 5-glycolneuraminic acid, etc.
  • the altered carbohydrate modifications modulate one or more of the following: solubilization of the antibody, facilitation of subcellular transport and secretion of the antibody, promotion of antibody assembly, conformational integrity, and antibody-mediated effector function.
  • the altered carbohydrate modifications enhance antibody mediated effector function relative to the antibody lacking the carbohydrate modification.
  • Carbohydrate modifications that lead to altered antibody mediated effector function are well known in the art (for example, see Shields, R.L. et al.
  • a humanized antibody is a derivative.
  • Such a humanized antibody can include amino acid residue substitutions, deletions or additions in one or more non-human CDRs.
  • the humanized antibody derivative may have substantially the same 25 45659820.1 binding, better binding, or worse binding when compared to a non-derivative humanized antibody.
  • one, two, three, four, or five amino acid residues of the CDR have been substituted, deleted or added (i.e., mutated).
  • the disclosed antibodies encompass modification of framework residues of the humanized antibodies. Framework residues in the framework regions may be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., U.S. Patent No.5,585,089; and Riechmann, L. et al. (1988) “Reshaping Human Antibodies For Therapy,” Nature 332:323- 327).
  • a derivative antibody or antibody fragment can be modified by chemical modifications using techniques known to those of skill in the art, including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc. In one embodiment, an antibody derivative possess a similar or identical function as the parental antibody.
  • an antibody derivative exhibits an altered activity relative to the parental antibody.
  • a derivative antibody or fragment thereof
  • Derivatized antibodies can be used to alter the half-lives (e.g., serum half-lives) of parental antibodies in a mammal, preferably a human.
  • such alteration will result in a half-life of greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.
  • substitutions, additions or deletions in the derivatized antibodies may be in the Fc region of the antibody.
  • the Fc portion of an antibody be varied by isotype or subclass, can be a chimeric or hybrid, and/or can be modified, for example to improve effector functions, control of half-life, tissue accessibility, augment biophysical characteristics such as stability, and improve efficiency of production (and less costly).
  • Many modifications useful in construction of disclosed proteins and methods for making them are known in the art, see for example Mueller, J.P. et al. (1997) “Humanized Porcine VCAM-Specific Monoclonal Antibodies With Chimeric Igg2/G4 Constant Regions Block Human Leukocyte Binding To Porcine Endothelial Cells,” Mol.
  • the Fc region is the native IgG1, IgG2, or IgG4 Fc region.
  • the Fc region is a hybrid, for example a chimeric consisting of IgG2/IgG4 Fc constant regions.
  • Modifications to the Fc region include, but are not limited to, IgG4 modified to prevent binding to Fc gamma receptors and complement, IgG1 modified to improve binding to one or more Fc gamma receptors, IgG1 modified to minimize effector function (amino acid changes), IgG1 with altered/no glycan (typically by changing expression host), and IgG1 with altered pH-dependent binding to FcRn.
  • the Fc region can include the entire hinge region, or less than the entire hinge region.
  • the antibodies can also be modified by the methods and coupling agents described by Davis et al. (See U.S. Patent No.4,179,337). Thus provided are antibodies with a heterologous molecule fused, conjugated, or otherwise attached thereto.
  • heterologous molecules may be detectable substance, enzymes, hormones, cell surface receptors, drug moieties, such as: toxins, etc.
  • ADCs antibody-drug conjugates
  • heterologous molecules are polypeptides having at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids.
  • antibodies or their antigen-binding fragments that are conjugated to a diagnostic or therapeutic agent or any other molecule.
  • the antibodies can be used diagnostically (in vivo, in situ or in vitro) to, for example, monitor the development or progression of a disease, disorder or infection as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.
  • the detectable substance may be coupled or conjugated either directly to the antibody or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No.4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to, various enzymes, enzymes including, but not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic group complexes such as, but not limited to, streptavidin/biotin and avidin/biotin; fluorescent materials such as, but not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent material such as, but not limited to, luminol; bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; radioactive material such as, but not limited to, bismuth (213Bi), carbon (14C), chromium (51Cr
  • the molecules of the disclose can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No.4,676,980.
  • Such heteroconjugate antibodies may additionally bind to haptens (such as fluorescein, etc.), or to cellular markers.
  • Bispecific and multispecific antibodies that bind the disclosed targets and e.g., a second cancer antigen or immune cell antigen are provided. Any of the molecules can be fused to marker sequences, such as a peptide, to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, the hemagglutinin “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I.A. et al. (1984) “The Structure Of An Antigenic Determinant In A Protein,” Cell, 37:767-778) and the “flag” tag (Knappik, A. et al. (1994) “An Improved Affinity Tag Based On The FLAG Peptide For The Detection And Purification Of Recombinant Antibody Fragments,” Biotechniques 17(4):754-761).
  • the molecules of the disclosure can be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen or of other molecules that are capable of binding to target antigen that has been immobilized to the support via binding to an antibody or antigen-binding fragment of the present invention.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • nucleic acid molecules DNA or RNA
  • vector molecules such as plasmids
  • nucleic acids can be single-stranded, double-stranded, may contain both single-stranded and double-stranded portions. 29 45659820.1 D. Nucleic Acids and Host Cells Nucleic acids and vectors encoding or expressing the disclosed polypeptides are also described. As used herein, “isolated nucleic acid” refers to a nucleic acid that is separated from other nucleic acid molecules that are present in a mammalian genome, including nucleic acids that normally flank one or both sides of the nucleic acid in a mammalian genome.
  • an isolated nucleic acid can be, for example, a DNA molecule, provided one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent.
  • an isolated nucleic acid includes, without limitation, a DNA molecule that exists as a separate molecule independent of other sequences (e.g., a chemically synthesized nucleic acid, or a cDNA or genomic DNA fragment produced by PCR or restriction endonuclease treatment), as well as recombinant DNA that is incorporated into a vector, an autonomously replicating plasmid, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or into the genomic DNA of a prokaryote or eukaryote.
  • a virus e.g., a retrovirus, lentivirus, adenovirus, or herpes virus
  • an isolated nucleic acid can include an engineered nucleic acid such as a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid.
  • Nucleic acids can be in sense or antisense orientation, or can be complementary to a reference sequence provided herein.
  • Nucleic acids can be DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • Modifications at the base moiety can include deoxyuridine for deoxythymidine, and 5-methyl-2’- deoxycytidine or 5-bromo-2’-deoxycytidine for deoxycytidine.
  • Modifications of the sugar moiety can include modification of the 2’ hydroxyl of the ribose sugar to form 2’-O-methyl or 2’-O-allyl sugars.
  • the deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev.7:187-195; and Hyrup et al. (1996) Bioorgan. Med. Chem.4:5-23.
  • deoxyphosphate backbone can be replaced with, for 30 45659820.1 example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
  • the nucleic acids can be operably linked to one or more expression control sequences.
  • “operably linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest. Examples of expression control sequences include promoters, enhancers, and transcription terminating regions.
  • a promoter is an expression control sequence composed of a region of a DNA molecule, typically within 100 nucleotides upstream of the point at which transcription starts (generally near the initiation site for RNA polymerase II). To bring a coding sequence under the control of a promoter, it is necessary to position the translation initiation site of the translational reading frame of the polypeptide between one and about fifty nucleotides downstream of the promoter. Enhancers provide expression specificity in terms of time, location, and level. Unlike promoters, enhancers can function when located at various distances from the transcription site. An enhancer also can be located downstream from the transcription initiation site.
  • a coding sequence is “operably linked” and “under the control” of expression control sequences in a cell when RNA polymerase is able to transcribe the coding sequence into mRNA, which then can be translated into the protein encoded by the coding sequence.
  • Nucleic acids such as those described above, can be inserted into vectors for expression in cells.
  • a “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • Vectors can be expression vectors.
  • an “expression vector” is a vector that includes one or more expression control sequences
  • an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalo virus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses.
  • An expression vector can include a tag sequence.
  • Tag sequences are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere 31 45659820.1 within the polypeptide including at either the carboxyl or amino terminus.
  • vectors containing nucleic acids to be expressed can be transferred into host cells.
  • host cell is intended to include prokaryotic and eukaryotic cells into which a recombinant expression vector can be introduced.
  • transformed and transfected encompass the introduction of a nucleic acid molecule (e.g., a vector) into a cell by one of a number of techniques.
  • Prokaryotic cells can be transformed with nucleic acids by, for example, electroporation or calcium chloride mediated transformation.
  • Nucleic acids can be transfected into mammalian cells by techniques including, for example, calcium phosphate co-precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, or microinjection.
  • Host cells e.g., a prokaryotic cell or a eukaryotic cell
  • Biomarkers of Aberrantly Expressed CDK5 and Uses Thereof can be used as biomarkers for MTC diagnosis and/or other diseases and disorders characterized by aberrantly activated Cdk5, identifying the corresponding subject as a target for treatment, and guiding such treatment selection. They can also be used to increase the positive predictive value of current screening modalities and for selection and monitoring the efficacy of treatment regimens. Such compositions and methods are disclosed herein.
  • the methods typically include detecting one or more of P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1 in cells of a sample from a subject.
  • the cells can be suspected of having aberrantly expressed CDK5.
  • Aberrant Cdk5 activity can be characterized by the simultaneous activation of multiple pathways regulating cell proliferation and invasion.
  • aberrant Cdk5 activity includes over or unregulated expression of Cdk5, hyper-phosphorylation of Cdk5 substrates including, but not limited to, P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1, or a combination thereof.
  • the cells are suspected or known to be cancer cells. Any of the methods can further include detecting one or more cancer antigen(s) or other disease markers 32 45659820.1 to further characterize the sample.
  • cancer antigen(s) or other disease markers 32 45659820.1 for example, overexpression of Cdk5 co-activators i.e., p35 / p25 have been detected in several cancers (colorectal, prostate, pituitary adenoma, pancreatic) that may serve as additional diagnostic markers of aberrant Cdk5 disease.
  • p35 is subjected to rapid proteasomal degradation in its membrane-bound form. Particularly neuroendocrine tumors are characterized by enhanced proteolytic cleavage of p35 to p25.
  • the disclosed biomarkers are proteins. Some embodiments provide these biomolecules in isolated form such as in a biological sample.
  • the preferred biological source for detection of the biomarkers is tissue (e.g., tissue suspected of being cancerous) including biopsy material from a tumor, or cells thereof.
  • the cells are blood cells such as white blood cells such as immune cells, or plasma cells.
  • intact cells are subjected to biomarker detection.
  • a sample may be obtained and processed using well-known and routine clinical methods.
  • the biological sample includes a plurality of cells.
  • the biological sample includes fresh or frozen tissue.
  • the biological sample includes formalin fixed, paraffin embedded tissue.
  • the cells are permeabilized.
  • a cell lysate or homogenate is subjected to biomarker detection.
  • the disclosed biomarkers for aberrant expression of Cdk5 are typically detected using one or more of the antibodies or other antigen binding molecules provided herein.
  • the biomarkers can be detected by any suitable method utilizing the provided antibodies.
  • the biomarkers are detected and/or measured by an immunoassay. Immunoassays utilize biospecific capture reagents, such as antibodies, to capture or locate the biomarkers.
  • the immunobinding methods include obtaining a sample, and contacting the sample with an antibody specific for the protein to be detected, as the case may be, under conditions effective to allow the formation of immunocomplexes.
  • the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches. These methods are generally based upon the detection 33 45659820.1 of a label or marker, such as any of those radioactive, fluorescent, biological and enzymatic tags.
  • a secondary binding ligand such as a second antibody and/or a biotin/avidin ligand binding arrangement, as is known in the art.
  • the antibody employed in the detection may itself be linked to a detectable label (also referred to as a detectable substance or reporter), wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • a detectable label also referred to as a detectable substance or reporter
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a “secondary” antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays can be used for detecting the biomarkers.
  • the detection of the biomarker is carried out on slides of test material (e.g., immunohistochemistry), Western blotting, surface plasmon resonance (e.g. Biacore), or by flow cytometry (FACS) analysis), e.g., as exemplified in the experiments below.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FIA fluoroimmunoassay
  • CLIA chemiluminescent immunoassay
  • CIA counting immunoassay
  • EMIT enzyme immunoassay
  • ARIS apoenzyme reactivation immunoassay
  • dipstick immunoassays dipstick immunoassays
  • immuno- chromatography assays Most assays now use nonradioactive labels.
  • Enzyme immunoassays can use enzymes as labels, such as, for example, horseradish peroxidase or alkaline phosphatase.
  • Chemiluminescent immunoassays can use luminol.
  • Fluorimetric immunoassays use fluorescent compounds (e.g., fluorescein) as labels.
  • the assays can be homogenous or heterogeneous assays, competitive and non- competitive assays.
  • sandwich, competitive, direct, and 34 45659820.1 indirect assays are four main kinds of ELISA: sandwich, competitive, direct, and 34 45659820.1 indirect assays. These methods differ in how the antibody or antigen is attached to the solid plate, and how the signal is detected.
  • an antibody is immobilized on a plate.
  • the sample containing the target antigen is added, which binds to the antibody and so is immobilized on the plate.
  • a second type of antibody is added, which also binds to the target antigen on the plate, forming a ‘sandwich’ with the target antigen in the middle.
  • the second antibody is linked to an enzyme, called a reporter enzyme, which allows the binding reaction to be measured by creating a color signal.
  • a reporter enzyme an enzyme, called a reporter enzyme, which allows the binding reaction to be measured by creating a color signal.
  • To create this signal first any unbound antibody is washed away, and a colorimetric substrate is added. The enzyme catalyzes a reaction of the substrate, creating a color change. A stronger color signal indicates more target antigen is present.
  • the first or second antibody is one of the disclosed antibodies, and the other antibody is one that detects the protein of interest or the biomarker (e.g., FAM53C, LARP6, RBL1, H1.5, and SUV39H1), but may or may not detect its phosphorylated state, and thus may target a different antigen of the protein (e.g., a non-phosphorylated antigen).
  • the assay is in the form of a sandwich assay, which is a noncompetitive immunoassay, wherein the molecule to be detected and/or quantified is bound to a first antibody and to a second antibody.
  • the first antibody may be bound to a solid phase, e.g., a bead, a surface of a well or other container, a chip or a strip
  • the second antibody is an antibody which is labeled, e.g. with a dye, with a radioisotope, or a reactive or catalytically active moiety.
  • the amount of labeled antibody bound to the analyte is then measured by an appropriate method.
  • the general composition and procedures involved with “sandwich assays” are well-established and known to the skilled person.
  • Immunohistochemistry is a process of localizing antigens (e.g., proteins) in tissue utilizing antigen-specific antibodies.
  • the antigen-binding antibody can be conjugated or fused to a tag that allows its detection, e.g., via visualization.
  • the tag is an enzyme that can catalyze a color-producing reaction, such as alkaline phosphatase or horseradish peroxidase.
  • the enzyme can be fused to the antibody or non-covalently bound, e.g., using a biotin-avidin system.
  • the antibody can be tagged with a fluorophore, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor.
  • the antigen-binding antibody can be directly tagged or it can itself be recognized by a detection antibody that carries the tag. Quantitative immunochemical techniques can also be used.
  • the Quantitative Tissue Biomarker Platform from HistoRx and/or measuring immunofluorescence level(s) can be used to quantify levels of biomarkers.
  • 35 45659820.1 Western blotting can be used and can be quantitative or qualitative.
  • a typical Western blotting procedure includes the steps of immunoprecipitating a target protein from a lysate of cells expressing the protein, performing an SDS-PAGE with said protein, transferring the protein to a nitrocellulose membrane, incubating the nitrocellulose membrane with said antibody, detecting said antibody with a secondary antibody conjugated to a fluorescent or chromogenic compound (e.g.
  • peroxidases such as horseradish peroxidase (HRP), alkaline phosphatase (AP), IRDye near-infrared (NIR) fluorescent dyes), and quantifying the respective signal of said compound (e.g. fluorescence, luminescence, chromogenic enzyme substrate).
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • NIR near-infrared fluorescent dyes
  • the ratio of two signals generated by Western blotting employing the same antibody but two different samples can be calculated, thereby determining how much more/less (fold- change) of the biomarker is present in one sample compared to another.
  • the methods provided herein involve determining the presence, absence, and/or concentration of biomarker in a cell, and/or the number of biomarker positive cells in sample by fluorescence activated cell sorting (FACS) using a flow cytometry device (e.g., Beckman Coulter Z2 Coulter Counter, Beckman Coulter Inc.).
  • a FACs-based method include the step of preparing the output composition for detection by flow cytometry before the biomarker can be detected. For example, the output composition can be incubated with a fluorescently labeled antibody that is specific for one or more biomarkers, and then the sample can be analyzed using a flow cytometer.
  • the cells are permeabilized to facilitate antibody access to intracellular biomarker.
  • flow cytometry cells bound by fluorescently labeled affinity reagents are carried in a fluidic stream, are separated based on size and/or fluorescent signal and are subsequently analyzed and counted using a FACS software program (e.g., FlowJo software).
  • the number or approximate number of cells can be determined by detection of the fluorescent signal, which optionally can be determined or processed by the FACS software program to provide the total or approximate number of particles in the output composition.
  • a sample is analyzed by means of a biochip.
  • Biochips generally include solid substrates and have a generally planar surface, to which a capture reagent (also called an adsorbent or affinity reagent) is attached. Frequently, the surface of a biochip includes a plurality of addressable locations, each of which has the capture reagent bound there.
  • a capture reagent also called an adsorbent or affinity reagent
  • Protein biochips are biochips adapted for the capture of polypeptides. Many protein biochips are described in the art. These include; for example, protein biochips produced by 36 45659820.1 Ciphergen Biosystems, Inc.
  • Photonic biosensors can be used in label-free assays.
  • photonic biosensors combine photonic sensing with bio recognition technology to create label-free testing on-chip. Instead of moving electrons around on silicon chips, light is moved around on silicon chips via waveguides. This technology has allowed the development of miniature lab-on-a-chip label- free immunoassay (LFIA) devices. These devices are functionalized with capture antibodies and have a resonance condition of light.
  • LFIA label-free immunoassay
  • This resonance wavelength will be shifted by a reaction between the capture antibody and the target antigen due to the change in refractive index. Measuring the shift in resonance wavelength provides a readout of a binding event.
  • Label-free assays therefore enable the detection of antigen-antibody binding without the use of an additional label, resulting in increased assay sensitivity and decreased working time.
  • Any of the detection methodologies can be multiplexed to detect two, three, four, or all five of the disclosed biomarker.
  • the methodology is or includes a multiplex immunoassay utilizing Luminex microbead technology.
  • the biomarkers can be used in diagnostic tests to assess cancer and/or other Cdk5- related disease and disorder status in a subject, e.g., to distinguish between normal cells and diseased cells, and disease status.
  • disease status includes, without limitation, the presence or absence of disease (e.g., cancer v. non-cancer), characterization of cells including cancer cells (e.g., level of aberrant Cdk5 activity), the risk of developing disease, the stage of the disease (e.g., non-invasive or early-stage cancer v. invasive or metastatic cancer), the progress of disease (e.g., progress of disease or remission of disease over time) and the effectiveness or response to treatment of disease.
  • disease status includes, without limitation, the presence or absence of disease (e.g., cancer v. non-cancer), characterization of cells including cancer cells (e.g., level of aberrant Cdk5 activity), the risk of developing disease, the stage of the disease (e.g., non-invasive or early-stage cancer v.
  • the biomarkers discussed herein can be present and/or expressed in cancer including but not limited to MTC, and, therefore, each is individually useful in aiding in the determination of cancer and/or other Cdk5-related diseases and disorders.
  • the method involves, first, measuring the selected biomarker in a subject sample using the methods described herein, and, second, comparing the measurement with a diagnostic amount or cut-off that distinguishes a positive cancer and/or other Cdk5-related disease and disorder status from 37 45659820.1 a negative cancer and/or other Cdk5-related disease and disorder status.
  • the diagnostic amount represents a measured amount of a biomarker above which a subject is classified as having a particular status. For example, because the biomarker is up-regulated compared to normal during cancer and/or other Cdk5-related diseases and disorders, then a measured amount above the diagnostic cutoff provides a diagnosis or status of the cancer and/or other Cdk5-related diseases and disorders. As is well understood in the art, by adjusting the particular diagnostic cut-off used in an assay, one can increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician.
  • the particular diagnostic cut-off can be determined, for example, by measuring the amount of the biomarker in a statistically significant number of samples from subjects with the different cancer statuses and drawing the cut-off to suit the diagnostician's desired levels of specificity and sensitivity.
  • Combinations of Markers While individual biomarkers are useful diagnostic biomarkers, a combination of biomarkers may provide greater predictive value of a particular status than single biomarkers alone. Specifically, the detection of a plurality of biomarkers in a sample can increase the sensitivity and/or specificity of the test. Thus, in one embodiment, two or more, three or more, four or more or even all five of the biomarkers can be detected and used to assess the status of cancer and/or other Cdk5-related disease and disorder in a subject.
  • Biomarker amounts or patterns can be characteristic of various risk states, e.g., high, medium, or low.
  • the risk of developing a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and/or pattern of biomarkers that is associated with the particular risk level.
  • E. Determining Stage of Disease Another embodiment provides methods for determining the stage of disease in a subject. Each stage of the disease can have a characteristic amount of a biomarker or relative amounts of a set of biomarkers (a pattern).
  • the stage of a disease is determined by measuring the relevant biomarker or biomarkers and then either submitting them to a classification algorithm or comparing them with a reference amount and/or pattern of biomarkers that is associated with the particular stage. 38 45659820.1 F. Determining Course (Progression/Remission) of Disease Still another embodiment provides methods for determining the course of disease in a subject. Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes.
  • This method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons. Similarly, this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.
  • Subject Management In certain embodiments of the method including the detection and/or analysis of one or more biomarkers further include managing subject treatment based on the status. Such management includes the actions of the physician or clinician subsequent to determining cancer and/or other Cdk5-related disease and disorder status.
  • a physician makes a diagnosis of cancer and/or other Cdk5-related disease and disorder
  • a certain regime of treatment such as prescription or administration of chemotherapy, radiation, immunotherapy, including, but not limited to administration of the compositions discussed in more detail below
  • a diagnosis of non-cancer or benign tumor might be followed with further testing to determine a specific disease that the patient might be suffering from.
  • further tests may be required.
  • One embodiment provides a method for selecting a subject for treatment for cancer and/or other Cdk5-related disease and disorder by detecting the presence or quantity of one or more biomarkers provided herein in a sample from a subject suspected of having cancer and/or other Cdk5-related disease and disorder, comparing the levels of biomarker in the sample to a predetermined standard, wherein the patient is selected for treatment for cancer and/or other Cdk5-related disease and disorder if certain biomarkers or levels of biomarkers are detected in the sample.
  • Such treatments can be those known to be effective and/or preferred for treating subjects with aberrant Cdk5-postive conditions.
  • the methods additionally or alternatively include identifying the subject as not having a Cdk5-related disease and disorder, when the test is negative.
  • the subject may have a cancer or another diseases or disorder
  • the subject can be 39 45659820.1 identified as negative for aberrant Cdk5-related cancer and other diseases and disorder.
  • Such embodiments may lead to selection of alternative treatments and may avoid treatments known to be effective or preferred for treating subjects with aberrant Cdk5-postive conditions, and/or may include treatments that are known not to be effective and/or preferred for treating subjects with aberrant Cdk5-postive conditions. Additional embodiments relate to the communication of assay results or diagnoses or both to technicians, physicians or patients, for example.
  • computers will be used to communicate assay results or diagnoses or both to interested parties, e.g.: physicians and their patients.
  • the assays will be performed or the assay results analyzed in a country or jurisdiction which differs from the country or jurisdiction to which the results or diagnoses are communicated.
  • a diagnosis based on the presence or absence in a test subject of any of the disclosed biomarkers is communicated to the subject as soon as possible after the diagnosis is obtained.
  • the diagnosis may be communicated to the subject by the subject's treating physician.
  • the diagnosis may be sent to a test subject by email or communicated to the subject by phone.
  • a computer may be used to communicate the diagnosis by email or phone.
  • the message containing results of a diagnostic test may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications.
  • all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.
  • the biomarkers can be used to screen for compounds that modulate the expression of the biomarkers in vitro or in vivo, which compounds in turn may be useful in treating or preventing cancer and/or other Cdk5-related disease and disorder in patients.
  • Compounds suitable for therapeutic testing may be screened initially by identifying compounds which reduce the presence of one or more biomarkers in the cancer and/or other Cdk5-related disease and disordered cells.
  • Test compounds capable of modulating the presence and/or expression of any of the biomarkers in cancer cells or other Cdk5-related disease and disorder may be administered to patients who are suffering from or are at risk of developing cancer having the biomarkers.
  • the administration of a test compound that decreases the activity of a particular 40 45659820.1 biomarker may decrease the risk of cancer and/or other Cdk5-related disease and disorder in a patient if the increased activity of the biomarker is responsible or indicative, at least in part, for the onset of the cancer and/or other Cdk5-related disease and disorder.
  • screening a test compound includes obtaining samples from test subjects before and after the subjects have been exposed to a test compound. The levels in the samples of one or more of the biomarkers can be measured and analyzed to determine whether the levels of the biomarkers change after exposure to a test compound.
  • the samples can be analyzed by any appropriate means known to one of skill in the art including e.g., by the means described herein.
  • the changes in the level of expression of one or more of the biomarkers can be measured using in vitro methods and materials.
  • human tissue cultured cells which express, or are capable of expressing, one or more of the biomarkers may be contacted with test compounds.
  • Subjects who have been treated with test compounds will be routinely examined for any physiological effects which may result from the treatment.
  • the test compounds will be evaluated for their ability to decrease disease likelihood in a subject.
  • test compounds are administered to subjects who have previously been diagnosed with cancer and/or other Cdk5-related disease and disorder, test compounds will be screened for their ability to slow or stop the progression of the disease.
  • Disease course refers to changes in disease status over time, including disease progression (worsening) and disease regression (improvement). Over time, the amounts or relative amounts (e.g., the pattern) of the biomarkers changes.
  • this method involves measuring one or more biomarkers in a subject at least two different time points, e.g., a first time and a second time, and comparing the change in amounts, if any. The course of disease is determined based on these comparisons.
  • this method is useful for determining the response to treatment. If a treatment is effective, then the biomarkers will trend toward normal, while if treatment is ineffective, the biomarkers will trend toward disease indications.
  • the biomarkers can be used in studies to determine if the subject is at risk for developing cancer and/or other Cdk5-related disease and disorder. 41 45659820.1 IV. Methods of Treatment Any of the disclosed methods can be coupled to a method of treating a subject in need thereof.
  • any of the disclosed methods can further include treating a positive for cancer and/or other Cdk5-related disease and disorder with a treatment known to be effective and/or preferred for treating subjects with aberrant Cdk5-postive conditions.
  • the method includes treating a subject with a disease or disorder, but negative for aberrant Cdk5 activity with a treatment not to be effective and/or preferred for treating subjects with aberrant Cdk5-postive conditions and/or selecting against treatments known to be effective or preferred for treating subjects with aberrant Cdk5-postive conditions.
  • the compositions are administered systemically, locally, or regionally.
  • the compositions are taken orally, injected, topically applied, or otherwise administered directly into the vasculature or onto vascular tissue at or adjacent to a site of cancerous growth.
  • local administration causes an increased localized concentration of the compositions, which is greater than that which can be achieved by systemic administration.
  • A. Conditions to Be Diagnosed and Treated can have a disease, disorder, or condition such as but not limited to, cancer, neurological diseases (such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Cerebral ischemia, Traumatic brain injury, and addiction), an immune system disorder such as autoimmune disease, an inflammatory disease, an infectious disease, or combinations thereof.
  • the disease, disorder, or condition is characterized by aberrant (e.g., increase) Cdk5 activity.
  • the disease, disorder, or condition can be associated with an elevated expression or specific expression of an antigen, e.g., P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and/or P-S391 SUV39H1.
  • an antigen e.g., P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and/or P-S391 SUV39H1.
  • the cancer or other disease or disorder is analyzed for one or more of P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1, increased presence of which indicates the subject has a disease or disorder associated aberrant Cdk5 activity.
  • the subject is previously, contemporaneously, or subsequently analyzed for one or more additional indicators for cancer.
  • additional indicators can be, for example, morphological or molecular (e.g., other biomarkers).
  • the subject is analyzed for one or more cancer antigens were detected on the cancer cells, 42 45659820.1 optionally leading to a cancer diagnosis.
  • the cancer was already diagnosed and/or characterized prior to or as an adjunct to treatment.
  • the subjects to be treated have a proliferative disease, such as a benign or malignant tumor.
  • the subjects to be treated have been diagnosed with stage I, stage II, stage III, or stage IV cancer.
  • the term cancer refers specifically to a malignant tumor.
  • compositions and methods are useful for treating subjects having benign or malignant tumors by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting, or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.
  • Malignant tumors that may be treated are classified according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands.
  • the compositions are particularly effective in treating carcinomas.
  • Sarcomas which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Tumors may develop in various organs or tissues throughout the body to establish malignant cancer.
  • the types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as colorectal cancer, peritoneal carcinomatosis, pancreatic cancer, multiple myeloma, sarcomas, brain, breast, esophageal, liver, lung, stomach, and uterine.
  • the compositions are used to treat multiple cancer types concurrently.
  • the compositions can also be used to treat metastases or tumors at multiple locations.
  • the cancer is one in which Cdk5 is up-regulated. Cdk5 has emerged as an important instigator of tumorigenic signaling in several types of neuroendocrine tumors (NETs) including pancreatic, adrenal, thyroid, and gastrointestinal tumors.
  • NETs neuroendocrine tumors
  • Non- neuroendocrine malignancies such as colorectal cancer and melanomas are also afflicted with pathological activation of Cdk5 signaling.
  • Cdk5 has been shown to promote tumor growth and 43 45659820.1 invasion by regulating the activity of proteins involved in metabolic reprogramming, cytoskeletal remodeling, and epithelial-mesenchymal transition.
  • Cdk5 is reported to be over-expressed or hyperactivated in various cancer tissues and tumor cell lines. For example, in patients with lung cancer it is associated with clinical pathological characteristics and poorer prognoses.
  • Cdk5 In head and neck squamous cell carcinoma, aberrant over-expression of Cdk5 significantly induces tumor cell motility and epithelial mesenchymal transition (EMT), which is considered as a pivotal process of cancer metastasis.
  • EMT epithelial mesenchymal transition
  • HCC hepatocellular carcinoma
  • over-expression and hyperactivation of Cdk5 play an oncogenic role by inducing proliferation and clonogenic growth of HCC.
  • pancreatic cancer cells Cdk5 is widely active.
  • both p35 and Cdk5 are up-regulated and hyperactivated, leading to subsequent EMT and cancer cell motility by phosphorylating Fak, which is known to be involved in cellular adhesion and spreading processes.
  • Cdk5 and p35 are also significantly increased in clinical breast cancer tissues and in breast cancer cell lines exposed to paclitaxel at transcriptional and translational levels. See, e.g., Liu, et al., Molecular Cancer volume 16, Article number: 60 (2017), 9 pages, and references cited therein.
  • results provided in the experiments below not only illustrate the importance of aberrant Cdk5 expression in Medullary Thyroid Carcinoma (MTC), but also highlight its involvement in metabolic dysregulations instilling aggressive MTC phenotype.
  • MTC Medullary Thyroid Carcinoma
  • An increasing number of studies show that malignant tumors are highly dependent on lipid metabolism and fatty acid synthesis for growth and survival [58, 59].
  • Cdk5 is an emerging candidate entangled in several metabolic conditions including cancer, diabetes, and obesity [60] [61].
  • Cdk5-dependent phosphorylation of PPAR ⁇ and PRKAG2 impairs key metabolic sensors such as adipsin, adiponectin, and AMPK kinase [12, 62].
  • the subject has a non-cancer metabolic disorder such as diabetes or obesity.
  • Therapeutic treatment can involve administering to a subject a therapeutically effective amount of a treatment after cancer or another condition has been diagnosed. 44 45659820.1 B.
  • therapies In some embodiments, patients are also subject to one or more therapies or procedures for the treatment of the disease or disorder. When two or more therapies or procedures are used, they can be simultaneous or sequential combination therapy.
  • the therapy is a conventional treatment for cancer, more preferably a conventional treatment for the particular cancer type, e.g., prostate or breast cancer.
  • the additional therapy or procedure is surgery, a radiation therapy, or chemotherapy.
  • the conventional cancer therapy is in the form of one or more active agents. Therefore, in some embodiments, the methods administer compositions in combination with one or more additional active agents.
  • active agent can be, for example, chemotherapeutic agents, cytokines, chemokines, radiation therapy, or immunotherapy.
  • the majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents.
  • Therapeutics include monoclonal antibodies and the tyrosine kinase inhibitors e.g., imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).
  • the therapy is a chemotherapeutic agent.
  • chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, camptothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllotoxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine, hydroxycarb amide, idarubicin, ifosfamide, innotecan, leucovorin, liposomal doxorubicin, liposomal daunorubici , lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, met
  • pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2)5, and combinations thereof. 45 45659820.1
  • the treatment is or includes immunotherapy such as inhibition of checkpoint proteins such as components of the PD-1/PD-L1 axis or CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and/or a cancer vaccine.
  • immunotherapy such as inhibition of checkpoint proteins such as components of the PD-1/PD-L1 axis or CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists), adoptive T cell therapy, and/or a cancer vaccine.
  • immune checkpoint modulators
  • Exemplary immune checkpoint modulators used in immunotherapy include Pembrolizumab (anti-PD1 mAb), Durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), Atezolizumab (anti-PDL1 mAb), Nivolumab (anti-PD1 mAb), Tremelimumab (anti-CTLA4 mAb), Avelumab (anti-PDL1 mAb), and RG7876 (CD40 agonist mAb).
  • the treatment is or includes adoptive T cell therapy. Methods of adoptive T cell therapy are known in the art and used in clinical practice.
  • adoptive T cell therapy involves the isolation and ex vivo expansion of tumor-specific T cells to achieve greater number of anti-tumor T cells than what could be obtained by vaccination alone.
  • the tumor-specific T cells are then infused into patients with cancer in an attempt to give their immune system the ability to overwhelm remaining tumor via T cells, which can attack and kill the cancer.
  • Several forms of adoptive T cell therapy can be used for cancer treatment including, but not limited to, culturing tumor infiltrating lymphocytes or TIL; isolating and expanding one particular T cell or clone; and using T cells that have been engineered to recognize and attack tumors.
  • the T cells are taken directly from the patient's blood.
  • the treatment is or includes a cancer vaccine.
  • Vaccination typically includes administering a subject an antigen (e.g., a cancer antigen) together with an adjuvant to elicit therapeutic T cells in vivo.
  • the cancer vaccine is a dendritic cell cancer vaccine in which the antigen is delivered by dendritic cells primed ex vivo to present the cancer antigen.
  • compositions and methods are used prior to or in conjunction with surgical removal of tumors, for example, in preventing primary tumor 46 45659820.1 metastasis.
  • the compositions and methods are used to enhance the body’s own anti-tumor immune functions.
  • the treatment targets Cdk5.
  • SCH727965 Dinaciclib (formerly SCH727965), a potent and selective small molecule inhibitor of Cdk2, Cdk5, Cdk1 and Cdk9, is the first Cdks inhibitor to enter the clinic trail. Its promising anti-cancer results and acceptable safety profile have been respectively proven in preclinical studies and human phase I trial. In preclinical model of ovarian cancer, SCH727965 has been shown to synergize with cisplatin in killing cancer cells. In phase I trial of SCH727965, subjects with advanced malignancies experienced acceptable and tolerable dose- limiting toxicities, including orthostatic hypotension, elevated uric acid, nausea, anemia, neutropenia, etc.
  • the treatment is a functional nucleic acid that targets Cdk5.
  • Functional nucleic acid molecules can be divided into the following non-limiting categories: antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, and other gene editing compositions.
  • antisense molecules siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, external guide sequences, and other gene editing compositions.
  • Such compositions can be used to reduce expression of Cdk5.
  • Cdk5 also plays an important role in treatment of chemo-resistant cancers. For example, in cervical cancer the expression of cyclin I is up-regulated by cisplatin treatment, which in turn confers cancer cells resistance to cisplatin by activating Cdk5 and its anti- apoptosis effect.
  • Cdk5 with siRNA can significantly increase the sensitivity to cisplatin in Hela cell lines with over-expressed cyclin I [76]. Similar results are also observed in Cdk5-inhibited HCC cells [66] and Cdk5-depleted ovarian cancer cell lines [77], in which cancer cells exhibit higher sensitivity to DNA damaging agents. Thus, some treatments include simultaneous reduction of Cdk5 activity, and chemotherapy. In some embodiments, the treatment is or includes roscovitine.
  • Roscovitine [CY-202, (R)-Roscovitine, Seliciclib] is a small molecule that inhibits cyclin-dependent kinases (CDKs) through direct competition at the ATP-binding site (Cicenas, et al., Ann Transl Med.2015 Jun;3(10):135. doi: 10.3978/j.issn.2305-5839.2015.03.61. PMID: 26207228; PMCID: PMC4486920.). It is a broad-range purine inhibitor, which inhibits CDK1, CDK2, CDK5 and 47 45659820.1 CDK7, but is a poor inhibitor for CDK4 and CDK6.
  • CDKs cyclin-dependent kinases
  • Roscovitine is widely used as a biological tool in cell cycle, cancer, apoptosis and neurobiology studies. Moreover, it is currently evaluated as a potential drug to treat cancers, neurodegenerative diseases, inflammation, viral infections, polycystic kidney disease and glomerulonephritis.
  • the treatment is or includes Indolinone A.
  • Indolinone A Selective Cdk5 inhibition by Indolinone A (IndoA) blocked all human NE cancer cell growth more potently than it affected normal human fibroblasts or rat INS cells (Carter, et al., PNAS, 117 (31) 18401-18411 (2020) doi:10.1073/pnas.2010103117.
  • the treatment is or includes MRT3-007.
  • MRT3-007 is a potent Cdk5 inhibitor that can reverse the phospho-cascade, invoking a senescence-like phenotype, a therapeutic approach that halted tumor progression in vivo (Gupta, et al., Cell Rep.2022 Aug 16;40(7):111218. doi: 10.1016/j.celrep.2022.111218. PMID: 35977518; PMCID: PMC9822535).
  • the treatment is or includes the peptide inhibitor TFP5/TP5.
  • TP5 has been shown to decrease glioblastoma cell viability and tumor growth by blocking cell cycle and increasing apoptosis through the inhibition of ATM phosphorylation, and act synergistically with radiotherapy and temozolomide by impairing DNA damage repair (Tabouret, et al., Cancers (Basel).2020 Jul 17;12(7):1935. doi: 10.3390/cancers12071935. PMID: 32708903; PMCID: PMC7409269.).
  • C. Controls The effect of the compositions can be compared to a control. Suitable controls are known in the art and include, for example, an untreated subject, or a placebo-treated subject.
  • a typical control is a comparison of a condition or symptom of a subject prior to and after administration of the targeted agent.
  • the condition or symptom can be a biochemical, molecular, physiological, or pathological readout, including but not limited to, the presence of level of the biomarkers provided herein.
  • the effect of a treatment on a particular cancer can be compared to an untreated subject, or the condition of the subject prior to treatment.
  • the symptom, pharmacologic, or physiologic indicator is measured in a subject prior to treatment, and again one or more times after treatment is initiated.
  • the control is a reference level, or average determined based on measuring the symptom, pharmacologic, or physiologic indicator in one or more subjects that do not have the disease or condition to be treated (e.g., healthy subjects).
  • the effect of the treatment is compared to a conventional treatment that is known 48 45659820.1 in the art.
  • an untreated control subject suffers from the same disease or condition as the treated subject.
  • kits can include a single dose or a plurality of doses of a treatment in a pharmaceutically acceptable carrier for shipping and storage and/or administration, and instructions for administering the compositions.
  • the instructions direct that an effective amount of the composition be administered to an individual with a particular condition/disease as indicated.
  • the composition can be formulated as described above with reference to a particular treatment method and can be packaged in any convenient manner. The present invention can be further understood by reference to the following numbered paragraphs: 1.
  • the antigen binding domain includes six complementarity-determining regions (CDRs), wherein the CDRs include one, two, three, four, five, or six consensus CDRs of the CDRs of: (i) anti-RBL1 antibody RBL1-1; (ii) anti-H1.5 antibody H1-5-1; (iii) anti-LARP6 antibody LARP6-1; (iv) anti-SUV39H1 antibody SUV39H1-1; or (v) anti-FAM53C antibody FAM53C-1. 49 45659820.1 3.
  • CDRs complementarity-determining regions
  • the CDRs include one, two, three, four, five, or six consensus CDRs of the CDRs of: (i) a heavy chain variable region including the amino acid sequence of SEQ ID NO:2 and/or a light chain variable region including the amino acid sequence of SEQ ID NO:3; (ii) a heavy chain variable region including the amino acid sequence of SEQ ID NO:15 and/or a light chain variable region including the amino acid sequence of SEQ ID NO:16; (iii) a heavy chain variable region including the amino acid sequence of SEQ ID NO:28 and/or a light chain variable region including the amino acid sequence of SEQ ID NO:29; (iv) a heavy chain variable region including the amino acid sequence of SEQ ID NO:41 and/or a light chain variable region including the amino acid sequence of SEQ ID NO:42; or (v) a heavy chain variable region including the amino acid sequence of SEQ ID NO:54 and/or a light chain variable region including the amino acid sequence of SEQ ID
  • the CDRs include one, two, three, four, five, or six including the amino acid sequence(s) of: (i) SEQ ID NOS:4-9 or a variant thereof with at least 70% sequence identity thereto; (ii) SEQ ID NOS:17-22 or a variant thereof with at least 70% sequence identity thereto; (iii) SEQ ID NOS:30-35 or a variant thereof with at least 70% sequence identity thereto; (iv) SEQ ID NOS:41-48 or a variant thereof with at least 70% sequence identity thereto; or (v) SEQ ID NOS:56-59, 34, and 60 or a variant thereof with at least 70% sequence identity thereto. 5.
  • the CDRs include the six CDRs of: (i) SEQ ID NOS:4-9 in the same order and orientation as presented in SEQ ID NOS:2 and 3, respectively; 50 45659820.1 (ii) SEQ ID NOS:17-22 in the same order and orientation as presented in SEQ ID NOS:15 and 16, respectively; (iii) SEQ ID NOS:30-35 in the same order and orientation as presented in SEQ ID NOS:28 and 29, respectively; (iv) SEQ ID NOS:41-48 in the same order and orientation as presented in SEQ ID NOS:41 and 42, respectively; or (v) SEQ ID NOS:56-59, 34, and 60 in the same order and orientation as presented in SEQ ID NOS:54 and 55, respectively.
  • the antibody or other molecule of any one of paragraphs 1-5 including (i) a heavy chain variable region including the amino acid sequence of SEQ ID NOS:2 or a variant thereof with at least 70% sequence identity thereto and/or a light chain variable region including the amino acid sequence of SEQ ID NOS:3 or a variant thereof with at least 70% sequence identity thereto; (ii) a heavy chain variable region including the amino acid sequence of SEQ ID NOS:15 or a variant thereof with at least 70% sequence identity thereto and/or a light chain variable region including the amino acid sequence of SEQ ID NOS:16 or a variant thereof with at least 70% sequence identity thereto; (iii) a heavy chain variable region including the amino acid sequence of SEQ ID NOS:28 or a variant thereof with at least 70% sequence identity thereto and/or a light chain variable region including the amino acid sequence of SEQ ID NOS:29 or a variant thereof with at least 70% sequence identity thereto; (iv) a heavy chain variable region including the amino acid sequence of SEQ ID NOS:
  • any one of paragraphs 1-6 including (i) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:2 and a light chain variable region wherein the light 51 45659820.1 chain variable region includes the amino acid sequence of SEQ ID NOS:3 or a variant thereof with at least 70% sequence identity thereto; (ii) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:15 and a light chain variable region wherein the light chain variable region includes the amino acid sequence of SEQ ID NOS:16 or a variant thereof with at least 70% sequence identity thereto; (iii) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:28 and a light chain variable region wherein the light chain variable region includes the amino acid sequence of SEQ ID NOS:29 or a variant thereof with at least 70% sequence identity thereto; (iv) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NO
  • any one of paragraphs 1-6 including (i) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:2 and a light chain variable region wherein the light chain variable region includes the amino acid sequence of SEQ ID NOS:3 or a variant thereof with at least 70% sequence identity thereto, without any variation in the CDRs of SEQ ID NOS:2 and 3; (ii) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:15 and a light chain variable region wherein the light chain variable region includes the amino acid sequence of SEQ ID NOS:16 or a variant thereof with at least 70% sequence identity thereto, without any variation in the CDRs of SEQ ID NOS:15 and 16; (iii) a heavy chain variable region wherein the heavy chain variable region includes the amino acid sequence of SEQ ID NOS:28 and a light chain variable region wherein the light chain variable region includes the amino acid sequence of SEQ ID NOS:29 or
  • the antibody or other molecule of any one of paragraphs 1-8 including: (i) the amino acid sequences of SEQ ID NOS:10 and/or 11, with or without the cleavable signal sequence; (ii) the amino acid sequences of SEQ ID NOS:23 and/or 24, with or without the cleavable signal sequence; (iii) the amino acid sequences of SEQ ID NOS:36 and/or 37, with or without the cleavable signal sequence; (iv) the amino acid sequences of SEQ ID NOS:49 and/or 50, with or without the cleavable signal sequence; or (v) the amino acid sequences of SEQ ID NOS:61 and/or 62, with or without the cleavable signal sequence. 10.
  • the antibody or other molecule of any one of paragraphs 1-9 includes an amino acid sequence encoded by the nucleic acid sequence of: (i) SEQ ID NOS:12 and/or 13; (ii) SEQ ID NOS:25 and/or 26; (iii) SEQ ID NOS:38 and/or 39; (iv) SEQ ID NOS:51 and/or 52; or (v) SEQ ID NOS:63 and/or 64.
  • the antibody or molecule of any one of paragraphs 1-10 including a heavy chain constant region. 53 45659820.1 12.
  • the antibody or molecule of paragraph 11, wherein the heavy chain constant region includes the amino acid sequence of SEQ ID NO:65 or a variant thereof with at least 70% sequence identity to SEQ ID NO:65. 13.
  • the antibody or molecule of any one of paragraphs 1-12 including a light chain constant region.
  • the light chain constant region includes the amino acid sequence of SEQ ID NO:66 or 67 or a variant thereof with at least 70% sequence identity to SEQ ID NO:66 or 67.
  • the functional fragment or fusion protein is selected from Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments optionally single chain variable fragments (scFv), and single domain antibodies optionally selected from sdAb, sdFv, and nanobody fragments. 18.
  • the antibody or molecule of any one of paragraphs 15-17 wherein the antibody is selected from intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, and multispecific antibodies optionally selected bispecific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. 19.
  • a method of detecting Cdk5 activity including detecting the level of phosphorylation of one or more of P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1 by contacting a biological sample with one or more antibodies of any one of paragraphs 1-20, and detecting binding between the antibody or antibodies and the one or more of FAM53C, LARP6, RBL1, H1.5, and SUV39H1. 54 45659820.1 22.
  • the method of paragraph 21 further including determining that the sample includes aberrant Cdk5 activity if the level of detected binding is higher in the biological sample than in a control.
  • the immunoassay is selected from an enzyme immunoassay (EIA), radioimmunoassay (RIA), fluoroimmunoassay (FIA), chemiluminescent immunoassay (CLIA) and counting immunoassay (CIA), homogeneous enzyme-multiplied immunoassays (“EMIT”), apoenzyme reactivation immunoassay (“ARIS”), dipstick immunoassays, or immuno-chromatography assays.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • FFIA fluoroimmunoassay
  • CLIA chemiluminescent immunoassay
  • CIA counting immunoassay
  • EMIT enzyme-multiplied immunoassays
  • ARIS apoenzyme reactivation immunoassay
  • dipstick immunoassays dipstick immunoassays
  • immuno-chromatography assays or immuno-chromatography assays.
  • the method of paragraph 25 wherein the cells or cell lysate or fraction thereof are derived from a biopsy from a subject.
  • the biopsy contains or is suspected of containing tumor cells.
  • the biopsy is a tumor biopsy. 29.
  • a method of diagnosing a subject with a Cdk5-related disease or disorder including detecting aberrant Cdk5 according to the method of any one of paragraphs 22-28. 30.
  • the Cdk5-related disease or disorder is cancer, diabetes, obesity, an immune system disorder, an inflammatory disease, an infectious disease, or a neurological disease optionally selected from Alzheimer's disease, Parkinson's disease, Huntington's disease, Cerebral ischemia, Traumatic brain injury, and addiction.
  • the Cdk5-related disease or disorder is a cancer selected from neuroendocrine cancers, colorectal cancer, melanomas, lung cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma (HCC), pancreatic cancer, breast cancer. 32.
  • the neuroendocrine cancer is Medullary Thyroid Carcinoma (MTC), pancreatic, adrenal, thyroid, and gastrointestinal cancer. 33.
  • MTC Medullary Thyroid Carcinoma
  • pancreatic adrenal
  • thyroid and gastrointestinal cancer.
  • the treatment includes a therapy effective for treating diseases and disorders characterized by aberrant Cdk5 activity. 35.
  • the treatment includes administering the subject an effective amount of Dinaciclib (SCH727965), a proteasome inhibitor optionally Bortezomib (Velcade), Indolinone A (IndoA), MRT3-007, TFP5/TP5, or a functional nucleic acid that reduces expression of the Cdk5 gene, mRNA, or protein, optionally wherein the functional nucleic acid is siRNA, miRNA, RNAi, or shRNA.
  • Dinaciclib SCH727965
  • a proteasome inhibitor optionally Bortezomib
  • IndoA Indolinone A
  • MRT3-007 TFP5/TP5
  • TFP5/TP5 a functional nucleic acid that reduces expression of the Cdk5 gene, mRNA, or protein
  • the functional nucleic acid is siRNA, miRNA, RNAi, or shRNA.
  • p25OE was controlled by doxycycline administration (Dox, 0.1 g/L) dissolved in drinking water. Doxycycline was removed at three weeks of age inducing tumors to grow for ⁇ 10 weeks. Tumors were arrested by re- administration of doxycycline. At the end of the experiments, bilateral tumors were harvested and snap-frozen for sequencing experiments, immunoblotting, and fixed for immunohistological staining. All mice were group-housed on a 12 h light/dark cycle with access to food and water ad libitum. All animal procedures were performed under protocols approved by the UAB Institutional Animal Care and Use Committee.
  • the pups carrying the NSE-tTA transgene were confirmed using the following primers – tTA 1080R: TTT CTG TAG GCC GTG TAC CTA; tTA 906F: GAT GTT AGA TAG GCG CCC TAC TCA C; Gdf5- D1: GGA GCA CTT CCA CTA TGG GAC & Gdf5-D2: AAA GAG TGA GGA GTT TGG GGA G.
  • the Tet-op p25 gene was evaluated by the following primers – HS 18: CCA TCG ATC TAG TAC AGC TCG TCC ATG C; HS 28: AAG GAC GAC GGC AAC TAC; Gdf5-D1: GGA GCA CTT CCA CTA TGG GAC & Gdf5-D2: AAA GAG TGA GGA GTT TGG GGA G.
  • Bitransgenic mice were positive for both the NSE or CGRP and p25-GFP alleles while control littermates were positive only for one of the two alleles. All reactions were carried out using the 2X master mix from Promega.
  • Magnetic Resonance Imaging MRI was performed with a Bruker Biospec 9.4 Tesla instrument using Paravision 5.1 software (Bruker Biospin, Billerica, MA).
  • a Bruker 72 mm ID volume coil was used for excitation and a custom 24 mm surface coil for signal reception (Doty Scientific Inc., Columbia, SC).
  • Mice were anesthetized with isoflurane gas and respiration observed with a MRI-compatible physiological monitoring system (SA Instruments Inc., Stony Brook, NY). Animals were imaged in supine position on a Bruker animal bed system with circulating heated water to maintain body temperature.
  • a 2D T2-weighted RARE sequence was used for imaging of the abdomen.
  • TR/TE 2000/25ms
  • echo spacing 12.5ms
  • ETL 4, 2 averages
  • matrix 300x300 for an in-plane resolution of 100 ⁇ m.
  • Prospective respiratory gating was used to minimize motion artifacts.
  • Tumor volumes were quantitated using ImageJ software. Immunoblotting and immunohistological staining Cells and tumor tissues were lysed in 1% SDS plus 50 mM NaF.
  • Samples were sonicated briefly, spun at 20,000 g for 5 min, and supernatant combined with Laemmli buffer for analysis by SDS-PAGE followed by transfer to nitrocellulose membrane and subsequent detection of target proteins using a Li-Cor Odyssey imaging system.
  • Immunoblotting was performed using antibodies for Cdk5 (Rockland 200-301-163; 1:1000), GFP (Cell Signaling Technology 2956; 1:2000), P-T202 LARP6 (disclosed herein; 1:1000), LARP6 (Invitrogen PA5-41881; 1:1000), P-S17 H1.5 (disclosed herein; 1:1000), H1.5 (Santa Cruz sc-247158; 1:1000), P-S988 RBL1 (disclosed herein; 1:1000), RBL1 (Santa Cruz sc-318; 1:500), P-S391 57 45659820.1 SUV39H1 (disclosed herein; 1:1000), SUV39H1 (sc-377112; 1:1000), P-T143 FAM53C (disclosed herein; 1:1000), FAM53C (Invitrogen PA5-114093; 1:1000) and ⁇ -actin (Invitrogen AM4302; 1:5000).
  • samples were fixed in formalin, embedded in paraffin, and sliced into 5 ⁇ m sections for placement on glass slides. Samples were deparaffinized and subjected to high temperature antigen retrieval in citrate buffer (pH 6.0).
  • citrate buffer pH 6.0
  • samples were permeabilized in 0.3% Triton X-100, blocked with 5% normal goat serum, and then incubated overnight at 4 ⁇ C in primary antibodies to GFP (Cell Signaling 2956; 1:200), ChrA (Abcam ab15160; 1:1000), P-T202 LARP6 (disclosed herein; 1:200), P-S17 H1.5 (disclosed herein; 1:50), and P-S988 RBL1 (disclosed herein; 1:100) diluted in 5% normal goat serum and 0.3% Tween 20.
  • GFP Cell Signaling 2956; 1:200
  • ChrA Abcam ab15160; 1:1000
  • P-T202 LARP6 disclosed herein; 1:200
  • P-S17 H1.5 disclosed
  • Sections were then incubated in 0.3% hydrogen peroxide and biotinylated secondary antibodies (Pierce 31820 or 31800; 1:500) applied to slides for 1 h at room temperature followed by 30 min of streptavidin-HRP. Slides were then incubated with DAB Chromogen (Dako Liquid DAB+ substrate K3468) and counter-stained with hematoxylin. Standard procedures were used for H&E staining (Feldman and Wolfe, Methods Mol Biol. 2014;1180:31-43. doi: 10.1007/978-1-4939-1050-2_3. PMID: 25015141.). Archival human tissues and tissue microarrays were obtained in accordance with UAB IRB protocol IRB- 300002147.
  • RNA sequencing RNA sequencing
  • WES Whole exome sequencing
  • RNA-Seq RNA sequencing
  • Total DNA was extracted from the frozen tumors using DNeasy Blood and tissue kit (Qiagen) according to the manufacturer’s instructions, and RNA was purified using RNEasy Plus Mini Kit (Qiagen).
  • exome capture was performed using the Agilent SureSelect Mouse All Exome QXT capture kit (Agilent). Briefly, the genomic DNA was subjected to tagmentation reactions inserting adaptor sequences randomly throughout the genome. The DNA was PCR amplified and then incubated with biotin labeled RNA capture probes complementary to every exon.
  • RNA Integrity Number (RIN) of ⁇ 7.0 was used for sequencing library 58 45659820.1 preparation.
  • CopywriteR (v2.6.1.216) [19], which extracts DNA copy number information from off-target reads, was used to call CNVs. Custom Python (v.3.10) and Shell scripts were used for downstream analysis and visualization. RNA Seq– To remove low-quality reads from raw sequences, fastp (v0.21.0) was used [20]. Sequence alignment was performed using STAR v2.7.3a-GCC-6.4.0-2.28 aligner[21] and GRCm39 assembly. Using the accepted alignment hits, gene counts were obtained using HTSeq (HTSeq v0.12.3-foss-2018b-Python-3.6.6)[22].
  • the enriched gene sets and pathways were analyzed using GSEApy (0.13.0), Enrichr, Shiny GO, and Metascape.
  • Cell proliferation Cell proliferation assay was performed on mouse MTC cells[10] in the presence or absence of doxycycline using CyQUANTTM Direct Cell Proliferation Assay following the manufacturer’s protocol (Thermo Fisher Scientific)[24].
  • Generation and purification of monoclonal antibodies The rabbit monoclonal antibodies were developed directly from isolated B cells of immunized animals without the use of hybridomas.
  • an antigen peptide containing the phosphorylated site of interest is synthesized with an N-terminal cysteine and conjugated via the thiol- group to carrier proteins.
  • At least two rabbits are immunized with the peptide.
  • peripheral blood is drawn and the titer of the antiserum 59 45659820.1 against the antigen is determined via indirect ELISA assays against the phosphorylated peptide.
  • the rabbit with the highest titer and desired activities was used for the isolation of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • Antigen-specific B cells are cultured in vitro in multi-well plates and supernatant samples are screened to identify desired antibodies.
  • the cDNAs encoding the heavy (H) and light (L) chains of the antibodies were obtained by reverse transcription-polymerase chain reaction (RT-PCR) of RNA samples isolated from positive B cell clones.
  • H and L cDNAs cloned into mammalian expression vectors and were transiently transfected into Chinese hamster ovary (CHO) cells.
  • Recombinant monoclonal antibodies were generated from the expressed heavy and light chains. Before purification, recombinant antibodies were screened by ELISA and additional application assays. For antibodies that are specific for phosphorylated sites on proteins, the non-phosphorylated antigens were used for counter-screening assays (monoclonal rabbit antibodies by Excel BioPharm LLC).
  • NE cell-specific promoter-driven tetracycline transactivator induces p25 overexpression (p25OE) resulting in tumor development at the orthotopic site.
  • p25OE p25 overexpression
  • the resultant Cdk5/p25 interaction facilitates pro-neoplastic signaling compared to its physiological counterpart, i.e. Cdk5/p35 ( Figure 1A).
  • p25OE in calcitonin-secreting C cells is controlled by the neuron-specific enolase (NSE) promoter developed bilateral MTCs in mice [10] ( Figure 1B).
  • NSE neuron-specific enolase
  • Calcitonin gene-related peptide is a splice variant of the calcitonin gene which translates into a neuropeptide localized in neuronal and neuroendocrine cells.
  • CGRP promoters are more efficient in restricting the transgene expression in calcitonin-secreting C cells in comparison to neuronal cells [25, 26].
  • the CGRP promoter was exploited in this transgenic system to induce stringent p25OE in thyroid C cells while preventing leaky expression in off-target tissues (Figure 1C).
  • both NSE and CGRP promoter-driven 71 45659820.1 p25OE developed MTC tumors ( Figure 1D).
  • NSE-p25OE and human tumors The top significantly enriched biological processes common between NSE-p25OE and human tumors include ‘G1/S transition of mitotic cycle’, and ‘mitotic cell cycle phase transition’ ( Figure 3B and 9B-9C). Moreover, pathways linked to mutated genes were highly clustered 73 45659820.1 across FOXO3A signaling, NOTCH-NFkB signaling, and G1/S phase transition, consistent with those previously reported in MTC patients ( Figure 3B) [39-41]. Conversely, CGRP-p25OE mouse vs. human tumor comparison revealed five intersecting genes harboring mutations in RPS6KA2, GPM6A, PATZ1, HACD4, and CBFA2T3 ( Figure 3C, 10A).
  • CGRP-p25OE revealed a total of 4348 DEGs, of which 2079 were upregulated and 2269 were down-regulated (Figure 4B).
  • Intersection size of unique and common DEGs up- and down-regulated in NSE and CGRP tumors was determined by upset plots.
  • both NSE/CGRP tumors showed upregulated transcripts of Cdk5R1 and Cdk5RAP2 indicating augmented transcriptional regulation of Cdk5 signaling components in these models ( Figure 4C).
  • Pathway and process analyses performed on DEGs were clustered based on similarities of enriched terms (p-value ⁇ 0.01, gene count, and enrichment factor > 1.5). Network analysis of enriched nodes with a similarity score of >0.3 was connected by edges.
  • upregulated DEGs in NSE tumors show hallmark clusters involved in processes such as cell 74 45659820.1 adhesion, positive regulation of cell migration, cell motility, actin filament polymerization, cytokine signaling, T-cell activation, and lymphocyte proliferation (Figure 4D).
  • the downregulated DEG in NSE tumors clustered cell morphogenesis, ion transport, receptor kinases, MAPK, and PI3K activities (Figure 4E).
  • processes such as tissue morphogenesis, ion transport, cytokine signaling, and extracellular matrix organization were afflicted both with mutations and transcriptional dysregulation in NSE tumors.
  • TRRUST database uncovered the transcriptional factors (TF) namely, c-Jun, Sp1, and NFkB as putative regulators of DEGs in NSE-p25OE ( Figure 11A).
  • TF transcriptional factors
  • c-Jun the transcriptional factors
  • Sp1 the transcriptional factor
  • NFkB the transcriptional factor that influences the transcription of genes involved in the cell cycle progression [43].
  • Rho GTPases which in turn drives the transcription of genes involved in the cell cycle progression [43].
  • the Sp1 is a known regulator of ion transport in the thyroid cancer [44] while the enrichment of NFkB signifies a plausible contribution to cytokine and inflammatory responses in NSE-p25 tumors [45].
  • the main protein phosphorylation substrates of this aberrantly active kinase included P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, and P-S391 SUV39H1 [48].
  • Both NSE/CGRP models manifest aberrant Cdk5 activation as indicated by increased Cdk5- dependent phosphorylation in growing (p25OE) versus arrested tumors (p25OFF) (Figure. S5A), indicating these phosphosites may serve as biomarkers for the detection of Cdk5-driven human tumors.
  • LARP6 (La Ribonucleoprotein 6), a translational regulator shuttles between cytoplasm and nucleus promotes nucleic acid binding while linker histone H1.5 facilitates chromatin compaction.
  • the immunocytochemical staining of TT cells displayed nuclear localization of these phosphoproteins similar to that observed in mouse MTC cells, consistent with the known function of these proteins in chromatin structure modulation. Having established selective detection of these aberrant Cdk5 effectors in human cells, histological tumor sections from a small cohort of MTC patients were obtained to assess the presence of these sites. Interestingly, the immunohistochemical results showed varying degrees of phospho-site staining across MTC patients confirming a characteristic heterogeneous expression profile of MTC tumors (Figure 6E).
  • Quantification of phospho-site expression indicated by optical density (OD), shows 27% and 44% of patients with elevated P-RBL1 and P-LARP6 in TMA1 (stage: 77 45659820.1 pT1bNX, pT2 N0) while 28% exhibited an increase in both P-LARP6/P-RBL1 (n 25; TMA1).
  • OD optical density
  • Cdk5 is aberrantly activated in several NETs including MTC, contributing to tumor development.
  • Cdk5 was used as a tool to generate conditional transgenic mice that develops ‘slow’ and ‘rapid’ onset human-like MTC tumors.
  • Aberrant Cdk5 can be characterized by the simultaneous activation of multiple pathways regulating cell proliferation and invasion. Hence it is important to capture the complexity of signal transduction downstream of hyperactive Cdk5 to identify distinctive markers of aggressiveness and targets for therapeutic intervention.
  • NSE- p25OE and CGRP-p25OE mediating mild and aggressive onset of the disease.
  • exomic and RNA sequencing was performed in tumors derived from the respective models.
  • Computational analysis identified key pathways associated with the altered genes in mouse tumors and those intersecting with human tumors.
  • important genes were prioritized based on changes in the gene and transcript levels.
  • Major pathways associated with altered genes in NSE-p25OE tumors were involved in actin filament-based processes.
  • Actin-dependent enrichment included mitotic cell cycle, cell adhesion, Rho GTPase, actomyosin, and extracellular matrix organization, processes known 78 45659820.1 for their role in cancer cell proliferation [50-53].
  • Cdk5 is known to regulate actin microtubule cytoskeleton, indicating a Cdk5-dependent phenotype is acquired by the NSE model [55, 56].
  • the main overlapping mutated genes and pathways altered in NSE-p25OE and human tumors included RB1, AKT1, SMARCA2, PLD1, FOXO3a, Notch, NFkB, and G1/S phase transition.
  • Cdk5 is an emerging candidate entangled in several metabolic conditions including cancer, diabetes, and obesity [60] [61]. Cdk5-dependent phosphorylation of PPAR ⁇ and PRKAG2 impairs key metabolic sensors such as adipsin, adiponectin, and AMPK kinase [12, 62].
  • Cdk5-mediated phosphorylation of acetyl-CoA synthetase 2 was shown to promote glioblastoma growth by regulating lipid production [63].
  • Cdk5-mediated phosphorylation of acetyl-CoA synthetase 2 was shown to promote glioblastoma growth by regulating lipid production [63].
  • transcriptomic analysis of CGRP-p25OE tumors also revealed alteration in metabolic pathways including lipid metabolism, arachidonic acid metabolism, DNA metabolism, and metabolism of vitamins/ cofactors. Apart from metabolism, cell cycle, and cell division mRNA clusters were enriched in CGRP tumors. Periodic expression of cell cycle regulatory genes facilitates tumor cell proliferation [64].
  • cell cycle regulatory genes such as PTTG1, AURKA, or loss of CDK/RB, p18, and p27 are known for promoting aggressiveness in MTC [65, 66]. It is believed that dysregulation of lipid 79 45659820.1 metabolism and cell cycle processes in orchestration with aberrant Cdk5 contributes to the aggressive phenotype in CGRP-p25OE tumors. The transcriptional correlates of mutated genes were also examined to prioritize important genes in mice models. The principal mutations that induced changes in the mRNA expression of NSE-p25OE were in the leading edge of mitotic spindle assembly and Notch signaling components.
  • HMMR hyaluronan- mediated motility receptor
  • Both RPS6KA2 and PDK2 are components of PI3K/Akt/mTOR pathway involved in the regulation of cell cycle progression and metabolic sensing in glycolytic cancers [70] [71] [72]. Alterations at the genetic and transcriptional levels of certain genes within a pathway can influence other genes eliciting interactions of multiple pathways.
  • the findings reinforce the need to classify patients based on aberrant Cdk5 activity, and further subclassify them into mild or aggressive forms based on the activation of distinct molecular pathways as described here.
  • Biomarker detection agents in the form of recombinant monoclonal antibodies that can distinguish Cdk5-dependent tumors were also developed and tested. These biomarkers can predict anti-Cdk5 therapy responsiveness in patient-derived xenografts [48].
  • transgenic mouse models that recapitulate both slow and rapid-onset human-like MTC tumors driven by aberrant Cdk5 activity were established.
  • the CGRP- p25OE model mimics aggressive tumors primarily characterized by alterations in metabolic pathways.
  • NSE-p25OE mice develop slow growing tumors characterized by dysregulation of mitotic spindle assembly and Notch signaling components. Findings encourage the development of personalized therapeutic strategies involving Cdk5 inhibitors in combination with patient-specific signaling modulators such as those targeting fatty acid metabolism or cell cycle regulators.
  • References 1. Modigliani, E., et al., Prognostic factors for survival and for biochemical cure in medullary thyroid carcinoma: results in 899 patients. The GETC Study Group.
  • Puig-Oliveras, A., et al., Expression-based GWAS identifies variants, gene interactions and key regulators affecting intramuscular fatty acid content and composition in porcine meat. Sci Rep, 2016.6: p.31803. 47. Motamed, M., et al., Steroid receptor coactivator 1 is an integrator of glucose and NAD+/NADH homeostasis. Mol Endocrinol, 2014.28(3): p.395-405. 48. Carter, A.M., et al., Phosphoprotein-based biomarkers as predictors for cancer therapy. Proc Natl Acad Sci U S A, 2020.117(31): p.18401-18411. 49.

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Abstract

Des anticorps améliorés pour la détection de RBL1 phosphorylée, H1.5, LARP6, SUV39H1 et FAM53C ont été mis au point. L'invention concerne également des dosages les utilisant pour détecter les biomarqueurs : P-T143 FAM53C, P-T202 LARP6, P-S988 RBL1, P-S17 H1.5, et P-S391 SUV39H1, ainsi que des applications diagnostiques et pronostiques qui en découlent.
PCT/US2024/033266 2023-06-08 2024-06-10 Compositions et méthodes de détection d'expression aberrante de cdk5 Pending WO2024254596A1 (fr)

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CN111410693B (zh) * 2020-04-15 2020-12-22 山西农业大学 一种抗cdk5纳米抗体及其应用
US20230026259A1 (en) * 2019-03-08 2023-01-26 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
WO2023004338A2 (fr) * 2021-07-20 2023-01-26 Epicrispr Biotechnologies, Inc. Systèmes et méthodes de régulation de gènes cibles

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230026259A1 (en) * 2019-03-08 2023-01-26 Obsidian Therapeutics, Inc. Ca2 compositions and methods for tunable regulation
CN111410693B (zh) * 2020-04-15 2020-12-22 山西农业大学 一种抗cdk5纳米抗体及其应用
WO2023004338A2 (fr) * 2021-07-20 2023-01-26 Epicrispr Biotechnologies, Inc. Systèmes et méthodes de régulation de gènes cibles

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