EP4665387A2 - Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins - Google Patents

Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins

Info

Publication number
EP4665387A2
EP4665387A2 EP24757619.2A EP24757619A EP4665387A2 EP 4665387 A2 EP4665387 A2 EP 4665387A2 EP 24757619 A EP24757619 A EP 24757619A EP 4665387 A2 EP4665387 A2 EP 4665387A2
Authority
EP
European Patent Office
Prior art keywords
peptides
redn
cancer
bump
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24757619.2A
Other languages
German (de)
English (en)
Inventor
Stephen Albert Johnston
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Calviri Inc
Original Assignee
Calviri Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Calviri Inc filed Critical Calviri Inc
Publication of EP4665387A2 publication Critical patent/EP4665387A2/fr
Pending legal-status Critical Current

Links

Classifications

    • G01N33/57585—
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • sequence listing is provided as a file entitled “2024-02- 12_SequenceListing-CALV037WO.xml”, created February 12, 2024, which is 229,957,632 bytes in size.
  • the information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
  • the present disclosure relates to the field of cancer diagnostics, therapeutics, and vaccines. More particularly, the present disclosure relates to cancer diagnostics, therapeutics, and vaccines related to RNA error derived neoantigen (REDN) peptides.
  • REDN RNA error derived neoantigen
  • Embodiments provided herein relate to RNA error derived neoantigen (REDN) peptides and methods of using such REDN peptides in diagnostics, therapeutics, and vaccines against cancer.
  • the disclosure relates to vaccine compositions, therapeutic compositions, methods of diagnosis, and methods of treatment using REDN peptides disclosed herein.
  • the REDN peptides provided herein are tryptophan (W) bump REDN peptides (W-bump REDN peptides).
  • W-bump REDN peptides tryptophan
  • Embodiments provided herein relate to REDNs produced at the RNA level used as a rich source for diagnostics, therapeutics, and vaccines.
  • compositions included a plurality of RNA error derived neoantigen (REDN) peptides.
  • the plurality of REDN peptides include tryptophan bump REDN peptides.
  • the composition is formulated as a vaccine.
  • the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart.
  • the plurality of REDN peptides include one or more peptides having a sequence or part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the compositions further includes an adjuvant.
  • the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide
  • the peptide arrays include a plurality of RNA error derived neoantigen (REDN) peptides.
  • the plurality of REDN peptides include tryptophan bump FSPs.
  • the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart.
  • the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296. In some embodiments, the plurality of REDN peptides is fixed on a substrate.
  • the substrate includes glass, silica, composite, resin, or combination thereof.
  • the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.
  • the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.
  • the plurality of REDN peptides include two or more pooled REDN peptides.
  • the REDN peptides are spaced between about 3 and about 9 pm apart.
  • the array is used to predict a response to an immunotherapy, to predict adverse responses to immunotherapy, to diagnose a cancer, to develop a vaccine, or to develop a therapeutic.
  • the array is used to detect binding of one or more antibodies against W-bump REDN peptides.
  • the therapeutic compounds bind to one or more W-bump REDN peptides.
  • the therapeutic compound is an antibody or a synthetic antibody.
  • Some embodiments provided herein relate to methods of treating or preventing a disorder in a subject.
  • the methods include administering a composition as a vaccine.
  • the composition is any composition as described herein.
  • the subject is a mammal.
  • the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.
  • the disorder is a cancer.
  • the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pit
  • Some embodiments described herein relate to methods of detecting a disorder in a subject.
  • the methods include: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides include tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.
  • the disorder is a cancer.
  • the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons are less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.
  • the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • the plurality of REDN peptides is fixed on a substrate.
  • the substrate include glass, silica, composite, resin, or combination thereof.
  • the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio- Layer Interferometry, and surface plasmon resonance.
  • the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.
  • the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.
  • the biological sample includes an antibody.
  • the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.
  • the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T- cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pit
  • the plurality of REDN peptides include two or more pooled REDN peptides.
  • the detecting the binding of the biological sample to the at least one peptide in the peptide array includes fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, or surface plasmon resonance assay.
  • Some embodiments provided herein relate to methods of measuring an immune response to a neoantigen peptide in a subject.
  • the methods include: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides including tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.
  • REDN RNA error derived neoantigen
  • the plurality of REDN peptides include peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons. In some embodiments, the two closely spaced tryptophan codons arc less than 8 codons apart. In some embodiments, the plurality of REDN peptides include one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361. In some embodiments, the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.
  • the plurality of REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • the plurality of REDN peptides is fixed on a substrate.
  • the substrate includes glass, silica, composite, resin, or combination thereof.
  • the peptide array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance.
  • the peptide array includes between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.
  • the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.
  • the biological sample includes an antibody.
  • the subject is a mammal.
  • the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a pig.
  • the subject has or is suspected of having a cancer.
  • the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pit
  • Some embodiments provided herein relate to methods of predicting a response of a subject to an immunotherapy.
  • the methods include (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides include tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern prior to immunotherapy.
  • REDN RNA error derived neoantigen
  • Some embodiments provided herein relate to methods of predicting adverse immune responses of a subject to an immunotherapy.
  • the methods include (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein: the plurality of REDN peptides include peptides including tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern indicative of adverse immune responses.
  • REDN RNA error derived neoantigen
  • Some embodiments provided herein relate to methods of treating a disorder in a subject.
  • the methods include screening therapeutic compounds that bind to one or more W-bump REDN peptides; and administering a therapeutic compound that binds to one or more W-bump REDN peptides to the subject.
  • the therapeutic compound is an antibody or a synthetic antibody.
  • Figure 1 depicts a comparison of normal translation of mRNA across tryptophan codons versus the situation in a tumor.
  • interferon gamma leads to uncharged tryptophan codons leading to a RNA error derived neoantigen .
  • Figure 2 depicts screening of predicted REDN peptides that can result from mis-translation at W-bumps, which have potential in diagnostic, therapeutic, and vaccines.
  • Figure 3 illustrates a table depicting W-bump REDN coverage of an initial cohort of cohort of dogs diagnosed with pre-stage 1, stage 1 or stage 2 hemangiosarcoma (HSA) cancer.
  • HSA hemangiosarcoma
  • a phrase in the form “A/B” or in the form “A and/or B” means (A), (B), or (A and B).
  • a phrase in the form “at least one of A, B, and C” means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C).
  • a phrase in the form “(A)B” means (B) or (AB) that is, A is an optional element.
  • the term “array” refers to an arrangement of molecules, such as biological macromolecules (such as peptides), in addressable locations on or in a substrate.
  • a “microarray” is an array that is miniaturized so as to require or be aided by microscopic examination for evaluation or analysis.
  • the array of molecules (“features”) makes it possible to carry out an exceptionally large number of analyses on a sample at one time.
  • each arrayed sample is addressable, in that its location can be reliably and consistently determined within at least two dimensions of the array.
  • the feature application location on an array can assume different shapes.
  • the array can be regular (such as arranged in uniform rows and columns) or irregular.
  • ordered arrays the location of each sample is assigned to the sample at the time when it is applied to the array, and a key may be provided in order to correlate each location with the appropriate target or feature position.
  • ordered arrays are arranged in a symmetrical grid pattern, but samples can be arranged in other patterns (such as in radially distributed lines, spiral lines, or ordered clusters).
  • Addressable arrays usually are computer readable, in that a computer can be programmed to correlate a particular address on the array with information about the sample at that position (such as hybridization or binding data, including for instance signal intensity).
  • the subject features in the array are arranged regularly, for instance in a Cartesian grid pattern, which can be correlated to address information by a computer.
  • the methods provided herein involve multiplexed arrays in which a plurality of peptides or polypeptides attached to a solid support are contacted to a biological sample (for example, blood or other bodily tissue obtained from a subject).
  • the peptide array is a plurality of short linear peptides immobilized on a solid surface (for example, a polystyrene or other solid substrate).
  • a solid surface for example, a polystyrene or other solid substrate.
  • peptide and polypeptide refer to a polymer in which the monomers are alpha amino acids joined together through amide bonds. Peptides are two or often more amino acid monomers long. Standard abbreviations for amino acids are used herein (see Stryer, 1988, Biochemistry, Third Ed., incorporated herein by reference). In certain embodiments, random- sequence peptide arrays are used.
  • substrate refers to any type of solid support to which the peptides are immobilized.
  • substrates include, but are not limited to, microarrays; beads; columns; optical fibers; wipes; nitrocellulose; nylon; glass; quartz; diazotized membranes (paper or nylon); silicones; polyformaldehyde; cellulose; cellulose acetate; paper; ceramics; metals; metalloids; semiconductive materials; coated beads; magnetic particles; plastics such as polyethylene, polypropylene, and polystyrene; gel-forming materials; silicates; agarose; polyacrylamides; methylmethacrylate polymers; sol gels; porous polymer hydrogels; nanostructured surfaces; nanotubes (such as carbon nanotubes); and nanoparticles (such as gold nanoparticles or quantum dots).
  • the peptides When bound to a substrate, the peptides can be directly linked to the support, or attached to the surface via a linker.
  • the solid substrate and/or the peptides can be derivatized using methods known in the art to facilitate binding of the peptides to the solid support, so long as the dcrivatization docs not eliminate detection of binding between the peptides and antibodies in the sera.
  • in-situ synthesis refers to synthesis of peptides or polypeptides in situ on an array. This could be done with photoactivatable amino acids as done by Nimble Therapeutics (maskless photolithography), PEPperPRINT, a standard mask-based system (much like Intel uses to lay down circuits), BOC or FMOC peptide chemistry, or other synthesis methods known in the art.
  • the term “detect,” “detection,” “detectable,” or “detecting” is understood both on a quantitative and a qualitative level, as well as a combination thereof. It thus includes quantitative, semi-quantitative, and qualitative measurements of measuring a cancer, using the methods and compositions as disclosed herein.
  • the term “mammal” includes both human and non-human mammals.
  • the term “subject” includes both human and veterinary subjects, including dogs.
  • a subject in need thereof means a human or non-human mammal that exhibits one or more symptoms or indications of cancer, , and/or who has been diagnosed with cancer.
  • a cancer may include a solid tumor and treatment for the same.
  • the term “subject” may be interchangeably used with the term “patient”.
  • a human subject may be diagnosed with a primary or a metastatic tumor and/or with one or more symptoms or indications including, but not limited to, unexplained weight loss, general weakness, persistent fatigue, loss of appetite, fever, night sweats, bone pain, shortness of breath, swollen abdomen, chest pain/pressure, enlargement of spleen, and elevation in the level of a cancer-related biomarker.
  • malignancy refers to a non-benign tumor or a cancer.
  • cancer refers to the broad class of disorders characterized by hyperproliferative cell growth, either in vitro (for example, transformed cells) or in vivo.
  • Cancers appropriate for treatment with checkpoint inhibitor therapy include without limitation a variety of neoplasms, including benign or malignant tumors, a variety of hyperplasias, and the like.
  • Exemplary cancers include carcinomas, sarcomas, leukemias, and lymphomas.
  • Cancer includes primary malignant tumors (for example, those whose cells have not migrated to sites in the subject’s body other than the site of the original tumor) and secondary malignant tumors (for example, those arising from metastasis, the migration of tumor cells to secondary sites that arc different from the site of the original tumor).
  • primary malignant tumors for example, those whose cells have not migrated to sites in the subject’s body other than the site of the original tumor
  • secondary malignant tumors for example, those arising from metastasis, the migration of tumor cells to secondary sites that arc different from the site of the original tumor.
  • Non-limiting examples of cancer may include gastric, myeloid, colon, nasopharyngeal, esophageal, and prostate tumors, glioma, neuroblastoma, breast cancer, lung cancer, ovarian cancer, colorectal cancer, thyroid cancer, leukemia (for example, adult T-cell leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, myelogenous leukemia, lymphocytic leukemia, acute myelogenous leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), T-lineage acute lymphoblastic leukemia or T-ALL chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), hairy cell leukemia), lymphoma (Hodgkin’s lymphoma (HL), non-Hodgkin’s lymphoma (NHL)), multiple myelo
  • a frameshift mutation is a mutation causing a change in the frame of the protein.
  • a frameshift variant peptide is a peptide in which a frame has changed due to a frameshift mutation.
  • RNA error derived neoantigen refers to a neoantigen that results from an RNA error, for example a frameshift, RNA mis-processing (including RNA mis- splicing), and RNA mis-translation.
  • a “W-bump” REDN peptide refers to a REDN peptide that is a result of tryptophan stalling.
  • the W-bump frameshift peptides include any one or more of the more than 66,000 REDN peptides mentioned by Bartok.
  • the W-bump REDN peptides include any one or more of the 177,361 REDN peptides as generated and disclosed herein (SEQ ID NOs: 1-177,361).
  • an array is prepared having one or more REDN peptides immobilized thereon having a sequence as set forth in SEQ ID NOs: 1-177,361.
  • a signature is detected on the array.
  • sample means non-biological samples and biological samples.
  • Non-biological samples include those prepared in vitro including varying concentrations of a target molecule of interest in solution.
  • Biological samples include, without limitation, blood, lymph, urine, saliva, sputum, other bodily secretions, cells, and tissue specimens and dilutions of them. Any suitable biological sample can be used.
  • a biological sample can be a specimen obtained from a subject ⁇ for example, a mammal such as a human, canine, mouse, rat, pig, guinea pig, cow, monkey, or ape) or can be derived from such a subject.
  • a subject can provide a plurality of biological sample, including a solid biological sample, from for example, a biopsy or a tissue.
  • a sample can be a tissue section or cells that are placed in or adapted to tissue culture.
  • a biological sample also can be a biological fluid such as urine, blood, plasma, serum, saliva, tears, or mucus, or such a sample absorbed onto a paper or polymer substrate.
  • a biological sample can be further fractionated, if desired, to a fraction containing particular cell types.
  • a sample can be a combination of samples from a subject (for example, a combination of a tissue and fluid sample). In some cases, sera are obtained from the individual using techniques known in the art.
  • a subject can, for example, use a “fingerstick”, or “fingerprick” to draw a small quantity of blood and add it to a surface, such as a filter paper or other absorbent source, or in a vial or container and optionally dried.
  • a biological sample obtained, for example, from a drop of a subject’s blood and placed on a filter paper can be directly mailed to a provider of the methods of the invention without a processing of the sample.
  • a biological sample provided by a subject can be concentrated or dilute.
  • binding refers to an association between two substances or molecules, such as the association of an antibody with a peptide. Binding can be detected by any procedure known to one skilled in the ail, such as by physical or functional properties of the formed complexes, such as a target/antibody complex.
  • control means a sample or standard used for comparison with an experimental sample, such as a tumor sample obtained from a patient with a particular type of cancer.
  • the control can be a sample obtained from a healthy patient or a non-tumor tissue sample obtained from a patient diagnosed with a particular type of cancer.
  • a control can also be a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of cancer patients with poor prognosis, or group of samples that represent baseline or normal values).
  • a difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference.
  • a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.
  • the methods provided herein are sensitive and involve small quantities of biological samples from a subject.
  • biological samples from a subject are too concentrated and require a dilution prior to being contacted with an array of the invention.
  • a plurality of dilutions can be applied to a biological sample prior to contacting the sample with an array of the invention.
  • a dilution can be a serial dilution, which can result in a geometric progression of the concentration in a logarithmic fashion.
  • a ten-fold serial dilution can be 1 M, 0.01 M, 0.001 M, and a geometric progression thereof.
  • a dilution can be, for example, a one-fold dilution, a two-fold dilution, a three-fold dilution, a four-fold dilution, a five-fold dilution, a six-fold dilution, a seven-fold dilution, an eight-fold dilution, a nine-fold dilution, a ten-fold dilution, a sixteen-fold dilution, a twenty-five-fold dilution, a thirty -two-fold dilution, a sixty-four-fold dilution, and/or a one-hundred-and-twenty-five-fold dilution.
  • the binding of a molecule to an array in accordance with certain embodiments of the methodology disclosed herein creates a pattern of binding that can be associated with a condition.
  • the affinity of binding of a molecule to a peptide in the array can be mathematically associated with a condition.
  • the off-target binding pattern of an antibody to a plurality of different peptides of the invention can be mathematically associated with a condition.
  • the avidity of binding of a molecule to a plurality of different peptides can be mathematically associated with a condition.
  • the peptide array can be contacted with the biological sample (for example, sera) under any suitable conditions to promote binding of antibodies in the sample to peptides immobilized on the array.
  • the methods presented herein are not limited by any specific type of binding conditions employed. Such conditions will vary depending on the array being used, the type of substrate, the density of the peptides arrayed on the substrate, desired stringency of the binding interaction, and nature of the competing materials in the binding solution. In certain embodiments, the conditions include a step to remove unbound antibodies from the addressable array.
  • any suitable detection technique can be used in the methods provided herein to detect binding of antibodies (“antibody reactivity”) in the biological sample to peptides on the array. Such reactivity may be measured or estimated in any operable way, such as, for example, by ELISA or by microarray assay.
  • any type of detectable label can be used to label peptides on the array, including but not limited to radioisotope labels, fluorescent labels, luminescent labels, and electrochemical labels (for example, ligand labels with different electrode mid-point potential, where detection includes detecting electric potential of the label).
  • bound antibodies can be detected, for example, using a detectably labeled secondary antibody.
  • Immunogenicity refers to the ability of a substance, such as a peptide, to elicit an immune response, such as an antibody response or a T cell response, when administered to an individual, for example, in a vaccine formulation.
  • an immune response means a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. For individuals with cancer, it is the immune response to the tumor.
  • a peptide that reacts with an antibody or elicits T cell activity in a biological sample from an individual is not immunogenic when administered in a vaccine formulation.
  • a peptide that reacts with an antibody or elicits T cell activity in a biological sample from an individual is immunogenic when administered in a vaccine formulation.
  • Immunogenicity is determined by methods of those of skill in the art including in animal model testing and using in silico prediction of immunogenicity. In silico immunogenicity prediction tools are available for free to the public, for example at the Immune Epitope Database and Analysis Resource (www.iedb.org).
  • an immunogenic peptide is a peptide which includes an allele- specific motif or other sequence such that the peptide will bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”) response, or a B cell response (for example, antibody production) against the antigen from which the immunogenic peptide is derived.
  • CTL cytotoxic T lymphocyte
  • B cell response for example, antibody production
  • Immunogenic peptides include synthetic embodiments of peptides described herein.
  • analogs non-peptide organic molecules
  • derivatives chemically functionalized peptide molecules obtained starting with the disclosed peptide sequences
  • variants homologs of these proteins
  • Each polypeptide of this disclosure is made of a sequence of amino acids, which may be either L- and/or D-amino acids, naturally occurring and otherwise.
  • Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein can be provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form a Cl -Cl 6 ester, or converted to an amide of formula NR1R2 wherein R1 and R2 are each independently H or Cl -Cl 6 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring.
  • Amino groups of the peptide can be in the form of a pharmaceutically acceptable acid addition salt, such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically acceptable acid addition salt such as the HC1, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains may be converted to Cl -Cl 6 alkoxy or to a C1-C16 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes.
  • Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • protecting groups such as acetamide groups.
  • Peptidomimetic and organomimetic embodiments are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of an immunogenic Brachyury polypeptide having measurable or enhanced ability to generate an immune response.
  • a pharmacophore is an idealized three- dimensional definition of the structural requirements for biological activity.
  • Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD).
  • the term “diagnose” or “diagnostic” means identifying the presence or nature of a pathologic condition. Diagnostic methods differ in their sensitivity and specificity.
  • the “sensitivity” of a diagnostic assay is the percentage of diseased subjects who test positive (percent of true positives).
  • the “specificity” of a diagnostic assay is 1 minus the false positive rate, where the false positive rate is defined as the proportion of those without the disease who test positive. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.
  • “Prognostic” means predicting the probability of development (for example, severity) of a pathologic condition.
  • the term “treat” or “treatment” refers to a method of reducing the effects of a disease or condition. Treatment can also refer to a method of reducing the disease or condition itself rather than just the symptoms.
  • the treatment can be any reduction from native levels and can be but is not limited to the complete ablation of the disease, condition, or the symptoms of the disease or condition.
  • a disclosed method for reducing the effects of a cancer is considered to be a treatment if there is a 10% reduction in one or more symptoms of the disease (for example, tumor size) in a subject with the disease when compared to native levels in the same subject or control subjects.
  • the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • treatment can refer to any reduction in the progression of a disease or cancer.
  • methods of reducing the effects of a cancer are considered to be a treatment if there is a 10% reduction in the tumor growth rate relative to a control subject or tumor growth rates in the same subject prior to the treatment.
  • the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • immunotherapeutic refers to a compound that is used to, in this case, treat cancer by inducing, enhancing, or suppressing the immune response.
  • Immunotherapeutic s encompass immune checkpoint inhibitors, antibody-drug conjugates (ADCs), monoclonal antibodies, T-cell therapy, small molecules, and bispecific antibodies (bsAbs).
  • ADCs antibody-drug conjugates
  • bsAbs bispecific antibodies
  • Antibody-drug conjugates include monoclonal antibodies linked to biologically active drugs to combine the targeting ability of antibodies as well as the cytotoxic ability of the drug.
  • T-cell therapy involves reprogramming a patient’s own immune T cells to attack tumors.
  • chimeric antigen receptor refers to a fusion protein of the membrane or intracellular signaling region of T-cell activating proteins (for example, CD3-zeta chain, CD28, 41BBL, 0X40, ICOS, high-affinity receptor for IgE (FcsRI) and other T-cell activating proteins) and the antigen-binding site (for example, a single-chain Fv fragment) of a cancer antigen- specific antibody.
  • T-cell activating proteins for example, CD3-zeta chain, CD28, 41BBL, 0X40, ICOS, high-affinity receptor for IgE (FcsRI) and other T-cell activating proteins
  • the antigen-binding site for example, a single-chain Fv fragment
  • Bispecific antibodies are recombinant proteins that can bind to two different types of antigens at the same time.
  • a bsAb can be engineered to bind a cytotoxic cell and a target tumor cell. That way, the b
  • Immune checkpoint inhibitor therapy is a form of cancer immunotherapy.
  • the therapy targets immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets.
  • Checkpoint therapy can block inhibitory checkpoints, restoring immune system function.
  • Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1.
  • PD- 1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274).
  • PD-L1 on the cell surface binds to PD1 on an immune cell surface, which inhibits immune cell activity.
  • PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to cither PD-1 or PD-L1 and therefore block the interaction may allow the T cells to attack the tumor.
  • Immune checkpoint inhibitors such as anti-PD-1 antibodies have been approved to treat different types of cancer (for example, bladder, lung, kidney, melanoma, head, neck, Hodgkin’s lymphoma, and solid tumors).
  • PD-1 inhibitors include nivolumab, pembrolizumab, cemiplimab and spartalizumab.
  • Additional CPIs include CTLA-4 blockage (for example, ipilimumab, such as for treatment of melanoma) and PD-LI inhibitors (for example, atezolizumab, avelumab, or durvalumab, such as for treatment of bladder cancer).
  • the FDA for the first time gave tumor-type, agnostic approval to treat any latestage cancer that is MSI-H. This was based on the remarkably positive responses to treatment of not only cancers with frequent MSI-H phenotypes (colon, endometrial and stomach), but rare MSI-H patients in other cancers. For example, a woman with triple negative, metastatic breast cancer who was MSI-H had a complete remission, while most breast cancers have been unresponsive to CPI treatment.
  • immunotherapeutic examples include Tremelimumab (CTLA-4 blocking antibody), 0X40 agonists (for example, agonist antibodies), antibodies to B7 ligands (for example, anti-B7-Hl, anti-B7-H3, anti-B7-H3, anti-B7-H4), durvalumab (MEDI4736, anti-PD-Ll antibody), MK-3475 (PD-1 blocker), Nivolumab (anti-PD-1 antibody), Pembrolizumab (anti-PD-1 antibody), Pidilizumab/CT-Oi 1, BY55 monoclonal antibody, AMP224 (anti-PD-Ll antibody), BMS- 936559 (anti-PD-Ll antibody), MPLDL3280A (anti- PD-Ll antibody), MSB0010718C (anti-PD-Ll antibody), and Yervoy/ipilimumab (anti- CTLA-4 checkpoint inhibitor). Many new inhibitor targets are being investigated. In some cases, IT treatment includes a
  • checkpoint inhibitor and “checkpoint pathway inhibitor” are used interchangeably and refer to negative regulatory molecules, usually antibodies, that block or inhibit anti-T cell anti-tumor function to enhance tumor killing.
  • Checkpoint inhibitors include, without limitation, CTLA-4, PD-LI, PD-L2, PD-1, B7-H3, B7- H4, BTLA, HVEM, TEVI3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1, CHK2, A2aR, and a B- 7 family ligand such as B7- 1, B7-2, B7-DC, B7-H1, B7-H2, B7- H3, B7-H4, B7-H5, B7-H6 and B7-H7 (or any combination thereof), or a combination thereof (for example, a combination of CTLA-4 and PD-L1 or PD-L2).
  • the term “adverse event, “adverse reaction,” “adverse response,” “adverse immune response,” “side effect” or “side effects” refers to the unacceptable or undesirable adverse symptoms resulting from or associated with the administration of a particular treatment such as an immunotherapeutic therapy. Side effects specifically to immunotherapeutic s are termed “immune related adverse events” (irAE). While side effects vary by the type of therapy, common side effects of immunotherapeutic therapies include, without limitation fatigue, infusion related reactions, dermatological toxicity, diarrhea/colitis, hepatotoxicity, pneumonitis, hyper- and hypothyroidism.
  • responsiveness to treatment by an immunotherapeutic is measured by at least one criterion selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.
  • the methods described herein can be carried out using a computer programmed to receive data (for example, data from a W-bump REDN peptide array indicating whether a subject has a binding signature associated with cancer, associated with responsiveness to immunotherapeutic therapy, or associated with adverse reactions to immunotherapeutic therapy).
  • the computer can output for display information related to a subject’s biomarkers, and the likelihood of the duration of time that the subject will be responsive to an immunotherapeutic therapy, suffer a side-effect, or the prognosis of survival.
  • a professional can take one or more actions that can affect patient care (for example, administer a new treatment or modify an existing treatment).
  • a medical professional can record the information in a subject’s medical record and/or in an electronic database.
  • a medical professional can record that the subject is likely or not likely to respond to an immunotherapeutic therapy, or otherwise transform the patient’s medical record, to reflect the patient’s medical condition.
  • a medical professional can review and evaluate a patient’ s medical record, and can assess multiple treatment strategies for clinical intervention of a patient’s condition.
  • the signature may indicate watchfulness or pre-treatment for a sideeffect or recommendation for a different treatment.
  • a professional can communicate information regarding biomarker analysis to a subject or a subject’s family.
  • a professional can provide a subject and/or a subject’s family with information regarding an immunotherapeutic therapy, including treatment options and potential side effects.
  • a professional can provide a copy of a subject’s medical records to communicate information regarding biomarker analysis and/or disease states to a specialist.
  • a professional can apply information regarding a subject’s biomarkers to advance research into immunotherapeutic therapy. For example, a researcher can compile data on the presence of a particular signature with information regarding the efficacy of an immunotherapeutic therapy, or side effects associated with an immunotherapeutic therapy.
  • a research professional can obtain a subject’ s biomarker information to evaluate the subject’s enrollment, or continued participation in a research study or clinical trial.
  • a research professional can communicate a subject’ s biomarker information to a medical professional, or can refer a subject to a medical professional for clinical assessment and/or treatment.
  • Any appropriate method can be used to communicate information to another person (for example, a professional), and information can be communicated directly or indirectly.
  • a laboratory technician can input biomarker information or cancer diagnosis information into a computer-based record.
  • information can be communicated by making a physical alteration to medical or research records.
  • a medical professional can make a permanent notation or flag a medical record for communicating information to other medical professionals reviewing the record.
  • Any type of communication can be used (for example, mail, e-mail, telephone, and face-to-face interactions).
  • Information also can be communicated to a professional by making that information electronically available to the professional.
  • information can be placed on a computer database such that a medical professional can access the information.
  • information can be communicated to a hospital, clinic, or research facility serving as an agent for the professional.
  • the term “vaccine” means a composition that elicits a prophylactic or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • a vaccine elicits an antigen-specific immune response to an antigen of a pathogen, for example, a bacterial or viral pathogen, or to a cellular constituent correlated with a pathological condition, such as cancer.
  • a vaccine may include a polynucleotide, a peptide or polypeptide, a virus, a bacterium, a cell or one or more cellular constituents.
  • the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent.
  • the immunogenic material may include live-attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them.
  • the vaccine is a subunit vaccine, which is an immunizing agent that has been treated to remove traces of nucleic acid (such as viral nucleic acid) so that only protein subunits remain. The subunits have less risk of causing adverse reactions.
  • the vaccine can also be a live vaccine, which is a vaccine prepared from living attenuated organisms or from viruses that have been attenuated but can still replicate in the cells of the host organism.
  • the immunogenic material for a cancer vaccine may include, for example, a protein or peptide expressed by a tumor or cancer cell. Vaccines may elicit both prophylactic (preventative) and therapeutic responses.
  • vector means a virus, bacterium, or other microbe, or a nucleic acid, used to deliver an antigen or a gene for an antigen, as part of a vaccine.
  • a nucleic acid vector is a nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements known in the art.
  • Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.
  • nucleic acid refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof,
  • nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral- methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like.
  • oligonucleotide typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (for example, A, T, G, C), this also includes an RNA sequence (for example, A, U, G, C) in which “U” replaces “T.” ’’Nucleotide” includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.
  • nucleotide sequences the left-hand end of a single- stranded nucleotide sequence is the 5 ‘-end; the left-hand direction of a double- stranded nucleotide sequence is referred to as the 5’-direction.
  • the direction of 5’ to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction.
  • the DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5’- end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3’ to the 3’ end of the coding RNA transcript are referred to as ’’downstream sequences.”
  • “cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • encode(s) or “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (for example, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription
  • a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • Recombinant nucleic acid refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors including an amplified or assembled nucleic acid, which can be used to transform a suitable host cell. A host cell that includes the recombinant nucleic acid is referred to as a ’’recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, such as a “recombinant polypeptide.”
  • a recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome-binding site) as well.
  • adjuvant means a vehicle used to enhance antigenicity; such as a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in- oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund’s incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund’s complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages).
  • adjuvants also include immuno stimulatory molecules, such as cytokines, costimulatory molecules, and for example, immuno stimulatory DNA or RNA molecules.
  • composition also can be formulated to contain an adjuvant in order to enhance the immunological response.
  • adjuvants include, but arc not limited to, lysolecithin, pluronic polyols, polyanions, other peptides, oil emulsions, and potentially useful human adjuvants such as Bacillus Calmette Guerin (BCG) and Corynebacterium parvum.
  • Adjuvants for inclusion in the inventive composition desirably are safe, well tolerated, such as QS-21 , Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-1 , GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, and MF59 (as described in, for example, Kim et al., Vaccine, 18: 597 (2000)).
  • adjuvants that can be administered to a mammal include lectins, growth factors, cytokines, and lymphokines (for example, alpha- interferon, gamma-interferon, platelet derived growth factor (PDGF), gCSF, gMCSF, TNF, epidermal growth factor (EGF), IL- 1, IL-2, IL- 4, IL-6, IL-8, IL- 10, and IL- 12), ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma in
  • Cosolvents may be added to a composition or formulation.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Supplementary compounds for example, preservatives, antioxidants, antimicrobial agents including biocides and bio stats such as antibacterial, antiviral, and antifungal agents
  • Pharmaceutical compositions may therefore include preservatives, antioxidants, and antimicrobial agents.
  • Preservatives can be used to inhibit microbial growth or increase stability of ingredients thereby prolonging the shelf life of the pharmaceutical formulation.
  • Suitable preservatives include, for example, EDTA, EGTA, benzalkonium chloride or benzoic acid or benzoates, such as sodium benzoate.
  • Antioxidants include, for example, ascorbic acid, vitamin A, vitamin E, tocopherols, and similar vitamins or provitamins.
  • the methods and compositions disclosed herein may include a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carriers of use are conventional. Remington’s Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, PA, 19 th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the compositions disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Any route of administration can be used to deliver the vaccine composition to the subject. Indeed, although more than one route can be used to administer the composition, a particular route can provide a more immediate and more effective reaction than another route.
  • Exemplary routes of administration for contact or in vivo delivery which a composition can optionally be formulated include inhalation, respiration, intranasal, intubation, intrapulmonary instillation, oral, buccal, intrapulmonary, intradermal, topical, dermal, parenteral, sublingual, subcutaneous, intravascular, intrathecal, intraarticular, intracavity, transdermal, iontophoretic, intraocular, ophthalmic, optical, intravenous (i.v.), intramuscular, intraglandular, intraorgan, or intralymphatic.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions or emulsions of the active compound, which preparations are typically sterile and can be isotonic with the blood of the intended recipient.
  • Non-limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable, or synthetic oils.
  • the composition is administered via intramuscular injection, for example, using a syringe or needleless delivery device.
  • this disclosure also provides a syringe or a needleless delivery device including the composition.
  • the composition also can be applied or instilled into body cavities, absorbed through the skin (for example, via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.
  • the composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant.
  • Implants such as an implantable device, for example, a mechanical reservoir or an implant or a device made of a polymeric composition, are particularly useful for administration of the composition.
  • the composition also can be administered in the form of a sustained-release formulation including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl- terephthalate (BHET), and/or a polylactic-glycolic acid. It can also be administered using a gene gun via microparticles.
  • a sustained-release formulation including, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl- terephthalate (BHET), and/or a polylactic-
  • the dose of the composition administered will depend on a number of factors, including the size of a target tissue, the extent of any side-effects, the particular route of administration, and the like.
  • the dose ideally includes an “effective amount” of the composition, for example, a dose of composition, which provokes a desired immune response in the subject.
  • the term “effective amount” includes an amount of agent, such as an agent that is sufficient to generate a desired response, such an immune response.
  • an “effective amount” is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease, for example to treat and/or prevent cancer in a subject.
  • an effective amount is a therapeutically effective amount.
  • an effective amount is an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with cancer.
  • the desired immune response can entail production of antibodies, protection upon subsequent challenge, immune tolerance, immune cell activation, and the like.
  • One dose or multiple doses of the composition can be administered to a mammal to elicit an immune response with desired characteristics, including the production of specific antibodies, or the production of functional T cells.
  • Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms/disorder are/is affected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex, and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counter indications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, for example, Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389.
  • a typical daily dosage of the antibody used alone might range from about 1 mg/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • a disclosed composition such as a vaccine or an antibody
  • the efficacy of the therapy or prophylaxis can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the ail will understand that a composition, such as a vaccine or an antibody, disclosed herein is efficacious in treating, inhibiting, or preventing a cancer in a subject by observing that the composition reduces tumor growth or prevents a further increase in tumor size.
  • inhibitor refers to decreasing an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
  • antibodies is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a naturally occurring antibody. Thus, these antigen-binding fragments are also intended to be designated by the term “antibody.” Antibodies may be chosen for their ability to interact with tumor- associated W-bump REDN peptides or other novopeptides or targets of interest, and in embodiments may be used such that tumor growth is inhibited. The antibodies can be tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities may be tested according to known clinical testing methods.
  • antibody encompasses, but is not limited to, whole immunoglobulin (for example, an intact antibody) of any class.
  • Native antibodies are usually heterotetrameric glycoproteins, composed of two identical light (L) chains and two identical heavy (H) chains.
  • L light chain
  • H heavy chain
  • each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes.
  • Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • Each heavy chain has at one end a variable domain (V(H)) followed by a number of constant domains.
  • V(H) variable domain
  • Each light chain has a variable domain at one end (V(L)) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains.
  • the light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.
  • immunoglobulins can be assigned to different classes.
  • IgA human immunoglobulins
  • IgD immunoglobulins
  • IgE immunoglobulins
  • IgG immunoglobulins
  • IgG-1 immunoglobulin-1
  • IgG-2 immunoglobulin-2
  • IgG-3 immunoglobulin-3
  • IgG-4 immunoglobulins-1
  • IgA-1 and IgA-2 immunoglobulins 1 and 2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
  • variable is used herein to describe certain portions of the variable domains that differ in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
  • variability is not usually evenly distributed through the variable domains of antibodies. It is typically concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions both in the light chain and the heavy chain variable domains.
  • CDRs complementarity determining regions
  • FR framework
  • the variable domains of native heavy and light chains each include four FR regions, largely adopting a beta sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies (see Kabat E. A. et al., “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md.).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • antibody or fragments thereof encompasses chimeric antibodies and hybrid antibodies, with dual or multiple antigen or epitope specificities, and fragments, such as F(ab')2, Fab', Fab, sFv, scFv, and the like, including hybrid fragments.
  • fragments of the antibodies that retain the ability to bind their specific antigens are provided.
  • antibody or fragment thereof Such antibodies and fragments can be made by techniques known in the art and can be screened for specificity and activity according to the methods set forth in the Examples and in general methods for producing antibodies and screening antibodies for specificity and activity (See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988)).
  • antibody or fragments thereof conjugates of antibody fragments and antigen binding proteins (single chain antibodies).
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen. Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, for example, the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sd. USA, 81 :6851-6855 (1984)).
  • nucleic acid approaches for antibody delivery also exist. Broadly neutralizing antibodies and antibody fragments can also be administered to patients or subjects as a nucleic acid preparation (for example, DNA or RNA) that encodes the antibody or antibody fragment, such that the patient’s or subject’s own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • the delivery of the nucleic acid can be by any operable means, such as, for example, those disclosed herein.
  • Some embodiments provided herein relate to methods and compositions for preventing, treating, and diagnosing cancer, and predicting response to immunotherapies.
  • the methods and compositions provided herein relate to tryptophan bump (W-bump) REDN peptides.
  • RNA error derived neoantigen generated by RNA mis-processing, mis-transcription, or mis-translation in tumors are a rich source for creating cancer diagnostics, therapeutics, and vaccines.
  • REDN peptides are highly immunogenic, resulting in generation of antibodies and cellular responses against such REDN peptides in a subject.
  • REDN errors are recurrent in different tumors and different patients, unlike most neoantigens produced from mutations in DNA that are personal.
  • the raising of antibodies and the recurrence are the basis of creating diagnostics for cancer and predicting responses to treatments.
  • Antibodies to the REDN peptide can be used as therapeutics to treat cancer.
  • the REDN peptides themselves can be used as vaccines to treat, or to prevent cancer.
  • W-bump REDN peptides tryptophan (W) bump REDN peptides
  • the W-bump REDNs may be used to diagnose cancer , treat cancer with vaccine, or prevent cancer with a vaccine.
  • RNA error derived neoantigens RNA error derived neoantigens
  • FS variants On average at least 20% of the FS variants would encode a new peptide of 9 or more amino acids. Since at least 8 amino acids are required to bind in the MHC I pocket for presentation to T cells (for example, 8, 9, 10, or 11 residues), many of the frameshift valiants could be presented. It will be seen that even short frameshift variants will present new 9-residue peptides by virtue of the fusion of wild-type and frameshift sequences.
  • Mutated proteins associated with frameshifts tend to be highly immunogenic and are expressed predominantly (if not exclusively) in tumor cells, making frameshift REDNs ideal vaccine candidates.
  • an insertion or deletion of a nucleic acid sequence that is a multiple of three will produce an in-frame deletion or insertion. These will also lead to the production of novopeptides since the junction points will be new peptide sequence.
  • Relative to oncogenesis there are two classes of mutated proteins to consider, whether produced by frameshifts or other mechanisms: the first class, “oncogenic- related variants,” are those that result in or contribute to tumor formation or progression.
  • the second class, “bystander variants,” are those that are not involved in oncogenesis but that happen to be altered simply because the cellular machinery is operating inefficiently. From the point of view of developing a vaccine, both are viable as vaccine candidates.
  • W-bump REDN peptides Bartok termed these REDN peptide at W codons “W-bump” REDN peptides.
  • W-bump REDN peptides are presented on a tumor cell and may be recognized by T-cells, which then kill the tumor cell. At least some of these W-bump REDN peptides are recurrently produced in different patients, though the numbers assayed were low. Bartok reported approximately 66,000 sequences identifies that could potentially be produced by this process.
  • the present disclosure extends further to provide herein compositions and methods that utilize these W-bump REDN peptides in cancer vaccines, diagnostics, and treatment.
  • the W-bump REDN peptides are used in combination with other antigens, including, for example, REDN peptide antigens produced by other RNA processing errors.
  • REDN peptide vaccines in dogs reduce deaths from cancer.
  • REDN peptide vaccines are used to reduce the size of a tumor.
  • the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular' carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer
  • a process for making a composition for treating cancer may begin by identifying one or more W-bump REDN peptide sequences.
  • W-bump REDN peptide sequences By informatic analysis, all REDN peptides that may be produced by switching frames at W codons were determined herein.
  • a peptide array of all possible predicted W-bump REDN peptides was designed. All possible REDN peptides downstream of W codons 10 aa or longer were bioinformatically determined.
  • W-bump REDN peptides may be bioinformatically determined by identifying sequences that include two W codons within 8 amino acids of one another and are 10 aa or longer. All possible REDN peptides may include peptides with a (-2), (-1), (+1), or (+2) frameshift.
  • the ribosome is much more likely to make an insertion (+1 REDN peptide) than a deletion (-1 REDN peptide) at these sites.
  • 177,194 such peptides were identified, as set forth in the accompanying sequence listing (SEQ ID NOs: 1-177,194). The restriction of the peptide being longer than 10 amino acids for both sources of REDN peptides was applied. Each REDN peptide antigen that was longer than 15 aa was divided into 15 aa, non-overlapping peptides. Peptides that shared more than 10 aa identical sequences with any human reference proteins were excluded to avoid autoimmunity as a vaccine.
  • W-bump REDN peptide sequences may be determined by using sequence information from publicly available human genome databases, and applying the informatic analysis described herein. All such sequences are incorporated herein by reference. Such sequences are described in the sequence listing.
  • a REDN peptide array may be prepared and processed to determine an IgG signal of each array protocols known to those of skill in the art.
  • a peptide array that includes all of the identified W-bump REDN peptides may be created to identify W-bump REDN peptides as potential neoantigens for inclusion in an anti-cancer therapeutic composition.
  • Figure 2 A biological sample may be obtained from a subject and contacted with the REDN peptide array. The sample may be blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.
  • the REDN peptide array may be analyzed to identify W-bump REDN peptides that recurrently bind with antibodies in the biological sample.
  • Each array may be normalized to its median florescence for analysis. Reactivity may be compared between the biological sample compared to a non-canccr control(s). Total reactivity on the arrays can be evaluated using at least two methods. The first method includes comparing the number of significant peptides in the biological sample and control samples using fold change and p- values. The second method uses a scoring method for each FS peptide. A peptide was scored as positive (red) if it was higher than six times the standard deviation (6SD) from the mean value of the control sample for the same peptide. W- bump peptides meeting these criteria may be forwarded for use in diagnostics, therapeutics, and vaccines.
  • 6SD standard deviation
  • the methods include contacting a W-bump REDN peptide array including a plurality of W-bump REDN peptides with a first biological sample obtained from a first individual known to have a cancer, measuring binding of the first biological sample to the W-bump REDN peptide array, contacting the same type of W-bump REDN peptide array with a second biological sample obtained from a second individual, measuring binding of the second biological sample to the W-bump REDN peptide array, comparing the binding of the two biological samples to the W-bump REDN peptide array, and identifying one or more W-bump REDN peptides that are immunogenic, cancer-specific, and/or shared across cancers.
  • the term “same type of W-bump REDN peptide array” refers to two or more W-bump FSP peptide arrays that include the same W-bump REDN peptides.
  • the same type of W-bump REDN peptide array may be the same physical unit, or it may be a different physical unit with the same W-bump REDN peptides included on the array.
  • measuring binding includes detecting antibody reactivity to the plurality of W-bump REDN peptides.
  • the second individual is a control individual without cancer. Comparison to this control screens out W- bump REDN peptides in which antibody reactivity is normal and unrelated to cancer.
  • the second individual is also known to have a cancer.
  • the second individual is known to have the cancer of the first individual. Use of a second individual with the same cancer as the first individual may provide confirmation that a “shared” W-bump REDN peptides (for example, a W-bump REDN peptide that is bound by samples of both the first and second individual) is common to a that particular type of cancer.
  • the second individual and the first individual have different types of cancer.
  • comparison of binding between the first individual and the second individual may identify W-bump REDN peptides that are disease- specific because they are not shared by individuals with different cancers and/or may identify W-bump REDN peptides that are shared across multiple diseases when binding is observed with both the first and second individuals’ biological samples.
  • These methods may be combined in that the binding of a first individual may be compared to a second individual who is a control with no cancer, to a third individual who is known to have cancerof the same type as the first individual, and/or a fourth individual who is known to have a different type of cancer.
  • comparison between groups is quantitative and/or qualitative.
  • each group are represented by multiple members.
  • the methods further include determining a nucleic acid sequence of said W-bump REDN peptides to identify one or more nucleic acids encoding W-bump REDN peptides that are immunogenic, cancer- specific, and/or shared across cancers.
  • determining a nucleic acid sequence of said W-bump REDN peptides includes identifying one or more W-bump REDN peptides that are immunogenic, cancerspecific, and/or shared across cancers, identifying the amino acid sequence of a W-bump REDN peptide, and then working backwards to determine a nucleic acid sequence that encodes said amino acid sequence, according to known mRNA codons which encode amino acids.
  • the identified W-bump REDN peptides may be used as a neoantigen in the anti-cancer therapeutic composition.
  • one or more W-Bump REDN peptides may be selected as a neoantigen based on the size of the W-bump REDN peptide or a predicted epitope binding.
  • Two or more W-bump REDN peptides may be selected as neoantigens.
  • the W-bump REDN peptide sequence may be manufactured into a vaccine using the W-bump REDN peptide and/or the W-bump REDN peptide sequence.
  • the anti-cancer therapeutic may be the resulting vaccine and can be administered to the subject in a therapeutically effective dose to treat cancer.
  • the disclosed arrays have a variety of potential uses.
  • the array is used to identify one or more W-bump REDN peptides of interest, such as W-bump REDN peptides that are immunogenic, cancer- specific, and/or shared across cancers.
  • the array may include a large number of W-bump REDN peptides, for example 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, or 200,000 or more W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values.
  • This broad coverage of W-bump REDN peptides would allow for the potential of many W-bump REDN peptides to be explored and discovered.
  • the array is used to diagnose cancer.
  • the array may include W-bump REDN peptides which have been observed across many different cancers, in order to provide a broad panel coverage.
  • the arrays are used to predict response to immunotherapy.
  • the arrays are used to predict adverse responses to immunotherapy.
  • the array may include W-bump REDN peptides which have been statistically correlated to a positive (desirable) or negative (undesirable) response to immunotherapy.
  • the array comprises one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 1-177,361, or a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361, and/or a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • the W-bump REDN peptides are used to design therapeutic or prophylactic vaccines.
  • the arrays are used to determine targets for therapeutics to treat cancer.
  • Certain embodiments of the arrays relate to arrays used in designing a personal vaccine, such as a cancer vaccine.
  • Certain embodiments of the arrays relate to arrays used in designing a universal vaccine, such as a cancer vaccine.
  • the arrays include at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 REDN peptides, or an amount within a range defined by any two of the aforementioned values.
  • the arrays include a range of peptides ranging from about 100 to about 2.5M peptides.
  • a plurality of W-bump REDN peptides are in-situ synthesized on the array.
  • the plurality of W- bump REDN peptides are fixed on a substrate.
  • the substrate includes glass, silica, composite, resin, or combination thereof.
  • the array is configured to detect binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. For example, after a biological sample is contacted with the array, binding of the biological sample to the array may be detected and/or measured by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the array is configured to facilitate detection of binding by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, Bio-Layer Interferometry, and surface plasmon resonance. In some embodiments, the plurality of W- bump REDN peptides include two or more pooled W-bump REDN peptides.
  • the arrays described herein may be combined with other embodiments.
  • the arrays described may be used in methods of identifying W-bump REDN peptides that are immunogenic, cancer-specific, and/or shared across cancers; methods of detecting cancer; methods of predicting immunotherapy response; methods of designing a cancer vaccine; methods of treating a subject with a vaccine; and methods of producing a therapeutic molecule designed to bind W-bump REDN peptides.
  • vaccine composition comprises one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 1-177,361.
  • the vaccine compositions include one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361, and/or a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • the vaccine compositions further include an adjuvant.
  • the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, Alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, Freund's complete, Freund's incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank's Balanced Salt Solution), IL- 12, IL-2, imiquimod, interferon-gamma, ISCOM, lipid core peptide (LCP), Lipofectin, lipopolysaccharide (LPS), liposomes, MF59, MLP+TDM, monophosphoryl lipid A, Montanide IMS-1313, Montanide
  • melanoma tumors have a high mutational level with an average of 200 neoepitope mutations. This provides a substantial number to algorithmically screen for optimal antigenic presentation.
  • Embodiments provided herein relate to W-bump REDN peptides produced by errors in RNA translation as a source of cancer neoantigens and a simple system to detect and screen them.
  • W-bump REDN peptides produced at the RNA level in tumor cells may be a reliable source of neoantigens for vaccines for several reasons.
  • these W-bump REDN peptides produce neoantigens which are more likely to be immunogenic than neo-epitopes encoded by single nucleotide mutations.
  • W-bump REDN peptides are a particularly attractive source as are a limited number of possible variants (REDN peptides are downstream of W codons and can be further screened by length of peptide, +1 or -1 frame), thus reducing the search space for W-bump REDN peptides.
  • a peptide array to screen for immune reactive neoantigens. Not all of the predicted W-bump REDN peptides are expected to be immunogenic and only a subset of these have use as diagnostics, therapeutic targets, or vaccine components. As described above, the creation of the peptide arrays can be used to assess which of the W-bump REDN peptides are useful for these purposes. This approach is much simpler than sequencing tumor DNA obtained from a biopsy. Fourth, because any expressed gene has the potential to produce neoantigens, it may not be necessary to limit the vaccine to oncological driver genes.
  • a W-bump REDN peptide array may be created and used to identify W-bump REDN peptides that recurrently bind to antibodies from biological samples obtained from a subject. From, the identified W-bump REDN peptides, a vaccine may be created and administered to a subject in a therapeutically effective amount to treat cancer. In some embodiments, the vaccine may be administered in two or more doses across a time period.
  • the vaccine compositions include one or more W- bump REDN peptides.
  • biological samples, such as blood, from cancer patients are applied to the W-bump REDN peptide arrays described herein to determine reactivity of peptides for each patient.
  • W-bump REDN peptides unique to the patient are used in a personal vaccine.
  • W-bump REDN peptides shared between different patients are used for off-the-shelf therapeutic or preventative vaccines.
  • the vaccine compositions include W-bump REDN peptides resulting from translation of ncRNA.
  • RNA W-bump REDN peptides would be produced in any cancer type, even if the DNA mutation level is low.
  • the vaccine compositions further include an adjuvant.
  • the adjuvant is ABM2, AS01B, AS02, AS02A, Adjumer, Adjuvax, Algammulin, alum, aluminum phosphate, aluminum potassium sulfate, Bordetella pertussis, calcitriol, chitosan, cholera toxin, CpG, dibutyl phthalate, dimethyldioctadecylammonium bromide (DDA), Freund’s adjuvant, Freund’s complete, Freund’s incomplete (IFA), GM-CSF, GMDP, gamma inulin, glycerol, HBSS (Hank’s Balanced Salt Solution), polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose (poly-ICLC, also known as Hiltonol), IL- 12, IL-2, imiquimod, interferon-gamma,
  • the W-bump REDN peptide array can be used to detect the antibody response in a patient to the W-bump REDN peptides as a direct readout of the W-bump REDN peptide’s clinical potential as diagnostics, therapeutic targets, or vaccines.
  • Methods, systems, and compositions provided herein are related to the analysis of a subject’s ability to generate antibodies to REDN peptide made from mis-processing/translating mRNA and are capable of making high density, high numbers of peptide arrays.
  • the methods include obtaining a biological sample from a subject with cancer, contacting the biological sample to an array including a plurality of W-bump REDN peptides, and measuring binding of the biological sample to the plurality of W-bump REDN peptides.
  • measuring binding includes detecting antibody reactivity to at least one peptide of the array.
  • sera samples from patients may be collected before treatment with an immunotherapy, such as the compositions described herein. The sera may be applied to the arrays containing the W-bump peptides, and the signatures for each patient may be analyzed. Based on the patient’s antibody reactivity to 400 W bump REDN peptide, a therapy response may be predicted.
  • the methods further include predicting a response of the subject to an immunotherapy. In some embodiments, the methods further include analyzing the binding to predict whether immunotherapy would be effective in treating the subject’s cancer. In some embodiments, the methods further include analyzing the binding to predict whether immunotherapy would elicit an adverse immune response to immunotherapy in the subject. In some embodiments, comparison is quantitative and/or qualitative. In some embodiments, analyzing includes comparing the binding of the biological sample to binding of another subject who responded positively to immunotherapy or experienced an adverse immune response in response to immunotherapy. In some embodiments, the binding is compared to another subject who had no response to immunotherapy (for example, immunotherapy was not effective in treating the subject’s cancer).
  • the binding is compared to another subject who did not have an adverse event in response to an immunotherapy.
  • the subject is classified as being likely to respond (positively) to treatment with the immunotherapeutic, or as being likely to have an adverse event in response to immunotherapy.
  • Certain embodiments relate to methods of predicting the response of a subject with cancer to an immunotherapy, and as described herein, can include, for example, obtaining the subject’s binding signature to a W-bump REDN peptides array using one or more biological samples obtained from the subject to determine whether the sample contains one or more indicators of favorable or unfavorable responses (for example, unfavorable side effects) to immunotherapeutic (“IT”) therapy.
  • binding signature binding signature
  • binding profile or “W-bump REDN peptide signature” refers the observed binding of a subject’s biological sample to a W-bump REDN peptides array.
  • a binding signature or binding profile may include qualitative information about whether a subject’s biological sample was observed to bind to a particular W-bump REDN peptide on a W-bump REDN peptide array and may include quantitative information such as the level or strength of observed binding to particular W-bump REDN peptides on a W-bump REDN peptide array.
  • binding signature or binding profile The correlation between a binding signature or binding profile and responsiveness to IT therapy can be established by obtaining binding signatures for subjects having a known favorable response to IT treatment and for subjects that were unresponsive or had an unfavorable response to treatment using sera (or other bodily samples) collected before each subject received treatment.
  • a control includes non-disease sera contacted with an identical array under the same experimental conditions.
  • the breadth of the binding profile can be quantified in multiple ways including, for example the number of motifs, the percentage of signature represented, and/or total immune reactivity.
  • cancer patients can be classified according to a method provided herein by quantifying a subject’s signature for responsiveness to IT treatment, prognosis, or likelihood of experiencing serious side-effects of IT treatment.
  • the methods are useful for determining a subject’s responsiveness for IT treatment of a tumor (including early- stage tumor formation) associated with W-bump REDN peptide expression.
  • a W-bump REDN peptide signature is established by using a biological sample (for example, blood, sera, plasma) that may contain antibodies having affinity to peptides on the W-bump REDN peptide array. As further described herein, antibody reactivity to at least one peptide of the array may be detected. As described herein, antibodies are employed as biomarkers of disease, thus taking advantage of the immune system’s expansive antibody repertoire to identify a statistically significant pattern of peptides, each with specific binding values having predictive, prognostic, and diagnostic potential. In some cases, the biological sample is diluted. The sample is incubated long enough to allow cognate binding to approach equilibrium - usually overnight.
  • a patient s W- bump REDN peptide signature for IT response, prognosis, or side-effects is determined by comparing two or more groups of interest. For example, a comparison may be made between patients who responded well to IT therapy and those that did not. Such comparisons are used to establish the classifier of interest.
  • the difference in groups may be determined directly by quantifying total binding to the W-bump REDN peptides.
  • the plurality of W-bump REDN peptides are fixed on a substrate.
  • the substrate includes glass, silica, composite, resin, or combination thereof.
  • the plurality of W-bump REDN peptides are in- situ synthesized on the array.
  • binding is detected by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, electro-interference, Bio-Layer Interferometry, and surface plasmon resonance.
  • the array includes at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values.
  • the arrays include a range of peptides ranging from about 100 to about 2.5M peptides.
  • the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof. Tn some embodiments, the biological sample includes antibodies.
  • the subject is a mammal. In some embodiments, the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a Pig-
  • the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt’s lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin’s lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,
  • the array may include a plurality of W-bump REDN peptides which have previously been identified as being immunogenic, cancer-specific, or shared across cancers according to the methods disclosed herein.
  • the methods include obtaining a biological sample from a subject, contacting the biological sample from the subject with an array including a plurality of W-bump REDN peptides, measuring binding of the biological sample to the plurality of W-bump REDN peptides, and analyzing the binding to predict whether the subject has a cancer.
  • the W-bump REDN peptides include one or more peptides discovered by methods described in Zhao et al. (Translation of noncoding RNAs and cancer, Cancer Letters 497 (2021) 89-99, which is incorporated by reference in its entirety).
  • the W-bump REDN peptides result from non-convention al translation of a W-bump REDN peptide.
  • measuring binding includes detecting antibody reactivity to at least one peptide of the array.
  • binding is detected by at least one of fluorescence, luminescence, calorimetry, chromatography, radioactivity, electrointerference, Bio-Layer Interferometry, and surface plasmon resonance.
  • analyzing the binding to predict whether the subject has cancer includes comparing the binding to previously measured binding patterns of other subjects who are known to have had cancer, or other subjects who did not have cancer (for example, positive and negative controls). For example, in some embodiments, binding is compared quantitatively and/or qualitatively to binding of a second subject, who is known to have cancer, to the same type of array. Sufficient similarity of binding is associated with having cancer, and it is predicted that the first subject has cancer.
  • the plurality of W-bump REDN peptides are fixed on a substrate.
  • the substrate includes glass, silica, composite, resin, or combination thereof.
  • the plurality of W-bump REDN peptides are in- situ synthesized on the array.
  • the array includes at least about 100, about 200, about 300, about 500, about 1,000, about 2,000, about 3,000, about 5,000, about 7,500, about 10,000, about 12,500, about 15,000, about 17,500, about 20,000, about 22,500, about 25,000, about 27,500, about 30,000, about 32,500, about 35,000, about 37,500, about 40,000, about 50,000, about 100,000, about 200,000, about 300,000, about 400,000, about 500,000, about 600,000, about 700,000, about 800,000, about 900,000, about 1,000,000, about 1,500,000, about 2,000,000, about 2,500,000, about 3,000,000, about 3,500,000, about 4,000,000, about 4,500,000, or about 5,000,000 W-bump REDN peptides, or an amount within a range defined by any two of the aforementioned values.
  • the arrays include a range of peptides ranging from about 100 to about 2.5M peptides.
  • the biological sample includes blood, serum, plasma, cerebrospinal fluid, saliva, urine, or combinations thereof.
  • the biological sample includes antibodies.
  • the subject is a mammal.
  • the subject is a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a cow, or a Pig- [0136]
  • the subject is suspected of having a cancer.
  • the cancer is selected from the group consisting of acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt’ s lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin’s lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian
  • a subject’s biological sample may be contacted with a W-bump REDN peptide array, and binding of the sample to one or more peptides of the W-bump REDN peptide array may be used to simultaneously detect cancer, predict response to immunotherapy, identify immunogenic peptides for use in a preventative or therapeutic vaccine, and/or identify antibodies for use in treatment with a therapeutic molecule designed to bind a W-bump REDN peptide.
  • the W-bump REDN peptide array may include W-bump REDN peptides which have previously been identified as being immunogenic, cancer-specific, or shared across cancers according to the methods disclosed herein.
  • a W-bump REDN peptide is recurrent across different patients and/or tumor types, it affords the potential to make generally useful diagnostics, therapeutics, or vaccines. For example, if 20% of breast cancer patients generate antibodies to one W-bump REDN peptide, another 30% to another, and etc., then the collection of these W-bump REDN peptides could be used as a diagnostic for breast cancer. An antibody generated against a W-bump REDN peptide present in 30% of the breast cancer patients could be used as a therapeutic for these patients.
  • composition comprising a plurality of RNA error derived neoantigen
  • REDN tryptophan bump REDN peptides
  • composition of alternative 1, wherein the composition is formulated as a vaccine is formulated as a vaccine.
  • REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.
  • REDN peptides comprise one or more peptides having a sequence or part of a sequence as set forth in SEQ ID NOs: 1-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296
  • REDN peptides comprise two or more pooled REDN peptides.
  • a peptide array comprising a plurality of RNA error derived neoantigen
  • REDN tryptophan bump REDN peptides
  • a method of treating or preventing a disorder in a subject comprising administering a composition according to any one of alternatives 1-10 as a vaccine.
  • the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ovarian
  • a method of detecting a disorder in a subject comprising:
  • a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN) peptides, wherein the plurality of REDN peptides comprise tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.
  • REDN RNA error derived neoantigen
  • REDN peptides comprise peptides created by uncharged tryptophan tRNA and ribosome stalling at sites on mRNA with two closely spaced tryptophan codons.
  • REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • REDN peptides is fixed on a substrate.
  • the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.
  • 500000 REDN peptides such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100
  • cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, ova
  • REDN peptides comprise two or more pooled REDN peptides.
  • a method of measuring an immune response to a neoantigen peptide in a subject comprising: (a) contacting a biological sample obtained from a subject to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; and (b) detecting binding of the biological sample to at least one peptide in the peptide array.
  • REDN RNA error derived neoantigen
  • REDN peptides comprise one or more peptides having a sequence or a part of a sequence as set forth in SEQ ID NOs: 1-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,195-177,361.
  • REDN peptides comprise one or more peptides having a sequence as set forth in any one of SEQ ID NOs: 177,284-177,296.
  • REDN peptides is fixed on a substrate.
  • the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70000, 80000, 90000, 100000, 150000, 200000, 250000, 300000, 350000, 400000, 450000, or 500000 REDN peptides.
  • the peptide array comprises between about 100 and 500000 peptides, such as at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, 20000, 22500, 25000, 27500, 30000, 32500, 35000, 37500, 40000, 45000, 50000, 60000, 70
  • the cancer is acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adenocarcinoma, adult T-cell leukemia, astrocytoma, bladder cancer, bone cancer, brain tumor, breast cancer, Burkitt's lymphoma, carcinoma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, colorectal cancer, endometrial cancer, glioblastoma multiforme, glioma, hepatocellular carcinoma, Hodgkin's lymphoma, inflammatory breast cancer, kidney cancer, leukemia, lung cancer, lymphoma, malignant mesothelioma, medulloblastoma, melanoma, multiple myeloma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer,
  • REDN peptides comprise two or more pooled REDN peptides.
  • a method of predicting a response of a subject to an immunotherapy comprising: (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern prior to immunotherapy.
  • REDN RNA error derived neoantigen
  • a method of predicting adverse immune responses of a subject to an immunotherapy comprising: (a) contacting a biological sample obtained from the subject following immunotherapy treatment to a peptide array comprising a plurality of RNA error derived neoantigen (REDN), wherein: the plurality of REDN peptides comprise peptides comprise tryptophan bump REDN peptides; (b) detecting binding of the biological sample to at least one peptide in the peptide array; and (c) comparing a binding pattern following immunotherapy to a binding pattern indicative of adverse immune responses.
  • REDN RNA error derived neoantigen
  • a method of treating a disorder in a subject comprising: screening therapeutic compounds that bind to one or more W-bump REDN peptides; and administering a therapeutic compound that binds to one or more W-bump REDN peptides to the subject.
  • the REDN peptide array was prepared and processed to determine an IgG signal of each array using standard protocols.
  • the specific IgG reactivities were analyzed in 64 non-cancer control samples and a total of 85 cancers from 5 different late-stage cancer types with 17 samples each (LC: lung cancer, BC: breast cancer, GBM: glioblastoma, GC: gastric cancer, PC: pancreatic cancer) and 12 stage I pancreatic cancer samples.
  • the purpose was to broadly screen against different cancers to determine which peptides were of value. Typically, only about 10-20% of REDN peptides resulting from W-bumps were immunoreactive.
  • Total reactivity on the arrays was evaluated in the 5 cancer types and noncancer samples with at least two methods.
  • the first method compared the number of significant peptides in the cancer and control samples using fold change and p-values.
  • the second method used a scoring method for each REDN peptide. A peptide was scored as positive (red) if it was higher than six times the standard deviation (6SD) from the mean value of non-cancers for the peptide. All 5 cancer types had more positive REDN peptides than the non-cancer controls. W-bump peptides meeting these criteria were then forwarded to testing in diagnostics, therapeutics, and vaccines.
  • REDN peptides in duplicate were produced.
  • Human serum samples (cancer and non-cancer) were incubated with arrays at 1:1000 dilution for overnight at 4°C.
  • Human IgG binding was detected by the Dy light 550 labeled goat anti human IgC Fc antibody with 1:5000 dilution for 45 minutes at room temperature.
  • the slides were scanned by InnoScan 910 instrument, and the relative fluorescence unit (RFU) for each peptide was extracted using Mappix software for subsequent analysis.
  • 89 W-Bump REDNs (10‘ 3 ) had positive reactivity with the sera antibodies in cancer samples but not in non-cancer samples.
  • the 89 W-bump REDNs are listed below in Table 1:
  • Arrays were synthesized displaying 1.2 million dog cancer REDN peptides. 277,000 of these were W-bump REDN peptides. Dog serum samples (21 non-cancer and 22 hemangiosarcoma (HSA) cancer) were incubated with arrays at 1:400 dilution for 24 hours at
  • FIG. 3 As shown in Figure 3, using a threshold of 20,000 RFUs for positivity, a set of 13 W-bump REDN peptides were identified that were positive in at least 7 of the HSA serum samples and none of the control samples. Figure 3 depicts detection of immunoreactivity of the 13 peptides across the 22 HSA samples.
  • Pep_l through Pep_13 refer to SEQ ID NOs: 177284-177296 in Table 2 below.
  • W-bump REDN peptides that recurrently bind to antibodies from sera of cancer patients are encoded on a plasmid in a standard genetic immunization vector and introduced with a gene gun. IxlO 5 B16F10 tumor cells are injected and animals vaccinated 4 weeks later.
  • the DNA fragments encoding W-bump REDNs are cloned as C-terminal fusions into the genetic immunization vectors pCMVi-UB and pCMVi-LSrCOMPTT with Bgl II and Hind III and mixed with a 1:1 ratio as the vaccine antigen.
  • Three adjuvants are encoded by genetic immunization vectors.
  • the pCMVi-mGM-CSF vector expresses the adjuvant mouse granulocyte/macrophage colony-stimulating factor (mGM-CSF) under the control of the human cytomegalovirus (CMV) promoter.
  • LT AB indicates immunization with a 1:5 ratio by weight of two plasmids, pCMVi-LTA and pCMVi-LTB, expressing the heat-labile enterotoxins LTA and LTB from Escherichia coli. These plasmids express LTA and LTB as C-terminal fusions to the secretion leader sequence from the human al antitrypsin gene. Additional adjuvants are the class A CpG 2216 single- stranded oligodeoxynucleotide obtained from Sigma and alum from Pierce.
  • DNA-gold micronanoplexes are prepared by combining, per bullet, 57 pL of cysteamine-gold solution with precipitated DNA ( ⁇ 10 pg) that has been resuspended in ⁇ 15 pL of water, and then vortexing for 10 min.
  • 6 pL/bullet of a freshly made solution of PEI-micron gold 167 mg/mL in 0.1 M MES, pH 6, without NaCl is added.
  • the pelleted micronanoplexes are washed with ethanol prior to resuspension in n-butanol (55 pL/bullet), followed by bullet formation under nitrogen gas.
  • antigen W-bump REDN and non-protective Cowpox viral antigen CPV 172 (31)
  • pCMVi vectors plus the adjuvants pCMVi-mGM-CSF (0.5 pg) and CpG 2216 (5 pg) for each shot. All of the mice are challenged with IxlO 5 B16-F10 cells 4 weeks after the immunization.
  • mice are genetically immunized in the ear by Gene Gun at 8 weeks of age (2 shots/mouse, 60 ng pooled antigens plus 0.25 pg LTAB and 2.5 pg CpG2216 as the adjuvant for each shot) and boosted twice (two days apart) in three weeks with 1 pg pooled antigens plus the same adjuvants dosage. All mice are boosted again in two weeks with 50 pg KLH conjugated W-bump REDN peptides with 50 pg CpG 2216 and 50 pl alum in total 100 pl PBS. The negative groups are immunized with the empty vectors and KLH protein with the same dosage. All mice are challenged with 5xl0 3 4T1 cells two weeks after the last immunization.
  • mice are genetically immunized in the ear by Gene Gun at 8 weeks of age (2 shots/mouse, 1 pg antigen plus 0.25 pg LTAB and 2.5 pg CpG2216 as the adjuvant for each shot), and boosted in two weeks with KLH conjugated W-Bump REDN peptide plus 50 pg Poly:IC (Sigma) in 100 pl PBS. The same regime is repeated in two weeks.
  • the negative groups are immunized with the empty vectors and KLH protein with the same dosage. All mice arc challenged with 5xl0 3 4T1 cells 4 weeks after the last immunization.
  • CD8 and CD4 T cell depletion stalls 2 weeks after the last immunization by i.p injection of 100 pg antibody (anti CD8, clone 2.43; anti CD4, clone GK 1.5; BioXCell, West Riverside, NH) every 3 days until the end of the experiment.
  • mice are genetically immunized by Gene Gun at 4-6 weeks with 100 ng of antigen(s) in pCMVi vectors, boosted twice (3-4 days apart) at 9-10 weeks with 1 pg of the same antigen(s), and boosted once at 13-14 weeks with protein.
  • Genetic immunizations include adjuvants LTAB (0.5 pg) and CpG 2216 (5 pg).
  • the protein boost includes 50 pg CpG 2216 and 50 pl alum in 100 pl PBS as the adjuvant.
  • Peptides used in the ELISPOT assays are synthesized in-house.
  • the Mouse IFN y ELISPOT Set (BD Biosciences) is used according to the manufacturer’s directions except that blocking is at 37°C.
  • Tumor volume is monitored and compared to control mice receiving a mock vaccination.
  • the vaccine confers significant retardation of tumor growth.
  • the pooled vaccine confers an even greater retardation of tumor grown than vaccines which include only one W- Bump REDN peptide.
  • Serum is diluted 1: 100 in binding buffer (0.01M Tris- HC1, pH 7.4, 1% alkali- soluble casein, 0.05% Tween-20), and 150 pl diluted samples are loaded into each compartment of the 12-plex array and incubated overnight at room temperature or 4 °C. After sample binding, the arrays are washed 3X in wash buffer (lx TBS, 0.05% Twccn-20), 10 minutes per wash. Primary sample binding is detected via Alexa Fluor® 647 -conjugated goat anti-human IgG secondary antibody (Jackson ImmunoResearch # 109- 605- 098).
  • the secondary antibody is diluted 1:10,000 (final concentration 0.15 ng/pl) in secondary binding buffer (lx TBS, 1% alkali-soluble casein, 0.05% Tween-20). Arrays are incubated with the secondary antibody for 3 hours at room temperature, washed 3X in wash buffer (10 minutes per wash), 30 seconds in reagent-grade water, and then dried by centrifuging at 690 RPM for 5 minutes. All washes and centrifugations are done on a Little Dipper 650C Microarray Processor (SciGene) with preset programs. The fluorescent signal of the secondary antibody is detected by scanning at 635 nm at 2 pm resolution and 15% gain, using an MS200 microarray scanner (Roche NimbleGen).
  • the binding of the samples to the array is compared for cancer and non-cancer samples, and among samples from different types of cancer.
  • Sets of W-bump REDN peptides are identified that have different levels of binding between cancer/non-cancer groups, and/or between different types of cancer.
  • the identified W-bump REDN peptides may be used to classify various cancers from non-cancer and distinguish cancer types.
  • the identified W-bump REDN peptides are included in diagnostics arrays for the detection of cancer and/or diagnosis of cancer type.
  • Example 6 Using W bump REDN peptides to predict response to immunotherapy
  • a physician could take a small sample of blood from a patient before treatment and determine with high accuracy whether the patient is likely to respond to that particular therapy. If the likelihood is low, a different therapy is recommended. If the patient is predicted to be a responder but likely to have an immune-related adverse event (irAE), W-bump signature analysis is integrated into the treatment plan, and a different therapy is recommended or the treatment plan is modified.
  • irAEs are generally graded from 1-4. Grades 3 and 4 are considered serious and can require immunosuppression treatment. Patients with irAEs are just as likely to have a positive response to treatment. The occurrence of a Grade 3 or 4 event can prohibit the patient from further checkpoint therapy.
  • mice Following intravenous (i.v.) injection of cells of the K7M2 osteosarcoma cell line, mice are treated with 3 dosages of anti-PDLl plus anti-CTLA4, and 2 additional dosages of anti-PLDl treatment. Treatments are spaced three days apart. Binding of samples from the mice to peptide arrays including W-bump REDN peptides (W-bump signatures) are analyzed at the following time-points: (1) prior to the tumor injection and the treatment, (2) right after the treatment, and (3) at the end of the experiment after non-responder mice die from lung metastasis. At each time point, W-bump signatures significantly distinguish the responder mice from the non-responder mice.
  • W-bump signatures significantly distinguish the responder mice from the non-responder mice.
  • W-bump signatures can predict the response even before the tumor is injected.
  • Further study of immunotherapeutic (“IT”) response prediction is performed using a mammary tumor mouse model and an array to measure specific binding to W-bump REDN peptides (W-bump signatures).
  • the 4T-1 mammary tumor cell line is used.
  • Four groups of mice are assayed. Group 1: No Treatment (28 mice).
  • Group 2 Early treatment group (1 st treatment at 16 weeks) (16 mice).
  • Group 3 Treat at first palpable tumor (1 st treatment at -33 weeks).
  • Group 4 Late treatment group (1st treatment at 24-26 weeks) (15 mice).
  • IT treatment is 100 pg anti-CTLA4 (UC10-4F10-11) plus 200 pg anti-PD-Ll (10F.9G2). Five doses are administered, with each dose administered every 3 days, and then two additional doses, with one every week. Palpable tumors are monitored following the treatment period.
  • W-bump REDN peptides are identified that are associated with an immune response in most of the patients that are tested, for example W-bump REDN peptides on the array which most samples from breast cancer patients react to, and most samples from patients without cancer don’t have binding to.
  • Monoclonal antibodies to a chosen set of these immunoreactive peptides are selected.
  • the criteria for choice includes frequency of reactivity in the patients or the level of antibody reactivity, such as compared to healthy control samples.
  • the monoclonal antibodies are tested in vitro for their ability to bind and or kill cancer cells (cell lines or tumor cells) that express the W-bump REDN peptides.
  • Antibody candidates which show in vitro activity are further screened in mouse tumor models for therapeutic effects when injected. Based on this data, one or more of the antibodies are produced on large scale by Good Manufacturing Practice to enter into clinical trials in patients with the relevant cancer. If the antibody is shown to be safe and efficacious it is used to treat cancer in patients having a cancer.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne des peptides néo-antigènes dérivés d'erreur d'ARN (REDN), et un système et des procédés d'utilisation de ceux-ci, les peptides de REDN étant le résultat du blocage du tryptophane, conduisant à une "bosse" de tryptophane (peptides REDN bosse-W ("W-bump")). Les systèmes concernent des réseaux de peptides, et la composition et les systèmes sont utilisés dans des procédés d'identification de néoantigènes et de traitement et de prévention du cancer. L'invention concerne également des procédés de diagnostic du cancer à l'aide de peptides REDN bosse-W ("W-bump"), des procédés de prédiction de réponse à une immunothérapie à l'aide de peptides REDN bosse-W ("W-bump"), des procédés de prédiction de réponses immunitaires défavorables à une immunothérapie à l'aide de peptides REDN bosse-W ("W-bump"), des procédés de traitement et de prévention du cancer à l'aide de vaccins composés de peptides REDN bosse-W ("W-bump"), ou de composés thérapeutiques conçus pour lier des peptides REDN bosse-W ("W-bump"). L'invention concerne également des compositions de vaccins qui comprennent des peptides REDN bosse-W ("W-bump") et des composés thérapeutiques qui se lient à des peptides REDN bosse-W ("W-bump").
EP24757619.2A 2023-02-17 2024-02-14 Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins Pending EP4665387A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363485735P 2023-02-17 2023-02-17
PCT/US2024/015795 WO2024173551A2 (fr) 2023-02-17 2024-02-14 Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins

Publications (1)

Publication Number Publication Date
EP4665387A2 true EP4665387A2 (fr) 2025-12-24

Family

ID=92420716

Family Applications (1)

Application Number Title Priority Date Filing Date
EP24757619.2A Pending EP4665387A2 (fr) 2023-02-17 2024-02-14 Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins

Country Status (2)

Country Link
EP (1) EP4665387A2 (fr)
WO (1) WO2024173551A2 (fr)

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060014178A1 (en) * 2004-05-26 2006-01-19 Whitson Robert H Use of Mrf-2 for screening and therapies
US20080280774A1 (en) * 2005-02-16 2008-11-13 Wyeth Methods and Systems for Diagnosis, Prognosis and Selection of Treatment of Leukemia
GB201710812D0 (en) * 2017-07-05 2017-08-16 Francis Crick Inst Ltd Method
US20230390371A1 (en) * 2020-10-16 2023-12-07 Stichting Het Nederlands Kanker Instituut-Antoni van Leeuwenhoek Ziekenhuis Anti-tumor immunity induces the presentation of aberrant peptides

Also Published As

Publication number Publication date
WO2024173551A2 (fr) 2024-08-22
WO2024173551A3 (fr) 2024-10-10

Similar Documents

Publication Publication Date Title
Ng et al. Antibodies against endogenous retroviruses promote lung cancer immunotherapy
AU2018338441B2 (en) Tumor mutational load and checkpoint immunotherapy
US20220235324A1 (en) Multiple antigen specific cell therapy methods
US11471519B2 (en) Methods of cancer treatment using tumor antigen-specific T cells
JP2017504324A (ja) 免疫療法に対する癌応答の決定因子
CN108700592A (zh) 新表位的迭代发现及其适应性免疫疗法和方法
WO2015108907A2 (fr) Compositions et méthodes pour l'identification, l'évaluation, la prévention, et le traitement d'un mélanome à l'aide d'isoformes de pd-l1
KR20230118713A (ko) 종양을 치료하는 방법에 사용하기 위한 항-pd-1 항체
US20220389102A1 (en) Hla class i sequence divergence and cancer therapy
US20210223257A1 (en) Method and system for determining microsatellite instability
EP3876971A1 (fr) Peptides, compositions et vaccins pour le traitement de tumeurs hypermutées à instabilité des microsatellites et leurs méthodes d'utilisation
CN113271970A (zh) 治疗方法
WO2024173551A2 (fr) Peptides néo-antigènes de tryptophane pour diagnostics, agents thérapeutiques et vaccins
WO2024206399A2 (fr) Antigènes peptidiques d'arn non codants pour vaccins contre le cancer, agents thérapeutiques et diagnostics
WO2020136879A1 (fr) Procédé de présentation de l'efficacité d'un agent immunothérapeutique pour le traitement d'une tumeur maligne et agent immunothérapeutique pour le traitement d'une tumeur maligne
WO2018057858A1 (fr) Déterminants de la réponse d'un cancer à l'immunothérapie
HK40098129A (en) Tumor mutational load and checkpoint immunotherapy
JP2017534054A (ja) 抗原提示を検出する方法

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250827

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR