WO2020076568A1 - Procédés et compositions pour le traitement du cancer - Google Patents

Procédés et compositions pour le traitement du cancer Download PDF

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WO2020076568A1
WO2020076568A1 PCT/US2019/054188 US2019054188W WO2020076568A1 WO 2020076568 A1 WO2020076568 A1 WO 2020076568A1 US 2019054188 W US2019054188 W US 2019054188W WO 2020076568 A1 WO2020076568 A1 WO 2020076568A1
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cell line
tumor
atcc
cells
subject
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Eckhard R. Podack
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University Of Miami
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70532—B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/99—Coculture with; Conditioned medium produced by genetically modified cells

Definitions

  • the invention relates to compositions comprising genetically modified tumor cell lines and methods for their use in treating immuno!ogically cold tumors.
  • Cancer immunotherapy is a rapidly evolving field. Despite promising results, long- lasting response rates remain around 10-20% for patients receiving single agent therapy.
  • Tumors may be divided into‘hot * (T cell inflamed) or‘cold’ (T cell non-inflamed) according to the presence of immune cells in the tumor microenvironment with a particular emphasis on adaptive immune cells.
  • CPI checkpoint inhibitor
  • the mechanism of recruitment of adaptive immune cells to the tumor microenvironment can be complicated and influenced by tumor intrinsic mechanisms (for instance neoantigen generation) and tumor extrinsic mechanisms specific to the generation of proteins important for leukocyte migration.
  • Chemokines can attract T cells to the tumor site and tumor intrinsic pathways can influence the composition of local chemokines.
  • Tumor-induced vasculature can hamper T cell migration.
  • Other immune cells and tumor-derived molecules can block T cell proliferation and survival. Enhancing T cell infiltration may increase response rates to immunotherapy and increase survival.
  • the present invention overcomes previous shortcomings in the art by providing compositions and methods that provide enhanced T cell infiltration and improved cancer treatment therapies.
  • One aspect of the invention relates to a method of treating an immunologically cold tumor in a subject in need thereof, comprising administering to the subject an effective amount of a tumor cell line genetically modified to express a nucleic acid encoding CD80
  • HLA human leukocyte antigen
  • a second aspect of the invention relates to a method of increasing infiltration of immune cells into an immunologically cold tumor in a subject in need thereof comprising administering to the subject an effective amount of a tumor ccl 1 line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA), thereby increasing infiltration of immune cells into the immunologically cold tumor of the subject.
  • a tumor ccl 1 line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA)
  • a third aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an immunogenic amount of a tumor cell line genetically modified to express a nucleic acid encoding B7.1 (CD80) and a nucleic acid encoding a human leukocyte antigen (HLA), and a
  • tumor cell line is not an adenocarcinoma lung cancer cell line.
  • SEQ ID NO:2 - B7.I (CD80) protein sequence Homo sapiens
  • SEQ ID NO:4 El protein sequence ( Bovine papillomavirus )
  • SEQ ID NO:5 E2 protein sequence ( Bovine papillomavirus )
  • SEQ ID NO:6 Beta-lactamase protein sequence ( Escherichia coif)
  • “about X” where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measureable value may include any other range and/or individual value therein.
  • phrases such as“between X and Y” and“between about X and Y” should be interpreted to include X and Y.
  • phrases such as“between about X and Y” mean“between about X and about Y” and phrases such as“from about X to Y” mean “from about X to about Y.”
  • transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.
  • the term“consisting essentially of’ when used in a claim of this invention is not intended to be interpreted to be equivalent to“comprising.”
  • the terms“increase,”“increasing,”“increased,”“enhance,” “enhanced,”“enhancing,” and“enhancement” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.
  • the terms“reduce,”“reduced,”“reducing,”“reduction,”“diminish,” and“decrease” describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% as compared to a control.
  • the reduction can result in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.
  • a“therapeutically effective” amount refers to an amount is an amount that provides some improvement or benefit to the subject.
  • a“therapeutically effective” amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject (e.g., in the case of cancer, reduction in tumor burden, prevention of further tumor growth, prevention of metastasis, or increase in survival time).
  • an effective amount of a tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) may be an amount sufficient to reduce the tumor burden of an immunologically cold tumor in a subject, prevent further growth of the tumor and/or increase survival time of a subject Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.
  • the therapeutically effective amount of the agent may vary depending on the tumor being treated and its severity as well as the age, weight, etc., of the patient to be treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions of the present invention may also be administered in combination with one or more additional therapeutic compounds (e.g., one or more checkpoint inhibitors).
  • the immunotherapeutic cell lines of the invention may be administered to a subject having one or more signs or symptoms of a disease or disorder (e.g., an immunologically cold tumor).
  • “treat,”“treating,” or“treatment of,” it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.
  • antibody as used herein include polyclonal, monoclonal, single chain, chimeric, humanized and human antibodies, prepared according to conventional methodology.
  • tumorigenicity refers primarily to the tumor status of a cell or cells (e.g., the extent of neoplastic transformation of a cell, the malignancy of a cell, the propensity for a cell to form a tumor and/or have characteristics of a tumor, or simply the presence or absence of tumor cells in a patient or tissue/organ), which is reflective of a change of a cell or population of cells from a normal to malignant state.
  • Tumorigenicity indicates that tumor cells are present in a sample, and/or that the transformation of cells from normal to tumor cells is in progress, as may be confirmed by any standard of measurement of tumor development.
  • the change typically involves cellular proliferation at a rate which is more rapid than the growth observed for normal cells under the same conditions, and which is typically characterized by one or more of the following traits: continued growth even after the instigating factor (e.g., carcinogen, virus) is no longer present; a lack of structural
  • a tumor therefore, is most generally described as a proliferation of cells (e.g., a neoplasia, a growth, a polyp) resulting from neoplastic growth and is most typically a malignant tumor.
  • a neoplasia is malignant or is predisposed to become malignant.
  • Malignant tumors are typically characterized as being anaplastic (primitive cellular growth characterized by a lack of differentiation), invasive (moves into and destroys surrounding tissues) and/or metastatic (spreads to other parts of the body).
  • nucleic acid As used herein,“nucleic acid,”“nucleotide sequence,” and“polynucleotide” are used interchangeably and encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA and chimeras of RNA and DNA.
  • polynucleotide, nucleotide sequence, or nucleic acid refers to a chain of nucleotides without regard to length of the chain.
  • the nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand.
  • the nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.
  • the present invention further provides a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid, nucleotide sequence, or polynucleotide of this invention.
  • an“isolated polynucleotide” is a nucleotide sequence (e.g., DNA or RNA) that is not immediately contiguous with nucleotide sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • an isolated nucleic acid includes some or all of the 5’ non-coding (e.g., promoter) sequences that are immediately contiguous to a coding sequence.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g. , a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment), independent of other sequences. It also includes a recombinant DNA that is part of a hybrid nucleic acid encoding an additional polypeptide or peptide sequence.
  • polynucleotide that includes a gene is not a fragment of a chromosome that includes such gene, but rath» includes the coding region and regulatory regions associated with the gene, but no additional genes naturally found on the chromosome.
  • isolated can refer to a nucleic acid, nucleotide sequence or polypeptide that is substantially free of cellular material, viral material, and/or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized).
  • an“isolated fragment” is a fragment of a nucleic acid, nucleotide sequence or polypeptide that is not naturally occurring as a fragment and would not be found in the natural state.“Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to provide the polypeptide or nucleic acid in a form in which it can be used for the intended purpose.
  • an isolated cell refers to a cell that is separated from other components with which it is normally associated in its natural state.
  • an isolated cell can be a cell in culture medium and/or a cell in a pharmaceutically acceptable carrier of this invention.
  • an isolated cell can be delivered to and/or introduced into a subject.
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then returned to the subject.
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then administered to a different subject (allogeneic).
  • an isolated cell can be a cell that is removed from a subject and manipulated as described herein ex vivo and then administered to the same subject (autologous).
  • a cell useful with the invention may be a cell that is from a subject and administered to the same subject
  • a cell useful with the invention may be a cell from a known cell line.
  • a“genetically modified cell” refers to a cell that has been genetically modified to express an exogenous nucleic acid, for example, by transfection or transduction.
  • a cell may be genetically modified to express, for example, a nucleic acid encoding CD80 (B7.1) and/or a nucleic acid encoding an HLA antigen, as disclosed herein.
  • CD80 B7.1
  • HLA antigen HLA antigen
  • a cell is to be genetically modified to express more than one polypeptide, for example, CD80 (B7.1) and an HLA antigen, it is understood that the polypeptides may be encoded on separate nucleic acids or on the same nucleic acid.
  • Methods of genetically modifying a cell are well known to those skilled in the art.
  • a genetically modified cell may be developed from a cell from a subject or it may be developed from a cell line.
  • An“exogenous” or a“recombinant” polynucleotide is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.
  • A“vector” is any nucleic acid molecule for the cloning of and/or transfer of a nucleic acid into a cell.
  • a vector may be a replicon to which another nucleotide sequence may be attached to allow for replication of the attached nucleotide sequence.
  • A“replicon” can be any genetic element (e.g., plasmid, phage, cosmid, chromosome, viral genome) that functions as an autonomous unit of nucleic acid replication in vivo , i.e., capable of replication under its own control.
  • the term“vector” includes both viral and nonviral (e.g.
  • plasmid nucleic acid molecules for introducing a nucleic acid into a cell in vitro , ex vivo , and/or in vivo .
  • a large number of vectors known in the art may be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc.
  • the insertion of the nucleic acid fragments corresponding to response elements and promoters into a suitable vector can be accomplished by ligating the appropriate nucleic acid fragments into a chosen vector that has complementary cohesive termini.
  • the ends of the nucleic acid molecules may be enzymatically modified or any site may be produced by ligating nucleotide sequences (linkers) to the nucleic acid termini.
  • Such vectors may be engineered to contain sequences encoding selectable markers that provide for the selection of cells that contain the vector and/or have incorporated the nucleic acid of the vector into the cellular genome. Such markers allow identification and/or selection of host cells that incorporate and express the proteins encoded by the marker.
  • A“recombinant” vector refers to a viral or non- viral vector that comprises one or more heterologous nucleotide sequences (i.e., transgenes), e.g., two, three, four, five or more heterologous nucleotide sequences.
  • Viral vectors have been used in a wide variety of gene delivery applications in cells, as well as living animal subjects.
  • Viral vectors that can be used include, but arc not limited to, retrovirus, lentivirus, adeno-associated virus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpes vims, Epstein- Barr virus, and adenovirus vectors.
  • Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), nucleic acid-protein complexes, and biopolymers.
  • a vector may also comprise one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (delivery to specific tissues, duration of expression, etc.).
  • Vectors may be introduced into the desired cells by methods known in the art, e.g., transfection, electroporation, microinjeclion, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a nucleic acid vector transporter (see, e.g., Wu et al ., J. Biol. Chem. 267: 963 (1992); Wu et al , J. Biol. Chem. 263: 14621 (1988); and Hartmut et al. , Canadian Patent Application No.
  • a polynucleotide of this invention can be delivered to a cell in vivo by lipofection.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo transfection of a nucleotide sequence of this invention (Feigner et ah, Proc: Natl. Acad. Set. USA 84: 7413 (1987); Mackey, et al. Proc. Natl. Acad. Set. U.S.A. 85: 8027 (1988); and Ulmer et al., Science 259:1745 (1993)).
  • the use of cationic lipids may promote
  • a nucleic acid in vivo can be used for facilitating delivery of a nucleic acid in vivo , such as a cationic oligopeptide (e.g. , W095/21931), peptides derived from nucleic acid binding proteins (e.g., WO96/25508), and/or a cationic polymer (e.g., W095/21931).
  • a cationic oligopeptide e.g. , W095/21931
  • peptides derived from nucleic acid binding proteins e.g., WO96/25508
  • a cationic polymer e.g., W095/21931
  • transfection means the uptake of exogenous or heterologous nucleic acid (RNA and/or DNA) by a cell.
  • a cell has been“transfected” or “transduced” with an exogenous or heterologous nucleic acid when such nucleic acid has been introduced or delivered inside the cell.
  • a cell has been“transformed” by exogenous or heterologous nucleic acid when the transfected or transduced nucleic acid imparts a phenotypic change in the cell and/or a change in an activity or function of the cell.
  • the transforming nucleic acid can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell or it can be present as a stable plasmid.
  • the term“immunological ly cold tumor” refers to a tumor that is lacking or has very few immune cells; a poorly T-cell infiltrated tumor, a tumor that blocks/prevents T-cells from penetrating into the tumor, a tumor that does not attract T-cell infiltration, a tumor having a microenvironment that is lacking in immune cells, e.g., a“T- cell excluding tumor.”
  • the term“immunotogically cold tumor” may be used to refer to tumors having a lower mutation burden, and/or a lower level of PD-L1 or PD-1 expression, which may result in a tumor that is lacking or has very few immune cells.
  • Solid tumors do not typically express both peptide MHC and a co-stimulatory molecule such as B7. Therefore, the tumor cells themselves are not capable of expanding T cells. Instead, tumor cells first must die and have their antigens taken up locally by professional antigen-presenting cells (e.g., antigen-presenting cells that express MHC class II molecules along with co-stimulatory molecules and pattern recognition receptors).
  • professional antigen-presenting cells e.g., antigen-presenting cells that express MHC class II molecules along with co-stimulatory molecules and pattern recognition receptors.
  • compositions of the present invention arenetically modified tumor cells expressing exogenous HLA and B7, which when administered to a subject comprising an immunologtcaliy cold tumor directly stimulate T cells, either via a specific peptide/MHC tumor context or via an allograft context
  • T cells are induced to proliferate and these T cells can start the process of recruiting other T cells and B cells to the tumor.
  • the process can result in a positive feedback loop of recruitment of T cells to the tumor site by the elaboration of chemokines by T cells after stimulation via the T cell receptor.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an immunogenic amount of a tumor cell line genetically modified to express a nucleic acid encoding B7.1 (CD80) and a nucleic acid encoding a human leukocyte antigen (HLA), and a pharmaceutically acceptable excipient, carrier, or diluent.
  • a pharmaceutically acceptable excipient, carrier, or diluent may be an adjuvant.
  • HLA antigens include, but are not limited to, HLA A1 , HLA A2, HLA A3, HLA A27, and the like.
  • the HLA antigen may be HLA A1 or HLA A2.
  • nucleic acid sequences encoding HLA antigens which may be used according to the invention without departing from the same (see below).
  • a method of the invention may include matching the HLA antigen to the subject to which a genetically modified cancer cell line of the invention is to be administered.
  • Methods of determining HLA haplotypes arc well known to those skilled in the art, for example, using well known serological assays using antibodies to HLA alleles or the mixed lymphocyte reaction.
  • Polynucleotides encoding HLA and CD80 for use in producing the genetically modified tumor cell lines of the invention are known in the art Non-limiting examples include the nucleotide sequences of SEQ ID NO:l, SEQ P> NO:8, SEQ ID NO:10, and/or
  • GenBank No. U02935.2 GenBank No. U02935.2
  • GenBank No.: X00492.1 GenBank No.: Z30341.1
  • polypeptides encoded by polynucleotides useful with the invention include the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, and/or SEQ ID NO:ll.
  • a tumor cell line may be a sarcoma cell line or a carcinoma cell line.
  • a tumor cell line may include, but is not limited to, a colorectal cancer cell line, a pancreatic cancer cell line, a breast cancer cell line, a prostate cancer cell line, a bladder cancer cell line, an endometrial cancer cell line, a kidney cancer cell line, a liver cancer cell line, a melanoma cell line, a brain cancer cell line, and/or a head and neck cancer cell line.
  • the tumor cell line is not a lung cancer cell line. In some embodiments, the tumor cell line is not a non-small cell lung cancer cell line.
  • the tumor cell line is not an adenocarcinoma lung cancer cell line.
  • Human colorectal cell lines include, but are not limited to, RKO (ATCC ® No. CRJL- 2577TM), RKO-AS45-1 (ATCC ® No. CRL-2579TM), IIT-29 (ATCC ® No. IITB-38TM), SW1417 (ATCC ® No. CCL-238TM), SW948 (ATCC ® No. CC1-237TM), DLD-1 (ATCC ® No.
  • HCT-15 ATCC ® No. CCL-225TM
  • SW403 ATCC ® No. CCL- 230TM
  • SW48 ATCC ® No. CCL-231TM
  • HCT-8 ATCC ® No. CCL-244TM
  • HCT 116 ATCC ® No. CCL-247TM
  • LS123 ATCC ® No. CCL-255TM
  • LS 180 ATCC ® No. CL- 187TM
  • HXpmOSO xeno] ATCC ® No. CRL-12011TM
  • HP[HT1080 poly] ATCC ® No. CRL-12012TM
  • CCD-18Co ATCC ® No.
  • CRL-1459TM CCD-33Co (ATCC ® No. CRL- 1539TM), CCD-112C0N (ATCC ® No. CRL-1541TM), CCD 841 CoN (ATCC ® No. CRL- 1790TM), CCD 841 CoTr (ATCC ® No. CRL-1807TM), FHC (ATCC ® No. CRL-1831TM), Ramos .2G6.4C 10 (ATCC ® No. CRL-1923TM), RKO-E6 (ATCC* No. CRL-2578TM),
  • ATRFLOX[Mutatect] (ATCC ® No. CRL-2780TM), Hs 255.T (ATCC ® No. CRL-7213TM), Hs 257.T (ATCC ® No. CRL-7214TM), Caco-2 (ATCC ® No. HTB-37TM), SK-CO- 1 (ATCC ® No. HTB-39TM), COLO 201 (ATCC ® No. CCL-224TM), COLO 205 (ATCC ® No. CCL-222TM), Hs 698.T (ATCC ® No. CRL-7435TM), LoVo (ATCC ® No. CCL-229TM), T84 (ATCC ® No. CCL-248TM), CW620[SW-620] (ATCC ® No. CCL-227TM), and/or SNU-C1 (ATCC ® No. CRL-5972TM).
  • Human pancreatic cancer cell lines include, but are not limited to, Capan2 (ATCC ® No. HTB-80TM), Pane 10.05 (ATCC ® No. CRL-2547TM), CFPAC1 (ATCC ® No. CRL- 1918TM), HPAF1I (ATCC ® No. CRL-1997TM), SW 1990 (ATCC* No. CRL-2172TM), BxPC3
  • Human breast cancer cell lines include, but are not limited to, 184B5 (ATCC No. CRL-8799TM), AU-565 (ATCC ® No. CRL-2351TM), BT-20 (ATCC ® No. HTB-19TM), BT- 474 (ATCC ® No. HTB-20TM), BT-483 (ATCC ® No. HTB-121TM), BT-549 (ATCC ® No. HTB-122TM), CAMA-1 (ATCC® No. HTB-21TM), DU4475 (ATCC ® No. HTB-123TM), HCC38 (ATCC ® No. CRL-2314TM), HCC70 (ATCC* No. CRL-2315TM), HCC202 (ATCC ® No.
  • HCC1187 (ATCC ® No. CRL-2322TM), HCC1395 (ATCC ® No. CRL- 2324TM), HCC1419 (ATCC ® No. CRL-2326TM), HCC1428 (ATCC ® No. CRL-2327TM), HCC1500 (ATCC ® No. CRL-2329TM), HCC1569 (ATCC* No. CRL-2330TM), HCC1599 (ATCC* No. CRL-2331TM), HCC1806 (ATCC ® No. CRL-2335TM), HCC1937 (ATCC ® No. CRL-2336TM), HCC1954 (ATCC ® No.
  • HCC2157 ATCC ® No. CRL-2340TM
  • HCC2218 ATCC ® No. CRL-2343TM
  • Hs 578Bst ATCC ® No. HTB-125TM
  • Hs 578T HCC2157
  • HCC2218 ATCC ® No. CRL-2343
  • Hs 578Bst ATCC ® No. HTB-125TM
  • HTB-30TM T47D (ATCC ® No. HTB-133TM), UACC-812 (ATCC ® No. CRL-1897TM), UACC-893 (ATCC ® No. CRL-1902TM), ZR-75-I (ATCC* No. CRL-1500TM), and/or ZR-75- 30 (ATCC ® No. CRL-1504TM).
  • Human bladder cancer cell lines include, but arc not limited to, HT-1376 (ATCC ® No. CRL-1472TM), 5637 (ATCC ® No. HTB-9TM), TCCSUP (ATCC ® No. HTB-5TM), scAber (ATCC ® No. HTB-3TM), UM-UC-3 (ATCC ® No. CRL-1749TM), SW780 (ATCC ® No. CRL- 2169TM), J82 (ATCC ® No. HTB -1), T24 (ATCC ® No. HTB-4TM), RT4 (ATCC ® No. HTB- 2TM), HT-1997 (ATCC ® No. CRL-1473TM), Hs 228.T (ATCC* No.
  • Human endometrial cancer cell lines include, but are not limited to, SK-UT-1 B (ATCC ® No. HTB-115TM), HEC-l-A (ATCC ® No. HTB-112TM), HEC-l-B (ATCC ® No. HTB-113TM), KLE (ATCC ® No. CRL-1622TM), LN3 CA (ATCC ® No. HTB-111TM), SK- UT-1 (ATCC ® No. HTB-114TM), and/or RL95-2 (ATCC ® No. CRL-1671TM).
  • SK-UT-1 B ATCC ® No. HTB-115TM
  • HEC-l-A ATCC ® No. HTB-112TM
  • HEC-l-B ATCC ® No. HTB-113TM
  • KLE ATCC ® No. CRL-1622TM
  • LN3 CA ATCC ® No. HTB-111TM
  • SK- UT-1 ATCC ® No. HTB-114TM
  • Human kidney cancer cell lines include, but are not limited to, 769-P (ATCC ® No. CRL-I933TM), Caki-1 (ATCC ® No. HTB-46TM), 786-0 (ATCC ® No. CRL-1932TM),
  • ProPakA.6 (ATCC* No. CRL-12006TM), Caki-2 (ATCC* No. HTB-47TM), G-402 (ATCC ® No. CRL-1440TM), ACHN (ATCC ® No. CRL-1611TM), ProPak-A.52 Clone #52 (ATCC ® No. CRL-12479TM), Hs 835.T (ATCC ® No. CRL-7569TM), Hs 891. T (ATCC ® No. CRL-7629TM), SW 156 (ATCC* No, CRL-2175TM), HS 926.T (ATCC ® No. CRL-7678TM), A-704 (ATCC No. HTB-45TM), and/or A-498 (ATCC ® No. HTB-44TM).
  • Human liver cancer cell lines include, but are not limited to, Capan-1 (ATCC ® No. HTB-79TM), SK-HEP-1 (ATCC ® No. HTB-52TM), DMS (ATCC ® No. CEL-2064TM),
  • Human melanoma cell lines include, but are not limited to, SK-MEL-l (ATCC ® No.
  • HTB-67TM HTB-67TM
  • A375 ATCC ® No. CRL-1619TM
  • G-361 ATCC ® No. CRL-1424TM
  • SK- MEL-3 ATCC* No. HTB-69TM
  • SH-4 ATCC ® No. CRL-7724TM
  • SK-MEL-24 ATCC ® No. HTB-71TM
  • RPMI-7951 ATCC ® No. HTB-66TM
  • Human brain cancer cell lines include, but are not limited to, CCF-STTG1 (ATCC » No. CRL-1718TM), SW 1088 (ATCC ® No. HTB-12TM), SW 1783 (ATCC ® No. HTB-13TM), CHLA-02-ATRT (ATCC ® No. CRL-3020TM), A172 (ATCC ® No. CRL-1620TM), U-138 MG (ATCC ® No. HTB-16TM), LN-18 (ATCC ® No. CRL-2610TM), LN-229 (ATCC ® No. CRL- 2611TM), U-87 MG (ATCC* No. HTB-14TM), U-l 18 MG (ATCC ® No. HTB-15TM), T98G (ATCC® No.
  • Hs 683 (ATCC* No. HTB-138TM), CHLA-01-MED (ATCC® No, CRL-3021TM), CHP-212 (ATCC ® No. CRL-2273TM), H4 (ATCC ® No. HTB-148TM), D341 Med (ATCC ® No. HTB-187TM), Daoy (ATCC ® No. HTB-186TM), PFSK-1 (ATCC® No. CRL-2060TM), DBTRG-05MG (ATCC ® No. CRL-2020TM), M059K (ATCC ® No. CRL- 2365TM), M059J (ATCC ® No. CRL-2366TM), and/or IMR-32 (ATCC ® No. CCL-127TM).
  • Human head and neck cell lines include, but are not limited to, Detroit 562 (ATCC ® No. CCL-138TM), FaDu (ATCC ® No. HTB-43TM), SCC-15 (ATCC ® No. CRL-1623TM), SCC-4 (ATCC ® No. CRL-1624TM), SCC-25 (ATCC ® No. CRL-1628TM), SCC-9 (ATCC ® No. CRL-1629TM), CAL27 (ATCC ® No. CRL-2095TM), 006 (ATCC* No. CRL-2718TM), 0019 (ATCC ® No. CRL-2721TM), 029 (ATCC ® No. CRL-2723TM), FDC-1 (ATCC ® No. CRL-2729TM), and/or Hsl57.Tg (ATCC ® No. CRL-7107TM).
  • Cell lines useful with the invention may be selected from known tumor cell lines or may be from a subject. Criteria for selection of a cell line for genetic modification with a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) may include the ease of working with the cells from that cell line. A cell line that is useful may also have a reasonable growth rate. Additionally, ease of administration of the cell line to a subject may also be used as a basis for selection. Other factors that may be used to determining if a cell is useful with the methods of the invention include the ability of the irradiated tumor cells to induce an immune response by CD8+T cells. The induced immune response may be compared to the response of a control.
  • the control may be an irradiated non-cancer (normal) cell line.
  • the control may be pancreatic cells from normal pancreatic tissue.
  • a control may be normal cells from the same patient
  • a tumor cell line may be allogeneic to a subject being treated.
  • an“allogeneic cell” or“allogeneic tumor cell line” refers to a cell or cell line that is not derived from the individual to which the cell or cell line is to be administered but is from the same species, that is, an“allogeneic cell” or“allogeneic tumor cell line” has a different genetic constitution than the individual.
  • the allogeneic cell can be a human cell, as disclosed herein, for administering to a human subject such as a cancer patient.
  • an“allogeneic tumor cell” refers to a tumor cell that is not derived from the individual to which the allogeneic cell is to be administered.
  • a tumor cell line may be autologous to a subject being treated.
  • An“autologous cell” or“autologous tumor cell line” refers to a cell or cell line that is taken from the individual to which the cell or cell line is to be administered and thus, has the same genetic constitution than the individual.
  • a pharmaceutical composition can be in a unit dosage form.
  • a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about 1 x 10 5 to about 1 x10 9 cells.
  • a unit dosage form may comprise about 1x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , I c IO 7 , 2x10 7 ,
  • a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about I x 10 5 to about 5x 10 8 cells, and any range or value therein.
  • a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about 1 c 10 s to about 1 x10 8 cells, and any range or value therein.
  • a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about 1 x10 7 to about 1 x10 8 cells, and any range or value therein.
  • a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about 1 x 10 7 to about 5x 10 7 cells, and any range or value therein. In some embodiments, a unit dosage form comprising a pharmaceutical composition of the invention may comprise a unit dosage form comprising about 4.5 x 10 7 cells.
  • a vaccine that comprises the pharmaceutical composition of the invention and a pharmaceutically acceptable adjuvant.
  • a unit dosage form of the present invention may include, but is not limited to, a vial, a container (e.g., an IV solution bag), and/or a filled-syringe.
  • a unit dosage form is a filled-syringe.
  • a pharmaceutical composition of the present invention may further comprise additional therapeutic agents, e.g., an immune checkpoint inhibitor.
  • an immune checkpoint inhibitor useful with this invention may inhibit transmembrane programmed cell death 1 protein (PDCD1, PD-1, CD279), PD-1 ligand, and/or cytotoxic T lymphocyte-associated molecule-4 (CTLA-4).
  • an immune checkpoint inhibitor may be an antibody that inhibits PDCD1 , PD- 1 , CD279, PD- L1 , CD274, and/or CLTA-4.
  • an antibody useftil for blocking PD-1 may include, but is not limited to, pembrolizumab, nivolumab, spartalizumab, lislelizumab, and/or cemiplimab.
  • an antibody useful for blocking PD-L1 may include, but is not limited to atezolizumab, aveiumab, and/or durvalumab.
  • an antibody useful for blocking CTLA-4 may include, but is not limited to, ipilimumab.
  • an antibody useful with the invention may be a monoclonal antibody, an antibody fragment, or a modified antibody (e.g, a single-chain variable fragment (scFv)).
  • a monoclonal antibody an antibody fragment
  • a modified antibody e.g, a single-chain variable fragment (scFv)
  • pharmaceutically acceptable it is meant a material that is not toxic or otherwise undesirable, i.e., the material may be administered to a subject without causing any undesirable biological effects.
  • An adjuvant refers to a substance which, when added to an immunogenic agent of the invention such as tumor cell genetically modified to express CD80 and an HLA antigen, nonspecifica!ly enhances or potentiates an immune response to the agent in the recipient host upon exposure to the mixture.
  • Adjuvants can include, for example, oil-in-water emulsions, water-in oil emulsions, alum (aluminum salts), liposomes and microparticles, such as, polysytrene, starch, polyphosphazene and polylactide/polyglycosides.
  • Adjuvants can also include, for example, squalene mixtures(SAF-I), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophospboryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. Nature 344: 873-875 (1990).
  • SAF-I squalene mixtures
  • IFA Incomplete Freund's Adjuvant
  • Various appropriate adjuvants are well known in the art (see, for example, Warren and Chedid, CRC Critical Reviews in Immunology 8: 83(1988); Allison and Byars, in Vaccines: New Approaches to Immunological Problems, Ellis, ed., Butterworth-Heinemann, Boston (1992)).
  • Additional pharmaceutically acceptable adjuvants may include, but arc not limited to, a bacterial derived adjuvant (e.g., TLR-2/4 ligands (e.g., bacillus Calmette-Gu6rin (BCG), lipopolysaccharidcs, DETOX (containing cell wall skeleton of Mycobacterium pMei (CWS) and monophosphoryl lipid A from Salmonella minnesota (MPL), and the like); TLR- 3 agonists (e.g., poiyriboinosinic-polyribocytidytic acid (Poly I:C)); TLR-7/8 ligands (e.g., low-molecular weight imidazoquinolines); and/or TLR-9 ligands (e.g., CpG
  • TLR-2/4 ligands e.g., bacillus Calmette-Gu6rin (BCG), lipopolysaccharidcs, DETO
  • a further aspect of the invention is a method of treating an immunologically cold tumor in a subject in need thereof or a method of increasing infiltration of immune cells into an immunologically cold tumor in a subject in need thereof, comprising administering to a subject a pharmaceutical composition of the invention comprising cells of a transgenic tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount.
  • a pharmaceutical composition of the invention comprising cells of a transgenic tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount.
  • methods of treating an immunologically cold tumor in a subject in need thereof and/or methods of increasing infiltration of immune cells into an immunologically cold tumor in a subject in need thereof comprising administering to the subject an effective amount of a tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA), thereby increasing infiltration of immune cells into the immunologically cold tumor of the subject and/or treating the immunologically cold tumor in the subject.
  • a tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA)
  • a“subject” of the invention includes any animal that has or is suspected of having an immunologically cold tumor as described herein.
  • a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., cattle, pig, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g. , cat, dog, ferret, gerbil, hamster etc.).
  • the subject may be a non-human primate subject (e.g.
  • an immunologically cold tumor may be a sarcoma or carcinoma.
  • an immunologically cold tumor may include but is not limited to colorectal cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, endometrial cancer, kidney cancer, liver cancer, melanoma, brain cancer, and/or head and neck cancer.
  • a tumor cell line may be allogeneic.
  • a tumor cell line may be the same or different from the immunologically cold tumor to be treated and may include but is not limited to, is a colorectal cancer cell line, a pancreatic cancer cell line, abreast cancer cell line, a prostate cancer cell line, a bladder cancer cell line, an endometrial cancer cell line, a kidney cancer cell line, a liver cancer cell line, a melanoma cell line, a testicular cancer cell line, an ovarian cancer cell line, a cervical cancer cell line, a skin cancer cell line, a renal cell cancer cell line, a retinoblastoma cell line cell line, a brain cancer cell line, and/or head and neck cancer cell line.
  • an effective amount of the tumor cell line that may be administered may be in a range of about 1x10 5 to about 1 x10 9 cells.
  • an effective amount of the tumor cell line that may be administered may comprise about 1 x10 5 , 2x10 5 , 3x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1 x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1 x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1 x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 2x10 8 , 3x
  • an effective amount of the tumor cell line may comprise about 1x10 5 to about 5x 10 8 cells, and any range or value therein. In some embodiments, an effective amount of the tumor cell line may comprise about 1 x10 6 to about 1 x10 8 cells, about 1 x10 7 to about 1 x10 8 cells, about 1 x10 7 to about 5x 10 7 cells, and any range or value therein. In some embodiments, an effective amount of the tumor cell line may comprise about 4x10 7 cells, about 4.5x10 7 cells and/or about 5x10 7 cells.
  • the genetically modified tumor cell lines of the present invention can be by any means known in the art for administering iimnunotherapculic cell lines.
  • the genetically modified tumor cell lines of the invention can be formulated for administration in a pharmaceutical carrier in accordance with known techniques.
  • a tumor cell line that is administered may be formulated as a vaccine comprising a pharmaceutically acceptable adjuvant.
  • the administering step may include, but is not limited to, parenteral injection (e.g., subcutaneous or intramuscular) or intradermal injection.
  • the genetically modified tumor cell lines may be administered intradennally.
  • administering may comprise one or more than one
  • administration e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times or more per treatment regimen.
  • the genetically modified tumor cell lines may be administered bi-weekly, weekly, or monthly.
  • a booster e.g., boosting injection
  • one or more boosters may be administered about 3 months to 3 years after ending the initial treatment regimen (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36 months, and the like, or any range or value therein).
  • a booster may comprise repeating an entire regime (e.g., the entire regimen carried out previous to the booster) or it may be less (e.g., just a single
  • administering the pharmaceutical compositions of the invention to a subject stimulates an immune response against the immunologically cold tumor in the subject, thereby treating the tumor.
  • an immune response may be indicated by an increase in the amount of mterferon-g secreting CDS* T cells of the subject.
  • an increase in the amount of interferon-7 secreting CD8 + T cells may be in the tumor, around the tumor, and/or an increase in the amount of interferon-g secreting CDS* T cells may be an increase in the amount of circulating interferon-g secreting CDS* T cells.
  • CPI checkpoint inhibitor therapy
  • a method of treating an immunologically cold tumor in a subject in need thereof, or a method of increasing infiltration of immune cells into an immunologicaily cold tumor in a subject in need thereof may further comprise administering to the subject an effective amount of an immune checkpoint inhibitor.
  • a method of the present invention may further comprise administering to the subject an additional therapeutic agent, e.g., an immune checkpoint inhibitor.
  • an immune checkpoint inhibitor useful with this invention may be a compound or molecule that inhibits transmembrane programmed cell death 1 protein (PDCDI, PD-1, CD279), PD-1 ligand, and/or CTLA-4.
  • an immune checkpoint inhibitor may be an antibody that inhibits PDCDI, PD-1 , CD279, PD-L1, CD274, and/or CLTA-4.
  • an antibody useful for blocking PD-1 may include, but is not limited to, pembrolizumab, nivoiumab, spartaiizumab, tislelizumab, and/or cemiplimab.
  • an antibody useful for blocking PD-L1 may include, but is not limited to, atezolizumab, avelumab, and/or durvalumab.
  • an antibody useful for blocking CTLA-4 may include, but is not limited to, ipilimumab.
  • an antibody useful with the invention may be a monoclonal antibody, an antibody fragment, or a modified antibody (e.g, a single- chain variable fragment (scFv)).
  • a monoclonal antibody an antibody fragment
  • a modified antibody e.g, a single- chain variable fragment (scFv)
  • the dosage of an immune checkpoint inhibitor depends on the choice of the route of administration; the nature of the formulation; the nature of the patient's illness; the subject’s size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. Wide variations in the needed dosage are to be expected in view of the variety of molecules available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by i.v. injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization as is well understood in the art. Administrations can be single or multiple (e.g., 2-, 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold).
  • Encapsulation of an immune checkpoint inhibitor in a suitable delivery vehicle may increase the efficiency of delivery, particularly for oral delivery or delivery into or nearby the location of a tumor, e.g., the peritoneal or pleural cavity.
  • the dose of an immune checkpoint inhibitor may be from about 0.01 mg/kg body weight per day to about 500 mg/kg body weight per day, e.g., about 0.1 mg/ kg body weight per day to about 20 mg/kg per day, about 0.1 mg/kg body weight per day to about 30 mg/kg per day, about 0.1 mg/kg body weight per day to about 40 mg/kg per day, about 0.1 mg/kg body weight per day to about 50 mg/kg per day, about 0.1 mg/kg body weight per day to about 75 mg/kg per day, about 0.1 mg/kg body weight per day to about 100 mg/kg per day, about 1 mg/kg per day to about 100 mg/kg per day, about 1 mg/kg per day to about 150 mg/kg per day, about 1 mg/kg per day to about 200 mg/kg par day, about 1 mg/kg per day to about 250 mg/kg per day, about 1 mg/kg per day to about 300 mg/kg per day, about 1 mg/kg per day to about 350 mg/kg per day, about 1 mg//kg
  • the dose may be from about 0.1 mg/kg to about 50 mg/kg per day. In some embodiments, the dose may be from about 0.1 mg/kg to about 40 mg/kg per day. In some embodiments, the dose may be from about 0.1 mg/kg to about 30 mg/kg per day. In some embodiments, the dose may be from about 0.1 mg/kg to about 20 mg/kg per day. In some embodiments, the dose does not exceed about 50 mg per day.
  • the dose of an immune checkpoint inhibitor may be from about 0.5 mg/kg body weight of a subject per week to about 350 mg/kg body weight per week, inclusive of all values and ranges therebetween, including endpoints. In some embodiments, the dose may be about 0.5 mg/kg per week. In some embodiments, the dose may be about 1 mg/kg per week. In some embodiments, the dose may be about 2 mg/kg per week. In some embodiments, the dose may be about 5 mg/kg per week. In some
  • the dose may be about 10 mg/kg per week. In some embodiments, the dose may be about 20 mg/kg per week. In some embodiments, the dose may be about 30 mg/kg per week. In some embodiments, the dose may be about 40 mg/kg per week. In some embodiments, the dose may be about 50 mg/kg per week. In some embodiments, the dose may be about 60 mg/kg per week. In some embodiments, the dose may be about 70 mg/kg per week. In some embodiments, the dose may be about 80 mg/kg per week. In some embodiments, the dose may be about 90 mg/kg per week. In some embodiments, the dose may be about 100 mg/kg per week. In some embodiments, the dose may be about 110 mg/kg per week.
  • the dose may be about 120 mg/kg per week. In some embodiments, the dose may be about 130 mg/kg per week. In some embodiments, the dose may be about 140 mg/kg per week. In some embodiments, the dose may be about 150 mg/kg per week. In some embodiments, the dose may be about 160 mg/kg per week. In some embodiments, the dose may be about 170 mg/kg per week. In some embodiments, the dose may be about 180 mg/kg per week. In some embodiments, the dose may be about 190 mg/kg per week. In some embodiments, the dose may be about 200 mg/kg per week. In some embodiments, the dose may be about 210 mg/kg per week. In some embodiments, the dose may be about 220 mg/kg per week.
  • the dose may be about 230 mg/kg per week. In some embodiments, the dose may be about 240 mg/kg per week. In some embodiments, the dose may be about 250 mg/kg per week. In some embodiments, the dose may be about 260 mg/kg per week. In some embodiments, the dose may be about 270 mg/kg per week. In some embodiments, the dose may be about 280 mg/kg per week. In some embodiments, the dose may be about 290 mg/kg per week. In some embodiments, the dose may be about 300 mg/kg per week. In some embodiments, the dose may be about 310 mg/kg per week. In some embodiments, the dose may be about 320 mg/kg per week. In some embodiments, the dose may be about 330 mg/kg per week. In some embodiments, the dose may be about 340 mg/kg per week. In some embodiments, the dose may be about 350 mg/kg per week.
  • administration of an immune checkpoint inhibitor may be pulsatile.
  • an amount of the immune checkpoint inhibitor may be administered about every 1 hour to about every 24 hours, for example, about every 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,
  • an amount of the immune checkpoint inhibitor may be administered about every 1 day, 2 days, 3 days, 4 days,
  • the administration of the immune checkpoint inhibitor may be of an indefinite duration, to be determined by the managing physician, and only terminated when the disease is judged to be either cured or in remission.
  • a method of the invention may comprise administering to a subject an effective amount of a tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) and chimeric antigen receptor (CAR) T cells designed to target an antigen on the surface of the immunologically cold tumor.
  • a tumor cell line genetically modified to express a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen (HLA) and chimeric antigen receptor (CAR) T cells designed to target an antigen on the surface of the immunologically cold tumor.
  • HLA human leukocyte antigen
  • CAR chimeric antigen receptor
  • methods of the present invention may be combined with other cancer treatments including, but not limited to, chemotherapy, radiotherapy, surgery, targeted therapy, hormone therapy, and the like.
  • cell lines useful with the invention may be selected from known tumor cell lines or may be from a subject. Criteria for selection of a cell line for genetic modification with a nucleic acid encoding CD80 (B7.I) and a nucleic acid encoding a human leukocyte antigen (HLA) may include the ease of working with the cells from that cell line. A cell line that is useful may also have a reasonable growth rate. Additionally, ease of administration of the cell line to a subject may also be used as a basis for selection. Other factors that may be used to determining if a cell is useful with the methods of the invention include the ability of the irradiated tumor cells to induce an immune response by CD8+T cells.
  • the induced immune response would be compared to the response of a control.
  • the control may be an irradiated non-can ccr (normal) cell line.
  • the control may be pancreatic cells from normal pancreatic tissue.
  • a control may be normal cells from the same patient.
  • a selected pancreatic cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA.
  • Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and IILA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5x10 7 cells until use.
  • Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells.
  • Co-expression of HLA and B7.1 is observed in at least about 70% of the cells (e.g., about 70 to about 100%, about 70% to about 95%, about 75% to about 95%, about 80 to about 100%, about 80% to about 95%, about 80% to about 95%, about 85 to about 100%, about 85% to about 95%; e.g., about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%, any range or value therein).
  • 70% of the cells e.g., about 70 to about 100%, about 70% to about 95%, about 75% to about 95%, about 80 to about 100%, about 80% to about 95%, about 80% to about 95%, about 85 to about 100%, about 85% to about 95%; e.g.
  • a selected colorectal cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1 ) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5x 10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • a selected breast cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5x10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells. Example 5.
  • a selected prostate cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis,
  • the genetically modified cel to are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5* 10 7 cells until use.
  • Genetically modified cells that are unable to form colonics upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells.
  • Co-expression of HI A and B7.1 is observed in at least about 70% of tire cells.
  • a selected bladder cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5> ⁇ 10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • a selected melanoma cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA.
  • Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5*10 7 cells until use.
  • Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells.
  • Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • a selected brain cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5x10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • a selected retinoblastoma cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with about 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in about 10% DMSO in aliquots of about 5x10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • a selected ovarian cancer cell line is transfected with a plasmid comprising a nucleic acid encoding CD80 (B7.1) and a nucleic acid encoding a human leukocyte antigen HLA. Verification of correct sequences may be based on, for example, restriction analysis, sequence analysis, and the expression of the CD80 and HLA polypeptides.
  • the genetically modified cells are irradiated to prevent their replication, for example, with 12,000 Rads in a cobalt (Co) irradiator, and stored frozen in 10% DMSO in aliquots of 5* 10 7 cells until use. Genetically modified cells that are unable to form colonies upon replating in tissue culture indicate the inability of the cells to replicate. Cells that cannot replicate are considered safe for use as vaccine cells. Co-expression of HLA and B7.1 is observed in at least about 70% of the cells.
  • Intracutancous injections are given at multiple body sites to reduce the extent of local skin reactions.
  • the subject receives a total dose of about 5*10 7 irradiated cells (e.g., about 12,000 rad) divided into two to five aliquots for administration as two to five intradermal injections of each aliquot in an extremity, spaced at least about 5 cm at needle entry from the nearest neighboring injection.
  • About nine immunizations (4.5x108 cells) are given over the course of therapy, one every two weeks, provided that no tumor progression occurs under therapy.
  • the injection sites are rotated to different limbs in a clockwise manner.
  • One course of vaccination is about three biweekly injections.
  • CT Scans imaging evaluation
  • grade ⁇ 2 patients with evidence of stable disease or responding disease by imaging evaluation (CT Scans) and none to moderate toxicity (grade ⁇ 2) are treated with an additional course at the same dose.
  • a second course of injections is provide at about two weeks after the third vaccination that completes the first course.
  • a third course at the same dose of therapy is given, starting two weeks after the third vaccination of the second course of therapy.
  • Clinical toxicity and immunologic evaluations by blood tests prior to and after each course are performed. Patients are followed clinically weekly during the study, including monitoring blood counts and basic chemistries.

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Abstract

L'invention concerne des procédés de traitement d'une tumeur immunologiquement froide chez un sujet en ayant besoin, comprenant l'administration au sujet d'une quantité efficace d'une lignée de cellules tumorales génétiquement modifiée pour exprimer un acide nucléique codant CD80 (B7.1) et un acide nucléique codant pour un antigène leucocytaire humain (HLA). L'invention concerne en outre des compositions pharmaceutiques comprenant une quantité immunogène d'une lignée de cellules tumorales génétiquement modifiée pour exprimer un acide nucléique codant pour B7.1 (CD80) et un acide nucléique codant pour un antigène leucocytaire humain (HLA).
PCT/US2019/054188 2018-10-10 2019-10-02 Procédés et compositions pour le traitement du cancer WO2020076568A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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US11369668B1 (en) 2019-12-03 2022-06-28 Neuvogen, Inc. Tumor cell vaccines

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US6805869B2 (en) * 1996-06-12 2004-10-19 Shanghai Cp Guojian Pharmaceutical Co., Ltd. Cellular vaccines and immunotherapeutics and methods for their preparation
US20070166279A1 (en) * 2005-05-10 2007-07-19 Eyal Talor Method for modulating hla class ii tumor cell surface expression with a cytokine mixture
US20110171211A1 (en) * 2008-03-20 2011-07-14 Podack Eckhard R Heat shock protein gp96 vaccination and methods of using same
US20160045583A1 (en) * 2003-09-26 2016-02-18 University Of Miami Tumor Vaccine
US20160317631A1 (en) * 2003-06-11 2016-11-03 The University Of Chicago Increased t-cell tumor infiltration and eradication of metastases by mutant light
US20180244783A1 (en) * 2015-08-31 2018-08-30 Oncomed Pharmaceuticals, Inc. Combination therapy for treatment of disease

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Publication number Priority date Publication date Assignee Title
US6805869B2 (en) * 1996-06-12 2004-10-19 Shanghai Cp Guojian Pharmaceutical Co., Ltd. Cellular vaccines and immunotherapeutics and methods for their preparation
US20160317631A1 (en) * 2003-06-11 2016-11-03 The University Of Chicago Increased t-cell tumor infiltration and eradication of metastases by mutant light
US20160045583A1 (en) * 2003-09-26 2016-02-18 University Of Miami Tumor Vaccine
US20070166279A1 (en) * 2005-05-10 2007-07-19 Eyal Talor Method for modulating hla class ii tumor cell surface expression with a cytokine mixture
US20110171211A1 (en) * 2008-03-20 2011-07-14 Podack Eckhard R Heat shock protein gp96 vaccination and methods of using same
US20180244783A1 (en) * 2015-08-31 2018-08-30 Oncomed Pharmaceuticals, Inc. Combination therapy for treatment of disease

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11369668B1 (en) 2019-12-03 2022-06-28 Neuvogen, Inc. Tumor cell vaccines
US11684659B2 (en) 2019-12-03 2023-06-27 Neuvogen, Inc. Tumor cell vaccines

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