WO2014159435A1 - Compositions, procédés et systèmes informatiques associés à la production et l'administration de lymphocytes t modifiés - Google Patents

Compositions, procédés et systèmes informatiques associés à la production et l'administration de lymphocytes t modifiés Download PDF

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WO2014159435A1
WO2014159435A1 PCT/US2014/023650 US2014023650W WO2014159435A1 WO 2014159435 A1 WO2014159435 A1 WO 2014159435A1 US 2014023650 W US2014023650 W US 2014023650W WO 2014159435 A1 WO2014159435 A1 WO 2014159435A1
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antigens
specific
tissue
antigen
subject
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PCT/US2014/023650
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English (en)
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Roderick A. Hyde
Wayne R. Kindsvogel
Gary L. Mcknight
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Elwha Llc
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Priority claimed from US13/826,803 external-priority patent/US20140274801A1/en
Priority claimed from US13/804,224 external-priority patent/US9587237B2/en
Priority claimed from US13/827,960 external-priority patent/US9499855B2/en
Application filed by Elwha Llc filed Critical Elwha Llc
Priority to EP14773595.5A priority Critical patent/EP2971288A4/fr
Publication of WO2014159435A1 publication Critical patent/WO2014159435A1/fr

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    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464404—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464469—Tumor associated carbohydrates
    • A61K39/46447—Mucins, e.g. MUC-1
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/30—Detection of binding sites or motifs
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Definitions

  • compositions, methods, and computer systems for constructing T cell clone(s) exhibiting an artificial Chimeric Antigen Receptor(s) as part of a library relate to administering the T cell clone cell line(s) from the library to a subject in need thereof.
  • FIG. 1A is a partial view of an embodiment related to engineering an artificial T cell line.
  • FIG. IB is a partial view of an embodiment related to engineering an artificial T cell line.
  • FIG. 2A is a partial view of an embodiment related to constructing a multi-specific chimeric antigen receptor.
  • FIG. 2B is a partial view of an embodiment related to constructing a multi-specific chimeric antigen receptor.
  • FIG. 2C is a partial view of an embodiment related to constructing a multi-specific chimeric antigen receptor.
  • FIG. 2D is a partial view of an embodiment related to constructing a multi-specific chimeric antigen receptor.
  • FIG. 3 is a partial view of an embodiment related to engineering an artificial T cell line, and computer systems thereof.
  • FIG. 4 is a partial view of an embodiment related to engineering an artificial T cell line, and computer systems thereof.
  • FIG. 5 is a partial view of an embodiment related to engineering an artificial T cell line and computer systems thereof.
  • FIG. 6 is a partial view of an embodiment related to engineering an artificial T cell line and computer systems thereof. DETAILED DESCRIPTION
  • the T CAR cells represent a universal T cell clone that has been manipulated to artificially and specifically recognize a particular target antigen (e.g., a disease specific antigen).
  • the T CAR cells provide a tool for immunotherapy of a subject afflicted with a disease or disorder.
  • computer systems utilized in conjunction with the methods of making or administering the T CAR cell libraries to a subject.
  • a Universal Subject Disease Specific Library of artificial cytolytic T cells is developed that bear a multi-specific artificial chimeric antigen receptor (CAR), as described in detail herein.
  • CAR multi-specific artificial chimeric antigen receptor
  • the development of a multi- specific T cell CAR clone Universal Subject Disease Specific Library provides an HLA- independent recognition of antigen.
  • the library provides a rapid and effective treatment of immune -related disorders and diseases.
  • the library is utilized to make a disease specific therapeutic T cell line.
  • Such CAR is engineered, for example, by using available sequence data or expression data derived from population studies of multiple tissue types from multiple subjects.
  • antigens are identified from all presently identified cell surface antigens and secreted proteins for a particular species of subject (e.g., human), or from all identified cell surface antigens and secreted proteins for a specific tissue type (e.g. breast cancer tissue), based on tumor or diseased tissue vs. normal tissue.
  • This differential expression screen identifies antigens specifically associated with various tissues, of either normal or diseased state.
  • the expression data are determined by screening primary tumor tissue against normal tissue. In an embodiment, the expression data are determined by screening primary tumor tissue against secondary tumor tissue. In an embodiment, the expression data are determined by screening primary tumor tissue or secondary tumor tissue against metastasized tissue.
  • a subject includes a mammal, bird, fish, reptile, or amphibian. In an embodiment, a subject includes a vertebrate or invertebrate. In an embodiment, a subject includes a plant. In an embodiment, a subject includes a human. In an
  • the subject includes a fetus in utero.
  • the genetic expression data are derived from multiple tumors from many subjects in order to provide an array of disease specific antigens.
  • multiple disease sites from multiple diseased subjects are utilized for sampling of genetic expression data, or multiple cell samples from a diseased subject.
  • the expression data are derived from multiple disease sites (e.g., joint synovial fluid and/or cell samples) from multiple subjects afflicted with rheumatoid arthritis or systemic lupus erthematosus.
  • the expression data are derived from multiple central nervous system (e.g., spinal tap) fluid and/or cell samples, from multiple subjects afflicted with multiple sclerosis.
  • the expression data are derived from multiple colon biopsies, from multiple subjects afflicted with Crohn's disease or Inflammatory Bowel Disease.
  • the expression data are derived from multiple sputum or saliva samples, from multiple subjects afflicted with asthma.
  • the expression data are derived from multiple blood samples, from multiple subjects afflicted with diabetes, Graves' disease, Hashimoto's thyroiditis, Myasthenia gravis, vasculitis, or other immune related disorder.
  • tissues including lymph, blood, saliva, urine, or other bodily fluid and/or cell sample
  • various tissues may be utilized for testing for cell surface expression of antigens and secreted proteins related to a particular disease or disorder.
  • a library of T cell CAR clones is constructed for each particular tissue type or state of diseased tissue type.
  • a library can be constructed for primary tumors in breast cancer, a separate library can be constructed for secondary tumors in breast cancer, and a separate library can be constructed for metastatic breast cancer.
  • a library can be constructed for multiple sclerosis, a separate library for Alzheimer's disease.
  • a library can be constructed for early stage Alzheimer's disease, and a separate library constructed for later state Alzheimer's disease. It is understood that these are non- limiting examples and can readily be applied to other tissue types or diseases.
  • the tissue type for purposes described herein includes but is not limited to, breast tissue, prostate tissue, colon tissue, stomach tissue or other gastrointestinal tissue, uterine tissue, eye tissue, ear tissue, skin tissue, blood, nasal tissue, mouth or throat tissue (such as oral mucosa, pharyngeal or laryngeal tissue, tongue) joint tissue, bone tissue, bone marrow tissue, scalp tissue, muscle tissue, ovarian tissue, testicular tissue, or fetal tissue.
  • a computer algorithm is used to prioritize the most commonly expressed cell surface antigens for targeting in the engineering of the artificial T CAR cell clones or the spatial or temporal expression of one or more antigens.
  • the T CAR cell clones are utilized for adoptive T cell therapy as a treatment modality for immunotherapy for tumor regression in cancer, or other immunological illnesses or afflictions.
  • a T CAR library is generated with each CAR recognizing only one antigen (with or without recognizing multiple epitopes of the only one antigen), then the library is
  • a computing device or computing system running an algorithm or computer program is used to select the top several most-commonly expressed cell surface antigens and secreted proteins specified for a particular target tissue.
  • the cell surface antigens and secreted proteins can be determined by FACS, gene microarray, RT-PCR, etc. or other standard techniques.
  • a computer algorithm can be used to select antigens based on their structural formation, post-translational modification, Kd value based on previously identified antibodies, genetic sequence, mR A sequence, peptide sequence, or other characteristic(s).
  • the expression data can be analyzed with Significance analysis of microarrays (SAM) developed by Stanford, ANOVA, ANOSVA (for analysis of splice variation), Bonferroni, Bayesian probability, Markov chains, Monte Carlo methods, or Gaussian distributions.
  • SAM Significance analysis of microarrays
  • ANOSVA for analysis of splice variation
  • Bonferroni for analysis of splice variation
  • Bayesian probability Markov chains
  • Monte Carlo methods or Gaussian distributions.
  • TSA tumor specific antigen
  • CNA CNA
  • T CAR cells are constructed with single chain antibodies providing specific binding to one or more cell surface antigens on human cells.
  • single chain variable region fragments scFvs
  • scFvs single chain variable region fragments
  • multi-specific T CAR cells are engineered for each of the presently identified human cell surface antigens (approximately 3700 proteins), and optionally various combinations thereof.
  • a T CAR recognizes only one antigen.
  • a T CAR recognizes multiple antigens.
  • a series of Western blots is conducted between normal tissue and diseased tissue from multiple subjects, or arrays, RT-PCR (optionally quantitative real-time, etc.), and other techniques are utilized for detecting glycosylation or post-translational modifications.
  • the target antigens are identified based on differences in genetic sequence, mRNA sequence, or post-translational modification (e.g., glycosylation), and from the identified target antigens, T CAR cells are constructed.
  • mRNA isolated from a disease site in a subject is sequenced and the sequence information compared by computer system with the identified sequences.
  • such a differential screening out of the normal sequences and identification of abnormal gene expression sequences is utilized to identify the target antigens for the disease of the subject.
  • an antigen includes a substance capable of the lock-and-key interaction of one molecule (the antigen) with another (the antibody or lymphocyte receptor). It is further understood that an antigen includes peptides, glycopeptides, peptidoglycans, lipids, proteoglycans, glycoproteins, glycolipids, and the like. It is understood that antigen specificity includes the ability of a lymphocyte (e.g., T CAR cell) to recognize an antigen specifically as a unique molecule and discern it from another antigen with a high level of precision. Antigen specificity is attributed to the primary, secondary, and tertiary structure, including side-chain conformations of the antigen. Moreover, an epitope includes the portion of the antigen that provides the recognition of the antigen by the lymphocyte receptor (e.g., T CAR cell).
  • an array of single chain variable fragments are identified through the use of identified antibodies against the particular antigens, or by way of screening the scFvs for the antigen(s) if antibodies are not yet readily available.
  • the selection of scFvs is conducted for example, by way of phage display, as described herein.
  • the selection of scFvs is conducted by way of ribosome display. See, for example, Lee et al. JIM, 284 (2004) 147-157, which is incorporated herein by reference.
  • the selection of scFvs is conducted by way of expression in transgenic organism (e.g., plant or animal).
  • a pair of scFvs will be developed for each pair of antigens and the scFvs will be incorporated into a CAR, an artificial T cell Chimeric Antigen Receptor.
  • the corresponding sequences are converted into a retroviral (e.g., lentiviral) vector, for example, according to standardized techniques.
  • the expression vector(s) and utilized to infect T cells thus resulting in a universal T cell clone that has been manipulated to artificially and specifically recognize a particular target antigen.
  • the summation of all such target-specific T CAR cell clones comprises the T CAR cell library of clones.
  • each T cell clone bears a multi-specific CAR that elicits a polyclonal response by targeting multiple epitopes of the same antigen, which is usually much stronger than a monoclonal reaction between a T cell and an antigen.
  • the polyclonal response of the T CAR cell may be more efficacious in a clinical setting.
  • the T cell CAR is engineered to include two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more target sequence specific binding sites.
  • This multi-specific CAR provides a higher level of specificity, and thus increased binding strength, for the target tissue.
  • the data expression analysis determines a distinctive antigen profile for a specific tissue disease or disease state. For example, utilizing heterogeneous expression profile analysis, it was found that among tumor-specific antigens, serum autoantibodies against seven candidate targets were detected in 4-11% of patients with lung and stomach cancers. See, for example, Xu et al. Cancer Res. 72(24): 1-11 (2012), which is
  • a differential screen is performed by comparing cell expression data of diseased cells (e.g., tumor cells) from multiple cell samples (e.g., multiple tissues or organs) of multiple subjects, with normal tissue from multiple cell samples of multiple subjects.
  • the parameters for normal tissue expression can be adjusted for example, in order to reduce background noise or fine-tuned in order to deduce a true differential expression for a particular target antigen.
  • the resulting data from this differential screen is manipulated by a computer (based on quantity of expression of antigen(s), quality of expression of antigen(s), Kd of binding of antigen(s) to cognate receptor(s) or antibody, or other characteristics) and derives the Universal Subject Disease Specific Antigens (UDSA) for a particular disease or disorder.
  • the UDSA are measured or categorized quantitatively and/or qualitatively.
  • the quality of the expression in the diseased tissue e.g., genetic or mR A sequence, glycosy
  • the UDSA and gene expression information related thereto are cataloged or organized based on one or more of a genetic or mRNA mutation, abnormal post-translational modification, Kd value that varies for antigen-receptor binding, methylation/acetylation of nucleic acids, lipid modifications, or other structural characteristics.
  • the gene expression information includes any of the qualitative or quantitative characteristics disclosed herein.an
  • glycosylation or other post-translational modifications are analyzed by way of sequencing, linkage analysis (e.g., enzymatic cleavage or otherwise), carbohydrate separation methods (e.g., high-performance liquid chromatography), and other means.
  • linkage analysis e.g., enzymatic cleavage or otherwise
  • carbohydrate separation methods e.g., high-performance liquid chromatography
  • genetic or mRNA sequence aberrations or mutations can be detected by sequencing, enzymatic cleavage, or other standard techniques.
  • the UDSA and gene expression information related thereto are cataloged or organized based on one or more of the total number of antigens expressed in a diseased tissue versus normal tissue, the relative number of antigens expressed in a diseased tissue versus normal tissue, a cluster expression of several particular antigens in a diseased tissue versus normal tissue, the absence of expression of a normal tissue antigen in a diseased tissue, cell surface density of a single antigen, cell surface concentration of a single antigen, or number of copies of an antigen per cell.
  • the UDSA are then utilized to make a Universal Subject Disease Specific Library of multi-specific CARs that recognize disease specific antigens found most commonly in the general population of subjects. Details of the molecular biology of making the Universal Subject Disease Specific Library are discussed further herein.
  • a subject afflicted with the particular disease for which the UDSAs have been identified has its own diseased cell samples analyzed for cell expression.
  • a computer system is utilized to select multi-specific clones from the Universal Subject Disease Specific Library that match the subject's own diseased cell expression profile. In this way, the subject receives multi-specific clones designed specifically for its individual treatment based on its own diseased cell sample expression data.
  • multiple libraries are generated each based on various stages of disease (e.g., progressive chronic diseases, metastatic cancer, etc.) compared with normal tissue or other disease states of the tissue.
  • a differential screen is performed by comparing cell expression data of the subject's own diseased cells (e.g., tumor cells), with normal tissue from the same subject.
  • the expression analysis is conducted with the primary tumor vs. normal tissue antigen profile.
  • the expression analysis includes the individual's specific disease analysis of primary tumor vs.
  • normal tissue further includes analysis to select antigens from a universal metastasis antigen profile, even if no metastases is present or detected in this particular subject. In that way, a prophylactic treatment to target metastases is conducted in conjunction with the specific primary disease antigen profile.
  • the expression analysis includes the individual's primary disease site antigen profile as well as secondary disease sites (e.g., metastases). In this way, multiple targets of disease are attacked efficiently and effectively.
  • a multi-specific CAR specifically recognizes two or more epitopes of the same antigen.
  • the physical proximity of the multi- specific CAR that specifically recognizes two or more epitopes of the same antigen provides a benefit in that the likelihood of receptor binding is increased and clustering of multiple separate antigen receptors at the T cell surface is not needed since the multi- specific receptors are physically located on the same CAR.
  • a computing device or computing system running an algorithm or computer program is used to analyze the Kd binding strengths of the multi-specific CAR, for example, by analysis of the binding strength of each selected antigen binding site with its target sequence, as well as the coordinated binding strength of the entire multi-specific receptor.
  • the spatial pattern of the multiple specific target sites is optimized based on the sequence in which the specific antigen sites occur in the CAR, or based on the spatial binding relationship with the particular targets.
  • the multi-specific CAR provides at least 2 or more, at least 3 or more, at least 4 or more, at least 5 or more, at least 6 or more antigen binding sites (indicating receptor specificity) (e.g., scFvs).
  • one or more multi-specific CARs are included on a single T cell clone.
  • two or more, three or more, or four or more multi-specific CARs are included on a single T cell clone.
  • the T cell clone is constructed by assembling the specific nucleotide sequences selected by, for example, splice overlap PCR and standard cloning methods. See, for example, U.S. Patent No. 6,410,319, which is incorporated herein by reference.
  • RNA sequence analysis is confirmed, for example, by DNA sequence analysis of the final product.
  • a mammalian expression vector e.g., pcDNAneo or similar vector, under the control of a CMV immediate-early or similar promoter.
  • T cells are then transfected with the artificial CAR construct according to standard techniques, and are analyzed (e.g., by FACS and/or Western Blot and/or Fluorescence in situ hybridization (FISH)) for confirmation that the cells harbor the non-naturally occurring construct.
  • FISH Fluorescence in situ hybridization
  • T cells expressing the multi-specific CAR are tested for in vitro stimulation of cytokine production, for cytolytic activity, and (where appropriate) induction of any apoptotic or "suicide" genes optionally included in the artificial CAR construct.
  • the T cell clone includes a conditionally inducible suicide gene, such as thymidylate kinase, inducible caspase 9, CD20, thymidine kinase, or modified FAS.
  • a conditionally inducible suicide gene such as thymidylate kinase, inducible caspase 9, CD20, thymidine kinase, or modified FAS.
  • ELIS A assays for IL-2 can be utilized to test for in vitro stimulation of cytokine production (IL-2 is commonly tested since it induces cell proliferation among lymphocytes).
  • Chromium Release Assays can be used for measuring the cytolytic activity of the clones by allowing a target cell to take up radioactive supernatant, followed by washing residual radioactivity away and incubating with the T cell clone presumed to be cytolytic. Measurement of release of the radioactivity indicates killing of the cells by the T cell clone.
  • In vitro cell proliferation assays utilized to measure the clones' ability to proliferate themselves can also be measured by standard techniques, including, for example, my measuring tritiated thymidine (thymidine is incorporated into the DNA of dividing cells). See U.S. Patent No. 6,410,319, incorporated herein by reference, for more details on some of these standardized techniques.
  • a subject afflicted by or having symptoms of a disease or disorder for example an immune-related disease or disorder (such as cancer) is treated with the appropriate multi- specific T cell clone(s) selected from the Universal Subject Disease Specific Library based on analysis of a cell sample (e.g., biopsy) the subject's specifically selected target antigens (such as tumor specific antigens) identified by use of a microarray, FACS, or other standard technique.
  • the multi-specific T cell clone Universal Subject Disease Specific Library is analyzed (optionally by computing device or computing system running an algorithm to identify the optimal T cell clone based on information determined from expression studies of the subject's tissue (e.g., tumor tissue)) and the optimal T cell clones are selected for the subject.
  • the subject is treated by infusing one or more therapeutically effective dose of the cytolytic T cell clones, each expressing the particular multi-specific artificial CAR.
  • a sample of cells e.g., from a biopsy
  • T cell CAR clones expected to be used for therapy.
  • the relative cytolysis is determined, allowing for a more accurate dosing range and schedule for the subject's therapy without causing undue cytotoxicity.
  • dosing and scheduling are performed as for any standard clinical trials or testing, and may include one or more parameters specific to the individual test subject.
  • an Individual Subject Disease Specific Library of cytolytic T cells expressing an artificial chimeric antigen receptor (CAR), as described in detail herein, is personalized specifically to an individual subject's own disease (e.g., tumor) antigen expression data or expression patterns, rather than population expression studies.
  • an individual subject's own disease e.g., tumor
  • antigens specific for the individual subject's diseased tissue are identified and isolated.
  • this information provides the basis for determining and/or constructing appropriate T CAR cell clones for therapeutic administration to the individual subject.
  • a database of previously identified antigen sequences is screened with the Individual
  • Subject's diseased tissue and/or sequences therefrom in order to determine the specific disease antigens associated with the Individual Subject's disease.
  • the disease specific library is utilized to generate a disease specific therapeutic T cell line.
  • CAR is engineered, for example, as described herein for the Universal Subject Disease Specific Library, except that in order to generate the CAR for the Individual Subject Disease Specific Library, sample cells (e.g., from a biopsy) from the subject afflicted with cancer or another immune disease are subjected to, for example, Next Generation Sequencing (NGS) which is a standard technique that provides a fast, high-quality sequence of the mRNA transcriptome of the sample cells.
  • NGS Next Generation Sequencing
  • normal cell mRNA can be conducted for an individual subject with specific disease.
  • the standard techniques of constructing the T CAR cell clones and library described herein for the Universal Subject library can be utilized for the Individual Subject Library.
  • a computer algorithm is used to prioritize the most commonly expressed cell surface antigens and secreted proteins for targeting in the engineering of the artificial cytolytic T CAR cells, or the spatial or temporal expression of one or more antigens (e.g., Protease-activated receptors (PAR receptors) are a G protein-coupled receptors that are activated by cleavage of part of their extracellular domain).
  • PAR receptors Protease-activated receptors
  • the T cell CAR clones are utilized for adoptive T cell therapy as a treatment modality for immunotherapy for tumor regression in cancer, or other immunological illnesses or afflictions.
  • the remainder of the process of constructing and utilizing the cytolytic T cell CAR clones is carried out in the same manner as described for the Universal Subject Disease Specific Library herein.
  • a multi-specific T cell CAR clone library is constructed in a similar manner as described for the Universal Subject Disease Specific Library herein.
  • the resulting data from this differential screen is manipulated by a computer (based on quantity of expression of antigen(s), quality of expression of antigen(s), Kd of binding of antigen(s) to cognate receptor(s) or antibody, or other characteristics) and derives the Individual Disease Specific Antigens (IDSA) for the individual subject's particular disease or disorder.
  • IDSA Individual Disease Specific Antigens
  • an Individual Subject is afflicted with multiple diseases simultaneously or sequentially
  • one or more Individual Subject Disease Specific Libraries are generated— each for use with one particular disease or disorder.
  • the IDSA are measured or categorized quantitatively and/or qualitatively, similarly as described herein for the Universal Subject Disease Specific Library.
  • the IDSA are then utilized to make an Individual Subject Disease Specific Library of multi-specific CARs to disease specific antigens found most commonly in the subject's own diseased cells. Details of the molecular biology of making the Individual Subject Disease Specific Library are discussed further herein.
  • a computer system is utilized to select multi- specific clones from the Library for use in various treatment sequences or dosages. In this way, the subject receives customized treatment for its own cell expression based on its diseased cells.
  • the multi-specific CAR is genetically engineered as an artificial T cell receptor specifically targeting the computer-selected IDSA or UDSA.
  • the CAR is a cell surface molecule including one or more extracellular domain (the specific targeting region of the receptor), one or more intracellular signaling domain, and one or more transmembrane domain.
  • the CAR genetically disrupts the innate T cell receptor of the engineered cell.
  • the genetic disruption of the innate T cell receptor results in the complete elimination of the innate T cell receptor function. This elimination of innate T cell receptor function operates to prevent allogeneic response (e.g., graft vs. host disease) by the T CAR cells on the subject receiving them during therapy.
  • individual components of the T CARs are produced from nucleic acid molecules using standard molecular biology methods.
  • nucleic acid molecules are inserted into a vector that is able to express the fusion polypeptide when introduced into an appropriate host cell (e.g., bacteria, yeast, insect, mammalian cells, or other cells).
  • an appropriate host cell e.g., bacteria, yeast, insect, mammalian cells, or other cells.
  • any of the many standard technical methods can be used for insertion of DNA fragments into a vector, as well as for constructing an expression vector encoding the fusion polypeptides described herein and under the control of transcriptional or translational control signals.
  • one or more of these methods may be used for in vitro recombinant DNA or synthetic techniques or in vivo recombination.
  • expression of the fusion polypeptides may be regulated by a second nucleic acid sequence wherein the polypeptide(s) is expressed in a host
  • the individual T cell clones are deficient in at least one Human
  • HLA Leukocyte Antigen
  • HLA II- Leukocyte Antigen
  • HLA class I- the HLA class II-
  • HLA DR, HLA DQ or HLA DP regions for HLA class II-
  • Standard methods of gene inactivation that can be utilized for this particular component include enforced methylation, siRNA, or shRNA.
  • the HLA I- and HLA II- conditions operates to prevent host T cell elimination of the engineered T cell clone, whereas the forced expression of HLA E or HLA G operates to prevent NK cell elimination of the engineered T cells once administered to a subject.
  • individual T cells used for generating T CAR cells can be CD4+/CD8-, CD4-/CD8+, CD4-/CD8-, or CD4+/CD8+.
  • the T cells can be a mixed population of CD4+ and CD8+ cells, or can be a population propagated from a single clone.
  • the T cells are a mixed population of CD4+ cells that produce IL-2 or other cytokines when their receptor engages the specific IDSA or UDSA, and CD8+ cells that lyse the target cells when their receptor engages the specific IDSA or UDSA.
  • T cells that have been modified as described herein are referred to as T CAR cells.
  • the T cell includes a memory T cell with a phenotype of CD62L+ CD45RA-.
  • the individual T cells are primary T CAR cells, such as from peripheral blood mononuclear cells (PBMC).
  • PBMC peripheral blood mononuclear cells
  • the individual T CAR cells are engineered from stem cells from the bone marrow, peripheral blood, hepatic, spleen, or other stem cell compartment.
  • the T CAR cells are engineered from hematopoietic stem cells.
  • the T cell CAR clones are expanded prior to or subsequent to differentiation into T cells.
  • the target antigen(s) described herein include one or more isolated nucleic acid molecule(s).
  • the one or more isolated nucleic acid molecules include at least one of single stranded or double stranded DNA or RNA
  • the sequence corresponds to either a full-length or partial gene sequence.
  • the sequence includes a small inhibitory RNA.
  • the T CAR cells are utilized in a method of treating a subject afflicted with a disease or disorder related to the IDSA or UDSA. Such method of treatment is described in detail, and includes administering specific T CAR cells to the subject in a therapeutically effective amount.
  • the T CAR cells administered with one or more cytokines including but not limited to IL-2, IL-4, IL-8, IL- 6, TGF-beta, IL-10, BAFF, APRIL-, IL-23, IL-17, an Interferon member, or similar cytokine.
  • cytokines including but not limited to IL-2, IL-4, IL-8, IL- 6, TGF-beta, IL-10, BAFF, APRIL-, IL-23, IL-17, an Interferon member, or similar cytokine.
  • modified T cells used in adoptive therapy are less effective if not stimulated with IL-2 to proliferate. See U.S. Patent No.
  • the method further includes a computer-selected panel of cytokines for administration before, during, or after administration of the T CAR cells to a particular subject.
  • the computer-selected panel of cytokines includes one or more cytokines, including the non-limiting examples set forth herein.
  • a subject is treated through administration of a therapeutically effective dose of T CAR cells.
  • the therapeutically effective dose includes approximately 10 6 , approximately 10 7 , approximately 10 8 , approximately 10 9 , approximately 10 10 , approximately 10 11 , approximately 10 12 , or any value there between, T CAR cells per square meter of body surface (cells/m ).
  • one or more cytokines are infused before, during, or subsequent to administration of the T CAR cells.
  • the one or more cytokines are administered in an amount of about 10 , about 10 4 , about 10 5 , about 10 6 , or any value therebetween, units per kilogram body weight.
  • the dosing schedule is determined based on standard adoptive T cell therapy schedules, modified as needed for efficacy, cytotoxicity, and other parameters for clinical trials.
  • the CAR is contained in a plasmid expression vector in proper orientation for expression, and is transfected (by electroporation, calcium phosphate, or other means) directly into the T cell.
  • the CAR is contained in a viral vector and the T cells are infected with the artificial construct.
  • construction of the CAR is conducted, for example, by standard PCR techniques such as splice overlap. Assembly of each of one or more of: target receptor sequence, ribosome binding sequence, signal peptide, variable regions (VL, VH, etc.), a linker region, hinge region, transmembrane domain, and/or zeta chain is conducted with PCR techniques and primers either commercially available or derived from the sequence of the target IDSA or UDSA, when necessary.
  • the final constructs are flanked by restriction sites for directional subcloning into expression vectors.
  • the CAR is chromosomally integrated at the innate T cell receptor alpha locus.
  • the CAR is chromosomally integrated in other active chromatin regions (for example the CAR can be linked to the innate T cell receptor-like promoter region, such as the active T cell receptor V alpha or V beta region).
  • the T CAR is integrated at the rearranged site of the T cell receptor V beta or V alpha promoter in the engineered cell line.
  • each T CAR cell line is propagated with the corresponding artificial antigen presenting cell to generate the memory T CAR cell phenotype of CD62L+CD45RA-.
  • the memory T CAR cells are enriched by cell sorting (e.g., by magnetic or antibody column sorting, FACS, or other technique).
  • cell sorting e.g., by magnetic or antibody column sorting, FACS, or other technique.
  • in vitro propagation cell lines are utilized, such as but not limited to, Jurkat, Daudi, P815, K562, 2c, and others.
  • cells are propagated by standard cell culture techniques.
  • the T CAR cells further include one or more magnetic particles.
  • the T CAR cells include one or more magnetic nanoparticles.
  • one or more magnetic particles are administered to a tumor or other disease site of a subject prior to, during, or subsequent to administration of one or more T CAR cells including one or more magnetic particles. In this way, the T CAR cells will be further directed to the disease site by way of the magnetic attraction by way of the magnetic particles both in the T CAR cells as well as at the disease site.
  • the T CAR cells further include one or more radio frequency identification devices (RFIDs).
  • RFIDs radio frequency identification devices
  • a sample of cells e.g., from a biopsy
  • the selected T cell clones expected to be used for therapy is extracted and tested for cytolysis with the selected T cell clones expected to be used for therapy.
  • the relative cytolysis is determined, allowing for a more accurate dosing range and schedule for the subject's therapy without causing undue cytotoxicity.
  • one or more computer systems include computer-readable non-transitory media which provides methods to one or more users.
  • the user includes a human, or another computer.
  • a system includes a processor.
  • a system includes non-transitory medium memory coupled with a processor, the non-transitory medium memory storing a plurality of machine instructions that cause the processor to perform the steps described herein.
  • computer software products are employed, that include computer readable non-transitory media including computer-executable instructions for performing certain logic steps of various methods described herein.
  • suitable computer readable non-transitory media include but are not limited to floppy disc, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes, etc.
  • the computer executable instructions are written in a suitable computer language or combination of several languages.
  • the various computer software products are utilized for various aspects including but not limited to, data analysis, management of data, operation of one or more instruments, storage of data, and further comparison over time of stored data with new or other stored data.
  • a computer-readable non-transitory medium includes encoded programming code for analyzing gene expression.
  • the software includes code for performing the steps of the determination described herein, and a computer-readable non-transitory medium for storing the code.
  • the programming code applies one or more Clustering Analysis algorithms, including one or more of K-Means, principal component analysis (PCA), self- organizing maps (SOM), or iterative independent component analysis (ICA) to data including one or more measured signals to identify an optimum number of independent clusters into which the data may be grouped.
  • K-Means principal component analysis
  • SOM self- organizing maps
  • ICA iterative independent component analysis
  • the programming code includes code for one or more of: removing background "noise" from the data, using Cluster Analysis to cluster data from one or more gene expression signals into an optimal number (n) of independent groups, or determine if there is a positive correlation of expression of one gene in a first group is statistically correlated with the expression of a second gene in a second group (e.g., if positive gene expression of gene X in primary diseased tissue is correlated with the expression of Y gene in metastasized diseased tissue).
  • the programming code includes code that compiles data into a form suitable for computer analysis.
  • the differential screening includes removing "noise" or providing for normalization of the data.
  • hybridization conditions utilized can be adjusted to decrease the possibility of non-specific binding (or background noise).
  • the computer algorithm(s) utilized herein can provide the computational analysis such that the background noise is reduced or eliminated altogether.
  • the computational model can analyze intensity input of data for a comparative analysis in order to determine with a statistically high degree of certainty, that a particular receptor is bound to a target antigen with specificity.
  • cluster analysis primarily includes top-down or bottom-up analysis.
  • massive parallel gene expression monitoring systems and mathematical methods have been developed for utilization with nucleic acid gene array technology.
  • top-down analysis typically starts with a given number of clusters and proceeds to partition the data into these clusters. In this way, the algorithm randomly assigns centers to each cluster and partitions the nearest data into those clusters. The algorithm iteratively finds new centers of the clusters by averaging over the data in the cluster and reassigning data to new clusters as the centers change. This continues until the centers no longer change.
  • bottom-up clustering (or tree clustering) starts by grouping data at the lowest level and builds larger groups by bringing the smaller groups together at the next highest level.
  • SOMS operate as competitive neural networks, and group input data into nearest neighbors based on weights of each neuron receiving input data.
  • the neuron capable of capturing the data results in an update toward the data input.
  • principal component analysis includes a stepwise analysis that creates a new component axis at each step where variation is observed in the data.
  • each component explains the varying bases for the change in the data.
  • the analysis projects the data into a new space spanned by the principal components.
  • iterative independent component analysis reduces input data into components and treats each component as statistically independent from the others. See, for example, U.S. Patent App. Pub. No. 2006/0074566, which is incorporated herein by reference. In this way, data is clustered that allows for maximum correlation of individual members within a group.
  • the computer systems described herein include systems including a computer-readable non-transitory medium on which is encoded programming code for analyzing gene expression and/or determining clustering of gene expression, correlation of gene expression, or determining a gene expression profile that is utilized as an antigenic profile for a particular tissue and/or subject.
  • a gene function is derived from the information obtained from one or more biological assays, including gene expression assays.
  • the antigenic profile includes the qualitative or quantitative characteristics of the antigens present in a particular analysis group, such as a tissue type or disease studied.
  • a method for immunotherapy of a subject comprises determining the qualitative or quantitative differential gene expression between a non- diseased tissue type from one or more tissue samples of two or more non-diseased subjects of a population, and a diseased tissue type from one or more tissue samples of two or more diseased first subjects of a population; deducing the Universal Subject Disease-Specific Antigens pool for the diseased tissue type based on the determination of the differential gene expression; engineering a Universal Subject Disease-Specific Library of multi- specific T cells bearing Chimeric Antigen Receptors each specifically recognize one or more members of the Universal Subject Disease-Specific Antigens pool; obtaining at least one diseased tissue sample from a diseased second subject to be treated, wherein the diseased tissue sample from the diseased second subject to be treated corresponds to the same tissue type as the diseased tissue type from one or more tissue samples of two or more diseased first subjects of a population; analyzing the qualitative or quantitative gene expression of the diseased tissue sample from the diseased second subject to be treated and determining the Universal
  • the non-diseased tissue type from one or more samples of two or more subjects of a population, and the diseased tissue type from one or more tissue samples of two or more diseased second subjects of a population are the same or similar tissue types.
  • a method for immunotherapy of a subject comprises obtaining gene sequence information for at least one diseased tissue type antigen from a diseased first subject; comparing the gene sequence information for at least one diseased tissue type antigen from a diseased first subject with one or more previously identified non-diseased tissue type antigens from two or more second subjects, wherein the diseased tissue type from the diseased first subject corresponds to the tissue type of the non-diseased tissue type from the two or more second subjects; and
  • the method further includes selecting one or more multi-specific T cells bearing Chimeric Antigen Receptors from the Universal Subject Disease-Specific Library effective against one or more antigens of the diseased tissue from the diseased first subject to be treated based on the determination of the one or more antigen members in common to both pools.
  • the method further includes administering a
  • a method for immunotherapy of a subject comprises determining the qualitative or quantitative differential gene expression between a non- diseased tissue type from a subject, and the corresponding diseased tissue type from the subject; deducing the Individual Subject Disease-Specific Antigens pool based on the determination of the differential gene expression; engineering an Individual Subject Disease-Specific Library of multi-specific T cells bearing Chimeric Antigen Receptors each specifically recognize one or more members of the Individual Subject Disease- Specific Antigens pool; selecting one or more multi-specific T cells bearing Chimeric Antigen Receptors from the Individual Subject Disease-Specific Library based on one or more criteria as being the most effective against the diseased tissue type from the subject; administering a therapeutically effective amount of the selected one or more multi-specific T cells bearing Chimeric Antigen Receptors to the subject.
  • the most effective T CAR cells for a subject is determined based on several factors, including for example, avidity with which the target disease-associated antigen binds the T CAR, the expression level of the target disease-associated antigen on the target diseased cell(s), any clustering of gene expression and the resulting T CAR structural configurations (e.g., a T CAR exhibiting receptor specificity for two different epitopes of the same antigen on a single receptor molecule can be designed in order to have one receptor specificity with increased avidity for one epitope over the other epitope, depending on the avidity or level of expression of the epitope on the target diseased cell), or any of the qualitative or quantitative characteristics described herein (e.g., genetic or mRNA mutations, abnormal post-translational modifications, variant Kd value, methylation/acetylation of nucleic acids, lipid modifications, relative amount of antigens expressed in a diseased tissue versus normal tissue, a cluster expression of several particular
  • the functional avidity of the T CAR is engineered in order to increase or decrease the responsiveness of the T CAR to the target disease-associated antigen. For example, higher avidity T CARs are likely to exhibit broader recognition of a target disease-associated epitope, compared to a lower avidity T CAR.
  • the recognition domain includes the site at which the T cell receptor physically interacts with the epitope.
  • the avidity of a T cell receptor includes the affinity of the particular epitope with the T cell receptor, but also includes the TCR binding time with the target antigen/MHC complex. At various levels of TCR avidity, calcium mobilization occurs, target cell lysis occurs, or full activation with T cell proliferation occurs. Thus, avidity can be measured by chromium release assay, based on SD50 values (concentration of peptide needed to achieve maximal specific lysis).
  • the Kd values of the T CAR cell receptor to antigen binding affinity ranges from about 5.1 to about 6.1 to about 7.1 to about 8.1 to about 9.1 to about 10.1 to about 11.1 to about 12.1 to about 13.1 to about 14.1 to about 15.1 to about 16.1 to about 17.1 to about 18.1 to about 19.1 to about 20.1 nanomolar.
  • the functional avidity of the T CAR cell receptor to antigen ranges from about 1.0-4.0 log pg/mL for half the maximal inhibition efficiency.
  • the T CAR cell receptor functional avidity includes about 2.0, about 2.5, or about 3.0 log pg/mL for half the maximal inhibition efficiency.
  • the T CAR avidity is inversely proportional to the amount of antigen needed to stimulate a response.
  • the higher the level of antigen needed the lower the avidity of the T CAR, whereas if the avidity level of the T CAR is higher, then a lower amount of antigen is needed to stimulate a response.
  • the avidity of the T CAR can be modulated to increase the receptor affinity or time contacted with antigen, for overall increased functional avidity.
  • the avidity of the T CAR can be modulated to decrease the overall functional avidity, for example by adjusting the receptor affinity or time contacted with antigen, according to standard practices.
  • the disease-associated antigens are further compared with antigens from one or more vaccinated subjects.
  • a comparison is made between diseased and non-diseased tissues (whether from Universal or Individual Subject) and secondarily the output from that first comparison is further compared with vaccinated tissues (from a population or from an individual subject).
  • a comparison is made directly between diseased and vaccinated tissues (whether Universal or Individual Subject) without first comparing with non-diseased non- vaccinated tissues.
  • a comparison is made directly between non- diseased and vaccinated tissues (whether Universal or Individual Subject) without first comparing with diseased tissues.
  • a method for immunotherapy of a subject comprises:
  • a composition comprises: a Universal Subject Disease-Specific library of engineered T cells including one or more multi-specific Chimeric Antigen Receptors each with selected specificity for two or more epitopes of one or more
  • a composition comprises: a Universal Subject Disease-Specific library of engineered T cells including one or more multi-specific Chimeric Antigen Receptor with selected avidity for two or more epitopes of one or more Universal Subject Disease-Specific Antigens on a single receptor.
  • a composition comprises: an
  • Individual Subject Disease-Specific library of engineered T cells including one or more multi-specific Chimeric Antigen Receptor with selected avidity for two or more epitopes of one or more Individual Subject Disease-Specific Antigens on a single receptor.
  • a composition comprises: an Individual Subject Disease- Specific library of engineered T cells including one or more multi-specific Chimeric
  • Antigen Receptors each with selected specificity for two or more epitopes of one or more Individual Subject Disease-Specific Antigens on a single receptor.
  • a system comprises: one or more input/output devices having a non-transitory signal bearing medium operable to accept at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects; accept at least one input related to gene expression information from one or more diseased tissue antigens from two or more subjects;
  • the differential threshold is based on a pre-determined avidity of the T CAR; and generate a Universal Subject Disease-Specific Antigen dataset for the differential antigen values.
  • a system comprises: one or more input/output devices having a non-transitory signal bearing medium operable to accept at least one input related to gene expression information from one or more non-diseased tissue antigens from a diseased subject to be treated; accept at least one input related to gene expression information from one or more diseased tissue antigens from the diseased subject to be treated; compare the input related to gene expression information from the one or more non-diseased tissue antigens from the diseased subject with the input related to gene expression information from the corresponding one or more diseased tissue antigens from the diseased subject; generate a differential antigen value based on the comparison;
  • the differential threshold is based on a pre-determined avidity of the T CAR; and generate an Individual Subject Disease-Specific Antigen dataset for the differential antigen values.
  • an article of manufacture comprises: a structure for accepting data related to a subject; a structure for receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects; a structure for receiving at least one input related to gene expression information from one or more diseased tissue antigens from two or more subjects; a structure for comparing the input related to gene expression information from one or more non- diseased tissue antigens from two or more subjects with the input related to gene expression information from the corresponding one or more diseased tissue antigens from two or more subjects; a structure for generating a differential antigen value based on the comparison; a structure for reinitiating accepting at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects, and comparing with the input related to gene expression information from the corresponding gene of the one or more diseased tissue antigens until a differential threshold is satisfied in one embodiment the differential threshold is based on a pre- determined avidity of the T C
  • an article of manufacture comprises: a structure for accepting data related to a subject; a structure for receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from a diseased subject to be treated; a structure for receiving at least one input related to gene expression information from one or more diseased tissue antigens from the diseased subject to be treated; a structure for comparing the input related to gene expression information from the one or more non-diseased tissue antigens from the diseased subject with the input related to gene expression information from the corresponding one or more diseased tissue antigens from the diseased subject;
  • the differential threshold includes a value, such as a numerical value, and can include a relative value, of one or more qualitative or quantitative gene expression information characteristics. Such qualitative and quantitative gene expression information characteristics have been described herein.
  • the differential threshold also includes one or more components related to a predetermined avidity (T CAR affinity, time contacted to antigen, etc.) and in an embodiment the predetermined avidity is based on the functional avidity of the T CAR.
  • a method executed on a computing device comprises accepting data related to a subject; receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects; receiving at least one input related to gene expression information from one or more diseased tissue antigens from two or more subjects;
  • the differential threshold is based on a pre-determined avidity of the T CAR; and generating a Universal Subject Disease-Specific Antigen dataset for the differential antigen values.
  • a method executed on a computing device comprises accepting data related to a subject; receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from a diseased subject to be treated; receiving at least one input related to gene expression information from one or more diseased tissue antigens from the diseased subject to be treated; comparing the input related to gene expression information from the one or more non-diseased tissue antigens from the diseased subject with the input related to gene expression information from the corresponding one or more diseased tissue antigens from the diseased subject; generating a differential antigen value based on the comparison; reinitiating accepting at least one input related to gene expression information from one or more non-diseased tissue antigens from the diseased subject, and comparing with the input related to gene expression information of the corresponding gene of the one or more diseased tissue antigens until a differential threshold is satisfied in one embodiment the differential threshold is based on a pre-determined avidity of the T CAR; and generating an Individual Subject Disease-
  • a system comprises circuitry configured for accepting data related to a subject; circuitry configured for receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects; circuitry configured for receiving at least one input related to gene expression information from one or more diseased tissue antigens from two or more subjects;
  • circuitry configured for comparing the input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects with the input related to gene expression information from the corresponding one or more diseased tissue antigens from two or more subjects; circuitry configured for generating a differential antigen value based on the comparison; circuitry configured for reinitiating accepting at least one input related to gene expression information from one or more non-diseased tissue antigens from two or more subjects, and comparing with the input related to gene expression information from the corresponding gene of the one or more diseased tissue antigens until a differential threshold is satisfied in one embodiment the differential threshold is based on a pre-determined avidity of the T CAR; and circuitry configured for generating a Universal Subject Disease-Specific Antigen dataset for the differential antigen values.
  • circuitry configured for accepting data related to a subject; circuitry configured for receiving at least one input related to gene expression information from one or more non-diseased tissue antigens from a diseased subject to be treated; circuitry configured for receiving at least one input related to gene expression information from one or more diseased tissue antigens from the diseased subject to be treated; circuitry configured for comparing the input related to gene expression information from the one or more non-diseased tissue antigens from the diseased subject with the input related to gene expression information from the
  • circuitry configured for generating a differential antigen value based on the comparison; circuitry configured for reinitiating accepting at least one input related to gene expression information from one or more non-diseased tissue antigens from the diseased subject, and comparing with the input related to gene expression information of the corresponding gene of the one or more diseased tissue antigens until a differential threshold is satisfied in one embodiment the differential threshold is based on a pre-determined avidity of the T CAR; and circuitry configured for generating an Individual Subject Disease-Specific Antigen dataset for the differential antigen values.
  • Disease-Specific Antigens includes particular antigens that originate from viruses (in diseases caused by viruses), and also includes antigens that are not particular to any specific histology or tissue but instead are common to disease, such as tumors.
  • the Disease- Associated Antigens are protein structures that contain mutation sin their sequence or are exhibit aberrant expression on tumor or diseased tissue versus normal tissue.
  • some Disease-Specific Antigens include, but are not limited to tumor-associated antigens such as EBNA-3, E6, E7, carcinoembryonic antigen (CEA), her-2/neu, Muc-1, MART-1, gplOO, tyrosinase, p53, beta-catenin, CDK4,
  • tumor-associated antigens such as EBNA-3, E6, E7, carcinoembryonic antigen (CEA), her-2/neu, Muc-1, MART-1, gplOO, tyrosinase, p53, beta-catenin, CDK4
  • some Disease-Specific Antigens include, but are not limited to tumor-associated antigens such as O antigens (lipopolysaccharide molecules from bacterial membranes), H antigens (components of flagella), K antigens (polysaccharides from bacterial capsules), or "clumping factors" (molecules bound to the outer surface causing agglutination.
  • tumor-associated antigens such as O antigens (lipopolysaccharide molecules from bacterial membranes), H antigens (components of flagella), K antigens (polysaccharides from bacterial capsules), or "clumping factors" (molecules bound to the outer surface causing agglutination.
  • some Disease-Specific Antigens include, but are not limited to, viral envelope proteins or other viral proteins.
  • construction of a Universal Memory T cell Line is conducted, for example, by engineering T memory cells (Tcm) at the Cre/lox recombination site, as drawn 100, by standard homologous recombination techniques.
  • Tcm T memory cells
  • the nucleic acid molecule is placed into the host cell such that the innate T cell receptor constant region alpha chain is disrupted, 110, thereby prohibiting innate T cell receptor formation in the host cell.
  • the engineered cells are selected for CD3(-), CD8+ 120.
  • HLA gene expression is optionally altered such that innate host cell NK and T cell elimination of the engineered T CAR cells is reduced or eliminated.
  • silencing RNA silencing RNA (siRNA or shRNA) are utilized to prevent expression of HLA I and/or HLA II, while HLA-E is engineered into the vector, as well as selection markers (e.g., hygromycin, thymidine kinase) 130.
  • selection markers e.g., hygromycin, thymidine kinase
  • the plasmid expression vector is transfected into the Cre/lox Tcm cell line as described 140 and positive clones selected 150 based on the selection criteria.
  • the resulting Tcm cell clones are Universal Recipient Tcm cell lines as described 160.
  • the structure of a multi-specific CAR includes at least two scFVs each specific to two different epitopes of the same antigen, or two different antigens 200.
  • the artificially engineered multi-specific CAR is utilized for site-specific integration into the Universal recipient Tcm cell line, as described herein. For example, at the lox sites of the targeting vector 210, 220 with Cre recombinase expression vector 230 and CMV promoter, with DHFR selectable marker 240.
  • a multi-specific T CAR recognizes two or more different epitopes of one antigen. That is, a T CAR expressed as a single molecule includes multi-specific epitope recognition. Having recognition of two or more epitopes of a single antigen provides for a greater likelihood of the T CAR contacting the target antigen. Moreover, since the multi-specific epitope recognition sites are both on a single molecule, there is a greater chance of physical contact with the target antigen.
  • Figure 2C illustrates a diseased cell (e.g., tumor cell) exhibiting multiple epitopes of a particular antigen (Epitope 1 and Epitope 2 of Disease-Specific Antigen 1) that are specifically recognized by the multi-specific T CAR expressed on the T cell (also illustrated is the internal T CAR signaling domain for activation of the T CAR upon binding with the cognate antigens).
  • a diseased cell e.g., tumor cell
  • epitopes of a particular antigen epitopes of a particular antigen (Epitope 1 and Epitope 2 of Disease-Specific Antigen 1) that are specifically recognized by the multi-specific T CAR expressed on the T cell (also illustrated is the internal T CAR signaling domain for activation of the T CAR upon binding with the cognate antigens).
  • a multi-specific T CAR recognizes two different antigens. That is, a T CAR expressed as a single molecule includes multi- specific antigen recognition. Having recognition of two or more different antigens increases the likelihood of the T CAR contacting the target antigens. Furthermore, since the multi-specific antigen recognition sites are both on a single molecule, there is a great chance of physical contact with the target antigen (vs. having separate receptors for each antigen).
  • Figure 2D illustrates a diseased cell (e.g., tumor cell) exhibiting multiple antigens (Disease-Specific Antigen 1 and Disease-Specific Antigen 2) that are specifically recognized by the multi-specific T CAR expressed on the T cell (also illustrated is the internal T CAR signaling domain for activation of the T cell upon binding of the T CAR with the cognate antigens).
  • Disease-Specific Antigen 1 and Disease-Specific Antigen 2 that are specifically recognized by the multi-specific T CAR expressed on the T cell (also illustrated is the internal T CAR signaling domain for activation of the T cell upon binding of the T CAR with the cognate antigens).
  • Construction of a polyclonal Tcm library is constructed, for example, by obtaining the listing of all currently identified human cell surface antigens 300, and screening the surface antigens for specific receptor-antigen pairs with scFv genes 310.
  • the parameters for screening are set according to the desired outcome, and in one embodiment two scFvs that specifically recognize two different epitopes on a single antigen are selected 310. From this selection process, a database is created with the sequences, and a freezer stock maintained 310.
  • multi-specific CAR genes are constructed in the lox targeting vector as described herein, for all of the currently identified human cell surface antigens (-3700), and a database and freezer stock maintained 320.
  • the multi-specific CAR vectors are trans fected into the recipient Tcm cell line, resulting in Tcm cell lines expressing multi-specific CARs, and a database and freezer stock are maintained 330, producing a polyclonal Tcm library with bi-specific recognition of approximately 3700 human cell surface antigens 340.
  • an initial differential screening of gene expression data 400 of normal tissue cell surface proteins (antigenic profile over a population for a particular tissue type based on gene expression of non- diseased tissue from multiple samples and/or from multiple subjects) compared with diseased tissue antigens (antigenic profile over a population for a particular diseased tissue based on gene expression of diseased tissue from multiple samples and/or from multiple subjects) is utilized for a computer determination of Population Disease-Specific Antigens (PDSA) based on the differential screening 410.
  • PDSA Population Disease-Specific Antigens
  • a library is generated for the Universal Subject Disease-Specific Library of multi-specific (including bispecific) T CAR cells effective against the PDSA 420.
  • an individual subject with a specific disease is addressed 430.
  • a biopsy or other cell or fluid sample is obtained from the diseased subject and analysis of the sample is performed 440.
  • a computer determination of the antigenic profile of the Individual Subject's Disease Specific Antigens is made based on the analysis of the Individual Subject's diseased cells 450.
  • a computer determination of the selection of which multi- specific T CAR cells correspond to the Individual Subject's Disease-Specific Antigens is conducted 460.
  • T CAR cells specifically selected for the subject based on the analysis of the individual subject's diseased cells in the context of the Universal Subject Disease Specific Library 470.
  • a library can be constructed for any particular tissue type or disease type.
  • several libraries are established for various stages of disease progression (e.g., metastasis, or chronic disease such as Alzheimer's or multiple sclerosis).
  • Figure 4 also describes a flow of information in a system including the computer software and hardware utilized herein.
  • a computer device or system 486 such as appended to the flow chart of Figure 4, can be employed.
  • a CPU 482 or other computer processor includes, for example, digital logic processors capable of processing input, executing algorithms, and generating output as needed in response to the inputs received from an input device (e.g., keyboard, mouse, monitor, internet, scanner, etc. 484).
  • an input device e.g., keyboard, mouse, monitor, internet, scanner, etc. 484
  • Such processors may include a microprocessor, such as ASIC, and may include or be in communication with media, such as non-transitory media, including computer-readable media which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • data is stored or collected by memory (e.g., RAM) 480 includes for example, memory storage device, system memory, or cache memory, and can be stored on media, for example, CD-ROM, DVD-ROM, or DVD, floppy disc, etc. 481.
  • an I/O controller 483 is utilized to manage the data communications of the computer system.
  • the I/O controller 483 includes any of a variety of devices for accepting and processing information from a user (e.g.
  • an output controller of the I/O controller 483 includes, for example, a display device for presenting information to a user (e.g., human or machine; remote or local), and any of the various iterations includes network or other types of remote communication.
  • a transmitter and/or receiver includes circuitry configured for transmitting or receiving information, respectively, as well as any corresponding software.
  • a transmitter and/or receiver includes an antenna, BluetoothTM, or other wireless device, as well as any corresponding software.
  • an Individual Subject Disease-Specific library and Methods of Making and Administering are included.
  • an initial differential screening of gene expression data 500 of normal tissue cell surface proteins (antigenic profile for a particular tissue type based on gene expression of non-diseased tissue from an individual subject) compared with diseased tissue antigens (antigenic profile for a particular tissue based on gene expression of diseased tissue from an individual subject) is utilized for a computer determination of Individual Subject Disease-Specific Antigens (ISDSA) based on the differential screening 510.
  • ISDSA Individual Subject Disease-Specific Antigens
  • a library is generated for the Individual Subject Disease-Specific Library of multi-specific (including bispecific) T CAR cells reactive to the ISDSA 520.
  • computer determination and selection of the T CAR cells most effective for the subject based on the multi-specificity of particular antigens is conducted 530.
  • the individual subject is treated with a therapeutically effective dose of T CAR cells 540.
  • one or more logic devices are included.
  • one or more computing devices are included.
  • Figure 5 also describes a flow of information in a system including the computer software and hardware utilized herein.
  • a computer device or system 594 such as appended to the flow chart of Figure 5, can be employed.
  • a CPU 570 or other computer processor includes, for example, digital logic processors capable of processing input, executing algorithms, and generating output as needed in response to the inputs received from an input device (e.g., keyboard, mouse, monitor, internet, scanner, etc. 590).
  • Such processors may include a microprocessor, such as ASIC, and may include or be in communication with media, such as non-transitory media, including computer-readable media which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • data is stored or collected by memory (e.g., RAM) 550 includes for example, memory storage device, system memory, or cache memory, and can be stored on media, for example, CD-ROM, DVD-ROM, or DVD, floppy disc, etc. 560.
  • an I/O controller 580 is utilized to manage the data communications of the computer system.
  • the I/O controller 580 includes any of a variety of devices for accepting and processing information from a user (e.g.
  • an output controller of the I/O controller 580 includes, for example, a display device for presenting information to a user (e.g., human or machine; remote or local), and any of the various iterations includes network or other types of remote communication.
  • a transmitter and/or receiver includes circuitry configured for transmitting or receiving information, respectively, as well as any corresponding software.
  • a transmitter and/or receiver includes an antenna
  • BluetoothTM or other wireless device, as well as any corresponding software.
  • a listing of gene sequences of all presently identified human cell surface antigens 600 is utilized for a computer
  • a Universal Subject Disease Specific Antigen Library of multi-specific T CAR cells reactive to the listing of gene sequences is generated 620, and an individual subject with a specific disease is addressed 630.
  • a biopsy or other cell or fluid sample is obtained from the individual subject at the disease site and analysis of the individual subject's diseased cells is conducted 640.
  • a computer determination of the Individual Subject's Disease Specific Antigens (ISDSA) based on the analysis of the individual subject's diseased cells 650.
  • computer determination of selection of multi-specific T CAR cells are conducted that correspond to the Individual Subject's Disease-Specific Antigens (ISDSA) 660.
  • the individual subject with specific disease is treated with a therapeutically effective dose of selected T CAR cells based on the analysis of the individual subject's disease-specific antigens in light of the library.
  • Figure 6 also describes a flow of information in a system including the computer software and hardware utilized herein.
  • a computer device or system 617 such as appended to the flow chart of Figure 6, can be employed.
  • a CPU 612 or other computer processor includes, for example, digital logic processors capable of processing input, executing algorithms, and generating output as needed in response to the inputs received from an input device (e.g., keyboard, mouse, monitor, internet, scanner, etc. 614).
  • an input device e.g., keyboard, mouse, monitor, internet, scanner, etc. 614.
  • Such processors may include a microprocessor, such as ASIC, and may include or be in communication with media, such as non-transitory media, including computer-readable media which stores instructions that, when executed by the processor, cause the processor to perform the steps described herein.
  • data is stored or collected by memory (e.g., RAM) 611 includes for example, memory storage device, system memory, or cache memory, and can be stored on media, for example, CD-ROM, DVD-ROM, or DVD, floppy disc, etc. 618.
  • an I/O controller 613 is utilized to manage the data communications of the computer system.
  • the I/O controller 613 includes any of a variety of devices for accepting and processing information from a user (e.g.
  • an output controller of the I/O controller 613 includes, for example, a display device for presenting information to a user (e.g., human or machine; remote or local), and any of the various iterations includes network or other types of remote communication.
  • a transmitter 615 and/or receiver 616 to transmit or receive data from one database to another or from one central data processing facility to one or more user facilities.
  • a transmitter and/or receiver includes circuitry configured for transmitting or receiving information, respectively, as well as any corresponding software.
  • a transmitter and/or receiver includes an antenna, BluetoothTM, or other wireless device, as well as any corresponding software.
  • the computing system includes, for example, at least one of a notebook computer, a work station, a personal data device, a desktop computer, a cluster of processors, a cluster of servers, a cloud computing center, a mobile telephone, or other computing device.
  • a computer or other processing unit is configured to receive or transmit information relating to receipt of information from a subject by, for example, a USB cable or wireless network. In an embodiment, a computer or other processing unit is configured for receiving or storing information.
  • the computer or other processing unit is configured to allow input or output of additional information or sharing of information, for example, with a subject's personal medical file or population medical health records. That is, in an embodiment, input or output of information related to two or more subjects or an individual subject can be shared with public health databases, or transferred to a subject's specific medical record (including electronic records). In an embodiment, the information shared with public health databases is anonymized such that identifying information related to the subject has been removed, altered, or encrypted.
  • Example 1 Construction of a universal recipient T cell clone for efficient CAR gene integration and T cell library construction.
  • a cloned universal recipient T cell with a targeted recombination site for site-specific introduction of chimeric antigen receptor (CAR) genes is constructed from human cytotoxic T lymphocytes (CTL) using a Cre/lox recombination system.
  • CTL cytotoxic T lymphocytes
  • CAR chimeric antigen receptor
  • Tcm central memory T cells
  • PBMNC peripheral blood mononuclear cells
  • CD8 and CD62L antibodies and cell sorter available from BD Biosciences, San Jose, CA.
  • Tcm T cell receptor alpha chain
  • Tcm with the targeting site vector DNA, including the lox recombination site, inserted in the TCR Ca gene are isolated by cell sorting for CD8 + , CD3 neg cells and selecting for drug-resistance (conferred by a drug resistance marker, e.g., neomycin resistance gene (neo) on the targeting DNA construct).
  • a drug resistance marker e.g., neomycin resistance gene (neo) on the targeting DNA construct.
  • CD3 neg Tcm cells with a lox recombination site disrupting the TCRa gene are expanded in vitro and subjected to additional recombinant DNA modifications to prevent recognition and killing of the universal recipient T cell clone by host T cells and NK cells.
  • the CD3 neg Tcm cells bearing a lox recombination site are genetically modified to prevent recognition by host (i.e., patient) T cells and NK cells.
  • Expression of HLA Class I and Class II genes is down-regulated to prevent T cell recognition of the universal Tcm cells. See Fig. IB 130.
  • short hairpin RNAs shRNA which bind to mRNA encoding Class I (HLA-A,-B, -C) and Class II (HLA -DR, -DQ, -DP) and prevent their translation into protein may be used to down- regulate HLA protein expression.
  • Various expression vectors and shRNA nucleotide sequences to produce shRNAs in T cells that inhibit HLA protein expression can be utilized with various embodiments described herein (see e.g., U.S. Patent Application No. 2007/0036773 by Cooper et al. published on Feb. 15, 2007 which is incorporated herein by reference).
  • a 58 nucleotide shRNA which inhibits HLA -A, -B and -C alpha chain expression may be encoded in a plasmid vector containing a selectable marker (see e.g., U.S. Patent Appl. No. 2007/0036773, Ibid.).
  • HLA-E a nonclassical HLA antigen, HLA-E, is expressed on the Tcm cell surface using an expression construct for HLA-E.
  • a DNA construct encoding HLA-E under the control of a constitutive promoter element and a selection/suicide gene, hygromycin phosphotransferase/thymidine kinase gene (Hy/TK) are described (see e.g., U.S. Patent Application No. 2007/0036773, Ibid.).
  • the Hy/TK gene may be used for positive (Hy) or negative (TK) selection, followed by transfection of Cre/lox Tcm cell line 140.
  • Thymidine kinase serves as a suicide gene in the Tcm cells in the event they need to be eliminated in vivo.
  • Administration of gancyclovir to Tcm cells expressing TK results in the production of toxic metabolites which kill the Tcm cells.
  • Universal Tcm cells which are: CD3 neg , HLA Class I neg , HLA Class II neg , resistant to G418 (i.e. ne ), resistant to hygromycin, and express HLA-E are selected and cloned 150. See Figure IB.
  • the universal recipient Tcm cells are used to create Tcm expressing CAR genes through a process of site-specific integration, as described in Figure IB 160.
  • Example 2 Efficient site-specific integration of CAR genes in the universal recipient Tcm cell line. As illustrated in Figure 1 A, 1 B, 2A, and 2B, multi-specific (at least two specific receptor recognition sites on the same CAR) CAR genes are integrated at the lox recombination site constructed in the universal recipient Tcm cell clone at the TCR Ca locus on chromosome 14(see Figs. 1A and IB).
  • a lox targeting vector which contains: lox recombination sites, a multi-specific CAR gene and a selection marker gene, e.g., dihydrofolate reductase (DHFR) which confers resistance to a cytotoxic drug, methotrexate (MTX).
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • Universal recipient Tcm cells are cotransfected with a vector which directs transient expression of the Cre recombinase 100 (see e.g., US Patent No. 6,130,364, Ibid .
  • Cre-mediated recombination between the lox sites in the Tcm cell and the lox targeting vector results in integration of the multi- specific CAR gene and DHFR at the TCRa locus on chromosome 14 110.
  • the transfected Tcm cells are selected in MTX-containing medium and selected for expression of the multi-specific CAR protein using flow cytometry. For example, fluorescent antibodies specific for human IgG Fc hinge are used to detect Tcm cells expressing a CAR gene. Site specific integration in approximately 16% of the recipient cells may be observed (see e.g., Araki et al, 1997, Ibid)
  • Example 3 Construction of polyclonal T cell library expressing CARs recognizing currently identified human cell surface proteins.
  • a library of CAR genes is constructed to specifically recognize each of all currently identified human cell surface antigens. See Figure 3. CARs are constructed with single chain antibodies providing specific binding to cell surface antigens. A
  • Single chain variable region fragments which bind to each cell surface antigen (based on the known sequences thereof) may be identified by screening phage display libraries comprised of human SCFv 310.
  • bacteriophage expressing a library of approximately 3 X 10 SCFv may be screened for binding to cell surface antigens (see e.g., De Kruif et al, Proc. Natl. Acad. Sci. USA 92: 3938-3942, 1995 and Rader et al, Current Opinion Biotechnology 8: 503-508, 1997 which are incorporated herein by reference).
  • Cell surface antigens to screen the SCFv phage library may be made using recombinant DNA methods to synthesize the corresponding genes and express them in vitro (see e.g., Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. which is incorporated herein by reference).
  • antibodies and SCFv recognizing cell surface antigens may be obtained from an online database of antibodies against human protein targets (see e.g., Antibodypedia Nature database online at: antibodypedia dot com.)
  • the identification numbers and locations of the bacteriophage-SCFv clones and the corresponding cell surface antigens they recognize are stored in a searchable computer database.
  • the SCFv clones are stored as bacteriophage frozen stocks which can be used to amplify the DNA sequences encoding the SCFvs and incorporate them in a CAR.
  • a gene encoding a CAR may contain: a SCFv, a human IgGl Fc hinge domain, a CD4 transmembrane domain, and portions from the cytoplasmic domains of CD28, CD137 and CD3 zeta chain (see e.g., Park et al. Trends in
  • a library of CAR genes is created in a lox targeting vector with specificities for each of all currently identified cell surface antigens using recombinant DNA methods (see e.g., Sambrook and Russell, Ibid.)
  • a library of multi-specific CAR genes is created by pairing SCFvs specific for surface antigens. For example, two different scFvs which recognize two different epitopes from a single cell surface protein may be paired to create a multi-specific CAR. See Figure 2A 200. Alternatively, scFvs recognizing two different cell surface proteins may be paired to construct a multi-specific CAR.
  • a searchable database is created with records for each multi-specific CAR vector including: antigen specificities, corresponding SCFvs and the freezer location of DNA stocks for each lox targeting vector.
  • a polyclonal Tcm cell library is constructed using the universal recipient Tcm cells and the CAR gene library created in the lox targeting vector. See Figure 2B 210-230, and Figure 3 330-340. Individual CAR genes are transfected into aliquots of approximately 10 6 recipient cells in individual wells of a multiwell plate (50-100 wells) and placed in selective media containing MTX. See Figure 2B 240. Surviving Tcm cells are screened using flow cytometry to identify cells expressing CAR on their cell surface.
  • fluorescent antibodies recognizing the IgGl hinge Fc segment on the CARs may be used for flow analysis.
  • Systems and methods for high throughput mammalian cell transfection and selection are described (see e.g., Muller-Hartmann et al, Expert Opin. Drug Discov. 2: 1453-1465, 2007 which is incorporated herein by reference).
  • a searchable database is created with records for each Tcm line detailing the location of the Tcm cell stock, the multi-specific CAR expressed, the antigens recognized and the SCFv clones employed 330.
  • a polyclonal Tcm library comprised of approximately 5000 Tcm lines is stored as separate frozen stocks for each Tcm line using standard cryogenic methods for
  • Tcm lines expressing multi-specific CARs specific for currently identified human cell surface proteins, viral antigens, and other pathogens may be included in the library.
  • Example 4 Treatment of a breast cancer patient with multiple Tcm lines expressing multi-specific CARs.
  • a human subject that is a patient with breast cancer is treated with multiple Tcm lines which are selected based upon the gene expression profile of the patient's tumor cells.
  • the patient's breast cancer tumor is surgically removed, and frozen sections are analyzed to obtain tumor cells for preparation of RNA.
  • the identity of all genes expressed by the tumor cells i.e., the transcriptome
  • Methods and instruments to isolate, amplify and determine the identity, i.e., nucleotide sequence, of all mRNAs expressed by a tumor cell are identified (see e.g., Curtis et al, Nature 486: 346-352, 2012 and the Technical Bulletin: Whole-Genome Expression Analysis... available from
  • Tcm lines expressing multi-specific CARs specific for cell surface antigens expressed by the patient's tumor cells are located from the searchable database and expanded.
  • a breast cancer tumor cell expressing the tumor associated antigens: HER-2/neu, MUC-1, and EGFR may be treated with three Tcm lines, expressing CAR specific for each of the surface antigens.
  • Multi-specific CARs provide increased efficacy.
  • multi-specific antibodies have been developed for targeting cells expressing two antigens (see e.g., U.S. Patent No. 5,601,819, and
  • Tcm lines expressing multi-specific CARs recognizing Her2 or MUCl or EGFR are retrieved from the library and expanded in vitro to obtain approximately 10 9 cells of each line.
  • the patient is infused with approximately 10 7 cells/kg of each Tcm line.
  • the transcriptome of metastatic tumor cells or circulating tumor cells may be determined. Tcm lines recognizing a new or altered set of surface antigens are selected from the library and administered to the patient.
  • metastatic breast cancer cells may lose expression of cell surface antigens ⁇ e.g., HLA-C) and gain expression of other antigens ⁇ e.g., CD51, integrin a v ) (see e.g., Kischel et al, Neoplasia 10: 1014-1020, 2008 which is incorporated herein by reference.
  • Tcm clones specific for the current set of surface antigens expressed by the metastatic tumor cells are selected from the Tcm library, expanded and administered to the patient.
  • an implementer may opt for a mainly hardware and/or firmware vehicle; alternatively, if flexibility is paramount, the implementer may opt for a mainly software implementation; or, yet again alternatively, the implementer may opt for some combination of hardware, software, and/or firmware.
  • any vehicle to be utilized is a choice dependent upon the context in which the vehicle will be deployed and the specific concerns (e.g., speed, flexibility, or predictability) of the implementer, any of which may vary.
  • Those skilled in the art will recognize that optical aspects of implementations will typically employ optically-oriented hardware, software, and/or firmware.
  • ASICs Integrated Circuits
  • FPGAs Field Programmable Gate Arrays
  • DSPs digital signal processors
  • ASICs Integrated Circuits
  • FPGAs Field Programmable Gate Arrays
  • DSPs digital signal processors
  • ASICs Integrated Circuits
  • computers e.g., as one or more programs running on one or more computer systems
  • processors e.g., as one or more programs running on one or more microprocessors
  • firmware e.g., as one or more programs running on one or more microprocessors
  • Examples of a signal-bearing medium include, but are not limited to, the following: a recordable type medium such as a floppy disk, a hard disk drive, a Compact Disc (CD), a Digital Video Disk (DVD), a digital tape, a computer memory, etc.; and a transmission type medium such as a digital and/or an analog communication medium (e.g., a fiber optic cable, a waveguide, a wired communications link, a wireless communication link, etc.).
  • electro-mechanical system includes, but is not limited to, electrical circuitry operably coupled with a transducer (e.g., an actuator, a motor, a piezoelectric crystal, etc.), electrical circuitry having at least one discrete electrical circuit, electrical circuitry having at least one integrated circuit, electrical circuitry having at least one application specific integrated circuit, electrical circuitry forming a general purpose computing device configured by a computer program (e.g., a general purpose computer configured by a computer program which at least partially carries out processes and/or devices described herein, or a microprocessor configured by a computer program which at least partially carries out processes and/or devices described herein), electrical circuitry forming a memory device (e.g., forms of random access memory), electrical circuitry forming a communications device (e.g., a modem, communications switch, or optical-electrical equipment), and any non-electrical analog thereto, such as optical or other analogs.
  • a transducer e.g., an actuator, a motor, a piezo
  • electromechanical as used herein is not necessarily limited to a system that has both electrical and mechanical actuation except as context may dictate otherwise.
  • electrical circuitry includes, but is not limited to, electrical circuitry having at least one discrete electrical circuit, electrical circuitry having at least one integrated circuit, electrical circuitry having at least one application specific integrated circuit, electrical circuitry forming a general purpose computing device configured by a computer program (e.g., a general purpose computer configured by a computer program which at least partially carries out processes and/or devices described herein, or a microprocessor configured by a computer program which at least partially carries out processes and/or devices described herein), electrical circuitry forming a memory device (e.g., forms of random access memory), and/or electrical circuitry forming a communications device (e.g., a modem, communications switch, or optical-electrical equipment).
  • a computer program e.g., a general purpose computer configured by a computer program which at least partially carries out processes and/or devices described herein, or a microprocessor configured by a computer program which at least partially carries out processes and/or devices described herein
  • electrical circuitry forming a memory device
  • examples of such other devices and/or processes and/or systems might include - as appropriate to context and application— all or part of devices and/or processes and/or systems of (a) an air conveyance (e.g., an airplane, rocket, hovercraft, helicopter, etc.), (b) a ground conveyance (e.g., a car, truck, locomotive, tank, armored personnel carrier, etc.), (c) a building (e.g., a home, warehouse, office, etc.), (d) an appliance (e.g., a refrigerator, a washing machine, a dryer, etc.), (e) a communications system (e.g., a networked system, a telephone system, a voice-over IP system, etc.), (f) a business entity (e.g., an Internet Service Provider (ISP) entity such as Comcast Cable, Quest, Southwestern Bell, etc.), or (g) a wired/wireless services entity (e.g., such as Sprint,
  • ISP Internet Service Provider (
  • a user may be representative of a human user, a robotic user (e.g., computational entity), and/or substantially any combination thereof (e.g., a user may be assisted by one or more robotic ).
  • a user as set forth herein, although shown as a single entity may in fact be composed of two or more entities.
  • a user in general, the same may be said of "sender” and/or other entity-oriented terms as such terms are used herein.
  • any two components so associated can also be viewed as being “operably connected”, or “operably coupled”, to each other to achieve the desired functionality, and any two components capable of being so associated can also be viewed as being “operably couplable”, to each other to achieve the desired functionality.
  • operably couplable include but are not limited to physically mateable and/or physically interacting components and/or wirelessly interactable and/or wirelessly interacting components and/or logically interacting and/or logically interactable components.

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Abstract

Des modes de réalisation décrits dans l'invention concernent des procédés, des dispositifs et des systèmes informatiques correspondants pour la dérivation de bibliothèques de CAR de lymphocytes T (sujet universel ou sujet individuel) pour un traitement personnalisé d'une maladie chez un sujet. Dans certains modes de réalisation, le criblage différentiel de données d'expression de tissu normal et de tissu malade est utilisé pour déterminer des antigènes spécifiques à une maladie et générer ainsi des lymphocytes T à CAR réactifs à ces antigènes pour former une bibliothèque spécifique à la maladie. Dans certains modes de réalisation, la détermination des clones de CAR de lymphocytes T les plus efficaces de la bibliothèque spécifique à la maladie est basée sur les propres antigènes spécifiques à la maladie du sujet. Dans certains modes de réalisation, un sujet est traité par une quantité thérapeutiquement efficace de clones de CAR de lymphocytes T.
PCT/US2014/023650 2013-03-14 2014-03-11 Compositions, procédés et systèmes informatiques associés à la production et l'administration de lymphocytes t modifiés WO2014159435A1 (fr)

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