CHIMERIC ANTIGEN RECEPTORS THAT BIND TO PROSTATE SPECIFIC MEMBRANE ANTIGEN

Abstract

The present invention relates to a novel chimeric antigen receptor (CAR) comprising an antigen-binding fragment which binds specifically to PSMA antigen, and a method of manufacturing high-quality CAR T cell products by transfection and/or transduction of T cells therewith, which allows to eradicate tumors in vivo alone or in combination with pharmaceutical drugs, such chemotherapies, biopharmaceutical drugs, such as antibodies, or small-molecule drugs, such as protein kinase inhibitors.

Claims

1-13. (canceled)

14. A chimeric antigen receptor comprising an antigen-binding fragment which binds specifically to a PSMA antigen, wherein said chimeric antigen receptor against PSMA is derived from the single-chain variable fragment D7 and includes a transmembrane domain and an intracellular signaling domain.

15. The chimeric antigen receptor according to claim 14, characterized in that said chimeric antigen receptor contains: at least three CDRs selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7, at least four CDRs selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:7, at least five CDRs selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, or at least six CDRs selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

16. The chimeric antigen receptor according to claim 14, characterized in that the antigen-binding fragment is humanized.

17. The chimeric antigen receptor according to claim 16, characterized in that said chimeric antigen receptor comprises amino acid residues 99 to 112 of SEQ ID NO: 1 (ARDGNFPYYAMDSW).

18. The chimeric antigen receptor according to claim 16, characterized in that said chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 7 (SQSTHVPT).

19. The chimeric antigen receptor according to claim 14, wherein said transmembrane domain is a CD3ζ cytoplasmatic domain and said intracellular signaling domain is a CD28 or 4-1BB cytoplasmatic domain or a combination thereof.

20. A nucleic acid coding for the chimeric antigen receptor according to claim 14.

21. A vector coding for the chimeric antigen receptor according to claim 14.

22. The vector according to claim 22, characterized in that said nucleic acid coding for said chimeric antigen receptor comprises SEQ ID NO:10, SEQ ID NO:11, or a sequence that is at least 95% identical to either SEQ ID NO:10 or SEQ ID NO:11.

23. An in vitro method of providing T cells comprising the chimeric antigen receptor according to claim 14, said method comprising the steps of: isolating said T cells from a donor by leukapheresis; transfecting/transducing said T cells with a vector coding for said chimeric antigen receptor; and isolating and amplifying the transfected/transduced T cells.

24. A T cell containing genetic information coding for the chimeric antigen receptor according to claim 14.

25. A method of treating prostate cancer or a prostate-derived tumor, said method comprising the step of administering an effective amount of a composition containing the chimeric antigen receptor according to claim 14 or a nucleic acid or vector encoding same to a patient in need thereof.

26. A method of treating a solid tumor expressing PSMA, said method comprising the step of introducing an agent having anti-tumor activity into said solid tumor, wherein said agent includes the chimeric antigen receptor according to claim 14 or a nucleic acid or vector encoding same.

27. The method according to claim 25, wherein said method further comprises the step of administering a chemotherapeutically active agent to said patient, whereby said chemotherapeutically active agent comprises a cytotoxic substance selected from the group consisting of taxol derivatives, 5-fluorouracil, cyclophosphamide, mitoxanthrione, docetaxel and capacitaxel.

28. The method according to claim 25, wherein said method further comprises the step of administering a biopharmaceutical selected from the group consisting of proteins, antibodies, vaccines, blood, blood components, allergenics, recombinant therapeutic proteins, gene therapies, somatic cells, tissues, cell therapies, enzyme inhibitors, and anti-genomic therapeutics.

29. The method of claim 25, wherein said biopharmaceutical is a protein kinase inhibitor.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0038] In the Figures and in the experiments the following abbreviations were used:

TABLE-US-00001 Abbreviation Explanation 4-1BB tumor necrosis factor receptor superfamily member 9 BLI bioluminescence imaging bw body weight C4-2 PSMA positive prostate cancer cell line CAR Chimeric Antigen Receptor CAR28 anti-PSMA CAR with CD28 co-stimulatory domain CAR41 anti-PSMA CAR with 4-1BB co-stimulatory domain CD28 cluster of differentiation 28 CD3ζ CD3 zeta region CD45RA cluster of differentiation 45 isoform RA CD62L cluster of differentiation 62, L-selectin CR complete remission DOC docetaxel DU 145 PSMA negative prostate cancer cell line EFS Short version of the elongation factor alpha gene promoter ELISA enzyme-linked immunosorbent assay i.v intravenously i.p. intraperitoneally s.c. subcutaneously Gr. A Granzyme A Gr. B Granzyme B. IFN-g Interferon-gamma LTR long terminal repeat of retroviral vector PR partial remission R R region of the retroviral long terminal repeat scFv single chain variable fragment SD standard deviation Tcm T cell central memory Teff T cell effector Tem T cell effector memory Tn, scm T cell naïve or T stem cell memory U5 U5 region of the retroviral long terminal repeat UT untransduced T cell WPRE woodchuck hepatitis virus post-transcriptional regulatory element XTT sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4- methoxy-6-nitro) benzene sulfonic acid hydrate ΔU3 deletion in the U3 region in the retroviral long terminal repeat

[0039] FIG. 1. Generation and quality assessment of PSMA targeting CAR T cells.

[0040] (A) Schematic of self-inactivating gamma-retroviral vectors to express 2.sup.nd generation anti-PSMA CARs. CAR expression is driven by an EFS promoter. The CAR consists of a single chain variable fragment D7 (derived from 3F/11 murine monoclonal antibody against PSMA), a hinge region (not shown), a transmembrane domain, a costimulatory domain either derived from 4-1BB (CAR41) or CD28 (CAR28), and an intracellular signaling domain derived from CD3ζ chain. The hinge region, which is included in both CARs, is derived from human IgG and provides a physical spacer between the D7-scFv and the transmembrane domain for optimal target recognition.

[0041] (B) CAR expression. T cells were activated for 2-3 days with anti-CD2/3/28 antibodies prior to retrovirus transduction. Following viral transduction, T cells were expanded for 8-9 days before cells were harvested and stained with anti-human IgG antibody (CAR) and CD3 to evaluate the transduction efficiencies.

[0042] (C) Qualitative CAR T cell phenotype characterization with flow cytometric analysis. T cell subsets were evaluated by assessing the expression of CD62L and CD45RA, pre-gated on CAR-CD3+ for the untransduced T cells (UT) (not shown) or CAR+CD3+ for both types of CAR T cells (not shown).

[0043] (D) Qualitative assessment of CAR T cell phenotype. Shown are the average percentages of the different T cell subsets from at least three independent experiments. PSMA, prostate specific membrane antigen; scFv, single chain variable fragment; EFS, elongation factor 1 alpha short promoter; Tn/Tscm, T cell naïve or T stem cell memory; Tcm, T cell central memory; Tem, T cell effector memory; Teff, T cell effector.

[0044] This characterization of the CAR T cell phenotype confirms that the chosen CAR design enables the generation of high-quality of CAR T cell products with a minimal amount of terminally differentiated T cell subsets.

[0045] FIG. 2. Cytotoxicity and cytokine release of PSMA-CAR T cells.

[0046] (A) Cytotoxicity profile of CAR T cells on PSMA positive C4-2 tumor cells. CAR T cells were co-cultured for 48 hours at different effector to target (E:T) ratios, with either antigen positive C4-2 tumor cells or PSMA negative cells (DU145) were used.

[0047] (B) Cytotoxicity of CAR T cells on PSMA-negative DU 145 tumor cells. CAR-T cells were co-cultured for 48 hours at different effector to target (E:T) ratios with DU145 cells.

[0048] Cytotoxicity was measured by an XTT ELISA based colorimetric assay (% cytotoxicity=100−% viability).

[0049] A comparison of the results as presented in FIG. 2A and FIG. 2B shows that the T cells transfected with the CAR, according to the invention, have a cytotoxic activity only with the cell line expressing the PSMA antigen since in panel A a PSMA expressing cell line (C4-2) was used. When the cell line does not express PSMA, as for example cell line DU 145, no cytotoxicity can be seen with the CAR T cells (FIG. 2B). Furthermore, the CAR28 and CAR41 cells of this invention showed superior cytotoxicity as compared to previously published data (PMID 16204083, PMID: 18026115, PMID: 19773745, PMID: 25358763, PMID: 23242161, PMID: 4174378, PMID: 25279468): up to 90% of PSMA expressing cells were eliminated (FIG. 2A), using a low E:T ratio of 1:1. The observed high in vitro cytotoxicity with a low E:T ratio of 1:1 is unique and was not described in any of the available PSMA-CARs so far, indicating the superior performance our CAR T cells based on the scFv D7.

[0050] (C) Quantification of pro-inflammatory cytokine release upon antigen stimulation. CAR T cells were stimulated with either PSMA-positive C4-2 cells or PSMA-negative DU 145 cells for 48 hours. Cytokines in supernatant were quantified by flow cytometry based cytokine-bead assay. Statistically significant differences are indicated by ***(P<0.001; n=3). UT; untransduced T cells, IFN-g; interferon gamma, Gr.A; Granzyme A, Gr.B; Granzyme B.

[0051] It can be seen from FIG. 2C that pro-inflammatory cytokine release occurred only when CAR T cells were co-cultured with cell lines expressing PSMA (cell line: C4-2). CAR28 were activated to a higher extend than CAR41 cells.

[0052] FIG. 3. CAR T cell phenotype and exhaustion profiles upon antigen specific stimulation.

[0053] (A) CAR T cell phenotype. CAR T cells were stimulated with PSMA-positive C4-2 tumor cells for 24 hours at a 1:1 effector-to-target (E:T) ratio before T cell phenotype profile was assessed based on the expression of CD62L and CD45RA. Cells were pre-gated on either CAR-CD3+ for UT control (not shown) or CAR+CD3+ for both types of CAR T cells (not shown).

[0054] (B) Quantitative assessment of CAR T cell phenotype. Shown are the average percentages of the different T cell subsets from three independent experiments. Statistical significant differences are indicated by ***(P<0.001) or *(P<0.05).

[0055] (C) T cell exhaustion profile. CAR T cells were stimulated with PSMA-positive tumor cells (C4-2) for 24 hours before T cell exhaustion profile was assessed by measuring the expression of CD223 (LAG-3) and CD279 (PD-1). Cells were pre-gated on CAR-CD3+ for UT control (not shown) or CAR+CD3+ for both types of CAR T cells (not shown).

[0056] (D) Quantitative assessment of CAR T cell exhaustion profiles. Shown are the average percentages of PD-1 or LAG-3 positive cells (three independent experiments). Statistically significant differences are indicated by ***(P<0.001). NS, not significant; UT, untransduced T cells; Tn/Tscm, T cell naïve or T stem cell memory; Tcm, T cell central memory; Tem, T cell effector memory; Teff, T cell effector; LAG-3, lymphocyte activation gene 3; PD-1, programmed cell death protein 1.

[0057] This set of experiments addressed the T cell phenotype and the exhaustion profiles of the CAR41 and CAR28 T cells upon antigen-specific stimulation. As seen in panels 3A and 3B, in contrast to CAR28 cells, a high percentage of CAR41 cells preserved an undifferentiated T cell phenotype upon antigen-specific stimulation. Furthermore, the CAR41 cells were less sensitive to exhaustion as compared to CAR28 T cells upon antigen-specific differentiation (FIGS. 3C and 3D).

[0058] FIG. 4: PSMA expression of the prostate cancer target cell line C4-2.sup.luc+.

[0059] For in vivo bioluminescence imaging, PSMA-positive prostate cancer C4-2 cells were transduced with a lentiviral vector encoding firefly luciferase, a neomycin resistance gene and green-fluorescent protein (GFP). The transduced cell line was named C4-2.sup.luc+.

[0060] (A) Binding of the anti-PSMA mAb 3/F11 to C4-2.sup.luc+ cells as determined by flow cytometry.

[0061] (B) Binding at a saturation concentration of 25 μg/ml 3/F11 to C4-2.sup.luc+ and PSMA-negative DU 145 cells as determined by flow cytometry.

[0062] Binding of the anti-PSMA mAb 3/F11, the parental mAb of the scFv D7, verified the PSMA expression on the surface of all C4-2.sup.luc+ prostate cancer target cells, while DU 145 prostate cancer control cells were shown to be PSMA negative.

[0063] FIG. 5: Bioluminescence of C4-2.sup.luc+ cells as determined by bioluminescence imaging (BLI) with the Living imaging system IVIS 200 (Xenogen VivoVision).

[0064] The luminescence of the C4-2.sup.luc+ target cells was tested using the In Vivo Imaging System IVIS 200 (Xenogen VivoVision) after incubation with luciferin. Compared to the non-transduced C4-2 cells, the C4-2.sup.luc+ cells showed bioluminescence, with an intensity (photons/sec/cm.sup.2) that was linear to the cell number. Thus, these cells were suitable for their use in the mouse tumor xenograft model with tumor detection by bioluminescence imaging (BLI). ROI, region of interest.

[0065] FIG. 6 Intratumoral CAR T cell therapy

[0066] (A) Schematic of intratumoral CAR T cell therapy. Tumor engraftment was established in animals by subcutaneous injection of 1.5×10.sup.6 cells. When the growing solid C4-2.sup.luc+ tumors have reached 60-80 mm.sup.3 volume, CAR T cells were intratumorally injected with single doses of 5×10.sup.6 CAR28 (n=5) or CAR41 T cells (n=5) at day 1 of treatment. Control mice were injected with the same number of UT cells (n=7) or left untreated (control, n=6). Tumors were visualized by bioluminescence imaging (BLI) until the end of treatment at day 22. D, day; i.t, intratumoral; s.c, subcutaneous.

[0067] (B) Antitumor activity of CAR28 and CAR41 T cells after intratumoral injection. Animals with subcutaneously growing solid C4-2.sup.luc+ tumors (60-80 mm.sup.3 volume) were intratumorally injected with single doses of 5×10.sup.6 CAR28 (n=5) or CAR41 T cells (n=5) on day 1 of treatment. Control mice were injected with the same number of UT cells (n=7) or left untreated (control, n=6). Tumors were visualized by BLI until the end of treatment on day 22. Tumor volumes, represented as Mean±SD were calculated from the BLI images using the formula Vol=d.sup.2×D/2, where d was the small diameter and D was the large diameter of the tumor. Statistical significant differences are indicated by ***p<0.001.

[0068] (C) Antitumor activity of intratumorally injected CAR28 and CAR41 T cells at the end of treatment (day 22). Tumor volumes were calculated from BLI images as described above. Tumor volumes are shown as Mean±SD. Statistical significant differences are indicated by ***p<0.001. Since ⅙ mice of the control group developed an ulcerating tumor, this mouse had to be euthanized on day 15 and was not included into the statistical analysis of day 22.

[0069] (D) Representative bioluminescence images of mice intratumorally treated on day 1 of treatment with single injections of CAR28 or CAR41 T cells compared to untreated animals (control) or animals treated with UT T cells. Two mice representing animals with large or small tumors of each treatment group are shown.

[0070] Intratumoral treatment of mice bearing solid C4-2.sup.luc+ tumors of 60-80 mm.sup.3 volume with one injection of 5×10.sup.6 CAR28 T cells on day 1 of treatment led to a significant inhibition of tumor growth and to 5/5 complete tumor remissions (CR) until day 15 (FIGS. 6B, C and D). One animal showed a recurrence of the tumor at day 22 with a tumor end volume of 0.55 mm.sup.3 (FIG. 6D, mouse pictured). For all mice of the group a mean tumor volume of 0.11±0.22 mm.sup.3 was calculated for day 22 (FIGS. 6B and C). Mice intratumorally treated with CAR41 T cells showed a different response. ⅕ CR and ⅕ partial remission (PR) were noted leading to a statistically significant inhibition of tumor growth with a mean tumor end volume of 397.3±393.6 mm.sup.3 on day 22 (FIGS. 6B and C). In contrast, all mice of the UT and control groups showed tumor progression leading to tumor end volumes of 1469.4±961.5 mm.sup.3 and 1289.1±636.8 mm.sup.3, respectively (FIGS. 6B and C).

[0071] FIG. 7: Changes in body weight of intratumorally treated mice at the end of treatment (day 22).

[0072] According to animal protection guidelines, a decrease in body weight is an essential sign of toxicity during treatment. No animal showed a critical decrease of its initial body weight of more than 20%. This evidences that the intratumoral treatment was tolerated well without apparent signs of off-target toxicity.

[0073] FIG. 8: Intravenous CAR T cell therapy.

[0074] (A) Schematic of intravenous CAR T cell therapy. Tumor engraftment was established in animals by subcutaneous injection of 1.5×10.sup.6 cells. When the growing solid C4-2.sup.luc+ tumors have reached 60-80 mm.sup.3 volume, CAR T cells were intravenously injected with single doses of 5×10.sup.6 CAR28 (n=5) or CAR41 T cells (n=5) on day 1 of treatment. Control mice were injected with the same number of UT cells (n=7) or left untreated (control, n=6). Tumors were visualized via BLI until the end of treatment on day 22. D, day; i.v, intravenous; s.c, subcutaneous.

[0075] (B) Antitumor activity of CAR28 and CAR41 T cells after intravenous injection. Animals with subcutaneously growing solid C4-2.sup.luc+ tumors (60-80 mm.sup.3 volume) were intravenously injected with single doses of 5×10.sup.6 CAR28 (n=5) or CAR41 T cells (n=5) at day 1 of treatment. Control mice were injected with the same number of untransduced T cells (UT cells) (n=5) or left untreated (control, n=6). Tumors were visualized via bioluminescence imaging until the end of treatment on day 22. Tumor volumes, represented as Mean±SD were calculated from the BLI images using the formula Vol=d.sup.2×D/2, where d was the small diameter and D was the large diameter of the tumor.

[0076] (C) Antitumor activity of intravenously injected CAR28 and CAR41 T cells at the end of treatment (day 22). Tumor volumes were calculated from BLI images as described above. Tumor volumes are shown as Mean±SD.

[0077] (D) Representative bioluminescence images of mice intravenously injected with single doses of CAR28 or CAR41 T cells. Two mice representing animals with large or small tumors of each treatment group are shown.

[0078] No inhibition of tumor growth could be measured after single intravenous injections of CAR28 or CAR41 T cells into mice bearing solid tumors with volumes of 60-80 mm.sup.3 (FIGS. 8B and C). As demonstrated in FIGS. 8C+D, animals of all treatment groups showed comparable tumor growth leading to tumor end volumes on day 22 of 1469.4±961.4 mm.sup.3 (control), 1275.7±544.5 mm.sup.3 (UT), 1427.3±448.5 mm.sup.3 (CAR28), and 1529.3±971.0 mm.sup.3 (CAR41).

[0079] FIG. 9: Combination therapy

[0080] (A) Schematic of combined chemotherapy with intravenous CAR T cell therapy. Tumor engraftment was established in animals by subcutaneous injection of 1.5×10.sup.6 cells. Chemotherapy had started at day 1 of the treatment for 3 days by intraperitoneal injection of docetaxel (DOC, 6 mg/kg bw). 48 hours after the last chemotherapy cycle, mice were injected intravenously with single doses of 5×10.sup.6 CAR28 (n=3) or CAR41 T cells (n=3). Control mice were injected with docetaxel alone (n=3) or left untreated (control, n=4). Tumors were visualized via BLI until the end of treatment at day 17. D, day; i.v, intravenous injection; i.p, intraperitoneal; s.c, subcutaneous.

[0081] (B) Antitumor activity of CAR28 and CAR41 T cells in combination with docetaxel chemotherapy. Mice with subcutaneously growing C4-2.sup.luc+ tumors (200-250 mm.sup.3) were intraperitoneally injected with docetaxel (DOC; 6 mg/kg bw) on days 1, 2, and 3 of treatment. 48 hours later mice were additionally injected with a single dose of 5×10.sup.6 CAR28 (n=3) or CAR41 T cells (n=3) intravenously. Tumor growth was monitored by BLI. Compared to the untreated control (n=4), DOC treatment led to a significant inhibition of tumor growth, which was enhanced by the addition of CAR28 or CAR41 T cells. 4/4 mice of the control group showed growing tumors during treatment, whereas in the DOC group ⅔ mice showed a PR. In the DOC+CAR28 group ⅓ animals showed a PR. In the DOC+CAR41 group ⅔ mice showed a CR and ⅓ mice a PR. A statistically significant difference between the DOC+CAR28 and DOC+CAR41 groups demonstrated the superior effects of the CAR41 T cells in this treatment scheme. Statistical significant differences are indicated by *p<0.05.

[0082] (C) Antitumor activity of CAR28 and CAR41 T cells in combination with docetaxel chemotherapy on day 17 of treatment. At the end of treatment on day 17 the mean tumor volume of mice of the control group was determined with 1817.5±165.5 mm.sup.3. The DOC group and the DOC+CAR28 group exhibited significantly lower volumes of 338.3±355.6 mm.sup.3 and of 559.9±317.2 mm.sup.3, respectively. Animals of the DOC+CAR41 group only had a mean tumor volume of 50.2±71.1 mm.sup.3. Statistical significant differences are indicated by *p<0.05.

[0083] (D) Representative bioluminescence images of mice pretreated with DOC and intravenously injected with single doses of CAR28 or CAR41 T cells. Two mice representing animals with large or small tumors of each treatment group are shown.

[0084] Taken together, pretreatment of tumors with docetaxel chemotherapy led to an antitumor activity of intravenously applicated CAR41 T cells.

[0085] FIG. 10: Sequences

[0086] FIG. 10 shows the amino acid sequence (“Frame1”) of the antigen-binding construct D7 which is derived from mice. Moreover, the coding nucleic acid sequence and the complementary strand thereof are provided. The CDR H1-H3 and CDR L1-L3 nucleic acid and amino acid sequences are marked with grey arrows.

[0087] The amino acid sequence of the ScFv fragment of D7 (SEQ ID NO:1) is essential for the present invention insofar as it is the starting sequence for the humanization of the antigen binding fragment. The humanized sequences have a homology of at least 80% to SEQ ID NO:1. In a more preferred embodiment the sequences have a homology of at least 90% and more preferred at least 95% to SEQ ID NO:1 and even more preferred the homology is at least 98% to SEQ ID NO:1. It should be noted that the CDR regions which are shown in FIG. 10 are conserved to a very high level which means that the CDR regions which are determined by the Kabat method have not more than three, preferably not more than one and preferably no amino acid exchange.

[0088] The sequences as disclosed in the present application are summarized as follows:

TABLE-US-00002 SEQ ID nucleic/amino NO: acid description 1 amino acid antigen binding fragment D7 FIG. 10 2 amino acid CDR-H1 FIG. 10 3 amino acid CDR-H2 FIG. 10 4 amino acid CDR-H3 FIG. 10 5 amino acid CDR-L1 FIG. 10 6 amino acid CDR-L2 FIG. 10 7 amino acid CDR-L3 FIG. 10 8 coding sequence D7 codon optimized for bacterial expresson 9 complementary strand D7 codon optimized for bacterial expression 10 coding strand CAR 28 FIG. 12 11 coding strand CAR 41 FIG. 13 12 nucleic acid coding strand D7 FIG. 10 13 nucleic acid complementary strand D7 FIG. 10 14 amino acid sequence chimeric antigen receptor FIG. 12 15 coding sequence {close oversize brace} with fragment of CD 28 FIG. 12 16 complementary strand FIG. 12 17 amino acid sequence chimeric antigen receptor FIG. 13 18 coding sequence {close oversize brace} with fragment of 4-1 BB FIG. 13 19 complementary strand FIG. 13 20 amino acid D7 VH murine FIG. 14a 21 amino acid hum D7 VH1 FIG. 14a 22 amino acid hum D7 VH2 FIG. 14a 23 amino acid hum D7 VH3 FIG. 14a 24 amino acid hum D7 VH4 FIG. 14a 25 amino acid hum D7 VH5 FIG. 14a 26 amino acid hum D7 VL1 FIG. 14b 27 amino acid hum D7 VL2 FIG. 14b 28 amino acid hum D7 VL3 FIG. 14b 29 amino acid hum D7 VL4 FIG. 14b 30 amino acid hum D7 VL5 FIG. 14b 31 amino acid D7 VL murine FIG. 14b 32 amino acid D7 VH murine FIG. 11 33 amino acid D7 VH hum 1 FIG. 11 34 amino acid D7 VH hum 2 FIG. 11 35 amino acid D7 VH hum 3 FIG. 11 36 amino acid D7 VH hum 4 FIG. 11 37 amino acid D7 VL murine FIG. 11 38 amino acid D7 VL hum 1 FIG. 11 39 amino acid D7 VL hum 2 FIG. 11 40 amino acid D7 VL hum 3 FIG. 11 41 amino acid D7 VL hum 4 FIG. 11

[0089] FIG. 11: Potential mutations in humanized sequences

[0090] In FIG. 11 the CDRs are shown. Usually the CDR-H1, CDR-H3 and CDR-L3 are without any amendments. It is possible, however, to replace one amino acid in CDR-L2, up to two amino acids in CDR-L1 and up to six amino acids in CDR-H3. The amino acids which can be replaced are designated as X1-X9 with the meaning shown in the legend to FIG. 11.

[0091] FIG. 12: Sequences of construct CAR28

[0092] FIGS. 12a-12d show the sequences of the construct designated as CAR-CD28.

[0093] FIG. 13: Sequence of construct CAR41

[0094] The sequence of the construct CAR-4-1BB is shown in FIGS. 13a-13d.

[0095] It should be noted that in FIGS. 10-13 the sequence information is provided together with an information which function the relevant parts of the sequences have. Therefore, it is understood that the person skilled in the art derives the information in the best manner from the sequences provided. Sequences 10-13 disclose preferred embodiments of the invention.

[0096] FIG. 14: Sequences of humanized ScFv derived from D7

[0097] (A) Shown are the murine sequence of the heavy chain of D7 and five humanized variants thereof whereby the locations of CDR-H1, CDR-H2 and CDR-H3 are shown according to the determination of Kabat and according to IMGT as well.

[0098] (B) Shown are the murine sequence of the light chain of D7 and of five humanized variants thereof, with CDR-L1, CDR-L2, and CDR-L3. It is understood that the heavy chains and the light chains can be combined with each other. It is for example possible to combine the heavy chain sequence of variant 1 (hum D7-VH1) with the light chain of variant 5 (hum D7-VL5).

[0099] FIG. 15: Comparison of CAR constructs according to the invention with constructs wherein the PSMA antigen binding fragments were already described in the prior art

[0100] (A) Schematic of the self-inactivating retroviral vector constructs to express 2.sup.nd generation anti-PSMA CARs. CAR expression is driven by an EFS promoter. The anti-PSMA CARs contain either the single chain variable fragment (scFv) 3D8 or J591 (both described in the prior art), or D7 (derived from 3F/11 murine monoclonal antibody against PSMA), which is fused to an Fc IgG1 derived hinge region, a transmembrane (tm) domain, a CD28 derived costimulatory domain, and an intracellular signaling domain derived from CD3ζ chain. The hinge region provides a physical spacer between the scFv and the tm domains for optimal target recognition. The CD28 co-stimulatory domain contains amino acid exchanges that prevent LCK binding and enhance anti-tumor activity in the presence of inhibitory regulatory T cells (Tregs). All generated retroviral constructs have the same scaffold but differ only in the scFv fragments allowing for a side-by-side comparison of the CAR constructs with respect to activity and cytotoxicity.

[0101] (B) CAR expression in Jurkat cells were transduced with retroviral particles coding for the three different PSMA-CARs. Following viral transduction, Jurkat cells were expanded for 16 days before cells were harvested and stained with anti-human IgG antibody (CAR) to evaluate the transduction efficiencies and the CAR expression levels.

[0102] (C) Antigen-specific-activation profile, CAR expressing Jurkat cells were stimulated for 24 h at a 1:1 effector-to-target ratio with either PSMA positive, PD-L1 negative C4-2 tumor cells (PSMA+/PDL1−), or PSMA positive, PD-L1 positive LNCaP tumor cells (PSMA+/PDL1+). As a negative control, CAR cells were co-cultured with PSMA negative DU145 tumor cells (PSMA−). Following 24 h stimulation, cells were harvested and activation profile was assessed by evaluating the percentage of cells that are positive for the activation marker CD69.

[0103] The antigen-specific activation profiles were compared in D7-based CAR T Jurkat cells side-by-side with J591 and 3D8-based CAR T Jurkat cells. As indicated in Panel C, the D7-based CAR and the J591-based CAR were able to mediate massive activation of the Jurkat cells upon antigen-specific sensitization, as measured by upregulation of the activation marker CD69 in about −70% of cells. Activation was not affected by the presence of the inhibitory ligand PD-L1. On the other hand, the 3D8-based CART cells were only weakly activated (up to 20% of CD69-positive cells).

[0104] (D) CAR surface expression in primary T cells. T cells were activated for 2-3 days with anti-CD2/CD3/CD28 antibodies prior to retrovirus transduction. After an expansion for 6-9 days, cells were harvested and stained with anti-human IgG antibody (CAR) and CD3 to evaluate the transduction efficiencies and CAR expression levels.

[0105] (E-G) Cytotoxicity profile of CAR T cells were generated by transducing primary T cells after 3 days of activation with anti.-CD3/CD28/CD2 antibodies. After an expansion for 8-9 days, the cytotoxicity profile was examined by co-culturing the CAR T cells for 48 hours at different effector-to-target (E:T) ratios, either C4-2 (PSMA+/PDL1−) tumor cells (E), LNCaP (PSMA+/PDL1+) tumor cells (F) or DU145 (PSMA−) tumor cells (G). Cytotoxicity was measured by an XTT ELISA based colorimetric assay (% cytotoxicity=100%−% viability). Statistically significant differences are indicated by *(P<0.05), **(P<0.01), or ***(P<0.001). UT, untransduced cells; PSMA, prostate specific membrane antigen; PD-L1, Programmed cell death ligand 1.

[0106] D7-based CART cells were compared side-by-side with J591-based CART cells. D7-based CAR T cells revealed a superior cytotoxicity profile as compared to J591-based CAR T cells on both tumor cell targets (E, F), as evidenced by the fact both tumor cell lines could be eliminated with lower effector-to-target ratios. Because of the weak activation profile (C) and the lack of efficient expression in T cells (D), 3D8-based CAR T cells were not further included in the comparison. In conclusion, both PSMA antigen-specific activation and cytotoxicity profile of the D7-based CAR T cells outperformed CAR T cells based on prior art CARs.

[0107] (H, I) Characterization of employed prostate cancer cell lines. The extent of PSMA target antigen expression (H) and PD-L1 (CD274) expression (I) was assessed on C4-2, LNCaP and DU145 prostate cell lines. For flow cytometric analysis, cells were either stained with 3/F11 antibody (anti-PSMA) or an anti-CD274 antibody. UT, untransduced T cells; PD-L1, Programmed cell death ligand 1; PSMA, prostate specific membrane antigen.

[0108] As shown in panel H, both C4-2 and LNCaP tumor cells express the PSMA antigen while the DU145 is negative for the PSMA antigen. Panel I shows that a large fraction of LNCaP and DU145 cells express PD-L1 while C4-2 cells were negative for PD-L1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] The results of the experiments shown in the Figures can be interpreted as follows:

[0110] In general, CARs are composed of an extracellular domain containing the antigen recognizing scFv, a hinge region, a transmembrane region, and one or more intracellular signaling domains that activate the T cell, including the CD3ζ chain. In 2.sup.nd or 3.sup.rd generation CARs, co-stimulatory domains, usually derived from CD28, 4-1BB, OX40, CD27 and/or ICOS are included (FIG. 1A). For the treatment of prostate cancer, many attempts had utilized CARs targeting PSMA epitopes and some of these strategies have already entered clinical trials (e.g. NCT01140373, NCT01929239). However, the potency of these CARs seems to be rather low both in vitro and in vivo. In particular, early studies based on 1.sup.st generation CARs based on the anti-PSMA scFv 3D8 or J591 (known as Pzl), have shown low potency of the resulting CAR T cells, as indicated by the need of having to employ high effector to target (E:T) ratios up to 100:1 to eliminate the tumor cells in vitro. The in vitro potency improved when 2.sup.nd or 3.sup.rd generation CARs based on either D2B or J591 derived scFvs were used. However, the potency of these CAR T cells remained low in xenotransplantion tumor mouse models, as indicated by the fact that the these PSMA targeting CAR T cells were only able to suppress tumor growth but not to eliminate the tumors in vivo, although very high CART cell doses, sometimes up to 20×10.sup.6 CART cells, or multiple infusions were applied (PMID 16204083, PMID: 18026115, PMID: 19773745, PMID: 25358763, PMID: 23242161, PMID: 4174378, PMID: 25279468). In view of these mixed results with rather inefficient PSMA-CAR T cells, it was intended to generate and validate novel, more efficient PSMA targeting CARs based on the scFv D7. Two different 2.sup.nd generation PSMA-CARs were designed which either harbor a CD28 (CAR28) or 4-1BB (CAR41) derived co-stimulatory domain (FIG. 1). The results demonstrate the high effectiveness of these newly developed D7-based PSMA-targeting CAR T cells both in vitro and in vivo. The manufactured CAR T cell products contained a high percentage of undifferentiated T cells, such as a naïve T cell, T stem cell memory and central memory T cell phenotypes (FIG. 1D). These D7-based CART cells completely eliminated PSMA-positive tumor cells in vitro at a low effector to target ratio and released the expected cytokines upon specific antigen stimulation (FIG. 2A-C).

[0111] CAR41 T cells maintained a more naïve phenotype and less exhaustion profile upon antigen specific stimulation as compared to CAR28 (FIG. 3).

[0112] Importantly, the D7-based CAR T cells eliminated solid PSMA-positive tumors in a mouse tumor model upon intratumoral application (FIG. 6). It can be seen that the control (untreated) and the control with untransduced (UT) T cells developed fast growing tumors. The two embodiments of the present invention (CAR28 T cells and CAR41 T cells), however, clearly blocked the tumor growth after an intratumoral single dose application and led to PR or CR. This proves that the concept works in vivo.

[0113] An intravenous single-dose injection of the CAR T cells of the present intervention did not cause growth inhibition of PSMA-positive solid tumors (FIG. 8). However, after chemotherapy with docetaxel, a single dose of intravenously injected CAR41 T cells led to a complete remission of large tumors (200-250 mm.sup.3) (FIG. 9).

[0114] D7-based CAR T cells were compared side-by-side with J591 and 3D8-based CAR T cells (FIG. 15). The antigen-specific activation profiles were compared in Jurkat cells transduced with expression vectors coding for D7, J591 and 3D8-based CARs. While the D7-based CAR and the J591-based CAR were able to mediate massive activation of the Jurkat cells upon antigen-specific sensitization (FIG. 15C), 3D8-CAR bearing cells were only weakly activated. Furthermore, upon transduction of primary T cells, D7-based CAR T cells revealed a superior cytotoxicity profile as compared to J591-based CAR T cells on two PSMA tumor cell lines (FIG. 15E, 15F), as evidenced by the fact that both tumor cell lines were eliminated with lower effector-to-target ratios.

[0115] In summary, it has been demonstrated that the D7-based anti-PSMA CAR T cells have unexpected properties that are superior to previously published PSMA-targeting CAR T cells, particularly in view of their superior in vitro cytotoxicity and high in vivo antitumor activity. They are hence promising tools for the development of novel immunotherapies for the treatment of advanced prostate cancer.

[0116] The present invention relates therefore to chimeric antigen receptors for T cells which comprise an antigen-binding fragment which binds specifically to the PSMA antigen. The antigen-binding fragment comprises preferably a V.sub.H and a V.sub.L fragment which are connected with a suitable linker. Moreover, the chimeric antigen receptor comprises preferably a spacer element, a transmembrane fragment and a CDR3ζ cytoplasmic domain. Furthermore, the chimeric antigen receptor preferably comprises a fragment from the CD28 (FIG. 12) and/or a 4-1BB (FIG. 13) cytoplasmic domain.

[0117] The chimeric antigen receptors of the present invention contain preferably at least three CDRs selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as shown in FIG. 10. The CDRs are shown by grey arrows above the amino acid sequence and the relevant nucleic acid sequence coding. In a preferred embodiment the chimeric antigen receptor comprises at least three (CDR-H1, CDR-H3, CDR-L3), preferably four (CDR-H1, CDR-H3, CDR-L2, CDR-L3), and more preferred at least five (CDR-H1, CDR-H3, CDR-L1, CDR-L2, CDR-L3) of the CDRs as shown in FIGS. 10 and 11, respectively.

[0118] The chimeric antigen receptors are preferably present in a humanized format. Such a humanized format can be obtained by inserting the at least three, preferably four, more preferred five or six CDRs into a suitable human antigen-binding scaffold having a high homology to the murine scaffold as shown in FIG. 11.

[0119] Usually the genetic information coding for the chimeric antigen receptor is introduced with the help of a suitable vector into the target immune cells (e.g. T cell). Such a vector can preferably be a lentivirus vector or a retroviral vector or a transposon or a plasmid. Alternatively, the genetic information can be introduced into the genome of the T cell in a targeted fashion with the help of designer nuclease technology, such as the CRISPR/Cas technology or the TALEN technology, as described herein.

[0120] In a further aspect the present invention relates to an in vitro method of providing T cells comprising a chimeric antigen receptor as described herein. In the first step T cells are isolated from a donor preferably by leukapheresis methods. This results in a substantial enrichment of the T cells. The T cells are then genetically modified by transfection with a suitable vector or by transduction with a viral vector containing the genetic information for the chimeric antigen receptor, respectively. Such genetically modified T cells can then be isolated and amplified whereby those T cells which do not contain the desired genetic information can be separated from the modified T cells or at least reduced. The transfected T cells can then be applied to the patient to be treated.

Example 1

[0121] Preparation of CAR Encoding Retroviral Particles

[0122] HEK293T cells were cultured at 37° C. in a humidified incubator with 5% CO.sub.2 in DMEM (Gibco, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin (100 U/ml), streptomycin (100 mg/L) and 10 mM HEPES (Sigma-Aldrich). One day prior to transfection, cells were seeded in 10 cm dishes at cell density of 5×10.sup.6 cells/dish. 24 h later, cells were transfected using polyethylenimine (PEI: 0.1 mg PEI/ml, Polysicence Inc., USA). Per 10 cm dish 3 μg of a plasmid encoding the VSV-G envelope, 6 μg of a gag/pol encoding plasmid, and 10 μg of a vector plasmid coding for either CAR28 or CAR41 were used. 48 and 72 h post-transfection, supernatants containing viral vectors were collected and concentrated using ultracentrifugation (WX ultra series; Thermo Scientific: 25,000 rpm for 2 hours at 4° C.). Concentrated vectors were suspended in 100 μl of cold PBS and kept at −80° C. until used. Biological titers of the vector preparations were determined by transducing the Jurkat T cell line, followed by staining of the transduced cells with anti-human IgG to determine CAR positive cells.

Example 2

[0123] Generation of PSMA-Targeting CAR T Cells

[0124] CAR T cells were generated from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated using phase separation (Ficoll, Sigma-Aldrich) according to the manufacture's recommendation and then frozen in liquid nitrogen until used. For generation of CAR T cells, PBMCs were thawed and let to recover for 24 h in RPMI complete medium [RPMI 1640 medium (Gibco, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (Biochrom, Berlin, Germany), penicillin (100 U/ml), streptomycin (100 mg/L) and 10 mM HEPES buffer (Sigma-Aldrich)]. Then, PBMCs were activated using anti-CD2/CD3/CD28 antibodies (Immunocult, StemCell Technologies) and cultured with RPMI complete medium supplemented with 100 U/ml of IL-2, 25 U/ml of IL-7 and 50 U/ml of IL-15 (all from Miltenyi Biotech) for 2 to 3 days before transduction with gamma-retroviral constructs encoding either CAR28 or CAR41 with MOIs ranging from 50-300. Transduced cells were cultured in wells coated with poly-D-lysin (PDL, Sigma-Aldrich) containing RPMI complete medium supplemented with 5 μg/ml of protamine sulfate (Sigma-Aldrich) and 1000 U/ml of IL-2, 25 U/ml of IL-7 and 50 U/ml of IL-15. After one day, medium was changed and cells were further expanded for 8-9 days in RPMI complete medium supplemented with 100 U/ml of IL-2, 25 U/ml of IL-7 and 50 U/ml of IL-15 before being frozen in liquid nitrogen until further use.

Example 3

[0125] Quality Assessment of CAR T Cells

[0126] To monitor CAR and TCR expression by flow cytometry (FACS Canto II or Accuri, BD Biosciences), cells were stained with anti-human IgG-PE (Southern Biotech) and CD3-APC (Miltenyi Biotec), respectively. As shown in FIG. 1B, up to 50% transduction efficiency was achieved for both CAR28 or CAR41. For quality assessment, CAR T cells were harvested and stained with anti-human CD62L-Bv421 (BD Biosciences), anti-human CD45RA-FITC (Biolegend), anti-human CD3-APC/H7 (BD Biosciences) and anti-human IgG-PE (CAR) (Southern Biotec) at the end of the expansion period (FIG. 1C, D). The T cell phenotype was determined based on the expression of CD62L and CD45RA. Cells were pre-gated on CD3+/CAR− for untransduced (UT) T cells or on CD3+/CAR+ for both types of CAR T cells (FIGS. 1C, D). Both the transduction and expansion protocol did not induce T cell differentiation, as high percentages of undifferentiated cells with naïve T cell (Tn), T stem cell memory (Tscm) or central memory T cell (Tcm) phenotypes, respectively, in combination with a low fraction of terminally differentiated effector T cells (Teff) were present (FIGS. 1C, D), indicating that the protocol allowed for the generation of high quality CAR T cell products.

Example 4

[0127] In Vitro Cytotoxicity of Manufactured CAR T Cells

[0128] The cytotoxic potential of the manufactured CAR T cells was determined by assessing cell viability using the XTT assay, as previously described. CAR T cells were co-cultured with either PSMA-positive C4-2 tumor cells or antigen-negative tumor control cells (Du145) in 96 well plates for 48 h at different effector to target ratios in a final volume of 200 μl/well of RPMI complete medium without any cytokines. To determine cell viability as a function of metabolic activity, 100 μl/well of medium was removed and replaced with 100 μl/well of XTT solution (Sigma-Aldrich) and cells incubated at 37° C. Colorimetric changes were quantified using an ELISA reader (Infinite F50, Tecan) at 450 nm. Cytotoxicity is indicated as the percentage of dead cells, which equals 100% minus the percentage of viable cells. Viability was calculated according to the equation [OD.sub.E+T−OD.sub.E only]/[OD.sub.T only−OD.sub.medium only] (E, effector cells=CAR T cells; T, target cells=tumor cells). As shown in FIG. 2A, CAR28 T cells eliminated ˜90% of target cells at an effector to target (E:T) ratio of 1:4, while CAR41 T cells demonstrated comparable activity at an E:T ratio of 1:1. Both types of CAR T cells revealed some minimal alloreactivity when co-cultured with PSMA-negative tumor cells (DU 145) (FIG. 2B). Together, the results demonstrate that both types of D7-based CAR T cells eliminate PSMA-positive target cells with high efficiency and specificity.

Example 5

[0129] Cytokine Release by Activated CAR T Cells

[0130] CAR T cells were co-cultured with either PSMA-positive C4-2 tumor cells or antigen negative DU 145 tumor cells for 48 h at an effector to target ratio of 1:1 in a final volume of 200 μl/well of RPMI complete medium without any cytokines. To evaluate the cytokines that were released from the CAR T cells during this time, supernatants were collected and evaluated by a multiplexed bead-based immunoassay (cytometric bead array, CBA assay, BD Biosciences) according to the manufacturer's recommendations. Three analytes, interferon gamma (IFN-g), granzyme A (Gr.A) and granzyme B (Gr.B), were determined (FIG. 2C).

[0131] Both types of the D7-based CAR T cells secreted the measured pro-inflammatory cytokines upon antigen stimulation, although CAR41 T cells released significantly less cytokines than CAR28 T cells.

Example 6

[0132] Cell Differentiation and Exhaustion Upon Antigen Stimulation

[0133] CAR T cells were stimulated with PSMA-positive C4-2 tumor cells for 24 h at an effector to target ratio of 1:1, before cells were harvested and stained for T cell phenotype and exhaustion profiles. Assessment of the CAR T cell phenotype upon antigen stimulation revealed that CAR41 cells were less prone to differentiate as compared to CAR28 cells, as indicated by the presence of a significantly higher number of undifferentiated T cell subtypes, such as Tn, scm and Tcm (FIG. 3A, B).

[0134] To monitor CAR T cell exhaustion upon specific antigen stimulation, CAR T cells were stimulated with PSMA-positive tumor cells (C4-2) for 24 h, before cells were harvested and stained with anti-human CD279-FITC (PD-1, BD Biosciences), anti-human CD223-eFluor710 (LAG-3, BD Biosciences), anti-human CD3-APC/H7 (BD Biosciences) and anti-human IgG-PE (Southern Biotec). The exhaustion profile was determined by flow cytometry (FACS Canto II) based on the expression of CD279 (PD-1) and CD223 (LAG-3). Cells were pre-gated either on CD3+/CAR− for UT T cells or CD3+/CAR+ for both types of CARs. Yet again, CAR41 cells displayed a different phenotype than CAR28 cells. The significantly reduced number of LAG-3 positive CAR41 T cells is indicative of a less exhausted T cell phenotype (FIG. 3C, D).

Example 7

[0135] PSMA expression of the prostate cancer target cells C4-2.sup.luc+

[0136] The PSMA expressing, androgen-independent prostate cancer cell line C4-2 was grown in RPMI 1640 medium (Gibco, Invitrogen, Karlsruhe, Germany) supplemented with penicillin (100 U/ml), streptomycin (100 mg/L) and 10% fetal calf serum (FCS, Biochrom, Berlin, Germany) at 37° C. in a humidified atmosphere of 5% CO.sub.2. For in vivo bioluminescence imaging, C4-2 cells were transduced with a lentiviral vector encoding firefly luciferase, a neomycin resistance gene, and green-fluorescent protein (GFP). The cells were named C4-2.sup.luc+ cells and selected with 2 mg/ml of the neomycin analog Geneticin (G-418 Sulfate) as previously described. C4-2.sup.luc+ cells were tested for PSMA expression by flow cytometry as described previously. In brief, 2×10.sup.5 C4-2.sup.luc+ cells/well in PBS with 3% FCS and 0.1% NaN.sub.3 were incubated with different concentrations of the anti-PSMA mAb 3/F11, the parental mAb of the scFv D7, for 1 h on ice. After washing, cells were incubated with 25 μl goat anti-mouse Ig-RPE (Becton-Dickinson, Mountain View, Calif.) for 40 min on ice. Cells were then washed repeatedly and suspended in 200 μl PBS containing 1 μg/ml propidium iodide, 3% FCS and 0.1% NaN.sub.3. Analysis of stained cells was performed using a FACSCalibur flow cytometer with CellQuest Pro software (BD Biosciences, Heidelberg, Germany).

[0137] As shown in FIG. 4A, the mAb 3/F11 bound with a binding constant (kd), defined as the half-maximal saturation concentration, of 0.96 μg/ml (about 6.3 nM) to the C4-2.sup.luc+ cells. This proved the high expression of PSMA on this cell line. C4-2.sup.luc+ cells were shown to be PSMA positive at saturation concentrations, while DU 145 prostate cancer control cells were shown to be PSMA negative (FIG. 4B).

Example 8

[0138] Bioluminescence of C4-2.sup.luc+ cells

[0139] Bioluminescence of the C4-2.sup.1″ cells was tested with help of the in vivo imaging system IVIS 200 (Xenogen VivoVision). For this, cells were seeded in different numbers into 96-well plates (Nunc Delta Surface, Thermo Fisher Scientific, Roskilde, Denmark) and incubated with 40 μl luciferin/well (BioSynth AG, Staad, Switzerland) as a substrate. Luminescence was analyzed using the software Living Image 3.0 within 10-30 min (Caliper Lifesciences).

[0140] Bioluminescence Imaging (BLI) showed that there was linearity between the number of C4-2.sup.luc+ cells and bioluminescence (quantified as region of interest (ROI). No luminescence signal could be detected with untransduced C4-2 cells and background staining was determined with ROI of max. 2.362×10.sup.6 photons/sec/cm.sup.2 (FIG. 5).

Example 9

[0141] Treatment of C4-2.sup.luc+ Tumor Xenografts by Intratumoral Injection of CAR T Cells

[0142] 5-6 week old male SCID CB17/lcr-Prkdc scid/Crl mice (20-25 g, Janvier Labs, St Berthevin Cedex, France) were injected with 1.5×10.sup.6 C4-2.sup.luc+ cells in PBS mixed with 50% Matrigel (Collaborative Biomedical Products, Chicago, Ill.) s.c. into the right flank. Tumor growth was monitored by BLI. For this, 200 μl of luciferin (BioSynth AG, Staad, Switzerland) were injected i.p. into the animals and BLI was done 10-30 min after injection under anesthesia using the Living imaging system IVIS 200 (Xenogen VivoVision). Tumor volumes were calculated from BLI pictures using the software Living Image 3.0 and the formula Volume=d.sup.2×D/2, where D was the large diameter and d the small diameter of the tumor. When tumors reached volumes of about 60-80 mm.sup.3, mice were injected intratumorally with only one dose of 5×10.sup.6 CAR28 (n=5) or CAR41 T cells (n=5), respectively (day 1 of treatment) (FIG. 6A). As controls, mice were injected with un-transduced T cells (UT, n=7) or left untreated (control, n=6). Tumor growth was monitored via BLI on days 1, 3, 8, 15, and 22 of treatment. Control groups (UT and untreated) showed a fast tumor growth. A reduced tumor growth was reached with the CAR41 T cells. A marked tumor regression was found in mice treated with CAR28 T cells (FIGS. 6B, C, and D). Until end of treatment at day 22, all animals of the control groups showed growing tumors. According to the animal protection guidelines, one mouse of the untreated group had to be killed due to an ulcerating tumor at day 15.

[0143] At day 22, mean tumor end volumes of 1469.4±961.5 mm.sup.3 (untreated control) and 1289.1±636.8 mm.sup.3 (UT) were reached, respectively. In contrast, 5/5 mice treated with CAR28 T cells showed a complete tumor remission (CR) from day 8 on and 4 mice remained tumor free until the end of the experiment. In the remaining animal a small tumor was detected at day 22 and verified by histological examination with a volume of 0.55 mm.sup.3. For all mice treated with CAR28, a mean tumor volume of 0.11±0.22 mm.sup.3 was calculated for day 22, which was statistically significant compared to the control groups (*p<0.05) (FIGS. 6B and 6D). Mice injected intratumorally with CAR41 T cells showed a different response to the treatment. One of the five animals treated showed a CR (FIGS. 6B and 6D). Another animal showed a PR, whereas the others had tumor progression until day 22 of treatment. In summary, a mean tumor volume in this group of 397.7±393.6 mm.sup.3 was determined (FIG. 6C).

Example 10

[0144] Adverse Side Effects

[0145] According to the animal protection guidelines, mice were observed for apparent signs of toxicity during treatment (loss of weight and appetite, changes in pelage, fever, tension, apathy, aggression, respiratory disorders, paralyses, death) and termination criteria were defined as follows: significantly reduced food intake, decrease of the initial body weight >20%, spasms, paralysis, abnormal breathing disorders, apathy, aggressiveness as a sign of severe pain, tumor diameter >20 mm, ulcerating tumor. With exception of one mouse of the untreated control group, which had to be killed at day 15 because of an ulcerating tumor, no mouse showed apparent signs of toxicity and no animal reached one or more determination criteria. FIG. 7 demonstrates that no animal showed a critical decrease of its initial body weight of >20%. Taken together, all animals have tolerated the intratumoral treatment with the anti-PSMA CAR T cells well without apparent signs of unspecific toxicity.

Example 11

[0146] Treatment of C4-2.sup.luc+ Tumor Xenografts by Intravenous Injection of CAR T Cells

[0147] Mice with C4-2.sup.luc+ tumor xenografts were intravenously injected with only one dose of 5×10.sup.6 CAR28 (n=5), CAR41 (n=5), or UT T cells (n=5) in 100 μl of PBS, respectively (day 1 of treatment) (FIG. 8A). Control mice (n=6) remained untreated. Tumor growth was monitored as described above. Compared to the control (tumor end volume: 1469.4±961.5 mm.sup.3) and the UT group (tumor end volume: 1275.7±544.5 mm.sup.3), mice injected with CAR28 T cells showed a similar tumor growth with a mean tumor volume of 1427.3±448.5 mm.sup.3 at the end of treatment at day 22 (FIGS. 8B and 8C). For animals treated with the CAR41 T cells a mean tumor volume of 1529.3±971.0 mm.sup.3 was determined (FIG. 8C). Tumor growth was not controlled in animals treated either with CAR41 or CAR28 intravenously (FIGS. 8B, C, and D).

[0148] In summary, intratumoral application of PSMA CAR T cells, both CAR28 and CAR41, completely eliminated tumors in vivo (FIG. 6). In contrast, intravenous injection of the CAR T cells did not lead to any antitumor activity (FIG. 8), suggesting that the intratumoral injection allowed the CAR T cells to bypass the TME, while upon intravenous injection the CAR T cells were likely inhibited by the TME.

Example 12

Treatment of C4-2.SUP.luc+ Tumor Xenografts by Combination of Chemotherapy and Intravenous Injection of CAR T Cells

[0149] Mice with large C4-2.sup.luc+ tumor xenografts (200-250 mm.sup.3) were intraperitoneally injected with 3 daily cycles of docetaxel (DOC, 6 mg/kg bw on days 1, 2, and 3 of treatment) (FIG. 9A). 48 hours after the last cycle, mice were intravenously injected with only one dose of 5×10.sup.6 CAR28 (n=3) CAR41 T cells (n=3), or PBS (n=3) respectively (FIG. 9A). Control animals (n=4) remained completely untreated. Tumor growth was monitored by BLI as described above. Compared to the control group, mice injected with DOC alone and with DOC+CAR28 showed an inhibition of tumor growth (FIG. 9B). Compared to the control group (1817.5±165.5 mm.sup.3) tumor end volumes of 338.2±355.6 mm.sup.3 for mice treated with DOC alone and of 599.9±317.2 mm.sup.3 for mice treated with DOC+CAR28 were reached on day 17 (FIG. 9C). In contrast, ⅔ animals treated with the DOC+CAR41 combination therapy showed CR and ⅓ mice a PR (FIG. 9B). A mean tumor volume of 50.2±71.0 mm.sup.3 was determined (FIG. 9C).

[0150] These results indicate that the D7-CAR T cells are effective in eliminating large tumors upon a single dose administration of CAR T cells in combination with chemotherapeutic agents, such as docetaxel (FIG. 9D).

Example 13

Humanization of Murine Sequences

[0151] In order to avoid later potential disadvantageous effects like antibodies generated in human patients against the murine part of the antigen binding fragment, the antigen binding fragment derived from the construct D7 was humanized.

[0152] In the course of humanizing the sequences the anti-PSMA binding sequence derived from the construct D7 was compared with the sequences of human stem cells. A sequence having a very high homology was selected and the area wherein the CDR binding sequences are located has been determined. For the determination of the CDR sequences several methods are known. The most preferred methods are the method according to Kabat and the IMGT method. IMGT, the International Immunogene Tics database, is a highly qualified integrated information system specializing in immunoglobulin molecules. Since the methods according to Kabat and IMGT provide somewhat different results, the sequences of different variants (hum D7 VH1-5 and hum D7 VL1-5) are shown in FIGS. 14 A and B. The relevant CDR regions according to Kabat and to IMGT are shown.

[0153] It is important to note that even by using the two different methods the sequence of CDRH3 having the amino acid sequence ARDGNFPYYAMDS (SEQ ID NO:11) and in CDRL3 having the sequence SQSTHVPT (SEQ ID NO:12) are identical according to both determination methods. It is therefore assumed that those two CDRs, namely CDRH3 and CDRL3 are important for the proper function of the construct. The humanized sequences as shown in FIGS. 14a and b correspond to SEQ ID NO:13 to 23.

Example 14

[0154] The biological activity of a preferred construct according to the present invention has been compared with a construct wherein the PSMA binding fragment is derived from the antibody J591. Furthermore, it was compared with a similar construct wherein the antigen binding construct was derived from another antibody (3D8).

[0155] The experiments and the results obtained therefrom are depicted in FIGS. 15A-15I.

[0156] FIG. 15A is a schematic representation of different anti-PSMA-CAR28 constructs which are identical except the PSMA binding fragment. In construct CAR28 (D7) the fragment according to the invention was used. In CAR28 (J591) the PSMA fragment was derived from the antibody J591 and in CAR28 (3D8) the PSMA fragment was derived from a PSMA binding antibody (3D8).

[0157] The constructs were introduced into Jurkat T-cell lines and the expression of the constructs was measured. In FIG. 15B the control is designated as UT (untransduced). FIG. 15B shows that all CAR constructs measured in flow cytometry are properly expressed in the Jurkat T-cell line except the control, which is designated as UT (untransduced).

[0158] In FIG. 15C the antigen-specific activation profile of the transduced JURKAT cells is measured by monitoring the expression of the activation marker upon antigen stimulation. As opposed to D7 and J591 based CARs the 3D8 based CAR mediated only weak activation of the transduced Jurkat cells upon expansion to PSMA expressed tumor cells.

[0159] FIG. 15D shows the CAR expression in primary T-cells. It can be seen that the construct according to the invention CAR28 (D7) is better expressed (42%) compared with CAR28 (J591) (30%). The expression of the construct wherein the PSMA binding fragment is derived from 3D8 is very low (4%). A construct which is not expressed to a sufficient extent in primary T-cells is not suitable for later therapeutic use.

[0160] The cytotoxicity profiles of the CAR28 T-cells on indicator cells, namely C4-2 (PSMA+/PDL1−); LNCap (PSMA+/PDL1+) and DU145 (PSMA−/PDI1+) is shown in in FIG. 15E-15G. To assess the cytotoxicity profile of the CAR T cells different ratios of effector (CAR28 T-cells):Target cells (different tumor cells, namely C4-2, LNCaP and DU145) have been determined. The construct according to the invention is superior to constructs having the antigen binding fragment derived from the construct J591, as can be seen from FIG. 15E and FIG. 15F. At lower effector:target ratios the construct according to the invention is always superior than the comparative construct. The effector target ratio is a very important aspect for the clinical use since very high effector:target ratios cannot be provided in real life. The lower the effector:target ratio is, the better the construct is.

[0161] FIG. 15H and FIG. 15I show the PSMA and PD-L-1 expression on the prostate cancer cell lines LNCaP, C4-2 and DU145.

Example 15

[0162] In an additional experiment it could be shown that a combination of chemotherapy together with CAR therapy is superior to chemotherapy alone.

[0163] The experiments are shown in FIG. 16A and FIG. 16B. FIG. 16A is a schematic of combination therapy of mice bearing subcutaneous C4-2.sup.luc xenografts with docetaxel (DOC) plus CAR T cells. Mice with 150-200 mm.sup.3 tumors were injected with 2 doses of 6 mg/kg bw DOC on days 1 and 2 followed by i.v. injection of CAR28 or CAR41 T cells on day 8. Tumor growth was monitored by palpation and BLI until day 22.

[0164] The results of the experiments are shown in FIG. 16B: in vivo antitumor activity of CAR28 and CAR41 T cells in combination with DOC in mice bearing subcutaneous C4-2.sup.luc xenografts. Statistically significant differences in tumor volume between DOC+CAR28 treated animals and untreated control were determined on days 15, 17, and 22 of treatment. Statistically significant differences in tumor volume between DOC+CAR28 treated animals and mice, which were only treated with DOC, were determined on days 17 and 22 of treatment. Moreover, tumor volume of animals treated with DOC+CAR41 was significantly smaller than that of the control group on day 22. This proves that combination treatment was superior to chemotherapy alone. Unpaired t-test, *p<0.05.

[0165] From the experiments it can be clearly seen that there is a synergistic effect of the CAR28 and CAR 41 constructs according to the invention together with a chemotherapeutic agent whereby the effect has been exemplified with docetaxel.