A TARGETING MODULE COMPRISING PD-L1 AND/OR PD-L2 FOR USE IN A METHOD FOR STIMULATING A CHIMERIC ANTIGEN RECEPTOR MEDIATED IMMUNE RESPONSE IN A MAMMAL

Abstract

The present invention relates to a targeting module comprising at least one PD-L1 and/or PD-L2 binding domain, and a tag-binding domain or a tag for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, a nucleic acid, a vector or a cell comprising a nucleotide sequence encoding the targeting module, a pharmaceutical composition and a kit comprising the targeting module and a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor.

Claims

1. A targeting module comprising i) at least one PD-L1 and/or PD-L2 binding domain, ii) a tag-binding domain or a tag, for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, wherein the targeting module is administered in combination with a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or a tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

2. The targeting module according to claim 1 is monovalent or bivalent.

3. The targeting module according to claim 1 or 2, wherein the PD-L1 and/or PD-L2 binding domain is BMS-936559, Atezolizumab, Durvalumab, Avelumab, 3F2.1 or a fragment thereof.

4. The targeting module according to one of the claims 1 to 3, wherein the tag is a peptide epitope tag.

5. The targeting module according to claim 4, wherein the peptide epitope tag is a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, a leucine zipper sequence or a short linear peptide sequence from a human nuclear protein, preferably from the human La protein.

6. The targeting module according to one of the claims 1 to 5 further comprising an effector cell binding domain, wherein the effector cell binding domain specifically binds an epitope on a human CD3 chain, a human T cell receptor (TCR) or human CD16.

7. The targeting module according to one of the claims 1 to 6, wherein the variable regions of the at least one PD-L1 and/or PD-L2 binding domain and/or the effector cell binding domain comprise a humanized amino acid sequence.

8. The targeting module according to one of the claims 1 to 7, wherein the length is in the range of 20 to 1600 amino acids.

9. The targeting module according to one of the claims 1 to 8 for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, wherein the targeting module is administered in combination with a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor and at least one further targeting module, wherein the at least one further targeting module comprises at least one target cell binding domain and a tag-binding domain or a tag, wherein the at least one target cell binding domain is an antibody, antibody fragment, a protein, a peptide or a low molecular weight organic ligand that binds to surface antigens selected from the group comprising CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD25, CD30, CD33, CD38, CD44, CD44v6 CD52, CD90, CD99, CD123, CD133, CD150 CD181, CD182, CD184, CD223, CD229, CD269 (BCMA), CD273, CD274, CD276, CD279, CD319, CD366 and CD371, interleukin receptors, especially preferred IL-8Rα (CXCR1), IL-8Rβ (CXCR2), IL-11Rα, IL-11Rβ, IL-13Rα1 and 2, CXCR4, c-Met, mesothelin, members of the epidermal growth factor receptor family and mutants thereof, especially preferred ErbB1, ErbB2, ErbB3, ErbB4 or mutants thereof, members of the tumor necrosis factor receptor superfamily, ephrins, ephrin receptors, especially preferred EphA1-10, EphA5 or EphB1-6, prostate specific antigens prostate stem cell antigen (PSCA) and prostate specific membrane antigen (PSMA), embryonic antigens carcinoembryonic antigen (CEA) and fetal acethylcholine receptor, members of the vascular endothelia growth factor family, epithelia cell adhesion molecule EpCAM, alphafetoprotein AFP, members of the intercellular adhesion molecule family, members of the mucin protein family, follicle stimulating hormone receptor (FSHR), the human high molecular weight-melanoma-associated antigen (HMW-MAA), folate binding protein FBP, folate receptors, somatostatin receptors, ligands of the NKG2D receptor, cytokine receptors, members of the epithelia glycoprotein family, diasialogangliosides, glypicans, G protein-coupled receptors, members of the carbonic anhydrase family, members of the carbohydrate antigen family, Notch ligands, melanoma-associated chondroitin sulfate proteoglycan (MCSP), glycoprotein A33, guanylatecyclase 2C and tumor-specific glycans, including mutants and analogues of the named antibodies, antibody fragments, proteins, peptides or low molecular weight organic ligands, wherein the targeting module according to one of the claims 1 to 8 and the at least one further targeting module comprise different target cell binding domains, and identical tag-binding domains or a tags.

10. A nucleic acid, a vector or a cell comprising a nucleotide sequence encoding a targeting module according to one of the claims 1 to 9 for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, wherein the nucleic acid, vector or cell is administered in combination with a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or a tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

11. The nucleic acid, vector or cell according to claim 10 further comprising an inducible expression system, preferably selected from Tet-On, Tet-On Advanced, Tet-On 3G, T-REx or a promoter responsive to nuclear factor of activated T cells (NFAT).

12. The nucleic acid, vector or cell according to claim 10 or 11 further comprising a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

13. A nucleic acid, a vector or a cell comprising an inducible expression system, preferably selected from Tet-On, Tet-On Advanced, Tet-On 3G, TREx or a promoter responsive to NFAT, and a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, and a nucleotide sequence encoding a targeting module according to one of the claims 1 to 9, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

14. A pharmaceutical composition comprising the targeting module according to one of the claims 1 to 9, the nucleic acid, vector or cell according to one of the claims 10 to 13 for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, wherein the pharmaceutical composition is administered in combination with a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or a tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

15. A kit comprising a) a targeting module according to one of the claims 1 to 9, the nucleic acid, vector or cell according to one of the claims 10 to 13 and b) a vector or a cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor, wherein the switchable chimeric antigen receptor comprises three domains, wherein the first domain is a tag-binding domain or tag, the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, wherein the tag-binding domain of the targeting module binds to the tag of the switchable chimeric antigen receptor or the tag of the targeting module binds to the tag-binding domain of the switchable chimeric antigen receptor.

16. The kit according to claim 15, wherein the tag is a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, a leucine zipper sequence or a short linear peptide sequence from a human nuclear protein, preferably from the human La protein.

17. The kit according to claim 15 or 16, wherein the tag-binding domain is an antibody or an antibody fragment binding to a myc-tag, a His-tag, a short linear peptide sequence from yeast transcription factor GCN4, a leucine zipper sequence or a short linear peptide sequence from a human nuclear protein, preferably from a human La protein.

18. The kit according to one of the claims 15 to 17, wherein the extracellular hinge and transmembrane domain is selected from hinge and transmembrane domains of human CD28 molecule, CD8a chain NK cell receptors, preferably natural killer group NKG2D; or parts of the constant region of an antibody and combinations thereof.

19. The kit according to one of the claims 15 to 18, wherein the signal transduction domain is selected from cytoplasmic regions of CD28, CD137 (4-1BB), CD134 (OX40), CD278 (ICOS), DAP10 and CD27, programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), cytoplasmic regions of CD3 chains, DAP12, CD122 (interleukin-2 receptor β), CD132 (interleukin-2 receptor γ), CD127 (interleukin-7 receptor α), CD360 (interleukin-21 receptor), activating Fc receptors and mutants thereof.

20. The kit according to one of the claims 15 to 19, wherein the targeting module and/or the vector or cell comprising a nucleotide sequence encoding a switchable chimeric antigen receptor are in the form of a pharmaceutical composition.

21. The kit according to one of the claims 15 to 20 for use in a method for stimulating a chimeric antigen receptor mediated immune response in a mammal, preferably for use in the treatment of cancer, infectious disease or autoimmune disease.

Description

[0300] FIG. 1 shows a schema of a switchable chimeric antigen receptor (CAR) with three domains, wherein the first domain is a tag-binding domain or a tag (exemplified as scFv), the second domain is an extracellular hinge and a transmembrane domain and the third domain is a signal transduction domain, and the optional fourth domain is a short peptide linker in the extracellular portion of the receptor.

[0301] FIG. 2 shows results of flow-cytometry based in vitro binding assays of three different PD-L1 and/or PD-L2 binding TMs to PD-L1 expressing cells. Data are presented as mean ± SD of at least four independent experiments.

[0302] FIG. 3 shows results of flow-cytometry based in vitro killing assays of switchable chimeric antigen receptor T cells redirected by three different PD-L1 and/or PD-L2 binding TMs to PD-L1 expressing cells. Target cell numbers were determined after incubation for A) 24 h and B) 48 h. Data are presented as mean ± SD of four independent experiments with individual T cell donors.

[0303] FIG. 4 shows results of live-cell microscopy in vitro killing assays of switchable chimeric antigen receptor T cells redirected by two different anti-PD-L1 TMs to interferon-y treated PC3-PSCA cells. Data are presented as mean ± SD of four independent experiments with individual T cell donors.

[0304] FIG. 5 shows results of in vivo treatment of immunodeficient mice subcutaneously transplanted with PC3-PSCA tumors and treated with switchable chimeric antigen receptor T cells alone or in different combinations with an anti-PSCA TM, an anti-PD-L1 TM based on Durvalumab or Durvalumab. Data are presented as mean ± SEM of eleven mice per group.

[0305] FIG. 6 shows a schema of combined UniCAR and TetOn-inducible immune checkpoint specific targeting module expression cassette (TRE-tetracycline response element, EF1a-elongation factor 1a, rtTA-reverse tetracycline transactivator, iRFP-infrared fluorescent protein, eGFP-enhanced green fluorescent protein).

[0306] FIG. 7 shows the frequency of gene modified primary human T cells obtained by lentiviral gene transfer: doxycycline- inducible gene expression of PD-L1 binding targeting modules under the control of a TetOn-cassette combined with a UniCAR. Data shown represent at least six batches obtained from individual human T cell donors.

[0307] FIG. 8 shows targeting module-dependent cytotoxic response of human T cells genetically engineered to express UniCAR and a PD-L1 and/or PD-L2 binding TM under control of a doxycycline-responsive TetOn-cassette A) after 48 h and B) after 120 h. Mean ± SD of results obtained with six independent human donors are shown.

[0308] FIG. 9 shows flow cytometry-based cytotoxicity assay to assess lysis of PD-L1 positive Nalm-6 cells mediated by UniCAR-T modified to secrete PD-L1 and/or PD-L2 binding TM A) after 48 h and B) after 120 h. Data are presented as mean ± SD of at least four independent experiments with individual T cell donors, respectively.

[0309] FIG. 10 shows a cytotoxicity assay of human T cells expressing UniCAR and a PD-L1 and/or PD-L2 binding TM under control of doxycycline-responsive TetOn-cassette against intrinsically PD-L1 up-regulating PC3-PSCA cells A) after 48 h and B) after 120 h. Data are shown as mean ± SD of three repetitive experiments with individual T cell donors, respectively.

[0310] FIG. 11 shows the quantification of TM-PD-L1-scFv1 concentrations secreted by gene-engineered human T cells upon doxycycline induction. Data are presented as mean ± SD of five repetitive experiments with five individual T cell donors.

[0311] FIG. 12 shows kinetics of On/Off switch of doxycycline-inducible TM-PD-L1-scFv1 secretion. Data are presented as mean ± SD of four individual T cell donors and experiments.

[0312] FIG. 13 shows the quantification of UniCAR molecules on the surface in dependence on activation by doxycycline-induced expression of TM-PD-L1-scFv1. Mean ± SD of three individual T cell donors are shown, statistical significance was calculated by paired t-test, ** p < 0.01.

[0313] FIG. 14 shows that basal expression of TM-PD-L1-scFv under control of Tet On-cassette is below threshold for cytotoxicity induction. Human T cells genetically modified to express a switchable chimeric antigen receptor and to secret TM-PD-L1-scFv in response to doxycycline were cultured in standard culture medium in absence (SN w/o) or presence of 1000 ng/ml doxycycline (SN +) for 72 hours. Supernatant was harvested, diluted 1:5, and added to Nalm-6-PD-L1 target cells and human T cells genetically modified to express a switchable chimeric antigen receptor alone. As additional controls, cell mix was incubated with 1 nM purified TM-PD-L1-scFv1 (+TM) or plain cell culture medium (w/o TM). After incubation for 24 h, lysis of target cells was analyzed using flow cytometry. Data presented as mean ± SD of four individual T cell donors. Statistical significance was calculated by One-way ANOVA; **** p<0.0001.

[0314] FIG. 15 shows stability of TM-PD-L1-scFv1 in culture medium at 37° C. for 72 h followed by flow cytometry-based cytotoxicity assay.

[0315] FIG. 16 shows the in vivo efficacy of human T cells genetically engineered to express UniCAR and TM-PD-L1-scFv under control of TetOn-cassette in a xenotransplantation model of prostate cancer (PCa). A) Schematic illustration of the experimental set-up. B) Measured tumor volume plotted as a function of time as mean ± SEM of at least nine mice.

[0316] FIG. 17 shows the surface plasmon resonance (SPR) measurements of the binding of TM-PD-L1-scFv1 to recombinant human Fc-tagged PD-L1.

[0317] FIG. 18 shows the surface plasmon resonance (SPR) measurements of the binding of TM-PD-L1-scFv2 to recombinant human Fc-tagged PD-L1.

[0318] FIG. 19 shows the in vitro elimination of the acute myeloid leukemia cell line MOLM-13 expressing recombinant PD-L1 by UniCAR and UniCAR/PD-1 switch-modified T cells. A) MOLM-13 cells were engineered to express recombinant PD-L1 and analyzed for PD-L1 surface expression via flow cytometry. B) UniCAR-T cells (CD137/CD3-ζ, UC04) or UniCAR-PD-1 switch T cells were co-cultured with MOLM-13-PD-L1 at an effector to target cell (E:T) ratio of 2:1 in absence or presence of a CD33-specific targeting module (TM CD33). Cytotoxic reactivity against MOLM-13-PD-L1 was assessed after 48 h and 120 h via flow cytometry (mean ± SD). C) Analysis of T cell activation by surface staining of the T cell activation marker CD25 after 120 h of co-cultivation with MOLM-13-PD-L1 (mean ± SD). Statistical significance for n = 3 independent T cell donors was assessed by the Wilcoxon-signed rank-test (* P<0.05, ns = not significant).

[0319] FIG. 20 shows pharmacokinetic studies of TM-PDL1 variants in NSG mice by determining plasma concentrations of TM-PD-L1 variants via ELISA: A) TM-PD-L1-scFv2, B) TM-PD-L1-scFv3 and C) TM-PD-L1-scFv4. Data is shown for three to four mice per time point analyzed.

[0320] FIG. 21 shows target-mediated internalization of PD-L1-specific TMs on PD-L1-expressing cell lines: In order to determine the PD-L1-mediated pharmacodynamic half-life of PD-L1-specific soluble adapters, the PD-L1-positive target cell lines (A) DU-145 and (B) U-251 were stained with 1000 nM TM-PD-L1-scFv3 (TMPD-L1.sub.v3) or TM-PD-L1-scFv5 (TMPD-L1.sub.v5) and incubated for indicated time points at either 4° C. or 37° C. Data is shown as % binding of max by normalizing the obtained MFI to the highest MFI measured. Additionally, the pharmacodynamic half-life (t.sub.½) was calculated for each TM at 37° C. Data is shown as mean ± SD of one experiment measured in technical triplicates.

PRODUCTION OF THE SWITCHABLE CAR CELL

[0321] The immune cells can be genetically engineered to express switchable CARs and/or inducible TMs directed against PD-L1 and/or PD-L2 by various methods. A polynucleotide vector encoding the switchable CAR and all necessary elements to ensure its expression in the genetically engineered immune cell is transferred into the immune cell. The transfer of the vector can be performed by electroporation or transfection of nucleic acids or the help of viral vector systems like adeno-, adeno-associated, retro-, foamy- or lentiviral viral gene transfer.

[0322] The lentiviral gene transfer is applied for stable expression of switchable CARs in immune cells by first constructing a lentiviral vector encoding for a selected switchable CAR. The lentiviral vector is pLVX-EF1alpha UniCAR 28/ζ (Clontech, Takara Bio Group), in which the lentiviral parts of the vector are derived from the human immunodeficiency virus (HIV) and the MSC/IRES/ZxGreenl portion was replaced by the switchable CAR construct.

[0323] The lentiviral particles are produced by transient transfection of human embryonal kidney (HEK) 293T (ACC 635) cells with the switchable CAR encoding lentiviral vector plasmid and cotransfection with a group specific antigen (gag) and Polymerase (pol) encoding plasmid (psPAX2) plus a plasmid encoding for an envelope (pMD2.G). After transfection, the packaging plasmid expresses Gag and Pol protein of HIV-1. The plasmid MD2.G encodes the glycoprotein of the vesicular stomatitis virus (VSV-G). VSV-G protein is used to lentiviral vectors to transduce a broad range of mammalian cells. Various envelopes from different virus species can be utilized for this purpose. Lentiviral vectors can successfully pseudotype with the envelope glycoproteins (Env) of amphotropic murine leukemia virus (MLV) or the G protein of vesicular stomatitis virus (VSV-G), a modified envelope of the prototypic foamy virus (PFV) or chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV.

[0324] Supernatants from transfected HEK293T cells are harvested 24 h to 96 h after transfection and virus particles are concentrated from the supernatant by ultracentrifugation or other methods. For lentiviral transduction of immune cells, peripheral blood mononuclear cells (PBMC) or isolated T cells are activated with mab specific for the CD3 complex, e.g. clone OKT3 or UCHT1, either given in solution or coated to plastic cell culture dishes or magnetic beads or a biodegradable polymer matrix. Activation of PBMC or isolated T cells is further enhanced by stimulating costimulatory pathways with mabs or ligands specific for CD27, CD28, CD134 or CD137 either alone or in combinations coated to plastic cell culture dishes or magnetic beads or a biodegradable polymer matrix and the supply with exogenous recombinant cytokines like interleukin (IL)-2, IL-7, IL-12, IL-15 and IL-21. Concentrated or non-concentrated virus particles are added to PBMC or T cell cultures 24 h to 96 h after initial administration of activating CD3 specific antibodies and/or antibodies specific for costimulatory receptors CD27, CD28, CD134 or CD137 and/or recombinant cytokines as single or multiple doses. T cell electroporation, transduction and expansion may be performed in open cell culture systems by manual handling or in closed partially or fully automated systems.

[0325] Stable transduction of T cells may be determined by flow cytometry after staining with tag-containing molecules for surface expression of switchable CARs or mabs directed against the fourth domain of switchable CARs from day 3 onwards after final administration of virus supernatant. Switchable CAR transduced T cells can be propagated in vitro by culturing them under supply of recombinant cytokines and activating anti-CD3 mabs.

[0326] In case the switchable CAR harbors the optional fourth domain, a peptide sequence forming a linear epitope for a mab, immune cells genetically modified to express switchable CARs can be specifically propagated in vitro by coating a mab or antibody fragments thereof binding to the fourth switchable CAR domain to the surface of culture dishes or to beads of any kind or a biodegradable polymer matrix, which are added to the cell culture at a defined ratio. The binding of surface-coated mabs to the switchable CAR peptide domain induces cross-linkage of cell-surface expressed switchable CARs and formation of an immune synapse, which leads to the activation of signal pathways specifically triggered by the signal domain of the switchable CAR. Depending on the signal pathways induced, this may lead to enhance proliferation and sustained resistance against activation-induced cell death of the switchable CAR carrying immune cells and therefore enrichment of switchable CAR genetically modified immune cells in a mixed population.

[0327] The optional fourth domain, a peptide sequence forming a linear epitope for a mab, can be further utilized to enrich and purify switchable CAR expressing immune cells from mixed populations. Enrichment and purification is performed with the help of a mab or antibody fragment thereof binding to the fourth switchable CAR domain to either mark switchable CAR expressing cells for cell sorting or to transiently link the switchable CAR expressing immune cell to small particles, which can be utilized for cell isolation. In one aspect, switchable CAR engrafted immune cells are incubated with the mab recognizing the fourth domain. Next, magnetic beads are added, which are conjugated with antibodies or fragments thereof directed against the species- and isotype specific heavy and light chains of the mab binding to the optional fourth domain. Thus, switchable CAR expressing immune cells and magnetic beads are linked and are trapped and separated from other immune cells in a magnetic field.

Design of Targeting Modules According to the Invention

[0328] In one example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of Atezolizumab according to SEQ ID No. 57, comprising SEQ IDs No. 6 and 7 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14 (TM-PD-L1-scFv1).

[0329] In an alternative example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of Atezolizumab according to SEQ ID No. 65, comprising SEQ IDs No. 6 and 7 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14.

[0330] In further examples, the PD-L1 and/or PD-L2 binding TM comprises an scFv of Durvalumab according to SEQ ID No. 58, comprising SEQ IDs No. 8 and 9 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14 (TM-PD-L1-scFv2).

[0331] In an alternative example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of Durvalumab according to SEQ ID No. 66, comprising SEQ IDs No. 8 and 9 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14.

[0332] In a further example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 86, comprising SEQ IDs No. 4 and 5 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14 (TM-PD-L1-scFv3).

[0333] In an alternative example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 85, comprising SEQ IDs No. 4 and 5 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14.

[0334] In a further example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 97, comprising SEQ IDs No. 77 and 78 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14 (TM-PD-L1-scFv4).

[0335] In an alternative example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 98, comprising SEQ IDs No. 77 and 78 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14.

[0336] In a further example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 87, comprising SEQ IDs No. 67 and 68 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14 (TM-PD-L1-scFv5).

[0337] In an alternative example, the PD-L1 and/or PD-L2 binding TM comprises an scFv of BMS-936559 according to SEQ ID No. 88, comprising SEQ IDs No. 67 and 68 connected by a linker and the human La epitope E5B9 according to SEQ ID No. 14.

[0338] In further examples, the PD-L1 and/or PD-L2 binding TM is bivalent and comprises two PD-1 extracellular domains, each of which is a fragment of SEQ ID No. 3 and the human La epitope E5B9 according to SEQ ID No. 14 (TM-tdPD1). TM-tdPD-1 is encoded by SEQ ID No. 59.

Characterization of the Functionality of the Targeting Modules According to the Invention

[0339] The functionality of the PD-L1 and/or PD-L2 binding TMs can be confirmed in binding assays to human and murine PD-L1 using surface plasmon resonance as well as in cell-based cytotoxicity assays.

[0340] The results of flow-cytometry based in vitro binding assays of three different PD-L1 and/or PD-L2 binding TMs to Nalm-6 cells transduced to express PD-L1 are shown in FIG. 2. Data are presented as mean ± SD of at least four independent experiments.

[0341] The results of flow-cytometry based in vitro killing assays of switchable chimeric antigen receptor T cells (SEQ ID No. 60) redirected by three different PD-L1 and/or PD-L2 binding TMs to Nalm-6 cells transduced to express PD-L1 are shown in FIG. 3. Effector to target ratio (E:T) was 1:1. Target cell numbers were determined after incubation for A) 24 h and B) 48 h. Data are presented as mean ± SD of four independent experiments with individual T cell donors.

[0342] The results of live-cell microscopy based in vitro killing assays of switchable chimeric antigen receptor T cells (SEQ ID No. 60) redirected by two different anti-PD-L1 TMs to interferon-y treated PC3-PSCA cells are shown in FIG. 4. Effector to target ratio (E:T) was 1:1. Target cell numbers were determined after incubation for 96 h. Data are presented as mean ± SD of four independent experiments with individual T cell donors.

[0343] The results of in vivo treatment of immunodeficient mice subcutaneously transplanted with PC3-PSCA tumors and treated with switchable chimeric antigen receptorT cells (SEQ ID No. 60) alone or in different combinations with an anti-PSCA TM, an anti-PD-L1 TM based on Durvalumab or Durvalumab are shown in FIG. 5. The switchable chimeric antigen receptor T cells were injected intravenously at the indicated time point. The TMs were injected twice daily intraperitoneally and Durvalumab was injected once intraperitoneally during each of the indicated treatment periods (T). Data are presented as mean ± SEM of eleven mice per group.

Design of a Combined Switchable CAR and Inducible, Targeting Module Expression Cassette (FIG. 6)

[0344] The lentiviral gene expression plasmid contains an EF1a promoter for continuous polycistronic transcription of rtTA and UniCAR, separated by a Thosea asigna virus derived peptide cleavage site (2pA). In addition, a PD-L1 and/or PD-L2 binding targeting module (TM) is under the control of a TRE3G promoter, which will only be activated by binding of rtTA in presence of doxycycline. This arrangement allows a doxycyclin-dependent inducible and reversible gene-expression.

Characterization of the Functionality of the Combined Switchable CAR and Inducible, Targeting Module Expression Cassette

[0345] The frequency of gene modified primary human T cells obtained by lentiviral gene transfer is shown in FIG. 7. T cells isolated from voluntary healthy human donors were transduced with lentiviral supernatant transferring genetic information for doxycycline-inducible gene expression of PD-L1 and/or PD-L2 binding targeting modules (TM-PD-L1-scFv-1, TM-PD-L1-scFv2 and TM-tdPD1) under the control of a TetOn-cassette combined with UniCAR. Transduction efficiency at day 14 after transduction was analyzed via immunofluorescence staining of surface displayed UniCAR using a primary mouse a7B6-mAb (10 .Math.g/ml) directed against the extracellular E7B6-tag integrated into the UniCAR hinge. An AlexaFluor 647 conjugated goat-a-mouse Ab was used as secondary Ab.

[0346] The TM-dependent cytotoxic response of human T cells genetically engineered to express UniCAR and the TM according to the invention under control of a doxycycline-responsive TetOn-cassette is shown in FIG. 8. T cells isolated from voluntary healthy human donors were genetically modified to express UniCAR and a PD-L1 and/or PD-L2 binding targeting module, in particular TM-PD-L1-scFv2, under the control of a TetOn-cassette. The ability of the UniCAR modified cells to mediate TM-dependent cytotoxic response against target cancer cells was evaluated in a flow-cytometry based cytotoxicity assay. Nalm-6-PSCA cells were cultured with human UniCAR expressing T cells at a total T cell to target cell ratio of 1:1 in the presence of increasing concentrations of a PSCA binding TM comprising a PSCA binding domain according to EP 2 990 416 B1; and the human La epitope E5B9 according to SEQ ID No. 14. At indicated time points the number of living target cells was determined. Specific lysis was calculated relative to a control without addition of TM.

[0347] The results of the cytotoxicity assay to assess lysis of PD-L1 positive Nalm-6 cells mediated by UniCAR-T modified to secrete PD-L1 specific TM are shown in FIG. 9. Nalm-6-PD-L1 target cells were cultured with genetically modified human T cells in a total T cell to target cell (E:T) ratio of 1:1 in presence of decreasing concentrations of doxycycline ranging from 1,000 to 1 ng/ml. T cells were engineered to express UniCAR and a doxycycline-responsive TetOn-cassette to induce expression and secretion of TM-PD-L1-scFv-1, TM-PD-L1-scFv2 or TM-tdPD1 in presence of doxycycline. Specific lysis of target cells was analyzed after A) 48 h and B) 120 h of incubation and normalized to background lysis without doxycycline.

[0348] FIG. 10 shows the results of the cytotoxicity assay of human T cells expressing UniCAR and a PD-L1 specific TM under control of doxycycline-responsive TetOn-cassette against intrinsically PD-L1 up-regulating PC3-PSCA cells. PC3-PSCA cells were treated with 25 ng/ml IFN-y to induce PD-L1 up-regulation. Subsequently, PD-L1 presenting PC3-PSCA were incubated with genetically modified T cells expressing UniCAR and a PD-L1-specific TM, in particular TM-PD-L1-scFv1, under the control of doxycycline inducible TetOn-cassette in a total T cell to target cell (E:T) ratio of 1:1 for A) 48 h and B) 120 h. Doxycycline was added in decreasing concentrations from 1000 to 1 ng/ml and specific lysis was calculated relative to lysis of target cells without doxycycline.

[0349] The quantification of TM-PD-L1-sFv1 concentrations secreted by gene-engineered human T cells upon doxycycline induction (FIG. 11) was carried out by incubating PD-L1 positive target cells and human T cells engineered to express UniCAR and a TetOn-cassette for doxycycline inducible TM-PD-L1-scFv1 in a total T cell to target cell (E:T) ratio of 1:1 with decreasing concentration of doxycycline for 120 h. Cell-free supernatant was harvested and secreted TM-PD-L1-scFv1 concentrations were analyzed by ELISA. Doxycycline concentration is blotted against TM concentration in ng/ml and resulting molar concentration, respectively.

[0350] The kinetics of On/Off switch of doxycycline-inducible TM-PD-L1-scFv1 secretion is shown in FIG. 12. For each indicated time point of the ON-phase 1×10.sup.5 human T cells genetically engineered to express UniCAR and TM-PD-L1-scFv1 under the control of doxycycline (dox) inducible TetOn-cassette were incubated in 500 .Math.l culture medium in the presence of 1000 ng/ml doxycycline. Likewise, for the OFF-phase a pool of the engineered T cells were first cultured with the same amount of doxycycline for 24 h, washed thoroughly afterwards and cultured as described for the ON-phase, but without addition of doxycycline for additional 4, 6, 8,12 and 24 h. For each time point analyzed, cell-free supernatant was harvested and TM-PD-L1-scFv1 levels were quantified using ELISA.

[0351] The quantification of UniCAR molecules on the surface in dependence on the activation by doxycycline-induced expression of TM-PD-L1-scFv1 is shown in FIG. 13. The number of UniCAR surface molecules on engineered T cells was quantified using the calibration curve generated with the DAKO QIFlKIT®. Immunofluorescence staining of gene-engineered T cells was performed using anti7B6-mAb for UniCAR detection and AlexaFluor 647 goat-a-mouse Ab as secondary Ab was applied to pre-stained T cells but calibration beads as well. Genetically engineered human T cells expressing UniCAR and TetOn-cassette for doxycycline-inducible TM-PD-L1-scFv1 were cultured in absence or presence of 1,000 ng/ml doxycycline for 24 hours.

[0352] FIG. 14 shows that the basal expression of TM-PD-L1-scFv under control of TetOn-cassette is below threshold for cytotoxicity induction. Human T cells genetically modified to express UniCAR and to secret TM-PD-L1-scFv in response to doxycycline were cultured in standard culture medium in absence (SN w/o) or presence of 1,000 ng/ml doxycycline (SN +) for 72 hours. Supernatant was harvested, diluted 1:5, and added to Nalm-6-PD-L1 target cells and human T cells genetically modified to express UniCAR alone. As additional controls, cell mix was incubated with 1 nM purified TM-PD-L1-scFv1 (+TM) or plain cell culture medium (w/o TM) instead. After incubation for 24 h lysis of target cells was analyzed using flow cytometry.

[0353] In order to verify stability of TM-PD-L1-scFv1 at 37° C., purified TM was incubated in culture medium at 37° C. for 72 h and potency was analyzed in a standard flow cytometry-based cytotoxicity assay using human T cells genetically modified to express UniCAR alone and Nalm-6-PD-L1 target cell in a total T cell to target cell (E:T) ratio of 1:1 for 24 h (FIG. 15).

[0354] The in vivo efficacy of human T cells genetically engineered to express UniCAR and TM-PD-L1-scFv under control of TetOn-cassette in a xenotransplantation model of prostate cancer (PCa) is shown in FIG. 16. The mice were transplanted subcutaneously with 1×10.sup.6 tumor cells (PC3-PSCA) (FIG. 16 A). Twenty-five days later 2 million T cells (of which 28.7% were genetically engineered to express UniCAR and TM-PD-L1-scFv under control of TetOn-cassette) were administered and doxycycline-feed was started the same day. Feeding of doxycycline was continued for two weeks in a row with a break of two days in between. Second treatment cycle was started two weeks later and performed in the same way. The tumor volume was measured once a week with a digital caliper and is blotted as a function of time as mean ± SEM of at least 9 mice (FIG. 16 B).

Combination of a Switchable CAR Redirected With a Targeting Module Against CD33 With a PD-L1:CD28 Switch Receptor

[0355] The in vitro elimination of the acute myeloid leukemia cell line MOLM-13 expressing recombinant PD-L1 by UniCAR and UniCAR/PD-1 switch-modified T cells was examined by MOLM-13 cells engineered to express recombinant PD-L1 and analyzed for PD-L1 surface expression via flow cytometry (see FIG. 19 A).

[0356] UniCAR-T cells (CD137/CD3-ζ, UC04) or UniCAR-PD-1 switch T cells were co-cultured with MOLM-13-PD-L1 at an effector to target cell (E:T) ratio of 2:1 in absence or presence of a CD33-specific targeting module (TM CD33). Cytotoxic reactivity against MOLM-13-PD-L1 was assessed after 48 h and 120 h via flow cytometry (see FIG. 19 B, mean ± SD).

[0357] The T cell activation was analyzed by surface staining of the T cell activation marker CD25 after 120 h of co-cultivation with MOLM-13-PD-L1 (see FIG. 19 C, mean ± SD). Statistical significance for n = 3 independent T cell donors was assessed by the Wilcoxon-signed rank-test (* P<0.05, ns = not significant).

[0358] The PD-L1:CD28 switch receptor in combination with UniCAR (4-1BB-CD3zeta UniCAR) exhibits a high targeting module-independent lysis of MOLM-13 PD-L1 cells, pointing to limitations of such an approach.

Pharmacokinetic Studies of TM-PDL1 Variants

[0359] For the pharmacokinetic studies of TM-PDL1 variants in NSG mice, TM-PD-L1 was administered via intravenous tail vein injection of 1 .Math.g/g.sub.BW of NSG mice. Peripheral blood was taken via retroorbital puncture at indicated time points. Plasma concentrations of TM-PD-L1 variants were determined via ELISA utilizing PDL1-Fc chimera capture protein and anti-penta-His HRPconjugate detection antibody (lower limit of quantification 0.03-0.22 ng/ml assay concentration). In FIG. 20 A), B), C) data is shown for three to four mice per time point analyzed. Indicated pharmacokinetic parameters were defined utilizing PK solver2.0 (Zhang et al. 2010) applying weighted (1/Y.sup.2) two-compartmental analysis. Slope of the terminal phase was calculated from the last three or four time points (t.sub.½β). In FIG. 20 the concentration of A) TM-PD-L1-scFv2, B) TM-PD-L1-scFv3 and C) TM-PD-L1-scFv4 is shown and Tab. 1 shows the calculated pharmacokinetic (PK) parameters.

TABLE-US-00003 Results from pharmacokinetic (PK) studies of TM-PDL1 variants: TM-PD-L1-scFv2 (TM-PD-L1.sub.v2), TM-PD-L1-scFv3 (TM-PD-L1.sub.v3) and TM-PD-L1-scFv4 (TM-PD-L1.sub.v4). PK parameter TM-PD-L1.sub.V2 TM-PD-L1.sub.V3 TM-PD-L1.sub.V4 t.sub.½α [min] 6.18 3.9 10.58 t.sub.½β [min] 112.08 75.4 65.26 AUC.sub.0-∞ [.Math.g/ml.sup.∗h] 1166.23 169.34 248.04

[0360] In FIG. 21 target-mediated internalization of PD-L1-specific TMs on PD-L1-expressing cell lines is shown. In order to determine the PD-L1-mediated pharmacodynamic half-life of PD-L1-specific soluble adapters, the PD-L1-positive target cell lines (A) DU-145 and (B) U-251 were stained with 1000 nM TM-PD-L1-scFv3 (TMPD-L1.sub.v3) orTM-PD-L1-scFv5 (TMPD-L1.sub.v5). Subsequently, stained target cells were incubated for indicated time points at either 4° C. or 37° C. Binding of PD-L1-specific TMs was analyzed via flow cytometry using an PE-labeled α-His Ab. Additionally, the pharmacodynamic half-life (t1/2) was calculated for each TM at 37° C.

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TABLE-US-00004 Reference signs 1 first domain, a tag-binding domain or tag. 2 second domain, an extracellular hinge and a transmembrane domain. 3 third domain, a signal transduction domain. 4 optional fourth domain, a short peptide linker.