Modulation of fusobacterium nucleatum subspecies animalis clade 2 microbes

Abstract

Compositions and methods useful in preventing, reducing, delaying, diagnosing, and treating cancer and in studying the effects of cancer-associated microbes are described. The compositions and methods can be used to detect and study cancer-associated microbes and / or attenuate, modify, or utilize the effects of the cancer-associated microbes. Compositions and methods to modulate the effects of the cancer-associated microbes include the use of vaccines, inhibitors, genetic engineering, immunotherapy, cell therapy, and chemotherapeutic treatments.

Description

MODULATION OF FUSOBACTERIUM NUCLEATUM SUBSPECIES ANIMALIS CLADE 2 MICROBESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Patent Application Nos. 63 / 508,753 filed June 16, 2023 and 63 / 564,152 filed March 12, 2024, both of which are incorporated herein by reference in their entirety as if fully set forth herein.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under CA229984, and DE027850 awarded by the National Institutes of Health. The government has certain rights in the invention.REFERENCE TO SEQUENCE LISTING

[0003] The Sequence Listing associated with this application is provided in XML format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the file containing the Sequence Listing is 3936350.xml. The file is 180,053 bytes, was created on June 14, 2024, and is being submitted electronically via Patent Center.FIELD OF THE DISCLOSURE

[0004] The current disclosure describes compositions and methods useful in reducing, diagnosing, treating, and studying the effects of cancer-associated microbes. The present disclosure provides compositions and methods to detect and study cancer-associated microbes and to attenuate, modify, or utilize the effects of the cancer-associated microbes Methods to modulate the effects of the cancer-associated microbes include the use of vaccines, inhibitors, genetic engineering, immunotherapy, cell therapy, and chemotherapeutic treatments. The cancer-associated microbes described herein include Fusobacterium nucleatum subspecies animalis clade 2 microbes.BACKGROUND OF THE DISCLOSURE

[0005] Cancer causes millions of deaths a year worldwide with rates rising as more people live to an older age. In 2024, it is estimated that over 2 million new cases of cancer will be diagnosed in the United States and over 600,000 people will die from the disease. Cancer is considered not one disease but several diseases and is considered a multifactorial disease, resulting from a combination of genetic and environmental factors, contributing to tumor heterogeneity.

[0006] Infections by viruses, bacteria, or fungi are among many risk factors for cancer development with 15% of cancers being caused by oncogenic pathogens worldwide. Thus, the study of the mechanisms of infection-mediated cancer is of particular interest, aimed at both preventing cancer and improving current treatments.

[0007] Fusobacterium nucleatum (Fn), for example, a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal (Gl) tract of healthy individuals, is enriched in human colorectal cancer (CRC) tumors. High intratumoral Fn loads are associated with recurrence, metastases, and poorer patient prognosis and exogenous Fninfection in animal and cellular models supports a cancer-promoting role for this bacterium. However, strain-to-strain variation in Fn genotypic and phenotypic features has raised challenges with reproducing Fn cancer-inducing phenotypes in some animal and cellular models, suggesting that a select group of Fn strains may possess carcinogenic properties. Thus, there is a need to better understand which Fn strains promote cancer and the mechanisms by which they act. Harnessing this knowledge can accommodate methods of either preventing or reducing the occurrence of cancer and infection and improving treatments.SUMMARY OF THE DISCLOSURE

[0008] Through a combination of bioinformatic tools, it was determined that Fusobacterium nucleatum subspecies animalis (Fna) includes two distinct clades that differ in their genetic and protein-coding content and are distinguishable by a multitude of genetic, epigenetic, and physical attributes. Of these, a single clade of Fna (Fna C2) is significantly associated with the colorectal cancer (CRC) tumor niche (p=0.0000012).

[0009] The present disclosure describes compositions and methods useful in preventing, reducing, delaying, diagnosing, and treating cancer or infection and also useful in studying the effects of cancer-associated microbes. Disclosed compositions and methods to modulate the effects of cancer-associated microbes include the use of vaccines, inhibitors, genetic engineering, immunotherapy, cell therapy, and chemotherapeutic treatments. Prophylactic and therapeutic treatments of microbe-associated cancers or infections are provided.

[0010] In particular embodiments, the cancer-associated microbe includes Fusobacterium nucleatum (Fn). In particular embodiments, the Fn includes Fna. In particular embodiments, the Fna includes Fna clade 2 (C2). In particular embodiments, the cancer includes a cancer of the gastrointestinal tract. In particular embodiments, the cancer includes colorectal cancer. In particular embodiments, the cancer includes oral squamous cell carcinoma. In particular embodiments, the infection includes an infection of the gastrointestinal tract.

[0011] Particular embodiments utilize a vaccine to modulate Fna C2 within a subject. In particular embodiments, a vaccine includes a Fna C2-associated antigen or a sequence encoding the Fna C2-associated antigen and a pharmaceutically acceptable carrier. In particular embodiments, the Fna C2-associated antigen includes fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. The Ena C2-associated antigen can be free floating or fused to a delivery scaffold. In particular embodiments, a delivery scaffold includes a scaffold protein, virus-like particle, or nanoparticle. The vaccine can also include a nucleic acid vaccine. In particular embodiments, the nucleic acid sequence (e.g., mRNA) encoding a Fna C2-associated antigen is delivered as a vaccine. Alternatively, the vaccine can include dead bacteria, attenuated bacteria, or genetically engineered bacteria wherein the Fna C2- associated antigen is expressed on the surface of the dead bacteria, attenuated bacteria, or genetically engineered bacteria.

[0012] Particular embodiments utilize genetically engineered bacteria strains to support delivery of a payload to tumors. In particular embodiments, the genetically engineered bacteria include non-pathogenic bacteria. In particular embodiments, the non-pathogenic bacteria include a backbone of bacterial taxa or includes a bacterial strain that doesnot harbor known virulence genes in the context of the planned administration or delivery route. In particular embodiments, the non-pathogenic bacteria include Fna C1 or attenuated Fna C2. In alternative embodiments Fna C1 is a pathogenic bacteria (e.g., in periodontal disease). In particular embodiments, the bacteria strains are genetically engineered to include a sequence encoding a heterologous eut operon, a heterologous pdu operon, and / or a heterologous gdar operon. In particular embodiments, the eut operon, pdu operon, and / or gdar operon provide competitive growth advantages in the tumor microenvironment. In particular embodiments, the bacteria strains are genetically engineered to further include a payload including a therapeutic payload or marker. The genetically engineered bacteria strains can be useful for invading tumors, particularly those which include cells expressing phosphatidylethanolamine (PE), ethanolamine (EA), or 1 ,2-propanediol (1,2-PD). In particular embodiments, the gdar operon provides competitive growth advantages in high acidic environments. Once the tumors have been invaded, the genetically engineered bacteria strains can release a therapeutic payload to treat the tumor. In particular embodiments, the genetically engineered bacteria include a mechanism to control their presence following administration, for example, a suicide switch.

[0013] Particular embodiments include a targeted therapeutic including a binding domain that binds a Fna C2- associated antigen. In particular embodiments, the Fna C2-associated antigen includes fusobacterial apoptosis protein- 2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. In particular embodiments, the targeted therapeutic includes an antibody, multi-domain binding molecule, an antibody conjugate, or a recombinant receptor. Methods of treating a subject are provided by administering the therapeutics described above. Additionally, an antibody, multidomain binding molecule, or an antibody conjugate with a binding domain that binds an Fna C2-associated antigen can be used to detect the C2 clade as a basis for guiding patient treatment.

[0014] Furthermore, in the presence of Fn, including Fna, cancer cells are halted in the G0-G1 phase of the cell cycle. Because many chemotherapeutic treatments target cancer cells in the S phase, traditional therapeutic methods may be rendered ineffective. Therefore, in particular embodiments, a method of treatment includes administering a Fn inhibitor or a drug that kills cancer cells in the G0-G1 phase of the cell cycle. In particular embodiments, the compositions and methods can include a combination treatment wherein the method includes administering i) a Fn inhibitor or a drug that kills cancer cells in the G0-G1 phase of the cell cycle and II) a second drug that inhibits cancer cells (e.g., chemotherapeutics targeting the S phase of the cell cycle).

[0015] Compositions and methods for spatially profiling a tumor microenvironment to assess the role of tumor microbiota are also described herein. In particular embodiments, a method of spatially profiling a tumor microenvironment includes obtaining a tissue sample from the tumor microenvironment; isolating individual cells; capturing nucleic acid sequences from each isolated cell; tagging nucleic acid sequences with an oligo sequence including a location barcode; analyzing captured nucleic acid sequences; determining expression profile at each location within the tissue sample. A platform for implementing these methods described herein is also provided.BRIEF DESCRIPTION OF THE FIGURES

[0016] Some of the drawings submitted herewith may be better understood in color. Applicant considers the color versions of the drawings as part of the original submission and reserves the right to present color images of the drawings in later proceedings.

[0017] FIGs. 1 A-1 G. Fusobacterium nucleatum (Fn) niche features. 1 A, Schematic of Fusobacterium strain collection (n=146) and sequencing strategy of unique strains. 1 B, Column graph depicts the proportion of Fusobacterium genomes, subset by species, within the CRC and oral niche. Inset panel shows all non-Fn species. 1C, Composition of Fusobacterium nucleatum (Fn) pangenome subset by niche. Anvi'o (Eren et al. 817 PeerJ 3, e1319, 2015) gene cluster (GC) prevalence was used to define core (>95%), accessory (> 5% and <95%), and rare (<5%) features. Disparate features are those that do not fall into any of the other noted bins. 1 D, Proportion of niche-enriched GCs across CRC-associated and oral-associated Fn genomes. 1 E, Ortholog mapping of CRC-enriched and oral-enriched GCs using KofamKOALA Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (Aramaki et al. Bioinformatics 36, 2251-2252, 2020). 1 F, Column graph depicts the proportion of Fn genomes, grouped by subspecies, within the CRC and oral niche. Statistical analysis was performed using a two sample Z test, two-tailed. NS, not significant. 1G, Presence / absence heatmap of canonical Fn virulence factors (fadA (Han et al. J. Bacteriol. 187, 5330-5340, 2005; Xu, M. et al. J. Biol. Chem. 282, 25000-25009, 2007; and Umana et al. J. Bacteriol. 201 , e00273-19, 2019), fap2 (Coppenhagen-Glazer et al. Infect. Immun. 83, 1104-1113, 2015), radD (Kaplan et al., Mol. Microbiol. 71 , 35—47, 2009; and Engevik et al. Gastroenterology 160, 1301-1314.e8, 2021), aim1 (Kaplan et al. J. Dent. Res. 84, 700-704, 2005), cmpA (Lima et al., MicrobiologyOpen 6, e00444, 2017), fusolisin (Brokstad et al. FEMS Microbiol. Lett. 66, 235-238, 1990; Bachrach et al. Oral Microbiol. Immunol. 19, 155-159, 2004; Ogawa et al. J Endod. 32, 521-523, 2006; and Doron et al. PLoS ONE 9, e111329, 2014) across Fn subspecies.

[0018] FIG. 2. Table showing strain collection, with genome and methylome accession numbers.

[0019] FIGs. 3A-3E. Fn genetic characterization by niche and subspecies. 3A, Size of the Fn pangenome split by the core genome (>95%) (labeled “Fn core”) and accessory (<95%) genome (labeled “Fn accessory"). n=10,000 random subsamplings of 135 Fn genomes. Data is plotted as median ± standard deviation (s.d.) 3B, Size of the Fn pangenome split by CRC-associated and oral-associated niche origin, with respective core and accessory genomes labeled. n- 0,000 random subsamplings of 55 Fn CRC-associated and 80 Fn oral-associated genomes. Data is plotted as median ± s.d. 3C, Size of the Fn pangenome split by Fn subspecieswith respective core and accessory genomes labeled. n=10,000 random subsamplings of 75 Fna, 17 Fnn, 33 Fnp, and 10 Fnv genomes. Data is plotted as median ± s.d. 3D, 3E, Maximum-likelihood dendrograms of 3D, fadA and 3E, fplA nucleotide and amino acid sequences. For each genome, tree end points indicate Fn subspecies. 3F, Column graph depicts the proportion of Fn genomes containing canonical Fn virulence factors, subset by subspecies. Statistical analysis performed using two sample Z test, two-tailed.

[0020] FIGs. 4A-4F. Phylogenetics and genomics of Fna clades. 4A, Maximum-likelihood phylogenetic trees of Fnsingle marker genes, 16S rRNA, rpoB, zinc protease, nusA and nusG. For each genome (n=135), tree end points indicate niche origin (CRC or oral) Fn subspecies, and Fna clade 4B, Genes-in-genomes map (GiG-map) visualization of protein coding gene content across Fna genomes. Previously published NCBI genomes are labeled by strain name. 4C, Size of the Fna pangenome split by Fna clade, Fna C1 and Fna C2, with respective core and accessory genomes labeled. n=10,000 random subsamplings of 24 Fna C1 and 51 Fna C2 genomes. Data is plotted as median ± s.d. 4D, Composition of Fna pangenome subset by clade. Anvi’o gene cluster (GC) prevalence was used to define core (>95%), accessory (> 5% and <95%), and rare (<5%) features conserved in both Fna C1 and Fna C2 strains ("Fna core” (>95% in all Fna strains), “Fna cloud” (> 5% and <95% in all Fna strains), “Fna rare” (<5% in all strains) or unique for strains from each clade. Disparate features are those that do not fall into any of the other noted bins. Plot box shows 25thpercentile, median, and 75thpercentile. Plot whiskers indicate minima and maxima. 4E, Column graph indicates chromosome sizes in Fna C1 (n=24) and Fna C2 (n=51 ). Data is plotted as mean ± standard error of mean (s.e.m). Statistical analysis performed using Welch’s T-test, two-tailed. 4F, Column graph depicts the proportion of Fna genomes containing innate bacterial genetic defense systems, subset by Fna clades. Statistical analysis performed using two sample Z test, two-tailed. 4G, Graph shows the percent relative abundance of Fna C1 and Fna C2 in paired saliva (circle) or tumor biopsy (triangle) samples from 39 patients with colorectal adenocarcinomas (Russo et al., Neoplasia 40, 100901 , 2023). Data is plotted as mean ±s.e.m. Statistical analysis performed using Welch's T-test, paired.

[0021] FIGs. 5A-5F. Genetic and epigenetic characteristics of Fna clades. 5A, kSNP50maximum-likelihood wholegenome phylogenetic tree. For each Fn genome (n=135), tree end points indicate niche origin, CRC or oral, and Fn subspecies is indicated. Within Fna, Fna clades are indicated. 5B, Average nucleotide identity (ANI) matrix. Fn subspecies And Fna clades are indicated. 5C, Genes-in-Genomes (GIG) map visualization of protein coding gene content across Fn genomes. Inset panel on the right highlights groups of protein coding genes, that are distinct between Fna C1 and Fna C2. Interactive GiG-Map dataset can be found at fredhutch.github.io / fusopangea / . 5D, Principal component analysis (PCA) of Anvi’o gene clusters by presence and absence in each genome. Ellipses drawn to 95% confidence. 5E, PCA of Fna genome-wide methyl-modified nucleotide sequences. Ellipses drawn to 95% confidence. The PCA biplot is overlayed such that the top five nucleotide motifs that are driving Fna clade bifurcation are labeled. Table indicates the distribution of each motif across Fna clades. 5F, Column graph depicts the proportion of Fna CRC-associated and Fna oral-associated genomes, subset by Fna clade. Statistical analysis was performed using a two sample Z test, two-tailed.

[0022] FIGs. 6A-6F. Column graph depicting the proportion of Fna genomes containing canonical Fn virulence factors, subset by clade. Statistical analysis performed using two sample Z test, two-tailed. 6B, Computational confocal analysis of colon cancer epithelial cells (HCT116; grey) co-incubated with representative Fna C1 or Fna C2 strains. Image scale bar is 4 p.m. Bar plot demonstrating percent of HCT116 cells with intracellular Fna; n=three biological replicates with three analyzed z-stacks. Data is plotted as mean ± s.e.m. Statistical analysis performed using Welch'sT-test, two-tailed. 6C, PPanGGOLiN (Aramaki, et al. Bioinformatics 36, 2251-2252(2020)) map of Fna pangenome. Each node represents a gene group, syntenic nodes represent neighboring genes, size indicates relative presence across Fna genomes, and color depicts pangenome partition (Fna core (red), Fna C1 accessory genome (green), Fna C2 accessory genome). White arrows and boxes indicate Fna C2-associated putative ethanolamine utilization (eut) and 1 ,2-propanediol utilization (pdu) operons. 6D, Schematics of these Fna C2 operons. Interactive PPanGGOLiN map available at fredhutch.github.io / fusopangea / . 6E, 6F, Differentially expressed genes (Iog2-transformed fold change > 0.58 and < -0.58 with -loglO(p-value) > 1.30) in a representative Fna C2 strain, SB010, exposed to ethanolamine (EA) or 1 ,2-propanediol (1 ,2-PD) as compared to unexposed SB010 control. To highlight SB010-unique content, genes also differentially expressed under the same exposure conditions in a representative Fna C1 strain, KCOM 3764, have been removed (FIGs. 8B-8E). Vertical dotted lines indicate the threshold of significant gene expression, defined as Iog2- transformed fold change > 0.58 and < -0.58. Statistical analysis performed using glmQLFTest, 2-sided. Data points in black reperesnt Fna cloud (present in > 5% and <95% in all Fna strains) genes. Starred data points indicate eut and pdu operon genes.

[0023] FIGs. 7A-7E. Morphological and genomic differences between Fna clades. Representative Fna C1 and Fna C2 strains co-cultured with human colon cancer cells (HCT116). 7A-7B, Computational analysis of confocal imaging. Independent masks for cancer epithelial cells (grey), and intracellular bacterial cells (Fna C1 green; Fna C2 lavender) were generated. Masks were used to calculate the percent of HCT116 cells with intracellular Fna (FIG. 6B) (see Methods). Scale bar is 20 p.m. 7C, Bacterial aerotolerance was assessed through serial dilution plating at start, midpoint, and endpoint of co-culture. Graph shows resulting bacterial colony forming units per milliliter, standardized to start point for each strain. Dashed line indicates normalization equal to one. Statistical analysis performed using a Welch’s T-test, two-tailed. 7D, Bar plots indicate Fna cell length and cell width as measured from confocal microscopy images, subset by Fna clades, Fna C1 or Fna C2; n=45 cells per Fna clade. Data is plotted as mean ± s.e.m. Statistical analysis performed using Welch's T-test, two-tailed. 7E, KofamKOALA KEGG (Aramaki et al., Bioinformatics 36, 2251 — 2252, 2020) ortholog mapping of Fna clade-enriched gene clusters.

[0024] FIGs. 8A-8D. Fna clade transcriptomic responses to intestinal metabolites. 8A, Bar plots demonstrate the proportion of stool metagenomic samples from patients with CRC or healthy controls in which putative eut and pdu operons were detected. Statistical analysis performed using two sample Z test, two-tailed. Cohort sample sizes are indicated at the bottom of each panel. 8B, 8C, Differentially expressed genes (with Iog2-transformed fold change > 0.58 and < -0.58 with -logW(p-value) > 1.30) of a representative Fna C1 strain (KCOM 3764) and a representative Fna C2 strain (SB010) under (8B) ethanolamine (EA) or (8C) 1 ,2-propanediol (1,2-PD) exposure as compared to their respective unexposed controls. Top five significant (-loglO(p-value) > 1.30) upregulated and downregulated genes are labeled. 8D, Differentially expressed genes (with Iog2-transformed fold change > 0.58 and < -0.58 with -Iog10(p-value) > 1.30) in SB010 under Vitamin B12 exposure alone as compared to unexposed control. All differentially expressedgenes labeled. For 8B-8D, vertical dotted lines indicate the threshold of significant gene expression, defined as Iog2- transformed fold change > 0.58 and < -0.58. Statistical analysis performed using glmQLFTest, 2-sided. Data points indicate whether a gene is categorized as part of the Fna core genome, Fna C1 -associated accessory genome, or Fna C2-associated accessory genome by PPanGGOLiN.

[0025] FIG. 9. Table showing Fna PPanGGOLiN pangenome analysis. Abbreviations: Secondary (2°); Amino acid (AA); Carbohydrate (Carb), Gastrointestinal niche adaptation (Gl Niche Ad); Transcriptional alterations (Transc Alt); and Nutrient acquisition (Nut Aq).

[0026] FIGs. 10A-10C. Fna C2 impact on intestinal tumorigenesis and metabolism. 10A, Schematic of study with six- to-eight week old ApcMin+ / - mice receiving streptomycin and dextran sodium sulfate (DSS) treatment to clear the native microbiome and induce colitis, respectively. Mice were orally gavaged with vehicle control (Arm 1) or a mix of three representative Fna C1 (Arm 2) and Fna C2 (Arm 3) strains. A strain mix was used to capture a higher proportion of Fna clade-specific accessory genes (FIG. 4D). Mice monitored until endpoint at six weeks post-gavage when mice reached fifteen to seventeen weeks of age. 10B, Plot indicates the number of intestinal adenomas by treatment arm, vehicle control, Fna C1 -treated, or Fna C2-treated; n=8 mice per arm. Data is plotted as mean ± s.e.m. Statistical analysis performed using one-way ANOVA. 10C, Partial least squares-discriminant analysis (PLS-DA) of detected intestinal metabolites (n=1 ,296). Treatment arms are labeled.

[0027] FIGs. 11 A-11 H. Intestinal metabolite changes in Fna treated mice. Scatter plots shows fold change for 1 ,296 detected metabolites from 11 A, Fna 02 treated mice compared to control, 11 B, Fna 02 treated mice compared to Fna 01 treated mice, and 110, Fna 01 treated mice compared to control colored by metabolic categorization. Top five characterized metabolites are labeled. Plots below indicate the pathway enrichment score all enriched pathways. Bars are colored by metabolic categorization. Altered metabolites in Fna treated mice. 11D, Plots show the Iog2-transformed fold change in metabolite level between treatment arms by the -logW(p-value). Points indicate individual metabolites colored by their metabolic categorization. Dotted lines indicate the threshold of significant gene expression, defined as Iog2-transformed fold change > 0.58 and < -0.58 (vertical lines) and a -log10(p-value) > 1.30 (horizontal line). Statistical analysis performed on natural log-transformed values using one-sided T-test. 11 E, Clustered heatmap of top fifty metabolites across study arms. Dendogram groups individual samples by similarity of metabolite profile. Treatment arm (vehicle control, Fna C1 -treated, or Fna C2-treated) is indicated. 11 F, Schematic of glutathione metabolic pathway. 11 G, Plot demonstrates the ratio between oxidized (GSSG) and reduced (GSH) glutathione levels for each treatment arm is indicated; n=4 mice per arm. Data is plotted as mean ± s e.m Statistical analysis performed using one-way ANOVA. 11 H, Schematic of eicosanoid metabolic pathway

[0028] FIG. 12. Significantly altered intestinal metabolites in Fna C2-treated mice. Plots of individual, characterized metabolites, with metabolic categories having > 10 significantly altered metabolites shown. Each plot shows the Iog2- transformed log change between Fna C2-treated mice compared to Fna C1 -treated mice versus the Iog2-transformed log change between Fna C2-treated mice compared to control mice. Dashed lines indicate the threshold of significantlyaltered metabolites, defined as Iog2-transformed fold change (FC) > 0.58 and < -0.58. Upper right quadrant indicates metabolites that are significantly elevated in Fna C2-treated mice as compared to both Fna C1-treated and control mice. Lower left quadrant indicates metabolites that are significantly lower in Fna C2-treated mice as compared to both Fna C1 -treated and control mice. Top metabolites in each of these quadrants are labeled.

[0029] FIG. 13. Table of Fna C1 and Fna C2 amplicon sequence variant (ASV) sequences.

[0030] FIGs. 14A-14D. Fn in human tissue microbiome and stool metagenomic specimens. 14A, Plot shows the relative abundance for Fusobacterium species (left panel), Fusobacterium nucleatum (Fn) subspecies, and Fn subspecies animalis (Fna) clades using microbial 16S rRNA gene sequencing of paired tumor (grey) and normal adjacent (black) tissue (n=62 patients). Amplicon sequence variants (Callahan et al., ISME J. 11, 2639-2643 (2017)) were used to obtain Fna clade resolution (FIGs. 15A-15J). Data is plotted as mean ±s.e.m. Statistical analysis was performed using a T-test, paired. 14B, Plots show the relative abundance for Fna C1 and Fna C2 within patient primary colorectal tumor tissue from two independent cohorts (Cohort 1 n=116, Cohort 2 n=86). Data is plotted as mean ±s.e.m. Statistical analysis was performed using a T-test, paired. 14C, Fna C1 and Fna C2 detection in stool metagenomic data from patients with CRC and healthy controls. Left plot shows the pooled effect sizes for Fna C1 and Fna C2 calculated using a meta-analysis of standardized mean differences and a random effects model on MetaPhlAn4 (Blanco-Miguez et al. Nature Biotechnology 41 , 1633-1644, 2023) species-level genome bins (SGB) abundances on all CRC (n=627) and healthy control (n=619) samples. Right plot shows the effect sizes for Fna C1 and Fna C2 calculated using the same approach, but here samples in which Fna C1 co-occurred with Fna C2 were excluded. Data is plotted as mean ± s.e.m. All p-values are corrected via the Benjamini-Yakuteli method. 14D, Bars indicate individual stool samples from patients with CRC (n=627) and are colored by Fna C1 and Fna C2 detection (Fna C1 detected, Fna C2 detected, Fna undetected: light grey). The lower brackets indicate the number of stool samples where Fna C1 occurred independently (n=5), Fna C2 occurred independently (n=147), Fna clades co-occurred (n=31 ), or Fna clades were not detected (n=444).

[0031] FIGs. 15A-15K. Detection of Fn subspecies and Fna clades in human stool metagenomes. Detection of 15A, Fn subspecies (Fnn, Fnv, Fnp) and 15A-15K, Fna clades (Fna C1 and Fna C2) in stool metagenomic data from previously published independent cohorts from patients with CRC and healthy controls. 15A, Plot shows the percent relative abundance of each Fn subspecies and Fna clade in stool samples from patients with CRC (n=627) or healthy controls (n=619). Data is plotted as mean ± s.e.m and statistical analysis performed using a one-way ANOVA. 15B- 15K, Samples are plotted both by (15B-15K) individual cohorts and (15K) pooled results. In each panel, top plot shows the percent relative abundances of Fna C1 and Fna C2 in each sample Data is plotted as mean ± s.e.m and statistical analysis performed using Welch’s T-test, paired. Bottom plot demonstrates the proportion of stool samples from patients with CRC and the proportion of stool samples from healthy controls in which Fna C1 and Fna C2 were detected. Data is plotted as mean + s.d. and statistical analysis performed using two sample Z test, two-tailed. Cohort sample sizes are indicated at the bottom of each panel.

[0032] FIG. 16. Meta-analysis of Fna in human stool metagenomes. Fna C1 and Fna C2 detection in stool metagenomic data from previously published independent cohorts from patients with CRC and healthy controls plotted by individual cohorts. Left plot shows the effect sizes for Fna C1 and Fna C2 calculated across all samples (CRC n=627, healthy control n=619) using a meta-analysis of standardized mean differences and a random effects model on MetaPhlAn4 (Blanco-Miguez et al. Nature Biotechnology 41 , 1633-1644, 2023) species-level genome bins (SGB) abundances. Right plot shows the effect sizes for Fna C1 and Fna C2 calculated using the same approach but with samples where Fna C1 co-occurred with Fna C2 excluded (CRC n=596, healthy control n=616). Data is plotted as mean ± s.e.m. Statistical significance assessed by Wald test, two-sided. All p-values are corrected via the Benjamini- Yakuteli method.

[0033] FIG. 17. Table of meta-analysis of stool sample metagenomes from colorectal cancer (CRC) patients and healthy controls.

[0034] FIGs. 18A-18M. Table of meta-analysis of stool sample metagenomes from CRC patients and healthy controls, samples with Fna C1 and Fna C2 co-occurrence excluded. Differences in pH preference and acid resistance across Fna clades. 18B, Schematic of potential gastrointestinal route from the oral primary niche to the CRC tumor secondary niche. 18C, Plot indicates growth activity as measured in Biolog PM10 plates for representative Fna C1 and Fna C2 strains. Data is plotted as the normalized average across duplicates. Statistical analysis at each pH performed using Welch’s T-test, two-tailed. NG= no growth. 18D, PPanGGOLiN (Gautreau, G. et al. PLOS Comput. Biol. 16, e1007732 (2020)) map of Fna pangenome. Each node represents a gene group, syntenic nodes represent neighboring genes, size indicates relative presence across Fna genomes, and color depicts elements in the Fna C1-associated accessory genome (green), and the Fna C2-associated accessory genome (lavender). White arrow indicates putative glutamate-dependent acid resistance (gdar) operon. 18E, Schematic depicts mechanism of GDAR acid resistance system. 18F, Qualitative and 18G, quantitative measurements of colorimetric assay measuring pH change indicative of conversion of glutamine to glutamate and conversion of glutamate to y-aminobutyric acid (GABA) in the presence of representative Fna C1 and Fna C2 strains. n=3 technical triplicates of 3 biological replicates of 3 strains per Fna clade. Data is plotted as mean ± s.e.m and statistical analysis performed using an ANOVA. 18H, Schematic of experiment testing the effects of pH stress by exposure to simulated gastric fluid (SGF) at pH 3 or SGF supplemented with 10mM glutamate at pH 3. Plates show resulting growth for a representative Fna C1 and a representative Fna C2 strain over the course of an hour (hr) exposure, as compared to tryptic soy broth (TSB) control at pH 6.7. 181, Bar plots demonstrate the proportion of stool metagenomic samples from patients with CRC or healthy controls in which a putative gdar operon was detected. Statistical analysis performed using two sample Z test, two-tailed Cohort sample sizes are indicated at the bottom of each panel. Intestinal adenoma burden and fecal Fusobacterium load in Fna treated mice. Schematic of study with ApcMin'h'- mice orally gavaged with vehicle control (Arm 1) or representative Fna C1 (Arm 2) and Fna C2 (Arm 3) strains can be found in FIG. 10A. 18J, 18K, Plots indicate the number of adenomas in 18J, small intestine and 18K, large intestine by treatment arm, vehicle control, Fna C1-treated, or Fna C2-treated. Data isplotted as mean ± s.e.m; n=8 mice per treatment arm. Statistical analysis performed using one-way ANOVA. 18L, Fusobacterium- argeted qPCR was performed on fecal pellets from one-day post-gavage (PG) to study endpoint. Fn presence in each sample is plotted as Fn copies per ng of fecal DNA with a detection limit (D.L.) of 1 Fn copy. Each sample was run in triplicate, and samples with ^2 duplicates with detectable signal are included. 18M. ANI ranges between Fusobacterium nucleatum subspecies and F. nucleatum subspecies animalis clades. Table shows the range of ANI scores between each Fusobacterium nucleatum {Fn) subspecies and each Fna clade as derived by Anvi’o.

[0035] FIG. 19. Fn colonizes microniches in human CRC RNAscope CISH Inset images: (Bac+) bacteria positive and (Bac-) negative regions.

[0036] FIG. 20. Presence / absence of eut & pdu across Fna clades (absent in oral-clade).

[0037] FIGs. 21 A-21 B. 21 A) Bacterial microcompartments (BMCs) in Fna, self-assembling organelles that include an enzymatic core encapsulated by a selectively permeable protein shell and 21 B) metabolic processes involved in respective BMC.

[0038] FIG. 22. 1,2-PD Induction of Fna forms BMCs in CRC-clade but not oral-clade, and RNAseq indicates that while pdu is upregulated, eut is repressed, suggesting networked coordination.

[0039] FIG. 23. Putative Identification Fna EA / 1 ,2-PD BMCs during intracellular invasion of HT29 cell.

[0040] FIG. 24. Computational confocal analysis facilitates quantification of Fna adhesion and invasion of cancer epithelial cells.

[0041] FIG. 25. Confocal of Fna selective invasion of cancer epithelial cells, but not normal / primary.

[0042] FIGs. 26A, 26B Different morphologies of SB050 (with phasevarion) B) PPanGGOLiN partitioned graph of phasevarion. Nodes represent genes, and size is proportional to conservation across Fna CRC-clade genomes. Diversity in HsdS genes (unlabeled nodes), but conservation in REase / MTase is shown.

[0043] FIG. 27. First 2 dimensions of PCA using methylome as predictors of Fna. Motif points a- scaled to 50%.

[0044] FIG. 28. Fna CRC-clade colonization of mouse colon 8- weeks post oral gavage. Confocal, RNAscope fluorescent in situ hybridization (FISH).

[0045] FIG. 29. Presence of bacteria-colonized microniches in human tumors throughout the gastrointestinal tract. RNA scope analysis with a targeted eubacteria probe shows the presence of heavily bacteria colonized regions.

[0046] FIG. 30. F. nucleatum promotes G0 / G1 cell cycle arrest. FUCCI constructs for CT26wt cancer epithelial cells in the presence and absence of F. nucleatum (left). Cells are colored computationally. Plot on the right demonstrates the proportion of cells in each stage of cell cycle.

[0047] FIG. 31. Actions of antineoplastic agents and other drugs within the cell cycle. Potential drugs actin on each phase of the cell cycle is shown for GO, G1 : Duplicate cellular content, S: DNA synthesis, G2: Double check repair, and M: Mitosis. Example topoisomerase inhibitors include etoposide, teniposide, irinotecan, and topotecan. Example antimetabolites include cladribine, cytarabine, 5-fluorouracil, hydroxyurea, methotrexate, pentostatin, and thiopurines. Example alkylating agents include nitrogen mustards, nitrosoureas, alkyl sulfonates, triazenes, ethylenimines andmethylmelamines, and platinum compounds.

[0048] FIGs. 32A-32C. (32A) RNAScope of patient CRC tumor demonstrates tissue invasive F. nucleatum. (32B) NanoString GeoMx in-situ analysis of CRC tumors based on F. nucleatum location. (32C) Anvi'o Pangenomic analysis of F. nucleatum genomes to identify core genes

[0049] FIGs. 33A-33E. Bacterial and Human Spatial Single-Cell Transcriptomics of a Patient Colorectal Tumore. (33A) Targeted RNAscope FISH of patient CRC 001 tumor. The white boxes indicate the regions of the tissue selected for CosMx Field of View (FOV) analysis. (33B) Immunohistochemistry for PanCk, CD68, CD45, and DNA on a 5um sequential tissue section to (33A) with FOV regions shown. This section was also used for the CosMX SMI analysis (>1000 human genes and custom designed 4 bacterial gene probes). (33C) Napari generated single cell type spatial analysis of the tissue section noted in (33B). Different cell types are noted on the right based on gene expression profiles of cell type specific genes. Transcript counts for the custom bacterial spike in probes (Bacteria 16S, Treponema, Porphyromonas and Fusobacterium) are colored, each dot represents a transcript count. The location of bacterial transcripts detected via CosMx SMI are concordant with the location of bacterial RNAscope signal in (33A), validating this approach. (33C) CosMx analysis showing the spatial co-localization of bacterial transcripts (left) with neutrophils (right) within this patient’s tumor tissue. € Inset FOV from (33D) demonstrating the capability of CosMx SMI to provide single cell spatial resolution. Cell types corresponding to (33C) are shown. Gene expression spatial distribution for IL8 (each dot represents a transcript), bacterial 16S, Fusobacterium and Treponema. This demonstrates the neutrophils spatially associated with intratumoral bacteria are upregulating IL-8 (example analysis). (33E) Subset of FIG. 33D.

[0050] FIGs. 34A, 34B. Tables of Fusobacterium nucleatum inhibitors.

[0051] FIGs. 35A-35C. Reverse translational approach to develop Fna CRC-clade as a microbial cellular therapy (MCT) platform. 35A) Graphical representation of a resected human colon adenocarcinoma, colonized by Fna CRC- clade and this human tumor tissue visualized via RNAscope FISH. 35B) Primary isolation (along with cryopreservation and whole genome / methylome sequencing) of Fna CRC-clade strain SB010 from this same human adenocarcinoma specimen. 35C) Administration of Fna CRC-clade strain SB010 (5x107CFU), along with RNAscope FISH imaging of harvested organs at end of trial. Upon single intravenous (IV) tail vein injection, Fna CRC-clade strain SB010 reached tumors and spread diffusely within the tumor microenvironment (TME) by 48 hrs and persisted in distinct microniche regions even after 1 week post IV. Fna CRC-clade SB010 was not observed in other organs including liver and spleen tissue specimens.

[0052] FIGs. 36A-36D. Establishing a genetic system for engineering Fna CRC-clade as an MCT platform within this proposal. 36A) SynDNA based editing of replicative vector pHS30 to remove superfluous regions (harboring gene artifacts and open reading frames (ORFs) of unknown function) and generate minimal pFn plasmid capable of reaching high transformation efficiencies. 36B) Impact of successive plasmid edits on Fna CRC-clade strain SB010 transformation efficiencies and colony morphologies observed for SB010 transformed with pFn plasmid. 36C)Schematic detailing the generation of SynDNA based Tn cassette for SB010, and amplification with unique barcodes for transpososome assembly (EzTn5 kit). 36D) Generation of a pooled Fna CRC-clade SB010 Tn mutant library, and colony morphologies observed for SB010 transformed with Tn (transpososomes). Fn morphologies validated by genome sequencing.

[0053] FIGs. 37A-37E. 37A) Pangenomic approaches to identify enriched genes in Fna CRC clade. 37B) PPanGGOLIN partitioned graph of Fna pangenome. Each node represents a gene group, syntenic nodes show neighboring genes on chromosomes. Elements in the Fna core, CRC-clade accessory, and oral-clade accessory genome are indicated and 37C) Fna CRC-clade accessory genome is shown 37D) Overlay of SB010 genes found to be essential for growth in vitro by Tn-seq. 37E) Schematic detailing approach to identify genes essential for growth (colonization) within murine tumors via BarSeq.

[0054] FIGs. 38A-38C. Data showing tumor Ag-expressing Staphylococcus epidermidis (S. epi) 38A) reduces subcutaneous tumor growth, 38B) increases systemic tumor-specific T cells, and 38C) increases specific tumorinfiltrating T cells.

[0055] FIG. 39. Example immunogenic peptide sequences.DETAILED DESCRIPTION

[0056] Cancer causes millions of deaths a year worldwide with rates rising as more people live to an older age. In 2024, it is estimated that over 2 million new cases of cancer will be diagnosed in the United States and over 600,000 people will die from the disease. Cancer is considered not one disease but several diseases and is considered a multifactorial disease, resulting from a combination of genetic and environmental factors, contributing to tumor heterogeneity.

[0057] Infections by viruses, bacteria, or fungi are among many risk factors for cancer development with 15% of cancers being caused by oncogenic pathogens worldwide. Thus, the study of the mechanisms of infection-mediated cancer is of particular interest, aimed at both preventing cancer and improving current treatments.

[0058] Fusobacterium nucleatum {Fn , for example, a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal (Gl) tract of healthy individuals, is enriched in human colorectal cancer (CRC) tumors. High intratumoral Fn loads are associated with recurrence, metastases, and poorer patient prognosis and exogenous Fn infection in animal and cellular models supports a cancer-promoting role for this bacterium. However, strain-to-strain variation in Fn genotypic and phenotypic features has raised challenges with reproducing Fn cancer-inducing phenotypes in some animal and cellular models, suggesting that a select group of Fn strains may possess carcinogenic abilities. Thus, there is a need to better understand which Fn strains promote cancer and the mechanisms by which they act. Harnessing this knowledge can accommodate methods of either preventing cancer or improving treatments.

[0059] Through a combination of bioinformatic tools, it was determined that Fusobacterium nucleatum subspecies animalis {Fna), currently a single subspecies, includes two distinct clades that differ in their genetic and protein-coding content and are distinguishable by a multitude of genetic, epigenetic, and physical attributes. Of these, a single cladeof Fna (Fna clade 2 or Fna C2) is significantly associated with the CRC tumor niche (p=0.0000012). Functional enrichment analysis identified 870 significantly enriched genetic factors in Fna C2 strains with putative functions in genetic defense mechanisms, amino acid transport, and virulence. Fna C2 represents a clade of high-risk strains with an increased pathogenic potential in the colonic tumor niche and is a novel target for microbiome modulation for the treatment of Fusobacterium-associated CRC.

[0060] The present disclosure describes compositions and methods useful in preventing, delaying, reducing, diagnosing, and treating cancer and also in studying the effects of cancer-associated microbes. Disclosed compositions and methods to modulate the effects of cancer-associated microbes include the use of vaccines, inhibitors, genetic engineering, immunotherapy, cell therapy, and chemotherapeutic treatments. Prophylactic and therapeutic treatments of microbe-associated cancers are provided.

[0061] In particular embodiments, the cancer-associated microbe includes Fn. In particular embodiments, the Fn includes Fusobacterium nucleatum subspecies animalis (Fna). In particular embodiments, the Fna includes Fna C2. In particular embodiments, the cancer includes a cancer of the gastrointestinal tract. In particular embodiments, the cancer includes colorectal cancer. In particular embodiments, the cancer includes oral squamous cell carcinoma.

[0062] Particular embodiments utilize a vaccine to modulate Fna C2 within a subject. In particular embodiments, a vaccine includes a Fna C2-associated antigen or a sequence encoding the Fna C2-associated antigen and a pharmaceutically acceptable carrier. In particular embodiments, the Fna C2-associated antigen includes fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. The Fna C2-associated antigen can be free floating or fused to a delivery scaffold. In particular embodiments, a delivery scaffold includes a scaffold protein, virus-like particle, or nanoparticle. The vaccine can also include a nucleic acid vaccine. In particular embodiments, the nucleic acid sequence (e.g., mRNA) encoding a Fna C2-associated antigen is delivered as a vaccine. Alternatively, the vaccine can include dead bacteria, attenuated bacteria, or genetically engineered bacteria wherein the Fna C2- associated antigen is expressed on the surface of the dead bacteria, attenuated bacteria, or genetically engineered bacteria.

[0063] Particular embodiments utilize genetically engineered bacteria strains to support delivery of a payload to tumors In particular embodiments, the genetically engineered bacteria include non-pathogenic bacteria. In particular embodiments, the non-pathogenic bacteria includes a backbone of bacterial taxa or includes a bacterial strain that does not harbor known virulence genes in the context of the planned administration or delivery route. In particular embodiments, the non-pathogenic bacteria include Fna C1 or attenuated Fna C2. In alternative embodiments Fna 01 is a pathogenic bacteria (e.g., in periodontal disease). In particular embodiments, the bacteria strains are genetically engineered to include a sequence encoding a heterologous eut operon, a heterologous pdu operon, and / or a heterologous gdar operon. In particular embodiments, the eut operon, pdu operon and / or gdar operon provide competitive growth advantages in the tumor microenvironment. In particular embodiments, the bacteria strains are genetically engineered to further include a payload including a therapeutic payload or marker. The geneticallyengineered bacteria strains can be useful for invading tumors, particularly those which include cells expressing phosphatidylethanolamine (PE), ethanolamine (EA), or 1 ,2-propanediol (1,2-PD). The gdar operon can provide a growth advantage in high acidic environments. Once the tumors have been invaded, the genetically engineered bacteria strains can release a therapeutic payload to treat the tumor. In particular embodiments, the genetically engineered bacteria include a mechanism to control their presence following administration, for example, a suicide switch.

[0064] Particular embodiments include a targeted therapeutic including a binding domain that binds a Fna C2- associated antigen. In particular embodiments, the Fna C2-associated antigen includes fusobacterial apoptosis protein- 2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. In particular embodiments, the targeted therapeutic includes an antibody, multi-domain binding molecule, an antibody conjugate, or a recombinant receptor (e.g., chimeric antigen receptor). Methods of treating a subject are provided by administering the therapeutics described above. Additionally, an antibody, multi-domain binding molecule, or an antibody conjugate with a binding domain that binds an Fna C2-associated antigen can be used to detect the C2 clade as a basis for guiding patient treatment.

[0065] Furthermore, in the presence of Fn, including Fna, cancer cells are halted in the G0-G1 phase of the cell cycle. Because many chemotherapeutic treatments target cancer cells in the S phase, traditional therapeutic methods may be rendered ineffective. Therefore, in particular embodiments, a method of treatment includes administering a Fn inhibitor or a drug that kills cancer cells in the G0-G1 phase of the cell cycle. In particular embodiments, the compositions and methods can include a combination treatment wherein the method includes administering i) a Fn inhibitor or a drug that kills cancer cells in the G0-G1 phase of the cell cycle and ii) a second drug that inhibits cancer cells (e.g., chemotherapeutics targeting the S phase of the cell cycle). In particular embodiments, the drug that kills cancer cells in the G0-G1 phase of the cell cycle includes nitrosureas, asparaginase, or actinomycin D. In particular embodiments, the drug that kills cancer cells in the G0-G1 phase of the cell cycle includes a phase-nonspecific cancer drug. In particular embodiments, the second drug includes a drug that kills cancer cells in the S phase of the cell cycle. In particular embodiments, the second drug includes a phase-nonspecific cancer drug.

[0066] Similarly, this method of cancer treatment can be used on other cancers associated with a cancer-promoting bacteria. In particular embodiments, a method of treating a cancer associated with a cancer-promoting bacteria includes administering a bacteria inhibitor (e.g., Fn inhibitor) and / or a cancer drug, wherein the bacteria inhibitor destroys and / or inhibits the cancer-promoting bacteria. In particular embodiments, the cancer drug will be a phasespecific drug or a phase-nonspecific drug.

[0067] Compositions and methods for spatially profiling a tumor microenvironment to assess the role of tumor microbiota are also described herein. In particular embodiments, a method of spatially profiling a tumor microenvironment includes obtaining a tissue sample from the tumor microenvironment; isolating individual cells; capturing nucleic acid sequences from each isolated cell; tagging nucleic acid sequences with an oligo sequence including a location barcode; analyzing captured nucleic acid sequences; determining expression profile at eachlocation within the tissue sample. A platform for implementing these methods described herein is also provided.

[0068] Aspects of the current disclosure are now described in more supporting detail as follows: (i) Fusobacterium; (ii) Detection and Characterization of Fn and Fn Genes; (iii) Methylation Detection Assays; (iv) Genetic Engineering Techniques; (v) Vaccines; (vi) Binding Molecules and Targeted Therapeutics; (vii) Single Cell Transcriptome Spatial Profiling; (viii) Compositions and Formulations; (ix) Methods of Use; (x) Reference Levels Derived from Control Populations; (xi) Kits; (xii) Exemplary Embodiments; (xiii) Examples; and (xix) Closing Paragraphs. These headings are provided for organizational purposes only and do not limit the scope or interpretation of the disclosure.

[0069] (i) Fusobacterium. Fusobacterium organisms are anaerobic, non-motile, gram-negative bacilli and include F. necrophorum, F. nucleatum, F. mortiferu, F. varium, F. gonidaformans, F. alocis, F. pseudonecrophorum, F. salci, and F. ulcerans. Microscopically, they are characterized by slender or fusiform rods with tapered ends, though some species may be pleomorphic. Fusobacterium is included in the genera of anaerobic, gram-negative, non-spore-forming bacteria, which include Bacteroides, Prevotella, and Porphyromonas. Fusobacterium can be differentiated from these other gram-negative, obligate anaerobes by its ability to produce significant amounts of butyric acid from glucose, giving cultured colonies a characteristic odor. Identification in the laboratory is made by morphology and biochemical assays.

[0070] Fusobacterium species are normal inhabitants of mucosal surfaces, including the mouth, upper respiratory tract, gastrointestinal tract, and urogenital tract. Worldwide, F. nucleatum is the most common Fusobacterium species found in clinical infections, while F. necrophorum is the most virulent. The species is generally susceptible to penicillin, clindamycin, and chloramphenicol and resistant to erythromycin and macrolides.

[0071] Fusobacterium nucleatum (Fn) has gained elevated attention as an emerging cancer-associated bacterium, particularly in human colorectal cancer (CRC) where an enrichment of Fn in CRC is found relative to non- cancerous colorectal tissues. Fn can be further differentiated by the subspecies level including the subspecies: animalis (Fna), nucleatum (Fnri), polymorphum (Fnp) and vincentii / fusiforme (Fnv). In particular embodiments, Fna is associated with colonization and virulence in cancer. In particular embodiments, Fna clade 2 (C2) is associated with colonization and virulence in cancer. Herein, Fusobacterium nucleatum refers to any or all of the subspecies within the Fusobacterium nucleatum species including, for example: F. nucleatum subsp. nucleatum, F. nucleatum subsp. animalis (including Fna 01 and Fna 02), F. nucleatum subsp. polymorphum, and / or F. nucleatum subsp. vincintii.

[0072] In particular embodiments Fna 02 expresses antigens on its surface. In particular embodiments, Fna 02 surface antigens include fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin.

[0073] Fna 02 also includes Fna C2-associated putative operons: eut and pdu. The molecules are involved in ethanolamine (EA) metabolism (eut) and 1 ,2-propanediol (1 ,2- PD) metabolism (pdu). EA and 1 ,2-PD are known to be critical virulence determinants in the pathogenic lifecycles of many enteric pathogens. In the human lower gastrointestinal tract, EA is released by phosphodiesterase activity on phosphatidylethanolamine, an abundantphospholipid in cellular membranes that is constantly shed from luminal surfaces by epithelial cell exfoliation. Similarly, 1 ,2-PD is liberated from its precursor fucose, present as a terminal sugar on intestinal mucin, by commensal anaerobic bacteria. Enteric pathogens not only gain a competitive growth advantage through direct metabolism of EA and 1,2- PD, but also exploit their intestinal specificity; whereby sensing of these molecules through eut and pdu activates global regulators of virulence and induces transcriptional profiles consistent with gastrointestinal niche adaptation. In particular embodiments, eut and pdu are not expressed by Fna C1 .

[0074] Fna C2 also includes a putative glutamate-dependent acid resistance (GDAR) system. This is absent in Fna C1. The GDAR system, found in pathogenic and commensal gut bacteria, is one of the most potent acid resistance mechanisms, with glutamate being the component for the system to operate at pH 3. Analysis of stool metagenomic datasets indicated that gdar operons are significantly enriched in patients with CRC as compared to healthy controls. In particular embodiments, the gdaroperon can provide a competitive growth advantage in highly acidic environments.

[0075] In particular embodiments, Fna C2 includes virulence factors. In particular embodiments, the virulence factors are not expressed in Fna C1. In particular embodiments, Fna C2 virulence factors include eut, pdu, radD, aim1 , cmpA, fusolisin, and fap2.

[0076] An inhibitor is a substance or molecule that slows down or prevents a particular chemical reaction or process or which reduces the activity of a particular reactant, enzyme, or cell. A bacterial inhibitor destroys or suppresses a bacterial cell's viability, growth, and / or ability to reproduce.

[0077] In particular embodiments, a Fusobacterium nucleatum inhibitor includes bacterial 50S ribosomal subunit inhibitor; bacterial DNA gyrase inhibitor; (17beta)-17-ethynyl-3-oxoestr-4-en-17-yl heptanoate; (1E)-1-(2-hydroxy-5- methylphenyl)-1-dodecanone oxime; (9Z)-2-hydroxy-9-octadecenoic acid; 4-Methylumbelliferone (4-MU); 5-BDBD; 9(E),11 (Z)-Octadecadienoic acid; acetylcholine receptor allosteric modulator; adenosine 5-monophosphate; adenylyl cyclase activator; AGN 194310; angiotensin receptor antagonist; ATM kinase inhibitor; bacterial DNA gyrase inhibitor; bacterial ribosomal inhibitor; BCL inhibitor; bromodomain inhibitor; candesartan cilexetil; carbonic anhydrase inhibitor; carboxamide antiepileptic; catechol 0 methyltransferase inhibitor; CO chemokine receptor antagonist; CDK inhibitor, breast cancer; chelating agent; clindamycin palmitate hydrochloride ; cytochrome P450 inhibitor; daunorubicin HCI (Daunomycin HCI); EGFR inhibitor; emodin; epirubicin HCI; estrogen receptor antagonist; evan's blue; FOXM1 inhibitor, protein synthesis inhibitor; GABA receptor antagonist; gamma secretase modulator; gemcitabine (elaidate); gentian violet; glucagon receptor antagonist, autotaxin inhibitor; GW 3965 hydrochloride; GW4064; HDAC inhibitor; hypericin; idarubicin HCI; integrin inhibitor; JNK inhibitor; laxative; macrophage migration inhibiting factor inhibitor; matrix metalloprotease inhibitor; mitoxantrone hydrochloride; MK 0893; monoamine oxidase inhibitor; mTOR inhibitor; nadide; nemorubicin; neuropeptide receptor antagonist; NFkB pathway inhibitor; non-nucleoside reverse transcriptase inhibitor; NU7441 (KU-57788); obatoclax mesylate (GX15-070); otilonium bromide; PERK inhibitor; phosphofructokinase inhibitor; Piceatannol; pimonidazole; PLK inhibitor; polarization inhibitor; potassium channel activator; progestogen hormone; protein tyrosine kinase activator; PSB 06126; purinergic receptor antagonist; retinoidreceptor antagonist; rifampin; RNA synthesis inhibitor; serine protease inhibitor; serine / threonine kinase inhibitor; serotonin receptor agonist; serotonin receptor antagonist; serotonin receptor inverse agonist; serum / glucocorticoid regulated kinase inhibitor; SHIP2 phosphatase inhibitor; siponimod; SIRT inhibitor; sortilin inhibitor; src inhibitor; sterol demethylase inhibitor; superoxide dismutase inhibitor; tachykinin antagonist; TC-0 9311; thyroid hormone receptor agonist; ticagrelor; tropomyosin receptor kinase inhibitor; ubiquitin-conjugating enzyme inhibitor; VEGFR inhibitor; WNT5a peptide mimetic; or ZLN005.

[0078] (ii) Detection and Characterization of Fn and Fn Genes. Exemplary methods known in the art for determining levels of gene expression include cDNA arrays, cDNA fragment fingerprinting, cDNA sequencing, clone hybridization, northern blotting, in situ hybridization, RNAse protection assays, differential display, differential screening, fluorescence resonance energy transfer (FRET) detection, liquid microarrays, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time RT-PCR analysis with TaqMan assays, target amplification methods, molecular beacons, microelectric arrays, oligonucleotide arrays, polynucleotide arrays, serial analysis of gene expression (SAGE), and / or subtractive hybridization. Other methods known in the art may be used. In particular embodiments, nucleic acid sequences that correspond to nucleic acids encoding biomarkers can be used to construct primers and probes for detecting and / or measuring biomarker nucleic acids.

[0079] The terms “hybridization” and “hybridize” refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing. Hybridization technologies that may be used with assays and detections methods described herein are described in, for example, US 5, 143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661 ,028; and 5,800,992 as well as PCT WO 95 / 21265; WO 96 / 31622; WO 97 / 10365; WO 97 / 27317; EP 373 203; and EP 785 280.

[0080] In particular embodiments, to measure the level of expression of biomarkers, nucleic acids can be extracted from the biological sample. Nucleic acids can be extracted and purified from biological samples using any suitable technique. A number of techniques are known in the art, and kits to practice the techniques are commercially available (e.g., FormaPure® nucleic acid extraction kit, Beckman Coulter, Inc., Brea, CA, High Pure FFPE RNA Micro Kit, Roche Applied Science, Indianapolis, Ind.). RNA particularly can be extracted from frozen tissue sections using TRIzol (Invitrogen, Carlsbad, CA) and purified using RNeasy FFPE Kit (Qiagen, Valencia, CA). RNA can be further purified using DNAse I treatment (Ambion, Austin, Tex.) to eliminate any contaminating DNA. RNA concentrations can be made using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Rockland, Del.). RNA can be further purified to eliminate contaminants that interfere with cDNA synthesis by cold sodium acetate precipitation. RNA integrity can be evaluated by running electropherograms, and RNA integrity number (RIN, a correlative measure that indicates intactness of mRNA) can be determined using the RNA 6000 PicoAssay for the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).

[0081] Amplification. In particular embodiments in which nucleic acids are extracted, the nucleic acids can besubjected to preparative reactions. These preparative reactions can include in vitro transcription (IVT), labeling, fragmentation, amplification and other reactions. For example, mRNA can first be treated with reverse transcriptase and a primer to create cDNA prior to detection, quantitation and / or amplification; this can be done in vitro with purified mRNA or in situ, e.g., in cells or tissues affixed to a slide.

[0082] “Amplification" refers to any process of producing at least one copy of a nucleic acid and in many cases produces multiple copies. An amplification product can be RNA or DNA, and may include a complementary strand to an expressed target sequence. DNA amplification products can be produced initially through reverse translation and then optionally from further amplification reactions. The amplification product may include all or a portion of a target sequence, and may optionally be labeled. A variety of amplification methods are suitable for use, including polymerase- based methods and ligation-based methods. Exemplary amplification techniques include PCR, lipase chain reaction (LCR), ribozyme-based methods, self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), the use of Q Beta replicase, reverse transcription, nick translation, and the like.

[0083] Asymmetric amplification reactions may be used to preferentially amplify one strand representing a target sequence that is used for detection. In some cases, the presence and / or amount of the amplification product itself may be used to determine the expression level of a given target sequence. In other instances, the amplification product may be used to hybridize to an array or other substrate including sensor polynucleotides which are used to detect and / or quantitate target sequence expression.

[0084] The first cycle of amplification in polymerase-based methods typically forms a primer extension product complementary to the template strand. If the template is single-stranded RNA, a polymerase with reverse transcriptase activity is used in the first amplification to reverse transcribe the RNA to DNA, and additional amplification cycles can be performed to copy the primer extension products. The primers for a PCR must, of course, be designed to hybridize to regions in their corresponding template that can produce an amplifiable segment; thus, in particular embodiments, each primer must hybridize so that its 3' nucleotide is paired to a nucleotide in its complementary template strand that is located 3' from the 3' nucleotide of the primer used to replicate that complementary template strand in the PCR.

[0085] The target nucleic acids can be amplified by contacting one or more strands of the target nucleic acids with a primer and a polymerase having suitable activity to extend the primer and copy the target nucleic acids to produce full- length complementary nucleic acids or smaller portions thereof. Any enzyme having a polymerase activity that can copy the target nucleic acids can be used, including DNA polymerases, RNA polymerases, reverse transcriptases, and / or enzymes having more than one type of polymerase or enzyme activity. The enzyme can be thermolabile or thermostable. Mixtures of enzymes can also be used. Exemplary enzymes include: DNA polymerases such as DNA Polymerase I (Pol I), the Klenow fragment of Pol I, T4, T7, Sequenase® (GE Healthcare, Limited, UK) T7, Sequenase® Version 2.0 T7, Tub, Taq, Tth, Pfic, Pfu, Tsp, Tfl, Tli and Pyrococcus sp GB-D DNA polymerases; RNA polymerases such as E. coll, SP6, T3 and T7 RNA polymerases; and reverse transcriptases such as AMV, M-MuLV, MMLV, RNAse H MMLV (SuperScript® (Life Technologies Corporation, Carlsbad, CA), SuperScript® II, ThermoScript (Invitrogen,Carlsbad, CA), HIV-1 , and RAV2 reverse transcriptases. All of these enzymes are commercially available. Exemplary polymerases with multiple specificities include RAV2 and TH (exo-) polymerases. Exemplary thermostable polymerases include Tub, Taq, Tth, Pfic, Pfu, Tsp, Tfl, Til and Pyrococcus sp. GB-D DNA polymerases.

[0086] Suitable reaction conditions are chosen to permit amplification of the target nucleic acids, including pH, buffer, ionic strength, presence and concentration of one or more salts, presence and concentration of reactants and cofactors such as nucleotides and magnesium and / or other metal ions (e.g., manganese), optional cosolvents, temperature, thermal cycling profile for amplification schemes including PCR, and may depend in part on the polymerase being used as well as the nature of the sample. Cosolvents include formamide (typically at from 2 to 10%), glycerol (typically at from 5 to 10%), and DMSO (typically at from 0.9 to 10%). Techniques may be used in the amplification scheme in order to minimize the production of false positives or artifacts produced during amplification. These include “touchdown" PCR, hot-start techniques, use of nested primers, or designing PCR primers so that they form stem-loop structures in the event of primer-dimer formation and thus are not amplified.

[0087] Techniques to accelerate PCR can be used, for example centrifugal PCR, which allows for greater convection within the sample, and including infrared heating steps for rapid heating and cooling of the sample. One or more cycles of amplification can be performed. An excess of one primer can be used to produce an excess of one primer extension product during PCR; preferably, the primer extension product produced in excess is the amplification product to be detected. A plurality of different primers may be used to amplify different target nucleic acids or different regions of particular target nucleic acids within the sample.

[0088] Reverse transcription can be performed by any method known in the art. For example, reverse transcription may be performed using the Omniscript kit (Qiagen, Valencia, CA) or the Superscript III kit (Invitrogen, Carlsbad, CA), for RT-PCR. Target-specific priming can be performed in order to increase the sensitivity of detection of target sequences and generate target-specific cDNA.

[0089] TaqMan RT-PCR can be performed using Applied Biosystems Prism (ABI) 7900 HT instruments (Foster City, CA). Primers and probe concentrations for TaqMan analysis can be added to amplify fluorescent amplicons using PCR cycling conditions such as 95°C for 10 minutes (min) for one cycle, 95°C for 20 seconds, and 60°C for 45 seconds for 40 cycles. A reference sample can be assayed to ensure reagent and process stability. Negative controls (e.g., no template) can be assayed to monitor any exogenous nucleic acid contamination.

[0090] An amplification reaction can be performed under conditions which allow an optionally labeled sensor polynucleotide to hybridize to the amplification product during at least part of an amplification cycle. When the assay is performed in this manner, real-time detection of this hybridization event can take place by monitoring for light emission or fluorescence during amplification, as known in the art.

[0091] Where the amplification product is to be used for hybridization to an array or microarray, a number of suitable commercially available amplification products are available. Amplification kits that are available include the WT- Ovation® (NuGen, San Carlos, CA) System, WT-Ovation® System v2, WT-Ovation® Pico System, WT-Ovation®FFPE Exon Module, RiboAmp® (Life Technologies Corporation, Carlsbad, CA), and RiboAmp®Plus / RNA Amplification Kits, alone or in combination. Amplified nucleic acids may be subjected to one or more purification reactions after amplification and labeling, for example using magnetic beads (e.g., RNACIean magnetic beads, Beckman Coulter, Inc. Brea, CA).

[0092] Primers and probes to measure the level of expression of biomarkers of Fn are commercially available.

[0093] Biomarker detection can utilize a probe. A probe is a molecule that specifically binds a target, either directly or indirectly. The target can be a biomarker, a fragment of the biomarker, or any molecule that is to be detected. In the case of antibody-antigen interactions, antibodies or antibody fragments specifically bind to the antigen or epitope within the antigen and does not bind to another entity.

[0094] In particular embodiments, binds means that two molecules bind with a dissociation constant or Kd (k0ff / k0n) of not less than .05 M, not less than .025 M, not less than .02 M, not less than .01 M, not less than 5 X 103M, not less than 2.5 X 103M, not less than 2 X 10'3M, not less than 1.5 X 103M, not less than 10-3M, not less than 5 X 10'4M, not less than 2.5 X 10‘4M, not less than 2 X 10’4M, not less than 1.5 X 10’4M, not less than 10’4M, not less than 5 X 105M, not less than 2.5 X 10'5M, not less than 2 X 105M, not less than 1.5 X 10'5M, not less than 10'5M, not less than 5 X 10’6M, not less than 2.5 X 10'6M, not less than 2 X 10’6M, not less than 1.5 X 10'6M, not less than 10'6M, or not less than 10 M, or in a range of .05 M to 10 M, in a range of 5 x 103M to 10’6M, or in a range of 10'4M to 10-7M.

[0095] In particular embodiments, the probe includes a nucleic acid. In particular embodiments, protein is measured to determine the level of expression of select biomarkers. "Protein" detection includes detection of full-length proteins, mature proteins, pre-proteins, polypeptides, isoforms, mutations, post-translationally modified proteins and variants thereof encoded by the genes described herein, and can be detected in any suitable manner. Proteins can be extracted from solid tissue through homogenization, protein extract buffer (e.g., Radioimmunoprecipitation Assay (RIPA)), protease inhibitors (e.g., leupeptin, edetic acid (EDTA), and aprotinin), sonication, and / or centrifugation.

[0096] In particular embodiments, the level of expression of target proteins can be measured using immunoassays. Examples of suitable immunoassays include immunoblotting, immunoprecipitation, immunofluorescence, chemiluminescence, electro-chemiluminescence (ECL), and / or enzyme-linked immunoassays (ELISA).

[0097] In particular embodiments, a probe includes a protein. As an example, a protein probe can be an antibody. An antibody can be a whole antibody or a binding fragment of an antibody. Antibodies can be conjugated to a solid support suitable for a diagnostic assay (e.g., beads such as protein A or protein G agarose, microspheres, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as passive binding. Antibodies can be conjugated to detectable labels or groups such as radiolabels (e.g.,35S,125l,1311), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein, Alexa, green fluorescent protein, rhodamine) in accordance with known techniques. Antibodies are commercially available to measure or detect Fusobacterium nucleatum, for example ABIN4888518 and ANT0084.

[0098] In particular embodiments, biomarker levels can be assessed by a protein activity assay. Exemplary protein activity assays include protease assays, kinase assays, phosphatase assays, and reductase assays, among many others.

[0099] In particular embodiments, levels or amounts of biomarkers of the present disclosure can be measured by chromatography, a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase. In particular embodiments, the chromatography is liquid chromatography (LC), a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s). Liquid chromatography includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC). In HPLC the degree of separation is increased by forcing the mobile phase under pressure through a stationary phase, typically a densely packed column.

[0100] In particular embodiments, the chromatography is gas chromatography (GC), a process in which a sample mixture is vaporized and injected into a stream of carrier gas (such as nitrogen or helium) moving through a column containing a stationary phase composed of a liquid or a particulate solid and is separated into its component compounds according to the affinity of the compounds for the stationary phase.

[0101] In particular embodiments, the chromatography is thin layer chromatography. Thin layer chromatography separates compounds on a thin layer of adsorbent material typically including a coating of silica gel on a glass plate or plastic sheet.

[0102] In particular embodiments, levels or amounts of biomarkers of the present disclosure can be measured by ultraviolet (U V) spectroscopy, which measures the attenuation of a beam of light after it passes through a sample or after the light is reflected from the sample surface. The absorption measurements can be at a single wavelength of light or can be over a spectral range.

[0103] In particular embodiments, levels or amounts of biomarkers of the present disclosure can be measured by capillary electrophoresis, which separates molecules in submillimeter diameter capillaries, microfluidic, or nanofluidic channels containing electrolyte solutions under the influence of an electric field. Capillary electrophoresis can include gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. Separated molecules appear as peaks with different retention times in an electropherogram, which reports detector response as a function of time.

[0104] In particular embodiments, levels, amounts, or ratios of biomarkers of the present disclosure can be measured by mass spectrometry. Mass spectrometry (MS) refers to an analytical technique to identify compounds by their mass. MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compound and calculating a mass-to-charge ratio (m / z). The compound may be ionized and detected by any suitable means. A "mass spectrometer” generally includes an ionizer and an ion detector. See, e.g., US 6,204,500; 6, 107,623; 6,268,144; 6, 124, 137; Wright et al., Prostate Cancer and Prostatic Diseases. 1999, 2:264-76; and Merchant and Weinberger, Electrophoresis. 2000, 21 : 1164-67.

[0105] Samples may be processed or purified to obtain preparations that are particularly suitable for analysis by mass spectrometry. Such purification will usually include chromatography, such as liquid chromatography, and may also often involve an additional purification procedure that is performed prior to chromatography. Various procedures may be used for this purpose depending on the type of sample or the type of chromatography. Examples include filtration, extraction, precipitation, centrifugation, delipidization, dilution, combinations thereof and the like.

[0106] Probes for nucleic acids or proteins can be linked to chips, such as microarray chips. See, for example, US 5,143,854; 6,087,112; 5,215,882; 5,707,807; 5,807,522; 5,958,342; 5,994,076; 6,004,755; 6,048,695; 6,060,240; 6,090,556; and 6,040,138. Microarray refers to a solid carrier or support that has a plurality of molecules bound to its surface at defined locations. The solid carrier or support can be made of any material. As an example, the material can be hard, such as metal, glass, plastic, silicon, ceramics, and textured and porous materials; or soft materials, such as gels, rubbers, polymers, and other non-rigid materials. The material can also be nylon membranes, epoxy-glass and borofluorate-glass. The solid carrier or support can be flat, but need not be and can include any type of shape such as spherical shapes (e.g., beads or microspheres). The solid carrier or support can have a flat surface as in slides and micro-titer plates having wells. The probes can be attached to one of a variety of solid substrates capable of withstanding the reagents and conditions necessary for use of the array. Examples include polymers, such as (poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene, polycarbonate, polypropylene and polystyrene; ceramic; silicon; silicon dioxide; modified silicon; (fused) silica, quartz or glass; functionalized glass; paper, such as filter paper; diazotized cellulose; nitrocellulose filter; nylon membrane; and polyacrylamide gel pad. Substrates that are transparent to light are useful for arrays that may be used in an assay that involves optical detection

[0107] Examples of array formats include membrane or filter arrays (for example, nitrocellulose, nylon arrays), plate arrays (for example, multiwell, such as a 24-, 96-, 256-, 384-, 864- or 1536-well, microtitre plate arrays), pin arrays, and bead arrays (for example, in a liquid "slurry”). Arrays on substrates such as glass or ceramic slides are often referred to as chip arrays or “chips.” Such arrays are well known in the art.

[0108] A probe can be labeled with a detectable label. A “detectable label” refers to a molecule capable of detection, including fluorescers, chemiluminescers, dyes, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, semiconductor nanoparticles, chromophores, ligands (e.g., biotin, streptavidin, or haptens), and radioactive isotopes. The term "fluorescer” refers to a substance or a portion thereof which is capable of exhibiting fluorescence in a detectable range. Particular examples of labels which may be used include horseradish peroxidase (HRP), SYBR® (Molecular Probes, Inc. Eugene, OR) green, SYBR® (Molecular Probes Inc., Eugene, OR) gold, fluorescein, carboxyfluorescein (FAM), Alexa Fluor dyes, Cy3, Cy5, Cy7, 7-amino-4-methylcoumarin-3-aceticacid (AMCA), 5-(and-6)-carboxy-X-rhodamine, lissamine rhodamine, fluorescein-5-isothiocyanate (FITC), 7- diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)- carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein 5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionic acid, eosin-5-isothiocyanate; erythrosine-5- isothiocyanate, 5-(and-6)-carboxyrhodamine 6G, CASCADE blue aectylazide, CAL Fluor Orange 560, CAL Fluor Red 610, Quasar Blue 670, tetramethyl rhodamine (TAMRA), 2', 4', 5', 7'-tetrachloro-4-7-dichlorofluorescein (TET), rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, Pacific Blue, Pacific Orange, quantum dots, luminol, NADPH, and a-p-galactosidase.

[0109] In particular embodiments, binding to nucleic acids or proteins on microarrays can be detected by scanning the microarray with a variety of laser or CCD-based scanners, and extracting features with software packages, for example, Imagene (Biodiscovery, Hawthorne, CA), Feature Extraction Software (Agilent), Scanalyze (Eisen, M. 1999. SCANALYZE User Manual; Stanford Univ., Stanford, CA Ver 2.32.), or GenePix (Axon Instruments).

[0110] Embodiments disclosed herein can be used with high throughput screening (HTS). Typically, HTS refers to a format that performs at least 100 assays, at least 500 assays, at least 1000 assays, at least 5000 assays, at least 10,000 assays, or more per day. When enumerating assays, either the number of samples or the number of protein, nucleic acid, or other biomarkers assayed can be considered.

[0111] Generally HTS methods involve a logical or physical array of either the subject samples, or the protein or nucleic acid biomarkers, or both. Appropriate array formats include both liquid and solid phase arrays. For example, assays employing liquid phase arrays, e.g. , for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microtiter plates. Microtiter plates with 96, 384, or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general, the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis.

[0112] HTS assays and screening systems are commercially available from, for example, Zymark Corp. (Hopkinton, MA); Air Technical Industries (Mentor, OH); Beckman Instruments, Inc. (Fullerton, CA); Precision Systems, Inc. (Natick, MA), etc. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide HTS as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various methods of HTS.

[0113] The systems and methods provide a quantitative detection of whether the biomarker is present in the sample being assayed, i.e. , an evaluation or assessment of the actual amount or relative abundance of the biomarker in the sample being assayed. In such embodiments, the quantitative detection may be absolute or, when comparing two or more different biomarkers in a sample, relative. As such, the term "quantifying” when used in the context of quantifying a biomarker in a sample can refer to absolute or to relative quantification. Absolute quantification can be accomplishedby inclusion of known concentration (s) of one or more control biomarkers and referencing, e.g., normalizing, the detected level of the biomarker with the known control biomarkers (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different biomarkers to provide a relative quantification of each of the two or more biomarkers, e.g., relative to each other. The actual measurement of values of the biomarkers can be determined using any method known in the art. In some embodiments, a biomarker is detected by measuring the expression levels of gene transcripts.

[0114] (iii) Methylation Detection Assays. Methylation of the markers can be assessed using various methylation detection assays. A "methylation detection assay” refers to an assay, which can be commercially available, for distinguishing methylated versus unmethylated cytosine loci in DNA. Techniques for measuring cytosine methylation include bisulfite-based methylation assays. The addition of bisulfite to DNA results in the methylation of unmethylated cytosine and its ultimate conversion to the nucleotide uracil. Uracil has similar binding properties to thiamine in the DNA sequence. Previously methylated cytosine does not undergo similar chemical conversion on exposure to bisulfite. Bisulfite assays can thus be used to discriminate previously methylated versus unmethylated cytosine.

[0115] An exemplary quantitative methylation detection assay combines bisulfite treatment and restriction analysis COBRA, which uses methylation sensitive restriction endonucleases, gel electrophoresis, and detection based on labeled hybridization probes. (Ziong and Laird, Nucleic Acid Res. 199725; 2532-4). Another exemplary detection assay is the methylation specific polymerase chain reaction PCR (MSPCR) for amplification of DNA segments of interest. This assay can be performed after sodium bisulfite conversion of cytosine and uses methylation sensitive probes. Other detection assays include the Quantitative Methylation (QM) assay, which combines PCR amplification with fluorescent probes designed to bind to putative methylation sites; MethyLightTM (Qiagen, Redwood City, CA) a quantitative methylation detection assay that uses fluorescence based PCR (Eads, et al., Cancer Res. 1999; 59:2302-2306); and Ms-SNuPE, a quantitative technique for determining differences in methylation levels in CpG sites. As with other techniques, Ms-SNuPE also requires bisulfite treatment to be performed first, leading to the conversion of unmethylated cytosine to uracil while methyl cytosine is unaffected. PCR primers specific for bisulfite converted DNA are then used to amplify the target sequence of interest (see Table 1). The amplified PCR product is isolated and used to quantitate the methylation status of the CpG site of interest. (Gonzalgo and Jones Nuclei Acids Res1997; 25:252-31).

[0116] In particular embodiments, the INFI Nl UM® (llumina, Inc., San Diego California, USA) Human Methylation 450 Beadchip assay can be used. The Illumina assay can be used for genome wide quantitative methylation profiling. In particular embodiments, genomic DNA can be extracted from cells. Genomic DNA can be isolated and proteins or other contaminants can be removed from the DNA using proteinase K. The DNA can then be removed from the solution using available methods such as organic extraction, salting out, or binding the DNA to a solid phase support. As described above, and in the Infinium® Assay Methylation Protocol Guide, the DNA can be treated with sodium bisulfite. The bisulfite converted DNA can then be denatured and amplified. A next step can use enzymatic means to fragment the DNA. The fragmented DNA can then be precipitated using isopropanol and separated by centrifugation. Theseparated DNA can next be suspended in a hybridization buffer. The fragmented DNA can then be hybridized to beads that have been covalently limited to 50mer nucleotide segments at a locus specific to the cytosine nucleotide of interest in the genome. There are a total of over 500,000 bead types specifically designed to anneal to the locus where the particular cytosine is located, and the beads are bound to silicon-based arrays. There are two bead types designed for each locus, one bead type represents a probe that is designed to match to the methylated locus at which the cytosine nucleotide will remain unchanged. The other bead type corresponds to an initially unmethylated cytosine, which after sodium bisulfite treatment, is converted to uracil and ultimately a thiamine nucleotide. Unhybridized DNA (DNA not annealed to the beads) is washed away leaving only DNA segments bound to the appropriate bead and containing the cytosine of interest. If the cytosine of interest was unmethylated prior to the sodium bisulfite treatment, then it will match with the unmethylated or “U” bead probe. If the cytosine was methylated, single base mismatch will occur with the “U" bead probe oligomer. No further nucleotide extension on the bead oligomer occurs. This will lead to low fluorescent signal from the “U" bead. The reverse will happen on the “M” or methylated bead probe.

[0117] Lasers can then be used to stimulate fluorophores bound to the beads. The level of methylation at each cytosine locus is detected by the intensity of the fluorescence from the methylated compared to the unmethylated bead. Cytosine methylation level is expressed as “p" which is the ratio of the methylated-bead probe signal to total signal intensity at that cytosine locus.

[0118] In particular embodiments, pyrosequencing can be used to detect marker methylation. Pyrosequencing is a method of DNA sequencing that relies on detection of the release of pyrophosphates as DNA is synthesized (and is therefore a “sequencing by synthesis" technique). To assess methylation by pyrosequencing, a DNA sample can be incubated with sodium bisulfite, converting unmethylated cytosine to uracil. The presence of uracil will result in thymine incorporation during PCR amplification. Therefore, sequencing results that include thymine at a nucleotide position that is known to encode cytosine can be interpreted as unmethylated sites. In contrast cytosines present in the sequencing results indicate that the site was methylated in the original DNA sample, because methylation protects cytosine from conversion to uracil upon treatment. Bisulfite treatment can also be performed on control samples with known methylation patterns, to reduce or eliminate false positive results. Commercially available pyrosequencing machines include Pyro Mark Q96 (Qiagen, Hilden, Germany). For more details on methods to use pyrosequencing for measurement of methylation, see Delaney et al. Methods Mol Biol. 2015 1343: 249-264. Pyrosequencing is especially useful for detecting methylation in the CpG sites within genes.

[0119] Methylation also can be detected indirectly using, for example, cDNA arrays, cDNA fragment fingerprinting, cDNA sequencing, clone hybridization, differential display, differential screening, FRET detection, liquid microarrays, PCR, RT-PCR, quantitative RT-PCR analysis with TaqMan assays, molecular beacons, microelectric arrays, oligonucleotide arrays, polynucleotide arrays, serial analysis of gene expression (SAGE), and / or subtractive hybridization.

[0120] Further hybridization technologies that may be used are described in, for example, U.S. Pat. Nos. 5, 143,854;5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661 ,028; and 5,800,992 as well as WO 95 / 21265; WO 96 / 31622; WO 97 / 10365; WO 97 / 27317; EP 373 203; and EP 785 280.

[0121] Various methylation detection assays use nucleic acids and / or proteins linked to chips, such as microarray chips. See, for example, U.S. Pat. Nos. 5, 143,854; 6,087, 112; 5,215,882; 5,707,807; 5,807,522; 5,958,342; 5,994,076; 6,004,755; 6,048,695; 6,060,240; 6,090,556; and 6,040, 138. Binding to nucleic acids or proteins on microarrays can be detected by scanning the microarray with a variety of lasers or charge coupled device (CCD)-based scanners, and extracting features with software packages, for example, Imagene (Biodiscovery, Hawthorne, CA), Feature Extraction Software (Agilent), Scanalyze (Eisen, M. 1999. SCANALYZE User Manual; Stanford Univ., Stanford, Calif. Ver 2.32.), or GenePix (Axon Instruments).

[0122] Embodiments disclosed herein can be used with high throughput screening (HTS). Typically, HTS refers to a format that performs at least 100 assays, at least 500 assays, at least 1000 assays, at least 5000 assays, at least 10,000 assays, or more per day. When enumerating assays, either the number of samples or the number of markers assayed can be considered.

[0123] Generally, HTS methods involve a logical or physical array of either samples, or the nucleic acid or protein markers, or both. Appropriate array formats include both liquid and solid phase arrays. For example, assays employing liquid phase arrays, e.g., for hybridization of nucleic acids, binding of antibodies or other receptors to ligand, etc., can be performed in multiwell or microtiter plates. Microtiter plates with 96, 384, or 1536 wells are widely available, and even higher numbers of wells, e.g., 3456 and 9600 can be used. In general, the choice of microtiter plates is determined by the methods and equipment, e.g., robotic handling and loading systems, used for sample preparation and analysis.

[0124] HTS assays and screening systems are commercially available from, for example, Zymark Corp. (Hopkinton, MA); Air Technical Industries (Mentor, OH); Beckman Instruments, Inc. (Fullerton, CA); Precision Systems, Inc. (Natick, MA), etc. These systems typically automate entire procedures including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay. These configurable systems provide HTS as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for the various methods of HTS.

[0125] (iv) Genetic Engineering Techniques. Bacterial and mammalian cells can be genetically engineered to express desired genes.

[0126] As used herein, the term “engineered bacterial cell” or “engineered bacteria” refers to a bacterial cell or bacteria that have been genetically modified from their native state. For instance, an engineered bacterial cell may have nucleotide insertions, nucleotide deletions, nucleotide rearrangements, and nucleotide modifications introduced into their DNA. These genetic modifications may be present in the chromosome of the bacteria or bacterial cell, or on a plasmid in the bacteria or bacterial cell. Exemplary bacterial cells include Fusobacterium, Bacillus, Bacteroides, Bifidobacterium, Brevihacteria, Clostridium, Enterococcus, Escherichia coii, Lactobacillus, Lactococcus,Saccharomyces, and Staphylococcus.

[0127] Engineered bacterial cells disclosed herein may include exogenous nucleotide sequences on plasmids. Alternatively, engineered bacterial cells may include exogenous nucleotide sequences stably incorporated into their chromosome. In particular embodiments, bacterial cells are genetically engineered to express Fna C2-associated antigens. In particular embodiments, bacterial cells are genetically engineered to express pdu or eut operons. In particular embodiments, bacterial cells are genetically engineered to express gdar operons. In particular embodiments, bacterial cells are genetically engineered to express an acid resistance (AR2) system. In particular embodiments, bacterial cells include Fn. In particular embodiments, bacterial cells include Fna. In particular embodiments, Fna includes Fna C1 or Fna C2. In particular embodiments, Fna includes Fna 02. In particular embodiments, the bacterial cell includes Fna SB010.

[0128] In particular embodiments, mammalian cells are genetically engineered to express a recombinant receptor. In particular embodiments, mammalian cells include human cells. In particular embodiments, mammalian cells include T- cells, B cells, natural killer (NK) cells, NK-T cells, monocytes / macrophages, lymphocytes, hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPC), and / or a mixture of HSC and HPC (i.e., HSPC). In particular embodiments, genetically modified cells include T-cells.

[0129] Desired genes disclosed herein can be introduced into cells by any method known in the art, including transfection, electroporation, microinjection, lipofection, calcium phosphate mediated transfection, infection with a viral or bacteriophage vector including the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell- mediated gene transfer, spheroplast fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92) and may be used, provided that the necessary developmental and physiological functions of the recipient cells are not unduly disrupted. The technique can provide for the stable transfer of the gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferably heritable and expressible by its cell progeny.

[0130] The term “gene” refers to a nucleic acid sequence (used interchangeably with polynucleotide or nucleotide sequence) as described herein. This definition includes various sequence polymorphisms, mutations, and / or sequence variants wherein such alterations do not substantially affect the function of the encoded gene. The term “gene” may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splice sites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression of the desired molecule. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codonpreference in a specific cell type. Portions of complete gene sequences are referenced throughout the disclosure as is understood by one of ordinary skill in the art.

[0131] Gene sequences encoding desired molecules are provided herein and can also be readily prepared by synthetic or recombinant methods from the relevant amino acid sequences and other description provided herein. In embodiments, the gene sequence encoding any of these sequences can also have one or more restriction enzyme sites at the 5' and / or 3' ends of the coding sequence in order to provide for easy excision and replacement of the gene sequence encoding the sequence with another gene sequence encoding a different sequence. In embodiments, the gene sequence encoding the sequences can be codon optimized for expression in mammalian cells.

[0132] In bacterial cells, gene sequences can be present on a plasmid or bacterial chromosome. In addition, multiple copies of any gene sequence or regulatory region may be present in the bacterium, wherein one or more copies of the gene sequence or regulatory region may be mutated or otherwise altered as described herein. In some embodiments, the genetically engineered bacteria are engineered to include multiple copies of the same gene sequence or regulatory region in order to enhance copy number or to include multiple different components of a gene performing multiple different functions

[0133] "Encoding” refers to the property of specific sequences of nucleotides in a gene, such as a cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as a defined sequence of amino acids. Thus, a gene codes for a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. A "gene sequence encoding a protein" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence or amino acid sequences of substantially similar form and function.

[0134] Polynucleotide gene sequences encoding more than one portion of an expressed molecule can be operably linked to each other and relevant regulatory sequences. For example, there can be a functional linkage between a regulatory sequence and an exogenous nucleic acid sequence resulting in expression of the latter. For another example, a first nucleic acid sequence can be operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary or helpful, join coding regions, into the same reading frame.

[0135] A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors may be, e.g., plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids, bacterial artificial chromosomes, viruses, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0136] " Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus: including HIV type 1, and HIV type 2);equine infectious anemia virus; feline immunodeficiency virus (Fl V); bovine immune deficiency virus (Bl V); and simian immunodeficiency virus (S I V) .

[0137] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a selfinactivating lentiviral vector as provided in Milone et ah, Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include: the LENTIVECTOR® (Oxofrd BioMedical PLC, United Kingdom) gene delivery technology from Oxford BioMedica, the LENTIMAX vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. In particular embodiments, mammalian cells are genetically engineered to express a recombinant receptor using a lentivirus or lentiviral vector.

[0138] " Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

[0139] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. In such embodiments, the gene to be expressed is cloned into the retroviral vector for its delivery into cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequences necessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat (LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation of genomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al., 1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651 ; Kiem, et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141 ; and Grossman and Wilson, 1993, Curr. Opin in Genetics and Devel. 3: 1 IQ- 114. Adenoviruses, adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science 252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest. 91 :225-234; Walsh, et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; and Lundstrom, 1999, J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351- 359); liposomes (Tarahovsky and Ivanitsky, 1998, Biochemistry (Mose) 63:607-618); ribozymes (Branch and Klotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med. 75:267-282).

[0140] There are a large number of available viral vectors suitable within the current disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, 2001 , Ann. Rev. Genomics Hum. Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles are described in, e.g., US 8,119,772; Walchli, et al., 2011 , PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, et al., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol. Ther. 18: 1748; and Verhoeyen, et al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0141] Targeted genetic engineering approaches may also be utilized. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system used for genetic engineering that is based on a bacterial system. Information regarding CRISPR-Cas systems and components thereof are described in, for example, US8697359, US8771945, US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, US8932814, US8945839, US8993233 and US8999641 and applications related thereto; and WO2014 / 018423, WO2014 / 093595, WO2014 / 093622, WO2014 / 093635,WO2014 / 093655, 02014 / 093661, 02014 / 093694, WO2014 / 093701 , WO2014 / 093709, WO2014 / 093712,WO2014 / 093718, WO2014 / 145599, 02014 / 204723, WO2014 / 204724, WO2014 / 204725, WO2014 / 204726,WO2014 / 204727, WO2014 / 204728, WO2014 / 204729, WO2015 / 065964, WO2015 / 089351 , WO2015 / 089354,WO2015 / 089364, WO2015 / 089419, WO2015 / 089427, WO2015 / 089462, WO2015 / 089465, WO2015 / 089473 andWO2015 / 089486, W02016205711, WO2017 / 106657, WO2017 / 127807 and applications related thereto.

[0142] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs are a class of sitespecific nucleases engineered to bind and cleave DNA at specific positions. ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequence which enables the ZFNs to target unique sequences within a genome in a variety of different cells. A zinc finger is a domain of 30 amino acids within the zinc finger binding domain whose structure is stabilized through coordination of a zinc ion. Examples of zinc fingers include C2H2 zinc fingers, C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring in nature whose design / composition results principally from rational criteria, e.g., application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. A well-known example of a ZFN is a fusion of the Fokl nuclease with a zinc finger DNA binding domain. For additional information regarding ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261 ; 6,607,882; 6,746,838; 6,794, 136; 6,824,978; 6,866,997; 6,933, 113; 6,979,539; 7,013,219; 7,030,215; 7,220,719; 7,241 ,573; 7,241 ,574; 7,585,849; 7,595,376; 6,903, 185; 6,479,626; 2003 / 0232410 and 2009 / 0203140 as well as Gaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11 :636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764 (2003); Bibikova, et al. Genetics 161 , 1169-1175 (2002); Wolfe, et al. Annual review of biophysics and biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academy of Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO journal 4, 1609-1614 (1985).

[0143] Particular embodiments can use transcription activator like effector nucleases (TALENs) as gene editing agents. TALENs refer to fusion proteins including a transcription activator-like effector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genes and genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavage domain to dimerize and induce a DSB. For additional information regarding TALENs, see US 8,440,431 ;8,440,432; 8,450,471; 8,586,363; and 8,697,853; as well as Joung and Sander, Nat Rev Mol Cell Biol, 2013, 14(l):49- 55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4) :291-303; Gaj et al., Nat Methods, 2012, 9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186, 757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science 326, 1501 (2009).

[0144] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of a meganuclease. Meganucleases, also known as homing endonucleases, are single peptide chains that have both DNA recognition and nuclease function in the same domain. In contrast to the TALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity.

[0145] Particular embodiments can use transposon-based systems as gene editing agents to mediate the integration of a genetic construct into cells. Generally, such methods will involve introducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and (ii) a second vector encoding a desired genetic element that is flanked by transposon repeats. Transposons or transposable elements include a (short) nucleic acid sequence with terminal repeat sequences upstream and downstream thereof and encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences.

[0146] Several transposon / transposase systems have been adapted for genetic insertions of heterologous DNA sequences. Examples of such transposases include sleeping beauty (“SB”, e.g., derived from the genome of salmonid fish); piggyback (e.g., derived from lepidopteran cells and / or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince (e.g., derived from Rana pipiens); Toll ; Tol2 (e.g., derived from medaka fish); TcBuster (e.g., derived from the red flour beetle Tribolium castaneum), Helraiser, Himarl , Passport, Minos, Ac / Ds, PIP, Harbinger, Harbinger3-DR, HSmarl, and spinON. Transposases and transposon systems are further described in US 6,489,458; 7,148,203; 8,227,432; and 9,228,180. In particular embodiments, the transposase includes a Tn5 transposase.

[0147] Nanoparticles that result in selective in vivo genetic modification of targeted cell types have been described and can be used within the teachings of the current disclosure. In particular embodiments, the nanoparticles can be those described in WO2014153114, WO2017181110, and WO201822672.

[0148] In particular embodiments, bacterial cells are genetically engineered by introducing a replicative vector (e.g., plasmid) to the bacterial cell. In particular embodiments, the replicative vector introduces or replaces genes of interest via allelic exchange through homology arms.

[0149] In particular embodiments, bacteria are genetically engineered to support its growth in the tumor microenvironment. In particular embodiments, the bacteria are genetically engineered to include a sequence encoding a heterologous euf operon. In particular embodiments, the bacteria are genetically engineered to include a sequence encoding heterologous pdu operon. In particular embodiments, the bacteria are genetically engineered to include a sequence encoding a heterologous eut operon and a heterologous pdu operon. In particular embodiments, the bacteriaare genetically engineered to include a sequence encoding a heterologous gdar operon. In particular embodiments, the genetically engineered bacteria include non-pathogenic bacteria. In particular embodiments, the non-pathogenic bacteria include Fna C1 or attenuated Fna C2. In particular embodiments, the bacteria strains are genetically engineered to further include a payload including a therapeutic payload or marker. The genetically engineered bacteria strains can be useful for invading tumors, particularly those which include cells expressing phosphatidylethanolamine (PE), ethanolamine (EA), or 1 ,2-propanediol (1,2-PD). In particular embodiments, the gdar operon provides a competitive growth advantage in highly acidic environments. Once the tumors have been invaded, the genetically engineered bacteria strains can release a therapeutic payload to treat the tumor. In particular embodiments, the genetically engineered bacteria include a mechanism to control their presence following administration, for example, a suicide switch.

[0150] In particular embodiments, immune cells are genetically engineered to express a recombinant receptor described elsewhere herein. In particular embodiments, an immune cell includes a T-cell, a B cell, a natural killer (NK) cell, an NK-T cell, a monocyte / macrophage, a lymphocyte, a hematopoietic stem cell (HSC), and / or a hematopoietic progenitor cell (HPC).ln particular embodiments, a T cell includes a CD8+ T cell or a CD4+ T cell.

[0151] (v) Vaccines. The present disclosure provides compositions or vaccines capable of eliciting an immune response to Fusobacterium nucleatum. In particular embodiments, the Fusobacterium nucleatum includes Fna C2. In particular embodiments, the composition or vaccine includes a dead or attenuated Fna 02, an Fna C2-associated antigen, an Fna C2-associated epitope, or a sequence encoding the Fna C2-associated antigen or epitope. In particular embodiments, the composition or vaccine further includes a pharmaceutically acceptable carrier. Fna C2-associated antigen can include fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. An Fna C2-associated epitope includes an epitope on an Fna C2-associated antigen. The Fna C2-associated epitope, herein, includes Fna C2-associated antigens and epitopes thereof. In particular embodiments, the Fna C2-associated antigen includes fap2. In particular embodiments, the Fna C2-associated antigen includes cmpA. In particular embodiments, the Fna C2-associated antigen includes fusolisin. In particular embodiments, the Fna C2-associated epitope includes an epitope of fap2. In particular embodiments, the Fna C2-associated epitope includes an epitope of cmpA. In particular embodiments, the Fna C2-associated epitope includes an epitope of fusolisin.

[0152] An "epitope” includes any determinant capable of being bound by an antigen-binding protein, such as a B cell receptor (BCR). An epitope is a region of molecule that is bound by a binding protein that targets that region of a molecule, and when that region of a molecule is a protein, includes specific residues that directly contact the binding protein. In particular implementations, an "epitope” denotes the binding site on a protein target bound by a corresponding binding domain. The binding domain binds to a linear epitope, (e.g. , an epitope including a stretch of 5 to 12 consecutive amino acids), to a three-dimensional structure formed by the spatial arrangement of several short stretches of the protein target, or a combination thereof.

[0153] In particular embodiments, the composition or vaccine can include a dead bacteria or attenuated bacteria. Inparticular embodiments, the composition or vaccine can include a subunit vaccine. In particular embodiments, the composition or vaccine can include a nucleic acid encoding the Fna C2-associated antigen or epitope, such as in an RNA vaccine. Attenuated means having been reduced in force, effect, or value. Attenuated strains of disease-causing bacteria and viruses are often used as vaccines. The weakened strains are used as vaccines because they stimulate a protective immune response while causing no disease or mild disease in the person receiving the vaccine.

[0154] Non-pathogenic bacteria are bacteria that is not capable of causing a disease or a harmful response in a host.

[0155] Dead, Attenuated, or Genetically Engineered Vaccine. Inactivated vaccines use the killed version of the molecule or organism that causes a disease. Live-attenuated vaccines use a weakened (or attenuated) form of the molecule or organism that causes a disease.

[0156] Inactivated vaccines herein also refers to killed vaccines or dead vaccines. An inactivated vaccine contains bacteria that has been inactivated, killed or is dead. The bacteria can be killed by a chemical treatment or heat.

[0157] Live-attenuated vaccines are usually stronger than inactivated vaccines but can have drawbacks such as concerns with administration to patients with weakened immune systems and difficulties with storage. “Attenuation,” as used herein, refers to the state of the bacterium wherein the bacterium has been weakened from its wild-type fitness by some form of recombinant or physical manipulation. This includes altering the genotype of the bacterium to reduce its ability to cause disease. However, the bacterium's ability to colonize the host and induce immune responses is, preferably, not substantially compromised.

[0158] Methods of attenuation are known in the art. For example, attenuation may be accomplished by altering (e.g., deleting) native nucleic acid sequences found in the wild-type bacterium. In other examples, can be accomplished by use of a regulatable promoter, and deletion and / or insertion of a mutation or gene.

[0159] Vaccines can also be produced using genetically engineered cells. In particular embodiments, a cell (e.g., bacterium) can be genetically engineered to express a desired antigen. Genetic engineering techniques described elsewhere herein can be used. In particular embodiments, an Fna C1 bacterium is genetically engineered to express an Fna C2-associated antigen.

[0160] Subunit Vaccines. Vaccines can include a specific piece of the molecule or organism that causes a disease, like a protein, sugar, or capsid. Subunit vaccines can be acellular and can include one or more antigens or epitopes on the antigen. In particular embodiments, the antigen or epitope is free floating or is associated with a delivery scaffold. In particular embodiments, a delivery scaffold includes scaffold protein, a virus-like particle, or a nanoparticle.

[0161] Subunit vaccines can include a desired epitope fused to a delivery scaffold in order to maintain the function and structure of the epitope without affecting self-assembly of the epitope. A delivery scaffold can confer desired properties to the epitope, for example, a delivery scaffold may improve the immunogenicity of an epitope, e.g., by altering the structure of the epitope, by altering uptake and processing of the epitope, and / or by allowing the epitope to bind to a binding partner. The delivery scaffold can include a scaffold protein or peptide. The delivery scaffold and antigen can be attached by co-expression or by chemical means.

[0162] An epitope-scaffold protein is a chimeric protein that includes an epitope sequence fused to a heterologous "acceptor" scaffold protein. Design of the epitope-scaffold is performed, for example, computationally in a manner that preserves the native structure and conformation of the epitope when it is fused onto the heterologous scaffold protein. The use of such scaffold proteins is well known in the art and such methods and techniques are described in WO 2011 / 050168 and US 2010 / 0068217 and the skilled person can follow methods described therein and apply them to the present disclosure.

[0163] Scaffold proteins are useful for creating immunogens in that they hold contact residues in the immunogen in the proper spatial orientation to facilitate interaction between such residues and contact residues of the epitope. Epitope-scaffolds can be engineered by superposition, grafting, or de novo techniques known to those skilled in the art. Examples of scaffold proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), diphtheria toxoid (DT), tetanus toxoid (TT), human serum albumin (HSA), ovalbumin, meningococcal outer membrane protein complex (OMPC), Hemophilus influenzae protein D, or Pseudomonas aeruginosa exotoxin A (rEPA).

[0164] Virus-like particles (VLPs), are much larger than the small monomeric protein or peptides, independently serve as immunopotentiators generating a strong immune response by their presence. By displaying the desired epitope on their surface, this serves to focus or direct the immune response to these epitopes. A VLP is a non-replicating, viral shell, derived from any of several viruses. VLPs are generally composed of one or more viral proteins, such as, those proteins referred to as capsid, coat, shell, surface and / or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs lack the viral components that are required for virus replication and thus represent a highly attenuated form of a virus. The VLP can display a polypeptide (e.g. , a Treponema pallidum epitope) that is capable of eliciting an immune response when administered to a subject. VLPs and methods of their production are known and familiar to the person of ordinary skill in the art, and viral proteins from several viruses are known to form VLPs. VLPs can form spontaneously upon recombinant expression of the protein in an appropriate expression system. The formation of such VLPs can be detected by any suitable technique. Examples of suitable techniques known in the art for detection of VLPs in a medium include, e.g., electron microscopy techniques, dynamic light scattering (DLS), selective chromatographic separation (e.g., ion exchange, hydrophobic interaction, and / or size exclusion chromatographic separation of the VLPs) and density gradient centrifugation. Further, VLPs can be isolated by known techniques, e.g., density gradient centrifugation and identified by characteristic density banding. See, for example, Baker et a / . (1991) Biophys. J. 60:1445-1456; and Hagensee et al. (1994) J. Virol. 68:4503-4505; Vincente, J Invertebr Pathol., 2011 ; Schneider-Ohrum and Ross, Curr. Top. Microbiol. Immunol., 354: 53073, 2012).

[0165] In particular implementations, the virus-like particle is derived from a virus selected from adeno-associated virus (AAV), Budgerigar fledgling disease virus (BFDV), Bluetongue virus (BTV), Ebola, Enterovirus 71 , Goose hemorrhagic polyoma virus (GHPV), Hepatitis B virus (HBV), Hepatitis C virus (HCV), Hepatitis 5 virus (HDV), Hepatitis E virus (HEV), human immunodeficiency virus (HIV), Human papillomavirus (HPV), Infectious bursal disease virus (IBDV), influenza (e.g., Influenza A, Influenza A H1 N1 , Influenza A H3N2, avian influenza (H5N3)), Marburg virus(MVD), bacteriophage MS2, Indian peanut clump virus (IPCV), Newcastle disease virus (NDV), Norovirus (No), Norwalk virus (Nv), Physalis mottle virus (PhMV), Polyomavirus (e.g., human polyomavirus or JC polyomavirus), parvovirus (e.g., Porcine parvovirus (PPV), canine parvovirus), Rabbit hemorrhagic disease virus (RHDV), Rotavirus, Semliki-Forest virus, Sever acute respiratory syndrome (SARS), Simian immunodeficiency virus (SIV), Simian virus 40 (SV40), Swine vesicular disease virus (SVDV), and so on.

[0166] A VLP can be any size but viruses typically range in size from 20 nm - 200 nm. In particular implementations, a VLP can be 0 - 500 nm in size, 20-200 nm in size, 30-100 nm in size, or 50-60 nm in size.

[0167] Nanoparticles are broadly defined as objects which behave as a single, wholly contained unit with dimensions generally in the range of 1 to 1000 nanometers. Nanoparticles include particles of spherical, elliptical, elongated shape or irregular structure. A nanoparticle can include one or more materials, for instance. For example, a nanoparticle can be made of self-assembling proteins including ferritin, lumazine, and / or encapsulin. To construct nanoparticles including an Fna C2 associated antigen or an epitope therefrom, the antigen / epitope is linked to a subunit of the nanoparticle (such as ferritin, lumazine, or encapsulin). The fusion protein self-assembles into a nanoparticle under appropriate conditions In other implementations, the epitope can be linked to the nanoparticle using linker chemistry or a peptide linker.

[0168] The term “nanoparticle" can also be used to describe other types and materials of particles including nanoshells, nanobeads, or nanodots. Nanoparticles can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of epitopes disclosed herein to their surface. For example, the nanoparticle can be made of self-assembling proteins including ferritin, lumazine, and encapsulin. To construct nanoparticles including the disclosed epitope, the epitope is linked to a subunit of the nanoparticle (such as ferritin, lumazine, or encapsulin). The fusion protein self-assembles into a nanoparticle under appropriate conditions. In other implementations, the epitope can be linked to the nanoparticle using linker chemistry or a peptide linker.

[0169] In particular implementations, a self-assembling protein can be used in a nanoparticle. Self-assembling proteins include ferritin, lumazine, and encapsulin. Ferritin is a protein whose primary function is intracellular iron storage. Ferritin is a protein whose main function is intracellular iron storage. Ferritin is composed of 24 subunits, each composed of four alpha-helical bundles that self-assemble into quaternary structures with octahedral symmetry (Cho et al. J Mol Biol. 2009; 390: 83-98). Several high-resolution structures of ferritin were determined by confirming that Helicobacter pylori ferritin was composed of 24 identical protomers, whereas in animals it had ferritin light and heavy chains that could assemble alone or bind at different rates into 24 subunit particles. (Granier et al. J Biol Inorg Chem. 2003, 8:105-111 ; Lawson DM et al. Nature. 1991 , 349:541-544). Ferritin self-assembles into nanoparticles with strong thermal and chemical stability. Thus, ferritin nanoparticles are well suited for antigen delivery and exposure.

[0170] Lumazine synthetase (LS) is also well suited as a nanoparticle platform for antigen presentation. Responsible for the penultimate catalytic step in the biosynthesis of riboflavin, LS is an enzyme present in a variety of organisms including archaea, bacteria, fungi, and plants (Weber, Flavins and Flavoproteins. Methods and Protocols, Series:Methods in Molecular Biology. 2014). The LS monomer is 150 amino acids long and includes a beta-sheet with tandem alpha-helical flanking on its sides. A number of different quaternary structures have been reported for LS, showing morphological diversity ranging from homopentamers to symmetrical assembly of 12 pentamers forming 150 A diameter capsids. LS cages of more than 100 subunits have also been described (Zhang et al. J Mol Biol. 2006, 362:753-770).

[0171] Encapsulins, novel protein cage nanoparticles isolated from the thermophilic bacterium Thermotoga maritima, can also be used as a platform to present antigens on the surface of self-assembling nanoparticles. The encapsulin is assembled from 60 copies of the same 31 kDa monomer with a thin, icosahedral T = 1 symmetrical cage structure with inner and outer diameters of 20 and 24 nm, respectively (Sutter M. et al. Nat Struct Mol Biol. 2008, 15: 939-947). Although the exact function of the encapsulin in T. maritima is not yet clearly understood, its crystal structure has recently been resolved, and its function is related to the oxidative stress response of DyP (destaining peroxidase) and Flp (ferritin-like) proteins.

[0172] The term "nanoparticle” can also be used to describe other types and materials of particles including nanoshells, nanobeads, or nanodots. Nanoparticles can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of epitopes disclosed herein to their surface. Examples of suitable materials include polymers such as polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co -glycolic acid)), polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon crosslinked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Nucleic Acid Vaccine. In particular embodiments, a nucleic acid vaccine refers to a messenger RNA (mRNA) vaccine. mRNA vaccines make proteins in order to trigger an immune response. mRNA vaccines have several benefits compared to other types of vaccines, including shorter manufacturing times and, because they do not contain a live virus, no risk of causing disease in the person getting vaccinated.

[0173] Nucleic acid vaccines include nucleic acids encoding an Fna C2-associated antigen or epitope. Nucleic acids may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, ct-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2-amino-LNA having a 2- amino functionalization, and 2'-amino-ct-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or chimeras or combinations thereof.

[0174] In some embodiments, polynucleotides of the present disclosure function as messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequencerepresents RNA (e.g., mRNA), the “T"s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also include the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T" of the DNA sequence is substituted with “U.”

[0175] The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and / or structural design features, which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.

[0176] In some embodiments, an RNA polynucleotide of an RNA (e.g., mRNA) vaccine encodes 2, 3, 4, 5, 6, 7, 8, 9, or 10 antigenic polypeptides. In some embodiments, an RNA (e.g., mRNA) polynucleotide of a bacterial vaccine encodes at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 antigenic polypeptides. In some embodiments, an RNA (e.g., mRNA) polynucleotide of a vaccine encodes at least 100 or at least 200 antigenic polypeptides. In some embodiments, an RNA polynucleotide of a vaccine encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50, 1-100, 2-50 or 2-100 antigenic polypeptides.

[0177] Adjuvants. Vaccines are often administered with vaccine adjuvants The term "adjuvant” refers to material that enhances the immune response to an antigen or epitope and is used herein in the customary use of the term. Most adjuvants contain a substance designed to protect the antigen or epitope from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses.

[0178] Suitable adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N. J.); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; and Quil A.

[0179] Suitable adjuvants also include toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of gram-negative bacteria, polyphosphazenes, emulsions, virosomes, cochleates, poly (lactide-co-glycolides) (PLG) microparticles, poloxamer particles, microparticles, and liposomes.

[0180] Exemplary vaccine adjuvants, include any kind of Toll-like receptor ligand or combinations thereof (e.g. CpG, Cpg-28 (a TLR9 agonist), Polyriboinosinic polyribocytidylic acid (Poly(l:C)), Adjuplex (a biodegradable matrix of carbomer homopolymer-Carbopol-and nanoliposomes), a-galactoceramide, MPLA, Motolimod (VTX-2337, a novel TLR8 agonist developed by VentiRx), IMO-2055 (EMD1201081 ), TMX-101 (imiquimod), MGN1703 (a TLR9 agonist), Ribi (a TLR4 agonist), G100 (a stabilized emulsion of the TLR4 agonist glucopyranosyl lipid A), GLA-LSQ (a Glucopyranosyl lipid adjuvant in a liposomal formulation with QS21). Entolimod (a derivative of Salmonella flagellin also known as CBLB502), Hiltonol (a TLR3 agonist), and Imiquimod), and / or inhibitors of heat-shock protein 90 (Hsp90), such as 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0181] In particular embodiments a squalene-based adjuvant can be used. Squalene is part of the group of molecules known as triterpenes, which are all hydrocarbons with 30 carbon molecules. Squalene can be derived from certain plant sources, such as rice bran, wheat germ, amaranth seeds, and olives, as well as from animal sources, such as shark liver oil. In particular embodiments, the squalene-based adjuvant is MF59® (Novartis, Basel, Switzerland). An example of a squalene-based adjuvant that is similar to MF59® but is designed for preclinical research use is Addavax™ (InvivoGen, San Diego, CA). MF59 has been FDA approved for use in an influenza vaccine, and studies indicate that it is safe for use during pregnancy (Tsai T, et al. Vaccine. 2010. 17:28(7): 1877-80; Heikkinen T, et al. American Journal of Obstetrics and Gynecology. 2012. 207(3):177). In particular embodiments, squalene-based adjuvants can include 0.1 % -20% (v / v) squalene oil. In particular embodiments, squalene-based adjuvants can include 5%(v / v) squalene oil.

[0182] In particular embodiments the adjuvant alum can be used. Alum refers to a family of salts that contain two sulfate groups, a monovalent cation, and a trivalent metal, such as aluminum or chromium. Alum is an FDA approved adjuvant. In particular embodiments, vaccines can include alum in the amounts of 1-1000 g / dose or 0.1 mg- 10mg / dose In particular embodiments, the adjuvant Vaxfectin® (Vical, Inc., San Diego, CA) can be used. Vaxfectin® is a cationic lipid based adjuvant.

[0183] In particular embodiments, one or more STING agonists are used as a vaccine adjuvant. "STING" is an abbreviation of "stimulator of interferon genes", which is also known as "endoplasmic reticulum interferon stimulator (ERIS)", "mediator of IRF3 activation (MITA)", "MPYS" or "transmembrane protein 173 (TM173)". STING is a transmembrane receptor protein and is encoded by the gene TMEM173 in human. Activation of STING leads to production of Type I interferons (e.g., IFN-a and IFN-p), via the IRF3 (interferon regulatory factor 3) pathway; and to production of pro -inflammatory cytokines (e.g., TNF-a and IL-ip), via the NF-KB pathway and / or the NLRP3 inflammasome. Particular examples of STING agonists include c-AIMP; (3’,2’)c-AIMP; (2',2')c-AIMP; (2’,3’)c-AIMP; c- AIMP(S); c-(dAMP-dlMP); c-(dAMP-2’FdlMP); c-(2’FdAMP-2’Fdl MP); (2’,3’)c-(AMP-2’FdlMP); c-[2’FdAMP(S)- 2’FdlMP(S)]; c-[2’FdAMP(S)-2’FdlMP(S)](POM)2; and DMXAA. Additional examples of STING agonists are described in W02016 / 145102.

[0184] In particular implementations, an adjuvant can include a carbomer-lecithin-based adjuvant (e.g., Adjuplex™, Millipore Sigma, Burlington, MA; Wegmann et al. (2015) Clin Vaccine Immunol. CVI-00736).

[0185] Other immune stimulants can also be used as vaccine adjuvants. Additional exemplary small molecule immune stimulants include TGF-p inhibitors, SHP-inhibitors, STAT-3 inhibitors, and / or STAT-5 inhibitors. Exemplary siRNA capable of down-regulating immune-suppressive signals or oncogenic pathways (such as kras) can be used whereas any plasmid DNA (such as minicircle DNA) encoding immune-stimulatory proteins can also be used. In particular embodiments, the immune stimulant may be a cytokine and or a combination of cytokines, such as IL-2, IL- 12 or IL-15 in combination with IFN-a, IFN-p or IFN-y, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines. The above-identified cytokines stimulate TH1 responses, but cytokines thatstimulate TH2 responses may also be used, such as IL-4, IL-10, IL-11 , or any effective combination thereof.

[0186] The term "effective adjuvant amount" or "effective amount of adjuvant" will be well understood by those skilled in the art, and includes an amount of one or more adjuvants which is capable of stimulating the immune response to an administered antigen or epitope, i.e., an amount that increases the immune response of an administered antigen or epitope composition, as measured in terms of the IgA levels in the nasal washings, serum IgG or IgM levels, or B and T-Cell proliferation. Suitably effective increases in immunoglobulin levels include by more than 5%, preferably by more than 25%, and in particular by more than 50%, as compared to the same antigen or epitope composition without any adjuvant.

[0187] (vi) Binding Molecules and Targeted Therapeutics. In particular embodiments, a binding molecule includes a binding domain that binds a Fusobacterium nucleatum antigen. A binding molecule can include a binding domain (e.g., antibody), a multi-domain binding molecule, an antibody conjugate, or a recombinant receptor expressing the binding domain on its surface. A targeted therapeutic is a binding molecule used for therapeutic purposes such as treating a subject. A binding molecule can be used for therapeutic, diagnostic, or research purposes.

[0188] (v-a) Binding Domains. Binding domains include any substance that binds to a cellular marker to form a complex. The choice of binding domain can depend upon the type and number of cellular markers that define the surface of a target cell. Examples of binding domains include cellular marker ligands, receptor ligands, antibodies, peptides, peptide aptamers, receptors (e.g., T cell receptors), or combinations and engineered fragments or formats thereof.

[0189] Antibodies are one example of binding domains and include whole antibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that bind specifically a cellular marker. Antibodies or antigen binding fragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies.

[0190] Antibodies are produced from two genes, a heavy chain gene and a light chain gene. Generally, an antibody includes two identical copies of a heavy chain, and two identical copies of a light chain. Within a variable heavy chain and variable light chain, segments referred to as complementary determining regions (CDRs) dictate epitope binding. Each heavy chain has three CDRs (i.e., CDRH1 , CDRH2, and CDRH3) and each light chain has three CDRs (i.e., CDRL1, CDRL2, and CDRL3). CDR regions are flanked by framework residues (FR).

[0191] In particular embodiments, the fap2 binding domain includes an anti-fap2 antibody. In particular embodiments, the cmpA binding domain includes an anti-cmpA antibody. In particular embodiments, the fusolisin binding domain includes an anti-fusolisin antibody.

[0192] In some instances, it is beneficial for the binding domain to be derived from the same species it will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain to include a human antibody, humanized antibody, or a fragment or engineered form thereof. Antibodies from human origin or humanizedantibodies have lowered or no immunogenicity in humans and have a lower number of non-immunogenic epitopes compared to non-human antibodies. Antibodies and their engineered fragments will generally be selected to have a reduced level or no antigenicity in human subjects.

[0193] Antibodies with binding domains that specifically bind a cellular marker can be prepared using methods of obtaining monoclonal antibodies, methods of phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce antibodies as is known to those of ordinary skill in the art (see, for example, US 6,291 ,161 and 6,291 ,158). Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to a cellular marker. For example, binding domains may be identified by screening a Fab phage library for Fab fragments that specifically bind a cellular marker (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display libraries of human antibodies are also available. Additionally, traditional strategies for hybridoma development using a cellular marker as an immunogen in convenient systems (e.g., mice, HuMAb mouse® (GenPharm Inti. Inc., Mountain View, CA), TC mouse® (Kirin Pharma Co. Ltd., Tokyo, JP), KM-mouse® (Medarex, Inc., Princeton, NJ), llamas, chicken, rats, hamsters, rabbits, etc.) can be used to develop binding domains. Once identified, the amino acid sequence of the antibody and gene sequence encoding the antibody can be isolated and / or determined.

[0194] As indicated, antibodies can be used as whole antibodies or binding fragments thereof, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc) forms and fragments thereof that specifically bind a cellular marker.

[0195] In some instances, scFvs can be prepared according to methods known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions of an antibody together using flexible polypeptide linkers. If a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientations and sizes see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. USA 90:6444-6448, US 2005 / 0100543, 2005 / 0175606, 2007 / 0014794, and PCT W02006 / 020258 and W02007 / 024715. More particularly, linker sequences that are used to connect the VL and VH of an scFv are generally five to 35 amino acids in length. In particular embodiments, a VL-VH linker includes from five to 35, ten to 30 amino acids or from 15 to 25 amino acids. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. scFV are commonly used as the binding domains of CAR discussed below.

[0196] Additional examples of antibody-based binding domain formats include scFv-based grababodies and soluble VH domain antibodies. These antibodies form binding regions using only heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22: 1161 , 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008.

[0197] In particular embodiments, a VL region in a binding domain of the present disclosure is derived from or based on a VL of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one ormore (e.g, 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g, conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VL of the known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VL region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VL region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0198] In particular embodiments, a binding domain VH region of the present disclosure can be derived from or based on a VH of a known monoclonal antibody and can contain one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the VH of a known monoclonal antibody. An insertion, deletion or substitution may be anywhere in the VH region, including at the amino- or carboxy-terminus or both ends of this region, provided that each CDR includes zero changes or at most one, two, or three changes and provided a binding domain containing the modified VH region can still specifically bind its target with an affinity similar to the wild type binding domain.

[0199] In particular embodiments, a binding domain includes or is a sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a light chain variable region (VL) or to a heavy chain variable region (VH), or both, wherein each CDR includes zero changes or at most one, two, or three changes, from a monoclonal antibody or fragment or derivative thereof that specifically binds to a cellular marker of interest.

[0200] An alternative source of binding domains includes sequences that encode random peptide libraries or sequences that encode an engineered diversity of amino acids in loop regions of alternative non-antibody scaffolds, such as single chain (sc) T-cell receptor (scTCR) (see, e.g., Lake et al., Int. Immunol.11 :745, 1999; Maynard et al., J. Immunol. Methods 306:51 , 2005; US 8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science 230: 1388, 1985), Kunitz domains (see, e.g., US 6,423,498), designed ankyrin repeat proteins (DARPins; Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin binding domains (adnectins or monobodies; Richards et al., J. Mol. Biol. 326: 1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381 :1238-1252), cysteine-knot miniproteins (Vita et al., 1995, Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408; Martin et al., 2002, Nat. Biotechnol. 21 :71 , 2002 and Huang et al. (2005) Structure 13:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 11 :497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161 , 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471 , 2003), lipocalin domains (see, e.g., WO 2006 / 095164, Beste et al. Proc. Nat'l. Acad. Sci. (USA) 96: 1898, 1999 and Schdnfeld et al. Proc. Nat'l. Acad. Sci. (USA) 106:8198, 2009), V-like domains (see, e.g, US 2007 / 0065431), C-type lectin domains (Zelensky and Gready, FEBS J. 272:6179, 2005; Beavil et al. Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al. Proc. Nat'l. Acad. Sci. (USA) 100:7779, 2003),mAb2 or Fc-region with antigen binding domain (Fcab™ (F-Star Biotechnology, Cambridge UK; see, e.g., WO 2007 / 098934 and WO 2006 / 072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21 : 1015, 2012; WO 2009 / 040338), affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibody, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013), or the like (Nord et al., Protein Eng. 8:601 , 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001 ; Binz et al., Nat. Biotechnol. 23: 1257, 2005; Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0201] Peptide aptamers include a peptide loop (which is specific for a cellular marker) attached at both ends to a protein scaffold. This double structural constraint increases the binding affinity of peptide aptamers to levels comparable to antibodies. The variable loop length is typically 8 to 20 amino acids and the scaffold can be any protein that is stable, soluble, small, and non-toxic. Peptide aptamer selection can be made using different systems, such as the yeast two-hybrid system (e.g., Gal4 yeast-two-hybrid system), or the LexA interaction trap system.

[0202] In particular embodiments, a binding domain is a scTCR including Va / p and Ca / p chains (e.g., Vo-Co, Vp-Cp, Va-V|3) or including a Va-Ca, Vp-Cp, Va-Vp pair specific for a cellular marker peptide-MHC complex.

[0203] In particular embodiments, engineered binding domains include Vo, Vp, Co, or Cp regions derived from or based on a Vo, Vp, Co, or Cp and includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the above-noted changes, when compared with the referenced Vo, Vp, Co, or Cp. An insertion, deletion or substitution may be anywhere in a VL, VH, Vet, Vp, Cct, or Cp region, including at the amino- or carboxy-terminus or both ends of these regions, provided that each CDR includes zero changes or at most one, two, or three changes and provides a target binding domain containing a modified Vet, Vp, Ca, or Cp region can still specifically bind its target with an affinity and action similar to wild type.

[0204] In particular embodiments, engineered binding domains include a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99 5%, or 100% identical to an amino acid sequence of a known or identified binding domain, wherein each CDR includes zero changes or at most one, two, or three changes, from a known or identified binding domain or fragment or derivative thereof that specifically binds to the targeted cellular marker.

[0205] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273: 927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262: 732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86: 9268-9272 (AbM numbering scheme); Lefranc M P et al. (2003) Dev Comp Immunol 27(1): 55-77 (IMGT numbering scheme); andHonegger and Pluckthun (2001) J Mol Biol 309(3): 657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, "30a," and deletions appearing in some antibodies. The two schemes place certain insertions and deletions ("indels") at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. In particular embodiments, the antibody CDR sequences disclosed herein are according to Kabat numbering.

[0206] In particular embodiments, a binding domain binds an Fna clade 2 antigen. In particular embodiments, an Fna clade 2 antigen includes fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin.

[0207] In particular embodiments, fap2 [Fusobacterium animalis] includes the sequence: MGNNLYKVEKDLRSIAKRYKSVKYSLGLAILFLMLGVSAFSEEVNTEQMKAIPTREEIVSSKENLKNSVGGLQTKINDA RLENEKALKGLRLELIQLMEQGNQWKSPWSSWQFGMNYFYDHWGSEYKGRGDKPQKYLFNGIYTRGNWKVRNA MDVAENQRVGGRPLTPGNDSLNSWKNTNNSSNGGVKIDRDNSISSSTNGNRSWGLVDLENLREPTNEVEILARISPK EVTKQAISLNITEPTVQTLEAPDVNPQVNEPLPAPVIGLPEVETVNISPLTINAPSAPTAPNAPSINIGIIAPTAPSAPTINV VPTSPSTPSAPTINIGIQPPSITPLSITTPDSVGTITINTPTINPVDFMLSPSGLSGDLTRKFNDRPYDIGSITINVTQNGFH GNYISTWGRVKNLDGISTRVNVNVEDTRAFMVDEGIKDDDTDIRPFRYTGTINLNKSKNVGIDVQGTHTSLSDGTNTG PGMNDIKNVANIKVINEGKIIGNGGSNVKNQVGFGFNNFDASSNNTRTEMINRGNGTITLGAPESAGIQLRPEDPWSS NNGPNRGLNMMTGENTGTVTINSYGSFGILTVKNRDLTYLKTHPSSTKLANTRDYTDYKVKTTSGGQIASRAQEANM SYIKNTGDINVNGDDSVGIGLLHNIQGVTIGGNVKIGDTDPSSFSWSNKAGGTAGKVEGAVGVYSEVRTRPILNGDYD DHALQNTSGKTLGTETVEVSGKVELGSYAEKSSGLRIKKEGSITLKSGGIVSVGGTNNYGAWNGEDYDYYRKTSATA YAENGTKYSDTGKINLESNSKINVTGKESIGYALLSGKGTNAGTITVTGHKDNTSSASKFKGSLGFYGEEGTFTNTNK GEISSSGNVAHAVALIGKAATGITFTNNGKINVTDKGNIGVYADGKYTFEHSGTNAKISVGSDSIGIYAKNTSGNLNINA PIEIDNSNKATTIGIYSDGDAKVKFGAGSKLKIGEKAVGLYSANPTKFNNTFKIETGKILDVELGKNSTFGLLNGNKTVTN SPLLSKYLNDNINGKINITSFGEGASLFYATLKAKAILDEDYKVTNADAASTSVLVANDGANVEIASGKKLETNTNVGLV ATKGAGASLSTSVAENKGTLISTRTDKGIGIYTYTANGENSGTIIMKNQNSVGILGSGGSNLKNTGKIELEAISSAGVYA ENSNMINSGTASEIWKKKASVGIYARETSISSVPKNVKNEGKIEIKADGDGKSAGIYSKKEGGAKLTIENTGNIEVAQK ASAGIYAKNESNQANTESWTNSGLIKMSAENSIGIMGEKSKVENSGTGTKGIEIAGKKSAGILVTKESEWNSGRILLA NNSILASSDGLVGISVDGSSTGENDASGEIKIDAAYSTGMLSSGGDITNAGKIDLEKKESIGIYATNANVTNNGATTKGI FIKDEKSVGIYSKINSSSTADKIVTNSGTIDIGGTSKTGSAGIYSIVKNGATKRLSTVSSGNITISQKESVGIYAKNKSSQA NTESWTNSSNGKIEVKNEGSAGILVEKSKVTNTAVGTNGIIVSAKKSAGIIGKLGSEIINSGIIKTETATPTAAADGWGI SLNASKATNDSNGTITLDTNYSTGMFGENSSTAINEGNITGTNKENIVGMAGDNSTVTNKNVITLNGKKATGVFGKNS STLLNETAGKITTKEEESVGMYSSLNSTATNKGTITTEKKTSAGMLGDKANIENDSSITTKEEISAGMYVKNGNSIATNKGTITTEKETSAGILAEIDEATGGTVSATNETAGTIWSDKTSAGMLGKVKSTVTASTAKLSLTNKKDININTKNSAGMMV VNESTAVGKGNVSAENTGTINLTASAATNEKNIGILADKATGINTGNINVNSKESIGILGQNASSITNNKTITLSGEKGIG MLAKDTNSTADNNDTINVNGKESLGMLGEDSGTVKNNKTIKVTAESGVGIFVRDNGTGSGVGENTSTGTISLENKESV GIFAKNNGTNDTAKNSGTINLGKADGSTSHESLIGMFAQAESGKTASVKNTKDININTKKSVGMYAKNNASNITDVDLE NTGDININSQESAGIYAPKSNISKVGTITLKNSTVSNGSSAVYVSNGGKVADTASAKINLGTVNQNRVAYYVNGKDSAL AGANIGKITGYGVGVYLQGASGDKATLDSNTSKLDYTAQGTGNGIIGLLLKGETDIQSYIKGIKVGNTVPRSNPSDKDK YAIGIYADAQGTAGTPYNITTPITAGKNGVGIFADKDSNINYTGNMEIGDGTTAGTGIFITKKNGTTRGKVTLGSNTIKLK GTKGWAIASEGTEFNGGTATIELEGKDVQGVGVYGKKGSDVDVSHWNFNNHGNSAEEVRSEEGKVHINGNKNLKP RMVLTHVINGETSIVTGKTVTSVNDGSITAKENIGLMAEGIKNHGMTIWQEGNFEAVNHGTIDFSAAEKSTAMFINSARGKNDGTIKVGKNSIGIYGFYNKDTRKYEGAPASPDPNKLEIETTSNSKISLGDASTGMYLINAEKIENKGGQITSESGAT KNVGIYAVNGQDSINANNKNLTMTTAANIALGNGSVGLYSKGQSYTVRNSVTNTGNITVGDKMTGSPSVAMYAENTN LTTDSKITVGKDGIAFYGKNSDITAKGSANFSNKGVLAYLENSKFISHLGNLGATQNTMLYLKNSIAQLDGAGTKVDMD VADGYTGAYIEGNSTLTGVKTIKLGQDSTGLFLKDANFVSNAESITGTKDKARGILATNSNLTNNSKISLSGAESIGIYSN ANNTKSWNNGELTIAGKKTLGVFLKGSQSFENKANINIADSANSLEPTIGIYTAEGSSNIKHTSGTIDVGQKSIAIYSTT NSNVEMNGGKIHVKDQGIGIYKQNGKATINGELDIDTHIATTKDSEPTGVYAVNGTEINDQASKISIGAKSYGFILNNTD VNKTNIYNNTNTGTVTMGNDSVFLYSNGKAIINNKRDISANNSNHLIAFYIKNGGDFTNNETIDFSTGKGNIGIYAPGGK ATNKGRILVGAADDIDPMTGKVYTDVSKIVYGIGMAADNGGHIVNEGEIRIYNNKSIGMYGKGVGTTVENKGTISLDGSRATATNKIQSMTGVYVDDGATFKNYGTITTTDSYAGRDGKVNENVTGLVGVAVMNGSTLENHGKIYIDADNSYGVIIR GKRDTKGNVERYAVIKNYGEIKVRGKGTWGISWKDVSQADIDELQKQINSKISSDPERQALREAEGTDKNYQGVTITV KDGKPTFLRNGVPISDSEVEQIGKLIGKESNLGMSDIGFYVDTLGRTKPIDIDGANPPINSQLIIGTEYSEKTNKKQWFV KGDVIKPFLDQIQGRNFKLTSIAGSLTWIATPVLDNYGQITGVAMAKLPYTSFVKKTDNAWNFTDGLEQRYDMNALDS VEKRIFNKLNGIGKNEEALLTQAYDEMMGHQYANVQQRVQVTGNILDKEFNHLRDSWSNPSKDSNKIKTFGMKGEYK TDTAGVIDYKYNAYGVAYVHENEDIKLGRGIGWYTGIVHNRFKFKDIGNSKEEQLQAKVGLFKSVPFDDNNSLNWTIS GDMFVGYNKMHRKFLWDEIFNAKSKYYTYGIGIKNEIGKEFRLSEGFSIRPYGALKVEYGRVSKIKEKSGEVRLEVKH NDYLSIKPEVGTELAYRHYFGIKTLRTSLEVAYENELGRVANGKNKAKVAYTNADWFNIRGEKEDRRGNVKFDLNVGLDNQRLGVTGNVGYDTKGHNVRGGVGLRVIF (SEQ ID NO: 120).

[0208] In particular embodiments, fusolisin [Fusobacterium animalis] includes the sequence: MRKEILKSKMMMIALASILFVSCGGGGGGGGGGSSNLPINPGTPSVPKPSTPSTPSNPEDNFPTVTNPLDSQKGNMS ALKASLYTAQKNSGVAIPNDTTEIDGSSVKVAILDANFVNAVRSGGSSAEDKDGHSVTRRRDKTLTDVYTDIDIIDESP NHPYIEKAVSGTENPTGLEHGEEVLEWRDLEYAPNNLANTYFPNNKPKNKIETILGSIGWDYNYMEGTSTKRKVAGI HPTQEMYEAAMAKFGNQSVKIFNQSFGSEDSYDDSKYRSYKGEGNLPLPFAKVHSTDSDKLMIPYFRDAINNKGGLF IWAAGNKNNTAATLEAGLPYFDKSLEKGWISWGVKPEKKSGGITTYNIIDGLSHAGSDAAYWSISADDSSIKKITSVNT VTGTVGYTIGYGSSYATPRVTRAAALVYDKYDWMTADQIRQTLFTTTDETNVTFRKPGRRVSSSPDSEYGWGMLNQ ARALKGPGAFMNVTSSSSASKIFNANIPAGKTSYFDNDIYGDGGLKKLGSGTLHLTGNNSFTGGSTVSAGTLEIHQVHASPin / GESGTLVLNPKAIVGYDSWAWGTIDTVNPQKITDSGlKVKNYGTVKFNGTTAIIGGDYVAYAGSNTQVGFKNS VKVLGKIRIENGNISVLSNDYVTKNEKSTIMEGKSFEGNIANVETNGMRTANVEVKDGKWATLSRQNPSEYLGENAE ASSKNVAENVEKVFEDLDQKVMSGTATKEEVLMGATVQSMSTMGFTSATEMMSGEIYASAQALTFSQAQNVNRDLS NRLSGLDNFKNSNKDSEVWFSVLGSAGKLRRDGYASADTRVTGGQFGADTKFSPTTTLGVALNYSYAKADFNRYAG ESKSDMVGVSLYGKQDLPYGFYTAGRLGLSHISSKVERELLTSTGDTVTGKINHHDKMLSAYVELGKKFGWFTPFIGY SQDYLRRGSFDESEASWGIKADKKNYRTSNFLVGARAEYVGNKYKLQAYVTQAINTDKRDLSYEGRFTGSSVKQKF YGVKQAKNTTWIGFGAFREITPVFGVYGNVDFRVEDKKWADSVFSAGLQYRF (SEQ ID NO: 121).

[0209] In particular embodiments, cmpA [Fusobacterium gonidiaformans] includes the sequence: MYRKLFVAFWITILFVSCGLEKVQDKKMKVGILSIADSGALFVAEKEQLFLKNGLDVELIPFGSAAEQSRAMEAGELDA MMTDAIVQNLVNQGENNLKEVLVALGDTAEHGKFLILASPTTEHNSLKNLSGAKLGISENTMMEFLVDSYFSLLNLEIH DVEKVNIPSLSLRMEMLLQGKIDLAILPEPLGDFAVLQGAKIVLDDTKLNENLSQSIIVFREAYIEKNFLEVKKFVKSYSE AAKMINEAPDKYKDYIFEMANIPEILKSSYRLPYYSIASVTDRQLFDKMQNWMIQKKLLTQTKDYSSSIDSRFIDIVGEE NDSVK (SEQ ID NO: 122).

[0210] In particular embodiments, the Fna 02 antigen includes an immunogenic peptide. In particular embodiments, an immunogenic peptide includes the sequence as set forth in SEQ ID NO: 120, SEQ ID NO: 121 , or SEQ ID NO: 122. In particular embodiments, an immunogenic peptide includes a fragment of the sequence as set forth in SEQ ID NO: 120, SEQ ID NO: 121 , or SEQ ID NO: 122. In particular embodiments, an immunogenic peptide includes a variant of the sequence as set forth in SEQ ID NO: 120, SEQ ID NO: 121 , or SEQ ID NO: 122. The fragment can include a 3000 amino acid (aa), 2500 aa, 2000 aa, 1500 aa, 1000 aa, 900 aa, 800 aa, 600 aa, 400 aa, 300 aa, 200 aa, 100 aa, 80 aa, 60 aa, 40 aa, 35 aa, 30 aa, 25 aa, 20 aa, 15 aa, 10 aa, 5 aa, 4 aa, 3 aa, 2 aa, or 1 aa truncation at the N-terminal, C- terminal, or both. In particular embodiments, the immunogenic peptide can include concatenated sequences. The term concatenate is broadly used to describe linking together into a chain or series. It is used to describe the linking together of nucleotide or amino acid sequences into a single nucleotide or amino acid sequence, respectively. In particular embodiments, a concatenated sequence can include multiple copies of the same immunogenic peptide, the immunogenic peptide with a multimerization domain, or the immunogenic peptide with a sequence that increases its immunogenicity

[0211] Example immunogenic peptides are provided in FIG. 39.

[0212] Binding domains can be engineered or may be commercially available.

[0213] (v-b) Multi-Domain Binding Molecules. Multi-domain binding molecules include at least two binding domains, wherein at least one binding domain includes a binding domain that binds an Fn antigen (expressed by Fn or a cell infected with Fn) disclosed herein. In particular embodiments, a multi-domain binding molecule includes at least one, at least two, at least three, or at least four binding domains that bind an epitope on the surface of Fn. In particular embodiments, at least one binding domain of a multi-domain binding molecule binds an immune cell.

[0214] Multi-domain binding molecules include bispecific antibodies which bind at least two epitopes wherein at leastone of the epitopes includes an Fn antigen. Multi-domain binding molecules include trispecific antibodies which bind at least 3 epitopes, wherein at least one of the epitopes includes an Fn antigen, and so on.

[0215] Bispecific antibodies can be prepared utilizing antibody fragments (for example, F(ab')2 bispecific antibodies). For example, WO 1996 / 016673 describes a bispecific anti-ErbB2 / anti-Fc gamma Rill antibody; US 5,837,234 describes a bispecific anti-ErbB2 / anti-Fc gamma Rl antibody; WO 1998 / 002463 describes a bispecific anti-ErbB2 / Fc alpha antibody; and US 5,821 ,337 describes a bispecific anti-ErbB2 / anti-CD3 antibody.

[0216] Some additional exemplary bispecific antibodies have two heavy chains (each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain), and two immunoglobulin light chains that confer antigen-binding specificity through association with each heavy chain. However, as indicated, additional architectures are envisioned, including bi-specific antibodies in which the light chain(s) associate with each heavy chain but do not (or minimally) contribute to antigen-binding specificity, or that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding of one or both of the heavy chains to one or both epitopes.

[0217] Two antibodies or fragments thereof can be linked through a linker to form a bispecific antibody. In particular embodiments, the two antibodies or fragments thereof can bind the same epitope or different epitopes. Examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013, 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0218] Commonly used flexible linkers include linker sequence with the amino acids glycine and serine (Gly-Ser linkers). In particular embodiments, the linker sequence includes sets of glycine and serine repeats such as from one to ten repeats of (GlyxSery)n, wherein x and y are independently an integer from 0 to 10 provided that x and y are not both 0 and wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). Particular examples include (Gly4Ser)n(SEQ ID NO:I), (Gly3Ser)n(Gly4Ser)n(SEQ ID NO: 2), (Gly3Ser)n(Gly2Ser)n(SEQ ID NO: 3), and (Gly3Ser)n(Gly4Ser)i (SEQ ID NO: 4). In particular embodiments, the linker is (Gly4Ser)4 (SEQ ID NO: 5), (Gly4Ser)3(SEQ ID NO: 6), (Gly4Ser)2(SEQ ID NO: 7), (Gly4Ser)i (SEQ ID NO: 8), (Gly3Ser)2(SEQ ID NO: 9), (Gly3Ser)i (SEQ ID NO: 10), (Gly2Ser)2(SEQ ID NO:I I) or (Gly2Ser)i, GGSGGGSGGSG (SEQ ID NO: 12), GGSGGGSGSG (SEQ ID NO: 13), or GGSGGGSG (SEQ ID NO: 14).

[0219] Linkers that include one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as CH3 alone or a CH2CH3 sequence can also be used. Additional examples of linkers can be found in Chen et al., Adv Drug Deliv Rev. 2013, 65(10): 1357-1369. Linkers can be flexible, rigid, or semi-rigid, depending on the desired functional domain presentation to a target.

[0220] In particular embodiments, binding domains disclosed herein can be used to create bi-, tri, (or more) specific immune cell engaging molecules. Immune cell engaging molecules have at least one binding domain that binds a receptor on an immune cell and alters the activation state of the immune cell. Examples of multi-domain immune cell engaging molecules include those which bind both an immune cell (e.g., T-cell or NK-cells) activating epitope and anFn antigen, with the goal of bringing immune cells to Fn antigen-expressing cells to destroy them. See, for example, US 2008 / 0145362. Such molecules are referred to herein as immune-activating multi-specifics or l-AMS). BiTEs® (Amgen, Thousand Oaks, CA) are one form of l-AMS. Immune cells that can be targeted for localized activation by I- AMS within the current disclosure include, for example, B-cells, T-cells, natural killer (NK) cells, and macrophages which are discussed in more detail herein.

[0221] l-AMS disclosed herein can target any T-cell activating epitope that upon binding induces T-cell activation. Examples of such T-cell activating epitopes are on T-cell markers including CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, and B7-H3.

[0222] Other forms of bispecific binding molecules include the single chain "Janusins” described in Traunecker et al. (Embo Journal, 10, 3655-3659, 1991). Bispecific binding molecules with extended half-lives are described in, for example, US Patent No. 8,921 ,528 and US Patent Publication No. 2014 / 0308285.

[0223] In particular embodiments, multi-domain binding molecules are multimers of an antibody disclosed herein. Multimerization strategies include formation of a fusion protein using protein linkers or use of IgA or IgM constant regions as a multimerization scaffold. In certain aspects, multimerization is achieved by linking antibodies or binding domains of antibodies in a fusion protein with protein linkers. Fusion proteins include different protein domains linked to each other directly or through intervening linker segments such that the function of each included domain is retained.

[0224] A “multimerization domain” is a domain that causes two or more proteins (monomers) to interact with each other through covalent and / or non-covalent association(s). Multimerization domains are highly conserved protein sequences that can include different types of sequence motifs such as a leucine zipper, helix loop-helix, ankyrin and PAS (Feuerstein et al, Proc. Natl. Acad. Sci. USA, 91 : 10655-10659, 1994). Multimerization domains present in proteins can bind to form dimers, trimers, tetramers, pentamers, hexamers, heptamers, etc., depending on the number of units / monomers incorporated into the multimer, and / or homomultimers or heteromultimers, depending on whether the binding monomers are the same type or a different type (US 10,030,065).

[0225] Multimerized antibodies and antibody-like molecules such as IgA and IgM antibodies have emerged as promising drug candidates in the fields of, e.g., immuno-oncology and infectious diseases allowing for improved specificity, improved avidity, and the ability to bind to multiple binding targets. See, e.g., US 9,951 , 134, 10,400,038, 9,938,347, US20190100597, US20180118814, US20180118816, US20190185570, and US20180265596, and PCT WO 2018 / 017888, WO 2018 / 017763, WO 2018 / 017889, WO 2018 / 017761 , and WO 2019 / 165340.

[0226] (v-c) Antibody Conjugates. Antibody conjugates include an Fn antigen binding domain disclosed herein linked to another molecule, other than an additional binding domain. Examples of antibody conjugates include antibody immunotoxins, antibody-drug conjugates (ADCs), antibody-detectable label conjugates, antibody radioisotope conjugates, and antibody-particle conjugates.

[0227] In particular embodiments, the Fn antigen binding domain can be formed as an antibody immunotoxin. Antibody immunotoxins include an Fn antigen binding domain disclosed herein conjugated to one or more cytotoxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof). A toxin can be any agent that is detrimental to cells. Frequently used plant toxins are divided into two classes: (1) holotoxins (or class II ribosome inactivating proteins), such as ricin, abrin, mistletoe lectin, and modeccin, and (2) hemitoxins (class I ribosome inactivating proteins), such as pokeweed antiviral protein (PAP), saporin, Bryodin 1 , bouganin, and gelonin. Commonly used bacterial toxins include diphtheria toxin (DT) and Pseudomonas exotoxin (PE). Kreitman, Current Pharmaceutical Biotechnology 2:313-325 (2001 ). The toxin may be obtained from essentially any source and can be a synthetic or a natural product.

[0228] Immunotoxins with multiple (e.g., four) cytotoxins per binding domain can be prepared by partial reduction of the binding domain with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA (diethylene triamine penta-acetic acid) in Dulbecco’s phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the binding domain can be measured using 5,5'-dithiobis(2- nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the linker-cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting immunotoxin mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting immunotoxin can then be sterile filtered, for example, through a 0.2 m filter, and can be lyophilized if desired for storage.

[0229] Antibody-drug conjugates allow for the targeted delivery of a drug moiety to a Fn-antigen-expressing cell, in particular embodiments intracellular accumulation therein, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells (Polakis P. (2005) Current Opinion in Pharmacology 5:382-387).

[0230] In particular embodiments, antibody-drug conjugates refer to targeted molecules which combine properties of both antibodies and cytotoxic drugs (e.g., chemotherapeutic drugs) by targeting potent cytotoxic drugs to antigenexpressing cells (Teicher, B. A. (2009) Current Cancer Drug Targets 9:982-1004), thereby enhancing the therapeutic index by maximizing efficacy and minimizing off-target toxicity (Carter, P. J. and Senter P. D. (2008) The Cancer Jour. 14(3):154-169; Chari, R. V (2008) Acc. Chem Res. 41 :98-107). See also Kamath & Iyer (Pharm Res. 32(11): 3470- 3479, 2015), which describes considerations for the development of antibody-drug conjugates.

[0231] The drug moiety (D) of an antibody-drug conjugate may include any compound, moiety or group that has a cytotoxic or cytostatic effect. Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding or intercalation, and inhibition of RNA polymerase, protein synthesis, and / or topoisomerase. Exemplary drugs include actinomycin D, anthracycline, auristatin, calicheamicin, camptothecin, CC1065, colchicin, cytochalasin B, daunorubicin, 1 -dehydrotestosterone, dihydroxy anthracinedione, dolastatin, doxorubicin, duocarmycin, elinafide, emetine, ethidium bromide, etoposide, gramicidin D, glucocorticoids, lidocaine, maytansinoid (including monomethyl auristatin E [MMAE]; vedotin), mithramycin, mitomycin, mitoxantrone,nemorubicin, PNU-159682, procaine, propranolol, puromycin, pyrrolobenzodiazepine (PBD), taxane, taxol, tenoposide, tetracaine, trichothecene, vinblastine, vinca alkaloid, vincristine, and stereoisomers, isosteres, analogs, and derivatives thereof that have cytotoxic activity.

[0232] The drug may be obtained from essentially any source; it may be synthetic or a natural product isolated from a selected source, e.g., a plant, bacterial, insect, mammalian or fungal source. The drug may also be a synthetically modified natural product or an analogue of a natural product.

[0233] In particular embodiments, the antibody-drug conjugates include an antibody conjugated, i.e., covalently attached, to the drug moiety. In particular embodiments, the Fn antigen binding domain is covalently attached to the drug moiety through a linker. A linker can include any chemical moiety that is capable of linking an antibody, antibody fragment (e.g., antigen binding fragments) or functional equivalent to another moiety, such as a drug moiety. Linkers can be susceptible to cleavage (cleavable linker), such as, acid-induced cleavage, photo-induced cleavage, peptidase- induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Alternatively, linkers can be substantially resistant to cleavage (e.g., stable linker or noncleavable linker). In some aspects, the linker is a procharged linker, a hydrophilic linker, or a dicarboxylic acidbased linker. The antibody-drug conjugate selectively delivers an effective dose of a drug to cells (e.g., cancer cells) whereby greater selectivity, i.e., a lower efficacious dose, may be achieved while increasing the therapeutic index (“therapeutic window”).

[0234] To prepare antibody-drug conjugates, linker-cytotoxin conjugates can be made by conventional methods analogous to those described by Doronina et al. (Bioconjugate Chem. 17: 114-124, 2006). Antibody-drug conjugates with multiple (e.g., four) drugs per antibody can be prepared by partial reduction of the antibody with an excess of a reducing reagent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 37°C for 30 min, then the buffer can be exchanged by elution through SEPHADEX G-25 resin with 1 mM DTPA in Dulbecco's phosphate-buffered saline (DPBS). The eluent can be diluted with further DPBS, and the thiol concentration of the antibody can be measured using 5,5'-dithiobis(2-nitrobenzoic acid) [Ellman's reagent]. An excess, for example 5-fold, of the linker- cytotoxin conjugate can be added at 4°C. for 1 hr, and the conjugation reaction can be quenched by addition of a substantial excess, for example 20-fold, of cysteine. The resulting ADC mixture can be purified on SEPHADEX G-25 equilibrated in PBS to remove unreacted linker-cytotoxin conjugate, desalted if desired, and purified by size-exclusion chromatography. The resulting ADC can then be sterile filtered, for example, through a 0.2 pm filter, and can be lyophilized if desired for storage. Methods used to produce immunotoxins can similarly be used to prepare antibodydrug conjugates.

[0235] Antibody-detectable label conjugates include a Fn antigen binding domain linked to a detectable label. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In particular embodiments, detectable labels can include fluorescent labels, chemiluminescent labels, spectral colorimetric labels, enzymaticlabels, and affinity tags.

[0236] Fluorescent labels can be particularly useful in cell staining, identification, imaging, and isolation uses. Exemplary fluorescent labels include blue fluorescent proteins (e.g. eBFP, eBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP, Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green (mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher Scientific)); Luciferase; orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRuby, mRFP1 , DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRedl, AsRed2, eqFP611 , mRaspberry, mStrawberry, Jred, Texas Red™ (Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); and tandem conjugates.

[0237] Antibody-radioisotope conjugates include an Fn antigen binding domain linked to a radioisotope for use in nuclear medicine. Nuclear medicine refers to the diagnosis and / or treatment of conditions by administering radioactive isotopes (radioisotopes or radionuclides) to a subject. Therapeutic nuclear medicine is often referred to as radiation therapy or radioimmunotherapy (RIT).

[0238] Examples of radioactive isotopes that can be conjugated to Fn antigen binding domains of the present disclosure include iodine-131 yttrium-90, arsenic-72, arsenic-74, iodine-131 , indium-1 1 1 , and lutetium-177, as well as alpha-emitting radionuclides such as astatine-211 , actinium-225, bismuth-212 or bismuth-213. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (DEC Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugatesissw, i25Xe,127Xe,133Xe,133mXe,135Xe,85mY,86Y,90Y,93Y,169Yb,175Yb,65Zn,71mZn,86Zr,95Zr, and / or97Zr. Radioisotopes can be used as a type of detectable label called a radiolabel. In particular embodiments, a radioisotope includes131l,90Y, and / or21 1At. In particular embodiments, a radioisotope is selected that includes a half-life (ti / 2) thatenables high-yield radiolabeling and drug delivery. In particular embodiments, a radioisotope is selected that includes a half-life (ti / 2) of 7.2 hrs. In particular embodiments, a radioisotope is selected that does not emit daughter radionuclides that cause organ toxicity.

[0241] Antibody-particle conjugates include an antibody linked to a particle. In particular embodiments, particles include microparticles, nanoparticles, nanoshells, nanobeads, microbeads, or nanodots. Particles can include, for example, latex beads, polystyrene beads, fluorescent beads, and / or colored beads, and can be made from organic matter and / or inorganic matter. They can be made of any suitable materials that allow for the conjugation of capture proteins, such as Fn antigen binding domains disclosed herein, to their surface. Examples of suitable materials include: ceramics, glass, polymers, and magnetic materials. Suitable polymers include polystyrene, poly-(methyl methacrylate), poly-(lactic acid), (poly-(lactic-co -glycolic acid)), polyesters, polyethers, polyolefins, polyalkylene oxides, polyamides, polyurethanes, polysaccharides, celluloses, polyisoprenes, methylstyrene, acrylic polymers, thoria sol, latex, nylon, Teflon cross- linked dextrans (e.g., Sepharose), chitosan, agarose, and cross-linked micelles. Additional examples include carbon graphited, titanium dioxide, and paramagnetic materials. See, e.g., "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. In particular embodiments, microparticles can be made of one or more materials. In particular embodiments, microparticles are paramagnetic microparticles. Particular embodiments utilize carboxymodified polystyrene latex (CML) flow cytometry beads and / or magnetic MagPlex® (Luminex, Austin, TX) flow cytometry beads. In particular embodiments, particles can carry a payload.

[0242] In particular embodiments, an antibody as disclosed herein can be linked to a conjugate by any method known in the art. In particular embodiments, the constant region can be modified to allow for site specific conjugation. Such techniques include the use of naturally occurring or engineered cysteine residues, disulfide bridges, poly-histidine sequences, glycoengineering tags, and transglutaminase recognition sequences. Antibody fragments can also be modified for site-specific conjugation, see for example, Kim et al., Mol Cancer Ther 2008;7(8).

[0243] (v-d) Recombinant Receptors. Binding domains can be used within recombinant receptors including chimeric antigen receptors (CAR), engineered T cell receptors (eTCR), or hybrids thereof.

[0244] CAR, when expressed by a cell, include several distinct subcomponents that allow the genetically modified cells (e.g , regulatory T cells) to recognize and kill cells expressing an antigen (e.g., Fn antigen). The subcomponents include at least an extracellular component and an intracellular component. The extracellular component includes a binding domain that specifically binds an Fn antigen epitope that is preferentially present on the surface of cells or in the area thereof. When the binding domain binds such epitopes, the intracellular component activates the cell to destroy the bound cell. CAR additionally include a transmembrane domain that directly or indirectly links the extracellular component to the intracellular component, and other subcomponents that can increase the CAR’s function. For example, the inclusion of a spacer region and / or one or more linker sequences can allow the CAR to have additional conformational flexibility, often increasing the binding domain's ability to bind the targeted epitope.

[0245] eTCR disclosed herein include Fn antigen binding domain disclosed herein linked to the Caand / or Cp chainsof a TCR. A TCR is a heterodimeric fusion protein that typically includes an a and p chain. Each chain includes a variable region (Vaand Vp) and a constant region (Caand Cp). In particular embodiments, an eTCR does not include the native TCR variable region but does include the native TCR constant region. In particular embodiments, the eTCR includes a Fn antigen binding domain as the variable region of the a and p chain. In particular embodiments, eTCR include a Caand / or Cp chain sequence that is at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% identical to an amino acid sequence of a known or identified TCR Caor Cp.

[0246] In particular embodiments, T cells are generated to react to F. nucleatum antigens (e.g., Fna C2-associated antigens). The T cells can be engineered based on Fna C2-associated antigen presentation via HLA I on cancer epithelial cells and macrophage. The Ana-specific TCR can be identified and the sequence can be inserted into immune cells, such that the immune cells express both TCRa and TCRp constructs that can be paired to the endogenous TCRa or p chains. In particular embodiments, the endogenous TCRa and TCRp genes (TRAC and TRBC) can be knocked out. In particular embodiments, the endogenous TCRa and TCRp genes are knocked out using CRISPR.

[0247] Particular embodiments of binding domains include a Fn antigen binding domain and / or the CDRs thereof.

[0248] Recombinant receptors can additionally include spacer regions, transmembrane domains, intracellular effector domains, transduction markers, selection cassettes, suicide switches, and / or tags.

[0249] Spacer regions are used to create appropriate distances and / or flexibility between sub-components of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 amino acids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 amino acids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region.

[0250] Transmembrane domains typically have a three-dimensional structure that is thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30 amino acids. The structure of a transmembrane domain can include an a helix, a p barrel, a p sheet, a p helix, or any combination thereof. Transmembrane domains can include at least the transmembrane region(s) of the a, p or chain of a T-cell receptor, CD28, CD27, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22; CD45, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0251] A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid within the extracellular region of the expressed protein (e.g., up to 15 amino acids of the extracellular region) and / or one or more additional amino acids within the intracellular region of the expressed protein (e.g., up to 15 amino acids of the intracellular components).

[0252] Intracellular effector domains activate the expressing cell when the binding domain binds antigen Fna C2- associated antigen, e.g., fap2, cmpA, or fusolisin). The term "effector domain” is thus meant to include any portion of the intracellular domain sufficient to transduce an activation signal.

[0253] An effector domain can include one, two, three or more intracellular signaling components (e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplaryeffector domains include signaling and stimulatory domains selected from: 4-1 BB (CD137), CD3y, CD35, CD3E, CD3(, CD27, CD28, DAP10, ICOS, LAG3, NKG2D, NOTCH1, 0X40, ROR2, SLAMF1 , TCRa, TCRp, TRIM, Wnt, Zap70, or any combination thereof. In particular embodiments, exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1 , lymphocyte function-associated antigen-1 (LFA- 1), LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD19, CD4, CD8o, CD8|3, IL2Rp, IL2Ry, IL7Ro, ITGA4, VLA1 , CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11 b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0254] Intracellular signaling component sequences that act in a stimulatory manner may include iTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include those derived from CD3y, CD35, CD3E, CD3 , CD5, CD22, CD66d, CD79a, CD79b, and common FcRy (FCER1 G), FcyRlla, FcRp (Fes Rib), DAP10, and DAP12. In particular embodiments, variants of CD3 retain at least one, two, three, or all ITAM regions.

[0255] A co-stimulatory domain is a domain whose activation can be required for an efficient lymphocyte response to cellular marker binding. Some molecules are interchangeable as intracellular signaling components or co-stimulatory domains. Examples of costimulatory domains include CD27, CD28, 4-1 BB (CD137), 0X40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, and a ligand that specifically binds with CD83.

[0256] In particular embodiments, genetic constructs can include a polynucleotide that encodes a self-cleaving polypeptide, wherein the polynucleotide encoding the self-cleaving polypeptide is located between the polynucleotide encoding the recombinant receptor and a control feature. Exemplary self-cleaving polypeptides include 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A), equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Further exemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example, Kim et al. (PLOS One 6:e18556 (2011).

[0257] Control features can include tag cassettes, transduction markers, selection cassettes, and / or suicide switches. "Tag cassette" refers to a unique synthetic peptide sequence affixed to, fused to, or that is part of a construct, to which a cognate binding molecule (e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the binding property can be used to activate, promote proliferation of, detect, enrich for, isolate, track, deplete and / or eliminate the tagged protein and / or cells expressing the tagged protein. Transduction markers can serve the same purposes but are derived from naturally occurring molecules and are often expressed using a skipping element that separates the transduction marker from the rest of the construct.

[0258] Tag cassettes that bind cognate binding molecules include, for example, His tag (HHHHHH; SEQ ID NO: 15), Flag tag (DYKDDDDK; SEQ ID NO: 16), Xpress tag (DLYDDDDK; SEQ ID NO: 17), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 18), Calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 19), Polyglutamate tag, HA tag(YPYDVPDYA; SEQ ID NO: 20), Myc tag (EQKLISEEDL; SEQ ID NO: 21), Strep tag (which refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 22), STREP® tag II (WSHPQFEK SEQ ID NO: 23 (I BA Institut fur Bioanalytik, Germany); see, e.g, US 7,981 ,632), Softag 1 (SLAELLNAGLGGS; SEQ ID NO: 24), Softag 3 (TQDPSRVG; SEQ ID NO: 25), and V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 26).

[0259] Conjugate binding molecules that specifically bind tag cassette sequences disclosed herein are commercially available. For example, His tag antibodies are commercially available from suppliers including Life Technologies, Pierce Antibodies, and GenScript. Flag tag antibodies are commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from suppliers including Pierce Antibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Pierce Antibodies. HA tag antibodies are commercially available from suppliers including Pierce Antibodies, Cell Signal and Abeam. Myc tag antibodies are commercially available from suppliers including Santa Cruz Biotechnology, Abeam, and Cell Signal. Strep tag antibodies are commercially available from suppliers including Abeam, Iba, and Qiagen.

[0260] Transduction markers may be selected from at least one of a truncated CD19 (tCD19; see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR or EGFRt; see Wang et al., Blood 118: 1255, 2011); an ECD of human CD34; and / or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1 (5 Pt 1); 448- 456, 2000) and CD20 antigens (see Philip et al, Blood 124: 1277-1278). In particular embodiments, cells are genetically modified to express EGFRt.

[0261] In particular embodiments, a selection cassette provides for positive selection or negative selection of a desired cell population. Negative selection is when several cell types are removed, leaving the cell type of interest. Positive selection involves targeting the desired cell population to only retain desired cells.

[0262] A selection cassette can encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Any number of selection systems may be used to recover transformed cells. In particular embodiments, a positive selection cassette includes resistance genes to thiamphenicol, neomycin, hygromycin, ampicillin, puromycin, phleomycin, zeomycin, blasticidin, or viomycin. In particular embodiments, a selection cassette includes the DHFR (dihydrofolate reductase) gene or DHFR double mutant (DHFRdm) gene providing resistance to methotrexate (MTX), the MGMT P140K gene responsible for the resistance to O6BG / BCNU, the HPRT (Hypoxanthine phosphoribosyl transferase) gene responsible for the transformation of specific bases present in the HAT selection medium (aminopterin, hypoxanthine, thymidine) or other genes for detoxification with respect to some drugs. In particular embodiments, the selection agent includes thiamphenicol, neomycin, hygromycin, puromycin, phleomycin, zeomycin, blasticidin, viomycin, ampicillin, O6BG / BCNU, MTX, tetracycline, aminopterin, hypoxanthine, thymidine kinase, DHFR, Gin synthetase, or ADA.

[0263] In particular embodiments, negative selection cassettes include a gene for transformation of a substrate present in the culture medium into a toxic substance for the cell that expresses the gene. These molecules include detoxification genes of diptheria toxin (DTA) (Yagi et al., Anal Biochem. 214(1)77-86, 1993; Yanagawa et al., Transgenic Res. 8(3):215-221, 1999), the kinase thymidine gene of the Herpes virus (HSV TK) sensitive to the presence of ganciclovir or Fl AU. The HPRT gene may also be used as a negative selection by addition of 6-th iog u an in e (6TG) into the medium, and for all positive and negative selections, a poly A transcription termination sequence from different origins, the most classical being derived from SV40 poly A, or a eukaryotic gene poly A (bovine growth hormone, rabbit p-globin, etc ).

[0264] In particular embodiments, a polynucleotide encoding an iCaspase9 construct (iCasp9) may be inserted into a recombinant receptor genetic construct as a suicide switch.

[0265] (vii) Single Cell Transcriptome Spatial Profiling. A single-cell nucleic acid (e.g., RNA) sequencing method for spatially profiling tumor microenvironments is also provided herein. The position of any given cell, relative to its neighbors and non-cellular structures, can provide helpful information for defining cellular phenotype, cell state, and ultimately cell and tissue function. Location can determine the signals to which cells are exposed. The nucleic acid sequences for these signals can be detected by transcriptomics.

[0266] In particular embodiments, a method of spatially profiling a tumor microenvironment includes obtaining a tissue sample from the tumor; capturing nucleic acid sequences and tagging said nucleic acid sequences with an oligo sequence; analyzing captured nucleic acid sequences containing the oligo sequence; and determining the expression profile. In particular embodiments, spatial profiling a tumor microenvironment includes isolating individual cells (e.g., bacterial cell, cancer cell, or bacteria-infected cell) from a tissue sample; permeabilizing each individual cell to release nucleic acid sequences; introducing beads tagged with oligo sequence, wherein each oligo sequence includes a PCR handle, barcode, and / or unique molecular identifier (UMI); and amplifying the nucleic acid sequences. In particular embodiments, the amplifying includes performing first-strand cDNA synthesis to create a first sequence including a complement sequence to the released nucleic acid sequence linked to a complement sequence to the oligo sequence; performing second-strand synthesis to create a second sequence that matches the nucleic acid sequences found in the cell linked to an oligo sequence; and performing cDNA synthesis to amplify the first and second sequences.

[0267] In some cases, tissue or sample preparation is necessary. For example, cells may need to be dissociated from the tissue. An example dissociation workflow includes mechanical dissociation / mincing of the tissue and enzymatic removal of extracellular matrix components, followed by washing and filtration to eliminate dead cells, debris, and cellular aggregates. Depending on the tissue type, its cell composition, extracellular matrix composition, and stiffness, the dissociation methods need to be optimized. This includes selecting the right enzymes for dissociation, optimizing enzymatic-digestion times, washing and centrifugation conditions, and selecting the right resuspension buffers. Resuspension buffers containing EDTA (>0.1 mM) and excess Mg2+ and Ca2+ ions interfere with reverse transcription (RT) reaction, thus reducing cDNA yield. The degradation of RNA during the sample processing stepscan be minimized by using nuclease-free reagents and by addition of RNase inhibitors.

[0268] Tissues can be fresh or fixed. Examples of fixed tissue samples include formalin-fixed paraffin-embedded tissue, cryopreserved tissue, or methanol fixed and frozen tissue. To prevent RNA contamination, methods to remove dead or dying cells from the tissue can be performed.

[0269] In particular embodiments, individual cells can be isolated using well loading or droplet loading. Droplet-based microfluidic technologies, including compartmentalization of cells into water-in-oil droplets for single-cell reactions in picoliter-scale volumes, provide many advantages in terms of throughput of cells as well as reaction efficiency. Current platforms provide the possibility to process thousands to up to tens of thousands of cells simultaneously in a single reaction. There are many commercially available platforms for performing droplet-based microfluidics and they typically start with encapsulation of single cells into water-in-oil droplets using microfluidic devices. Together with the cell, barcoded beads and reagents for cell lysis and RT are included in the droplet. Barcoded beads have also been used in combination with microwell plates where cells are captured by gravity. The barcodes on beads contain a common PCR handle. Following cell lysis in the droplets, mRNA gets captured onto the primers, while they are still attached to the microparticles, forming STAMPs (single-cell transcriptomes attached to microparticles). Here, cDNA is amplified exponentially by PCR via the PCR handle sequence and libraries are generated as per standard procedures. Most barcode systems used with beads share common features. They usually have a cell-specific barcode that aids in tracing the cell from which the mRNA transcripts originate. In addition, each captured transcript is labeled with a unique molecular identifier (UMI) to allow transcript counting and to reduce PCR amplification-induced biases. The barcodes frequently also have an oligo(dT) sequence, which positions the cell barcodes and UMI at the 3’ -ends of poly adenylated mRNA transcripts, thus making these techniques 3'-end specific

[0270] In certain embodiments, a transcriptome is sequenced. The transcriptome may be used to genotype nuclear and mitochondrial genomes in addition to determining gene expression. As used herein the term "transcriptome" refers to the set of transcripts molecules. In some embodiments, transcript refers to RNA molecules, e.g., messenger RNA (mRNA) molecules, small interfering RNA (siRNA) molecules, transfer RNA (tRNA) molecules, ribosomal RNA (rRNA) molecules, and complimentary sequences, e.g., cDNA molecules. In some embodiments, a transcriptome refers to a set of rRNA molecules. In some embodiments, a transcriptome refers to a set of mRNA molecules. In some embodiments, a transcriptome refers to cDNA generated from one or more of mRNA molecules, siRNA molecules, tRNA molecules, rRNA molecules, in a sample, for example, a single cell or a population of cells. In some embodiments, a transcriptome refers to 5%, 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of transcripts from a single cell or a population of cells.

[0271] Single cell RNA sequencing (scRNAseq) is well known by those of skill in the art (see, e.g., Kalisky, et al., Annual review of genetics 45, 431-445, (2011); Kaliskyet al., Nature Methods 8, 311-314 (2011); Islam, et al. Genome Research, (2011); Tang, et al. Nature Protocols 5, 516-535, (2010); Tang, et al. Nature Methods 6, 377-382, (2009); Ramskold, et al. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, et al., Cell Reports, CellReports, Volume 2, Issue 3, p 666-673, 2012). In brief, scRNAseq involves isolating single cells in, for example, oilbased emulsion, capturing transcript, performing reverse transcription, and performing molecular or cellular barcoding To track the location of the cell, a barcode oligo sequence can be attached to the captured transcript in order to indicate the coordinates from which the transcript was captured.

[0272] In certain embodiments, the disclosurse provides high-throughput single-cell RNA-seq where the RNAs from different cells are tagged individually, allowing a single library to be created while retaining the cell identity of each read. In this regard reference is made to Macosko et al., 2015, Cell 161 , 1202-1214; W02016 / 040476; Klein et al., 2015, Cell 161 , 1187-1201; WO2016168584; Zheng, et al., 2016, Nature Biotechnology 34, 303-311 ; Zheng, et al., 2017, Nat. Commun. 8, 14049; WO2014210353A2; Zilionis, et al., 2017, “Nat Protoc. January; 12(1 ):44-73; Cao et al., 2017, Science 357(6352): 661-667; Rosenberg et al., 2017, Science 360(6385: 176-182; Vitak, et al., Nature Methods, 14(3): 302-308, 2017; Cao, et al., Science, 357 (6352) :661-667, 2017; Gierahn et al., Nature Methods 14, 395-398 (2017); and Hughes, et al., Immunity, 2020, 53(4): 878-894.e7.

[0273] In particular embodiments, transcripts can be flanked with a unique molecular identifier (UMI) (see, e.g., Kivioja et al., 2012, Nat. Methods. 9 (1): 72-4 and Islam et al., 2014, Nat. Methods. 11 (2): 163-6) a unique sample barcode, a unique location barcode, a unique cell barcode (cell into the sequencing library, or a combination. The barcode as used herein refers to a short sequence of nucleotides (for example, DNA or RNA) that is used as an identifier for an associated molecule, such as a target molecule and / or target nucleic acid, or as an identifier of the source of an associated molecule, such as a sample or cell-of-origin. A barcode may also refer to any unique, non-naturally occurring, nucleic acid sequence that may be used to identify the originating source of a nucleic acid fragment.

[0274] Barcoding may be performed based on any of the compositions or methods disclosed in, for example, WO 2014047561. In certain embodiments barcoding uses an error correcting scheme (Moon, Error Correction Coding: Mathematical Methods and Algorithms (Wiley, New York, ed. 1, 2005)). Not being bound by theory, amplified sequences from different sources can be sequenced together and resolved based on the barcode associated with each sequencing read.

[0275] In particular embodiments, sequencing is performed using UMI. The term “unique molecular identifiers” (UMI) as used herein refers to a sequencing linker or a subtype of nucleic acid barcode used in a method that uses molecular tags to detect and quantify unique amplified products. A UMI is used to distinguish effects through a single clone from multiple clones. The term “clone” as used herein may refer to a single mRNA or target nucleic acid to be sequenced. Unique Molecular Identifiers may be short (usually 4-10 bp) random barcodes added to transcripts during reversetranscription. They enable sequencing reads to be assigned to individual transcript molecules and thus the removal of amplification noise and biases from RNA-seq data. The UMI may also be used to determine the number of transcripts that gave rise to an amplified product.

[0276] (viii) Compositions and Formulations. Compositions disclosed herein include binding domains, antibodies, multi-domain specific antibodies, proteins / antigens, genetic constructs, vectors, vaccines, nanoparticles, virus-likeparticles, recombinant receptors, antibody conjugates, or nucleic acid sequences. Formulations include mammalian cells, immune cells, bacterial cells, or populations of cells as disclosed herein. Formulations include genetically engineered mammalian cells, genetically engineered immune cells, genetically engineered bacterial cells, or populations of genetically engineered cells as disclosed herein. Compositions and / or formulations can be formulated alone or in combination into compositions for administration to subjects. Salts and / or pro-drugs of active ingredients can also be used. The nucleic acids, particle, protein vaccine antigens, and / or vaccine adjuvants disclosed herein (individually, collectively, or in grouped combinations referred to as "active ingredients”) can be provided as part of compositions formulated for administration to subjects.

[0277] A pharmaceutically acceptable salt includes any salt that retains the activity of the composition and / or formulation and is acceptable for pharmaceutical use. A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt.

[0278] Suitable pharmaceutically acceptable acid addition salts can be prepared from an inorganic acid or an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids.

[0279] Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine, lysine, arginine and procaine.

[0280] A prodrug includes an active ingredient which is converted to a therapeutically active compound after administration, such as by cleavage of a peptide or by hydrolysis of a biologically labile group.

[0281] Exemplary generally used pharmaceutically acceptable carriers include any and all absorption delaying agents, antioxidants (e.g., ascorbic acid, methionine, vitamin E), binders, buffering agents, bulking agents or fillers, chelating agents (e.g., EDTA), coatings, disintegration agents, dispersion media, gels, isotonic agents, lubricants, preservatives, salts, solvents or co-solvents, stabilizers, surfactants, and / or delivery vehicles.

[0282] Exemplary antioxidants include ascorbic acid, methionine, and vitamin E.

[0283] Exemplary buffering agents include citrate buffers, succinate buffers, tartrate buffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers, phosphate buffers, histidine buffers, and / or trimethylamine salts.

[0284] An exemplary chelating agent is EDTA.

[0285] Exemplary isotonic agents include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol, or mannitol

[0286] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, hexamethonium chloride, alkyl parabens such asmethyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

[0287] Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the active ingredient or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (i.e., <10 residues); proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers are typically present in the range of from 0.1 to 10,000 parts by weight based on therapeutic weight.

[0288] The compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection. For injection, formulation can be formulated as aqueous solutions, such as in buffers including Hanks' solution, Ringer's solution, or physiological saline, or in culture media, such as Iscove’s Modified Dulbecco's Medium (IMDM). The aqueous solutions can include formulatory agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the formulation can be in lyophilized and / or powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0289] For oral administration, the compositions can be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. For oral solid formulations such as powders, capsules and tablets, suitable excipients include binders (gum tragacanth, acacia, cornstarch, gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol and sorbitol; dicalcium phosphate, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate; cellulose preparations such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxy- methylcellulose, and / or polyvinylpyrrolidone (PVP); granulating agents; and binding agents. If desired, disintegrating agents can be added, such as corn starch, potato starch, alginic acid, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. If desired, solid dosage forms can be sugar-coated or enteric-coated using standard techniques. Flavoring agents, such as peppermint, oil of Wintergreen, cherry flavoring, orange flavoring, etc. can also be used.

[0290] Compositions can be formulated as an aerosol. In particular embodiments, the aerosol is provided as part of an anhydrous, liquid or dry powder inhaler. Aerosol sprays from pressurized packs or nebulizers can also be used with a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator may also be formulated including a powder mix of active ingredients and a suitable powder base such as lactose or starch.

[0291] Compositions and / or formulations can also be formulated as depot preparations. Depot preparations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0292] Additionally, compositions and / or formulations can be formulated as sustained-release systems utilizing semipermeable matrices of solid polymers including at least one active ingredient. Various sustained-release materials have been established and are well known by those of ordinary skill in the art. Sustained-release systems may, depending on their chemical nature, release the active ingredient following administration for a few weeks up to over 100 days. Depot preparations can be administered by injection; parenteral injection; instillation; or implantation into soft tissues, a body cavity, or occasionally into a blood vessel with injection through fine needles.

[0293] Depot formulations can include a variety of bioerodible polymers including poly(lactide), poly(glycolide), poly(caprolactone) and poly(lactide)-co(glycolide) (PLG) of desirable lactide:glycolide ratios, average molecular weights, polydispersities, and terminal group chemistries. Blending different polymer types in different ratios using various grades can result in characteristics that borrow from each of the contributing polymers.

[0294] The use of different solvents (for example, dichloromethane, chloroform, ethyl acetate, triacetin, N-methyl pyrrolidone, tetrahydrofuran, phenol, or combinations thereof) can alter microparticle size and structure in order to modulate release characteristics. Other useful solvents include water, ethanol, dimethyl sulfoxide (DMSO), N-methyl- 2-pyrrolidone (NMP), acetone, methanol, isopropyl alcohol (IPA), ethyl benzoate, and benzyl benzoate.

[0295] Exemplary release modifiers can include surfactants, detergents, internal phase viscosity enhancers, complexing agents, surface active molecules, co-solvents, chelators, stabilizers, derivatives of cellulose, (hydroxypropyl)methyl cellulose (HPMC), HPMC acetate, cellulose acetate, pluronics (e.g., F68 / F127), polysorbates, Span® (Croda Americas, Wilmington, Delaware), poly(vinyl alcohol) (PVA), Brij® (Croda Americas, Wilmington, Delaware), sucrose acetate isobutyrate (SAIB), salts, and buffers.

[0296] Excipients that partition into the external phase boundary of microparticles such as surfactants including polysorbates, dioctylsulfosuccinates, poloxamers, PVA, can also alter properties including particle stability and erosion rates, hydration and channel structure, interfacial transport, and kinetics in a favorable manner.

[0297] Additional processing of the disclosed sustained release depot formulations can utilize stabilizing excipients including mannitol, sucrose, trehalose, and glycine with other components such as polysorbates, PVAs, and dioctylsulfosuccinates in buffers such as Tris, citrate, or histidine. A freeze-dry cycle can also be used to produce very low moisture powders that reconstitute to similar size and performance characteristics of the original suspension.

[0298] Particular embodiments include formulation of active ingredients within hydrogels. Exemplary hydrogels include collagen hydrogels; type I collagen, fibrin, or a mixture thereof cross-linked, as the cross-linked state of these molecules in vivo; type I collagen hydrogels naturally cross-linked by lysyl oxidase-derived aldimine bonds (Sabeh etal., (2009) J Cell Biol 185:11-19); or other synthetic hydrogels as described in, for example, Rowe & Weiss (2008) Trends Cell Biol 18:560-574; Rowe & Weiss (2009) Annu Rev Cell Dev Biol 25:567-595; Egeblad et al., (2010) Curr Opin Cell Biol 22:697-706; Harunaga & Yamada (2011) Matrix Biol 30:363-368; Willis et al., (2013) J Microsc 251 :250- 260; and Gill et al. (2012) Cancer Res 72:6013-6023. In particular embodiments, a hydrogel refers to a network of polymer chains that are hydrophilic in which water or an aqueous medium is the dispersion medium. Particular embodiments may utilize a zwitterionic polymer as described in WC2016 / 040489.

[0299] Any composition or formulation disclosed herein can advantageously include any other pharmaceutically acceptable carriers which include those that do not produce significantly adverse, allergic, or other untoward reactions that outweigh the benefit of administration. Exemplary pharmaceutically acceptable carriers and formulations are disclosed in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, formulations can be prepared to meet sterility, pyrogenicity, general safety, and purity standards as required by US FDA Office of Biological Standards and / or other relevant foreign regulatory agencies.

[0300] In particular embodiments, active ingredients may be formulated to remain inert at an administration site. In particular embodiments, this feature can be achieved with a proteolytically cleavable blocking peptide that is cleaved by proteases, such as (i) serum proteases, (ii) proteases secreted by cancers or bacteria, and / or (iii) administered proteases.

[0301] Exemplary serum proteases include thrombin, Factor X, dipeptidyl peptidase IV, plasmin, and Hageman factor. Exemplary proteases secreted by cancers include ADAMTSs (A disintegrin and metalloprotease domains with thrombospondins motifs (ADAMTS-1 , ADAMTS-4 and ADAMTS-5), metalloproteinases or matrix-degrading proteases (MMP3 and MMP7), urokinase-type plasminogen activator (uPA), the serine protease prostate-specific antigen (PSA), and cathepsins including aspartic cathepsins (cathepsins D and E) and cysteine cathepsins (cathepsins B, C, F, H, K, L, O, S, V, X, and W).

[0302] In particular embodiments, active ingredients may be formulated to remain inert at an administration site by altering the pH of the environment and / or by using a crystalline form of an active ingredient, as previously applied to insulin.

[0303] In particular embodiments, the compositions include at least 0 1 % w / v or w / w of the active ingredient; at least 1 % w / v or w / w of active ingredient; at least 10% w / v or w / w of active ingredient; at least 20% w / v or w / w of active ingredient; at least 30% w / v or w / w of active ingredient; at least 40% w / v or w / w of active ingredient; at least 50% w / v or w / w of active ingredient; at least 60% w / v or w / w of active ingredient; at least 70% w / v or w / w of active ingredient; at least 80% w / v or w / w of active ingredient; at least 90% w / v or w / w of active ingredient; at least 95% w / v or w / w of active ingredient; or at least 99% w / v or w / w of active ingredient.

[0304] In particular embodiments, active ingredients within a combination therapy are formulated into separate individual compositions. In particular embodiments, active ingredient combinations may be formulated into compositions and / or formulations together. When formulated together, the active ingredient may be included in thesame amounts or in different amounts or ratios. For example, if two active ingredient are provided, these ingredients could be included in the following exemplary ratios: 1 :1 , 2:1 , 1 :2, 5:1 , 1 :5, 10:1 , 1 : 10, etc. If three active ingredients are provided, these ingredients could be included in the following exemplary ratios: a 1 :1 : 1 ratio, 2: 1 : 1 ratio, 1 :2:1 ratio, 1 :1 :2 ratio, 5: 1 :1 ratio, 1 :5: 1 ratio, 1 :1 :5 ratio, 10:1 :1 ratio, 1 : 10: 1 ratio, 1 :1 :10 ratio, 2:2: 1 ratio, 1 :2:2 ratio, 2:1 :2 ratio, 5:5:1 ratio, 1 :5:5 ratio, 5:1 :5 ratio, 10:10:1 ratio, 1 :10: 10 ratio, 10:1 :10 ratio, etc.

[0305] Compositions and / or formulations disclosed herein can be formulated for administration by, for example, injection, infusion, perfusion, or lavage. The compositions disclosed herein can further be formulated for intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal, intralesional, intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, intravesicular, oral and / or subcutaneous administration and more particularly by intravenous, intraarterial, intraperitoneal, intrarectal, intratumoral, intravesicular, and / or oral injection.

[0306] (ix) Methods of Use. Methods disclosed herein include treating subjects (e.g., humans, veterinary animals (dogs, cats, reptiles, birds) livestock (e.g., horses, cattle, goats, pigs, chickens) and research animals (e.g., monkeys, rats, mice, fish) with compositions disclosed herein. Treating subjects includes delivering therapeutically effective amounts. Therapeutically effective amounts include those that provide effective amounts, prophylactic treatments and / or therapeutic treatments.

[0307] An "effective amount" is the amount of a composition necessary to result in a desired physiological change in the subject. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause a statistically-significant effect in an animal model or in vitro assay relevant to the assessment of an infection or cancer’s development, progression, and / or resolution.

[0308] A "prophylactic treatment" includes a treatment administered to a subject who does not display signs or symptoms of an infection or cancer or displays only early signs or symptoms of an infection or cancer such that treatment is administered for the purpose of diminishing or decreasing the risk of developing the infection or cancer further. Thus, a prophylactic treatment functions as a preventative treatment against an infection or cancer. In particular embodiments, prophylactic treatments reduce, delay, or prevent the worsening of an infection or cancer.

[0309] Particular uses of the compositions include use as prophylactic vaccines. Vaccines increase the immunity of a subject against a particular infection. Therefore, "Fna vaccine" or “Fna C2 vaccine” can refer to a treatment that increases the immunity of a subject against Fna (e.g., Fna C2). In certain examples, a vaccine may be administered prophy lactically, for example to a subject that is immunologically naive (e.g., no prior exposure or experience with Fna C2). In particular embodiments, a vaccine may be administered therapeutically to a subject who has been exposed to Fna C2. Thus, a vaccine can be used to ameliorate a symptom and / or complication associated with Fna C2, examples of each of which are described elsewhere herein.

[0310] In particular embodiments, a Fna C2 vaccine is a therapeutically effective composition including binding domains derived from ai-mAb that bind VH3-21 / VL1 -40 B cell receptors (BCRs) that induce an immune response in a subject against Fna C2. The skilled artisan will appreciate that the immune system generally is capable of producingan innate immune response and an adaptive immune response. An innate immune response generally can be characterized as not being substantially antigen specific and / or not generating immune memory. An adaptive immune response can be characterized as being substantially antigen specific, maturing over time (e.g., increasing affinity and / or avidity for antigen), and in general can produce immunologic memory. Even though these and other functional distinctions between innate and adaptive immunity can be discerned, the skilled artisan will appreciate that the innate and adaptive immune systems can be integrated and therefore can act in concert.

[0311] In particular embodiments, administration of a Fna vaccine can further include administration of one or more adjuvants as described elsewhere herein.

[0312] " Immune response" refers to a response of the immune system to produce Fna C2 neutralizing antibodies in response to a vaccine as described herein. In particular embodiments, the immune response causes production of antibodies to neutralize Fna 02. In particular embodiments, an immune response to a vaccine can be an innate and / or adaptive response. In particular embodiments, an adaptive immune response can be a "primary immune response" which refers to an immune response occurring on the first exposure of a "naive" subject to Fna C2 or vaccine. For example, in the case of a primary antibody response, after a lag or latent period of from 3 to 14 days depending on, for example, the composition, dose, and subject, antibodies to Fna C2 can be produced. Generally, IgM production lasts for several days followed by IgG production and the IgM response can decrease. Antibody production can terminate after several weeks but memory cells can be produced. In particular embodiments, an adaptive immune response can be a "secondary immune response", "anamnestic response," or "booster response" which refer to the immune response occurring on a second and subsequent exposure of a subject to Fna C2 or vaccine. Generally, in a secondary immune response, memory cells respond to the Fna C2 or vaccine and therefore the secondary immune response can differ from a primary immune response qualitatively and / or quantitatively. For example, in comparison to a primary antibody response, the lag period of a secondary antibody response can be shorter, the peak antibody titer can be higher, higher affinity antibody can be produced, and / or antibody can persist for a greater period of time.

[0313] A "therapeutic treatment" includes a treatment administered to a subject who displays symptoms or signs of an infection or cancer and is administered to the subject for the purpose of diminishing or eliminating those signs or symptoms of the infection or cancer. The therapeutic treatment can reduce, control, or eliminate the presence or activity of the infection or cancer and / or reduce control or eliminate side effects of the infection or cancer.

[0314] Function as an effective amount, prophylactic treatment or therapeutic treatment are not mutually exclusive, and in particular embodiments, administered dosages may accomplish more than one treatment type.

[0315] In particular embodiments, therapeutically effective amounts provide anti-infection and / or anti-cancer effects. Anti-infection effects include a reducing or preventing a bacteria from infecting a tissue, decreasing the volume of infected tissue, increasing lifespan, increasing life expectancy, reducing or eliminating infection-associated symptoms, such as fever, sore throat, or inflammation. .

[0316] Anti-cancer effects include a decrease in the number of cancer cells, decrease in the number of metastases,a decrease in tumor volume, an increase in life expectancy, induced chemo- or radiosensitivity in cancer cells, inhibited angiogenesis near cancer cells, inhibited cancer cell proliferation, inhibited tumor growth, prevented or reduced metastases, prolonged subject life, reduced cancer-associated pain, and / or reduced relapse or re-occurrence of cancer following treatment.

[0317] A "tumor" is a swelling or lesion formed by an abnormal growth of cells (called neoplastic cells or tumor cells). A "tumor cell" is an abnormal cell that grows by a rapid, uncontrolled cellular proliferation and continues to grow after the stimuli that initiated the new growth cease. Tumors show partial or complete lack of structural organization and functional coordination with the normal tissue, and usually form a distinct mass of tissue, which may be benign, pre- malignant or malignant.

[0318] In particular embodiments, therapeutically effective amounts induce an immune response. The immune response can be against a Fusobacterium nucleatum or a cell infected with Fusobacterium nucleatum. In particular embodiments, the Fusobacterium nucleatum includes Fna C2.

[0319] Examples of disorders that can be treated with the vaccines, compositions, inhibitors, binding domains (e.g., antibodies), multi-domain binding molecules, antibody conjugates, and recombinant receptors disclosed herein include cancer and infections. Examples of cancers that can be treated using approaches described herein include colon cancer, mouth cancer, gastrointestinal cancer, prostate cancer, skin cancer, bladder cancer, breast cancer, ovarian cancer, lung cancer, and kidney cancer. In particular embodiments, the cancer includes colorectal cancer (CRC). In particular embodiments, the cancer includes oral squamous cell carcinoma (OSCC). In particular embodiments, the cancer includes melanoma, colon adenocarcinoma, or medullary breast adenocarcinoma.

[0320] In particular embodiments, a prophylactic treatment includes vaccinating a subject against Fusobacterium nucleatum anlmalis {Fna) clade 2. In particular embodiments, the vaccinating includes administering a therapeutically effective amount of a vaccine. In particular embodiments, the vaccine includes an Fna C2-associated antigen. In particular embodiments, the Fna C2-associated antigen is free floating or fused to a delivery scaffold. In particular embodiments, the Fna C2-associated antigen includes Fna C2-associated antigen includes fap2, cmpA, or fusolisin. In particular embodiments, vaccine includes dead bacteria or attenuated bacteria.

[0321] In particular embodiments, a cancer associated with a cancer-promoting bacteria can be treated with compositions disclosed herein. In particular embodiments, a method of treating a cancer associated with a cancerpromoting bacteria includes administering a bacteria inhibitor, wherein the bacteria inhibitor targets and inhibits the cancer-promoting bacteria; and administering a cancer drug. In particular embodiments, the cancer drug can be any molecule, drug, or cell that kills cancer. In particular embodiments, the bacteria inhibitor destroys bacteria or suppresses their growth or their ability to reproduce. A molecule to kill cancer can include antibodies, antibody-drug conjugates, peptides or proteins. A drug to kill cancer can include phase-specific drugs or phase non-specific drugs. A phase-nonspecific drug is a class of pharmaceuticals that can kill cells in all phases of the cell cycle and include alkylating agents, antitumor antibiotics, or other drugs. A phase-specific drug is a class of pharmaceuticals that targetand kill cells in a particular phase or phases of the cell cycle. Phase-specific drugs can target the G0-G1 phase of the cell cycle (e.g., nitrosureas, asparaginase, or actinomycin D), the S phase of the cell cycle (e.g., antimetabolite, antifolate, antipyrimidine, antipurine, or topoisomerase inhibitor), the G2 phase of the cell cycle, or the M phase of the cell cycle. Antimetabolites can include cladribine, cytarabine, 5-fluorouracil, hydroxyurea, methotrexate, pentostatin, or thiopurine. Topoisomerase inhibitors can include etoposide, teniposide, irinotecan, or topotecan. In particular embodiments, the cancer-promoting bacteria is Fusobacterium nucleatum and inhibitors thereof are described elsewhere herein. Bacteria inhibitors for other cancer-promoting bacteria are known in the art.

[0322] Herein, a G0-G1 phase cancer drug refers to any molecule or composition capable of killing cells in the GO- G1 phase. This includes molecules or compositions that specifically target cells in the G0-G1 phase of the cell cycle or any cell regardless of phase.

[0323] Some bacteria (e.g., Fna) arrests cells in the G0-G1 phase of the cell cycle and many chemotherapeutics target the S phase of the cell cycle. In particular embodiments, a method of treating a cancer associated with cancerpromoting bacteria includes administering a G0-G1 phase cancer drug and a second drug. In particular embodiments, the second drug is a chemotherapeutic targeting the S phase of the cell cycle or is a phase-nonspecific drug.

[0324] In particular embodiments, a cancer associated with a cancer-promoting bacteria can be treated with a targeted therapeutic described herein. In particular embodiments, the cancer-promoting bacteria includes Fna 02 and the method includes administering a therapeutically effective amount of a targeted therapeutic. In particular embodiments, the targeted therapeutic includes a binding molecule with a binding domain that binds a Fna 02- associated antigen. In particular embodiments, the Fna C2-associated antigen includes fap2, cmpA, or fusolisin. In particular embodiments, the targeted therapeutic includes an antibody or fragment thereof, a multi-domain binding molecule, an antibody conjugate, or a recombinant receptor. In particular embodiments, the recombinant receptor includes a chimeric antigen receptor and is expressed by an immune cell.

[0325] Additionally, in particular embodiments, bacteria can be engineered to have the ability to adapt to the tumor microenvironment. Methods to engineer bacteria are described elsewhere herein. In particular embodiments, bacteria are engineered to express an eut operon and / or a pdu operon. In particular embodiments, bacteria are engineered to express an gdar operon. In particular embodiments, bacteria are engineered to include a gene that provides growth adaptation to the tumor microenvironment and a therapeutic payload and / or marker.

[0326] For administration, therapeutically effective amounts (also referred to herein as doses) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician, veterinarian or researcher taking into account parameters such as physical and physiological factors including target, body weight, severity of condition, type of cancer or infection, stage of cancer or infection, previous or concurrent therapeutic interventions, idiopathy of the subject and route of administration.

[0327] Useful doses can range from 0.1 to 5 pig / kg or from 0.5 to 1 pig / kg. In other examples, a dose can include 1g / kg, 15 pg / kg, 30 pg / kg, 50 pg / kg, 55 pg / kg, 70 pg / kg, 90 pg / kg, 150 pg / kg, 350 pg / kg, 500 pg / kg, 750 pg / kg, 1000 pg / kg, 0.1 to 5 mg / kg or from 0.5 to 1 mg / kg. In other examples, a dose can include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0328] Therapeutically effective amounts of cell-based formulations can include 104to 109cells / kg body weight, or 103to 1011cells / kg body weight. Therapeutically effective amounts to administer can include greater than 102cells, greater than 103cells, greater than 104cells, greater than 106cells, greater than 106cells, greater than 107cells, greater than 108cells, greater than 109cells, greater than 1010cells, or greater than 1011.

[0329] Therapeutically effective amounts can be achieved by administering single or multiple doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, every 4 days, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months or yearly).

[0330] The pharmaceutical compositions described herein can be administered by, for example, injection, inhalation, infusion, perfusion, lavage, or ingestion. Routes of administration can include intravenous, intradermal, intraarterial, intraparenteral, intranasal, intralesional, intramuscular, oral, subcutaneous, and / or sublingual administration.

[0331] In addition to therapeutic uses, the methods disclosed herein can be used to diagnose that a subject having cancer has a bacteria-promoting cancer such as Fna C2. In particular embodiments, the method includes detecting Fna C2 within the subject and directing treatment dependent on whether Fna C2 is detected.

[0332] Furthermore, the single cell transcriptome spatial profiling described herein can be used to assess the role of tumor microbiota. In particular embodiments, a method of spatially profiling a tumor microenvironment includes obtaining a tissue sample from the tumor microenvironment; isolating individual cells; capturing nucleic acid sequences from each isolated cell; tagging nucleic acid sequences with an oligo sequence including a location barcode; analyzing captured nucleic acid sequences; determining expression profile at each location within the tissue sample.

[0333] (x) Reference Levels Derived from Control Populations. Obtained values for parameters associated with a therapy or diagnostic method described herein can be compared to a reference level derived from a control population, and this comparison can indicate whether a therapy or diagnosis described herein is effective for a subject in need thereof. Reference levels can be obtained from one or more relevant datasets from a control population. A "dataset" as used herein is a set of numerical values resulting from evaluation of a sample (or population of samples) under a desired condition. The values of the dataset can be obtained, for example, by experimentally obtaining measures from a sample and constructing a dataset from these measurements. As is understood by one of ordinary skill in the art, the reference level can be based on e.g., any mathematical or statistical formula useful and known in the art for arriving at a meaningful aggregate reference level from a collection of individual data points; e.g., mean, median, median of the mean, etc. Alternatively, a reference level or dataset to create a reference level can be obtained from a service provider such as a laboratory, or from a database or a server on which the dataset has been stored.

[0334] A reference level from a dataset can be derived from previous measures derived from a control population. A"control population" is any grouping of subjects or samples of like specified characteristics. The grouping could be according to, for example, clinical parameters, clinical assessments, therapeutic regimens, disease status, severity of condition, etc. In particular embodiments, the grouping is based on microniche environment and cancer status. In particular embodiments, a normal control population includes individuals that are age-matched to a test subject and do not have cancer. In particular embodiments, age-matched includes, e.g., 0-10 years old; 30-40 years old, 60-65 years old, 70-85 years old, etc., as is clinically relevant under the circumstances. In particular embodiments, a control population can include those that have a disorder (e.g., cancer) and have not been administered a therapeutically effective amount.

[0335] In particular embodiments, the relevant reference level for values of a particular parameter associated with a therapy or diagnostic method described herein is obtained based on the value of a particular corresponding parameter associated with a therapy or diagnosis in a control population to determine whether a therapy or diagnosis disclosed herein has been therapeutically effective for a subject in need thereof.

[0336] In particular embodiments, conclusions are drawn based on whether a sample value is statistically significantly different or not statistically significantly different from a reference level. A measure is not statistically significantly different if the difference is within a level that would be expected to occur based on chance alone. In contrast, a statistically significant difference or increase is one that is greater than what would be expected to occur by chance alone. Statistical significance or lack thereof can be determined by any of various methods well-known in the art. An example of a commonly used measure of statistical significance is the p-value. The p-value represents the probability of obtaining a given result equivalent to a particular data point, where the data point is the result of random chance alone. A result is often considered significant (not random chance) at a p-value less than or equal to 0 05. In particular embodiments, a sample value is "comparable to” a reference level derived from a normal control population if the sample value and the reference level are not statistically significantly different.

[0337] (xi) Kits. Also provided herein is a kit which may be used with the treatment / therapy, spatial profiling method, genetic engineering, detection, and / or diagnostic method described herein. The kit may include a positive control and / or a negative control. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and / or other material useful in sample processing, washing, or conducting any other step of the methods described herein. For example, depending on the aspect of the methods practiced, kits could include one or more of tissue samples (e.g., peripheral blood mononuclear cells (PBMCs), tissue biopsy, tumor, oral tissue, gut tissue, or other organ, and / or cells derived therefrom); genetic expression components (e.g., plasmids, genes for expression provided by vectors (e.g., lentiviral vector, retroviral vector), CRISPR components, ZFNs, TALENs, MegaTALs, targeted viral vectors and / or nanoparticles); dead or attenuated bacteria; Fna C2-associated antigen (e.g., fap2, cmpA, fusolisin); epitopes; vaccine adjuvants; nucleic acids encoding a recombinant receptor (e.g., CAR or eTCR); lentiviral artificial expression construct; a nucleic acid encoding an antibody; a nucleic acid encoding an scFv; cells (e.g., immune cells); antibody conjugates; bacterial cells (e.g., Fna C1 , Fna C2, Fnn , cell formulation or activationcomponents (e.g., saline, buffered saline, phosphate buffered saline (PBS), biocompatible buffers, Vitamin K1 , Vitamin B12, physiological saline, water, Hanks' solution, Ringer's solution, antibiotics, human serum components, fetal bovine serum, defibrinated horse blood, stabilizers, preservatives, glycerol); combination therapy components (e.g., local anesthetics, chemotherapeutic agents); an antibody tagged with a fluorescent molecule; PCR amplification sequences; culture vessels (e.g., culture plates); reference levels, animal models; primer pairs; GAPDH; IFN-y enzyme-linked immunosorbent assay (ELISA); etc.

[0338] The kit according to the present disclosure may also include instructions for carrying out the therapeutic or diagnostic method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.

[0339] The Exemplary Embodiments and Example below are included to demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art should recognize in light of the present disclosure that many changes can be made to the specific embodiments disclosed herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0340] (xii) Exemplary Embodiments.1 . A method for identifying a Fusobacterium nucleatum subspecies animalis (Fna) as an Fna clade 1 (C1 ) or an Fna clade 2 (C2), the method including: determining a characteristic of the Fna; and comparing the characteristic to a reference characteristic that identifies the Fna as Fna C1 or Fna C2, thereby identifying the Fna as Fna C1 or Fna C2.2. The method of embodiment 1 , wherein the reference characteristic includes a coding sequence, an operon, DNA methylation, a growth condition, or a dimension.3. The method of embodiment 2, wherein the reference characteristic includes the coding sequence, the operon, or DNA methylation.4. The method of embodiment 3, wherein the method further includes sequencing DNA of the Fna for presence of the reference characteristic.5. The method of embodiment 4, wherein the reference characteristic is within a target region of the DNA.6. The method of embodiment 5, wherein the target region include a virulence factor, a metabolic operon, or an acid resistance operon.7. The method of any of embodiments 4-6, wherein the sequencing includes amplifying the DNA.8. The method of embodiment 7, wherein the amplifying the DNA includes amplifying the target region of the DNA.The method of embodiments 7 or 8, wherein the amplifying the DNA includes polymerase chain reaction (PCR). The method of any of embodiments 6-9, wherein the virulence factor includes fap2, cmpA, or fusolisin. The method of embodiment 10, wherein presence of the virulence factor indicates that the Fna is Fna C2. The method of embodiment 10, wherein absence of the virulence factor indicates that the Fna is Fna 01. The method of any of embodiments 6-12, wherein the metabolic operon includes pdu or eut. The method of embodiment 13, wherein presence of pdu or eut indicates that the Fna is Fna C2. The method of any of embodiments 6-14, wherein the acid resistance operon includes gdar. The method of embodiment 15, wherein presence of gdar indicates that the Fna is Fna C2 The method of any of embodiments 4-16, wherein the sequencing includes detecting methyl-modified DNA motifs. The method of any of embodiments 4-17, wherein the sequencing the DNA includes bisulfite sequencing to detect methyl-modified DNA motifs. The method of embodiments 17 or 18, wherein the methyl-modified DNA motifs include GTNm6AC, GCm6AG, or Gm6ANTC. The method of embodiment 19, wherein presence of GTNm6AC or GCm6AG indicates that the Fna is Fna C1 The method of embodiment 19, wherein presence of Gm6ANTC indicates that the Fna is Fna C2. The method of any of embodiments 2-21, wherein the reference characteristic includes the growth condition. The method of embodiment 22, wherein the growth condition includes pH. The method of any of embodiments 1-23, wherein the method includes adjusting pH and measuring growth of the Fna. The method of embodiment 24, wherein higher growth at pH 9.5 to pH 10 compared to pH 5.5 to pH 8.5 indicates that the Fna is Fna C1. The method of embodiment 24, wherein higher growth at pH 5.5 to pH 8.5 compared to pH 9.5 to pH 10 indicates that the Fna is Fna C2. The method of any of embodiments 2-26, wherein the reference characteristic includes the dimension. The method of embodiment 27, wherein the method includes measuring the dimension. The method of embodiment 28, wherein the dimension is a length of 1-3.5 pm and the Fna is Fna C1 . The method of embodiment 28, wherein the dimension is a length of 3.6-11 pm and the Fna is Fna C2. A method of identifying Fna immunogenic peptides, the method including: co-culturing Fna with antigen presenting cells (APC); lysing Fna-infected APC to create a lysed sample, creating a lysate; collecting MHC or HLA molecules from the lysed sample; and sequencing the MHC or HLA molecules, thus identifying Fna immunogenic peptides. The method of embodiment 31 , wherein the Fna includes Fna C2.The method of embodiments 31 or 32, wherein the APC include macrophages, dendritic cells, or B cells. The method of any of embodiments 31-33, wherein the APC include macrophages. The method of embodiment 34, wherein the macrophages include THP1 cells. The method of any of embodiments 31-35, wherein the HLA molecules include HLA-I molecules. The method of any of embodiments 31-36, wherein the collecting includes purifying the lysate with pan-HLA antibody covalently bound to Protein-A Sepharose beads. The method of any of embodiments 31-37, further including determining the immunogenicity of the identified Fna immunogenic peptides. The method of embodiment 38, wherein the determining the immunogenicity of the identified Fna immunogenic peptides includes administering the Fna immunogenic peptides to immune cells and measuring CD44, CD25, CD69, TNFa, and / or IFNy. Use of an Fna immunogenic peptide identified according to the method of any of embodiments 31-39 as a vaccine antigen. The use of embodiment 40, wherein the use includes administering the vaccine antigen to a subject. Use of an Fna immunogenic peptide identified according to the method of any of embodiments 31-39 as an antigen to generate a binding domain. The use of embodiment 42, wherein the use includes administering the antigen to an animal to generate antibodies against the antigen. The use of embodiment 43, wherein the animal is a mouse or llama. The use of embodiments 43 or 44, further including isolating the generated antibodies. The use of embodiment 45, further including sequencing the isolated antibodies. The use of any of embodiments 42-46, wherein the use includes in silico modeling to generate antibodies against the antigen. The use of any of embodiments 42-47, wherein the use includes contacting the antigen with an immune cell and collecting immune cells that respond to antigen. The use of embodiment 48, wherein the immune cell is a T cell. The use of embodiments 48 or 49, wherein the immune cell is an antigen-reactive T cell. The use of any of embodiments 48-50, further including sequencing a T cell receptor that bound the antigen. A nucleic acid sequence encoding an immunogenic peptide identified by the method of any of embodiments 31- 39. The nucleic acid sequence of embodiment 52, wherein the nucleic acid sequence includes DNA or RNA. The nucleic acid sequence of embodiments 52 or 53, wherein the nucleic acid sequence includes cDNA. The nucleic acid sequence of embodiments 52 or 53, wherein the nucleic acid sequence includes RNA. The nucleic acid sequence of embodiment 55, wherein the RNA is synthetic RNA.The nucleic acid sequence of embodiment 56, wherein the synthetic RNA is in vitro-transcribed RNA. A cell expressing an immunogenic peptide identified by the method of any of embodiments 31-39. A mutant Fna C2 protein including at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the sequences as set forth in SEQ ID NOs: 120-146. The mutant Fna C2 protein of embodiment 59, wherein the nucleic acid encoding the mutant Fna C2 protein includes the nucleic acid of any of embodiments 52-57. The mutant Fna C2 protein of embodiments 59 or 60, wherein the mutant Fna C2 protein is expressed on a delivery scaffold. The mutant Fna C2 protein of embodiment 61 , wherein the delivery scaffold includes a scaffold protein, a viruslike particle, or a nanoparticle. A composition including (i) the mutant Fna C2 protein of any of embodiments 59-62 or a nucleotide encoding the mutant Fna C2 protein of any of embodiments 59-62 and ii) a pharmaceutically acceptable carrier. The composition of embodiment 63, further including an adjuvant. The composition of embodiment 64, wherein the adjuvant includes a TLR agonist, a squalene-based adjuvant, an alum-based adjuvant, a STING agonist, a carbomer-lecithin-based adjuvant, a small molecule immune stimulant, or a cytokine. The composition of embodiment 65, wherein the TLR agonist includes monophosphoryl lipid A (MPLA). The composition of embodiment 65, wherein the STING agonist includes c-AIMP; (3’,2’)c-AI MP; (2’,2’)c-AIMP; (2’,3’)c-AIMP; c-AIMP(S); c-(dAMP-dlMP); c-(dAMP-2’FdlMP); c-(2’FdAMP-2’Fdl MP); (2’,3’)c-(AMP-2’FdlMP); c- [2' FdAMP(S)-2’FdlMP(S)]; c-[2’FdAMP(S)-2’Fdl MP(S)](POM)2; or DMXAA. The composition of embodiment 65, wherein the small molecule immune stimulant includes a TGF-p inhibitor, a SHP-inhibitor, a STAT-3 inhibitor, or a STAT-5 inhibitor. The composition of embodiment 65, wherein the cytokine includes IL-2, IL-4, IL-10, IL-11 , IL-12, IL-15, IFN-a, IFN-p or lFN-y, or GM-CSF. A method of genetically modifying a cell to express a eut operon, a pdu operon, and / or a gdar operon, the method including contacting the cell with an artificial expression construct or series of artificial expression constructs including a sequence encoding a heterologous eut operon, a heterologous pdu operon, and / or a heterologous gdar operon. The method of embodiment 70, wherein the cell includes a bacterial cell. The method of embodiment 71 , wherein the bacterial cell includes a non-pathogenic bacterium. The method of embodiments 71 or 72, wherein the bacterial cell includes Fusobacterium nucleatum. The method of any of embodiments 71-73, wherein the bacterial cell includes Fusobacterium nucleatum subspecies animalis (Fna). The method of embodiment 74, wherein the Fna includes a Fna clade 1 (C1).The method of embodiment 74, wherein the Fna includes an attenuated Fna clade 2 (C2). The method of any of embodiments 70-76, wherein the artificial expression construct or series of artificial expression constructs include the sequence encoding the heterologous eut operon and the sequence encoding the heterologous pdu operon. The method of any of embodiments 70-77, wherein the artificial expression construct or series of artificial expression constructs include the sequence encoding the heterologous eut operon, the sequence encoding the heterologous pdu operon, and the sequence encoding the heterologous gdar operon. The method of any of embodiments 70-78, wherein the artificial expression construct includes a plasmid. The method of any of embodiments 70-79, wherein the method further includes providing a transposase. The method of embodiment 80, wherein the transposase includes a Tn5 transposase. The method of any of embodiments 70-81, wherein the contacting includes high-volume microfluidic electroporation. The method of any of embodiments 70-82, wherein the artificial expression construct or series of artificial expression constructs include a sequence encoding a payload. The method of embodiment 83, wherein the payload includes a therapeutic payload or a marker. The method of embodiment 84, wherein the therapeutic payload includes an antibody, a multi-domain binding molecule, or a therapeutic molecule. The method of embodiment 84, wherein the marker includes a fluorescent label. The method of any of embodiments 70-86, wherein the artificial expression construct or series of artificial expression constructs include a sequence encoding a transduction marker, tag cassette, selection cassette, or suicide switch. A method of treating a subject in need thereof, wherein the subject has a cancer associated with Fusobacterium nucleatum subsp. animalis (Fna) clade 2 (C2)including: administering a therapeutically effective amount of a cancer drug; and administering a therapeutically effective amount of a bacteria inhibitor, wherein the bacteria inhibitor destroys or suppresses growth or proliferation of the Fusobacterium nucleatum subsp. animalis (Fna) clade 2 (C2), thereby treating the subject in need thereof. The method of embodiment 88, further including diagnosing that the cancer is associated with Fusobacterium nucleatum subsp. animalis (Fna) clade 2 (C2). The method of embodiment 89, wherein the diagnosing includes detecting Fusobacterium nucleatum subsp. animalis (Fna) clade 2. The method of any of embodiments 88-90, wherein the bacteria inhibitor includes a Fusobacterium nucleatum inhibitor. The method of embodiment 91 , wherein the Fusobacterium nucleatum inhibitor includes bacterial 50S ribosomalsubunit inhibitor; bacterial DNA gyrase inhibitor; (17beta)-17-ethynyl-3-oxoestr-4-en-17-yl heptanoate; (1 E)-1-(2- hydroxy-5-methylphenyl)-1-dodecanone oxime; (9Z)-2-hydroxy-9-octadecenoic acid; 4-Methylumbelliferone (4- MU); 5-BDBD; 9(E), 11 (Z)-Octadecadienoic acid; acetylcholine receptor allosteric modulator; adenosine 5- monophosphate; adenylyl cyclase activator; AGN 194310; angiotensin receptor antagonist; ATM kinase inhibitor; bacterial DNA gyrase inhibitor; bacterial ribosomal inhibitor; BCL inhibitor; bromodomain inhibitor; candesartan cilexetil; carbonic anhydrase inhibitor; carboxamide antiepileptic; catechol 0 methyltransferase inhibitor; CC chemokine receptor antagonist; CDK inhibitor, breast cancer; chelating agent; clindamycin palmitate hydrochloride; cytochrome P450 inhibitor; daunorubicin HCI (Daunomycin HCI); EGFR inhibitor; emodin; epirubicin HCI; estrogen receptor antagonist; evan's blue; FOXM1 inhibitor, protein synthesis inhibitor; GABA receptor antagonist; gamma secretase modulator; gemcitabine (elaidate); gentian violet; glucagon receptor antagonist, autotaxin inhibitor; GW 3965 hydrochloride; GW4064; HDAC inhibitor; hypericin; idarubicin HCI; integrin inhibitor; JNK inhibitor; laxative; macrophage migration inhibiting factor inhibitor; matrix metalloprotease inhibitor; mitoxantrone hydrochloride; MK 0893; monoamine oxidase inhibitor; mTOR inhibitor; nadide; nemorubicin; neuropeptide receptor antagonist; NFkB pathway inhibitor; non-nucleoside reverse transcriptase inhibitor; NU7441 (KU-57788); obatoclax mesylate (GX15-070); otilonium bromide; PERK inhibitor; phosphofructokinase inhibitor; Piceatannol; pimonidazole; PLK inhibitor; polarization inhibitor; potassium channel activator; progestogen hormone; protein tyrosine kinase activator; PSB 06126; purinergic receptor antagonist; retinoid receptor antagonist; rifampin; RNA synthesis inhibitor; serine protease inhibitor; serine / threonine kinase inhibitor; serotonin receptor agonist; serotonin receptor antagonist; serotonin receptor inverse agonist; serum / glucocorticoid regulated kinase inhibitor; SHIP2 phosphatase inhibitor; siponimod; SIRT inhibitor; sortilin inhibitor; src inhibitor; sterol demethylase inhibitor; superoxide dismutase inhibitor; tachykinin antagonist; TC-09311 ; thyroid hormone receptor agonist; ticagrelor; tropomyosin receptor kinase inhibitor; ubiquitin-conjugating enzyme inhibitor; VEGFR inhibitor; WNT5a peptide mimetic; or ZLN005. The method of any of embodiments 88-92, wherein the cancer drug targets the G0-G1 phase of the cell cycle. The method of any of embodiments 88-93, wherein the cancer drug includes nitrosureas, asparaginase, or actinomycin D. The method of any of embodiments 88-92, wherein the cancer drug includes a phase-nonspecific drug. The method of embodiment 95, wherein the phase-nonspecific drug includes an alkylating agent, an antitumor antibiotic, or other drug. The method of embodiment 96, wherein the alkylating agent includes nitrogen mustard, nitrosourea, alkyl sulfonate, triazene, ethylenimine, methylmelamine, or a platinum compound. The method of any of embodiments 88-92, wherein the cancer drug targets cancer cells in the S phase of the cell cycle. The method of any of embodiments 88-98, wherein the cancer drug includes an antimetabolite, antifolate,antipyrimidine, antipurine, or topoisomerase inhibitor. The method of embodiment 99, wherein the antimetabolite includes a cladribine, cytarabine, 5-fluorouracil, hydroxyurea, methotrexate, pentostatin, or thiopurine. The method of embodiment 99, wherein the topoisomerase inhibitor includes etoposide, teniposide, irinotecan, or topotecan. The method of any of embodiments 88-101, wherein the cancer includes colorectal cancer (ORC) or oral squamous cell carcinoma (OSCC). A cell genetically modified to include a sequence encoding a heterologous eufoperon, a heterologous pdu operon, and / or a heterologous gdar operon. The cell of embodiment 103, wherein the cell includes a bacterial cell. The cell of embodiment 104, wherein the bacterial cell includes a non-pathogenic bacterium. The cell of embodiments 104 or 105, wherein the bacterial cell includes Fusobacterium nucleatum. The cell of any of embodiments 104-106, wherein the bacterial cell includes Fusobacterium nucleatum subspecies animalis {Fna). The cell of embodiment 107, wherein the Fna includes a Fna clade 1 (C1). The cell of embodiment 107, wherein the Fna includes an attenuated Fna clade 2 (C2). The cell of any of embodiments 103-109, wherein the cell includes the sequence encoding the heterologous eut operon and the heterologous pdu operon. The cell of any of embodiments 103-110, wherein the cell includes a the sequence encoding the heterologous gdar operon. The cell of any of embodiments 103-111, wherein the cell includes a the sequence encoding the heterologous eut operon, the heterologous pdu operon, and the heterologous gdar operon. The cell of any of embodiments 103-113, further including a sequence encoding a payload. The cell of embodiment 113, wherein the payload includes a therapeutic payload or a marker. The cell of embodiment 114, wherein the therapeutic payload includes an antibody, a multi-domain binding molecule, or a therapeutic molecule. The cell of embodiment 114, wherein the marker includes a fluorescent label. The cell of any of embodiments 103-116, further including a sequence encoding a transduction marker, tag cassette, selection cassette, or suicide switch. A binding molecule including a binding domain that binds a Fusobacterium nucleatum subsp. animalis (Fna)- associated antigen. The binding molecule of embodiment 118, wherein the Fna-associated antigen includes an Fna clade 2 (C2)- associated antigen. The binding molecule of embodiment 119, wherein the Fna C2-associated antigen includes fusobacterial apoptosisprotein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin. The binding molecule of any of embodiments 118-120, wherein the binding molecule includes a recombinant receptor, an antibody or antigen binding fragment, multi-domain binding molecule, or antibody conjugate. The binding molecule of any of embodiments 118-121, wherein the binding domain includes a single chain variable fragment (scFv). The binding molecule of any of embodiments 118-122, wherein the binding molecule is a recombinant receptor including, when expressed by a cell, an extracellular component including the binding domain that binds the Ana-associated antigen; an intracellular component including an effector domain; and a transmembrane domain. The binding molecule of embodiment 123, wherein the recombinant receptor includes a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof. The binding molecule of any of embodiments 123 or 124, wherein the recombinant receptor is a CAR. The binding molecule of any of embodiments 118-122, wherein the binding molecule is an antibody or antigen binding fragment thereof. The binding molecule of any of embodiments 118-121 , wherein the binding molecule includes a multi-domain binding molecule having at least a second binding domain. The binding molecule of embodiment 127, wherein the second binding domain binds an immune cell. The binding molecule of embodiments 127 or 128, wherein the second binding domain binds CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3. The binding molecule of any of embodiments 118-122, the binding molecule includes an antibody conjugate. The binding molecule of embodiment 130, wherein the antibody conjugate includes an immunotoxin, an antibodydrug conjugate, an antibody-detectable label conjugate, an antibody-radioisotope conjugate, or an antibodyparticle conjugate. The binding molecule of any of embodiments 118-131 , wherein the binding molecule includes a targeted therapeutic. A cell expressing the binding molecule of any of embodiments 118-132. A composition including the i) nucleic acid sequence of any of embodiments 52-57 or the binding molecule of any of embodiments 118-132 and ii) a pharmaceutically acceptable carrier. A formulation including the cell of embodiments 58 or 133 and ii) a pharmaceutically acceptable carrier. A method of detecting Fusobacterium nucleatum subsp. animalis (Fna) clade 2 including contacting the binding molecule of any of embodiments 118-132 with a sample and detecting the binding molecule, thereby detecting Fna C2.137. The method of embodiment 136, wherein the binding molecule includes an antibody or an antibody conjugate.138. The method of embodiments 136 or 137, wherein the binding molecule includes an antibody and the method further includes contacting the antibody with a secondary antibody conjugated to a detectable label.139. The method of embodiments 137 or 138, wherein the antibody conjugate includes an antibody-detectable label conjugate.140. A method of treating a subject in need thereof including administering a therapeutically effective amount of: the nucleic acid sequence of any of embodiments 52-57, the mutant Fna C2 protein of any of embodiments 59-62, the composition of any of embodiments 63-69 or 134, and / or the formulation of embodiment 135; thereby treating the subject in need thereof.141. The method of embodiment 140, wherein the subject in need thereof has cancer or an infection.142. The method of embodiment 141 , wherein the cancer includes colorectal cancer (CRC), or oral squamous cell carcinoma (OSCC).143. The method of embodiments 141 or 142, wherein the cancer includes CRC.144. The method of embodiments 141 or 142, wherein the cancer includes OSCC.145. The method of any of embodiments 141-144, wherein the infection includes infection with Fna clade 2.146. The method of any of embodiments 140-145, wherein the method stimulates an immune response in the subject in need thereof.147. The method of any of embodiments 140-146, wherein the treating reduces or eliminates Fna C2 bacteria and / or symptoms of Fna C2 bacteria.148. The method of any of embodiments 140-147, wherein the formulation includes a cell that is capable of invading a tumor site.149. The method of embodiment 148, wherein the cell invades environments in which cells express phosphatidylethanolamine (PE), ethanolamine (EA), or 1 ,2-propanediol (1 ,2-PD).150. The method of embodiments 148 or 149, wherein the cell invades environments with high acidity.151. The method of any of embodiments 140-150, wherein the administering includes intravenous, intraarterial, intraperitoneal, intrarectal, intratumoral, intravesicular, and / or oral administration.

[0341] (xiii) Examples.

[0342] Example 1 . A distinct Fusobacterium nucleatum clade dominates the colorectal cancer tumor niche. Abstract. Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal (Gl) tract of healthy individuals (Segata, et al., Genome Biol. 13, R42 (2012) ("Segata 2012”)), is enriched in human colorectal cancer (CRC) tumors (Kostic, et al., Genome Res. 22, 292-298 (2012) (“Kostic 2012”); Kostic, et al., Cell Host Microbe 14, 207-215 (2013) (“Kostic 2013”); Flanagan, et al., Eur. J. Clin. Microbiol. Infect. Dis.33, 1381-1390 (2014); and Mima, et al., Gut 65, 1973-1980 (2016) (“Mima 2016”)). High intratumoral Fn loads are associated with recurrence, metastases, and poorer patient prognosis (Mima 2016; Bullman 2017; Yu, et al., Cell 170, 548-563.e16 (2017) (“Yu 2017”); and Serna, etal., Ann. Oncol. 31 , 1366-1375 (2020)). To delineate Fn genetic factors facilitating tumor-colonization, closed genomes for 135 Fn strains were generated; 80 oral strains from non-cancer individuals and 55 unique cancer strains cultured from 51 CRC patient tumors. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumor-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, Fna C2 (over Fna C1) dominates the tumor niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the Gl tract. Supporting this, Fna C2-treated mice had increased intestinal adenomas and altered metabolites. Microbiome analysis of human tumor tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that Fna C2 is tumor-enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy controls. Collectively, results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC, and reveal the genetic underpinnings of Fna C2's tumor pathoadaptation.

[0343] Introduction. Patients with CRC tumors harboring high levels of Fn have poorer survival (Mima 2016). Fusobacterlum colonizes regions of patient tumors with immune and epithelial cell functions supportive of cancer progression (Galeano Nino 2022), it persists in metastatic disease (Bullman 2017), and microbiome modulation targeting Fn could change the course of this disease (Bullman 2017; and LaCourse, et al., Cell Rep. 41, 111625 (2022) (“LaCourse 2022”)). Moreover, exogenous Fn infection in animal and cellular models has supported a cancerpromoting role for this bacterium (Bullman 2017; LaCourse 2021; Galeano Nino 2022; LaCourse 2022; Allen-Vercoe, et al., Gut Microbes 2, 294-298 (2011) (“Allen-Vercoe 2011”); McGuire, et al., mBio 5, e01864-14 (2014) (“McGuire 2014”); and Holt & Cochrane, Gastroenterology 152, 694-696 (2017) (“Holt & Cochrane 2017”). However, considerable strain-to-strain variation in Fn genotypic and phenotypic features has been described (Allen-Vercoe 2011 ; McGuire 2014; Holt & Cochrane 2017; Ponath, et al., Proc. Natl. Acad. Sci. 119, e2201460119 (2022); and Queen, et al., MBio 2021 Feb 22131e0299121 13, 17 (2022) (“Queen 2022”)). Such heterogeneity has raised challenges with reproducing Fn cancer-inducing phenotypes in animal and cellular models (Queen 2022; and Tomkovich, et al., Cancer Res. 77, 2620-2632 (2017) (“Tomkovich 2017”)) and it has been suggested that a select group of Fn strains may possess carcinogenic abilities (Tomkovich 2017).

[0344] Leveraging a comprehensive collection of human CRC Fn strains and performing extensive comparative genomics, a select clade within Fn subspecies was revealed to predominate the CRC niche. In vitro and in vivo functional studies demonstrate that this clade is highly virulent in the context of CRC Subsequent mechanistic studies of Fn pathogenicity in CRC can be useful and may be helpful in the development of targeted inhibitors.

[0345] Niche-enriched Fn genes and subspecies. Classical microbiology culture approaches have reemerged asvaluable tools to functionally assess members of tissue-associated microbiomes. Fusobacterium isolation was performed and cultured from 130 human colorectal cancer (CRC) tumors from which 65 Fusobacterium CRC- associated strains were obtained from 59 unique patients. Given that Fn is predominantly an oral pathobiont, 81 Fusobacterium strains isolated from the oral cavity of non-cancer individuals were included, as a control group. These oral strains were obtained from the American Type Culture Collection (ATCC) and the Korean Collection for Oral Microbiology (KCOM) repositories. Using PacBio long-read single-molecule real-time (SMRT) (Eid, et al., Sci. 2009 Jan 23235910133-8323, 7 (2009)) sequences for complete and closed genomes were generated, with corresponding epigenetic methylomes, for 146 unique Fusobacterium strains (FIGs. 1A and 2), 92% of which belonged to the species Fusobacterium nucleatum {Fn) (n=135; n=55 CRC-associated and n=80 oral-associated) (FIG. 1 B). As Fn is the most frequently detected species in CRC tumors (Kostic 2012; and Kostic 2013), analysis was therefore focused on a comparison of these 55 CRC-associated and 80 oral-associated Fn genomes.

[0346] Given that Fn strains in human CRC tumors are predicted to originate from the human oral cavity (Komiya, et al., Gut 68, 1335-1337 (2019); and Abed, etal., Infect. Microbiol. 10, 400 (2020) ("Abed 2020”)), but are rare members of the lower gastrointestinal (Gl) tract microbiota of non-cancer patients (Segata 2012), it was suggested that CRC- associated Fn strains harbor an additional genetic repertoire to facilitate their colonization in human CRC tumors. To test this, 135 Fn genomes were examined using the analysis and visualization platform for (omics data (Anvi’o) workflow for microbial pangenomics (Eren, et al., PeerJ 3, e1319 (2015)). Pangenomic analysis identifies all genes present in a species (“pangenome’’) and discerns between gene content conserved amongst most members (“core genome”) and gene content shared amongst subsets of members (“accessory genomes”) (Tettelin, et al., Proc Natl Acad Sci U 2005 Sep 271023913950-5 7 (2005); and The Pangenome: Diversity, Dynamics and Evolution of Genomes, Springer International Publishing, 2020). It was observed that accessory genome size increases as the number of sampled Fn genomes increases, supportive of previous proposals that Fn has an open pangenome (Ang, etal., Genome Biol. Evol. 8, 2928-2938 (2016)) (FIG. 3A). To account for uneven sampling (Horesh, et al., Microb. Genomics 7, (2021)) between CRC (n=55) and oral (n=80) genomes, the analysis was subset based on niche and found that CRC-associated Fn strains have a smaller accessory genome as compared to oral-associated strains (FIGs. 1C, 2, and 3B). Functional enrichment analysis (Shaiber, et al., Genome Biol. 21 , 292 (2020)) identified 483 and 241 gene clusters significantly enriched (q<0.05) in CRC and oral strains, respectively (FIG. 1D). KEGG ortholog analysis (Aramaki, et al., Bioinformatics 36, 2251-2252 (2020)) of the 724 niche-enriched gene clusters revealed that mapped gene clusters (31 .2%) were predominantly involved in putative metabolic functions and pathways (FIG. 1 E).

[0347] Previously published studies using orthogonal approaches have observed a differential distribution of Fn subspecies in tumor tissue and mucosal biopsy specimens from CRC patients (Borozan, et al., Cancer Epidemiol. Biomarkers Prev. 31, 210-220 (2022) (“Borozan 2022”); and Strauss, et al. Inflamm. Bowel Dis. 17, 1971-1978 (2011) (“Strauss 2011”)). As there is sufficient genetic heterogeneity between the four Fn subspecies that reclassification into separate species has been proposed (Kook, ef al., Curr. Microbiol. 74, 1137-1147 (2017)), the resolution of theanalyses was increased to the subspecies level (animalis (Fna), nucleatum (Fnn), polymorphum (Fnp) and vincentii / fusiforme (Fnv)) (see Methods of Example 1). It is notable that studies seeking to delineate the contributions of Fn in CRC predominately use the model strains Fnn ATCC 25586 (Yu 2017), an oral isolate, and Fnn ATCC 23726 (Abed 2020; and Abed, et al., Cell Host Microbe 20, 215-225 (2016) ("Abed 2016”)), a urogenital isolate, and Fna 7_1 (Strauss 2011), an inflammatory bowel disease patient isolate. Here, analysis of the proportion of Fn subspecies by niche found that of the four Fn subspecies, Fna is the subspecies significantly associated with the CRC niche (two sample Z test, two-tailed, p=0.0232) (FIG. 1 F), validating previous studies (Borozan 2022; and Strauss 2011), while Fnn is instead significantly enriched in the oral niche (two sample Z test, two-tailed, p=0.00932) (FIG. 1 F). Thus, the repertoire of genetic factors associated with colonization or virulence in the CRC niche would not be fully represented in Fnn model strains and indeed show that Fnn ATCC 25586 only contains 17.60% of CRC-enriched gene clusters. This supports the use of Fna strains for mechanistic studies, such as Fna 7_1 , which has previously been shown to induce colonic tumors in murine models (Kostic 2013). Interestingly, comparison of Fn subspecies pangenomes showed that Fna has the smallest core genome as compared to other subspecies, suggestive of further unresolved Fna genetic heterogeneity (FIG. 3C).

[0348] Since Fna is enriched in the CRC tumor niche, it was tested whether Fn virulence factors previously described as important for host colonization are more prevalent in Fna as compared to other Fn subspecies. Fn type VA autotransporter virulence factors include Fusolisin (Bachrach, et al., Oral Microbiol. Immunol. 19, 155-159 (2004)), a serine protease that damages host tissue and inactivates immune effectors; FplA (Casasanta, et al., J. Biol. Chem. 292, 20240-20254 (2017)), a phospholipase autotransporter that binds to host phosphoinosite-signaling lipids; and such as Fap2 (Coppenhagen-Glazer, et al., Immun. 83, 1104-1113 (2015)), Aim1 (Kaplan, et al., J. Dent. Res. 84, 700-704 (2005)), RadD (Kaplan, et al., Mol. Microbiol. 71, 35-47 (2009)), and CmpA (Lima, et al., Microbiology Open 6, e00444 (2017)). Since the role of fplA, aim1, radD and cmpA in CRC remains unclear, to ensure a comprehensive analysis the presence of these virulence factors was queried across the Fn genome collection. An additional adhesin, FadA (Han, et al., J. Bacteriol. 187, 5330-5340 (2005) ("Han 2005”); and Xu, et al., J. Biol. Chem. 282, 25000-25009 (2007) ("Xu 2007”)), mediates Fn attachment to and invasion of host epithelial cells (Fardini, et al., Mol. Microbiol. 82, 1468-1480 (2011); and Rubinstein, et al., Cell Host Microbe 14, 195-206 (2013)), with two additional FadA homologs recently identified (Umana, et al., J. Bacteriol. 201 , e00273-19 (2019) ("Umana 2019”)). Previously analyzed fadA distribution in a limited number of Fusobacterium genomes has suggested fadA absence from passively invading species and increased incidence in highly invasive species (Manson McGuire, et al., mBio 5, e01864-14 (2014) (“Manson McGuire 2014”)), although this distribution does not always coincide with in vivo invasion assays (Han 2005; Gursoy, et al., J. Periodontol. 81 , 1084-1091 (2010); Manson McGuire 2014; Umana 2019). Intriguingly, it was found that although fplA and fadA are well conserved in Fn, their nucleotide and amino acid sequences segregate by Fn subspecies, perhaps indicative of variable interactions with host-ligands (FIGs. 3D, 3E). The results show that none of these canonical virulence factors are significantly associated with Fna as compared to other Fn subspecies, suggestingthat additional unknown genetic factors facilitate the enrichment of Fna in CRC (FIG. 3F). However, it was observed that a subset of Fna strains lacked fap2, cmpA, and fusolisin and, by rpoB gene analysis, these Fna strains appeared to form a distinct Fna clade (FIG. 1 G).

[0349] An Fna clade enriched in the CRC niche. To further examine the observation that Fna strains form two distinct clades, phylogenetic trees of housekeeping genes previously used for Fn subspecies typing (Borozan 2022; and Kim, et al., J. Clin. Microbiol. 48, 545-553 (2010)) were compared. Analysis of these single-marker genes supported the observation that two distinct Fna clades exist, which are called Fna Clade 1 Fna C1) and Fna Clade 2 {Fna C2) (FIG. 4A). Beyond these single-marker genes, genome-wide differences between Fna clades are supported by a kSNP (Gardner, et al., Bioinformatics 31, 2877-2878 (2015) ("Gardner 2015”) reference-free whole-genome phylogeny (FIG. 5A). To quantify the relatedness of Fna C1 to Fna C2, the average nucleotide identity (ANI), a well-established index that measures the percent similarity between genomes, were compared with an established 95% species threshold (Richter & Rossello-Mora, Proc. Natl. Acad. Sci. 106, 19126-19131 (2009)). Between Fna clades the ANI ranged from 91.61 % to 93.11%, comparable to the ANI between other Fn subspecies (FIGs. 5B, 18M). Further, the patterns of protein-coding genes present across Fna genomes were visualized using the Genes in Genomes-Map (GiG-map) tool and found that Fna C1 and Fna C2 have distinct protein-coding gene content (FIG. 5C). This was further supported by principal component analysis (PCA) of Anvi'o gene cluster presence / absence (FIG. 5D). Notably, the frequently used Fna 7_1 strain groups with Fna C2 (FIG. 4B).

[0350] Therefore, the genetic, epigenetic, and ecological properties of Fna were reassessed as two genetically distinct clades. Comparison of the Fna clade pangenomes showed that Fna C1 and Fna C2 had similar core genome sizes, while Fna C2 had a larger accessory genome (FIGs. 4C, 4D), suggesting that Fna C2 strains harbor additional genetic factors which may be beneficial during colonization of CRC tumors. Consistent with this, comparisons of individual genome size and content indicated that Fna C2 strains have significantly larger chromosome sizes (Welch's T-test, two-tailed, p<0.00001) (FIG. 4E), more extrachromosomal plasmids (FIG. 2), and a greater number of innate genetic defenses and mobile genetic elements (FIG. 4F) as compared to Fna C1 strains. PCA analysis of Fna methylomes indicated that Fna C1 and Fna C2 are also epigenetically distinct (FIG. 5E). The methyl-modified DNA motifs most influencing this epigenetic bifurcation are GTNm6AC (100% Fna C1 , 0% Fna C2), GCm6AG (100% Fna C1 , 0% Fna C2), and Gm6ANTC (0% Fna C1 , 63% Fna C2) (FIG. 5E). Surprisingly, while both Fna clades are present in the oral cavity with non-significant differences, Fna C2 (not Fna C1) is significantly associated with the CRC niche (two sample Z test, two-tailed, p<0.00001) (FIG. 5F). This observation was further tested on publicly available 16S rRNA gene sequencing data from paired tumor tissue and saliva samples from patients with CRC (Russo, et al., Neoplasia 40, 100901 (2023)). In order to gain resolution to the Fna clade level, Fna clade-specific amplicon sequence variants (ASVs) (Callahan, et al., ISME J. 11 , 2639-2643 (2017)) were identified (see Methods of Example 1, FIG. 13). Supportive of the observations, Fna C2 is significantly enriched in tumor samples as compared to Fna C1 (T-test, paired, p=0.047) (FIG. 4G). However, there was no statistically significant difference between Fna clades in paired oralsamples, indicating that while both Fna clades are present in the oral cavity of patients with CRC, Fna C2 is enriched in the tumor niche (FIG. 4G).

[0351] Human lower Gl tract Fna C2 enrichment. Pangenome analysis revealed that Fna is composed of two distinct clades but only Fna C2 is enriched in the CRC niche. Low levels of Fna C1 in this niche could be due to poor tumor colonization potential, an inherent lack of virulence or tumor-supportive factors that are possessed by Fna C2, or that Fna C1 is unable to evade immune clearance. To interrogate these possibilities and reveal Fna clade-specific genetic factors, a comprehensive inter-Fna clade comparative analysis was applied across all 75 Fna genomes (24 Fna C1, 51 Fna C2). Interestingly, canonical Fn virulence factors including adhesins radD, aim1, and fadA2 were significantly enriched in Fna C1 as compared to Fna C2 (two sample Z test, two-tailed, p<0.00001) (FIGs. 1 G, 6A) suggesting their role may be particularly important in the oral cavity. Conversely, as noted, fap2, cmpA, and fusolisin are absent from Fna C1 and significantly associated with Fna C2 (two sample Z test, two-tailed, p<0.00001) (FIGs. 1G, 6A). Given the reported epithelial (Abed 2016) and immune cell (Gur, et al., Immunity 42, 344-355 (2015)) interactions of Fap2 in CRC, its association with Fna C2 supports its increased adherence and invasion potential in this niche. Co-culture of Fna strains from each clade with a human colon cancer cell line (HCT 116) demonstrate that Fna C2 has significantly higher cancer epithelial cell invasion compared to Fna C1 strains (FIGs. 6B, 7A, and 7B) (Welch's T-test, two-tailed, p=0.0113), indicative of differential invasion potential and / or aerotolerance of individual strains (FIG. 7C). Further, Fna clades are morphologically distinct whereby Fna C2 cells are significantly longer (Fna C1 : 2.01 pim average, Fna C2: 5.26 pim average) and thinner (Fna C1 : 0.39 pim average, Fna C2: 0.33 pim average) than Fna C1 cells (FIG. 7D) (Welch’s T-test, two-tailed, p<0.00001 length, p<0.00001 width). As bacterial morphology can affect colonization of host niches and susceptibility to host defenses (Yang, et al., Microbiol. Mol. Biol. Rev. 80, 187-203 (2016)), physical differences between Fna clade cells are noteworthy.

[0352] Delineation of Fna clade-unique genome content could reveal hitherto unknown genetic factors enabling Fna C2 transit to and survival within the human colonic niche. Predominant genetic differences between Fna clades are consistent with Fna C2 having increased nutrient scavenging mechanisms and enhanced metabolic potential (FIG 7E). As functionally related bacterial genes often form co-regulated units (operons), the Partitioned PanGenome Graph of Linked Neighbors tool (PPanGGOLiN) (Gautreau, et al., PLOS Comput. Biol. 16, e1007732 (2020)) was implemented to assess if Fna clade-unique genetic factors formed putative operons (FIG. 6C). Consistent with Anvi'o analysis, Fna C2 syntenic blocks were predominantly associated with metabolic mechanisms (FIG. 9). Thus, pathoadaptation of Fna C2 to the CRC niche is multifactorial, and in addition to canonical Fn virulence factors, is likely facilitated by enhanced metabolic capabilities.

[0353] To validate the inter-Fna clade pangenomic approach, two Fna C2-associated putative operons consistent with ethanolamine (EA) metabolism (euf) and 1 ,2-propanediol (1,2-PD) metabolism (pdu) (FIG. 6D) were focused on. These operons include 20% of Fna C2-unique gene content (FIG. 9). Enteric pathogens not only gain a competitive advantage through direct metabolism of EA and 1 ,2-PD, but also exploit their intestinal specificity. Sensing through eutand pdu activates global regulators of virulence and induces transcriptional profiles consistent with Gl niche adaptation (Pacheco & Sperandio, Microbiol. Spectr 3, 3.3.09 (2015)). Analysis of stool metagenomic datasets from publicly available cohorts of patients with CRC (n=627) and healthy controls (n=619) indicates that eut and pdu operons are significantly enriched in patients with CRC (two sample Z test, two-tailed, eut p<0.00001 , pdu p<0.00001) (FIG. 8A).

[0354] Motivated by the conservation of eut and pdu in Fna C2, and their absence in Fna C1, global transcriptomic responses of Fna cells were assessed upon exposure to these intestinal-associated metabolites. RNA sequencing of representative Fna C1 and Fna C2 strains after exposure to EA or 1,2-PD indicated that both Fna clades have significant transcriptomic changes (FIGs. 8B-8D). As Fna C1 is deficient in both eut and pdu, significant transcriptomic changes upon exposure to EA / 1 ,2-PD in Fna C1 would be independent of these operons. Thus, through a subtractive approach, differentially expressed genes (T-test, two-tailed, p<0.05) in Fna C2 cells were focused on that were not differentially expressed (T-test, two-tailed, p>0.05) in Fna C1 cells. The results demonstrate that in Fna C2, eut and pdu genes are transcriptionally upregulated in response to EA / 1.2-PD, respectively (FIGs. 6E-6F).

[0355] Furthermore, Fna C2 cells exposed to EA or 1 ,2-PD significantly upregulated 13.02% of Fna C2-associated genes, including canonical Fn virulence factors. Although present in both clades, radD and aim1 are upregulated in the presence of EA in Fna C2 but not Fna C1 cells (FIG. 6E). Virulence factors uniquely present in Fna C2 are additionally upregulated when Fna C2 cells are exposed to EA cmpA, fusolisin, fap2) or 1 ,2-PD (fap2) (FIG. 6E, 6F). The upregulation of Fna C2-associated genes and virulence factors known to be involved in interactions with human epithelial cells suggests that upon transit to the human Gl tract, sensing of these molecules could induce Fna C2 transcriptional profiles consistent with extra-oral niche adaptation.

[0356] This also led to reconsideration of how Fna C2 might be translocating to extra-oral tumor niches. Prior studies suggest that oral Fusobacteria travel to CRC tumors through the bloodstream during transient bacteremia caused by activities such as daily hygiene practices or dental procedures (Abed 2020). The identification of transcriptionally active eut / pdu operons suggests that direct descent through the Gl tract, with subsequent infiltration of CRC tumors via the lumen, may be an additional pathway used by Fna C2. Yet, for Gl transit to be a viable route of dissemination, Fna C2 would need to overcome the deleterious effects of extreme acid stress encountered in the stomach (pH 1.5 - 3.5) (FIG. 18B) Assessing preferential growth pH, both Fna clades are sensitive to pH below 4.5 (FIG 18C). From pH 5.5-8.5, Fna C2 strains had significantly higher growth activity as compared to Fna C1, with maximum growth activity at pH 7 (FIG. 18C). Under basic conditions (pH 9.5-10), Fna C1 strains had significantly higher growth activity as compared to Fna C2, with maximum growth activity at pH 10 (FIG. 18C). Pangenome analysis also revealed a putative glutamatedependent acid resistance (GDAR) system conserved across all Fna C2, but absent in Fna C1 (FIGs. 9, 18D). The GDAR system, found in pathogenic and commensal gut bacteria, is one of the most potent acid resistance mechanisms (Biase & Pennacchietti, Mol. Microbiol. (2012)), with glutamate being the component for the system to operate at pH < 3 (FIG. 18E). Using a colorimetric pH change assay, the conversion of glutamine through glutamate into y-aminobutyric acid (GABA) was tested in the presence of Fna C1 and Fna C2 and found that this conversion is significantly higher inthe presence of Fna 02 strains (FIG. 18F, 18G).

[0357] To further mimic effects of pH stress during gastric transit, Fna clades were exposed to simulated gastric fluid (SGF) at pH 3. Both were non-viable after 10 min of SGF exposure. However, in the presence of supplemented glutamate, Fna C2 survived for an extended period (60 mins), which was not observed for Fna C1 lacking GDAR (FIG. 18H). Analysis of stool metagenomic datasets indicated that gdar operons are significantly enriched in patients with CRC as compared to healthy controls (two sample Z test, two-tailed, p<0.00001) (FIG. 181). Thus, in addition to active eut and pdu systems, differences in pH preference and acid resistance mechanisms may contribute to Fna C2’s ability to access the Gl and tumor niches.

[0358] Fna C2 impacts intestinal tumorigenesis. As Fna 02 enriched gene clusters were predominantly associated with enhanced metabolic potential (FIGs. 7E, 9), a goal was to determine if Fna treatment of the dextran sodium sulfate (DSS) induced colitis ApcMin+Amurine model (Tanaka, et al., Int. J. Cancer 118, 25-34 (2006)) of CRC impacted intestinal tumorigenesis and metabolic pathways in vivo (FIG. 10A). To capture a higher proportion of Fna clade-specific accessory genes (FIG. 4D) a mix of three representative strains for each clade was used. Following a single oral gavage of either Fna C1 mix, Fna C2 mix, or vehicle control, a significant increase in intestinal adenomas in Fna C2- treated mice compared to both Fna C1 and vehicle control independently was observed (FIG. 10B) (ANOVA, p=0.0065 and p=0.0069, respectively), particularly in the large intestine (FIGs. 18J-18K) (ANOVA, p=0.0070 and p=0.0009, respectively). There was no significant difference in adenoma burden between Fna C1 treatment and vehicle control mice. Low level Fn was inconsistently detected during the course of the study (FIG. 18L). Liquid chromatographymass spectrometry (LC-MS) global metabolomics was performed on intestinal tissue from each treatment arm for comparative metabolite analysis. Partial least squares-discriminant analysis (PLS-DA) of measured intestinal metabolites demonstrated that Fna C2-treated mice formed a distinct cluster away from other treatment arms, suggesting a differential metabolic profile. However, intestinal metabolites from Fna C1 -treated and vehicle control mice had more similar metabolite profiles, clustering together (FIG. 10C).

[0359] Of 1 ,296 metabolites measured (FIGs. 11 A-11 C), comparative analysis demonstrated a significant enrichment in glutathione metabolism and gamma-glutamyl amino acid pathways in Fna C2 treated mice, as compared to both Fna C1 treated and vehicle control mice (FIGs. 11A-11D, 12) Specifically, a significant increase in the precursors to GSH synthesis, including cysteine and gamma-glutamylcysteine, decreased levels of glutathione in its reduced form (gamma-glutamyl-cysteinyl-glycine; GSH), and significantly higher levels of the GSH degradation product 5-oxoproline (FIGs. 11 E, 12) were observed. Consistent with gamma-glutamyl amino acid generation from reduced glutathione in the presence of gamma-glutamyl transpeptidase (GGT) increased levels of gamma-glutamyl amino acids and cysteine, glycine and cystei ny l-glyci ne levels (FIG. 11 E, 11 F) were also observed. GSH deficiency or an elevated ratio of oxidized (GSSG) to reduced (GSH) forms of glutathione increases the vulnerability of mammalian cells to oxidative stress, inflammation, and tumor progression (Kennedy, et al., Biomolecules 10, 1429 (2020)). The GSSG:GSH ratio of Fna C2-treated mice significantly increased by 3.5- and 3.0-fold compared to the control and Fna C1-treated groupsrespectively, suggesting increased oxidative stress (FIG. 11G) (one-way ANOVA, p=0.0031 and p=0.0047, respectively). Studies have demonstrated that metabolism of GSH by GGT can exert pro-oxidant effects. In cancer cells, this is a source of endogenous reactive oxygen species which can facilitate persistent oxidative stress and contribute to genomic instability (Pompella, et al., Biochem. Pharmacol. 71 , 231-238 (2006); and Hanigan, Advances in Cancer Research vol. 122 103-141 (Elsevier, 2014)). Consistent with this, significantly increased levels of other markers of oxidative stress were observed including cystine and cysteine-glutathione disulfide and significantly decreased levels of polyamines capable of scavenging reactive oxygen species, including putrescine, spermidine and spermine in Fna C2 treated mice (FIGs. 11 E, 12). Interestingly, recent work demonstrated that Fusobacterium was predominantly associated with epithelial cells harboring severe chromosomal abnormalities (Galeano Nino 2022), one of the most common forms of genomic instability in cancer.

[0360] In addition to their role in combating oxidative stress, polyamines are able to suppress inflammation through inhibition of macrophage cytokine synthesis (Rao, et al., Physiology 35, 328-337 (2020)). Consistent with increased inflammation, significantly higher levels of N-monomethylarginine and dimethylarginine in Fna C2 treated mice as compared to other treatment arms (FIG. 11 E) were observed. Both of these metabolites inhibit the synthesis of the anti-inflammatory agent nitric oxide. Furthermore, significantly higher levels of pro-inflammatory prostaglandins and ceramides were observed, including prostaglandin A2, N-palmitoyl-sphingosine and N-palmitoyl-sphingadienine (FIG. 11 E). Ceramides can also be metabolized by cancer cells to reduce tumor cell apoptosis and proliferation (Morad & Cabot, Nat. Rev. Cancer 13, 51-65 (2013)). Other metabolites that promote cancer cell proliferation and metastasis across a range of cancers include eicosanoids, which are similarly significantly increased in Fna C2 treated mice as compared to Fna C1 treated or vehicle control mice (FIGs. 11 A, 11 B, 11 H). This included increased levels of 6-keto prostaglandin F1 -alpha (6k-PGF1a) (FIG. 11E) via COX-2 metabolism of arachidonic acid. Interestingly, COX-2 (PTGS2) was previously reported to be one of the most upregulated genes in Fn-associated human colorectal tumors (Kostic 2013). Overall, the results demonstrate the ability of Fna C2, but not Fna C1 , to metabolically impact the intestinal milieu towards pro-oncogenic conditions.

[0361] Fna C2 enrichment in human CRC cohorts. As Fna C2 strains are both significantly enriched in the CRC niche (FIG. 5F) and increase intestinal tumorigenesis in the mouse model compared to Fna C1 (FIGs 10B, 18J-18K), the prevalence and abundance of these Fna clades in human tissue and stool specimens through culture-independent approaches were next determined. Bacterial 16S rRNA gene sequencing was performed on resected tumor tissue from 116 patients with treatment-naive colorectal cancer (CRC Cohort 1) and on adjacent normal tissue from 62 of these patients. Comparing the percentage relative abundance of different Fusobacterium species between paired tumor and adjacent normal tissue (n=62 patients), Fn was the Fusobacterium species significantly enriched in tumor tissue compared to adjacent normal (FIG. 14A), supportive of previous reports (Kostic 2012; and Castellarin, et al., Genome Res. 22, 299-306 (2012) ("Castellarin 2012”)) (T-test, paired, p=0.0022). However, using the Fna clade-specific ASV's to resolve Fn to a higher taxonomic resolution that includes Fna C1 , Fna C2 and non-Fna subspecies of Fn, only FnaC2 is significantly enriched in tumor compared to paired normal tissue (FIGs. 13, 14A) (T-test, paired, p=0.0093). As neither Fna C1 nor non-Fna subspecies of Fn are significantly enriched, this suggests that it is specifically Fna C2 that is driving the previously reported enrichment of Fn in human CRC tumors. Furthermore, across two independent patient cohorts (CRC Cohort 1 n=116 and CRC Cohort 2 n=86) it is demonstrated that within CRC tumor tissue, Fna C2 is significantly enriched compared to Fna C1 (FIG. 14B) (T-test, paired, Cohort 1 p=0.0009, Cohort 2 p=0.0014), supporting the observations at the Fna strain-level (FIG. 5F).

[0362] To determine if the prevalence of Fna clades differed between patients with CRC and healthy controls, stool metagenomic datasets were analyzed from publicly available cohorts of patients with CRC (n=627) and healthy controls (n=619) (FIGs. 15A-15K). Fna was detected in 29.2% of stool samples from patients with CRC and 4.8% of stool samples from healthy controls. Meta-analysis of standardized mean differences by random effects model for Fna C1 and Fna C2 demonstrated that both Fna clades have a significant pooled effect size associated with CRC (Fna C1 effect size=0.21 , 95% confidence interval (0.09,0.32), p=4.45e-04; Fna C2 effect size=0.45, 95% confidence interval (0.34, 0.56), p=5.55e-15) (FIGs. 14C, 16, 17). However, the effect size for Fna C2 was larger than for Fna C1. Importantly, in the absence of Fna C2 co-occurrence, Fna C1 was not significantly associated with CRC (FIGs. 14C, 16, 18A). Although synergistic interactions between CRC-enriched microbes have been previously reported (Dejea, et al., Science 359, 592-597 (2018)), it is not clear whether Fna C1 co-occurrence with Fna C2 results in a compounding pathogenic effect. Similar to the observation in CRC tumor tissue, Fna C2 is more prevalent and abundant in the stool of patients with CRC than Fna C1 (FIGs. 14D, 15A-15K) and is significantly enriched in the stool of patients with CRC as compared to healthy controls (FIG. 15A). These culture-independent human specimen analyses support the strainlevel genomic discovery that Fna C2 is the dominant CRC-associated Fna clade (FIGs. 5A-5F). This further highlights the significance of the in vitro (FIGs. 6A-6F) and in vivo (FIGs. 10A-10C) findings demonstrating the increased virulence and tumorigenic potential of Fna C2 as compared to Fna C1.

[0363] Discussion. Through large-scale culturing, sequencing, and comparative genomic analyses of human CRC and non-cancer oral Fn strains, the distinct CRC-enriched genetic factors of Fn were revealed. Further, it was identified that these CRC-enriched factors were predominantly present within a specific clade of Fn subsp. animalis (Fna). This was mirrored by the discovery that Fna is bifurcated into two distinct clades; Fna C1 which is largely restricted to the oral cavity, and Fna C2 which dominates the human CRC tumor niche. Intrigui ngly, Fna C2 induced tumors and altered intestinal metabolism towards increased oxidative stress within a CRC animal model. Further, comparative genomic analysis between Fna clades revealed the genetic elements that cumulatively engender Fna C2’s pathoadaptation to the CRC niche. Given the power of using Fna C1 as a comparative group for Fna C2, an interactive website was created to enable the exploration of Fna pangenomic datasets designated ‘The Fusobacterium Panqenome Atlas (Fusopangea)' at fredhutch.github.io / fusopangea / . Collectively this work demonstrates, in the context of CRC, that Fna C2 is a highly virulent subgroup of Fn.

[0364] Methods of Example 1. Fusobacterium strain isolation from colorectal cancer (CRC) patient tumor tissue.Fusobacterium strains from colorectal cancer (CRC) patient tumor tissue specimens from North America and Europe were isolated as previously described (Bullman 2017). Briefly, tissue sections were minced with a scalpel, and spread plated on selective fastidious anaerobe agar (FAA) plates (Oxoid, ThermoFisher Scientific) supplemented with 7% or 10% defibrinated horse blood (Lampire Biological Laboratories, Fisher Scientific) with josamycin, vancomycin, and norfloxacin at 3, 4, and 1 .g / ml respectively (Sigma Aldrich). Plates were incubated at 37°C in anaerobic conditions (AnaeroGen Gas Generating Systems, Oxoid, ThermoFisher Scientific) and inspected for growth every two days. Colonies were picked and streak-purified and colony PCR was carried out on selected bacterial colonies as previously described (Bullman 2017) with 16S rRNA gene universal primers (342F and 1492R). Colony PCR products were sent for Sanger sequencing and BLASTn analysis of trace sequences was used to confirm bacterial species identity. Cultures were suspended in tryptic soy broth and 40% glycerol and stored at -80°C.

[0365] Fusobacterium strain isolation from KCOM and ATCC ampoules. Fusobacterium strains from the Korean Collection for Oral Microbiology (KCOM) collection were isolated from the oral cavity as previously described44. Strains from the American Type Culture Collection (ATCC) and the KCOM repositories were grown from ampoules on 1) Schaedler agar plates supplemented with Vitamin K1 and 5% defibrinated sheep blood (Becton Dickinson) and 2) fastidious anaerobe agar (FAA) plates (Oxoid, ThermoFisher Scientific) supplemented with 7% defibrinated horse blood (Lampire Biological Laboratories, Fisher Scientific). Plates were incubated at 37°C in a Bactron600 anaerobic chamber (Sheldon Manufacturing) for 5-7 days. Cultures were suspended in Schaedler broth with Vitamin K1 and 30% glycerol and stored at -80°C.

[0366] High-molec...

Claims

CLAIMSWhat is claimed is:1 . A method for identifying a Fusobacterium nucleatum subspecies animalis (Fna) as an Fna clade 1 (01) or an Fna clade 2 (02), the method comprising: obtaining a sample containing Fna; quantifying fap2, cmpA, or fusolisin within the sample; and comparing a quantity of fap2, cmpA, or fusolisin to a threshold quantity, wherein the quantity of fap2, cmpA, or fusolisin above the threshold quantity indicates that the Fna is Fna C2, thereby identifying the Fna as Fna C1 or Fna 02.

2. A method for identifying a Fusobacterium nucleatum subspecies animalis (Fna) as an Fna clade 1 (01) or an Fna clade 2 (02), the method comprising: determining a characteristic of the Fna; and comparing the characteristic to a reference characteristic that identifies the Fna as Fna C1 or Fna 02, thereby identifying the Fna as Fna C1 or Fna 023. The method of claim 2, wherein the reference characteristic comprises a coding sequence, an operon, DNA methylation, a growth condition, or a dimension.

4. The method of claim 3, wherein the reference characteristic comprises the coding sequence, the operon, or DNA methylation.

5. The method of claim 4, wherein the method further comprises sequencing DNA of the Fna for presence of the reference characteristic.

6. The method of claim 5, wherein the reference characteristic is within a target region of the DNA.

7. The method of claim 6, wherein the target region comprise a virulence factor, a metabolic operon, or an acid resistance operon.

8. The method of claim 5, wherein the sequencing comprises amplifying the DNA.

9. The method of claim 8, wherein the amplifying the DNA comprises amplifying the target region of the DNA.10 The method of claim 8, wherein the amplifying the DNA comprises polymerase chain reaction (PCR).

11. The method of claim 7, wherein the virulence factor comprises fap2, cmpA, or fusolisin.

12. The method of claim 11, wherein presence of the virulence factor indicates that the Fna is Fna C2.

13. The method of claim 11, wherein absence of the virulence factor indicates that the Fna is Fna C1.

14. The method of claim 7, wherein the metabolic operon comprises pdu or eut.

15. The method of claim 14, wherein presence of pdu or eut indicates that the Fna is Fna 02.16 The method of claim 7, wherein the acid resistance operon comprises gdar17. The method of claim 16, wherein presence of gdar indicates that the Fna is Fna C2.

18. The method of claim 5, wherein the sequencing comprises detecting methyl-modified DNA motifs.

19. The method of claim 5, wherein the sequencing the DNA comprises bisulfite sequencing to detect methyl-modified DNA motifs.

20. The method of claims 18 or 19, wherein the methyl-modified DNA motifs comprise GTNm6AC, GCm6AG, or Gm6ANTC.

21. The method of claim 20, wherein presence of GTNm6AC or GCm6AG indicates that the Fna is Fna C1 .

22. The method of claim 20, wherein presence of Gm6ANTC indicates that the Fna is Fna C2.

23. The method of claim 3, wherein the reference characteristic comprises the growth condition.24 The method of claim 23, wherein the growth condition comprises pH25. The method of claim 2, wherein the method comprises adjusting pH and measuring growth of the Fna.

26. The method of claim 25, wherein higher growth at pH 9.5 to pH 10 compared to pH 5.5 to pH 8.5 indicates that the Fna is Fna C1.

27. The method of claim 25, wherein higher growth at pH 5.5 to pH 8.5 compared to pH 9.5 to pH 10 indicates that the Fna is Fna C2.28 The method of claim 3, wherein the reference characteristic comprises the dimension.

29. The method of claim 28, wherein the method comprises measuring the dimension.

30. The method of claim 29, wherein the dimension is a length of 1-3.5 pm and the Fna is Fna C1 .

31. The method of claim 29, wherein the dimension is a length of 3.6-11 pm and the Fna is Fna C2.

32. A method of identifying Fna immunogenic peptides, the method comprising: co-culturing Fna with antigen presenting cells (APC); lysing Fna-infected APC to create a lysed sample, creating a lysate; collecting MHC or HLA molecules from the lysed sample; and sequencing the MHC or HLA molecules, thus identifying Fna immunogenic peptides.

33. The method of claim 32, wherein the Fna comprises Fna C2.

34. The method of claim 32, wherein the APC comprise macrophages, dendritic cells, or B cells.35 The method of claim 32, wherein the APC comprise macrophages36. The method of claim 35, wherein the macrophages comprise THP1 cells.

37. The method of claim 32, wherein the HLA molecules comprise HLA-I molecules.

38. The method of claim 32, wherein the collecting comprises purifying the lysate with pan-HLA antibody covalently bound to Protein-A Sepharose beads.

39. The method of claim 32, further comprising determining the immunogenicity of the identified Fna immunogenic peptides.

40. The method of claim 39, wherein the determining the immunogenicity of the identified Fna immunogenic peptides comprises administering the Fna immunogenic peptides to immune cells and measuring CD44, CD25, CD69,TNFo, and / or IFNy.41 Use of an Fna immunogenic peptide identified according to the method of claim 32 as a vaccine antigen.

42. The use of claim 41 , wherein the use comprises administering the vaccine antigen to a subject.

43. Use of an Fna immunogenic peptide identified according to the method of claim 32 as an antigen to generate a binding domain.

44. The use of claim 43, wherein the use comprises administering the antigen to an animal to generate antibodies against the antigen.45 The use of claim 44, wherein the animal is a mouse or llama.

46. The use of claim 44, further comprising isolating the generated antibodies.

47. The use of claim 46, further comprising sequencing the isolated antibodies.

48. The use of claim 43, wherein the use comprises in silico modeling to generate antibodies against the antigen.

49. The use of claim 43, wherein the use comprises contacting the antigen with an immune cell and collecting immune cells that respond to antigen.50 The use of claim 49, wherein the immune cell is a T cell.

51. The use of claim 49, wherein the immune cell is an antigen-reactive T cell.

52. The use of claim 49, further comprising sequencing a T cell receptor that bound the antigen.

53. A nucleic acid sequence encoding an immunogenic peptide identified by the method of claim 32.

54. The nucleic acid sequence of claim 53, wherein the nucleic acid sequence comprises DNA or RNA.

55. The nucleic acid sequence of claim 53, wherein the nucleic acid sequence comprises cDNA.56 The nucleic acid sequence of claim 53, wherein the nucleic acid sequence comprises RNA.

57. The nucleic acid sequence of claim 56, wherein the RNA is synthetic RNA.

58. The nucleic acid sequence of claim 57, wherein the synthetic RNA is in vitro-transcribed RNA.

59. A cell expressing an immunogenic peptide identified by the method of claim 32.

60. A mutant Fna C2 protein comprising at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to any of the sequences as set forth in SEQ ID NOs: 120-146.61 The mutant Fna C2 protein of claim 60, wherein a nucleic acid encoding the mutant Fna C2 protein comprises the nucleic acid of claim 53.

62. The mutant Fna C2 protein of claim 60, wherein the mutant Fna C2 protein is expressed on a delivery scaffold.

63. The mutant Fna C2 protein of claim 62, wherein the delivery scaffold comprises a scaffold protein, a virus-like particle, or a nanoparticle.

64. A composition comprising (i) the mutant Fna C2 protein of claim 60 or a nucleotide encoding the mutant Fna 02 protein of claim 60 and ii) a pharmaceutically acceptable carrier.

65. The composition of claim 64, further comprising an adjuvant.

66. The composition of claim 65, wherein the adjuvant comprises a TLR agonist, a squalene-based adjuvant, analum-based adjuvant, a STING agonist, a carbomer-lecithin-based adjuvant, a small molecule immune stimulant, or a cytokine.

67. The composition of claim 66, wherein the TLR agonist comprises monophosphoryl lipid A (MPLA).

68. The composition of claim 66, wherein the STING agonist comprises c-AIMP; (3’,2’)c-AI MP; (2',2')c-AI MP; (2’,3’)c-AIMP; c-AIMP(S); c-(dAMP-dlMP); c-(dAMP-2’FdlMP); c-(2’FdAMP-2’Fdl MP); (2’,3’)c-(AMP-2’FdlMP); c- [2’ FdAMP(S)-2’FdlMP(S)]; c-[2’FdAMP(S)-2’Fdl MP(S)](POM)2; or DMXAA.

69. The composition of claim 66, wherein the small molecule immune stimulant comprises a TGF-P inhibitor, a SHP- inhibitor, a STAT-3 inhibitor, or a STAT-5 inhibitor.

70. The composition of claim 66, wherein the cytokine comprises IL-2, IL-4, IL-10, IL-11 , IL-12, IL-15, IFN-a, IFN-p or lFN-y, or GM-CSF.

71. A method of genetically modifying a cell to express a eut operon, a pdu operon, and / or a gdar operon, the method comprising contacting the cell with an artificial expression construct or series of artificial expression constructs comprising a sequence encoding a heterologous eut operon, a heterologous pdu operon, and / or a heterologous gdar operon.

72. The method of claim 71, wherein the cell comprises a bacterial cell.

73. The method of claim 72, wherein the bacterial cell comprises a non-pathogenic bacterium.

74. The method of claim 72, wherein the bacterial cell comprises Fusobacterium nucleatum.

75. The method of claim 72, wherein the bacterial cell comprises Fusobacterium nucleatum subspecies animalis (Fna).

76. The method of claim 75, wherein the Fna comprises a Fna clade 1 (C1 ).77 The method of claim 75, wherein the Fna comprises an attenuated Fna clade 2 (C2).

78. The method of claim 71, wherein the artificial expression construct or series of artificial expression constructs comprise the sequence encoding the heterologous eut operon and the sequence encoding the heterologous pdu operon.

79. The method of claim 71, wherein the artificial expression construct or series of artificial expression constructs comprise the sequence encoding the heterologous eut operon, the sequence encoding the heterologous pdu operon, and the sequence encoding the heterologous gdar operon.

80. The method of claim 71, wherein the artificial expression construct comprises a plasmid.

81. The method of claim 71 , wherein the method further comprises providing a transposase.

82. The method of claim 81, wherein the transposase comprises a Tn5 transposase.

83. The method of claim 71, wherein the contacting comprises high-volume microfluidic electroporation.

84. The method of claim 71, wherein the artificial expression construct or series of artificial expression constructs comprise a sequence encoding a payload.

85. The method of claim 84, wherein the payload comprises a therapeutic payload or a marker.

86. The method of claim 85, wherein the therapeutic payload comprises an antibody, a multi-domain binding molecule,or a therapeutic molecule.87 The method of claim 85, wherein the marker comprises a fluorescent label.

88. The method of claim 71, wherein the artificial expression construct or series of artificial expression constructs comprise a sequence encoding a transduction marker, tag cassette, selection cassette, or suicide switch.

89. A method of treating a subject in need thereof, wherein the subject has a cancer associated with Fusobacterium nucleatum subsp. animalis (Fna) clade 2 (C2)comprising: administering a therapeutically effective amount of a cancer drug; and administering a therapeutically effective amount of a bacteria inhibitor, wherein the bacteria inhibitor destroys or suppresses growth or proliferation of the Fusobacterium nucleatum subsp. animalis (Fna) clade 2 (C2), thereby treating the subject in need thereof.

90. The method of claim 89, further comprising diagnosing that the cancer is associated with Fusobacterium nucleatum subsp. animalis Fna) clade 2 (02).

91. The method of claim 90, wherein the diagnosing comprises detecting Fusobacterium nucleatum subsp. animalis (Fna) clade 292. The method of claim 89, wherein the bacteria inhibitor comprises a Fusobacterium nucleatum inhibitor.

93. The method of claim 92, wherein the Fusobacterium nucleatum inhibitor comprises bacterial 50S ribosomal subunit inhibitor; bacterial DNA gyrase inhibitor; (17beta)-17-ethynyl-3-oxoestr-4-en-17-yl heptanoate; (1 E)-1 -(2-hydroxy- 5-methylphenyl)-1-dodecanone oxime; (9Z)-2-hydroxy-9-octadecenoic acid; 4-Methylumbelliferone (4-MU); 5- BDBD; 9(E), 11 (Z)-Octadecadienoic acid; acetylcholine receptor allosteric modulator; adenosine 5- monophosphate; adenylyl cyclase activator; AGN 194310; angiotensin receptor antagonist; ATM kinase inhibitor; bacterial DNA gyrase inhibitor; bacterial ribosomal inhibitor; BCL inhibitor; bromodomain inhibitor; candesartan cilexetil; carbonic anhydrase inhibitor; carboxamide antiepileptic; catechol 0 methyltransferase inhibitor; CC chemokine receptor antagonist; CDK inhibitor, breast cancer; chelating agent; clindamycin palmitate hydrochloride; cytochrome P450 inhibitor; daunorubicin HCI (Daunomycin HOI); EGFR inhibitor; emodin; epirubicin HCI; estrogen receptor antagonist; evan's blue; FOXM1 inhibitor, protein synthesis inhibitor; GABA receptor antagonist; gamma secretase modulator; gemcitabine (elaidate); gentian violet; glucagon receptor antagonist, autotaxin inhibitor; GW 3965 hydrochloride; GW4064; HDAC inhibitor; hypericin; idarubicin HCI; integrin inhibitor; JNK inhibitor; laxative; macrophage migration inhibiting factor inhibitor; matrix metalloprotease inhibitor; mitoxantrone hydrochloride; MK 0893; monoamine oxidase inhibitor; mTOR inhibitor; nadide; nemorubicin; neuropeptide receptor antagonist; NFkB pathway inhibitor; non-nucleoside reverse transcriptase inhibitor; NU7441 (KU-57788); obatoclax mesylate (GX15-070); otilonium bromide; PERK inhibitor; phosphofructokinase inhibitor; Piceatannol; pimonidazole; PLK inhibitor; polarization inhibitor; potassium channel activator; progestogen hormone; protein tyrosine kinase activator; PSB 06126; purinergic receptor antagonist; retinoid receptor antagonist; rifampin; RNA synthesis inhibitor; serine protease inhibitor; serine / threonine kinase inhibitor; serotoninreceptor agonist; serotonin receptor antagonist; serotonin receptor inverse agonist; serum / glucocorticoid regulated kinase inhibitor; SHIP2 phosphatase inhibitor; siponimod; SIRT inhibitor; sortilin inhibitor; src inhibitor; sterol demethylase inhibitor; superoxide dismutase inhibitor; tachykinin antagonist; TC-09311 ; thyroid hormone receptor agonist; ticagrelor; tropomyosin receptor kinase inhibitor; ubiquitin-conjugating enzyme inhibitor; VEGFR inhibitor; WNT5a peptide mimetic; or ZLN005.

94. The method of claim 89, wherein the cancer drug targets the G0-G1 phase of the cell cycle.

95. The method of claim 89, wherein the cancer drug comprises nitrosureas, asparaginase, or actinomycin D.96 The method of claim 89, wherein the cancer drug comprises a phase-nonspecific drug.

97. The method of claim 96, wherein the phase-nonspecific drug comprises an alkylating agent, an antitumor antibiotic, or other drug.

98. The method of claim 97, wherein the alkylating agent comprises nitrogen mustard, nitrosourea, alkyl sulfonate, triazene, ethylenimine, methylmelamine, or a platinum compound.

99. The method of claim 89, wherein the cancer drug targets cancer cells in the S phase of the cell cycle.

100. The method of claim 89, wherein the cancer drug comprises an antimetabolite, antifolate, antipyrimidine, antipurine, or topoisomerase inhibitor.

101. The method of claim 100, wherein the antimetabolite comprises a cladribine, cytarabine, 5-fluorouracil, hydroxyurea, methotrexate, pentostatin, or thiopurine.

102. The method of claim 100, wherein the topoisomerase inhibitor comprises etoposide, teniposide, irinotecan, or topotecan.

103. The method of claim 89, wherein the cancer comprises colorectal cancer (CRC) or oral squamous cell carcinoma (OSCC).104.A cell genetically modified to comprise a sequence encoding a heterologous eut operon, a heterologous pdu operon, and / or a heterologous gdar operon.

105. The cell of claim 104, wherein the cell comprises a bacterial cell.

106. The cell of claim 105, wherein the bacterial cell comprises a non-pathogenic bacterium.

107. The cell of claim 105, wherein the bacterial cell comprises Fusobacterium nucleatum.

108. The cell of claim 105, wherein the bacterial cell comprises Fusobacterium nucleatum subspecies animalis (Fna).

109. The cell of claim 108, wherein the Fna comprises a Fna clade 1 (C1).

110. The cell of claim 108, wherein the Fna comprises an attenuated Fna clade 2 (C2).

111. The cell of claim 104, wherein the cell comprises the sequence encoding the heterologous eut operon and the heterologous pdu operon.

112. The cell of claim 104, wherein the cell comprises a the sequence encoding the heterologous gdar operon.

113. The cell of claim 104, wherein the cell comprises a the sequence encoding the heterologous eut operon, the heterologous pdu operon, and the heterologous gdar operon.

114. The cell of claim 104, further comprising a sequence encoding a payload.

115. The cell of claim 114, wherein the payload comprises a therapeutic payload or a marker.

116. The cell of claim 115, wherein the therapeutic payload comprises an antibody, a multi-domain binding molecule, or a therapeutic molecule.

117. The cell of claim 115, wherein the marker comprises a fluorescent label.

118. The cell of claim 104, further comprising a sequence encoding a transduction marker, tag cassette, selection cassette, or suicide switch.

119. A binding molecule comprising a binding domain that binds a Fusobacterium nucleatum subsp. animalis (Fna)- associated antigen.

120. The binding molecule of claim 119, wherein the Ana-associated antigen comprises an Fna clade 2 (C2)-associated antigen.

121. The binding molecule of claim 120, wherein the Fna C2-associated antigen comprises fusobacterial apoptosis protein-2 (fap2), coaggregation mediating protein A (cmpA), or fusolisin.

122. The binding molecule of claim 119, wherein the binding molecule comprises a recombinant receptor, an antibody or antigen binding fragment, multi-domain binding molecule, or antibody conjugate.

123. The binding molecule of claim 119, wherein the binding domain comprises a single chain variable fragment (scFv).

124. The binding molecule of claim 119, wherein the binding molecule is a recombinant receptor comprising, when expressed by a cell, an extracellular component comprising the binding domain that binds the Fna-associated antigen; an intracellular component comprising an effector domain; and a transmembrane domain.

125. The binding molecule of claim 124, wherein the recombinant receptor comprises a chimeric antigen receptor (CAR), an engineered T cell receptor (eTCR), or a hybrid thereof.

126. The binding molecule of claim 124, wherein the recombinant receptor is a CAR.

127. The binding molecule of claim 119, wherein the binding molecule is an antibody or antigen binding fragment thereof.128.The binding molecule of claim 119, wherein the binding molecule comprises a multi-domain binding molecule having at least a second binding domain.

129. The binding molecule of claim 128, wherein the second binding domain binds an immune cell.

130. The binding molecule of claim 128, wherein the second binding domain binds CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, 4-1 BB (CD137), 0X40, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, or B7-H3131. The binding molecule of claim 119, the binding molecule comprises an antibody conjugate.

132. The binding molecule of claim 131 , wherein the antibody conjugate comprises an immunotoxin, an antibody-drugconjugate, an antibody-detectable label conjugate, an antibody-radioisotope conjugate, or an antibody-particle conjugate.

133. The binding molecule of claim 119, wherein the binding molecule comprises a targeted therapeutic.

134. A cell expressing the binding molecule of claim 119.

135. A composition comprising the i) nucleic acid sequence of claim 53 or the binding molecule of claim 119 and ii) a pharmaceutically acceptable carrier.

136. A formulation comprising the cell of claims 59 or 134 and ii) a pharmaceutically acceptable carrier.

137. A method of detecting Fusobacterium nucleatum subsp. animalis (Fna) clade 2 comprising contacting the binding molecule of claim 119 with a sample and detecting the binding molecule, thereby detecting Fna C2.

138. The method of claim 137, wherein the binding molecule comprises an antibody or an antibody conjugate.

139. The method of claim 137, wherein the binding molecule comprises an antibody and the method further comprises contacting the antibody with a secondary antibody conjugated to a detectable label.

140. The method of claim 138, wherein the antibody conjugate comprises an antibody-detectable label conjugate.

141. A method of treating a subject in need thereof comprising administering a therapeutically effective amount of: the nucleic acid sequence of claim 53, the mutant Fna C2 protein of claim 60, the composition of claims 64 or 135, and / or the formulation of claim 136; thereby treating the subject in need thereof.

142. The method of claim 141 , wherein the subject in need thereof has cancer or an infection143. The method of claim 142, wherein the cancer comprises colorectal cancer (CRC), or oral squamous cell carcinoma (OSCC).

144. The method of claim 142, wherein the cancer comprises CRC.

145. The method of claim 142, wherein the cancer comprises OSCC.

146. The method of claim 142, wherein the infection comprises infection with Fna clade 2.

147. The method of claim 141 , wherein the method stimulates an immune response in the subject in need thereof.

148. The method of claim 141, wherein the treating reduces or eliminates Fna C2 bacteria and / or symptoms of Fna C2 bacteria.

149. The method of claim 141, wherein the formulation comprises a cell that is capable of invading a tumor site.

150. The method of claim 149, wherein the cell invades environments in which cells express phosphatidylethanolamine (PE), ethanolamine (EA), or 1 ,2-propanediol (1,2-PD).

151. The method of claim 149, wherein the cell invades environments with high acidity.

152. The method of claim 141 , wherein the administering comprises intravenous, intraarterial, intraperitoneal, intrarectal, intratumoral, intravesicular, and / or oral administration.

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