WO2021155034A1 - Compositions and methods for tcr reprogramming using muc16 specific fusion proteins - Google Patents
Compositions and methods for tcr reprogramming using muc16 specific fusion proteins Download PDFInfo
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- WO2021155034A1 WO2021155034A1 PCT/US2021/015542 US2021015542W WO2021155034A1 WO 2021155034 A1 WO2021155034 A1 WO 2021155034A1 US 2021015542 W US2021015542 W US 2021015542W WO 2021155034 A1 WO2021155034 A1 WO 2021155034A1
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- C12N2510/00—Genetically modified cells
Definitions
- cancer immunotherapy Most patients with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
- CAR chimeric antigen receptor
- CTL019 The clinical results with CD-19-specific CAR-T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g.
- TCR T cell receptor
- beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma.
- HLA-A2 HLA-A2.
- TCR T cell receptor
- TFP T cell receptor fusion proteins
- a pharmaceutical composition comprising a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: a TCR-integrating subunit comprising: at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain; and a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked.
- the intracellular signaling domain comprises a stimulatory domain from an intracellular signaling domain.
- a pharmaceutical composition comprising (I) a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: (a) a TCR-integrating subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a transmembrane domain; and (b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and (II) a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked.
- the TFP further comprises an intracellular domain.
- the MUC16 binding domain specifically binds membrane-bound MUC16. In some embodiments, the MUC16 binding domain specifically binds membrane-bound MUC16 in the presence of soluble MUC16. In some embodiments, the T cell exhibits increased cytotoxicity to a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
- the sequence encoding the MUC16 binding domain is connected to the sequence encoding the TCR extracellular domain by a sequence encoding a linker.
- the linker comprises (G4S) n , wherein G is glycine, S is serine, and n is an integer from 1 to 4.
- the MUC16 binding domain comprises mesothelin or a fragment thereof. In some embodiments, the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin. In some embodiments, the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKH KLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
- the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
- the pharmaceutical composition is substantially free of serum.
- the human T cell has greater than or more efficient cytotoxic activity than a CD8+ or CD4+ T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (a) the MUC16 binding domain, operatively linked to (b) at least a portion of an extracellular domain (c) a transmembrane domain (d) at least a portion of a CD28,
- CAR chimeric antigen receptor
- the T cell is a primary T cell. In some embodiments, the T cell is an iNKT cell. In some embodiments, the T cell is a human CD4+ T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is a human CD8+ T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is a human CD8+ T cell. In some embodiments, the T cell is an alpha beta (ab or ⁇ ) T cell. In some embodiments, the T cell is a gamma delta (gd or ⁇ ) T cell.
- the T cell further comprises a nucleic acid encoding a first polypeptide comprising at least a portion of an inhibitory molecule , wherein the at least a portion of an inhibitory molecule is associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
- the inhibitory molecule is PD-1.
- the second polypeptide comprises a costimulatory domain and primary signaling domain from a protein selected from the group consisting of CD28, CD27, ICOS,
- production of IL-2 or IFN ⁇ by the T cell is increased in the presence of a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
- the cell is a population of human T cells, wherein an individual T cell of the population comprises at least two TFP molecules, or at least two T cells of the population collectively comprise at least two TFP molecules; wherein the at least two TFP molecules comprise a MUC16 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and wherein at least one of the at least two TFP molecules functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof.
- the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the TCR-integrating subunit comprises a TCR intracellular domain.
- the intracellular domain comprises an intracellular domain from TCR alpha, TCR beta, TCR delta, or TCR gamma, or an amino acid sequence having at least one modification thereto.
- the intracellular domain comprises a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, or an amino acid sequence having at least one modification thereto.
- the TCR-integrating subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
- the TCR-integrating subunit is derived only from CD3 epsilon. In some embodiments, wherein the TCR-integrating subunit is derived only from CD3 gamma. In some embodiments, the TCR-integrating subunit is derived only from CD3 delta.
- the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
- the TFP further comprises a sequence encoding a costimulatory domain.
- the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
- the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma
- IT AM immunoreceptor tyrosine-based activation motif
- TCR subunit CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor
- the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon.
- the IT AM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
- a recombinant nucleic acid comprising a human T cell
- the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: a TCR-integrating subunit comprising: at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain; and a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked.
- the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain.
- a recombinant nucleic acid encoding a T cell receptor (TCR) fusion protein (TFP) comprising: (a) a TCR-integrating subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a transmembrane domain; and (b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked; and wherein the TFP functionally interacts with a TCR when expressed in the T cell.
- the TFP further comprises an intracellular domain.
- the recombinant nucleic acid encodes a TFP having any of the features of a TFP described herein.
- the recombinant nucleic acid further comprises a leader sequence.
- the nucleic acid is selected from the group consisting of a DNA and a RNA.
- the nucleic acid is a mRNA.
- the nucleic acid is a circRNA.
- the nucleic acid comprises a nucleotide analog.
- the nucleotide analog is selected from the group consisting of 2’ -O-methyl, 2’-O- methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-O-aminopropyl
- NMA NMA modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2 , -fluoroN3-P5’-phosphoramidite.
- LNA locked nucleic acid
- ENA ethylene nucleic acid
- PNA peptide nucleic acid
- HNA anhydrohexitol nucleic acid
- morpholino a methylphosphonate nucleotide
- thiolphosphonate nucleotide a thiolphosphonate nucleotide
- 2 , -fluoroN3-P5’-phosphoramidite a 2 , -fluoroN3-P5’-
- the recombinant nucleic acid further comprises a promoter.
- the recombinant nucleic acid is an in vitro transcribed nucleic acid.
- the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence.
- a recombinant polypeptide molecule encoded by any of the recombinant nucleic acids described herein.
- a vector comprising any of the recombinant nucleic acids described herein encoding any of the TFPs described herein.
- the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
- the vector further comprises a promoter.
- the vector is an in vitro transcribed vector.
- a nucleic acid sequence in the vector further comprises a poly(A) tail.
- a nucleic acid sequence in the vector further comprises a 3’UTR.
- a cell comprising any of the recombinant nucleic acids, TFPs, or vectors described herein.
- the cell is a human T-cell.
- the T-cell is a CD8+ or CD4+ T-cell.
- the T cell is a human alpha beta (ab or ⁇ ) T cell.
- the T cell is a human gamma delta (gd or ⁇ ) T cell.
- the cell further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
- the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
- a protein complex comprising a TFP encoded by any of the recombinant nucleic acids described herein, and at least one endogenous TCR subunit or endogenous TCR complex.
- a human CD8+ or CD4+ T-cell comprising at least two different TFP proteins of any of the protein complexes described herein.
- a human CD8+ or CD4+ T-cell comprising at least two different TFP molecules encoded by any of the recombinant nucleic acids described herein.
- a population of human CD8+ or CD4+ T-cells wherein the T-cells of the population individually or collectively comprise at least two TFP molecules encoded by any of the recombinant nucleic acids described herein.
- a method of making a cell comprising transducing a T-cell with any of the recombinant nucleic acids or vectors described herein.
- RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding any of the TFPs described herein.
- provided herein is a method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein.
- the cell is an autologous T-cell. In some embodiments, the cell is an allogeneic T-cell. In some embodiments, the mammal is a human.
- a method of treating a mammal having a disease associated with expression of MUC16 comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein.
- the disease associated with MUC16 expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, myelodysplasia, a myelodysplastic syndrome, a preleukemia, a non-cancer related indication associated with expression of MUC16.
- the disease is pancreatic cancer, ovarian cancer, breast cancer, or any combination thereof.
- the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-MUC16 chimeric antigen receptor (CAR). [0051] In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a TFP molecule
- TFP molecule are administered in combination with an agent that treats the disease associated with MUC16.
- a method of treating a mammal having a disease associated with expression of MUC16 comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein for use as a medicament, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-MUC16 chimeric antigen receptor (CAR).
- CAR anti-MUC16 chimeric antigen receptor
- compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein for use as a medicament are provided herein.
- FIGs. 1A and IB are a series of graphs showing detection of MUC16 on the surface of MUC16 expressing OVCAR3 cells.
- FIG. 1 A MUC16 expression by OVCAR3 cells is detected by R-phycoerythrin conjugated anti-MUC16 antibody.
- FIG. 1 A MUC16 expression by OVCAR3 cells is detected by R-phycoerythrin conjugated anti-MUC16 antibody.
- FIG. 2 is a series of graphs showing detection of MUC16.TFP (i.e., a TFP having the MSLN IAB peptide) on the surface of non-transduced (NT) and transduced (TFP) CD3 ⁇ knock out Jurkat cells by anti- CD3 ⁇ , anti-TCR ⁇ , and SSI scFv hFc.
- FIGs. 3A and 3B are a series of plots showing Jurkat cell activation mediated by
- FIG. 4 is a graph showing fold expansion of MUC16.TFP transduced and non-transduced T cells from three donors (A, B and C). Non-transduced and transduced primary T cells expanded normally in all three donors.
- FIG. 5 is a series of plots showing levels of surface expression of MUC16.TFP as measured by SSI scFv hFc in MUC16.TFP transduced and non-transduced control T cells from the three donors (A, B and C).
- FIGs. 6A and 6B are a series of plots showing expression of T cell activation markers CD69 and CD25 in MUC16.TFP transduced and non-transduced T cells from three donors (A, B and C) co-cultured with OVCAR3 MUC16 expressing cells or C30 control cells at an effectortarget ratio of 3 : 1. Donor A is shown in FIG. 6A and Donors B and C are shown in FIG. 6B.
- FIG. 7 is a series of graphs showing cytotoxicity of MUC16.TFP transduced or non- transduced T cells from three donors when co-cultured for 24 hours with OVCAR3 cells (Donors A-C) or C30 cells (Donors B and C) at an effectortarget cell ratio of 3 : 1 and 1:1.
- FIGs. 8A-8C are a series of graphs showing levels of IFN- ⁇ (FIG. 8 A), TNF- ⁇ (FIG. 8B), and GM-CSF (FIG. 8C) produced by T cells expressing MUC16.TFP or non-transduced control cells from three donors when co-cultured for 24 hours with OVCAR3 cells or C30 cells.
- the present disclosure encompasses recombinant DNA constructs encoding TFPs and variants thereof, wherein the TFP comprises a MUC16 binding domain that is not an antibody or antigen binding fragment thereof, wherein the sequence of the antigen or the fragment thereof is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR- integrating subunit or portion thereof.
- the MUC16 binding domain comprises a portion of the mesothelin protein, such as a portion of the extracellular domain of the mesothelin protein.
- the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
- the present disclosure also provides a TFP molecule or a TCR complex having the TFP molecule incorporated therein.
- the present disclosure also provides a cell (e.g., a T cell or a Treg) comprises the TFP or the recombinant nucleic acid molecule encoding the TFP.
- a cell e.g., a T cell or a Treg
- TFPs when expressed in a T-cell, can target MUC16 expressing cells.
- the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise.
- the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ⁇ one standard deviation of that value(s).
- subject or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g. , domesticated, agricultural or wild, animals, as well as birds, and aquatic animals.
- exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep.
- the subject is a human.
- “Patients” are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
- treating refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.
- treat or prevent is sometimes used herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely. Treatment can be performed both for prophylaxis and during the course of clinical pathology.
- Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
- preventing refers to the prevention of the disease or condition, e.g ., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
- the disease or condition e.g ., tumor formation
- a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered.
- therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols.
- a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
- a “TFP T cell” is a T cell that has been transduced according to the methods disclosed herein and that expresses a TFP, e.g., incorporated into the natural TCR.
- the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.
- the TFP T cell is an NK cell or a regulatory T cell.
- MUC16 also known as mucin 16 or CA125 (cancer antigen 125, carcinoma antigen 125, or carbohydrate antigen 125), refers to a protein that in humans is encoded by the MUC 16 gene.
- MUC16 is a member of the mucin family glycoproteins and has found application as a tumor marker or biomarker that may be elevated in the blood of some patients with specific types of cancers or other conditions that are benign.
- MUC 16 is used as a biomarker for ovarian cancer detection and has been found to be elevated in other cancers, including endometrial cancer, fallopian tube cancer, lung cancer, breast cancer and gastrointestinal cancer.
- MUC16 has also been shown to suppress the activity of natural killer cells in the immune response to cancer cells (see, e.g., Patankar et al., Gynecologic Oncology
- human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot.
- amino acid sequence of human MUC16 can be found as UniProt/Swiss-Prot Accession No. Q8WXI7.
- nucleotide sequence encoding human MUC16 can be found at Accession No.
- the nucleotide sequence encoding human MUC16 transcript variant XI can be found at Accession No. XM_017027486.
- the nucleotide sequence encoding human MUC16 transcript variant X2 can be found at Accession No. XM_017027487.
- the nucleotide sequence encoding human MUC16 transcript variant X3 can be found at Accession No. XM_017027488.
- the nucleotide sequence encoding human MUC16 transcript variant X4 can be found at Accession No. XM_017027489.
- the nucleotide sequence encoding human MUC16 transcript variant X5 can be found at Accession No. XM_ 017027490.
- the nucleotide sequence encoding human MUC16 transcript variant X6 can be found at Accession No. XM_017027491.
- the nucleotide sequence encoding human MUC16 transcript variant X7 can be found at Accession No. XM_017027492.
- the nucleotide sequence encoding human MUC16 transcript variant X8 can be found at Accession No. XM_ 017027493.
- the nucleotide sequence encoding human MUC16 transcript variant X9 can be found at Accession No. XM_017027494.
- the nucleotide sequence encoding human MUC16 transcript variant XI 0 can be found at Accession No.
- the nucleotide sequence encoding human MUC16 transcript variant XI 1 can be found at Accession No. XM_017027499.
- the nucleotide sequence encoding human MUC16 transcript variant X12 can be found at Accession No. XM_017027500.
- the nucleotide sequence encoding human MUC16 transcript variant X13 can be found at Accession No. XM_017027501.
- the binding portion of TFPs recognizes and binds an epitope within the extracellular domain of the MUC16 protein as expressed on a normal or malignant mesothelioma cell, ovarian cancer cell, pancreatic adenocarcinoma cell, or squamous cell carcinoma cell.
- the term “mesothelin” also known as MSLN or CAK1 antigen or Pre-pro- megakaryocyte-potentiating factor, refers to the protein that in humans is encoded by the MSLN (or Megakaryocyte-potentiating factor (MPF)) gene.
- MSLN Megakaryocyte-potentiating factor
- Mesothelin is a 40 kDa protein present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma.
- the mesothelin gene encodes a precursor protein that is processed to yield mesothelin which is attached to the cell membrane by a glycophosphatidylinositol linkage and a 31 -kDa shed fragment named megakaryocyte- potentiating factor (MPF).
- MPF megakaryocyte- potentiating factor
- Mesothelin may be involved in cell adhesion, but its biological function is not known.
- Mesothelin is a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium.
- Mesothelin is an antigenic determinant detectable on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and some squamous cell carcinomas (see, e.g ., Kojima et al., J. Biol. Chem. 270:21984-
- CA125/MUC16 see, e.g., Rump et al., J. Biol. Chem. 279:9190-9198(2004) and Ma et al., J.
- the human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot.
- amino acid sequence of human mesothelin can be found as UniProt/Swiss-Prot Accession No. Q13421.
- the human mesothelin polypeptide canonical sequence is UniProt Accession No. Q13421 (or Q13421-1):
- LVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO:2).
- the nucleotide sequence encoding human mesothelin transcript variant 1 can be found at Accession No. NM005823.
- the nucleotide sequence encoding human mesothelin transcript variant 2 can be found at Accession No. NMO 13404.
- the nucleotide sequence encoding human mesothelin transcript variant 3 can be found at Accession No. NMOOl 177355.
- Mesothelin is expressed on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and squamous cell carcinomas (see, e.g. , Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. Cancer Res. 12:4225-4231(2006)).
- binding domain refers to a protein, or polypeptide sequence, which specifically binds to a target.
- the target is a polypeptide.
- the target is MUC16.
- the target is cell-surface bound MUC16.
- the binding domain is not an antibody or fragment thereof.
- antibody refers to a protein, or polypeptide sequence, derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
- antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigenic determining variable region of an intact antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
- antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.
- sdAb single domain antibodies
- the TFP composition of the disclosure does not comprise an antibody or antibody fragment.
- antigen or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
- antigens can be derived from recombinant or genomic DNA.
- any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
- an antigen need not be encoded solely by a full length nucleotide sequence of a gene.
- an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide.
- a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
- anti-tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g ., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
- An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, and cells of the disclosure in prevention of the occurrence of tumor in the first place.
- affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a ligand) and its binding partner.
- affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., a ligand and its binding partner).
- the affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (K D ). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below.
- Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE ® ) or biolayer interferometry (e.g., FORTEBIO ® ).
- SPR surface plasmon resonance
- BIACORE ® BIACORE ®
- biolayer interferometry e.g., FORTEBIO ®
- binding means binding that is measurably different from a non-specific or non- selective interaction (e.g., with a non-target molecule).
- Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
- Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the binding domain to the target molecule is competitively inhibited by the control molecule.
- autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
- allogeneic refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
- xenogeneic refers to a graft derived from an animal of a different species.
- circRNA refers to a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. CircRNAs are 3-5’ covalently closed RNA rings, and circRNAs do not display Cap or poly(A) tails.
- cancer refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer, and the like.
- package insert is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
- therapeutic or diagnostic products e.g., kits
- cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
- a “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
- Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.
- tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- cancer refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
- cell proliferative disorder refers to disorders that are associated with some degree of abnormal cell proliferation.
- the cell proliferative disorder is a cancer.
- the tumor is a solid tumor.
- the tumor is a hematologic malignancy.
- composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
- modulate and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
- increase and activate refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
- reduce and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
- the term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor.
- An “agonist” is an entity that binds to and agonizes a receptor.
- the term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor.
- An “antagonist” is an entity that binds to and antagonizes a receptor.
- effector T cell includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells.
- CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
- CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol ., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.
- the term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells.
- the regulatory T cell has a CD4+CD25+Foxp3+ phenotype.
- the regulatory T cell has a CD8+CD25+ phenotype. See Nocentini et al., Br. J. Pharmacol ., 2012, 165:2089-2099, incorporated by reference in its entirety, for additional information on regulatory T cells expressing TARGET.
- the term “dendritic cell” refers to a professional antigen-presenting cell capable of activating a naive T cell and stimulating growth and differentiation of a B cell.
- the phrase “disease associated with expression of MUC16” includes, but is not limited to, a disease associated with expression of MUC16 or condition associated with cells which express MUC16 including, e.g ., proliferative diseases such as a cancer or malignancy or a precancerous condition.
- the cancer is a glioblastoma.
- the cancer is a mesothelioma.
- the cancer is a pancreatic cancer.
- the cancer is an ovarian cancer.
- the cancer is a brain cancer.
- the cancer is a stomach cancer.
- the cancer is a lung cancer.
- the cancer is an endometrial cancer.
- Non-cancer related indications associated with expression of MUC16 include, but are not limited to, e.g. , autoimmune disease, (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergy and asthma), and transplantation.
- conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the ligand (e.g., binding domain) containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a ligand (e.g., binding domain) of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
- one or more amino acid residues within a TFP of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
- stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
- a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
- Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
- the term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
- the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
- a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “IT AM”.
- IT AM immunoreceptor tyrosine-based activation motif
- Examples of an IT AM containing primary cytoplasmic signaling sequence that is of particular use in the disclosure includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
- the term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface.
- T cells may recognize these complexes using their T cell receptors (TCRs).
- TCRs T cell receptors
- intracellular signaling domain refers to an intracellular portion of a molecule.
- the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell.
- immune effector function e.g., in a TFP-expressing T cell
- examples of immune effector function, e.g., in a TFP-expressing T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
- the intracellular signaling domain can comprise a primary intracellular signaling domain.
- Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
- the intracellular signaling domain can comprise a costimulatory intracellular domain.
- Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
- a primary intracellular signaling domain can comprise an IT AM (“immunoreceptor tyrosine-based activation motif’).
- IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP 10 and DAP12.
- costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
- Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that may be required for an efficient immune response.
- Costimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla/CD18) and 4-1BB (CD137).
- a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
- a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
- Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.
- the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
- 4-1BB refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No.
- AAA62478.2 or the equivalent residues from a non-human species, e.g ., mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species, e.g. , mouse, rodent, monkey, ape and the like.
- the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g ., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
- a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
- Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
- nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
- the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
- an effective amount or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result.
- endogenous refers to any material from or produced inside an organism, cell, tissue or system.
- exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
- expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
- transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
- Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
- the term “transfer vector” includes an autonomously replicating plasmid or a virus.
- the term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
- Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
- expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
- An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
- Expression vectors include all those known in the art, including cosmids, plasmids ( e.g ., naked or contained in liposomes) and viruses (e.g ., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
- lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
- lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
- Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTORTM gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
- homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
- two nucleic acid molecules such as, two DNA molecules or two RNA molecules
- polypeptide molecules between two polypeptide molecules.
- a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
- the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
- isolated means altered or removed from the natural state.
- a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
- An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
- A refers to adenosine
- C refers to cytosine
- G refers to guanosine
- T refers to thymidine
- U refers to uridine.
- operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
- a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
- a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
- DNA sequences can be contiguous with each other and, e.g ., where necessary to join two protein coding regions, are in the same reading frame.
- parenteral administration of an immunogenic composition includes, e.g. , subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
- nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
- DNA deoxyribonucleic acids
- RNA ribonucleic acids
- degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
- peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
- a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
- Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
- the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
- Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
- a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
- promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, that can initiate the specific transcription of a polynucleotide sequence.
- promoter/regulatory sequence refers to a nucleic acid sequence which can be used for expression of a gene product operably linked to the promoter/regulatory sequence.
- this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
- the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
- constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
- inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
- tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
- linker and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
- the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3.
- the linkers include multiple repeats of (Gly 2 Ser), (GlySer) or (GlyNer) Also included within the scope of the disclosure are linkers described in WO2012/138475 (incorporated herein by reference).
- the linker sequence comprises a long linker (LL) sequence.
- the linker sequence comprises a short linker (SL) sequence.
- a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
- the 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other.
- RNA polymerase Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a cap- synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that may be required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
- the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
- in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
- the in vitro transcribed RNA is generated from an in vitro transcription vector.
- the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
- a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
- the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
- Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
- polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
- mRNA messenger RNA
- the 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
- poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
- Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
- the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
- the cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site.
- adenosine residues are added to the free 3’ end at the cleavage site.
- transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
- signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
- cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
- subject is intended to include living organisms in which an immune response can be elicited (e.g ., mammals, human).
- a “substantially purified” cell refers to a cell that is essentially free of other cell types.
- a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
- a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
- the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
- terapéutica as used herein means a treatment.
- a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
- prophylaxis means the prevention of or protective treatment for a disease or disease state.
- the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, glioblastoma, endometroid mucinous ovarian carcinoma, malignant pleural disease, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with MUC16 expression, and any combination thereof.
- transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
- a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
- the cell includes the primary subject cell and its progeny.
- the term “specifically binds,” refers to a specific ligand (e.g., a binding domain), which recognizes and binds a cognate binding partner (e.g., MUC16) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
- a specific ligand e.g., a binding domain
- MUC16 cognate binding partner
- Ranges throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range.
- a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%,
- compositions of matter and methods of use for the treatment of a disease such as cancer, using T cell receptor (TCR) fusion proteins having a non-antibody MUC16 binding domain are provided herein.
- TCR T cell receptor
- a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface target polypeptide or antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
- TFPs provide substantial benefits as compared to Chimeric Antigen Receptors.
- CAR Chimeric Antigen Receptor
- a CAR refers to a recombinant polypeptide comprising an extracellular binding domain, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “an intracellular signaling domains”) comprising a functional signaling domain derived from a stimulatory molecule as defined below.
- the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex.
- the CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.
- MUC16 (CA125) is a tumor associated antigen polypeptide, expressed by the human ocular surface epithelia in the mucosa of the bronchus, fallopian tube, and uterus. MUC16 is a large mucin-like glycoprotein present on the cell surface of tumor cells associated with beta- galactoside-binding, cell-surface lectins, which can be components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression.
- MUC16 can be to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces.
- Evidence suggests that expression of the cytoplasmic tail of MUC16 can enable tumor cells to grow, promote cell motility and may facilitate invasion. This appears to be due to the ability of the C-terminal domain of MUC16 to facilitate signaling that leads to a decrease in the expression of E-cadherin and increase the expression of N-cadherin and vimentin, which can be expression patterns consistent with epithelial-mesenchymal transition.
- MUC16 is a type I transmembrane protein composed of three domains, a large Ser-/Thr-rich N-terminal domain spanning 12,070 residues, a repeat domain of between eleven and more than 60 partially conserved tandem repeats of on average 156 amino acids each, and a C-terminal non-repeating domain containing a transmembrane sequence and a short cytoplasmic tail.
- the N-terminal domain and repeat domain are heavily O-glycosylated and N- glycosylated.
- MUC16 is expressed on the cell surface, and, in addition, soluble proteolytic fragments are also released into the extracellular space.
- mRNA encoding the MUC 16 polypeptide expressed from the MUC 16 gene can be significantly, reproducibly and detectably overexpressed in certain types of human cancerous ovarian, breast and pancreatic tumors as compared to the corresponding normal human ovarian, breast and pancreatic tissues, respectively.
- a variety of independent and different types of cancerous human ovarian tissue samples quantitatively analyzed for MUC16 expression show the level of expression of MUC16 in the cancerous samples can be variable, with a significant number of the cancerous samples showing an at least 6-fold (to as high as an about 580-fold) increase in MUC16 expression when compared to the mean level of ME1C16 expression for the group of normal ovarian tissue samples analyzed.
- detectable and reproducible MUC16 overexpression can be observed for ovarian cancer types; endometrioid adenocarcinoma, serous cystadenocarcinoma, including papillary and clear cell adenocarcinoma, as compared to normal ovarian tissue. Due to its overexpression in certain human tumors, the MUC16 polypeptide and the nucleic acid encoding that polypeptide are targets for quantitative and qualitative comparisons among various mammalian tissue samples. The expression profiles of MUC16 polypeptide, and the nucleic acid encoding that polypeptide, can be exploited for the diagnosis and therapeutic treatment of certain types of cancerous tumors in mammals.
- MUC16 is a serum marker used routinely to monitor patients with ovarian cancer.
- MUC16 is a mullerian duct differentiation antigen that is overexpressed in epithelial ovarian cancer cells and secreted into the blood, although its expression may not be entirely confined to ovarian cancer.
- MUC16 can be typical of serous ovarian adenocarcinoma, whereas it is not elevated in mucinous ovarian cancer. MUC16 may not be recommended for ovarian cancer screening because normal level may not exclude tumor. However, MUC16 detection can be a standard tool in monitoring clinical course and disease status in patients who have histologically confirmed malignancies.
- MUC16 levels Numerous studies have confirmed the usefulness of MUC16 levels in monitoring the progress of patients with EOC, and as a cancer serum marker. A rise in ME1C16 levels typically can precede clinical detection by about 3 months. During chemotherapy, changes in serum MUC16 levels can correlate with the course of the disease. MUC 16 can be used as a surrogate marker for clinical response in trials of new drugs. On the other hand, MUC 16 may not be useful in the initial diagnosis of EOC because of its elevation in a number of benign conditions. The MUC 16- specific antibody MAb-B43.13 (oregovomab, OvaRex MAb-B43.13) was in clinical trials for patients with ovarian carcinoma as an immunotherapeutic agent.
- MUC16 (CA-125) can play a role in advancing tumorigenesis and tumor proliferation by several different mechanisms.
- One way that MUC 16 helps the growth of tumors can be by suppressing the response of natural killer cells, thereby protecting cancer cells from the immune response.
- Further evidence that MUC 16 can protect tumor cells from the immune system may be the discovery that the heavily glycosylated tandem repeat domain of MUC 16 can bind to galectin-1 (an immunosuppressive protein).
- MUC 16 can participate in cell-to-cell interactions that enable the metastasis of tumor cells. This can be supported by evidence showing that MUC 16 can bind selectively to mesothelin, a glycoprotein normally expressed by the mesothelial cells of the peritoneum (the lining of the abdominal cavity).
- MUC 16 and mesothelin interactions may provide the first step in tumor cell invasion of the peritoneum.
- Mesothelin has also been found to be expressed in several types of cancers including mesothelioma, ovarian cancer and squamous cell carcinoma. Since mesothelin is also expressed by tumor cells, MUC 16 and mesothelial interactions may aid in the gathering of other tumor cells to the location of a metastasis, thus increasing the size of the metastasis.
- the extracellular domain of mature mesothelin binds MUC 16 and that amino acids 1-64, in particular, of mesothelin, have high affinity for MUC 16. In particular, mesothelin has higher affinity for membrane bound MUC 16 than for soluble MUC 16.
- the N-linked glycans of MUC 16 are necessary for binding to mesothelin.
- MUC 16 may also play a role in reducing the sensitivity of cancer cells to drug therapy.
- overexpression of MUC 16 can protect cells from the effects of genotoxic drugs, such as cisplatin.
- T cell receptor (TCR) fusion proteins (TFP) T cell receptor (TCR) fusion proteins (TFP)
- the present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises a polypeptide that binds specifically to MUC16, e.g ., human MUC16, wherein the sequence of the polypeptide is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR-integrating subunit or portion thereof.
- the TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
- the TFP of the present disclosure comprises a target-specific binding element (e.g., a MUC16-specific binding element) otherwise referred to as a binding domain (e.g., a MUC16 binding domain) that does not comprise an antibody or antibody fragment.
- a target-specific binding element e.g., a MUC16-specific binding element
- the TFP-mediated T cell response can be directed to a target of interest by way of engineering a binding domain into the TFP that specifically binds a desired target (e.g., MUC16).
- the portion of the TFP comprising the binding domain comprises a binding domain that targets MUC16.
- the binding domain targets human MUC16.
- the binding domain can be any domain that binds to the target other than an antibody or antibody fragment.
- the MUC16 binding domain provided herein can comprise a peptide sequence of a MUC 16-interacting protein or a fragment thereof (e.g., a functional fragment or fragment capable of interacting with MUC 16).
- the MUC 16 binding domain can specifically bind to a MUC 16 protein or portion thereof (e.g., the extracellular domain of MUC 16).
- the MUC 16 binding domain can bind to membrane-bound MUC 16.
- the MUC 16 binding domain can specifically bind to membrane-bound MUC 16 in the presence of soluble MUC 16.
- the MUC 16 binding domain can comprise a peptide sequence of mesothelin.
- the MUC 16 binding domain can comprise a full-length mesothelin (i.e., the full length mature mesothelin protein - amino acids 296-606 of the mesothelin precursor sequence) or a fragment thereof.
- the MUC 16 binding domain can comprise the mesothelin precursor protein or a fragment thereof.
- the MUC 16 binding domain can comprise a functional MUC 16 binding domain.
- the fragment of mesothelin can be a functional fragment.
- the functional fragment of mesothelin can comprise a minimum length of peptide sequence derived from mesothelin that can bind to MUC 16.
- the functional fragment of mesothelin can comprise an extracellular domain of mesothelin.
- the functional fragment of mesothelin can comprise at least 5, 6, 7, 8, 9,
- the MUC16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence of mesothelin or a fragment thereof.
- the MUC16 binding domain can comprise SEQ ID NO: 1 described herein.
- the MUC16 binding domain can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
- the MUC16 binding domain can comprise a truncation of SEQ ID NO: 1 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- EVEKT ACP S GKK AREIDE SLIF YKKWELE AC VD AALL AT QMDRVN AIPF T YEQL DVLKHKLDEL (SEQ ID NO: 1).
- the MUC16 binding domain can comprise mature mesothelin (SEQ ID NO: 2) described herein.
- the MUC16 binding domain can comprise at least 5, 6, 7, 8,
- the MUC16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence of mature mesothelin, for example, SEQ ID NO: 3 described herein.
- EVEKT ACP SGKKAPEIDESLIF YKKWELE AC VD AALL AT QMDRVNAIPFT YEQL DVLKHKLDEL YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMS PQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTC DPRQLD VL YPK ARL AF QNMN GSE YF VKIQ SFLGGAPTEDLK AL S Q QN V SMDL ATFMKL RTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPN GYLVLDLSMQEALS (SEQ ID NO: 3).
- the MUC16 binding domain provided herein can comprise a peptide sequence of a MUC 16-interacting protein other than mesothelin or a fragment thereof.
- MUC 16-interacting proteins include, but are not limited to, galectin 1 (LGALSl), immunoglobulin (CD79A) binding protein 1 (IGBP1), ubiquitin C (UBC), Double-strand-break repair protein rad21 homolog (RAD21), mini chromosome maintenance complex component 2 (MCM2), Nanog homeobox (NANOG), POU class 5 homeobox 1 (POU5F1), epidermal growth factor receptor (EGFR), tripartite motif containing 25 (TRIM25), Protein virilizer homolog (VIRMA), MUC1, MUC4,
- the MUC16 binding domain can comprise a peptide sequence selected from Table 1.
- the MUC 16 binding domain can comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
- the MUC 16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence selected from Table 1.
- the binding domain of the TFP comprises an amino acid sequence that is homologous to a binding domain amino acid sequence described herein, and the binding domain retains the desired functional properties of the binding domain described herein.
- the binding domain of the TFP is engineered by modifying one or more amino acids.
- binding domains of the present disclosure may further be modified such that they vary in amino acid sequence (e.g ., from wild-type), but not in desired activity.
- additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein.
- a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
- a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
- Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
- basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid
- Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 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%,
- identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
- sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
- sequence comparison algorithm When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g ., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.
- the algorithm parameters for using nucleotide BLAST to determine nucleotide sequence identity may use scoring parameters with a match/mismatch score of 1,-2 and wherein the gap costs are linear.
- the length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 28 for sequence alignment.
- the algorithm parameters for using protein BLAST to determine a peptide sequence identity may use scoring parameters with a BLOSUM62 matrix to assign a score for aligning pairs of residues, and determining overall alignment score, wherein the gap costs may have an existence penalty of 11 and an extension penalty of 1.
- the matrix adjustment method to compensate for amino acid composition of sequences may be a conditional compositional score matrix adjustment.
- the length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 6 for sequence alignment.
- the present disclosure contemplates modifications of the starting binding domain amino acid sequence that generate functionally equivalent molecules.
- the binding domain comprised in the TFP can be modified to retain at least about 70%, 71%. 72%.
- the present disclosure contemplates modifications of the entire TFP construct, e.g. , modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
- the TFP construct can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
- the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain.
- An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g ., the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, an extracellular domain may include at least the extracellular domain of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD279, or CD154.
- the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the extracellular domain comprises the IgC domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,
- the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
- the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
- the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
- the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C- terminus or at both the N- and C-terminus.
- the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
- the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
- the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- the extracellular domain can be a TCR extracellular domain.
- the TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit.
- the extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof.
- the extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- the extracellular domain can comprise a variable domain and the extracellular component of a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some cases, the extracellular domain may not comprise a variable domain.
- the extracellular domain can comprise the extracellular component of a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- the extracellular domain can comprise the extracellular component of a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- the extracellular domain can comprise a fragment (e.g., functional fragment) of the extracellular component of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- the extracellular domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the the extracellular component of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
- the TCR chain can be a murine or human TCR chain.
- the extracellular domain can comprise a constant domain of a murine TCR alpha chain, a murine TCR beta chain, a human TCR gamma chain or a human TCR delta chain.
- a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence.
- a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP.
- a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g ., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (at least 1, 2, 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, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3,
- the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used.
- the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
- the transmembrane domain is capable of homodimerization with another TFP on the TFP-
- the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
- the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
- the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein.
- a hinge e.g. , a hinge from a human protein.
- the hinge can be a human immunoglobulin (Ig) hinge, e.g. , an IgG4 hinge, or a CD8a hinge.
- the transmembrane domain comprises, or comprises at least 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, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
- the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
- the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- Linkers
- a short oligo- or polypeptide linker may form the linkage between the binding element and the TCR extracellular domain of the TFP.
- a glycine-serine doublet provides a particularly suitable linker.
- the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length.
- the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:37).
- the linker is encoded by a nucleotide sequence of GGT GGC GG AGGT TC T GG AGGT GG AGGT T C C (SEQ ID NO: 38).
- the cytoplasmic domain of the TFP can include an intracellular domain.
- the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
- the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
- An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
- effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
- intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
- intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
- intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
- the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
- the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
- the intracellular domain comprises a sequence having at least about 50%,
- the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- the intracellular domain comprises, or comprises at least 5,
- the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
- the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
- naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g ., a costimulatory domain).
- primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine- based activation motifs (ITAMs).
- ITAMs immunoreceptor tyrosine- based activation motifs
- ITAMs containing primary intracellular signaling domains that are of particular use in the present disclosure include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
- a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 epsilon, CD3 delta, or CD3 gamma.
- a primary signaling domain comprises a modified ITAM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
- a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
- a primary signaling domain comprises one, two, three, four or more ITAM motifs.
- the intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure.
- the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
- the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
- a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
- examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
- CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012;
- the intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order.
- a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8,
- the TFP- expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different binding domain, e.g., to the same target (MUC16) or a different target.
- a second TFP e.g., a second TFP that includes a different binding domain, e.g., to the same target (MUC16) or a different target.
- the binding domains of the different TFPs can be such that the binding domains do not interact with one another.
- a cell expressing a first and second TFP can have an binding domain of the first TFP that does not associate with the binding domain of the second TFP.
- the TFP-expressing cell described herein can further comprise one or more TCR constant domains, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
- the TCR subunit and the antibody can be operatively linked.
- the TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
- the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
- the TCR chain can be a murine or human TCR chain.
- the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.
- the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta, and further comprises the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
- TFP-expressing cell comprises a constant domain of a TCR alpha chain and a TCR beta chain.
- TFP- expressing cell comprises a constant domain of a TCR gamma chain and a TCR delta chain.
- the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR alpha chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR beta chain.
- the TCR beta constant domain can further comprise second binding domain that is operatively linked to the TCR beta constant domain.
- the second binding domain can be the same or different as the binding domain of the TFP.
- the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR beta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR alpha chain.
- the TCR alpha constant domain can further comprise second binding domain that is operatively linked to the TCR alpha constant domain.
- the second binding domain can be the same or different as the binding domain of the TFP.
- the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR gamma chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR delta chain.
- the TCR delta constant domain can further comprise a second binding domain that is operatively linked to the TCR delta constant domain.
- the second binding domain can be the same or different as the binding domain of the TFP.
- the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR delta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR gamma chain.
- the TCR gamma constant domain can further comprise a second binding domain that is operatively linked to the TCR gamma constant domain.
- the second binding domain can be the same or different as the binding domain of the TFP.
- the TFP-expressing cell described herein can further express another agent, e.g ., an agent which enhances the activity of a TFP-expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules e.g. , PD1
- Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFRbeta.
- the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g.
- the agent comprises a first polypeptide, e.g. , of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIRl, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4- 1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
- a costimulatory domain e.g., 4- 1BB, CD27 or CD28, e.g., as described herein
- a primary signaling domain e.g., a CD3 zeta signaling domain described herein.
- the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
- PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA.
- PD1 is expressed on activated B cells, T cells and myeloid cells (Agata et al.
- PD-L2 Programmed Death-Ligand 2
- Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.
- the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g ., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as CD28, 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP).
- the agent comprises the extracellular domain (ECD) and transmembrane domain of an inhibitory molecule, e.g. , Programmed Death 1 (PD1) that is fused to an intracellular signaling domain such as CD28, 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP).
- the PD1 TFP when used in combinations with an MUC16 TFP described herein, improves the persistence of the T cell.
- the TFP is a PD1 TFP comprising the extracellular domain of PD 1.
- TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the PD-L1 or PD-L2.
- the present disclosure provides a population of TFP-expressing T cells, e.g. , TFP-T cells.
- the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
- the population of TFP-T cells can include a first cell expressing a TFP having a MUC16 binding domain described herein, and a second cell expressing a TFP having a different MUC16 binding domain, e.g. , a MUC16 binding domain described herein that differs from the MUC16 binding domain in the TFP expressed by the first cell.
- the population of TFP- expressing cells can include a first cell expressing a TFP that includes a MUC16 binding domain, e.g. , as described herein, and a second cell expressing a TFP that includes a binding domain to a target other than MUC16 (e.g., another tumor-associated antigen).
- a target other than MUC16 e.g., another tumor-associated antigen
- the TFP of the present invention may be used in multi cistronic vectors or vectors expressing several proteins in the same transcriptional unit.
- Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit.
- Such peptides include 2A-peptides, or 2A- like sequences, from members of the Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589-594.
- the recombinant nucleic acid described herein encodes the TFP in frame with the agent, e.g., a second polypeptide, e.g., a second TFP, a constant domain, or a PD-1 fusion protein, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.
- the agent e.g., a second polypeptide, e.g., a second TFP, a constant domain, or a PD-1 fusion protein
- the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a MUC16 domain described herein, and a second cell expressing another agent, e.g. , an agent which enhances the activity of a TFP- expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules e.g. , can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
- the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g. , an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g. , an intracellular signaling domain described herein.
- the agent is a cytokine.
- the cytokine is IL-15.
- IL-15 increases the persistence of the T cells described herein.
- RNA encoding TFPs include methods for producing in vitro transcribed RNA encoding TFPs.
- the present disclosure also includes a TFP encoding RNA construct that can be directly transfected into a cell.
- a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
- RNA so produced can efficiently transfect different kinds of cells.
- the template includes sequences for the TFP.
- the MUC16 TFP is encoded by a messenger RNA (mRNA).
- mRNA messenger RNA
- the mRNA encoding the MUC16 TFP is introduced into a T cell for production of a TFP-T cell.
- the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
- the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
- DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
- the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
- the desired template for in vitro transcription is a TFP of the present disclosure.
- the DNA to be used for PCR contains an open reading frame.
- the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
- the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs).
- the nucleic acid can include exons and introns.
- the DNA to be used for PCR is a human nucleic acid sequence.
- the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs.
- the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
- An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
- PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
- Methods for performing PCR are well known in the art.
- Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
- “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
- the primers can be designed to be substantially complementary to any portion of the DNA template.
- the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs.
- the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
- the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs.
- Primers useful for PCR can be generated by synthetic methods that are well known in the art.
- “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
- Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
- reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
- Downstream is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
- Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
- the reagents and polymerase are commercially available from a number of sources.
- the RNA preferably has 5’ and 3’ UTRs.
- the 5’ UTR is between one and 3,000 nucleotides in length.
- the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.
- the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest.
- UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
- the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
- the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
- a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence.
- Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation.
- the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
- various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
- a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
- the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
- the promoter is a T7 polymerase promoter, as described elsewhere herein.
- Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
- the mRNA has both a cap on the 5’ end and a 3 ’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
- RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
- the transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
- phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res.,
- the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
- Poly(A) tails also provide stability to RNAs and reduce their degradation.
- the length of a poly(A) tail positively correlates with the stability of the transcribed
- the poly(A) tail is between 100 and 5000 adenosines.
- Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
- E-PAP E. coli polyA polymerase
- increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
- the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
- ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
- RNAs produced by the methods disclosed herein include a 5’ cap.
- the 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).
- RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
- IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
- RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation
- recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR- integrating subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, optionally comprising a stimulatory domain from an intracellular signaling domain; and (ii) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a T cell.
- TCR T cell receptor
- TFP T cell receptor fusion protein
- the TCR-integrating subunit and the binding domain are operatively linked by a linker sequence.
- the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, CD3 zeta, TCR alpha, TCR beta, TCR gamma or TCR delta.
- the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, or only CD3 delta.
- the TCR-integrating subunit comprises an intracellular domain.
- the intracellular domain is from TCR alpha, TCR beta, TCR gamma or TCR delta.
- the intracellular domain comprises a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, or an amino acid sequence having at least one modification thereto.
- the TCR-integrating subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
- the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the TCR-integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta chain TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD 137, CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
- the TCR-integrating subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, CD3 zeta, or an amino acid sequence having at least one modification thereto.
- the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- 1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
- the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain.
- the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
- the TCR-integrating subunit comprises an immunoreceptor tyrosine- based activation motif (IT AM) of a TCR-integrating subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional
- IT AM immunorecept
- the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon.
- the IT AM is selected from the group consisting of a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, and a CD3 delta subunit and replaces a different IT AM selected from the group consisting of a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, and a CD3 delta subunit.
- the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
- the binding domain comprises mesothelin or a fragment thereof.
- the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin.
- the functional MUC16 binding domain of mesothelin comprises the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of
- EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKH KLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
- the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
- the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the nucleic acid is a circular RNA.
- the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
- the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR delta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain and a sequence encoding a TCR delta transmembrane domain.
- the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases
- CRISPR® clustered regularly interspaced short palindromic repeats
- TALE transcription activator-like effector
- TALENs see, e.g., U.S. Patent No. 9,393,257
- ZFN zinc finger nuclease
- TAL repeats methods.
- a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also
- TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
- gene editing techniques are employed to disrupt an endogenous TCR gene.
- mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
- mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain.
- gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene.
- multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
- HLAs human leukocyte antigens
- PD1 programmed cell death protein 1
- nickase nucleases generate single-stranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
- ssDI single strand DNA incorporation
- ssTR single strand template repair
- Genetic modification of genomic DNA can be performed using site-specific, rare- cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome.
- ZFNs zinc-finger nucleases
- ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme.
- the zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre determined DNA sequence -18 basepairs in length.
- ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005), Nucleic Acids Res 33, 5978).
- TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
- a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9).
- the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
- Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762).
- a Compact TALEN comprises an engineered, site-specific TAL- effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease. Unlike Fokl, I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
- Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
- the CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
- a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
- a caspase effector nuclease typically microbial Cas9
- a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
- CRISPR systems There are two classes of CRISPR systems known in the art (Adli (2016) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the type II CRISPR- Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing.
- Cpfl Casl2a proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl) are particularly interesting.
- Homing endonucleases are a group of naturally-occurring nucleases that recognize 15-
- meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446).
- meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.
- chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446).
- a megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
- nucleases In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus. Vectors
- the instant invention provides vectors comprising the recombinant nucleic acid(s) encoding the TFP and/or additional molecules of interest (e.g., a protein or proteins to be secreted by the TFP T cell).
- the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
- the vector is an AAV6 vector. In some instances, the vector further comprises a promoter. In some instances, the vector is an in vitro transcribed vector.
- nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
- the gene of interest can be produced synthetically, rather than cloned.
- the present disclosure also provides vectors in which a DNA of the present disclosure is inserted.
- Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
- Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
- the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35).
- the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.
- the expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties).
- the present disclosure provides a gene therapy vector.
- the nucleic acid can be cloned into a number of types of vectors.
- the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
- Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
- the expression vector may be provided to a cell in the form of a viral vector.
- Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
- Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
- a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
- retroviruses provide a convenient platform for gene delivery systems.
- a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
- the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
- retroviral systems are known in the art.
- adenovirus vectors are used.
- a number of adenovirus vectors are known in the art.
- lentivirus vectors are used.
- Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
- these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
- the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
- tk thymidine kinase
- the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
- individual elements can function either cooperatively or independently to activate transcription.
- a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter.
- the native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
- the EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther. 17(8): 1453- 1464 (2009)).
- CMV immediate early cytomegalovirus
- This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
- other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
- SV40 simian virus 40
- MMTV mouse mammary tumor virus
- HSV human immunodeficiency virus
- inducible promoters are also contemplated as part of the present disclosure.
- the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
- inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
- the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
- the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells.
- Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
- Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
- a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
- Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
- the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
- the expression vector can be transferred into a host cell by physical, chemical, or biological means.
- Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection [00265] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
- Viral vectors and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.
- Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
- Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
- An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome
- nucleic acids e.g., an artificial membrane vesicle.
- Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
- an exemplary delivery vehicle is a liposome.
- lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
- the nucleic acid may be associated with a lipid.
- the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
- Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
- Lipids are fatty substances which may be naturally occurring or synthetic lipids.
- lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
- Lipids suitable for use can be obtained from commercial sources.
- DMPC dimyristyl phosphatidylcholine
- DCP dicetyl phosphate
- Choi cholesterol
- DMPG dimyristyl phosphatidylglycerol
- Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol.
- Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991
- compositions that have different structures in solution than the normal vesicular structure are also encompassed.
- the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
- lipofectamine-nucleic acid complexes are also contemplated.
- assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
- moleukin assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
- biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
- the present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule.
- a TFP vector can be directly transduced into a cell, e.g., a T cell.
- the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
- the vector is capable of expressing the TFP construct in mammalian T cells.
- the mammalian T cell is a human T cell.
- TFP T cells are transduced with an RNA molecule.
- the RNA is circular RNA.
- the circular RNA is exogenous.
- circular RNA is endogenous.
- circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or in vivo or ex vivo.
- Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5’ covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half- lives and may therefore improve the overall efficacy of mRNA in a variety of applications.
- Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al.,
- splice sites are used in reverse: downstream splice donors are
- RNA circularization To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed.
- IVT in vitro transcription
- Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
- precursor RNA was synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
- the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
- the group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998). When the td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995).
- the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.
- a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg 2+ as cofactors.
- This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro , these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5' to 3' linked circles.
- a sequence containing a full-length encephalomyocarditis virus such as EMCV) IRES, a gene encoding a TFP, a CAR, a TCR or combination thereof, two short regions corresponding to exon fragments (El and E2), and of the PIE construct between the 3' and 5' introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage or the permuted group I catalytic intron in the pre-tRNA gene of Anabaena.
- EMCV encephalomyocarditis virus
- the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5' and 3' ends of the precursor RNA with the aim of bringing the 5' and 3' splice sites into proximity of one another.
- the splicing reaction can be treated with RNase R.
- the TFP is encoded by a circular RNA.
- the circular RNA encoding the TFP is introduced into a T cell for production of a TFP-T cell.
- the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
- linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template.
- DNA of interest from any source can be directly converted by PCR into a template for in vitro RNA synthesis using appropriate primers and buffer and RNA polymerase and nucleotides modified or not.
- the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, digested DNA, synthetic DNA sequence or any other appropriate source of DNA.
- the desired template for in vitro transcription is a TFP of the present disclosure.
- the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism.
- the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs).
- the nucleic acid can include exons and introns.
- the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and
- the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
- An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein.
- the portions of DNA that are ligated together can be from a single organism or from more than one organism.
- PCR is used to generate a template for in vitro transcription of linear precursor RNA which is used for transfection.
- Methods for performing PCR are well known in the art.
- Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
- “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
- the primers can be designed to be substantially complementary to any portion of the DNA template.
- the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs.
- the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
- the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs.
- Primers useful for PCR can be generated by synthetic methods that are well known in the art.
- “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
- Upstream is used herein to refer to a location 5’ to the DNA sequence to be amplified relative to the coding strand.
- reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
- Downstream is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
- Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
- the reagents and polymerase are commercially available from a number of sources.
- the RNA preferably has 5’ and 3’ UTRs.
- the 5’ UTR is between one and 3000 nucleotides in length.
- the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal RNA stability or/and translation efficiency following transfection of the transcribed RNA.
- the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest.
- UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
- the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA.
- AU-rich elements in 3’UTR sequences can decrease the stability of mRNA whereas protein binding motif can increase the stabilitiy of mRNA and circular RNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
- the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
- a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some
- RNA transcripts but do not appear to be required for all RNAs to enable efficient translation.
- the requirement for Kozak sequences for many mRNAs is known in the art.
- the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
- various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
- a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
- the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
- the promoter is a T7 polymerase promoter, as described elsewhere herein.
- Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
- the RNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
- RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
- phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res ., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
- the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
- Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
- Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E.
- E-PAP coli polyA polymerase
- increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
- the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
- ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
- RNAs produced by the methods disclosed herein include a 5’ cap.
- the 5’ cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436- 444 (2001); Stepinski et al., RNA , 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun ., 330:958-966 (2005)).
- RNAs e.g. circular RNA
- IRES internal ribosome entry site
- the IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
- RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector®-II (Amaxa Biosystems, Cologne, Germany)), ECM® 830 (BTX) (Harvard Instruments, Boston, Mass.), Neon Transfection System (ThermoFisher), Cell squeezing (SQZ Biotechnologies) or the Gene Pulser® II (BioRad, Denver, Colo.),
- modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein. [00294] In some embodiments, the modified T cell comprises a functional disruption of an endogenous TCR.
- the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
- the endogenous TCR that is functionally disrupted is an endogenous
- the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
- the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain.
- the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
- the functional disruption is a disruption of a gene encoding the endogenous TCR.
- the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
- the T cell is a human T cell.
- the T cell is a CD8+ or CD4+ T cell.
- the T cell is an allogenic T cell.
- the T cell is a TCR alpha-beta T cell.
- the T cell is a TCR gamma-delta T cell.
- TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No.
- the modified T cells are ⁇ T cells.
- the ⁇ T cells are V ⁇ 1+ V ⁇ 2- ⁇ T cells.
- the ⁇ T cells are V ⁇ 1- V ⁇ 2+ ⁇ T cells.
- the ⁇ T cells are V ⁇ 1- V ⁇ 2- ⁇ T cells.
- a source of T cells Prior to expansion and genetic modification, a source of T cells is obtained from a subject.
- the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
- T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used.
- T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
- cells from the circulating blood of an individual are obtained by apheresis.
- the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
- the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
- the cells are washed with phosphate buffered saline (PBS).
- PBS phosphate buffered saline
- the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
- a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, the Oncol ogyCytoMate, or the
- the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free,
- Mg-free PBS PlasmaLyte A
- PlasmaLyte A or other saline solution with or without buffer.
- the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
- the T cells are alpha beta (ab or ⁇ ) T cells.
- the T cells are ⁇ T cells ⁇ T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.
- Suitable doses for a therapeutic effect would be at least 10 5 or between about 10 5 and about 10 10 cells per dose, for example, preferably in a series of dosing cycles.
- An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 10 5 cells on Day 0, for example increasing incrementally up to a target dose of about 10 10 cells within several weeks of initiating an intra-patient dose escalation scheme.
- Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.
- an effective amount or sufficient number of the isolated, T cells is present in the composition and introduced into the subject such that long-term, specific, anti -turn or responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment.
- the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.
- the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as to achieve the desired therapeutic response.
- the amounts of each active agent included in the compositions described herein e.g., the amount per each cell to be contacted or the amount per certain body weight
- T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL ® gradient or by counterflow centrifugal elutriation.
- a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques.
- T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g.
- the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours.
- TIL tumor infiltrating lymphocytes
- subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
- multiple rounds of selection can also be used in the context of this disclosure. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
- Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
- One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
- a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8.
- T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
- a T cell population can be selected that expresses one or more of IFN- ⁇ , TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g ., other cytokines.
- Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712, which is herein incorporated by reference.
- the concentration of cells and surface can be varied.
- it may be desirable to significantly decrease the volume in which beads and cells are mixed together e.g., increase the concentration of cells, to ensure maximum contact of cells and beads.
- a concentration of 2 billion cells/mL is used.
- a concentration of 1 billion cells/mL is used.
- greater than 100 million cells/mL is used.
- a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
- a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
- concentrations of 125 or 150 million cells/mL can be used.
- Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
- use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
- using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
- the concentration of cells used is 5xl0 6 /mL. In other aspects, the concentration used can be from about 1x10 5 /mL to 1x10 6 /mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.
- T cells for stimulation can also be frozen after a washing step.
- the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
- the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5%
- cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
- a blood sample or an apheresis product is taken from a generally healthy subject.
- a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
- the T cells may be expanded, frozen, and used at a later time.
- samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
- the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.
- agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-
- T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
- the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
- these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
- mobilization for example, mobilization with
- GM-CSF GM-CSF
- conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
- Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
- T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, and 7,572,631.
- the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
- an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
- T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g ., bryostatin) in conjunction with a calcium ionophore.
- a protein kinase C activator e.g ., bryostatin
- a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
- a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
- an anti-CD3 antibody and an anti-CD28 antibody To stimulate proliferation of either CD4+ T cells, CD8+ T cells, or CD4+CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody.
- an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999).
- T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS ® or Trans-Act ® beads, for a time period sufficient for activation of the T cells.
- the time period is at least 1, 2, 3, 4, 5, or 6 hours.
- the time period is 10 to 24 hours, e.g., 24 hours.
- T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others).
- T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-
- cells activated in the presence of an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction.
- the cells after transduction, are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody.
- the cells are subcultured every 1, 2, 3, 4, 5, or 6 days.
- cells are expanded for 4, 5, 6, 7,
- T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0,
- T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP orHMB-PP) or other structurutally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0,
- T cells 5.0, 7.5, 10, or 100 mM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
- feeder cells irradiated cancer cells, PBMCs, artificial antigen presenting cells.
- the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-
- the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days.
- the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g.,
- OKT3 in the presence of IL-2.
- the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.
- T cells that have been exposed to varied stimulation times may exhibit different characteristics.
- typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
- TH, CD4+ helper T cell population
- TC, CD8+ cytotoxic or suppressor T cell population
- CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
- CD4 and CD8 markers vary significantly, but in large part, reproducibly during the course of the cell expansion process.
- MUC16 TFP Once a MUC16 TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a MUC16 TFP are described in further detail below
- TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g ., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
- T cells (1:1 mixture of CD4 + and CD8 + T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions.
- TFPs are detected by Western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
- TFP + T cells following stimulation can be measured by flow cytometry.
- a mixture of CD4 + and CD8 + T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed.
- exemplary promoters include the CMV IE gene, EF-1 alpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters.
- GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
- a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence.
- mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1.
- mice can also be used to measure a TFP-T activity.
- xenograft model using human MUC16-specific TFP+ T cells to treat a cancer in immunodeficient mice see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
- mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1 : 1 ratio into NOD/SCID/ ⁇ -/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals.
- Peripheral blood MUC16+ cancer cell counts are measured in mice that are injected with alpha-MUC16-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test.
- absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/ ⁇ -/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for cancer at 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
- Dose dependent TFP treatment response can be evaluated (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
- peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood MUC16+ cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
- Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo.
- T cells are enumerated in cultures using CountBrightTM fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer.
- TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP-expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant MUC16 protein and a secondary avidin-PE conjugate.
- CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer’s instructions.
- Fluorescence is assessed using a FACScalibur flow cytometer, and data is analyzed according to the manufacturer’s instructions.
- Cytotoxicity can be assessed by a standard 51 Cr-release assay (see, e.g ., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, target cells are loaded with 51 Cr (as NaCrO 4 , New England Nuclear) at 37 °C for 2 hours with frequent agitation, washed twice in complete RPMI medium and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of triton-X 100 detergent (total release, TR) are also prepared.
- 51 Cr as NaCrO 4 , New England Nuclear
- % Lysis (ER-SR)/(TR-SR), where ER represents the average 51 Cr released for each experimental condition.
- Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g. , in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCTD/yc-/- (NSG) mice are injected IV with cancer cells followed 7 days later with T cells 4 hour after electroporation with the TFP constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence.
- therapeutic efficacy and specificity of a single injection of TFP+ T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with MUC16 TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated. [00322] Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the MUC16 TFP constructs of the present disclosure.
- the present disclosure provides methods for treating a disease associated with MUC16 expression.
- the present disclosure provides methods for treating a disease wherein part of the tumor is negative for MUC16 and part of the tumor is positive for MUC16.
- the TFP of the present disclosure is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of MUC16, wherein the subject that has undergone treatment for elevated levels of MUC16 exhibits a disease associated with elevated levels of MUC16.
- the present disclosure pertains to a vector comprising MUC16 TFP operably linked to promoter for expression in mammalian T cells.
- the present disclosure provides a recombinant T cell expressing the MUC16 TFP for use in treating MUC 16- expressing tumors, wherein the recombinant T cell expressing the MUC 16 TFP is termed a MUC16 TFP-T.
- the MUC16 TFP-T of the present disclosure is capable of contacting a tumor cell with at least one MUC 16 TFP of the disclosure expressed on its surface such that the TFP-T targets the tumor cell and growth of the tumor is inhibited.
- the present disclosure pertains to a method of inhibiting growth of a MUC 16-expressing tumor cell, comprising contacting the tumor cell with a MUC 16 TFP T cell of the present disclosure such that the TFP-T is activated in response to the target (MUC 16) and targets the cancer cell, wherein the growth of the tumor is inhibited.
- the present disclosure pertains to a method of treating cancer in a subject.
- the method comprises administering to the subject a MUC 16 TFP T cell of the present disclosure such that the cancer is treated in the subject.
- An example of a cancer that is treatable by the MUC16 TFP T cell of the disclosure is a cancer associated with expression of MUC16.
- the cancer is a mesothelioma.
- the cancer is a pancreatic cancer.
- the cancer is an ovarian cancer.
- the cancer is a stomach cancer.
- the cancer is a lung cancer.
- the cancer is an endometrial cancer.
- MUC 16 TFP therapy can be used in combination with one or more additional therapies.
- the present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
- the infused cell is able to kill tumor cells in the recipient.
- TFP- expressing T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
- the T cells administered to the patient, or their progeny persist in the patient for at least one month, two month, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty -two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
- the present disclosure also includes a type of cellular therapy where T cells are modified, e.g ., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
- the infused cell is able to kill tumor cells in the recipient.
- the T cells administered to the patient is present for less than one month, e.g. , three weeks, two weeks, or one week, after administration of the T cell to the patient.
- the anti-tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.
- the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the MUC16 antigen, resist soluble MUC16 inhibition, mediate bystander killing and/or mediate regression of an established human tumor.
- antigen-less tumor cells within a heterogeneous field of MUC 16-expressing tumor may be susceptible to indirect destruction by MUC16-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
- the human TFP -modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
- the mammal is a human.
- cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro ) with a vector expressing a TFP disclosed herein.
- the TFP- modified cell can be administered to a mammalian recipient to provide a therapeutic benefit.
- the mammalian recipient may be a human and the TFP-modified cell can be autologous with respect to the recipient.
- the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
- ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo.
- other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
- the modified T cells described herein are allogeneic T cells. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid disclosed herein, or the vector disclosed herein.
- compositions and methods for in vivo immunization to elicit an immune response directed against MUC16 in a patient In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against MUC16 in a patient.
- the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
- the TFP-modified T cells of the disclosure are used in the treatment of diseases, disorders and conditions associated with expression of MUC16.
- the cells of the disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of MUC16.
- the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of MUC16 comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP-modified T cells of the present disclosure.
- the TFP-T cells of the present disclosure may be used to treat a proliferative disease such as a cancer or malignancy or a precancerous condition.
- the cancer is a mesothelioma.
- the cancer is a pancreatic cancer.
- the cancer is an ovarian cancer.
- the cancer is a stomach cancer.
- the cancer is a lung cancer.
- the cancer is breast cancer.
- the cancer is a endometrial cancer.
- a disease associated with MUC16 expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing MUC16.
- Non-cancer related indications associated with expression of MUC16 include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma), inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery and transplantation.
- TFP-modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
- the present disclosure also provides methods for inhibiting the proliferation or reducing a MUC 16-expressing cell population, the methods comprising contacting a population of cells comprising a MUC 16-expressing cell with a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell.
- the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing MUC 16, the methods comprising contacting the MUC 16-expressing cancer cell population with a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell.
- the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing MUC 16, the methods comprising contacting the MUC 16-expressing cancer cell population with an a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell.
- the MUC16 TFP-T cell of the present disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model a cancer associated with MUC 16-expressing cells relative to a negative control.
- the subject is a human.
- the present disclosure also provides methods for preventing, treating and/or managing a disease associated with MUC 16-expressing cells (e.g., a cancer expressing MUC 16), the methods comprising administering to a subject in need a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell.
- the subject is a human.
- disorders associated with MUC 16-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, or endometrial cancer or atypical cancers expressing MUC 16).
- the present disclosure also provides methods for preventing, treating and/or managing a disease associated with MUC 16-expressing cells, the methods comprising administering to a subject in need a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16- expressing cell.
- the subject is a human.
- the present disclosure provides methods for preventing relapse of cancer associated with MUC 16-expressing cells, the methods comprising administering to a subject in need thereof a MUC16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell.
- the methods comprise administering to the subject in need thereof an effective amount of a MUC 16 TFP-T cell described herein that binds to the MUC 16-expressing cell in combination with an effective amount of another therapy.
- a TFP-expressing cell described herein may be used in combination with other known agents and therapies.
- Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g ., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
- the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
- the delivery of one treatment ends before the delivery of the other treatment begins.
- the treatment is more effective because of combined administration.
- the second treatment is more effective, e.g. , an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment.
- delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
- the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
- the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
- the “at least one additional therapeutic agent” includes a TFP- expressing cell.
- T cells that express multiple TFPs, which bind to the same or different targets or target antigens, or same or different epitopes on the same target antigen.
- populations of T cells in which a first subset of T cells express a first TFP and a second subset of T cells express a second TFP.
- a TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially.
- the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
- a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
- a TFP-expressing cell described herein may also be used in combination with a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg
- a TFP-expressing cell described herein may also be used in combination with a promoter of myeloid cell differentiation (e.g., all-trans retinoic acid), an inhibitor of myeloid-derived suppressor cell (MDSC) expansion (e.g., inhibitors of c-kit receptor or a VEGF inhibitor), an inhibition of MDSC function (e.g., COX2 inhibitors or phosphodiesterase-5 inhibitors), or therapeutic elimination ofMDSCs (e.g., with a chemotherapeutic regimen such as treatment with doxorubicin and cyclophosphamide).
- a promoter of myeloid cell differentiation e.g., all-trans retinoic acid
- MDSC myeloid-derived suppressor cell
- an inhibition of MDSC function e.g., COX2 inhibitors or phosphodiesterase-5 inhibitors
- therapeutic elimination ofMDSCs e.g., with a chemotherapeutic regimen such as treatment with doxorubicin and cyclophos
- Other therapeutic agents that may prevent the expansion ofMDSCs include amino-biphosphonate, biphosphanate, sildenafil and tadalafil, nitroaspirin, vitamin D3, and gemcitabine. (See, e.g.,
- the additional therapeutic agent comprises an immunostimulatory agent.
- the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof.
- the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), programmed cell death protein 1 (also PD-1 or CD279), programmed death ligand 1 (also PD-L1 or CD274), transforming growth factor beta (TGF ⁇ ), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 or HAVCR2 or CD366), neuritin, B- and T-lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof.
- CTL-4 cytotoxic T-lymphocyte-associated protein 4
- TGF ⁇ transforming growth factor beta
- LAG-3 lymphocyte-activation gene 3
- Tim-3 hepatitis A virus cellular receptor 2 or HAV
- the agent is selected from an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-Ll antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a) and 5 (CEACAM-5, also CD66e), vset immunoregulatory receptor (also VISR or VISTA), leukocyte-associated immunoglobulin-like receptor 1 (also LAIRl or CD305), CD160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof.
- an anti-PD-1 antibody e.g., pembrolizumab or nivolumab
- anti-PD-Ll antibody e.g., atezolizumab
- an anti-CTLA-4 antibody e.g., ipi
- the agent is pembrolizumab. In some aspects, the agent is nivolumab. In some aspects, the agent is atezolizumab. [00349] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic and a small molecule.
- the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine
- the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., ChemMed Chem , 2016, 14:1576 (DOI:
- the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and
- PD-L1 is administered after administration of an antibody provided herein.
- the agent that inhibits the interaction between PD-1 and PD-L1 is administered contemporaneously with an antibody provided herein, but the agent and antibody are administered in separate pharmaceutical compositions.
- the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell.
- the co-stimulatory receptor is selected from GITR, 0X40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, a toll-like receptor, RIG-1, and a NOD-like receptor.
- the agonist is an antibody.
- the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3 -di oxygenase, or the adenosine A2A receptor.
- the immunostimulatory agent is a cytokine.
- the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.
- the immunostimulatory agent is an oncolytic virus.
- the oncolytic virus is selected from a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, and a maraba virus.
- additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine).
- the additional therapeutic agent is folinic acid, 5- fluorouracil, and/or oxaliplatin.
- the additional therapeutic agent is 5- fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or
- the additional therapeutic agent may be administered by any suitable means.
- a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition.
- an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.
- administration of the TFP can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
- administration of a TFP provided herein, and the additional therapeutic agent occur within about one month of each other.
- administration of a TFP provided herein, and the additional therapeutic agent occur within about one week of each other.
- administration of a TFP provided herein, and the additional therapeutic agent occur within about one day of each other.
- administration of a TFP provided herein, and the additional therapeutic agent occur within about twelve hours of each other.
- administration of a TFP provided herein, and the additional therapeutic agent occur within about one hour of each other.
- the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell.
- Side effects associated with the administration of a TFP-expressing cell include, but are not limited to cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
- CRS cytokine release syndrome
- HHL hemophagocytic lymphohistiocytosis
- MAS Macrophage Activation Syndrome
- Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
- the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell.
- the soluble factor elevated in the subject is one or more of IFN- ⁇ , TNF ⁇ , IL-2, IL-6 and IL8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
- agents include, but are not limited to a steroid, an inhibitor of TNF ⁇ , and an inhibitor of IL-6.
- An example of a TNF ⁇ inhibitor is entanercept.
- An example of an IL-6 inhibitor is tocilizumab (toe).
- the subject can be administered an agent which enhances the activity of a TFP-expressing cell.
- the agent can be an agent which inhibits an inhibitory molecule.
- Inhibitory molecules e.g., Programmed Death 1 (PD1)
- PD1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
- inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3,
- an inhibitory nucleic acid e.g. , an inhibitory nucleic acid, e.g. , a dsRNA, e.g. , an siRNA or shRNA
- an inhibitory molecule can be used to inhibit expression of an inhibitory molecule in the TFP-expressing cell.
- the inhibitor is a shRNA.
- the inhibitory molecule is inhibited within a TFP-expressing cell.
- a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g. , all of the components, of the TFP.
- the inhibitor of an inhibitory signal can be, e.g. , an antibody or antibody fragment that binds to an inhibitory molecule.
- the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as
- MDX-010 and MDX-101 are marketed as YervoyTM; Bristol-Myers Squibb; tremelimumab
- the agent is an antibody or antibody fragment that binds to TIM3.
- the agent is an antibody or antibody fragment that binds to LAG3.
- the T cells may be altered (e.g., by gene transfer) in vivo via a lentivirus, e.g., a lentivirus specifically targeting a CD4+ or CD8+ T cell.
- a lentivirus e.g., a lentivirus specifically targeting a CD4+ or CD8+ T cell.
- the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
- the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
- the fusion protein is expressed by the same cell that expressed the TFP.
- the fusion protein is expressed by the same vector as the TFP.
- the fusion protein is expressed by a cell, e.g., a T cell that does not express a MUC16 TFP.
- compositions of the present disclosure may comprise a TFP-expressing cell, e.g ., a plurality of TFP-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
- Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
- Compositions of the present disclosure are in one aspect formulated for intravenous administration.
- compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
- the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
- the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
- a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
- the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
- an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
- T cells can be activated from blood draws of from 10 cc to 400 cc.
- T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
- compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
- the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection.
- the T cell compositions of the present disclosure are administered by i.v. injection.
- the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
- subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g ., T cells.
- T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP-expressing T cell of the present disclosure.
- Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
- subjects receive an infusion of the expanded TFP T cells of the present disclosure.
- expanded cells are administered before or following surgery.
- the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
- the scaling of dosages for human administration can be performed according to art-accepted practices.
- the dose for alemtuzumab will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
- the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766).
- the TFP is introduced into T cells, e.g. , using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
- more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the present disclosure are administered per week.
- the subject receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject.
- the subject receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
- the TFP T cells are administered every other day for 3 administrations per week.
- the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
- MUC16 TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way will have stable TFP expression.
- TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.
- Transient expression of TFPs can be affected by RNA TFP vector delivery.
- the TFP RNA is transduced into the T cell by electroporation.
- anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen-day break in exposure to antigen.
- TFP T cell infusion breaks should not last more than ten to fourteen days.
- Cytokine release syndrome is a form of systemic inflammatory response syndrome that arises as a complication of some diseases or infections, and is also an adverse effect of some monoclonal antibody drugs, as well as adoptive T cell therapies.
- TFP T cells can exhibit better killing activity than CAR-T cells.
- TFP T cells administered to a subject can exhibit better killing activity than CAR-T cells administered to a subject. This can be one of the advantages of TFP T cells over CAR-T cells.
- TFP T cells can exhibit less cytokine release CAR-T cells.
- a subject administered TFP T cells can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies.
- TFP T cells can exhibit similar or better killing activity than CAR-T cells and the TFP T cells can exhibit less cytokine release than the CAR-T cells.
- TFP T cells administered to a subject can exhibit similar or better killing activity than CAR-T cells administered to a subject and the subject can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies.
- the cytokine release of a treatment with TFP T cells is less than the cytokine release of a treatment with CAR-T cells. In some embodiments, the cytokine release of a treatment with TFP T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least
- cytokine release of a treatment with CAR-T cells 50%, at least 60%, at least 70%, at least 80%, or at least 90% less than the cytokine release of a treatment with CAR-T cells.
- Various cytokines can be released less in the T cell treatment with
- the cytokine is IL-2, IFN- ⁇ , IL-4, TNF- ⁇ ,
- the treatment with TFP T cells release less perforin, granzyme A, granzyme B, or a combination thereof, than the treatment with CAR-T cells.
- the perforin, granzyme A, or granzyme B released in a treatment with TFP T cells is at least 10%, at least
- the given cytokine comprises one or more cytokines selected from the group consisting of IL-2, IFN- ⁇ , IL-4, TNF- ⁇ , IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-1 ⁇ , MIP-1 ⁇ , and any combination thereof.
- the TFP T cells may exhibit similar or better activity in killing tumor cells than CAR-T cells.
- a tumor growth in the mammal is inhibited such that a size of the tumor is at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, or at most 60% of a size of a tumor in a mammal treated with T cells that do not express the TFP after at least 8 days of treatment, wherein the mammal treated with T cells expressing TFP and the mammal treated with T cells that do not express the TFP have the same tumor size before the treatment.
- the tumor growth in the mammal is completely inhibited.
- the tumor growth in the mammal is completely inhibited for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, or more.
- the population of T cells transduced with TFP kill similar amount of tumor cells compared to the CAR-T cells comprising the same binding domain.
- the TFP T cells can exhibit different gene expression profile than cells that do not express TFP. In some cases, the TFP T cells may exhibit similar gene expression profiles than
- the TFP T cells may exhibit different gene expression profiles than CAR-T cells.
- the population of T cells transduced with TFP have a different gene expression profile than the CAR-T cells comprising the same binding domain.
- an expression level of a gene is different in the T cells transduced with the
- the gene has a function in antigen presentation, TCR signaling, homeostasis, metabolism, chemokine signaling, cytokine signaling, toll like receptor signaling,
- MMP and adhesion molecule signaling or TNFR related signaling.
- the MUC 16-binding IAB peptide of mesothelin comprises amino acids 1-64 of mesothelin
- the expression of MUC16 by OVCAR3 cells, but not C30 cells was validated by anti-human MUC16 Ab (Clone X75, R-phycoerythrin conjugated) (FIG. 1 A).
- the binding of IAB to MUC16 was validated by testing allophycocyanin-conjugaed IAB hFc peptide (labeled by Zenon human IgG labeling reagents) against MUC 16-positive OVCAR3 cells and MUC 16-negative C30 cells. As shown in FIG. IB, binding of IAB hFc is detected for the MUC 16-positive OVCAR3 cells but not the MUC 16-negative C30 cells.
- MUC 16 TFP constructs can be engineered by cloning the DNA sequence of amino acids 1-64 of mesothelin
- MUC16 polypeptide canonical sequence is UniProt Accession No. Q8WXI7. Provided are polypeptides that are capable of specifically binding to the human MUC16 polypeptide, and fragments thereof.
- Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain.
- the CD3 episolon, CD3 delta, and CD3 gamma subunits have an intracellular domain.
- a human TCR complex contains the CD3-epsilon polypeptide, the CD3 -gamma poly peptide, the CD3 -delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide.
- TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide.
- the human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766.
- the human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693.
- the human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234.
- the human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963.
- the human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1.
- the human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.
- the human CD3-epsilon polypeptide canonical sequence is:
- the signal peptide of human CD3 ⁇ is:
- VMS VATIVIVDICITGGLLLLVYYW S (SEQ ID NO: 54).
- the intracellular domain of human CD3 ⁇ is: KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:55).
- the human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS GFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ GNQLRRN (SEQ ID NO:42).
- the signal peptide of human CD3 ⁇ is: MEQGKGLAVLILAIILLQGTLA (SEQ ID NO:56).
- the extracellular domain of human CD3 ⁇ is: QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO:57).
- the transmembrane domain of human CD3 ⁇ is: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO:58).
- the intracellular domain of human CD3 ⁇ is: GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:59).
- the human CD3-delta polypeptide canonical sequence is: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:43).
- the signal peptide of human CD3 ⁇ is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:60).
- the extracellular domain of human CD3 ⁇ is: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE STVQVHYRMCQSCVELDPATVA (SEQ ID NO:61).
- the transmembrane domain of human CD3 ⁇ is: GIIVTDVIATLLLALGVFCFA (SEQ ID NO:62).
- the intracellular domain of human CD3 ⁇ is: GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO:63).
- the human CD3-zeta polypeptide canonical sequence is: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:44).
- the human TCR alpha chain canonical sequence is: MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAG PLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:45).
- the human TCR alpha chain C region canonical sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:46).
- the human TCR alpha chain human IgC sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 69)
- the transmembrane domain of the human TCR alpha chain is: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:68).
- the intracellular domain of the human TCR alpha chain is: SS
- the human TCR alpha chain V region CTL-L17 canonical sequence is: MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDY FLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAK GAGTASKLTFGTGTRLQVTL (SEQ ID NO:47).
- the murine TCR alpha chain constant (mTRAC) region canonical sequence is: XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAW SNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLM TLRLWSS (SEQ ID NO: 75).
- the transmembrane domain of the murine TCR alpha chain is: MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 76).
- the intracellular domain of the murine TCR alpha chain is: SS (SEQ ID NO: 77)
- the human TCR beta 1 chain C region canonical sequence is: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF (SEQ ID NO:48).
- the human TCR beta 1 chain human IgC sequence is: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE (SEQ ID NO: 70)
- the transmembrane domain of the human TCR beta 1 chain is: ILLGKATLYAVLVSALVLMAM (SEQ ID NO:6).
- the human TCR beta 1 chain V region CTL-L17 canonical sequence is: MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTL GQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGL NQPQHFGDGTRLSIL (SEQ ID NO:49).
- the intracellular domain of the human TCR beta 1 chain is: VKRKDF (SEQ ID NO: 71)
- the human TCR beta chain V region YT35 canonical sequence is: MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTM MRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCS ANYGYTFGSGTRLTVV (SEQ ID NO:50).
- the murine TCR beta chain constant region canonical sequence is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 78).
- the transmembrane domain of the murine TCR beta chain is: ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 79).
- the intracellular domain of the murine TCR beta chain is: KRKNS (SEQ ID NO: 80) [00419]
- the human TCR beta 2 chain C region canonical sequence is: DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDP Q PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI V SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 81) [00420]
- the transmembrane domain of the human TCR beta 2 chain is: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 82) [00421]
- the intracellular domain of the human TCR beta 2 chain is: MAMVKRKDSRG (SEQ ID NO: 83) [00422]
- the human TCR beta gamma human IgC sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSA (SEQ ID NO: 72)
- the transmembrane domain of the human TCR gamma chain is: YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO:65).
- the intracellular domain of the human TCR gamma chain is: RRTAFCCNGEKS (SEQ ID NO: 73)
- the human TCR delta chain C region canonical sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:66).
- the human TCR delta gamma human IgC sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTV (SEQ ID NO: 74)
- the transmembrane domain of the human TCR delta chain is: LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO:67).
- the intracellular domain of the human TCR delta chain is: L Generation ofTFPs from TCR Domains and MU Cl 6-binding peptides
- the IAB peptide from MLSN that targets MUC16 was recombinantly linked to CD3- epsilon using the linker sequence (G 4 S) 3 to generate a TFP (MUC16.TFP) having the amino acid sequence of SEQ ID NO: 51 shown below.
- linker sequence G 4 S 3 to generate a TFP (MUC16.TFP) having the amino acid sequence of SEQ ID NO: 51 shown below.
- Various other linkers and MUC16-binding peptide configurations can be utilized as is described in Example 1.
- Expression vectors include: a promoter (eukaryotic elongation factor 1 alpha (EFla) promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g ., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
- a promoter eukaryotic elongation factor 1 alpha (EFla) promoter
- EFla eukaryotic elongation factor 1 alpha
- BGH Bovine Growth Hormone
- the TFP-encoding nucleic acid construct was cloned into the pLRPO lentiviral expression vector.
- the TFP.MUC16 lentiviral transfer vectors were used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles.
- Expi293F-cells were suspended in free-style (FS) media and allowed to incubate at 37 degrees C, 8% CO2, 150 rpm for 1-3 hours.
- the transfer DNA plasmid, Gag/Pol plasmid, Rev plasmid, and VSV-G plasmid were diluted in FS media. PEIpro was then diluted in FS media and added to the mixture of DNA and media.
- the incubated cells were added to this mixture and are incubated at 37 degrees C, 8% CO2, 150 rpm for 18-24 hours. The following day, the supernatant was replaced with fresh media and supplemented with sodium butyrate and incubated at 37°C for an additional 24 hours.
- the lentivirus containing supernatant was then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 30 minutes at 4°C, the cleared supernatant was filtered with a low-protein binding 0.45 pm sterile filter. The virus was subsequently concentrated by Lenti-X. The virus stock preparation was either used for infection immediately or aliquoted and stored at -80°C for future use.
- CD3 epsilon knock-out Jurkat cells were generated by knocking out CD3 ⁇ subunit from wild-type (WT) Jurkat cells with CRISPR technique, as described, e.g., in co-pending U.S. Patent Publication No. 2017-0166622.
- Jurkat CD3 ⁇ KO cells were incubated with virus at a multiplicity of infection (MOI) of five. Medium was replaced twenty-four-hours post incubation.
- TFP expression in Jurkat cells was validated based on detection of cell-surface TFP expression in CD3 epsilon knock-out Jurkat cells.
- CD3 epsilon knock-out the surface expression of TCR complex was absent on CD3 epsilon knock-out Jurkat cells.
- TFP.MUC16 surface expression on TFP.MUC16-transduced CD3 epsilon knock-out Jurkat cells was demonstrated by the significantly increased binding of an anti-CD3 epsilon antibody and an anti-TCR ⁇ antibody in comparison to the non-transduced control CD3 epsilon knock-out Jurkat cells (FIG. 2).
- TFP.MUC16-transduced (TFP) and non- transduced (NT) CD3 epsilon knock-out Jurkat cells were generated, validated for transduction (FIG. 2), and set up in co-culture with MUC16 positive OVCAR3 cells or MUC16 negative C30 cells (validated in FIGs. 1A and IB) at different effector-to-target ratios (3:1 or 1:1 effector to target cells).
- Jurkat cell activation was determined by measuring the expression of CD25 and CD69 at 24 hours after the co-culture by flow cytometry, with an Ab panel including anti-human CD25 (Clone BC96) and anti-human CD69 (clone FN50). As is shown in FIG. 3, increased activation was detected with TFP.MUC16-transduced Jurkat cells co-cultured with OVCAR3 target cells (FIG. 3A), but not the TFP.MUC16-transduced Jurkat cells co-cultured with C30 target cells (FIG. 3B) or non-transduced control Jurkat cells co-cultured with either OVCAR3 or C30 cells.
- T cells were purifried from healthy donor leukopak or PBMCs via positive selection of CD4+ and CD8+ T cells with CD4 and CD8 microbeads from Miltenyi Biotech.
- T cells freshly isolated or thawed from previously prepared frozen vials, were activated by MACS GMP T cell TransAct (Miltenyi Biotech), in the presence of human IL-7 and IL-15 (both from Miltenyi Biotech, premium grade).
- activated T cells were transduced with lentivirus encoding the MUC16.TFP.
- the cells were washed, subcultured in fresh medium with cytokines and then expanded up to day 10 by supplementing fresh medium every 2 days. At each day of subculture, fresh medium with cytokines were added to maintain the cell suspension at
- MUC16.TFP transduced T cells were measured similarly as for Jurkat cells. As described above, MUC16.TFP T cells or non-transduced T cells were generated from three donors. At day 10 of expansion, T cells were harvested and stimulated by co-culture with MUC 16-positive OVCAR3 cells or MUC 16-negative C30 cells at 1:1 (effectortumor) ratio for 24 hours. Expression of CD25 and CD69 were determined by flow cytometry with anti human CD25 (Clone BC96) and anti-human CD69 (clone FN50). As is shown in FIG. 6A,
- CD25 and CD69 expression was evaluated for the TFP- population and the TFP+ population (gated on SSI scFv hFc negative and positive populations, respectively) of MUC16.
- TFP transduced T cells
- NT non-transduced cells
- Dramatic increase in frequency of CD25+ and CD69+ cells was only observed for TFP+ population in MUC 16.
- TFP T cells co-cultured with MUC 16 positive OVCAR3 cells.
- Increase in frequency of CD25+ cells was observed for TFP- population in MUC16.
- TFP- populations in MUC.16 TFP transduced T cells co-cultured with MUC16 negative C30 cells, or between non-transduced T cells co-cultured with OVCAR3 cells or C30 cells. Taken together, the data indicates that IAB-MUC16 interaction specifically induce TFP+ T cell activation.
- the luciferase-based cytotoxicity assay assesses the cytotoxicity of TFP T cells by indirectly measuring the luciferase enzymatic activity in the residual live target cells after co culture.
- MUC 16-positive OVCAR3 and MUC 16-negative C30 cells were modified to overexpress firefly luciferase via transduction with firefly luciferase encoding lentivirus followed with antibiotic selection to generate stable cell line.
- the target cells were plated at 10000 cells per well in 96-well plate.
- TFP transduced or non-transduced T cells were added to the target cells at different effector-to-target ratios (3 : 1 or 1 : 1).
- the mixture of cells was then cultured for 24 at 37°C with 5 % CO2 before the luciferase enzymatic activity in the live target cells was measured by the Bright-Glo® Luciferase Assay System (Promega®, Catalogue number E2610).
- the cells were spun into a pellet and resuspended in medium containing the luciferase substrate.
- TFP transduced T cells demonstrated enhanced cytotoxicity towards MUC 16-positive OVCAR3 relative to untransduced T cells. No cytotoxicity was observed for MUC 16. TFP transduced T cells against MUC16 negative C30 cells.
- a measure of effector T-cell activation and proliferation associated with the recognition of cells bearing cognate antigen is the production of effector cytokines such as interferon-gamma (IFN- ⁇ ), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF- ⁇ ).
- IFN- ⁇ interferon-gamma
- GM-CSF granulocyte-macrophage colony-stimulating factor
- TNF- ⁇ tumor necrosis factor alpha
- Target-specific cytokine production including IFN- ⁇ , GM-CSF, and TNF- ⁇ by TFP T cells was measured from supernatants harvested 24 hours after the co-culture of T cells with MUC 16-positive OVCAR3 and MUC 16-negative C30 target cells using the U-PLEX® Biomarker Group I (hu) Assays (Meso Scale Diagnostics®, LLC, catalog number: K15067L-4).
- U-PLEX® Biomarker Group I (hu) Assays Meso Scale Diagnostics®, LLC, catalog number: K15067L-4.
- TFP.MUC16-transduced T cells from all three donors co-cultured with MUC 16- positive OVCAR3 target cells relative to TFP.MUC16-transduced T cells co-cultured with
- OVCAR3 or CD30 cells OVCAR3 or CD30 cells.
- Example 9 MUC16 specific proliferation of T cells expressing MUC16-TFP
- MUC 16 specific proliferation of MUC16-TFP T cells is determined by monitoring the dilution of T cell tracing signal (decrease in signal intensity of CellTraceTM) by flowcytometry analysis.
- T cells expressing MUC16-TFPs are labelled with CellTraceTM Far Red Proliferation Kit (Cat. #C34564ThermoFisher), then co-cultured with CD30 or OVCAR3 cells at 1-to-l ratio for 3 days.
- T cells expressing MUC16-TFPs labelled with CellTrace Far Red Proliferation kit were also stimulated with medium alone or with 1 ⁇ g/mL plate-bound anti-CD3 antibody (clone OKT-3, Cat #14-0037-82, Invitrogen) for 3 days.
- T cells expressing MUC16-TFPs will show MUC 16-specific proliferation, demonstrated by the decrease of CellTracer signal when co- cultured with OVCAR3 cells, but not CD30 cells.
- Example 10 In vivo activity of MUC16-TFP T cells
- T cells expressing MUC16-TFPs are evaluated in NSG mouse xenograft models of human ovarian carcinoma cell lines, OVCAR3 cells.
- OVCAR3 cells Six-week-old female NSG (NOD.Cg- Prkdc scld I12rg tm 1 Wjl /SzJ, The Jackson Laboratory, are intraperitoneally inoculated with OVCAR3 (5 x 10 6 cells/mouse), or subcutaneously with OVCAR3 cells (5 x 10 6 cells/mouse, 1-to-l mixture with Matrigel®).
- Tumor burden is determined by bioluminescence imaging (BLI) for the intraperitoneal models with the intraperitoneal injection of 0.2ml of luciferin substrate (VWR) diluted in PBS (150 mg/kg). Tumor burden of the subcutaneous model is measured as the tumor volume by Caliper.
- Example 11 Immunohistochemistry staining of normal human tissues using the Fc-fusion IAB mesothelin peptide
- Control materials and FFPE sections are stained with an IAB mesothelin peptide that is genetically fused to a mouse Fc region for detection using HRP conjugated anti-mouse Fc secondary antibody.
- the positive control consists of FFPE sections of human ovarian tumors from two donors.
- the negative control is an FFPE section of a human heart.
- the panel of tested tissues includes the following: blood cells, cerebellum or cerebral cortex, gastrointestinal tract (esophagus, small intestine, stomach, colon - as available), spleen, kidney (glomerulus, tubule), liver, lymph node, skin, placenta, testis and tonsil from one donor each.
- Results Two human ovarian carcinoma tissues from different donors are used as a positive control and will show positive staining. From the normal tissues, the human heart negative control will show negative staining. The staining will further show negative or limited expression of MUC16 in normal human tissues. This makes it an attractive target for cancer therapy of MUC16 positive malignancies.
- the MUC16-specific IAB mesothelin peptide will be able to bind and stain antigen positive tissues.
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Abstract
Provided herein are T cell receptor (TCR) fusion proteins (TFPs), T cells engineered to express one or more MUC16 TFPs, and methods of use thereof for the treatment of diseases, including cancer.
Description
COMPOSITIONS AND METHODS FOR TCR REPROGRAMMING USING MUC16
SPECIFIC FUSION PROTEINS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No. 62/967,391, filed on January 29, 2020, and U.S. Provisional Application No. 63/009,886, filed on April 14, 2020, each of which is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] Most patients with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
[0003] Recent developments using chimeric antigen receptor (CAR) modified autologous T cell therapy, which relies on redirecting genetically engineered T cells to a suitable cell-surface molecule on cancer cells, show promising results in harnessing the power of the immune system to treat B cell malignancies (see, e.g ., Sadelain et al., Cancer Discovery 3:388-398 (2013)). The clinical results with CD-19-specific CAR-T cells (called CTL019) have shown complete remissions in patients suffering from chronic lymphocytic leukemia (CLL) as well as in childhood acute lymphoblastic leukemia (ALL) (see, e.g. , Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368: 1509-1518 (2013)). An alternative approach is the use of T cell receptor (TCR) alpha and beta chains selected for a tumor-associated peptide antigen for genetically engineering autologous T cells. These TCR chains will form complete TCR complexes and provide the T cells with a TCR for a second defined specificity. Encouraging results were obtained with engineered autologous T cells expressing NY-ESO-1 -specific TCR alpha and beta chains in patients with synovial carcinoma. [0004] Besides the ability of genetically modified T cells expressing a CAR or a second TCR to recognize and destroy respective target cells in vitro!ex vivo , successful patient therapy with engineered T cells requires the T cells to be capable of strong activation, expansion, persistence over time, and, in case of relapsing disease, to enable a ‘memory’ response. High and manageable clinical efficacy of CAR-T cells is currently limited to BCMA- and CD- 19-positive
B cell malignancies and to NY-ESO-1 -peptide expressing synovial sarcoma patients expressing
HLA-A2. There is a clear need to improve genetically engineered T cells to more broadly act against various human malignancies.
SUMMARY
[0005] Provided herein are T cell receptor (TCR) fusion proteins (TFPs), T cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases.
[0006] According to an aspect, provided herein is a pharmaceutical composition comprising a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: a TCR-integrating subunit comprising: at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain; and a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked. In some embodiments, the intracellular signaling domain comprises a stimulatory domain from an intracellular signaling domain.
[0007] According to another aspect, provided herein is a pharmaceutical composition comprising (I) a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: (a) a TCR-integrating subunit comprising: (i) at least a portion of a TCR extracellular domain, and (ii) a transmembrane domain; and (b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and (II) a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked. In some embodiments, the TFP further comprises an intracellular domain.
[0008] In some embodiments, the MUC16 binding domain specifically binds membrane-bound MUC16. In some embodiments, the MUC16 binding domain specifically binds membrane-bound MUC16 in the presence of soluble MUC16. In some embodiments, the T cell exhibits increased cytotoxicity to a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
[0009] In some embodiments, the sequence encoding the MUC16 binding domain is connected to the sequence encoding the TCR extracellular domain by a sequence encoding a linker. In some embodiments, the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.
[0010] In some embodiments, the MUC16 binding domain comprises mesothelin or a fragment thereof. In some embodiments, the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin. In some embodiments, the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of
EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKH KLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In some embodiments, the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
[0011] In some embodiments, the pharmaceutical composition is substantially free of serum. [0012] In some embodiments, the human T cell has greater than or more efficient cytotoxic activity than a CD8+ or CD4+ T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (a) the MUC16 binding domain, operatively linked to (b) at least a portion of an extracellular domain (c) a transmembrane domain (d) at least a portion of a CD28,
4- IBB, 0X40, ICOS, CD27, and/or CD2 intracellular domain and (e) a CD3 zeta intracellular domain.
[0013] In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is an iNKT cell. In some embodiments, the T cell is a human CD4+ T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is a human CD8+ T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is a human CD8+ T cell. In some embodiments, the T cell is an alpha beta (ab or αβ) T cell. In some embodiments, the T cell is a gamma delta (gd or γδ) T cell.
[0014] In some embodiments, the T cell further comprises a nucleic acid encoding a first polypeptide comprising at least a portion of an inhibitory molecule , wherein the at least a portion of an inhibitory molecule is associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. In some embodiments, the inhibitory molecule is PD-1.
In some embodiments, the second polypeptide comprises a costimulatory domain and primary signaling domain from a protein selected from the group consisting of CD28, CD27, ICOS,
CD3ζ, 41-BB, 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, LFA-1, CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, and B7-H3.
[0015] In some embodiments, production of IL-2 or IFNγ by the T cell is increased in the presence of a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
[0016] In some embodiments, the cell is a population of human T cells, wherein an individual T cell of the population comprises at least two TFP molecules, or at least two T cells of the population collectively comprise at least two TFP molecules; wherein the at least two TFP molecules comprise a MUC16 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and wherein at least one of the at least two TFP molecules
functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof.
[0017] In some embodiments, the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
[0018] In some embodiments, the TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
[0019] In some embodiments, the TCR-integrating subunit comprises a TCR intracellular domain. In some embodiments, the intracellular domain comprises an intracellular domain from TCR alpha, TCR beta, TCR delta, or TCR gamma, or an amino acid sequence having at least one modification thereto. In some embodiments, the intracellular domain comprises a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, or an amino acid sequence having at least one modification thereto.
[0020] In some embodiments, the TCR-integrating subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
[0021] In some embodiments, the TCR-integrating subunit is derived only from CD3 epsilon. In some embodiments, wherein the TCR-integrating subunit is derived only from CD3 gamma. In some embodiments, the TCR-integrating subunit is derived only from CD3 delta.
[0022] In some embodiments, the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
[0023] In some embodiments, the TFP further comprises a sequence encoding a costimulatory domain. In some embodiments, the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
[0024] In some embodiments, the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma
TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor
2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32,
CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some embodiments, the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon. In some embodiments, the IT AM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
[0025] According to another aspect, provided herein is a recombinant nucleic acid comprising a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising: a TCR-integrating subunit comprising: at least a portion of a TCR extracellular domain, a transmembrane domain, and a TCR intracellular domain; and a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked. In some embodiments, the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain.
[0026] According to another aspect, provided herein is a recombinant nucleic acid encoding a T cell receptor (TCR) fusion protein (TFP) comprising: (a) a TCR-integrating subunit comprising (i) at least a portion of a TCR extracellular domain, and (ii) a transmembrane domain; and (b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked; and wherein the TFP functionally interacts with a TCR when expressed in the T cell. In some embodiments, the TFP further comprises an intracellular domain.
[0027] In some embodiments, the recombinant nucleic acid encodes a TFP having any of the features of a TFP described herein.
[0028] In some embodiments, the recombinant nucleic acid further comprises a leader sequence. [0029] In some embodiments, the nucleic acid is selected from the group consisting of a DNA and a RNA. In some embodiments, the nucleic acid is a mRNA. In some embodiments, the nucleic acid is a circRNA.
[0030] In some embodiments, the nucleic acid comprises a nucleotide analog. In some embodiments, the nucleotide analog is selected from the group consisting of 2’ -O-methyl, 2’-O- methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-O-aminopropyl
(2’-O-AP), 2'-O-dimethylaminoethyl (2’ -O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-
DMAP), T-O-dimethylaminoethyloxy ethyl (2’-O-DMAEOE), 2’-O-N-methylacetamido (2’-O-
NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2,-fluoroN3-P5’-phosphoramidite.
[0031] In some embodiments, the recombinant nucleic acid further comprises a promoter.
[0032] In some embodiments, the recombinant nucleic acid is an in vitro transcribed nucleic acid.
[0033] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3’UTR sequence.
[0034] In another aspect, provided herein is a recombinant polypeptide molecule encoded by any of the recombinant nucleic acids described herein.
[0035] In another aspect provided herein is a vector comprising any of the recombinant nucleic acids described herein encoding any of the TFPs described herein.
[0036] In some embodiments, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3’UTR.
[0037] In another aspect provided herein is a cell comprising any of the recombinant nucleic acids, TFPs, or vectors described herein.
[0038] In some embodiments, the cell is a human T-cell. In some embodiments, the T-cell is a CD8+ or CD4+ T-cell. In some embodiments, the T cell is a human alpha beta (ab or αβ) T cell. In some embodiments, the T cell is a human gamma delta (gd or γδ) T cell.
[0039] In some embodiments, the cell further comprises a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. In some embodiments, the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
[0040] In another aspect provided herein is a protein complex comprising a TFP encoded by any of the recombinant nucleic acids described herein, and at least one endogenous TCR subunit or endogenous TCR complex.
[0041] In another aspect, provided herein is a human CD8+ or CD4+ T-cell comprising at least two different TFP proteins of any of the protein complexes described herein.
[0042] In another aspect, provided herein is a human CD8+ or CD4+ T-cell comprising at least two different TFP molecules encoded by any of the recombinant nucleic acids described herein. [0043] In another aspect, provided herein is a population of human CD8+ or CD4+ T-cells, wherein the T-cells of the population individually or collectively comprise at least two TFP molecules encoded by any of the recombinant nucleic acids described herein.
[0044] In another aspect, provided herein is a method of making a cell comprising transducing a T-cell with any of the recombinant nucleic acids or vectors described herein.
[0045] In another aspect, provided herein is a method of generating a population of RNA- engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding any of the TFPs described herein.
[0046] In another aspect, provided herein is a method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein.
[0047] In some embodiments, the cell is an autologous T-cell. In some embodiments, the cell is an allogeneic T-cell. In some embodiments, the mammal is a human.
[0048] In another aspect, provided herein is a method of treating a mammal having a disease associated with expression of MUC16 comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein.
[0049] In some embodiments, the disease associated with MUC16 expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, myelodysplasia, a myelodysplastic syndrome, a preleukemia, a non-cancer related indication associated with expression of MUC16. In some embodiments, the disease is pancreatic cancer, ovarian cancer, breast cancer, or any combination thereof.
[0050] In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. In some embodiments, less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-MUC16 chimeric antigen receptor (CAR).
[0051] In some embodiments, the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. In some embodiments, the cells expressing a
TFP molecule are administered in combination with an agent that treats the disease associated with MUC16.
[0052] In another aspect, provided herein is a method of treating a mammal having a disease associated with expression of MUC16 comprising administering to the mammal an effective amount of any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein for use as a medicament, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-MUC16 chimeric antigen receptor (CAR).
[0053] In another aspect, provided herein is any of the pharmaceutical compositions, recombinant nucleic acids, recombinant polypeptides, vectors, or cells described herein for use as a medicament.
INCORPORATION BY REFERENCE
[0054] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS [0055] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0056] FIGs. 1A and IB are a series of graphs showing detection of MUC16 on the surface of MUC16 expressing OVCAR3 cells. In FIG. 1 A, MUC16 expression by OVCAR3 cells is detected by R-phycoerythrin conjugated anti-MUC16 antibody. In FIG. IB, binding of MSLN peptide IAB to MUC16 expressing OVCAR3 cells is confirmed by using allophycocyanin- conjugted IAB hFc peptide (via Zenon human IgG labeling reagents) (FIG. IB).
[0057] FIG. 2 is a series of graphs showing detection of MUC16.TFP (i.e., a TFP having the MSLN IAB peptide) on the surface of non-transduced (NT) and transduced (TFP) CD3ε knock out Jurkat cells by anti- CD3ε, anti-TCRαβ, and SSI scFv hFc.
[0058] FIGs. 3A and 3B are a series of plots showing Jurkat cell activation mediated by
MUC16.TFP in co-culture with MUC16 expressing OVCAR3 cells (FIG. 3 A), but not with
MUC16 negative C30 cells (FIG. 3B).
[0059] FIG. 4 is a graph showing fold expansion of MUC16.TFP transduced and non-transduced T cells from three donors (A, B and C). Non-transduced and transduced primary T cells expanded normally in all three donors.
[0060] FIG. 5 is a series of plots showing levels of surface expression of MUC16.TFP as measured by SSI scFv hFc in MUC16.TFP transduced and non-transduced control T cells from the three donors (A, B and C).
[0061] FIGs. 6A and 6B are a series of plots showing expression of T cell activation markers CD69 and CD25 in MUC16.TFP transduced and non-transduced T cells from three donors (A, B and C) co-cultured with OVCAR3 MUC16 expressing cells or C30 control cells at an effectortarget ratio of 3 : 1. Donor A is shown in FIG. 6A and Donors B and C are shown in FIG. 6B.
[0062] FIG. 7 is a series of graphs showing cytotoxicity of MUC16.TFP transduced or non- transduced T cells from three donors when co-cultured for 24 hours with OVCAR3 cells (Donors A-C) or C30 cells (Donors B and C) at an effectortarget cell ratio of 3 : 1 and 1:1.
[0063] FIGs. 8A-8C are a series of graphs showing levels of IFN-γ (FIG. 8 A), TNF-α (FIG. 8B), and GM-CSF (FIG. 8C) produced by T cells expressing MUC16.TFP or non-transduced control cells from three donors when co-cultured for 24 hours with OVCAR3 cells or C30 cells.
DETAILED DESCRIPTION
[0064] The present disclosure encompasses recombinant DNA constructs encoding TFPs and variants thereof, wherein the TFP comprises a MUC16 binding domain that is not an antibody or antigen binding fragment thereof, wherein the sequence of the antigen or the fragment thereof is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR- integrating subunit or portion thereof. In certain embodiments, the MUC16 binding domain comprises a portion of the mesothelin protein, such as a portion of the extracellular domain of the mesothelin protein. The TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
[0065] The present disclosure also provides a TFP molecule or a TCR complex having the TFP molecule incorporated therein. The present disclosure also provides a cell (e.g., a T cell or a Treg) comprises the TFP or the recombinant nucleic acid molecule encoding the TFP. Advantageously, such TFPs, when expressed in a T-cell, can target MUC16 expressing cells.
Definitions
[0066] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.
[0067] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.
[0068] As used herein, the term “comprising” also specifically includes embodiments “consisting of’ and “consisting essentially of’ the recited elements, unless specifically indicated otherwise. [0069] The term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ± 10%, ± 5%, or ± 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ± one standard deviation of that value(s).
[0070] As used herein the specification, “subject” or “subjects” or “individuals” may include, but are not limited to, mammals such as humans or non-human mammals, e.g. , domesticated, agricultural or wild, animals, as well as birds, and aquatic animals. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human. “Patients” are subjects suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein.
[0071] As used herein, “treating” or “treatment” refers to any indicia of success in the treatment or amelioration of the disease or condition. Treating can include, for example, reducing, delaying or alleviating the severity of one or more symptoms of the disease or condition, or it can include reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient. As used herein, “treat or prevent” is sometimes used
herein to refer to a method that results in some level of treatment or amelioration of the disease or condition, and contemplates a range of results directed to that end, including but not restricted to prevention of the condition entirely. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
[0072] As used herein, “preventing” refers to the prevention of the disease or condition, e.g ., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present disclosure and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
[0073] As used herein, a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non-beneficial event to the individual to whom the composition is administered. By “therapeutically effective dose” herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)) [0074] As used herein, a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell. A “TFP T cell” is a T cell that has been transduced according to the methods disclosed herein and that expresses a TFP, e.g., incorporated into the natural TCR. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell. In some embodiments, the TFP T cell is an NK cell or a regulatory T cell.
[0075] As used herein, the term “MUC16” also known as mucin 16 or CA125 (cancer antigen 125, carcinoma antigen 125, or carbohydrate antigen 125), refers to a protein that in humans is encoded by the MUC 16 gene. MUC16 is a member of the mucin family glycoproteins and has found application as a tumor marker or biomarker that may be elevated in the blood of some patients with specific types of cancers or other conditions that are benign. MUC 16 is used as a biomarker for ovarian cancer detection and has been found to be elevated in other cancers,
including endometrial cancer, fallopian tube cancer, lung cancer, breast cancer and gastrointestinal cancer. MUC16 has also been shown to suppress the activity of natural killer cells in the immune response to cancer cells (see, e.g., Patankar et al., Gynecologic Oncology
99(3); 704-13).
[0076] The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human MUC16 can be found as UniProt/Swiss-Prot Accession No. Q8WXI7.
[0077] The nucleotide sequence encoding human MUC16 can be found at Accession No.
NM 024690. The nucleotide sequence encoding human MUC16 transcript variant XI can be found at Accession No. XM_017027486. The nucleotide sequence encoding human MUC16 transcript variant X2 can be found at Accession No. XM_017027487. The nucleotide sequence encoding human MUC16 transcript variant X3 can be found at Accession No. XM_017027488. The nucleotide sequence encoding human MUC16 transcript variant X4 can be found at Accession No. XM_017027489. The nucleotide sequence encoding human MUC16 transcript variant X5 can be found at Accession No. XM_ 017027490. The nucleotide sequence encoding human MUC16 transcript variant X6 can be found at Accession No. XM_017027491. The nucleotide sequence encoding human MUC16 transcript variant X7 can be found at Accession No. XM_017027492. The nucleotide sequence encoding human MUC16 transcript variant X8 can be found at Accession No. XM_ 017027493. The nucleotide sequence encoding human MUC16 transcript variant X9 can be found at Accession No. XM_017027494. The nucleotide sequence encoding human MUC16 transcript variant XI 0 can be found at Accession No.
XM_0 17027495. The nucleotide sequence encoding human MUC16 transcript variant XI 1 can be found at Accession No. XM_017027499. The nucleotide sequence encoding human MUC16 transcript variant X12 can be found at Accession No. XM_017027500. The nucleotide sequence encoding human MUC16 transcript variant X13 can be found at Accession No. XM_017027501. In one example, the binding portion of TFPs recognizes and binds an epitope within the extracellular domain of the MUC16 protein as expressed on a normal or malignant mesothelioma cell, ovarian cancer cell, pancreatic adenocarcinoma cell, or squamous cell carcinoma cell.
[0078] As used herein, the term “mesothelin” also known as MSLN or CAK1 antigen or Pre-pro- megakaryocyte-potentiating factor, refers to the protein that in humans is encoded by the MSLN (or Megakaryocyte-potentiating factor (MPF)) gene. Mesothelin is a 40 kDa protein present on normal mesothelial cells and overexpressed in several human tumors, including mesothelioma and ovarian and pancreatic adenocarcinoma. The mesothelin gene encodes a precursor protein that is processed to yield mesothelin which is attached to the cell membrane by a glycophosphatidylinositol linkage and a 31 -kDa shed fragment named megakaryocyte-
potentiating factor (MPF). Mesothelin may be involved in cell adhesion, but its biological function is not known. Mesothelin is a tumor differentiation antigen that is normally present on the mesothelial cells lining the pleura, peritoneum and pericardium. Mesothelin is an antigenic determinant detectable on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and some squamous cell carcinomas (see, e.g ., Kojima et al., J. Biol. Chem. 270:21984-
21990(1995) and Onda et al., Clin. Cancer Res. 12:4225-4231(2006)). Mesothelin interacts with
CA125/MUC16 (see, e.g., Rump et al., J. Biol. Chem. 279:9190-9198(2004) and Ma et al., J.
Biol. Chem. 287:33123-33131(2012)).
[0079] The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human mesothelin can be found as UniProt/Swiss-Prot Accession No. Q13421. The human mesothelin polypeptide canonical sequence is UniProt Accession No. Q13421 (or Q13421-1):
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISS
L SPRQLLGFPC AE V S GL S TERVREL A V AL AQKNVKL S TEQLRCL AHRL SEPPEDLD ALPL
DLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPST
W S VSTMD ALRGLLPVLGQPIIRSIPQGIVAAWRQRS SRDPSWRQPERTILRPRFRREVEKT
ACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDEL
YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQ
VATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDP
RQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRT
DAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGY
LVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA (SEQ ID NO:2).
[0080] The nucleotide sequence encoding human mesothelin transcript variant 1 can be found at Accession No. NM005823. The nucleotide sequence encoding human mesothelin transcript variant 2 can be found at Accession No. NMO 13404. The nucleotide sequence encoding human mesothelin transcript variant 3 can be found at Accession No. NMOOl 177355. Mesothelin is expressed on mesothelioma cells, ovarian cancer cells, pancreatic adenocarcinoma cell and squamous cell carcinomas (see, e.g. , Kojima et al., J. Biol. Chem. 270:21984-21990(1995) and Onda et al., Clin. Cancer Res. 12:4225-4231(2006)).
[0081] The term “binding domain”, as used herein, refers to a protein, or polypeptide sequence, which specifically binds to a target. In some embodiments, the target is a polypeptide. In some embodiments, the target is MUC16. In some embodiments, the target is cell-surface bound MUC16. In some embodiments, the binding domain is not an antibody or fragment thereof.
[0082] The term “antibody,” as used herein, refers to a protein, or polypeptide sequence, derived from an immunoglobulin molecule, which specifically binds to an antigen. Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
[0083] The terms “antibody fragment” refer to at least one portion of an antibody, or recombinant variants thereof, that contains the antigenic determining variable region of an intact antibody that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments. The TFP composition of the disclosure does not comprise an antibody or antibody fragment.
[0084] The term “antigen” or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
[0085] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
[0086] The term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g ., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also
be manifested by the ability of the peptides, polynucleotides, and cells of the disclosure in prevention of the occurrence of tumor in the first place.
[0087] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., a ligand) and its binding partner. Unless indicated otherwise, as used herein, “affinity” refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., a ligand and its binding partner). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). The kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
[0088] The terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular binding partner (e.g., a polypeptide target) of a ligand (e.g., a binding domain) mean binding that is measurably different from a non-specific or non- selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the binding domain to the target molecule is competitively inhibited by the control molecule.
[0089] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
[0090] The term “allogeneic” refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
[0091] The term “xenogeneic” refers to a graft derived from an animal of a different species. [0092] The term “circularized RNA” or “circRNA” refers to a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. CircRNAs are 3-5’ covalently closed RNA rings, and circRNAs do not display Cap or poly(A) tails.
[0093] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include
but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer, and the like.
[0094] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
[0095] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
[0096] A “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer. Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.
[0097] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,”
“cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematologic malignancy.
[0098] The term “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
[0099] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
[00100] The terms “increase” and “activate” refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
[00101] The terms “reduce” and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
[00102] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.
[00103] The term “antagonize” refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor. An “antagonist” is an entity that binds to and antagonizes a receptor.
[00104] The term “effector T cell” includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells. CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol ., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.
[00105] The term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells. In some aspects, the regulatory T cell has a CD4+CD25+Foxp3+ phenotype. In some aspects, the regulatory T cell has a CD8+CD25+ phenotype. See Nocentini et al., Br. J. Pharmacol ., 2012, 165:2089-2099, incorporated by reference in its entirety, for additional information on regulatory T cells expressing TARGET. [00106] The term “dendritic cell” refers to a professional antigen-presenting cell capable of activating a naive T cell and stimulating growth and differentiation of a B cell.
[00107] The phrase “disease associated with expression of MUC16” includes, but is not limited to, a disease associated with expression of MUC16 or condition associated with cells which express MUC16 including, e.g ., proliferative diseases such as a cancer or malignancy or a precancerous condition. In one aspect, the cancer is a glioblastoma. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is a pancreatic cancer. In one aspect, the cancer is an ovarian cancer. In one aspect, the cancer is a brain cancer. In one aspect, the cancer is a stomach cancer. In one aspect, the cancer is a lung cancer. In one aspect, the cancer is an endometrial cancer. Non-cancer related indications associated with expression of MUC16 include, but are not limited to, e.g. , autoimmune disease, (e.g., lupus, rheumatoid arthritis, colitis), inflammatory disorders (allergy and asthma), and transplantation.
[00108] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the ligand (e.g., binding domain) containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a ligand (e.g., binding domain) of the disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a TFP of the disclosure can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
[00109] The term “stimulation” refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
[00110] The term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “IT AM”. Examples of an IT AM containing primary cytoplasmic signaling sequence that is of particular use in the disclosure includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
[00111] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.
[00112] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell. Examples of immune effector function, e.g., in a TFP-expressing T cell, include cytolytic activity and T helper cell activity, including the secretion of cytokines. In an embodiment, the
intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
[00113] A primary intracellular signaling domain can comprise an IT AM (“immunoreceptor tyrosine-based activation motif’). Examples of IT AM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP 10 and DAP12.
[00114] The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that may be required for an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP 10, DAP 12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla/CD18) and 4-1BB (CD137). A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof. The term “4-1BB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g ., mouse, rodent, monkey, ape and the like; and a “4- IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non-human species, e.g. , mouse, rodent, monkey, ape and the like. [00115] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined
sequence of nucleotides ( e.g ., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
[00116] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
[00117] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological or therapeutic result. [00118] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.
[00119] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.
[00120] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
[00121] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
[00122] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro
expression system. Expression vectors include all those known in the art, including cosmids, plasmids ( e.g ., naked or contained in liposomes) and viruses ( e.g ., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
[00123] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
[00124] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
[00125] The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
[00126] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[00127] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.
[00128] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression
of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked
DNA sequences can be contiguous with each other and, e.g ., where necessary to join two protein coding regions, are in the same reading frame.
[00129] The term “parenteral” administration of an immunogenic composition includes, e.g. , subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
[00130] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
[00131] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
[00132] The term “promoter” refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, that can initiate the specific transcription of a polynucleotide sequence.
[00133] The term “promoter/regulatory sequence” refers to a nucleic acid sequence which can be used for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
[00134] The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
[00135] The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
[00136] The term “tissue-specific” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
[00137] The terms “linker” and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (GlyNer) Also included within the scope of the disclosure are linkers described in WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)n, wherein n=l to 3.
[00138] As used herein, a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end
of a eukaryotic messenger RNA shortly after the start of transcription. The 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a cap- synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that may be required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
[00139] As used herein, “ in vitro transcribed RNA” refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
[00140] As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
[00141] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3’ end. The 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.
[00142] As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of
time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
[00143] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
[00144] The term “subject” is intended to include living organisms in which an immune response can be elicited ( e.g ., mammals, human).
[00145] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
[00146] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
[00147] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.
[00148] In some instances, the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, glioblastoma, endometroid mucinous ovarian carcinoma, malignant pleural disease, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with MUC16 expression, and any combination thereof. [00149] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.
[00150] The term “specifically binds,” refers to a specific ligand (e.g., a binding domain), which recognizes and binds a cognate binding partner (e.g., MUC16) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
[00151] Ranges: throughout this disclosure, various aspects of the present disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2,
2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%,
96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
DESCRIPTION
[00152] Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer, using T cell receptor (TCR) fusion proteins having a non-antibody MUC16 binding domain. As used herein, a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface target polypeptide or antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell. As provided herein, TFPs provide substantial benefits as compared to Chimeric Antigen Receptors. The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a recombinant polypeptide comprising an extracellular binding domain, a transmembrane domain, and cytoplasmic signaling domains (also referred to herein as “an intracellular signaling domains”) comprising a functional signaling domain derived from a stimulatory molecule as defined below. Generally, the central intracellular signaling domain of a CAR is derived from the CD3 zeta chain that is normally found associated with the TCR complex. The CD3 zeta signaling domain can be fused with one or more functional signaling domains derived from at least one co-stimulatory molecule such as 4-1BB (i.e., CD137), CD27 and/or CD28.
MUC16
[00153] MUC16 (CA125) is a tumor associated antigen polypeptide, expressed by the human ocular surface epithelia in the mucosa of the bronchus, fallopian tube, and uterus. MUC16 is a large mucin-like glycoprotein present on the cell surface of tumor cells associated with beta-
galactoside-binding, cell-surface lectins, which can be components of the extracellular matrix implicated in the regulation of cell adhesion, apoptosis, cell proliferation and tumor progression.
One proposed function of MUC16 can be to provide a protective, lubricating barrier against particles and infectious agents at mucosal surfaces. Evidence suggests that expression of the cytoplasmic tail of MUC16 can enable tumor cells to grow, promote cell motility and may facilitate invasion. This appears to be due to the ability of the C-terminal domain of MUC16 to facilitate signaling that leads to a decrease in the expression of E-cadherin and increase the expression of N-cadherin and vimentin, which can be expression patterns consistent with epithelial-mesenchymal transition.
Highly polymorphic, MUC16 is a type I transmembrane protein composed of three domains, a large Ser-/Thr-rich N-terminal domain spanning 12,070 residues, a repeat domain of between eleven and more than 60 partially conserved tandem repeats of on average 156 amino acids each, and a C-terminal non-repeating domain containing a transmembrane sequence and a short cytoplasmic tail. The N-terminal domain and repeat domain are heavily O-glycosylated and N- glycosylated. MUC16 is expressed on the cell surface, and, in addition, soluble proteolytic fragments are also released into the extracellular space. mRNA encoding the MUC 16 polypeptide expressed from the MUC 16 gene can be significantly, reproducibly and detectably overexpressed in certain types of human cancerous ovarian, breast and pancreatic tumors as compared to the corresponding normal human ovarian, breast and pancreatic tissues, respectively. A variety of independent and different types of cancerous human ovarian tissue samples quantitatively analyzed for MUC16 expression show the level of expression of MUC16 in the cancerous samples can be variable, with a significant number of the cancerous samples showing an at least 6-fold (to as high as an about 580-fold) increase in MUC16 expression when compared to the mean level of ME1C16 expression for the group of normal ovarian tissue samples analyzed. In particular, detectable and reproducible MUC16 overexpression can be observed for ovarian cancer types; endometrioid adenocarcinoma, serous cystadenocarcinoma, including papillary and clear cell adenocarcinoma, as compared to normal ovarian tissue. Due to its overexpression in certain human tumors, the MUC16 polypeptide and the nucleic acid encoding that polypeptide are targets for quantitative and qualitative comparisons among various mammalian tissue samples. The expression profiles of MUC16 polypeptide, and the nucleic acid encoding that polypeptide, can be exploited for the diagnosis and therapeutic treatment of certain types of cancerous tumors in mammals.
[00154] MUC16 is a serum marker used routinely to monitor patients with ovarian cancer. MUC16 is a mullerian duct differentiation antigen that is overexpressed in epithelial ovarian cancer cells and secreted into the blood, although its expression may not be entirely confined to
ovarian cancer. Serum MUC16 levels can be elevated in about 80% of patients with epithelial ovarian cancer (EOC) but in less than 1% of healthy women. High serum concentration of
MUC16 can be typical of serous ovarian adenocarcinoma, whereas it is not elevated in mucinous ovarian cancer. MUC16 may not be recommended for ovarian cancer screening because normal level may not exclude tumor. However, MUC16 detection can be a standard tool in monitoring clinical course and disease status in patients who have histologically confirmed malignancies.
Numerous studies have confirmed the usefulness of MUC16 levels in monitoring the progress of patients with EOC, and as a cancer serum marker. A rise in ME1C16 levels typically can precede clinical detection by about 3 months. During chemotherapy, changes in serum MUC16 levels can correlate with the course of the disease. MUC 16 can be used as a surrogate marker for clinical response in trials of new drugs. On the other hand, MUC 16 may not be useful in the initial diagnosis of EOC because of its elevation in a number of benign conditions. The MUC 16- specific antibody MAb-B43.13 (oregovomab, OvaRex MAb-B43.13) was in clinical trials for patients with ovarian carcinoma as an immunotherapeutic agent.
[00155] MUC16 (CA-125) can play a role in advancing tumorigenesis and tumor proliferation by several different mechanisms. One way that MUC 16 helps the growth of tumors can be by suppressing the response of natural killer cells, thereby protecting cancer cells from the immune response. Further evidence that MUC 16 can protect tumor cells from the immune system may be the discovery that the heavily glycosylated tandem repeat domain of MUC 16 can bind to galectin-1 (an immunosuppressive protein). MUC 16 can participate in cell-to-cell interactions that enable the metastasis of tumor cells. This can be supported by evidence showing that MUC 16 can bind selectively to mesothelin, a glycoprotein normally expressed by the mesothelial cells of the peritoneum (the lining of the abdominal cavity). MUC 16 and mesothelin interactions may provide the first step in tumor cell invasion of the peritoneum. Mesothelin has also been found to be expressed in several types of cancers including mesothelioma, ovarian cancer and squamous cell carcinoma. Since mesothelin is also expressed by tumor cells, MUC 16 and mesothelial interactions may aid in the gathering of other tumor cells to the location of a metastasis, thus increasing the size of the metastasis. The extracellular domain of mature mesothelin binds MUC 16 and that amino acids 1-64, in particular, of mesothelin, have high affinity for MUC 16. In particular, mesothelin has higher affinity for membrane bound MUC 16 than for soluble MUC 16. The N-linked glycans of MUC 16 are necessary for binding to mesothelin.
[00156] MUC 16 may also play a role in reducing the sensitivity of cancer cells to drug therapy. For example, overexpression of MUC 16 can protect cells from the effects of genotoxic drugs, such as cisplatin.
T cell receptor (TCR) fusion proteins (TFP)
[00157] The present disclosure encompasses recombinant DNA constructs encoding TFPs, wherein the TFP comprises a polypeptide that binds specifically to MUC16, e.g ., human MUC16, wherein the sequence of the polypeptide is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR-integrating subunit or portion thereof. The TFPs provided herein are able to associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
[00158] In one aspect, the TFP of the present disclosure comprises a target-specific binding element (e.g., a MUC16-specific binding element) otherwise referred to as a binding domain (e.g., a MUC16 binding domain) that does not comprise an antibody or antibody fragment. [00159] In one aspect, the TFP-mediated T cell response can be directed to a target of interest by way of engineering a binding domain into the TFP that specifically binds a desired target (e.g., MUC16).
[00160] In one aspect, the portion of the TFP comprising the binding domain comprises a binding domain that targets MUC16. In one aspect, the binding domain targets human MUC16. [00161] The binding domain can be any domain that binds to the target other than an antibody or antibody fragment. In some embodiments, the MUC16 binding domain provided herein can comprise a peptide sequence of a MUC 16-interacting protein or a fragment thereof (e.g., a functional fragment or fragment capable of interacting with MUC 16). The MUC 16 binding domain can specifically bind to a MUC 16 protein or portion thereof (e.g., the extracellular domain of MUC 16). The MUC 16 binding domain can bind to membrane-bound MUC 16. The MUC 16 binding domain can specifically bind to membrane-bound MUC 16 in the presence of soluble MUC 16.
[00162] The MUC 16 binding domain can comprise a peptide sequence of mesothelin. The MUC 16 binding domain can comprise a full-length mesothelin (i.e., the full length mature mesothelin protein - amino acids 296-606 of the mesothelin precursor sequence) or a fragment thereof. The MUC 16 binding domain can comprise the mesothelin precursor protein or a fragment thereof. The MUC 16 binding domain can comprise a functional MUC 16 binding domain. The fragment of mesothelin can be a functional fragment. The functional fragment of mesothelin can comprise a minimum length of peptide sequence derived from mesothelin that can bind to MUC 16. The functional fragment of mesothelin can comprise an extracellular domain of mesothelin. The functional fragment of mesothelin can comprise at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61
62, 63, 64, 65, 66, 67, 68, 69, 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 or more amino acid residues of the peptide sequence of mesothelin. The MUC16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence of mesothelin or a fragment thereof.
[00163] For example, the MUC16 binding domain can comprise SEQ ID NO: 1 described herein. The MUC16 binding domain can comprise at least 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, 37, 38, 39, 40, 41, 42,
43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more consecutive amino acid residues of SEQ ID NO: 1. The MUC16 binding domain can comprise a truncation of SEQ ID NO: 1 by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00164] EVEKT ACP S GKK AREIDE SLIF YKKWELE AC VD AALL AT QMDRVN AIPF T YEQL DVLKHKLDEL (SEQ ID NO: 1).
[00165] For another example, the MUC16 binding domain can comprise mature mesothelin (SEQ ID NO: 2) described herein. The MUC16 binding domain can comprise at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more consecutive amino acid residues of SEQ ID NO: 3. The MUC16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence of mature mesothelin, for example, SEQ ID NO: 3 described herein.
[00166] EVEKT ACP SGKKAPEIDESLIF YKKWELE AC VD AALL AT QMDRVNAIPFT YEQL DVLKHKLDEL YPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMS PQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTC DPRQLD VL YPK ARL AF QNMN GSE YF VKIQ SFLGGAPTEDLK AL S Q QN V SMDL ATFMKL RTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPN GYLVLDLSMQEALS (SEQ ID NO: 3).
[00167] The MUC16 binding domain provided herein can comprise a peptide sequence of a MUC 16-interacting protein other than mesothelin or a fragment thereof. MUC 16-interacting proteins include, but are not limited to, galectin 1 (LGALSl), immunoglobulin (CD79A) binding protein 1 (IGBP1), ubiquitin C (UBC), Double-strand-break repair protein rad21 homolog (RAD21), mini chromosome maintenance complex component 2 (MCM2), Nanog homeobox (NANOG), POU class 5 homeobox 1 (POU5F1), epidermal growth factor receptor (EGFR),
tripartite motif containing 25 (TRIM25), Protein virilizer homolog (VIRMA), MUC1, MUC4,
MUC6, MUC5AC, MUC5B, MUC12, MUC13, MUC15, MUC17, MUC20, and Cepl70-like protein. The MUC16 binding domain can comprise a peptide sequence selected from Table 1.
The MUC 16 binding domain can comprise at least 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, 37, 38, 39, 40, 41, 42, 43, 44
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more consecutive amino acid residues of a peptide sequence selected from Table 1. The MUC 16 binding domain can comprise a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity of a sequence selected from Table 1.
[00169] In one aspect, the binding domain of the TFP comprises an amino acid sequence that is homologous to a binding domain amino acid sequence described herein, and the binding domain retains the desired functional properties of the binding domain described herein.
[00170] In various aspects, the binding domain of the TFP is engineered by modifying one or more amino acids.
[00171] It will be understood by one of ordinary skill in the art that the binding domains of the present disclosure may further be modified such that they vary in amino acid sequence ( e.g ., from wild-type), but not in desired activity. For example, additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein. For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
[00172] Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). [00173] Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 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%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
[00174] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g ., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison,
Wis.), or by manual alignment and visual inspection (see, e.g. , Brent et al., (2003) Current
Protocols in Molecular Biology). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al.,
(1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The algorithm parameters for using nucleotide BLAST to determine nucleotide sequence identity may use scoring parameters with a match/mismatch score of 1,-2 and wherein the gap costs are linear. The length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 28 for sequence alignment. The algorithm parameters for using protein BLAST to determine a peptide sequence identity may use scoring parameters with a BLOSUM62 matrix to assign a score for aligning pairs of residues, and determining overall alignment score, wherein the gap costs may have an existence penalty of 11 and an extension penalty of 1. The matrix adjustment method to compensate for amino acid composition of sequences may be a conditional compositional score matrix adjustment. The length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 6 for sequence alignment.
[00175] In one aspect, the present disclosure contemplates modifications of the starting binding domain amino acid sequence that generate functionally equivalent molecules. For example, the binding domain comprised in the TFP can be modified to retain at least 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% identity of the starting binding domain. The present disclosure contemplates modifications of the entire TFP construct, e.g. ,
modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules. The TFP construct can be modified to retain at least 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% or
99% identity of the starting TFP construct.
Extracellular domain
[00176] The extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain. An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g ., the alpha, beta, gamma, delta, or zeta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, an extracellular domain may include at least the extracellular domain of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD279, or CD154. In some instances, the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some embodiments, the extracellular domain comprises the IgC domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
[00177] In some embodiments, the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of at
least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00178] In some embodiments, the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more consecutive amino acid residues of an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C- terminus or at both the N- and C-terminus.
[00179] In some embodiments, the extracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 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 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00180] The extracellular domain can be a TCR extracellular domain. The TCR extracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit or a CD3 delta TCR subunit. The extracellular domain can be a full-length TCR extracellular domain or fragment (e.g., functional fragment) thereof. The extracellular domain can comprise a variable domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The
extracellular domain can comprise a variable domain and the extracellular component of a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some cases, the extracellular domain may not comprise a variable domain.
[00181] The extracellular domain can comprise the extracellular component of a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The extracellular domain can comprise the extracellular component of a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. The extracellular domain can comprise a fragment (e.g., functional fragment) of the extracellular component of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. For example, the extracellular domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the the extracellular component of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
[00182] The TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species. The TCR chain can be a murine or human TCR chain. For example, the extracellular domain can comprise a constant domain of a murine TCR alpha chain, a murine TCR beta chain, a human TCR gamma chain or a human TCR delta chain.
Transmembrane Domain
[00183] In general, a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g ., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (at least 1, 2, 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, or more amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 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, or more amino acids of the intracellular region). In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the intracellular region. In one aspect, the transmembrane domain is one that is associated with one of the other domains of the TFP is used. In some instances, the
transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP-
T cell surface. In a different aspect the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
[00184] The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target. In some instances, the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. In some instances, the transmembrane domain can be attached to the extracellular region of the TFP, e.g. , the binding domain of the TFP, via a hinge, e.g. , a hinge from a human protein. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g. , an IgG4 hinge, or a CD8a hinge.
[00185] In some embodiments, the transmembrane domain comprises, or comprises at least 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, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
Linkers
[00186] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the binding element and the TCR extracellular domain of the TFP. A glycine-serine doublet provides a particularly suitable linker. In some cases, the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:37). In some embodiments, the linker is encoded by a nucleotide sequence of GGT GGC GG AGGT TC T GG AGGT GG AGGT T C C (SEQ ID NO: 38).
Cytoplasmic Domain
[00187] The cytoplasmic domain of the TFP can include an intracellular domain. In some embodiments, the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta. In some embodiments, the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, they are able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
[00188] Examples of intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same
functional capability. In some embodiments, the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the intracellular domain comprises a sequence having at least about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain. In some embodiments, the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00189] In some embodiments, the intracellular domain comprises, or comprises at least 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, 60, 61, or 62 or more consecutive amino acid residues of the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00190] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal is required. Thus, naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g ., a costimulatory domain). [00191] A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a
stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine- based activation motifs (ITAMs).
[00192] Examples of ITAMs containing primary intracellular signaling domains that are of particular use in the present disclosure include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one embodiment, a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 epsilon, CD3 delta, or CD3 gamma. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.
[00193] The intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure. For example, the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012;
119(3):696-706).
[00194] The intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8,
9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. [00195] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker. [00196] In one aspect, the TFP- expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different binding domain, e.g., to the same target
(MUC16) or a different target. In one embodiment, when the TFP-expressing cell comprises two or more different TFPs, the binding domains of the different TFPs can be such that the binding domains do not interact with one another. For example, a cell expressing a first and second TFP can have an binding domain of the first TFP that does not associate with the binding domain of the second TFP.
[00197] In some instances, the TFP-expressing cell described herein can further comprise one or more TCR constant domains, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain. The TCR subunit and the antibody can be operatively linked. The TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
[00198] The TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species. The TCR chain can be a murine or human TCR chain. For example, the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain. [00199] In some instances, the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain of CD3 epsilon, CD3 gamma, or CD3 delta, and further comprises the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain. In some embodiments, TFP-expressing cell comprises a constant domain of a TCR alpha chain and a TCR beta chain. In some embodiments, TFP- expressing cell comprises a constant domain of a TCR gamma chain and a TCR delta chain. [00200] In some instances, the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR alpha chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR beta chain.
The TCR beta constant domain can further comprise second binding domain that is operatively linked to the TCR beta constant domain. The second binding domain can be the same or different as the binding domain of the TFP.
[00201] In some instances, the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR beta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR alpha chain. The TCR alpha constant domain can further comprise second binding domain that is operatively
linked to the TCR alpha constant domain. The second binding domain can be the same or different as the binding domain of the TFP.
[00202] In some instances, the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR gamma chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR delta chain. The TCR delta constant domain can further comprise a second binding domain that is operatively linked to the TCR delta constant domain. The second binding domain can be the same or different as the binding domain of the TFP.
[00203] In some instances, the TFP-expressing cell described herein comprises a TFP comprising (i) a binding domain and (ii) the constant domain of a TCR delta chain (i.e., comprising at least a portion of a TCR extracellular domain, a TCR transmembrane domain, and a TCR intracellular domain), and further comprises the constant domain of a TCR gamma chain. The TCR gamma constant domain can further comprise a second binding domain that is operatively linked to the TCR gamma constant domain. The second binding domain can be the same or different as the binding domain of the TFP.
[00204] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g ., an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g. , PD1, can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIRl, CD160, 2B4 and TGFRbeta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g. , an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g. , an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g. , of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, BTLA, LAIRl, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4- 1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4,
ICOS, and BTLA. PD1 is expressed on activated B cells, T cells and myeloid cells (Agata et al.
1996 Int. Immunol 8:765-75). Two ligands for PD1, Programmed Death-Ligand 1 (PD-L1) and
Programmed Death-Ligand 2 (PD-L2) have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat
Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother
54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1.
[00205] In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g ., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as CD28, 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP). In one another, the agent comprises the extracellular domain (ECD) and transmembrane domain of an inhibitory molecule, e.g. , Programmed Death 1 (PD1) that is fused to an intracellular signaling domain such as CD28, 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP). In one embodiment, the PD1 TFP, when used in combinations with an MUC16 TFP described herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD1 TFP comprising the extracellular domain of PD 1. Alternatively, provided are TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the PD-L1 or PD-L2.
[00206] In another aspect, the present disclosure provides a population of TFP-expressing T cells, e.g. , TFP-T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP-T cells can include a first cell expressing a TFP having a MUC16 binding domain described herein, and a second cell expressing a TFP having a different MUC16 binding domain, e.g. , a MUC16 binding domain described herein that differs from the MUC16 binding domain in the TFP expressed by the first cell. As another example, the population of TFP- expressing cells can include a first cell expressing a TFP that includes a MUC16 binding domain, e.g. , as described herein, and a second cell expressing a TFP that includes a binding domain to a target other than MUC16 (e.g., another tumor-associated antigen).
[00207] The TFP of the present invention may be used in multi cistronic vectors or vectors expressing several proteins in the same transcriptional unit. Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead. For example, several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit. Such peptides include 2A-peptides, or 2A-
like sequences, from members of the Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589-594. In some embodiments, the recombinant nucleic acid described herein encodes the TFP in frame with the agent, , e.g., a second polypeptide, e.g., a second TFP, a constant domain, or a PD-1 fusion protein, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.
[00208] In another aspect, the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a MUC16 domain described herein, and a second cell expressing another agent, e.g. , an agent which enhances the activity of a TFP- expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g. , can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4 and TGFR beta. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g. , an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g. , an intracellular signaling domain described herein. In some embodiments, the agent is a cytokine. In some embodiments, the cytokine is IL-15. In some embodiments, IL-15 increases the persistence of the T cells described herein.
[00209] Disclosed herein are methods for producing in vitro transcribed RNA encoding TFPs. The present disclosure also includes a TFP encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the TFP.
[00210] In one aspect, the MUC16 TFP is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the MUC16 TFP is introduced into a T cell for production of a TFP-T cell.
In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a TFP of the present disclosure. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally
occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
[00211] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
[00212] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.
[00213] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. In one embodiment, the 5’ UTR is between one and 3,000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added
to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.
[00214] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’UTR sequences can decrease the stability of mRNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
[00215] In one embodiment, the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5’ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation.
In other embodiments the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
[00216] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
[00217] In a preferred embodiment, the mRNA has both a cap on the 5’ end and a 3 ’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
[00218] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res.,
13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
[00219] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3’ stretch without cloning highly desirable.
[00220] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation.
Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed
RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
[00221] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
[00222] 5’ caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5’ cap. The 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).
[00223] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
[00224] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation
(Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard
Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator
(Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001).
Recombinant Nucleic Acids Encoding a TFP
[00225] Disclosed herein, in some embodiments, are recombinant nucleic acids comprising (a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising (i) a TCR- integrating subunit comprising (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, optionally comprising a stimulatory domain from an intracellular signaling domain; and (ii) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the antibody are operatively linked, and wherein the TFP functionally incorporates into a TCR complex when expressed in a T cell.
[00226] In some instances, the TCR-integrating subunit and the binding domain are operatively linked by a linker sequence. In some instances, the linker sequence comprises (G4S)n, wherein n=l to 5.
[00227] In some instances, the transmembrane domain is a TCR transmembrane domain from CD3 epsilon, CD3 gamma, CD3 delta, CD3 zeta, TCR alpha, TCR beta, TCR gamma or TCR delta. In some instances, the intracellular domain is derived from only CD3 epsilon, only CD3 gamma, or only CD3 delta.
[00228] In some embodiments, the TCR-integrating subunit comprises an intracellular domain. In some embodiments, the intracellular domain is from TCR alpha, TCR beta, TCR gamma or TCR delta. In some embodiments, the intracellular domain comprises a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma, or CD3 delta, or an amino acid sequence having at least one modification thereto.
[00229] In some instances, the TCR-integrating subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
[00230] In some instances, the TCR extracellular domain comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR
subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
[00231] In some instances, the TCR-integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 zeta chain TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD 137, CD 154, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
[00232] In some instances, the TCR-integrating subunit comprises a TCR intracellular domain comprising a stimulatory domain of a protein selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, CD3 zeta, or an amino acid sequence having at least one modification thereto.
[00233] In some instances, the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain of a protein selected from a functional signaling domain of 4- 1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
[00234] In some instances, the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain. In some instances, the costimulatory domain comprises a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
[00235] In some instances, the TCR-integrating subunit comprises an immunoreceptor tyrosine- based activation motif (IT AM) of a TCR-integrating subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto. In some instances, the IT AM replaces an IT AM of CD3 gamma, CD3 delta, or CD3 epsilon. In some instances, the IT AM is selected from the group consisting of a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, and a CD3 delta subunit and replaces a different IT AM
selected from the group consisting of a CD3 zeta subunit, a CD3 epsilon subunit, a CD3 gamma subunit, and a CD3 delta subunit.
[00236] In some instances, the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP.
[00237] In some instances, the binding domain comprises mesothelin or a fragment thereof. In some embodiments, the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin. In some embodiments, the functional MUC16 binding domain of mesothelin comprises the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of
EVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKH KLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto. In some embodiments, the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
[00238] In some instances, the nucleic acid is selected from the group consisting of a DNA and an RNA. In some instances, the nucleic acid is an mRNA. In some instances, the nucleic acid is a circular RNA.
[00239] In some instances, the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
[00240] In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR delta transmembrane domain. In some instances, the recombinant nucleic acid further comprises a sequence encoding a TCR gamma transmembrane domain and a sequence encoding a TCR delta transmembrane domain.
Gene Editing Technologies
[00241] In some embodiments, the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Patent No. 8,697,359), transcription activator-like effector (TALE)
nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases
(endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev.
Genetics (2010) vl 1, 636-646), or megaTAL nucleases (a fusion protein of a meganuclease to
TAL repeats) methods. In this way, a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also
Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews
Immunol. 9.10: 704-716, each incorporated herein by reference. In some embodiments, one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a
TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
[00242] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as “genome editing. [00243] In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
[00244] Current gene editing technologies comprise meganucleases, zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a double-stranded DNA break (DSB). DSB may then be repaired by either non- homologous end joining (NHEJ) or -when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, nickase nucleases generate single-stranded DNA breaks (SSB). DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
[00245] Genetic modification of genomic DNA can be performed using site-specific, rare- cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest. Methods for producing engineered, site-specific endonucleases are known in the art. For example, zinc-finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme. The zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre determined DNA sequence -18 basepairs in length. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005), Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9). In this case, however, the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair. Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762). A Compact TALEN comprises an engineered, site-specific TAL- effector DNA-binding domain fused to the nuclease domain from the I-Tevl homing endonuclease. Unlike Fokl, I-Tevl does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
[00246] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). The CRISPR gene-editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA. A CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA" or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).
[00247] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the type II CRISPR-
Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing.
More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli
(2018) Nat. Commun. 9:1911). Among these, such as Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.
[00248] Homing endonucleases are a group of naturally-occurring nucleases that recognize 15-
40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima
(2017), Protein Eng Des Sel. 30(7): 503-522). Meganucleases (MN) are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446). In some embodiments, meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.
[00249] In addition to mentioned four major gene editing technologies, chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446). For example, A megaTAL is a single chimeric protein, which is the combination of the easy-to- tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases. [00250] In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus. Vectors
[00251] In some embodiments, the instant invention provides vectors comprising the recombinant nucleic acid(s) encoding the TFP and/or additional molecules of interest (e.g., a protein or proteins to be secreted by the TFP T cell). In some instances, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an
adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
In some instances, the vector is an AAV6 vector. In some instances, the vector further comprises a promoter. In some instances, the vector is an in vitro transcribed vector.
[00252] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
[00253] The present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
[00254] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.
[00255] The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties). In another embodiment, the present disclosure provides a gene therapy vector.
[00256] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
[00257] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional
in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
[00258] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art.
In one embodiment, lentivirus vectors are used.
[00259] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.
[00260] An example of a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther. 17(8): 1453- 1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present disclosure. The use of an inducible promoter provides a molecular switch capable of turning on
expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
[00261] In order to assess the expression of a TFP polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.
[00262] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
[00263] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.
[00264] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection
[00265] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
[00266] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome
(e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
[00267] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
[00268] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C.
Chloroform is used as the only solvent since it is more readily evaporated than methanol.
“Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991
Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.
[00269] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
[00270] The present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule. In one aspect, a TFP vector can be directly transduced into a cell, e.g., a T cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the TFP construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.
Circular RNA
[00271] In some embodiments, TFP T cells are transduced with an RNA molecule. In some embodiments, the RNA is circular RNA. In some embodiments, the circular RNA is exogenous. In other embodiments, circular RNA is endogenous. In other embodiments, circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or in vivo or ex vivo.
[00272] Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5’ covalently closed RNA rings, and circular RNAs do not display
Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half- lives and may therefore improve the overall efficacy of mRNA in a variety of applications.
Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al.,
2015). For circularization, splice sites are used in reverse: downstream splice donors are
“backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman,
2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
[00273] To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed. A method for generating circular RNA can involve in vitro transcription (IVT) of a precursor linear RNA template with specially designed primers. Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns. In preferred embodiments, precursor RNA was synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
[00274] The group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998). When the td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995). In preferred embodiments, the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.
[00275] In some exemplary embodiments, a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg2+as cofactors. This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro , these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5' to 3' linked circles. [00276] In one aspect, disclosed herein is a sequence containing a full-length encephalomyocarditis virus (such as EMCV) IRES, a gene encoding a TFP, a CAR, a TCR or combination thereof, two short regions corresponding to exon fragments (El and E2), and of the
PIE construct between the 3' and 5' introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage or the permuted group I catalytic intron in the pre-tRNA gene of Anabaena. In more preferred embodiments, the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5' and 3' ends of the precursor RNA with the aim of bringing the 5' and 3' splice sites into proximity of one another. To ensure that the major splicing product was circular, the splicing reaction can be treated with RNase R.
[00277] In one aspect the TFP is encoded by a circular RNA. In one aspect the circular RNA encoding the TFP is introduced into a T cell for production of a TFP-T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
[00278] In some aspects, linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro RNA synthesis using appropriate primers and buffer and RNA polymerase and nucleotides modified or not. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, digested DNA, synthetic DNA sequence or any other appropriate source of DNA. The desired template for in vitro transcription is a TFP of the present disclosure. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and
3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.
[00279] In some exemplary embodiments, PCR is used to generate a template for in vitro transcription of linear precursor RNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For
example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5’ to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
[00280] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.
[00281] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. In one embodiment, the 5’ UTR is between one and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths required to achieve optimal RNA stability or/and translation efficiency following transfection of the transcribed RNA.
[00282] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3’UTR sequences can decrease the stability of mRNA whereas protein binding motif can increase the stabilitiy of mRNA and circular RNA. Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
[00283] In one embodiment, the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5’ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by
adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some
RNA transcripts but do not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
[00284] To enable synthesis of linear precursor RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art. [00285] In some embodiments, the RNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
[00286] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res ., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).
[00287] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3’ stretch without cloning highly desirable.
[00288] The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
[00289] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
[00290] 5’ caps can also provide stability to RNA molecules. In some embodiments, RNAs produced by the methods disclosed herein include a 5’ cap. The 5’ cap is provided using techniques known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436- 444 (2001); Stepinski et al., RNA , 7:1468-95 (2001); Elango et al., Biochim. Biophys. Res. Commun ., 330:958-966 (2005)).
[00291] The RNAs (e.g. circular RNA) are produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
[00292] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector®-II (Amaxa Biosystems, Cologne, Germany)), ECM® 830 (BTX) (Harvard Instruments, Boston, Mass.), Neon Transfection System (ThermoFisher), Cell squeezing (SQZ Biotechnologies) or the Gene Pulser® II (BioRad, Denver, Colo.),
Multiporator® (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa et al. Hum Gene Ther ., 12(8):861-70 (2001). For additional information on TFP T cells produced by the methods above, see copending Provisional Application Serial No. 62/836,977, which is herein incorporated by reference. Modified T cells
[00293] Disclosed herein are modified T cells comprising the sequence encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the sequence encoding the TFP disclosed herein or a TFP encoded by the sequence of the nucleic acid disclosed herein.
[00294] In some embodiments, the modified T cell comprises a functional disruption of an endogenous TCR. In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous
TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain. In some instances, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain. In some instances, the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain. In some instances, the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell. In some instances, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some instances, the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell. In some instances, the T cell is a human T cell. In some instances, the T cell is a CD8+ or CD4+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the T cell is a TCR alpha-beta T cell. In some instances, the T cell is a TCR gamma-delta T cell.
In some instances, one or more of TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No.
WO2019173693, which is herein incorporated by reference.
[00295] In some embodiments, the modified T cells are γδ T cells. In some embodiments, the γδ T cells are Vδ1+ Vδ2- γδ T cells. In some embodiments, the γδ T cells are Vδ1- Vδ2+ γδ T cells. In some embodiments, the γδ T cells are Vδ1- Vδ2- γδ T cells.
Sources of T cells
[00296] Prior to expansion and genetic modification, a source of T cells is obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used. In certain aspects of the present disclosure, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™
separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one aspect of the disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative aspect, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, the Oncol ogyCytoMate, or the
Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free,
Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
[00297] In embodiments, the T cells are alpha beta (ab or αβ) T cells. In some embodiments, the T cells are γδ T cells γδ T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example. Suitable doses for a therapeutic effect would be at least 105 or between about 105 and about 1010 cells per dose, for example, preferably in a series of dosing cycles. An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 105 cells on Day 0, for example increasing incrementally up to a target dose of about 1010 cells within several weeks of initiating an intra-patient dose escalation scheme. Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir-access device), intraperitoneal, and direct injection into a tumor mass.
[00298] An effective amount or sufficient number of the isolated, T cells is present in the composition and introduced into the subject such that long-term, specific, anti -turn or responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.
[00299] Accordingly, the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as
to achieve the desired therapeutic response. Furthermore, the amounts of each active agent included in the compositions described herein (e.g., the amount per each cell to be contacted or the amount per certain body weight) can vary in different applications.
[00300] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL® gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g. , 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T or Trans-Act beads®, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti- CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this disclosure. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
[00301] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for
regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.
Alternatively, in certain aspects, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
[00302] In one embodiment, a T cell population can be selected that expresses one or more of IFN-γ, TNF-alpha, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g ., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712, which is herein incorporated by reference.
[00303] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 2 billion cells/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, greater than 100 million cells/mL is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
[00304] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5xl06/mL. In other aspects, the concentration used can be from about 1x105/mL to 1x106/mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.
[00305] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing
granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5%
DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5%
Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of
1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen. In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
[00306] Also contemplated in the context of the disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation. [00307] In a further aspect of the present disclosure, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or
improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with
GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
Activation and Expansion of T Cells
[00308] T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, and 7,572,631.
[00309] Generally, the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular,
T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator ( e.g ., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells, CD8+ T cells, or CD4+CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999). In some embodiments, T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS® or Trans-Act® beads, for a time period sufficient for activation of the T cells. In one aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours, e.g., 24 hours. In some embodiments, T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain (e.g., IL-2, IL-7, IL-12, IL-15, IL-21, and others). In some embodiments, T cells
are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-
CD3 antibody, anti-CD28 antibody in combination with the same cytokines. In some embodiments, cells activated in the presence of an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody. In some embodiments, the cells are subcultured every 1, 2, 3, 4, 5, or 6 days. In some embodiments, cells are expanded for 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, or 30 days.
[00310] The expansion of T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0,
3.0, 4.0, 5.0, 7.5, 10, or 100 mM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). The expansion of T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP orHMB-PP) or other structurutally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0,
5.0, 7.5, 10, or 100 mM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells). In some embodiments, the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-
3-butenyl-l -pyrophosphate in the presence of IL-2 for one-to-two weeks. In some embodiments, the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL-2, e.g., for approximately 14 days. In some embodiments, the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g.,
OKT3) in the presence of IL-2. In some embodiments, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.
[00311] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and
CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an
increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
[00312] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process.
Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.
[00313] Once a MUC16 TFP is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a MUC16 TFP are described in further detail below
[00314] Western blot analysis of TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, e.g ., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Very briefly, T cells (1:1 mixture of CD4+ and CD8+ T cells) expressing the TFPs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. TFPs are detected by Western blotting using an antibody to a TCR chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.
[00315] In vitro expansion of TFP+ T cells following stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 and APCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EF-1 alpha, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Alternatively, a mixture of CD4+ and CD8+ T cells are stimulated with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduced with TFP on day 1 using a bicistronic lentiviral vector expressing TFP along with eGFP using a 2A ribosomal skipping sequence. Cultures are re-stimulated with either MUC16+ K562 cells (K562-MUC16), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL- 3/28) following washing. Exogenous IL-2 is added to the cultures every other day at 100 IU/mL. GFP+ T cells are enumerated by flow cytometry using bead-based counting (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)).
[00316] Sustained TFP+ T cell expansion in the absence of re-stimulation can also be measured
(see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter following stimulation with alphaCD3/alphaCD28 coated magnetic beads on day 0, and transduction with the indicated TFP on day 1.
[00317] Animal models can also be used to measure a TFP-T activity. For example, xenograft model using human MUC16-specific TFP+ T cells to treat a cancer in immunodeficient mice (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Very briefly, after establishment of cancer, mice are randomized as to treatment groups. Different numbers of engineered T cells are coinjected at a 1 : 1 ratio into NOD/SCID/γ-/- mice bearing cancer. The number of copies of each vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for cancer at weekly intervals. Peripheral blood MUC16+ cancer cell counts are measured in mice that are injected with alpha-MUC16-zeta TFP+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD/SCID/γ-/- mice can also be analyzed. Mice are injected with cancer cells and 3 weeks later are injected with T cells engineered to express TFP by a bicistronic lentiviral vector that encodes the TFP linked to eGFP. T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for cancer at 1-week intervals. Survival curves for the TFP+ T cell groups are compared using the log-rank test.
[00318] Dose dependent TFP treatment response can be evaluated (see, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). For example, peripheral blood is obtained 35-70 days after establishing cancer in mice injected on day 21 with TFP T cells, an equivalent number of mock-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood MUC16+ cancer cell counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.
[00319] Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of TFP- mediated proliferation is performed in microtiter plates by mixing washed T cells with cells expressing MUC16 or CD32 and CD137 (KT32-BBL) for a final T cell:cell expressing MUC16 ratio of 2: 1. Cells expressing MUC16 are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures
using CountBright™ fluorescent beads (Invitrogen) and flow cytometry as described by the manufacturer. TFP+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked TFP-expressing lentiviral vectors. For TFP+ T cells not expressing GFP, the TFP+ T cells are detected with biotinylated recombinant MUC16 protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences) according the manufacturer’s instructions.
Fluorescence is assessed using a FACScalibur flow cytometer, and data is analyzed according to the manufacturer’s instructions.
[00320] Cytotoxicity can be assessed by a standard 51Cr-release assay (see, e.g ., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009)). Briefly, target cells are loaded with 51Cr (as NaCrO4, New England Nuclear) at 37 °C for 2 hours with frequent agitation, washed twice in complete RPMI medium and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37 °C, supernatant from each well is harvested. Released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, Mass.). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula: % Lysis=(ER-SR)/(TR-SR), where ER represents the average 51Cr released for each experimental condition.
[00321] Imaging technologies can be used to evaluate specific trafficking and proliferation of TFPs in tumor-bearing animal models. Such assays have been described, e.g. , in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCTD/yc-/- (NSG) mice are injected IV with cancer cells followed 7 days later with T cells 4 hour after electroporation with the TFP constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of TFP+ T cells in a cancer xenograft model can be measured as follows: NSG mice are injected with cancer cells transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with MUC16 TFP 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive cancer in representative mice at day 5 (2 days before treatment) and day 8 (24 hours post TFP+ PBLs) can be generated.
[00322] Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the MUC16 TFP constructs of the present disclosure.
Therapeutic Applications
MU Cl 6 Associated Diseases and/or Disorders
[00323] In one aspect, the present disclosure provides methods for treating a disease associated with MUC16 expression. In one aspect, the present disclosure provides methods for treating a disease wherein part of the tumor is negative for MUC16 and part of the tumor is positive for MUC16. For example, the TFP of the present disclosure is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of MUC16, wherein the subject that has undergone treatment for elevated levels of MUC16 exhibits a disease associated with elevated levels of MUC16.
[00324] In one aspect, the present disclosure pertains to a vector comprising MUC16 TFP operably linked to promoter for expression in mammalian T cells. In one aspect, the present disclosure provides a recombinant T cell expressing the MUC16 TFP for use in treating MUC 16- expressing tumors, wherein the recombinant T cell expressing the MUC 16 TFP is termed a MUC16 TFP-T. In one aspect, the MUC16 TFP-T of the present disclosure is capable of contacting a tumor cell with at least one MUC 16 TFP of the disclosure expressed on its surface such that the TFP-T targets the tumor cell and growth of the tumor is inhibited.
[00325] In one aspect, the present disclosure pertains to a method of inhibiting growth of a MUC 16-expressing tumor cell, comprising contacting the tumor cell with a MUC 16 TFP T cell of the present disclosure such that the TFP-T is activated in response to the target (MUC 16) and targets the cancer cell, wherein the growth of the tumor is inhibited.
[00326] In one aspect, the present disclosure pertains to a method of treating cancer in a subject. The method comprises administering to the subject a MUC 16 TFP T cell of the present disclosure such that the cancer is treated in the subject. An example of a cancer that is treatable by the MUC16 TFP T cell of the disclosure is a cancer associated with expression of MUC16. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is a pancreatic cancer. In one aspect, the cancer is an ovarian cancer. In one aspect, the cancer is a stomach cancer. In one aspect, the cancer is a lung cancer. In one aspect, the cancer is an endometrial cancer. In some embodiments, MUC 16 TFP therapy can be used in combination with one or more additional therapies.
[00327] The present disclosure includes a type of cellular therapy where T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, TFP-
expressing T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control. In various aspects, the T cells administered to the patient, or their progeny, persist in the patient for at least one month, two month, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty -two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
[00328] The present disclosure also includes a type of cellular therapy where T cells are modified, e.g ., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the T cells administered to the patient, is present for less than one month, e.g. , three weeks, two weeks, or one week, after administration of the T cell to the patient.
[00329] Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the MUC16 antigen, resist soluble MUC16 inhibition, mediate bystander killing and/or mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of MUC 16-expressing tumor may be susceptible to indirect destruction by MUC16-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
[00330] In one aspect, the human TFP -modified T cells of the present disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.
[00331] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells.
[00332] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro ) with a vector expressing a TFP disclosed herein. The TFP- modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the TFP-modified cell can be autologous with respect
to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
[00333] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art, therefore the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
[00334] In some instances, the modified T cells described herein are allogeneic T cells. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of an unmodified control T cell. In some instances, less cytokines are released in the subject compared a subject administered an effective amount of a modified T cell comprising the recombinant nucleic acid disclosed herein, or the vector disclosed herein.
[00335] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against MUC16 in a patient.
[00336] Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the TFP-modified T cells of the disclosure are used in the treatment of diseases, disorders and conditions associated with expression of MUC16. In certain aspects, the cells of the disclosure are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of MUC16. Thus, the present disclosure provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of MUC16 comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP-modified T cells of the present disclosure.
[00337] In one aspect the TFP-T cells of the present disclosure may be used to treat a proliferative disease such as a cancer or malignancy or a precancerous condition. In one aspect, the cancer is a mesothelioma. In one aspect, the cancer is a pancreatic cancer. In one aspect, the cancer is an ovarian cancer. In one aspect, the cancer is a stomach cancer. In one aspect, the cancer is a lung cancer. In one aspect, the cancer is breast cancer. In one aspect, the cancer is a endometrial cancer. Further a disease associated with MUC16 expression includes, but is not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or
proliferative diseases expressing MUC16. Non-cancer related indications associated with expression of MUC16 include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma), inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery and transplantation.
[00338] The TFP-modified T cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.
[00339] The present disclosure also provides methods for inhibiting the proliferation or reducing a MUC 16-expressing cell population, the methods comprising contacting a population of cells comprising a MUC 16-expressing cell with a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell. In some aspects, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing MUC 16, the methods comprising contacting the MUC 16-expressing cancer cell population with a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell. In one aspect, the present disclosure provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing MUC 16, the methods comprising contacting the MUC 16-expressing cancer cell population with an a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell. In certain aspects, the MUC16 TFP-T cell of the present disclosure reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model a cancer associated with MUC 16-expressing cells relative to a negative control. In one aspect, the subject is a human.
[00340] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with MUC 16-expressing cells (e.g., a cancer expressing MUC 16), the methods comprising administering to a subject in need a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell. In one aspect, the subject is a human. Non limiting examples of disorders associated with MUC 16-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as pancreatic cancer, ovarian cancer, stomach cancer, lung cancer, or endometrial cancer or atypical cancers expressing MUC 16).
[00341] The present disclosure also provides methods for preventing, treating and/or managing a disease associated with MUC 16-expressing cells, the methods comprising administering to a subject in need a MUC 16 TFP-T cell of the present disclosure that binds to the MUC 16- expressing cell. In one aspect, the subject is a human.
[00342] The present disclosure provides methods for preventing relapse of cancer associated with MUC 16-expressing cells, the methods comprising administering to a subject in need thereof a MUC16 TFP-T cell of the present disclosure that binds to the MUC 16-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of a MUC 16 TFP-T cell described herein that binds to the MUC 16-expressing cell in combination with an effective amount of another therapy.
Combination Therapies
[00343] A TFP-expressing cell described herein may be used in combination with other known agents and therapies. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g ., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g. , an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. [00344] In some embodiments, the “at least one additional therapeutic agent” includes a TFP- expressing cell. Also provided are T cells that express multiple TFPs, which bind to the same or different targets or target antigens, or same or different epitopes on the same target antigen. Also provided are populations of T cells in which a first subset of T cells express a first TFP and a second subset of T cells express a second TFP.
[00345] A TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
[00346] In further aspects, a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. A TFP-expressing cell described herein may also be used in combination with a peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg
108:963-971. In a further aspect, a TFP-expressing cell described herein may also be used in combination with a promoter of myeloid cell differentiation (e.g., all-trans retinoic acid), an inhibitor of myeloid-derived suppressor cell (MDSC) expansion (e.g., inhibitors of c-kit receptor or a VEGF inhibitor), an inhibition of MDSC function (e.g., COX2 inhibitors or phosphodiesterase-5 inhibitors), or therapeutic elimination ofMDSCs (e.g., with a chemotherapeutic regimen such as treatment with doxorubicin and cyclophosphamide). Other therapeutic agents that may prevent the expansion ofMDSCs include amino-biphosphonate, biphosphanate, sildenafil and tadalafil, nitroaspirin, vitamin D3, and gemcitabine. (See, e.g.,
Gabrilovich andNagaraj, Nat. Rev. Immunol , (2009) v9(3): 162-174).
[00347] In some embodiments, the additional therapeutic agent comprises an immunostimulatory agent.
[00348] In some embodiments, the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof. In some aspects, the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD152), programmed cell death protein 1 (also PD-1 or CD279), programmed death ligand 1 (also PD-L1 or CD274), transforming growth factor beta (TGFβ), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 or HAVCR2 or CD366), neuritin, B- and T-lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof. In some aspects, the agent is selected from an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-Ll antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a) and 5 (CEACAM-5, also CD66e), vset immunoregulatory receptor (also VISR or VISTA), leukocyte-associated immunoglobulin-like receptor 1 (also LAIRl or CD305), CD160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof. In some aspects, the agent is pembrolizumab. In some aspects, the agent is nivolumab. In some aspects, the agent is atezolizumab.
[00349] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic and a small molecule. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine
1, and sulfamethizole 2. In some embodiments, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., ChemMed Chem , 2016, 14:1576 (DOI:
10.1002/cmdc.201500566), incorporated by reference in its entirety. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and
PD-L1 is administered after administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered contemporaneously with an antibody provided herein, but the agent and antibody are administered in separate pharmaceutical compositions.
[00350] In some embodiments, the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell. In some aspects, the co-stimulatory receptor is selected from GITR, 0X40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, a toll-like receptor, RIG-1, and a NOD-like receptor. In some embodiments, the agonist is an antibody.
[00351] In some embodiments, the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3 -di oxygenase, or the adenosine A2A receptor.
[00352] In some embodiments, the immunostimulatory agent is a cytokine. In some aspects, the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.
[00353] In some embodiments, the immunostimulatory agent is an oncolytic virus. In some aspects, the oncolytic virus is selected from a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, and a maraba virus.
[00354] Further examples of additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or
gemcitabine). In some embodiments, the additional therapeutic agent is folinic acid, 5- fluorouracil, and/or oxaliplatin. In some embodiments, the additional therapeutic agent is 5- fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or
MEK-targeted agent.
[00355] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.
[00356] In embodiments where a TFP provided herein and the additional therapeutic agent are included in different pharmaceutical compositions, administration of the TFP can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent. In some aspects, administration of a TFP provided herein, and the additional therapeutic agent occur within about one month of each other. In some aspects, administration of a TFP provided herein, and the additional therapeutic agent occur within about one week of each other. In some aspects, administration of a TFP provided herein, and the additional therapeutic agent occur within about one day of each other. In some aspects, administration of a TFP provided herein, and the additional therapeutic agent occur within about twelve hours of each other. In some aspects, administration of a TFP provided herein, and the additional therapeutic agent occur within about one hour of each other.
[00357] In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell. Side effects associated with the administration of a TFP-expressing cell include, but are not limited to cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. Accordingly, the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-γ, TNFα, IL-2, IL-6 and IL8. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. Such agents include, but are not limited to a steroid, an inhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFα inhibitor is entanercept. An example of an IL-6 inhibitor is tocilizumab (toe).
[00358] In one embodiment, the subject can be administered an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD1), can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3,
VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFRbeta. Inhibition of an inhibitory molecule, e.g. , by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g. , an inhibitory nucleic acid, e.g. , a dsRNA, e.g. , an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the TFP-expressing cell. In an embodiment the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a TFP-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g. , all of the components, of the TFP. In one embodiment, the inhibitor of an inhibitory signal can be, e.g. , an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as
MDX-010 and MDX-101, and marketed as Yervoy™; Bristol-Myers Squibb; tremelimumab
(IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-
675,206)). In an embodiment, the agent is an antibody or antibody fragment that binds to TIM3.
In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3.
[00359] In some embodiments, the T cells may be altered (e.g., by gene transfer) in vivo via a lentivirus, e.g., a lentivirus specifically targeting a CD4+ or CD8+ T cell. (See, e.g., Zhou et al.,
J. Immunol. (2015) 195:2493-2501).
[00360] In some embodiments, the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the TFP. In some embodiments, the fusion protein is expressed by the same vector as the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express a MUC16 TFP.
Pharmaceutical Compositions
[00361] Pharmaceutical compositions of the present disclosure may comprise a TFP-expressing cell, e.g ., a plurality of TFP-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure are in one aspect formulated for intravenous administration.
[00362] Pharmaceutical compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
[00363] In one embodiment, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
[00364] When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 104 to 109 cells/kg body weight, in some instances 105 to 106 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
[00365] In certain aspects, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present disclosure, and reinfuse the patient with these activated and expanded T
cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain aspects, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
[00366] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present disclosure are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present disclosure are administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
[00367] In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g ., T cells. These T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the present disclosure may be introduced, thereby creating a TFP-expressing T cell of the present disclosure. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded TFP T cells of the present disclosure. In an additional aspect, expanded cells are administered before or following surgery.
[00368] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for alemtuzumab, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766).
[00369] In one embodiment, the TFP is introduced into T cells, e.g. , using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the present disclosure, and one or more subsequent administrations of the TFP T cells of the present disclosure, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the TFP T cells of the present disclosure are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T
cells of the present disclosure are administered per week. In one embodiment, the subject ( e.g ., human subject) receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the TFP T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells of the present disclosure are administered for at least two, three, four, five, six, seven, eight or more weeks.
[00370] In one aspect, MUC16 TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way will have stable TFP expression.
[00371] In one aspect, TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of TFPs can be affected by RNA TFP vector delivery. In one aspect, the TFP RNA is transduced into the T cell by electroporation. [00372] A potential issue that can arise in patients being treated using transiently expressing TFP T cells (particularly with murine scFv bearing TFP T cells) is anaphylaxis after multiple treatments.
[00373] Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e., anti-TFP antibodies having an anti-IgE isotype. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen-day break in exposure to antigen.
[00374] If a patient is at high risk of generating an anti-TFP antibody response during the course of transient TFP therapy (such as those generated by RNA transductions), TFP T cell infusion breaks should not last more than ten to fourteen days.
Cytokine Release
[00375] Cytokine release syndrome is a form of systemic inflammatory response syndrome that arises as a complication of some diseases or infections, and is also an adverse effect of some monoclonal antibody drugs, as well as adoptive T cell therapies. TFP T cells can exhibit better killing activity than CAR-T cells. TFP T cells administered to a subject can exhibit better killing activity than CAR-T cells administered to a subject. This can be one of the advantages of TFP T cells over CAR-T cells. TFP T cells can exhibit less cytokine release CAR-T cells. A subject administered TFP T cells can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies. TFP
T cells can exhibit similar or better killing activity than CAR-T cells and the TFP T cells can exhibit less cytokine release than the CAR-T cells. TFP T cells administered to a subject can exhibit similar or better killing activity than CAR-T cells administered to a subject and the subject can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies.
[00376] In some cases, the cytokine release of a treatment with TFP T cells is less than the cytokine release of a treatment with CAR-T cells. In some embodiments, the cytokine release of a treatment with TFP T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least
50%, at least 60%, at least 70%, at least 80%, or at least 90% less than the cytokine release of a treatment with CAR-T cells. Various cytokines can be released less in the T cell treatment with
TFP T cells than CAR-T cells. In some embodiments, the cytokine is IL-2, IFN-γ, IL-4, TNF-α,
IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-1α, MIR-1β, or a combination thereof. In some cases, the treatment with TFP T cells release less perforin, granzyme A, granzyme B, or a combination thereof, than the treatment with CAR-T cells. In some embodiments, the perforin, granzyme A, or granzyme B released in a treatment with TFP T cells is at least 10%, at least
20%, at least 30%, at least 40%, at least 50%, or at least 60% less than a treatment with CAR-T cells.
[00377] In some embodiments, for a given cytokine, at least 10% less amount of the given cytokine is released following treatment compared to an amount of the given cytokine of a mammal treated with a CAR-T cell comprising the same binding domain. In some embodiments, the given cytokine comprises one or more cytokines selected from the group consisting of IL-2, IFN-γ, IL-4, TNF-α, IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-1α, MIP-1β, and any combination thereof.
[00378] The TFP T cells may exhibit similar or better activity in killing tumor cells than CAR-T cells. In some embodiments, a tumor growth in the mammal is inhibited such that a size of the tumor is at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, or at most 60% of a size of a tumor in a mammal treated with T cells that do not express the TFP after at least 8 days of treatment, wherein the mammal treated with T cells expressing TFP and the mammal treated with T cells that do not express the TFP have the same tumor size before the treatment. In some embodiments, the tumor growth in the mammal is completely inhibited. In some embodiments, the tumor growth in the mammal is completely inhibited for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, or more. In some embodiments, the population of T cells transduced with TFP kill similar amount of tumor cells compared to the CAR-T cells comprising the same binding domain.
[00379] The TFP T cells can exhibit different gene expression profile than cells that do not express TFP. In some cases, the TFP T cells may exhibit similar gene expression profiles than
CAR-T cells. In some other cases, the TFP T cells may exhibit different gene expression profiles than CAR-T cells. In some embodiments, the population of T cells transduced with TFP have a different gene expression profile than the CAR-T cells comprising the same binding domain. In some embodiments, an expression level of a gene is different in the T cells transduced with the
TFP than an expression level of the gene in the CAR-T cells comprising the same binding domain. In some embodiments, the gene has a function in antigen presentation, TCR signaling, homeostasis, metabolism, chemokine signaling, cytokine signaling, toll like receptor signaling,
MMP and adhesion molecule signaling, or TNFR related signaling.
EXAMPLES
[00380] The present disclosure is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the present disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present disclosure and practice the claimed methods. The following working examples specifically point out various aspects of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.
Example 1: Characterization of IAB Peptide
[00381] The MUC 16-binding IAB peptide of mesothelin comprises amino acids 1-64 of mesothelin
(EVEKT ACP S GKK AREIDE SLIF YKKWELE AC VD A ALL AT QMDRVN AIPF T YEQLD VLK HKLDEL; SEQ ID NO: 1). The expression of MUC16 by OVCAR3 cells, but not C30 cells was validated by anti-human MUC16 Ab (Clone X75, R-phycoerythrin conjugated) (FIG. 1 A). The binding of IAB to MUC16 was validated by testing allophycocyanin-conjugaed IAB hFc peptide (labeled by Zenon human IgG labeling reagents) against MUC 16-positive OVCAR3 cells and MUC 16-negative C30 cells. As shown in FIG. IB, binding of IAB hFc is detected for the MUC 16-positive OVCAR3 cells but not the MUC 16-negative C30 cells.
Example 2: Generation of MUC16 TFP Constructs
[00382] MUC 16 TFP constructs can be engineered by cloning the DNA sequence of amino acids 1-64 of mesothelin
(EVEKT ACP S GKK AREIDE SLIF YKKWELE AC VDAALL AT QMDRVN AIPF T YEQLD VLK
HKLDEL; SEQ ID NO: 1) linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): A A AGGGGS GGGGS GGGGSLE (SEQ ID NO:39) or a long linker
(LL): A A AIE VMYPPP YLGGGGS GGGGS GGGGSLE (SEQ ID NO:40) into p510 vector
((System Biosciences (SBI)). Other vectors may also be used, for example, pLRPO vector.
[00383] Various other vector may be used to generate fusion protein constructs. The human
MUC16 polypeptide canonical sequence is UniProt Accession No. Q8WXI7. Provided are polypeptides that are capable of specifically binding to the human MUC16 polypeptide, and fragments thereof.
Source of TCR Subunits
[00384] Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain. The CD3 episolon, CD3 delta, and CD3 gamma subunits have an intracellular domain. A human TCR complex contains the CD3-epsilon polypeptide, the CD3 -gamma poly peptide, the CD3 -delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide. TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide. The human CD3-epsilon polypeptide canonical sequence is Uniprot Accession No. P07766. The human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693. The human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234. The human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963. The human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1. The human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.
[00385] The human CD3-epsilon polypeptide canonical sequence is:
MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHL SLKEF SELEQ S GY Y V C YPRGSKPED ANF YL YLR ARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGQRDL YSGLNQRRI (SEQ ID NO:41).
[00386] The signal peptide of human CD3ε is:
MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO:52).
[00387] The extracellular domain of human CD3ε is:
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEF SELEQ S GY Y VC YPRGSKPED ANF YLYLRARV CEN CMEMD (SEQ ID NO:53). [00388] The transmembrane domain of human CD3ε is:
VMS VATIVIVDICITGGLLLLVYYW S (SEQ ID NO: 54).
[00389] The intracellular domain of human CD3ε is:
KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:55). [00390] The human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS GFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ GNQLRRN (SEQ ID NO:42). [00391] The signal peptide of human CD3γ is: MEQGKGLAVLILAIILLQGTLA (SEQ ID NO:56). [00392] The extracellular domain of human CD3γ is: QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO:57). [00393] The transmembrane domain of human CD3 γ is: GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO:58). [00394] The intracellular domain of human CD3γ is: GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:59). [00395] The human CD3-delta polypeptide canonical sequence is: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:43). [00396] The signal peptide of human CD3δ is: MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:60). [00397] The extracellular domain of human CD3δ is: FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE STVQVHYRMCQSCVELDPATVA (SEQ ID NO:61). [00398] The transmembrane domain of human CD3δ is: GIIVTDVIATLLLALGVFCFA (SEQ ID NO:62). [00399] The intracellular domain of human CD3δ is: GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO:63). [00400] The human CD3-zeta polypeptide canonical sequence is: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:44).
[00401] The human TCR alpha chain canonical sequence is: MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAG PLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:45). [00402] The human TCR alpha chain C region canonical sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG FRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:46). [00403] The human TCR alpha chain human IgC sequence is: PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 69) [00404] The transmembrane domain of the human TCR alpha chain is: VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:68). [00405] The intracellular domain of the human TCR alpha chain is: SS [00406] The human TCR alpha chain V region CTL-L17 canonical sequence is: MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDY FLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAK GAGTASKLTFGTGTRLQVTL (SEQ ID NO:47). [00407] The murine TCR alpha chain constant (mTRAC) region canonical sequence is: XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAW SNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLM TLRLWSS (SEQ ID NO: 75). [00408] The transmembrane domain of the murine TCR alpha chain is: MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 76). [00409] The intracellular domain of the murine TCR alpha chain is: SS (SEQ ID NO: 77) [00410] The human TCR beta 1 chain C region canonical sequence is: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF (SEQ ID NO:48). [00411] The human TCR beta 1 chain human IgC sequence is:
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE (SEQ ID NO: 70) [00412] The transmembrane domain of the human TCR beta 1 chain is: ILLGKATLYAVLVSALVLMAM (SEQ ID NO:6). [00413] The human TCR beta 1 chain V region CTL-L17 canonical sequence is: MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTL GQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGL NQPQHFGDGTRLSIL (SEQ ID NO:49). [00414] The intracellular domain of the human TCR beta 1 chain is: VKRKDF (SEQ ID NO: 71) [00415] The human TCR beta chain V region YT35 canonical sequence is: MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTM MRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCS ANYGYTFGSGTRLTVV (SEQ ID NO:50). [00416] The murine TCR beta chain constant region canonical sequence is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 78). [00417] The transmembrane domain of the murine TCR beta chain is: ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 79). [00418] The intracellular domain of the murine TCR beta chain is: KRKNS (SEQ ID NO: 80) [00419] The human TCR beta 2 chain C region canonical sequence is: DLKNVFPPKVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDP Q PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI V SAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG (SEQ ID NO: 81) [00420] The transmembrane domain of the human TCR beta 2 chain is: TILYEILLGKATLYAVLVSALVL (SEQ ID NO: 82) [00421] The intracellular domain of the human TCR beta 2 chain is: MAMVKRKDSRG (SEQ ID NO: 83)
[00422] The murine TCR beta 2 chain C region canonical sequence is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 84) [00423] The transmembrane domain of the murine TCR beta 2 chain is: ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 85) [00424] The intracellular domain of the murine TCR beta 2 chain is: KRKNS (SEQ ID NO: 86) [00425] The human TCR gamma chain C region canonical sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:64). [00426] The human TCR beta gamma human IgC sequence is: DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSA (SEQ ID NO: 72) [00427] The transmembrane domain of the human TCR gamma chain is: YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO:65). [00428] The intracellular domain of the human TCR gamma chain is: RRTAFCCNGEKS (SEQ ID NO: 73) [00429] The human TCR delta chain C region canonical sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO:66). [00430] The human TCR delta gamma human IgC sequence is: SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTV (SEQ ID NO: 74) [00431] The transmembrane domain of the human TCR delta chain is: LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO:67). [00432] The intracellular domain of the human TCR delta chain is: L
Generation ofTFPs from TCR Domains and MU Cl 6-binding peptides
[00433] The IAB peptide from MLSN that targets MUC16 was recombinantly linked to CD3- epsilon using the linker sequence (G4S)3 to generate a TFP (MUC16.TFP) having the amino acid sequence of SEQ ID NO: 51 shown below. Various other linkers and MUC16-binding peptide configurations can be utilized as is described in Example 1.
1 MLLLVTSLLL CELPHPAFLL IPEVEKTACP SGKKAREIDE SLIFYKKWEL EACVDAALLA 61 TQMDRVNAIP FTYEQLDVLK HKLDELAAAG GGGSGGGGSG GGGSLEDGNE EMGGITQTPY 121 KVSISGTTVI LTCPQYPGSE ILWQHNDKNI GGDEDDKNIG SDEDHLSLKE FSELEQSGYY 181 VCYPRGSKPE DANFYLYLRA RVCENCMEMD VMSVATIVIV DICITGGLLL LVYYWSKNRK 241 AKAKPVTRGA GAGGRQRGQN KERPPPVPNP DYEPIRKGQR DLYSGLNQRR I (SEQ ID NO: 51)
TFP Expression Vectors
[00434] Expression vectors are provided that include: a promoter (eukaryotic elongation factor 1 alpha (EFla) promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes ( e.g ., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
[00435] The TFP-encoding nucleic acid construct was cloned into the pLRPO lentiviral expression vector. The TFP.MUC16 lentiviral transfer vectors were used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles. Expi293F-cells were suspended in free-style (FS) media and allowed to incubate at 37 degrees C, 8% CO2, 150 rpm for 1-3 hours. The transfer DNA plasmid, Gag/Pol plasmid, Rev plasmid, and VSV-G plasmid were diluted in FS media. PEIpro was then diluted in FS media and added to the mixture of DNA and media. The incubated cells were added to this mixture and are incubated at 37 degrees C, 8% CO2, 150 rpm for 18-24 hours. The following day, the supernatant was replaced with fresh media and supplemented with sodium butyrate and incubated at 37°C for an additional 24 hours. The lentivirus containing supernatant was then collected into a 50 mL sterile, capped conical centrifuge tube and put on ice. After centrifugation at 3000 rpm for 30 minutes at 4°C, the cleared supernatant was filtered with a low-protein binding 0.45 pm sterile filter. The virus was subsequently concentrated by Lenti-X. The virus stock preparation was either used for infection immediately or aliquoted and stored at -80°C for future use.
Example 3: Generation of T cell receptor fusion protein Jurkat Cells
Transduction of CD3 epsilon knock-out Jurkat cells
[00436] CD3 epsilon knock-out Jurkat cells were generated by knocking out CD3ε subunit from wild-type (WT) Jurkat cells with CRISPR technique, as described, e.g., in co-pending U.S.
Patent Publication No. 2017-0166622. Jurkat CD3ε KO cells were incubated with virus at a multiplicity of infection (MOI) of five. Medium was replaced twenty-four-hours post incubation. Validation of TFP Expression
[00437] Expression of the TFP in Jurkat cells was validated based on detection of cell-surface TFP expression in CD3 epsilon knock-out Jurkat cells. As a result of CD3 epsilon knock-out, the surface expression of TCR complex was absent on CD3 epsilon knock-out Jurkat cells.
Indirectly, TFP.MUC16 surface expression on TFP.MUC16-transduced CD3 epsilon knock-out Jurkat cells, was demonstrated by the significantly increased binding of an anti-CD3 epsilon antibody and an anti-TCRαβ antibody in comparison to the non-transduced control CD3 epsilon knock-out Jurkat cells (FIG. 2). Furthermore, direct evidence comes from the positive staining of TFP.MUC16-transduced CD3 epsilon knock-out Jurkat cells with SSI scFv hFc (e.g., anti- mesothelin SSI single chain Fv), a reagent derived from anti-MSLN antibody SSI that specifically binds to the LAB peptide of MSLN protein (see Kaneko et al., JBC, 2009, 284 (6):3739-3749).
Example 4: Measurement of Activation of Jurkat Cells by FACS
[00438] IAB-MUC16 interaction mediated activation of Jurkat cells expressing TFP constructs was measured by flow cytometry. As described above, TFP.MUC16-transduced (TFP) and non- transduced (NT) CD3 epsilon knock-out Jurkat cells were generated, validated for transduction (FIG. 2), and set up in co-culture with MUC16 positive OVCAR3 cells or MUC16 negative C30 cells (validated in FIGs. 1A and IB) at different effector-to-target ratios (3:1 or 1:1 effector to target cells). Jurkat cell activation was determined by measuring the expression of CD25 and CD69 at 24 hours after the co-culture by flow cytometry, with an Ab panel including anti-human CD25 (Clone BC96) and anti-human CD69 (clone FN50). As is shown in FIG. 3, increased activation was detected with TFP.MUC16-transduced Jurkat cells co-cultured with OVCAR3 target cells (FIG. 3A), but not the TFP.MUC16-transduced Jurkat cells co-cultured with C30 target cells (FIG. 3B) or non-transduced control Jurkat cells co-cultured with either OVCAR3 or C30 cells.
Example 5: Generation of T cell receptor fusion protein T Cells
T-cell activation, tranduction, and expansion
[00439] T cells were purifried from healthy donor leukopak or PBMCs via positive selection of CD4+ and CD8+ T cells with CD4 and CD8 microbeads from Miltenyi Biotech. On day 0, T cells, freshly isolated or thawed from previously prepared frozen vials, were activated by MACS GMP T cell TransAct (Miltenyi Biotech), in the presence of human IL-7 and IL-15 (both from
Miltenyi Biotech, premium grade). On day 1, activated T cells were transduced with lentivirus encoding the MUC16.TFP. On day 4, the cells were washed, subcultured in fresh medium with cytokines and then expanded up to day 10 by supplementing fresh medium every 2 days. At each day of subculture, fresh medium with cytokines were added to maintain the cell suspension at
1x106 cells/mL. The expansion of T cells from three donors, transduced with MUC16.TFP, and non-transduced controls, is shown in FIG. 4. No dramatic difference in expansion was observed between MUC16.TFP and non-transduced T cells for each tested donor.
[00440] Verification of TFP expression by cell staining
[00441] Following lentiviral transduction, expression of MUC16.TFPs by transduced T cells was confirmed by flow cytometry, using the SSI scFv hFc, on day 10 of cell expansion. T cells were washed three times in PBS and then re-suspended in PBS at 1x105 cells per well. For dead cell exclusion, cells were incubated with LIVE/DEAD® Fixable Aqua Dead Cell Stain (Invitrogen) for 30 minutes at 4°C in the dark. Cells were then washed twice with PBS and re suspended in 100 μL of staining buffer (PBS with 2% BSA). Cells were harvested, washed with PBS three times and blocked with human Fc block (BioLegend) for 10 minutes. Cells were then stained with 1 μg of R-phycoerythrin Zenon labeled SSI scFv hFc using Zenon Human Antibody Conjugation Kit (ThermoFisher) for 30 minutes at 4°C in the dark. Cells were then washed twice with staining buffer (PBS with 2% BSA) and submitted to data acquisition on LSR Fortessa™-X20 (BD Biosciences) using FACS Diva software. The TFP expression was analyzed, with FlowJo® (BD Biosciences), for live T cells (CD3+ alive cells). As is shown in FIG. 5, in all three donors, binding of the SSI scFv hFc was detected in TFP.MUC16-transduced T cells, but not in non-transduced control T cells from the same donors.
Example 6: Measurement of MUC16-Specific Activation of MUC16. TFP T Cells
[00442] Specific activation of the MUC16.TFP transduced T cells was measured similarly as for Jurkat cells. As described above, MUC16.TFP T cells or non-transduced T cells were generated from three donors. At day 10 of expansion, T cells were harvested and stimulated by co-culture with MUC 16-positive OVCAR3 cells or MUC 16-negative C30 cells at 1:1 (effectortumor) ratio for 24 hours. Expression of CD25 and CD69 were determined by flow cytometry with anti human CD25 (Clone BC96) and anti-human CD69 (clone FN50). As is shown in FIG. 6A,
CD25 and CD69 expression was evaluated for the TFP- population and the TFP+ population (gated on SSI scFv hFc negative and positive populations, respectively) of MUC16.TFP transduced T cells (TFP) as well as non-transduced cells (NT) from donor A. Dramatic increase in frequency of CD25+ and CD69+ cells was only observed for TFP+ population in MUC 16. TFP T cells co-cultured with MUC 16 positive OVCAR3 cells. Increase in frequency of CD25+ cells
was observed for TFP- population in MUC16.TFP co-culutred with OVCAR3 cells, likely due to the paracrine effect of the cytokines released by the TFP+ population in the same co-culture setup. No difference in CD25+ and CD69+ cells frequency was observed between TFP+ and
TFP- populations in MUC.16 TFP transduced T cells co-cultured with MUC16 negative C30 cells, or between non-transduced T cells co-cultured with OVCAR3 cells or C30 cells. Taken together, the data indicates that IAB-MUC16 interaction specifically induce TFP+ T cell activation.
Example 7: Luciferase-based cytotoxicity assay
[00443] The luciferase-based cytotoxicity assay assesses the cytotoxicity of TFP T cells by indirectly measuring the luciferase enzymatic activity in the residual live target cells after co culture. MUC 16-positive OVCAR3 and MUC 16-negative C30 cells were modified to overexpress firefly luciferase via transduction with firefly luciferase encoding lentivirus followed with antibiotic selection to generate stable cell line.
[00444] The target cells were plated at 10000 cells per well in 96-well plate. The MUC 16. TFP transduced or non-transduced T cells were added to the target cells at different effector-to-target ratios (3 : 1 or 1 : 1). The mixture of cells was then cultured for 24 at 37°C with 5 % CO2 before the luciferase enzymatic activity in the live target cells was measured by the Bright-Glo® Luciferase Assay System (Promega®, Catalogue number E2610). The cells were spun into a pellet and resuspended in medium containing the luciferase substrate. The percentage of tumor cell killing was then calculated with the following formula: % Cytotoxicity = 100% x [1 - RLU (Tumor cells + T cells) / RLU (Tumor cells)].
[00445] As is shown in FIG. 7, for all three donors, at both ratios of effector to target cell ratios, MUC 16. TFP transduced T cells demonstrated enhanced cytotoxicity towards MUC 16-positive OVCAR3 relative to untransduced T cells. No cytotoxicity was observed for MUC 16. TFP transduced T cells against MUC16 negative C30 cells.
Example 8: Cytokine Secretion measurement by MSP
[00446] A measure of effector T-cell activation and proliferation associated with the recognition of cells bearing cognate antigen is the production of effector cytokines such as interferon-gamma (IFN-γ), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-α).
[00447] Target-specific cytokine production including IFN-γ, GM-CSF, and TNF-α by TFP T cells was measured from supernatants harvested 24 hours after the co-culture of T cells with MUC 16-positive OVCAR3 and MUC 16-negative C30 target cells using the U-PLEX® Biomarker Group I (hu) Assays (Meso Scale Diagnostics®, LLC, catalog number: K15067L-4).
[00448] As is shown in FIGs. 8A-8C, increased levels of IFN-γ, GM-CSF, and TNF-α were observed in TFP.MUC16-transduced T cells from all three donors co-cultured with MUC 16- positive OVCAR3 target cells relative to TFP.MUC16-transduced T cells co-cultured with
MUC 16-negative C30 target cells or non-transduced control T cells co-cultured with either
OVCAR3 or CD30 cells.
Example 9: MUC16 specific proliferation of T cells expressing MUC16-TFP [00449] MUC 16 specific proliferation of MUC16-TFP T cells is determined by monitoring the dilution of T cell tracing signal (decrease in signal intensity of CellTrace™) by flowcytometry analysis. T cells expressing MUC16-TFPs are labelled with CellTrace™ Far Red Proliferation Kit (Cat. #C34564ThermoFisher), then co-cultured with CD30 or OVCAR3 cells at 1-to-l ratio for 3 days. T cells expressing MUC16-TFPs labelled with CellTrace Far Red Proliferation kit were also stimulated with medium alone or with 1 μg/mL plate-bound anti-CD3 antibody (clone OKT-3, Cat #14-0037-82, Invitrogen) for 3 days. T cells expressing MUC16-TFPs will show MUC 16-specific proliferation, demonstrated by the decrease of CellTracer signal when co- cultured with OVCAR3 cells, but not CD30 cells.
Example 10: In vivo activity of MUC16-TFP T cells
[00450] T cells expressing MUC16-TFPs are evaluated in NSG mouse xenograft models of human ovarian carcinoma cell lines, OVCAR3 cells. Six-week-old female NSG (NOD.Cg- PrkdcscldI12rgtm 1 Wjl/SzJ, The Jackson Laboratory, are intraperitoneally inoculated with OVCAR3 (5 x 106 cells/mouse), or subcutaneously with OVCAR3 cells (5 x 106 cells/mouse, 1-to-l mixture with Matrigel®). Tumor burden is determined by bioluminescence imaging (BLI) for the intraperitoneal models with the intraperitoneal injection of 0.2ml of luciferin substrate (VWR) diluted in PBS (150 mg/kg). Tumor burden of the subcutaneous model is measured as the tumor volume by Caliper. Once the tumor model is established (intraperitoneal models: BLI signal > 108; subcutaneous model: tumor volume > 75 mm3), T cells expressing MUC16-TFPs (MUC16 TFP1 and MUC 16 TFP2) or non-transduced T cells (NT), or vehicle (PBS) are injected intravenously at the dose of 107 T cells per mouse.
[00451] The in vivo efficacy of T cells expressing MUC 16 TFPs will be observed across intraperitoneal and subcutaneous models of OVCAR3 cells. In all tumor models, MUC 16 TFP will significantly delay tumor growth or clear the tumor cells from the mice.
Example 11: Immunohistochemistry staining of normal human tissues using the Fc-fusion IAB mesothelin peptide
[00452] The objective of the study is to obtain information on the MUC16 expression of normal human tissues using an Fc-fusion IAB mesothelin peptide
[00453] Control materials and FFPE sections are stained with an IAB mesothelin peptide that is genetically fused to a mouse Fc region for detection using HRP conjugated anti-mouse Fc secondary antibody. The positive control consists of FFPE sections of human ovarian tumors from two donors. The negative control is an FFPE section of a human heart. The panel of tested tissues includes the following: blood cells, cerebellum or cerebral cortex, gastrointestinal tract (esophagus, small intestine, stomach, colon - as available), spleen, kidney (glomerulus, tubule), liver, lymph node, skin, placenta, testis and tonsil from one donor each.
[00454] Results: Two human ovarian carcinoma tissues from different donors are used as a positive control and will show positive staining. From the normal tissues, the human heart negative control will show negative staining. The staining will further show negative or limited expression of MUC16 in normal human tissues. This makes it an attractive target for cancer therapy of MUC16 positive malignancies. The MUC16-specific IAB mesothelin peptide will be able to bind and stain antigen positive tissues.
Example 12: Clinical Studies
[00455] Patients with unresectable ovarian cancer with relapsed or refractory disease will be enrolled for clinical studies of T cells expressing MUC16-TFPs. The initial study will explore the safety profile of T cells expressing MUC16-TFPs and will explore cell kinetics and pharmacodynamics outcomes. Those results will inform the selection of dosages for further studies, which will then be administered to a larger cohort of patients with unresectable ovarian cancer to define the efficacy profile of T cells expressing MUC16-TFPs.
Endnotes
[00456] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
Claims
1. A pharmaceutical composition comprising
(I) a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR-integrating subunit comprising:
(i) at least a portion of a TCR extracellular domain,
(ii) a transmembrane domain, and
(iii) a TCR intracellular domain; and
(b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and
(II) a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked.
2. A pharmaceutical composition comprising
(I) a human T cell, wherein the T cell comprises a nucleic acid molecule encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR-integrating subunit comprising:
(i) at least a portion of a TCR extracellular domain, and
(ii) a transmembrane domain; and
(b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; and
(II) a pharmaceutically acceptable carrier; and wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked.
3. The pharmaceutical composition of claim 2, wherein the TFP further comprises an intracellular domain.
4. The pharmaceutical composition of any one of claims 1-3, wherein the MUC16 binding domain specifically binds membrane-bound MUC16.
5. The pharmaceutical composition of claim 4, wherein the MUC16 binding domain specifically binds membrane-bound MUC16 in the presence of soluble MUC16.
6. The pharmaceutical composition of any one of the preceding claims, wherein the T cell exhibits increased cytotoxicity to a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
7. The pharmaceutical composition of any one of the preceding claims, wherein the encoded
TFP molecule functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof when expressed in the T cell.
8. The pharmaceutical composition of any one of claims 1-7, wherein the sequence encoding the MUC16 binding domain is connected to the sequence encoding the TCR extracellular domain by a sequence encoding a linker.
9. The pharmaceutical composition of claim 8, wherein the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.
10. The pharmaceutical composition of any one of the preceding claims, wherein the MUC16 binding domain comprises mesothelin or a fragment thereof.
11. The pharmaceutical composition of claim 10, wherein the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin.
12. The pharmaceutical composition of claim 11, wherein the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of
E VEKT ACP S GKK AREIDESLIF YKKWELE AC VD AALL AT QMDRVN AIPF T YEQLD V LKHKLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
13. The pharmaceutical composition of claim 11, wherein the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
14. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition is substantially free of serum.
15. The pharmaceutical composition of any one of the preceding claims, wherein the human T cell has greater than or more efficient cytotoxic activity than a CD8+ or CD4+ T cell comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (a) the MUC16 binding domain, operatively linked to (b) at least a portion of an extracellular domain (c) a transmembrane domain (d) at least a portion of a CD28, 4- IBB, 0X40, ICOS, CD27, and/or CD2 intracellular domain and (e) a CD3 zeta intracellular domain.
16. The pharmaceutical composition of any one of claims 1-15, wherein the T cell is a primary T cell.
17. The pharmaceutical composition of any one of claims 1-15, wherein the T cell is an iNKT cell.
18. The pharmaceutical composition of any one of claims 1-15, wherein the T cell is a human
CD4+ T cell.
19. The pharmaceutical composition of any one of claims 1-15, wherein the T cell is a human CD8+ T cell.
20. The pharmaceutical composition of any one of claims 1-16, wherein the T cell is a human alpha beta (ab or αβ) T cell.
21. The pharmaceutical composition of any one of claims 1-16, wherein the T cell is a human gamma delta (gd or γδ) T cell.
22. The pharmaceutical composition of any one of the preceding claims, wherein the T cell further comprises a nucleic acid encoding a first polypeptide comprising at least a portion of an inhibitory molecule , wherein the at least a portion of an inhibitory molecule is associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
23. The pharmaceutical composition of claim 22, wherein the inhibitory molecule is PD-1.
24. The pharmaceutical composition of claim 22 or 23, wherein the second polypeptide comprises a costimulatory domain and primary signaling domain from a protein selected from the group consisting of CD28, CD27, ICOS, CD3ζ, 41-BB, 0X40, GITR, CD30,
CD40, ICOS, BAFFR, HVEM, LFA-1, CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80,
CD 160, and B7-H3.
25. The pharmaceutical composition of any one of the preceding claims, wherein production of IL-2 or IFNy by the T cell is increased in the presence of a cell expressing an antigen that specifically interacts with the MUC16 binding domain compared to a T cell not containing the TFP.
26. The pharmaceutical composition of any one of the preceding claims, wherein the cell is a population of human T cells, wherein an individual T cell of the population comprises at least two TFP molecules, or at least two T cells of the population collectively comprise at least two TFP molecules; wherein the at least two TFP molecules comprise a MUC16 binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and wherein at least one of the at least two TFP molecules functionally interacts with an endogenous TCR complex, at least one endogenous TCR polypeptide, or a combination thereof.
27. The pharmaceutical composition of any one of the preceding claims, wherein the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR
beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
28. The pharmaceutical composition of any one of the preceding claims, wherein the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
29. The pharmaceutical composition of any one of the preceding claims, wherein the TCR intracellular domain comprises an intracellular domain of TCR alpha, TCR beta, TCR delta, or TCR gamma, or an amino acid sequence having at least one modification thereto.
30. The pharmaceutical composition of any one of the preceding claims, wherein the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto.
31. The pharmaceutical composition of any one of the preceding claims, wherein the TCR- integrating subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
32. The pharmaceutical composition of any one of claims 1-31, wherein the TCR-integrating subunit is derived only from CD3 epsilon.
33. The pharmaceutical composition of any one of claims 1-31, wherein the TCR-integrating subunit is derived only from CD3 gamma.
34. The pharmaceutical composition of any one of claims 1-31, wherein the TCR-integrating subunit is derived only from CD3 delta.
35. The pharmaceutical composition of any one of claims 1-29, wherein the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
36. The pharmaceutical composition of any one of the preceding claims, further comprising a sequence encoding a costimulatory domain.
37. The pharmaceutical composition of claim 36, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4- 1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
38. The pharmaceutical composition of any one of the preceding claims, wherein the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
39. The pharmaceutical composition of claim 38, wherein the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
40. The pharmaceutical composition of claim 39, wherein the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
41. A recombinant nucleic acid encoding a T cell receptor (TCR) fusion protein (TFP) comprising:
(a) a TCR-integrating subunit comprising
(i) at least a portion of a TCR extracellular domain,
(ii) a transmembrane domain, and
(iii) a TCR intracellular domain; and
(b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked; and wherein the TFP functionally interacts with a TCR when expressed in the T cell.
42. A recombinant nucleic acid encoding a T cell receptor (TCR) fusion protein (TFP)
comprising:
(a) a TCR-integrating subunit comprising
(i) at least a portion of a TCR extracellular domain, and
(ii) a transmembrane domain; and
(b) a MUC16 binding domain that does not comprise an antibody or antigen binding fragment thereof; wherein the TCR-integrating subunit and the MUC16 binding domain are operatively linked; and wherein the TFP functionally interacts with a TCR when expressed in the T cell.
43. The recombinant nucleic acid of claim 42, wherein the TFP further comprises an intracellular domain.
44. The recombinant nucleic acid of any one of claims 41-43, wherein the MUC16 binding domain specifically binds membrane-bound MUC16.
45. The recombinant nucleic acid of claim 44, wherein the MUC16 binding domain specifically binds membrane-bound MUC16 in the presence of soluble MUC16.
46. The recombinant nucleic acid of any one of claims 41-45, wherein the sequence encoding the MUC16 binding domain is connected to the sequence encoding the TCR extracellular domain by a sequence encoding a linker.
47. The recombinant nucleic acid of claim 46, wherein the linker comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 4.
48. The recombinant nucleic acid of any one of claims 41-47, wherein the MUC16 binding domain comprises mesothelin or a fragment thereof.
49. The recombinant nucleic acid of claim 48, wherein the MUC16 binding domain comprises the functional MUC16 binding domain of mesothelin.
50. The recombinant nucleic acid of claim 49, wherein the functional MUC16 binding domain of mesothelin comprises an amino acid sequence of
E VEKT ACP S GKK AREIDESLIF YKKWELE AC VD AALL AT QMDRVN AIPF T YEQLD V LKHKLDEL (SEQ ID NO: 1), or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity thereto.
51. The recombinant nucleic acid of claim 49, wherein the functional MUC16 binding domain of mesothelin comprises a truncation of SEQ ID NO: 1 by at least 1, at least 2, or at least 3 amino acids at the N- or C-terminus or at both the N- and C-terminus.
52. The recombinant nucleic acid of any one of claims 41-51, wherein the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion
thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma
TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
53. The recombinant nucleic acid of any one of claims 41-52, wherein the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
54. The recombinant nucleic acid of any one of claims 41-53, wherein the TCR intracellular domain comprises an intracellular domain of TCR alpha, TCR beta, TCR delta, or TCR gamma.
55. The recombinant nucleic acid of claim 54, wherein the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon, or an amino acid sequence having at least one modification thereto.
56. The recombinant nucleic acid of any one of claims 41-55, wherein the TCR-integrating subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit.
57. The recombinant nucleic acid of any one of claims 41-56, wherein the TCR-integrating subunit is derived only from CD3 epsilon.
58. The recombinant nucleic acid of any one of claims 41-56, wherein the TCR-integrating subunit is derived only from CD3 gamma.
59. The recombinant nucleic acid of any one of claims 41-56, wherein the TCR-integrating subunit is derived only from CD3 delta.
60. The recombinant nucleic acid of any one of claims 41-56, wherein the TCR-integrating subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4- IBB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto.
61. The recombinant nucleic acid of any one of claims 41-60, further comprising a sequence encoding a costimulatory domain.
62. The recombinant nucleic acid of claim 61, wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
63. The recombinant nucleic acid of any one of claims 41-62, wherein the TFP includes an immunoreceptor tyrosine-based activation motif (IT AM) of a TCR subunit that comprises an IT AM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit,
TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b 1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta receptor 1 chain, TYROBP (DAP 12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications thereto.
64. The recombinant nucleic acid of claim 63, wherein the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon.
65. The recombinant nucleic acid of claim 63, wherein the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit.
66. The recombinant nucleic acid of any one of claims 41-65, further comprising a leader sequence.
67. The recombinant nucleic acid of any one of claims 41-66, wherein the nucleic acid is selected from the group consisting of a DNA and a RNA.
68. The recombinant nucleic acid of claim 67, wherein the nucleic acid is a mRNA.
69. The recombinant nucleic acid of claim 67, wherein the nucleic acid is a circRNA.
70. The recombinant nucleic acid of any one of claims 41-69, wherein the nucleic acid comprises a nucleotide analog.
71. The recombinant nucleic acid of claim 70, wherein the nucleotide analog is selected from the group consisting of 2’ -O-methyl, 2’-O-methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, T-deoxy-2’-fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O-dimethylaminoethyl (2’-O-
DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), T-O-dimethylaminoethyloxy ethyl ( 2’ -
O-DMAEOE), 2’-O-N-methylacetamido (2’-O-NMA) modified, a locked nucleic acid
(LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’-phosphoramidite.
72. The recombinant nucleic acid of any one of claims 41-71, further comprising a promoter.
73. The recombinant nucleic acid of any one of claims 41-72, wherein the nucleic acid is an in vitro transcribed nucleic acid.
74. The recombinant nucleic acid of any one of claims 41-73, wherein the nucleic acid further comprises a sequence encoding a poly(A) tail.
75. The recombinant nucleic acid of any one of claims 41-74, wherein the nucleic acid further comprises a 3’UTR sequence.
76. A recombinant polypeptide molecule encoded by the recombinant nucleic acid of any one of claims 41-75.
77. A vector comprising a recombinant nucleic acid encoding a TFP of any one of claims 41-75.
78. The vector of claim 77, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
79. The vector of claim 77 or 78, further comprising a promoter.
80. The vector of any one of claims 77-79, wherein the vector is an in vitro transcribed vector.
81. The vector of any one of claims 77-80, wherein a nucleic acid sequence in the vector further comprises a poly(A) tail.
82. The vector of any one of claims 77-81, wherein a nucleic acid sequence in the vector further comprises a 3’UTR.
83. A cell comprising the recombinant nucleic acid molecule of any one of claims 41-75, the polypeptide molecule of claim 76, or the vector of any one of claims 77-82.
84. The cell of claim 83, wherein the cell is a human T-cell.
85. The cell of claim 84, wherein the T-cell is a CD8+ or CD4+ T-cell.
86. The cell of claim 84, wherein the T cell is a human alpha beta (ab or αβ) T cell.
87. The cell of claim 84, wherein the T cell is a human gamma delta (gd or γδ) T cell.
88. The cell of any one of claims 83-87, further comprising a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain.
89. The cell of claim 88, wherein the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain.
90. A protein complex comprising
(a) a TFP encoded by the recombinant nucleic acid molecule of any one of claims 39- 71, and
(b) at least one endogenous TCR subunit or endogenous TCR complex.
91. A human CD8+ or CD4+ T-cell comprising at least two different TFP proteins per the protein complex of claim 90.
92. A human CD8+ or CD4+ T-cell comprising at least two different TFP molecules encoded by the recombinant nucleic acid of any one of claims 41-75.
93. A population of human CD8+ or CD4+ T-cells, wherein the T-cells of the population individually or collectively comprise at least two TFP molecules encoded by the recombinant nucleic acid of any one of claims 41-75.
94. A method of making a cell comprising transducing a T-cell with the recombinant nucleic acid of any one of claims 41-75 or the vector of any one of claims 77-82.
95. A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule of any one of claims 1-40.
96. A method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of the pharmaceutical composition of claims 1-40, the recombinant nucleic acid of any one of claims 41-75, the polypeptide molecule of claim 76, the vector of claims 77-82, or the cell of any one of claims 83-89 and 91-93.
97. The method of claim 96, wherein the cell is an autologous T-cell.
98. The method of claim 96, wherein the cell is an allogeneic T-cell.
99. The method of any one of claims 96-98, wherein the mammal is a human.
100. A method of treating a mammal having a disease associated with expression of MUC16
comprising administering to the mammal an effective amount of the pharmaceutical composition of claims 1-40, the recombinant nucleic acid of any one of claims 41-75, the polypeptide molecule of claim 76, the vector of claims 77-82, or the cell of any one of claims 83-89 and 91-93.
101. The method of claim 100, wherein the disease associated with MUC16 expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, myelodysplasia, a myelodysplastic syndrome, a preleukemia, a non-cancer related indication associated with expression of MUC16.
102. The method of claim 101, wherein the disease is pancreatic cancer, ovarian cancer, breast cancer, or any combination thereof.
103. The method of any one of claims 96-102, wherein the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule.
104. The method of any one of claims 96-103, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti- MUC16 chimeric antigen receptor (CAR).
105. The method of any one of claims 96-104, wherein the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule.
106. The method of any one of claims 96-105, wherein the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with MUC16.
107. The pharmaceutical composition of any one of claims 1-40, the recombinant nucleic acid of any one of claims 41-75, the polypeptide molecule of claim 76, the vector of claims 77-82, or the cell of any one of claims 83-89 and 91-93, for use as a medicament.
108. A method of treating a mammal having a disease associated with expression of MUC16 comprising administering to the mammal an effective amount of the pharmaceutical composition of claims 1-40, the recombinant nucleic acid of any one of claims 41-75, the polypeptide molecule of claim 76, the vector of claims 77-82, or the cell of any one of claims 83-89 and 91-93, wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-MUC16 chimeric antigen receptor (CAR).
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US11965012B2 (en) | 2015-05-18 | 2024-04-23 | TCR2 Therapeutics Inc. | Compositions and methods for TCR reprogramming using fusion proteins |
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