US20190247500A1 - Use of Triplex CMV Vaccine in CAR T Cell Therapy - Google Patents

Use of Triplex CMV Vaccine in CAR T Cell Therapy Download PDF

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US20190247500A1
US20190247500A1 US16/343,701 US201716343701A US2019247500A1 US 20190247500 A1 US20190247500 A1 US 20190247500A1 US 201716343701 A US201716343701 A US 201716343701A US 2019247500 A1 US2019247500 A1 US 2019247500A1
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Don J. Diamond
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City of Hope
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Definitions

  • Tumor-specific T cell based immunotherapies including therapies employing engineered T cells, have been investigated for anti-tumor treatment.
  • the T cells used in such therapies do not remain active in vivo for a long enough period.
  • non-Hodgkin lymphoma NBL
  • NHL non-Hodgkin lymphoma
  • Efforts to improve the survival of patients with recurrent lymphoma have focused mainly on the use of autologous hematopoietic cell transplant (HCT), which is curative in approximately half of good-risk patients, but confers a less than 15% 5-year event-free survival in patients with poor prognostic features.
  • HCT autologous hematopoietic cell transplant
  • Allogeneic HCT provides a tumor-free stem cell graft, cells that have not been damaged by prior chemotherapy and the opportunity for graft-versus-lymphoma (GVL) effect, and has been increasingly applied in patients with relapsed NHL.
  • VDL graft-versus-lymphoma
  • allogeneic HCT is associated with both significant risks of transplant-related complications and also disease recurrence.
  • new therapies that can consolidate the tumor cytoreduction achieved with autologous or allogeneic HCT by eradicating the limited number of tumor cells surviving after autologous myeloablative and reduced intensity allogeneic conditioning.
  • cytomegalovirus (CMV) Triplex Vaccine in combination with engineered T cells that both recognize a CMV antigen and express a chimeric antigen receptor target to an antigen expressed on normal B cells as well as on cancerous cells (CMV/CAR T cells) to treat a variety of cancers.
  • the methods entail administering CMV/CAR T cells which recognize a tumor antigen (e.g., CD19) in addition to a CMV antigen to a patient.
  • a CMV Triplex Vaccine is administered to the patient.
  • the vaccine can promote proliferation of the CMV/CAR T cells and enhance their anti-tumor activity.
  • the methods can improve T cell resistance and provide a means by which to re- stimulate CAR T cells after relapse.
  • the methods can provide more reliable engraftment and persistence in a low target-antigen setting (e.g., post-myeloablative HCT) with re-expansion of CAR T cells by CMV vaccine administration.
  • the methods described herein also permit in vivo expansion of CMV-specific CAR T cells, instead of or in addition to ex vivo expansion, avoiding excessive T cell exhaustion that results in some cases from ex vivo manufacturing.
  • the CMV/CAR T cells can be prepared by a method comprising: (a) providing PBMC from a cytomegalovirus (CMV)-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen (e.g., pp65 or a mixture of IE1/IE2 overlapping peptides); (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV (e.g., treating them to create a population of cells that is enriched for stimulated cells specific for CMV relative to the untreated population of cells); (d) transducing at least a portion of the enriched population of cells with a vector (e.g., a lentiviral vector) expressing a CAR, thereby preparing T cells specific for CMV and expressing a CAR.
  • a CMV vaccine for example, the CMV Triplex Vaccine, can be administered to the donor prior to harvest of the PBMC in order to increase the frequency of CMV
  • the CMV Triplex Vaccine is a recombinant MVA expressing a fusion protein of two CMV antigens, IE1-exon4 and IE2-exon5 and CMV antigen pp65.
  • the sequence encoding the fusion protein is inserted in the MVA deletion-II locus and the sequence encoding the CMV pp65 antigen is inserted into the MVA deletion-III locus.
  • the CMV Triplex Vaccine is described in greater detail in U.S. Pat. No. 8,580,276, hereby incorporated by reference.
  • the methods described herein include: a method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) either prior to or subsequent to administering the composition comprising a population of T cells to the patient.
  • CAR chimeric antigen receptor
  • CMV cytomegalovirus
  • Described herein is a method for treating a patient comprising: (a) providing a composition comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; (b) administering the composition to the patient; and (c) administering to the patient a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) either prior to or subsequent to administering the composition comprising a population of T cells to the patient.
  • CAR chimeric antigen receptor
  • CMV cytomegalovirus
  • the step of administering a viral vector to the patient comprises administering recombinant MVA virus; expression of (i) CMV pp65 and (ii) the fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) is under the control of mH5 promoter; the patient is immunocompromised; the patient is immunocompetent; the patient is CMV-seronegative prior to treatment; the patient is CMV-seropositive prior to treatment; the patient received hematopoietic stem cells (HSC) from a CMV-positive or CMV-negative donor prior to administering the comprising a population of T cells expressing both a chimeric antigen receptor (CAR) and a T cell receptor specific for a cytomegalovirus (CMV) antigen; and the CAR is targeted to CD19; administration of the viral vector occurs at least 5 days after treatment with the composition comprising a population of T cells; the viral vector is administered to the patient
  • the CAR is selective can be selective for any antigen, for example: CD19, CS1, CD123, 5T4, 8H9, ⁇ v ⁇ 6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor- ⁇ , G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high mole
  • the CAR is selective for an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13 ⁇ 2, PSA, HER-2, EGFRv3, CEA, and C7R;
  • the CAR comprises: a scFv selective for the selected non-CMV antigen; a hinge/linker region; a transmembrane domain; a co-signaling domain; and CD3 ⁇ signaling domain;
  • the co-signaling domain is selected from a CD28 co-signaling domain and a 4-IBB co-signaling domain;
  • transmembrane domain is selected from a CD28 transmembrane domain and a CD4 transmembrane domain.
  • the population of human T cells is autologous to the patient; the population of human T cells is allogeneic to the patient; the method reduces the risk of CMV infection; the method reduces CMV viremia and/or disease; the patient was CMV-immune prior to treatment and the method reduces the risk of CMV infection; the patient was not CMV-immune prior to treatment and the method reduces CMV viremia or disease.
  • the step of providing a population of T cells expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) providing PBMC or a T cell subpopulation from a CMV-seropositive human donor; (b) exposing the PBMC or the T cell subpopulation to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR.
  • the step of treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV comprises treating the stimulated cells to produce a population of cells enriched for cells expressing an activation marker.
  • the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a human donor to convert a CMV-seronegative human donor to one containing T cells responsive to CMV antigens pp65, IE1 and IE2; (b) obtaining PBMC from the CMV-seropositive human donor; (c) exposing the PBMC to at least one CMV antigen; (d) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (e) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for
  • the step of providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen comprises: (a) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.
  • the viral vector is administered to the patient or the hematopoietic stem cell transplant donor at least twice subsequent to the administration of the composition comprising a population of T cells, the hematopoietic stem cells were autologous to the patient; and the hematopoietic stem cells were allogenic to the patient.
  • Also described is a method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen comprising: (a)) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.
  • Also described is a method for preparing T cells expressing a CAR and a T cell receptor specific for a CMV antigen comprising: a)) administering a viral vector encoding: (i) CMV pp65 and (ii) a fusion protein comprising exon 4 of CMV protein IE1 (e4) and exon 5 of CMV protein IE2 (e5) to a CMV-positive human donor; (b) obtaining PBMC from the CMV-seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby providing a population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.
  • the method further comprises expanding the population of T cell expressing a CAR and a T cell receptor specific for a CMV antigen.
  • the activation marker is IFN- ⁇ or other activation marker such as CD137, CD107 or other cytokines;
  • the CMV antigen is pp65 protein or an antigenic portion thereof;
  • the CMV antigen comprises two or more different antigenic CMV pp65 peptides;
  • the step of transducing the enriched population of cells does not comprise CD3 stimulation;
  • the step of transducing the enriched population of cells does not comprise CD28 stimulation;
  • the step of transducing the enriched population of cells does not comprise CD28 stimulation or CD3 stimulation;
  • the step of transducing the enriched population of cells does not comprise exposing the cells to an anti-CD28 antibody or an anti-CD3 antibody;
  • the enriched population of cells is at least 40% IFN- ⁇ positive, at least 20% CD8 positive, and at least 20% CD4 positive;
  • the enriched population of cells are cultured for fewer than 10 days prior to the step of transducing the enriched population of cells with a vector encoding a CAR;
  • the method
  • the method includes a step of preparing T cells specific for cytomegalovirus (CMV) and expressing a chimeric antigen receptor (CAR), the method comprising: (a) providing T cells (e.g., PBMC) from a cytomegalovirus CMV seropositive human donor; (b) exposing the PBMC to at least one CMV antigen; (c) treating the exposed cells to produce a population of cells enriched for stimulated cells specific for CMV; (d) transducing at least a portion of the enriched population of cells with a vector expressing a CAR, thereby preparing T cells specific for CMV and expressing a CAR.
  • T cells e.g., PBMC
  • the step of treating the exposed cells (e.g., using a selection step) to produce a population of cells enriched for stimulated cells specific for CMV comprises treating the stimulated cells to produce a population of cells enriched for cells expressing an activation marker (e.g., IFN- ⁇ of IL-13);
  • the PBMC are cultured for less than 5 days (less than 4, 3, 2, 1 days) prior to exposure to the CMV antigen;
  • the cells are exposed to the CMV antigen for fewer than 3 days (fewer than 48 hrs, 36 hrs, 24 hrs) the CMV antigen is pp65 protein or an antigenic portion thereof;
  • the CMV antigen comprises two or more different antigenic CMV pp65 peptides;
  • the step of transducing the enriched population of cells does not comprise CD3 stimulation;
  • the step of transducing the enriched population of cells does not comprise CD28 stimulation;
  • the step of transducing the enriched population of cells does not comprise CD3 stimulation or CD28 stimulation;
  • the T cells are from a CMV positive donor and are exposed to a CMV antigen such as CMV pp65 or a mixture of CMV protein peptides (for example 10-20 amino acid peptides that are fragments of pp65) in the presence of IL-2 to create a population of stimulated cells.
  • a CMV antigen such as CMV pp65 or a mixture of CMV protein peptides (for example 10-20 amino acid peptides that are fragments of pp65) in the presence of IL-2 to create a population of stimulated cells.
  • the population of stimulated cells is treated to prepare a population of cells that express IFN- ⁇ .
  • the CMV/CAR T cells do not recognize an antigen from a second virus. For example, they do not recognize an Epstein-Barr virus antigen or an influenza virus antigen or an Adenovirus antigen.
  • the method further comprises expanding the CMV specific T cells expressing a CAR (CMV/CAR T cells) by exposing them an antigen that binds to the CAR.
  • the CMV/CAR T cells are not expanded ex vivo by exposure to an antigen that binds the CAR, by a CMV antigen or by exposure to exogenously added cytokines.
  • the step of expanding the CMV-specific T cells expressing a CAR comprises exposing the cells to T cells expressing the antigen that bind the CAR (e.g., the expansion takes place is the presence of at least one exogenously added interleukin (e.g., one or both of IL-1 and IL-15) and a T cell expressing the antigen recognized by the CAR.
  • the expansion takes place is the presence of at least one exogenously added interleukin (e.g., one or both of IL-1 and IL-15) and a T cell expressing the antigen recognized by the CAR.
  • the CAR is selective for an antigen selected from: CD19, CS1, CD123, 5T4, 8H9, ⁇ v ⁇ 6 integrin, alphafetoprotein (AFP), B7-H6, CA-125 carbonic anhydrase 9 (CA9), CD19, CD20, CD22, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD52, CD123, CD171, carcionoembryonic antigen (CEA), EGFrvIII, epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), ErbB1/EGFR, ErbB2/HER2/neu/EGFR2, ErbB3, ErbB4, epithelial tumor antigen (ETA), FBP, fetal acetylcholine receptor (AchR), folate receptor- ⁇ , G250/CAIX, ganglioside 2 (GD2), ganglioside 3 (GD3), HLA-A1, HLA-A2, high molecular
  • the CAR is selective for an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13 ⁇ 2, PSA, HER2, EGFRv3, CEA, and C7R.
  • the CAR comprises: a scFv selective for the selected non-CMV antigen; a hinge/linker region; a transmembrane domain; a co-signaling domain; and CD3 ⁇ signaling domain;
  • the chimeric antigen receptor further comprises a spacer sequence located between the co-signaling domain and the CD3 ⁇ signaling domain;
  • the co-signaling domain is selected from a CD28 co-signaling domain and a 4-IBB co-signaling domain;
  • the transmembrane domain is selected from a CD28 transmembrane domain and a CD4 transmembrane domain;
  • the vector expressing the CAR expresses a truncated human EGFR from the same transcript encoding the CAR, wherein the truncated human EGFR lacks a EGF ligand binding domain and lacks a cytoplasmic signaling domain;
  • the spacer sequence comprises or consists of 3-10 consecutive Gly;
  • the CMV/CAR T cell population is a population in which at least 20% of the cells in the population are CD4+, in which at least 20% of the cells in the population are CD8+, or in which at least 60% of the cells in the population are IFN ⁇ +.
  • the T cells are specific for CMV pp65; and the CAR binds an antigen selected from: CD19, CD123, CS1, BCMA, CD44v6, CD33, CD22, IL-13 ⁇ 2, PSA, HER2, EGFRv3, CEA, and C7R.
  • Also described is a method of treating a patient suffering from cancer comprising administering a composition comprising CMV/CAR T cells followed by administration of CMV Triplex Vaccine.
  • the population of human T cells are autologous to the patient; the population of human T cells are allogenic to the patient; the population of human T cells are autologous to the patient; the method further comprises administering to the patient at least two or at least three doses of a CMV Triplex Vaccine.
  • amino acid modification refers to an amino acid substitution, insertion, and/or deletion in a protein or peptide sequence.
  • An “amino acid substitution” or “substitution” refers to replacement of an amino acid at a particular position in a parent peptide or protein sequence with another amino acid.
  • a substitution can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping).
  • Amino acids with nonpolar R groups Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine
  • Amino acids with uncharged polar R groups Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamine
  • Amino acids with charged polar R groups negatively charged at pH 6.0: Aspartic acid, Glutamic acid
  • Basic amino acids positively charged at pH 6.0
  • Lysine, Arginine, Histidine at pH 6.0
  • Another grouping may be those amino acids with phenyl groups: Phenylalanine, Tryptophan, and Tyrosine.
  • FIGS. 1A-D depict the development of clinically feasible platform for derivation of CMV/CAR T cells and the schematic structure of a lentiviral vector expressing a CD19 CAR.
  • CMV-specific T cells from CMV immune HLA A2 donors were selected using IFN ⁇ capture after overnight stimulation with cGMP grade CMVpp65 protein. After selection, the cells were stained with antibodies specific to IFN ⁇ , CD4, and CD8. The frequency of each population is presented after exclusion of dead cells with DAPI.
  • B The selected cells were transduced with the second generation CD19CAR with a double mutation in the spacer, 24 hours after the IFN ⁇ capture.
  • CAR expression was defined by cetuximab-biotin and streptavidin (SA) APC-Cy7 staining. Percentages of CAR + cells are indicated in each histogram (filled gray), and based on subtraction of that stained with SA-APC-Cy7 alone (black line).
  • SA cetuximab-biotin and streptavidin
  • C Growth of total cell number was determined by Guava Viacount at different time points.
  • D Schematic diagram of 10039 nt lentiviral vector encoding a CD19 CAR.
  • CD19R:CD28:z(CO)-T2A-EGFRt construct Within the 3183 nucleotide long CD19R:CD28:z(CO)-T2A-EGFRt construct, the CD19-specific scFv, IgG4 Fc spacer, the CD28 transmembrane and cytoplasmic signaling domains, three-glycine linker, and CD3z cytoplasmic signaling domains of the CD19R:CD28:z(CO) CAR containing the 2 point mutations, L235E and N297Q, in the CH2 portion of the IgG4 spacer (CD19R(EQ)), as well as the T2A ribosome skip and truncated EGFR sequences are indicated.
  • the human GM-CSF receptor alpha signal sequences that drive surface translocation of the CD19R:CD28:z(CO) CAR and EGFRt are also indicated.
  • FIGS. 2A-2C depict the results of studies demonstrating that CMV/CAR T cells exhibit specific effector function after engagement with CD19 + and CMVpp65 + tumors.
  • A 7 days after the second CD19 Ag stimulation, T cells were stained with HLA A2 restricted pp65 tetramer, cetuximab-biotin, anti-CD8 and antibodies specific to central memory T cell surface markers. Percent positive cells are indicated after dead cell exclusion with DAPI, gating based on pp65 tetramer and cetuximab double-positivity, and isotype-matched stained samples.
  • CMV/CAR T cells (10 5 ) were activated overnight with 10 5 LCL-OKT3, LCL, or KG1a in 96-well tissue culture plates and 10 5 U251T and engineered pp65 expressing U251T cells (pp65U251T) in 24-well tissue culture plates. Supernatants were collected after overnight co-incubation of CMV/CAR T cells and stimulators. Cytokine levels with indicated stimulators (means ⁇ SEM of triplicate wells) were determined using cytometric bead array.
  • FIG. 2D depicts the results of studies examining cytokine levels in the serum of CMV/CAR T cell treated tumor bearing mice.
  • NSG mice were injected i.v. on day 0 with 2.5 ⁇ 106 GFPffluc+ LCL cells.
  • recipient mice were administered i.v. with 2 ⁇ 106 CMV/CAR cells that underwent 2 rounds of CD19 stimulation.
  • Vaccine was given by i.v. injection of peptide (pp65 or MP1) pulsed autologous T cells on day 14.
  • serum of recipient mice was collected and levels of human cytokines were determined by cytometric bead array. Cytokine levels in the serum of untreated mice was used as baseline. Mean and SEMs from triplicates are presented.
  • FIG. 3 depicts the mH5-IEfusion-pZWIIA (GUS) plasmid DNA sequence (SEQ ID NO: A.
  • FIG. 4 depicts the mH5-pp65-pLW51(GUS) plasmid DNA sequence (SEQ ID NO : B.
  • FIG. 5 depicts an IE1 antigen and the IE2 antigen fusion protein (SEQ ID NO:C).
  • T cells specific for CMV and CD19 are T cells specific for CMV and CD19. These CMV/CAR T cells were generated using a rapid and efficient method for generating and selecting CMV-specific T cells.
  • the method which employs IFN ⁇ capture of CMV-specific T cells, consistently and efficiently enriched CMV-specific T cells while preserving the broad spectrum of CMV repertoires.
  • the cells remained amenable to gene modification after a brief CMVpp65 stimulation, avoiding the need for CD3/CD28 bead activation prior to transduction. This is significant because CD3/CD28 activation can cause activation-induced cell death (AICD) of CMV-specific T cells.
  • AICD activation-induced cell death
  • CMV/CAR T cells described herein express a CAR targeted to CD19, the same methods can be used to generate CMV CAR T cells targeted to any desired antigen.
  • HLA human leukocyte antigen
  • CMV/CAR T cells can be enhanced as a consequence of proliferation following CMV peptide vaccination. This suggests that the cell dose of CMV/CAR T cells could be significantly decreased as compared to conventional CAR T cells, due to their potential to proliferate in vivo in response to vaccine, avoiding prolonged culture times and the risk of terminal differentiation.
  • the CMV/CAR T cells also express a truncated EGFR (EGFRt).
  • EGFRt truncated EGFR
  • Cells expressing EGFRt can be killed by administration of an antibody, such a cetuximab, targeted to EGFR. This permits control and reduction of potential on/off-target toxicity.
  • CMV Triplex Vaccine subsequent to treatment with CMV/CD19 CAR T cells can augment the antitumor activity of adoptively transferred CMV/CD19 CAR T cells in several scenarios: 1) to salvage patients not achieving complete remission or relapsing after CART cell therapy, 2) vaccine boost when CD19 CAR T cells are failing to persist regardless of tumor responses at that time, 3) planned vaccination on days post-administration CD19 CART cells.
  • MRD minimal residual disease
  • CMV Triplex Vaccine has the potential to profoundly impact the general field of adoptive T cell therapy, since by transducing a variety of tumor-directed CARs into CMV-specific T cells, it is possible to tailor this strategy to a wide range of malignancies and tumor targets.
  • CMV Triplex Vaccine is a recombinant MVA that expresses three CMV antigens, i.e., at least a portion or Immediate-Early Gene-1 (IE1), at least a portion of Immediate-Early Gene-2 (IE2) and at least a portion of pp65.
  • the IE1 antigen and the IE2 antigen can be expressed a fusion protein, for example, a protein encoded by the nucleotide sequence of SEQ ID NO: C.
  • Expression of the CMV antigens can be under the control of a modified H5 (mH5) promoter.
  • a CMV Triplex Vaccine is fully described in U.S. Pat. No. 8,580,276 and in Wang et al. ( Vaccine 28:1547, 2010)
  • the CMV Triplex Vaccine can express CMV pp65 and an CMV IE fusion protein (IEfusion).
  • the IEfusion can include an antigenic portion of IE1 (e.g., Exon 4) and an antigenic portion of IE2 (e.g., Exon 5), wherein the antigenic portions elicit an immune response when expressed by a vaccine.
  • the IEfusion is has the sequence encoded by SEQ ID NO: C or another nucleotide sequence that encodes the same amino acid sequence as SEQ ID NO: C.
  • the CMV Triplex Vaccine includes three of the best recognized antigens in the CD8 subset: pp65, IE1, and IE2. There is no region of homology greater than 5 amino acids between the major exons of both proteins. Individually, both antigens are recognized broadly by almost 70% of the general population (Sylwester et al. 2005). The divergent sequence of both IE1/e4 and IE2/e5 used here predicts an entirely different subset of HLA binding peptides using publicly available Class I and II motif algorithms (Peters and Sette 2007). Human subjects that were evaluated for recognition of both IE1 and IE2 antigens were found in many instances to recognize one or the other but not both.
  • the data also confirms that the IEfusion protein is processed and presented appropriately to stimulate existing T cell populations in a manner that maintains the phenotypic distribution as expected in the ex vivo analysis.
  • the most rigorous evaluation of the processing of the rMVA for T cell response is using PBMC from transplant patients. PBMC from HCT recipients in all three risk categories were evaluated and an equivalently strong recognition of both rMVAs was found.
  • the nucleic acid sequence encoding vaccinia mH5 promoter has a sequence containing nucleotides 3075-3168 of SEQ ID NO: A or 3022-3133 of SEQ ID NO: B
  • CAR include an extracellular antigen-binding domain (often a scFv derived from variable heavy and light chains of an antibody), a spacer domain, a transmembrane domain and an intracellular signaling domain.
  • the intracellular signaling domain usually includes the endodomain of a T cell co-stimulatory molecule (e.g., CD28, 4-1BB or OX-40) and the intracellular domain of CD3 ⁇ .
  • the CAR described herein can include a spacer region located between the cancer antigen targeting domain (e.g., a CD19 ScFv, e.g., the scFv portion can include the CD19 targeted scFv sequence of a CD19-targeted CAR such as that described in Wang et al. 2016 Blood 127:2980-2990) and the transmembrane domain.
  • a spacer region located between the cancer antigen targeting domain (e.g., a CD19 ScFv, e.g., the scFv portion can include the CD19 targeted scFv sequence of a CD19-targeted CAR such as that described in Wang et al. 2016 Blood 127:2980-2990) and the transmembrane domain.
  • spacer regions can be used. Some of them include at least portion of a human Fc region, for example a hinge portion of a human Fc region or a CH3 domain or variants thereof. Table 1 below provides various spacers that can
  • Some spacer regions include all or part of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CH1 and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge or a CD8 hinge.
  • Some spacer regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain.
  • the immunoglobulin derived sequences can include one ore more amino acid modifications, for example, 1, 2, 3, 4 or 5 substitutions, e.g., substitutions that reduce off-target binding.
  • the spacer region can also comprise a IgG4 hinge region having the sequence ESKYGPPCPSCP (SEQ ID NO:4) or ESKYGPPCPPCP (SEQ ID NO:3).
  • the spacer region can also comprise the sequence ESKYGPPCPPCP (SEQ ID NO:3) followed by the linker sequence GGGSSGGGSG (SEQ ID NO:2) followed by IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K (SEQ ID NO:12).
  • ESKYGPPCPPCP SEQ ID NO:3
  • linker sequence GGGSSGGGSG SEQ ID NO:2
  • IgG4 CH3 sequence GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG K (SEQ ID NO:12).
  • the entire spacer region can comprise the sequence: ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSLGK (SEQ ID NO:11).
  • the spacer has 1, 2, 3, 4 or 5 single amino acid changes (e.g., conservative changes) compared to those shown in Table 1.
  • the IgG4 Fc hinge/linker region that is mutated at two positions (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs).
  • transmembrane domains can be used in the.
  • Table 2 includes examples of suitable transmembrane domains. Where a spacer region is present, the transmembrane domain is located carboxy terminal to the spacer region.
  • the costimulatory domain can be any domain that is suitable for use with a CD3 ⁇ signaling domain.
  • the costimulatory domain is a CD28 costimulatory domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RSKRSR GG HSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO:23; LL to GG amino acid change double underlined).
  • the CD28 co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative and preferably not in the underlined GG sequence) compared to SEQ ID NO:23.
  • the co-signaling domain is a 4-1BB co-signaling domain that includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to:
  • the 4-1BB co-signaling domain has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:24.
  • the costimulatory domain(s) are located between the transmembrane domain and the CD3 ⁇ signaling domain.
  • Table 3 includes examples of suitable costimulatory domains together with the sequence of the CD3 ⁇ signaling domain.
  • the costimulatory domain is selected from the group consisting of: a costimulatory domain depicted in Table 3 or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a CD28 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications, a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications and an OX40 costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications.
  • a 4-1BB costimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications in present.
  • costimulatory domains there are two costimulatory domains, for example a CD28 co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions) and a 4-1BB co-stimulatory domain or a variant thereof having 1-5 (e.g., 1 or 2) amino acid modifications (e.g., substitutions).
  • the 1-5 (e.g., 1 or 2) amino acid modification are substitutions.
  • the costimulatory domain is amino terminal to the CD3 ⁇ signaling domain and in some cases a short linker consisting of 2-10, e.g., 3 amino acids (e.g., GGG) is positioned between the costimulatory domain and the CD3 ⁇ signaling domain.
  • the CD3 ⁇ Signaling domain can be any domain that is suitable for use with a CD3 ⁇ signaling domain.
  • the CD3 ⁇ signaling domain includes a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO:21).
  • the CD3 ⁇ signaling has 1, 2, 3, 4 of 5 amino acid changes (preferably conservative) compared to SEQ ID NO:21.
  • the CD3 ⁇ signaling domain can be followed by a ribosomal skip sequence (e.g., LEGGGEGRGSLLTCGDVEENPGPR; SEQ ID NO:27) and a truncated EGFR having a sequence that is at least 90%, at least 95%, at least 98% identical to or identical to: LVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHI LPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIR GRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRE CVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDG PHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYG
  • Example 1 Enrichment of CMV-Specific T Cells from PBMC of Healthy Donors after Stimulation with cGMP Grade CMVpp65 Protein
  • CMV-specific T cells were prepared from PBMC of healthy donors by stimulating the PBMC with cGMP grade CMVpp65 protein. Briefly, PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) from peripheral blood of consented healthy, HLA-A2 CMV-immune donors under a City of Hope Internal Review Board-approved protocol. PBMC were frozen for later use.
  • PBMC peripheral blood mononuclear cells
  • cGMP current good manufacturing practice
  • IFN ⁇ -positive T cells were consistently enriched from a baseline mean of 3.8% (range 1.8-5.6) to a post-capture mean of 71.8% (range 61-81) and contained polyclonal CD8 + (34%) and CD4 + T cells (37%) after selection ( FIG. 1A and FIG. 1C ).
  • the selected CMV-specific T cells included both CD4 and CD8 subsets and represented the entire spectrum of CMV-specificity, showing responsiveness to CMVpp65 pepmix stimulation with broad recognition.
  • IFN ⁇ -captured CMV-specific T cells were transduced 2 days after the selection, without OKT3 activation, using the second generation CD19RCD28EGFRt lentiviral construct containing the IgG4 Fc hinge region mutations (L235E; N297Q) that improve potency due to distortion of the FcR binding domain.
  • the complete amino sequence of the this CD19 CAR is depicted in FIG. 3 . Starting seven days post lenti-transduction, the cells were stimulated on a weekly basis with 8000 cGy-irradiated, CD19-expressing NIH3T3 cells at a 1:10 ratio (T cells: CD19NIH 3T3).
  • the percentage of CAR + cells detected by cetuximab increased from 8% post transduction to 46% after 2 rounds of stimulation with a 120-150-fold total cell increase ( FIG. 1B and FIG. 1D ). Further details regarding the lentiviral construct, the CD19-expressing NIH3T3 cells and other materials and techniques used in the studies described herein are presented below.
  • the expanded CMV/CAR T cells specifically lysed CD19 + LCLs with the same maximum killing levels as the OKT3-expressing LCL used as positive controls.
  • specific killing was also observed when pp65U251T cells were used as targets as compared to parental U251T cells ( FIG. 2B ).
  • IFN ⁇ secretion was observed after either CD19 or pp65 antigen stimulation as compared to antigen-negative stimulators such as KG1a and U251T parental cells ( FIG. 2C ).
  • Antibodies and Flow Cytometry Fluorochrome-conjugated isotype controls, anti-CD3, anti-CD4, anti-CD8, anti-CD28, anti-CD45, anti-CD27, anti-CD62L, anti-CD127, anti-IFN ⁇ , and streptavidin were obtained from BD Biosciences. Biotinylated cetuximab was generated from cetuximab purchased from the City of Hope pharmacy. The IFN- ⁇ Secretion Assay—Cell Enrichment and Detection Kit and CMVpp65 protein were purchased from Miltenyi Biotec (Miltenyi Biotec, Germany).
  • PE-conjugated CMV pp65 NLVPMVATV-HLA-A2*0201 iTAg MHC tetramer
  • PE-conjugated multi-allele negative tetramer was obtained from Beckman Coulter (Fullerton, Calif.).
  • Carboxyfluorescein diacetate succinimidyl ester (CFSE) was purchased from Invitrogen (Carlsbad, Calif.). All monoclonal antibodies, tetramers and CFSE were used according to the manufacturer's instructions.
  • Flow cytometry data acquisition was performed on a MACSQuant (Miltenyi Biotec, Germany) or FACScalibur (BD Biosciences), and the percentage of cells in a region of analysis was calculated using FCS Express V3 (De Novo Software).
  • EBV-transformed lymphoblastoid cell lines were made from peripheral blood mononuclear cells (PBMC) as previously described (16).
  • PBMC peripheral blood mononuclear cells
  • LCL-OKT3 allogeneic LCLs were resuspended in nucleofection solution using the Amaxa Nucleofector kit T, OKT3-2A-Hygromycin_pEK plasmid was added to 5 ⁇ g/107 cells, the cells were electroporated using the Amaxa Nucleofector I, and the resulting cells were grown in RPMI 1640 with 10% FCS containing 0.4 mg/ml hygromycin.
  • LCLs were transduced with lentiviral vector encoding eGFP-ffluc. Initial transduction efficiency was 48.5%, so the GFP+ cells were sorted by FACS for >98% purity.
  • parental NIH3T3 cells ATCC
  • the established cell line was further engineered to express CD19GFP by lentiviral transduction.
  • GFP+ cells were purified by FACS sorting and expanded for the use of stimulation of CMV/CAR T cells.
  • pp65 stimulator cells To generate pp65 stimulator cells, U251T cells derived from human glioblastoma cells from an HLA A2 donor (ATCC) were transduced with a lentiviral vector encoding full length pp65 fused to green fluorescent protein (GFP). pp65U251T cells were purified by GFP expression using flow cytometry. Banks of all cell lines were authenticated for the desired antigen/marker expression by flow cytometry prior to cryopreservation, and thawed cells were cultured for less than 6 months prior to use in assays.
  • ATC human glioblastoma cells from an HLA A2 donor
  • GFP green fluorescent protein
  • pp65 peptide NLVPMVATV HLA-A 0201 CMVpp65
  • MP1 GIGFVFTL peptide HLA-A 0201 influenza
  • pepMix HCMVA pp65 was purchased from JPT peptide Technologies (GmbH, Berlin Germany). All peptides were used according to the manufacturer's instructions.
  • Lentivirus vector construction The lentivirus CAR construct was modified from the previously described CD19-specific scFvFc: ⁇ chimeric immunoreceptor(18), to create a third-generation vector.
  • the CD19CAR containing a CD28 ⁇ co-stimulatory domain carries mutations at two sites (L235E; N297Q) within the CH2 region on the IgG4-Fc spacers to ensure enhanced potency and persistence after adoptive transfer ( FIG. 7 ).
  • the lentiviral vector also expressed a truncated human epidermal growth factor receptor (huEGFRt), which includes a cetuximab (ErbituxTM) binding domain but excludes the EGF-ligand binding and cytoplasmic signaling domains.
  • huEGFRt human epidermal growth factor receptor
  • a T2A ribosome skip sequence links the codon-optimized CD19R:CD28: ⁇ sequence to the huEGFRt sequence, resulting in coordinate expression of both CD19R:CD28: ⁇ and EGFRt from a single transcript (CD19CARCD28EGFRt) (19).
  • the CD19RCD28EGFRt DNA sequence (optimized by GeneArt) was then cloned into a self-inactivating (SIN) lentiviral vector pHIV7 (gift from Jiing-Kuan Yee, Beckman Research Institute of City of Hope) in which the CMV promoter was replaced by the EF-1 ⁇ promoter.
  • PBMCs were isolated by density gradient centrifugation over Ficoll-Paque (Pharmacia Biotech, Piscataway, N.J.) from peripheral blood of consented healthy, HLA-A2 CMV-immune donors under a City of Hope Internal Review Board-approved protocol. PBMC were frozen for later use.
  • PBMC peripheral blood mononuclear cells
  • cGMP current good manufacturing practice
  • CMVpp65 protein Miltenyi Biotec, Germany
  • RPMI 1640 Irvine Scientific, Santa Ana, Calif.
  • 2 mM L-glutamine Irvine Scientific
  • 25 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid HPES, Irvine Scientific
  • penicillin 0.1 mg/mL streptomycin (Irvine Scientific) in the presence of 5 U/ml IL-2 and 10% human AB serum.
  • CMV-specific T cells were selected using the IFN ⁇ capture (Miltenyi Biotec, Germany) technique according to the manufacturer's instructions.
  • CMV/CAR T cells The selected CMV-specific T cells were transduced on day 2 post IFN ⁇ capture with lentiviral vector expressing CD19CARCD28EGFRt at MOI 3. Seven to ten days after lenti-transduction, the CMV/CAR T cells were expanded by stimulation through CAR-mediated activation signals using 8000 cGy-irradiated CD19-expressing NIH 3T3 cells at a 10:1 ratio (T cells:CD19 NIH3T3) once a week as described (17) in the presence of IL-2 50 U/m1 and IL-15 1 ng/ml. After 2 rounds of expansion, the growth and functionality of the CMV/CAR T cells was evaluated in vitro and in vivo.
  • CMV/CAR T cells (10 5 ) were activated overnight with 105 LCL-OKT3, LCL, or KG1a cells in 96-well tissue culture plates, and with 10 5 U251T and engineered pp65-expressing U251T cells (pp65U251T) in 24-well tissue culture plates in the presence of Brefeldin A (BD Biosciences). The cell mixture was then stained using anti-CD8, cetuximab and streptavidin, and pp65Tetramer to analyze surface co-expression of CD8, CAR and CMV-specific TCR, respectively. Cells were then fixed and permeabilized using the BD Cytofix/Cytoperm kit (BD Biosciences). After fixation, the T cells were stained with an anti-IFN ⁇ .
  • BD Biosciences BD Cytofix/Cytoperm kit
  • CFSE Proliferation assays CMV/CAR T cells were labeled with 0.5 ⁇ M CFSE and co-cultured with stimulator cells LCL-OKT3, LCLs, and pp65 U251T for 8 days. Co-cultures with U251T and KG1a cells were used as negative controls. Proliferation of CD3- and CAR-positive populations was determined using multicolor flow cytometry.
  • Cytokine production assays T cells (10 5 ) were co-cultured overnight in 96-well tissue culture plates with 105 LCL-OKT3, LCL, or KG1a cells and in 24-well tissue culture plates with 105 U251T and engineered pp65-expressing U251T cells. Supernatants were then analyzed by cytometric bead array using the Bio-Plex Human Cytokine 17-Plex Panel (Bio-Rad Laboratories) according to the manufacturer's instructions.
  • Cytotoxicity assays 4-hour chromium-release assays (CRA) were performed as previously described (20) using effector cells that had been harvested directly after 2 rounds of CD19 Ag stimulations.
  • CRA chromium-release assays

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EP3589295A4 (fr) 2017-02-28 2020-11-04 Endocyte, Inc. Compositions et méthodes de thérapie par lymphocytes t car
JP7417542B2 (ja) 2018-01-22 2024-01-18 シアトル チルドレンズ ホスピタル （ディービーエイ シアトル チルドレンズ リサーチ インスティテュート） Ｃａｒ ｔ細胞の使用方法
CN109293773B (zh) * 2018-09-25 2020-09-04 上海邦耀生物科技有限公司 靶向cd38蛋白的抗体、嵌合抗原受体和药物
CN111544585A (zh) * 2019-02-11 2020-08-18 北京卡替医疗技术有限公司 一种可助推免疫细胞在体内扩增的佐剂
EP3941491A4 (fr) 2019-03-21 2023-03-29 Gigamune, Inc. Cellules modifiées exprimant des récepteurs de lymphocytes t anti-viraux et leurs méthodes d'utilisation
CN112239505B (zh) * 2020-10-13 2022-05-20 上海良润生物医药科技有限公司 重组抗cd171八价抗体及神经来源外泌体的捕获方法

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