Chimeric antigen receptors that bind to prostate-specific membrane antigen

Abstract

The present invention relates to novel chimeric antigen receptors (CARs) comprising antigen-binding fragments that specifically bind to the PSMA antigen, and methods for producing high-quality CAR T cell products by gene transfer and / or transduction of T cells with the same, which can be used to effectively treat tumors in vivo, either alone or in combination with pharmaceuticals such as biopharmaceuticals, including chemotherapy and small molecule drugs such as antibodies or protein kinase inhibitors.

Description

[Technical field]

[0001] The present invention relates to a chimeric antigen receptor that binds to a tumor antigen, and the antigen is prostate-specific membrane antigen (PSMA). The chimeric antigen receptor (hereinafter referred to as CAR) is introduced into immune cells, particularly T cells, NK cells, iNKT cells, and CIK cells, and specifically reacts with tumor cells that express PSMA, leading to the elimination of tumor cells. The construct of the present invention includes two main parts: one is an antigen-binding region that specifically binds to prostate-specific membrane antigen (PSMA), and the other is a costimulatory and activation domain derived from the receptor of immune cells that is involved in the signal transduction and activation of immune cells. [Background technology]

[0002] Prostate cancer is the second most frequently diagnosed cancer in men worldwide, with an estimated 1.1 million new cases per year. In addition, it is expected to cause 307,000 deaths, making it the fifth leading cause of cancer deaths. Although primary tumors can be successfully treated, there is no cure for advanced stages. Thus, new treatment options are urgently needed.

[0003] Prostate-specific membrane antigen (PSMA) is the best characterized antigen in prostate cancer for antibody-based diagnosis and therapeutic intervention. The protein is also known as glutamic acid carboxypeptidase II (EC 3.4.17.21), N-acetylglucosidase I (NAALADase), or folate hydrolase. PSMA is a type II membrane glycoprotein of 750 amino acids with a small intracellular domain of 19 amino acids, a transmembrane domain of 24 amino acids, and a large extracellular domain of 707 amino acids. The extracellular domain folds into three distinct domains: a protease domain (57-116 and 352-590 amino acids), an apical domain (117-351 amino acids), and a C-terminal domain (591-750 amino acids). It shows high structural similarity and identity to human transferrin receptor 1. PSMA is highly restricted to the surface of prostate cancer cells, is present on cancer cells at all stages of tumor development, and shows enhanced expression in androgen-independent and metastatic disease. PSMA is not secreted into the extracellular space, but undergoes constitutive internalization that is enhanced by binding of PSMA-specific antibodies. These characteristics make it an ideal candidate for targeted therapy of localized and advanced prostate cancer. Furthermore, PSMA has been found to be expressed on the neovascular endothelium of virtually all solid tumor types, without expression on normal vascular endothelium. It is therefore considered to be a unique antiangiogenic target.

[0004] Monoclonal antibodies (mAbs) are highly specific and versatile tools for cell targeting. In the last decades, they have attracted high interest in medical research and have become the fastest growing class of drugs for treating various human diseases, including cancer. The antibody 7E11 was the first published PSMA-specific mAb and was found to bind to the N-terminus of the intracellular domain of PSMA (MWNLLH). An in-labeled form of 7E11 (ProstaScint, Cytogen, Philadelphia, PA) was approved by the US Food and Drug Administration (FDA) for the detection and imaging of metastatic prostate cancer in soft tissues. However, since this antibody binds to an intracellular epitope, 7E11 cannot bind to viable cells. A positive signal in in vivo imaging with ProstaScint had to be traced back to the detection of dead or dying cells within the tumor mass. Thus, a novel class of anti-PSMA mAbs was created that specifically bind to an extracellular epitope of PSMA expressed by living cells.

[0005] EP 1 883 698 discloses three different mAbs, namely 3 / A12, 3 / E7, and 3 / F11, that show strong and specific binding to the extracellular portion of PSMA on the surface of prostate cancer cells and prostate tissue samples. In a direct comparison with mAb J591 (PMID: 18552139; PMID: 24135437; PMID: 25771365; PMID: 26175541), a clinically effective antibody for radioimmunotherapy, mAb 3 / A12 showed higher binding to PSMA expressing C4-2 prostate cancer target cells (K d (Average half-saturation concentration (K d ) = 14 nM; K d=16 nM). Furthermore, competitive binding studies demonstrated that mAb 3 / A12 binds to a different extracellular PSMA epitope than J591 (PMID:19938014). In immunohistological studies on a panel of human normal tissues, no binding of 3 / A12 mAb to PSMA-negative tissues (adrenal gland, bone marrow, cerebellum, cerebrum, pituitary gland, colon, esophagus, heart, kidney, liver, lung, pericardium, nerve, ovary, pancreas, skeletal muscle, skin, spleen, stomach, testis, thymus, thyroid, tonsil, and uterus) was detected. Only binding to secretory cells was observed in salivary gland and duodenal brush border cells, known to express PSMA (PMID:19938014). mAb 3 / A12 showed moderate immunoreactivity to acinar secretory epithelial cells of all tested normal prostate tissues. Stronger and more widespread staining was observed in almost all epithelial cells of adenocarcinoma and lymph node metastases. No immunohistochemical staining was detected on frozen sections of breast samples, whereas staining of breast ductal epithelium was detected with mAb J591 in accordance with other published data. PSMA expression in breast tissue was not detected by PCR or by Western blotting, so mAb J591 likely cross-reacts with other antigens.

[0006] Alzubi et al. gave an oral presentation in October 2018 in Lausanne, Switzerland (Conference on Transforming Modern Medicine - Stem Cell and Gene Therapy, Lausanne, Switzerland, October 16-19, 2018). The oral presentation only showed an overview without substantial disclosure. The antigen-binding fragment used in the CAR was derived from monoclonal antibody 3 / F11. The monoclonal antibody is different from monoclonal antibody 3 / A12. The antigen-binding fragment used in the present invention was developed therefrom.

[0007] The single chain variable fragment (scFv) A5 (disclosed in WO 2006 / 125481) was produced by phage display technology from mAb 3 / A12. The most important fragment for the specificity of the anti-PSMA scFv is V L and V HA5 is a moiety, which is preferably linked to a polyglycinin linker. d It binds to PSMA-expressing cells at approximately 33 nM.

[0008] According to the present invention, the A5 scFv was used in the construction of a chimeric antigen receptor (CAR) to provide a construct for use in immune cells to target cancer cells expressing PSMA.

[0009] Ma et al. (The Prostate (2014) 74, pp 286-296) disclose a second generation CAR against PSMA that contains a CD28 costimulatory domain, a CD3ζ signal domain, and an antigen-binding portion from the mouse anti-human PSMA monoclonal antibody 3D8, commercially available from Northwest Biopharmaceutics, Inc.

[0010] Santoro et al. (Cancer Immunol.Res (2015), pp 68-84) describe T cells bearing a chimeric antigen receptor for prostate-specific membrane antigen. The PSMA-binding moiety is derived from the antibody J591.

[0011] Zhong et al. (Molecular Therapy (2010), pp 413-420) also disclose a chimeric antigen receptor. The PSMA-binding fragment is also derived from the antibody J591. The sequence of J591 is well known in the art. WO 2009 / 017823 discloses its VH and VL domains.

[0012] Hassani et al. (J. Cell Biochem (2019) 120:10787-10795) disclose the construction of a chimeric antigen receptor with a nanobody against PSMA. H It was used in the publication due to its high homology with the family, which should reduce the risk of an immunogenic response of the human anti-mouse antibody response.

[0013] WO 2018 / 111340 discloses a method for measuring the titer and proliferative function of chimeric antigen receptor (CAR)-T cells.

[0014] Cartellieri et al. (Blood (2015) 126(23):5549) describe a chimeric antigen receptor platform that should overcome the limitations of conventional CAR T cells.

[0015] WO 2010 / 037836 discloses a cross-species specific PSMA x CD3 bispecific single chain antibody capable of binding to an epitope on human and non-chimpanzee primate CD3YJ.

[0016] The antigen-binding fragment is derived from a murine sequence. The murine sequence of the variable domain of the heavy chain (VH) of A5 is shown as SEQ ID NO:1, and the murine sequence of the variable domain of the light chain (VL) of A5 is shown as SEQ ID NO:5. For those closely related fragments, the CDR regions were identified as follows: CDR-H1 was identified as SEQ ID NO:2 having the amino acid sequence of GFTFSDYYM, CDR-H2 was identified as SEQ ID NO:3 having the amino acid sequence of IISDGGY, and CDR-H3 was identified as SEQ ID NO:4 having the amino acid sequence of GFPLLRHGAMDY. For the CDRs in the light chain, CDR-L1 (SEQ ID NO:6) has the amino acid sequence KASQNVDTNVA, CDR-L2 (SEQ ID NO:7) has the amino acid sequence SASYRYS, and CDR-L3 (SEQ ID NO:8) has the amino acid sequence QQYDSYPYT.

[0017] In the above paragraph, the CDRs are provided according to the Kabat numbering system, but the CDRs can also be determined according to the IMGT numbering. The following sequences of the CDRs were determined according to the IMGT numbering: CDR-H1 was determined as SEQ ID NO: 9: GFTFSDYY, CDR-H2 was determined as SEQ ID NO: 10: ISDGGYYT, and CDR-H3 was determined as SEQ ID NO: 11: TRGFPLLRHGAMDYWG.

[0018] For the light chain, the sequence was determined according to the IMGT numbering: CDR-L1 was determined to be: SEQ ID NO:12: QNVDTN, CDR-L2 was determined to be: SEQ ID NO:13: SAS, and CDR-L3 was determined to be: SEQ ID NO:14: QQYDSYPYT.

[0019] Humanization of mouse antibodies involves the transfer of beneficial properties (e.g., antigen-specific binding, avoidance of off-target effects due to non-cross-reactivity with other antigens) from one antibody to another in order to reduce immunogenicity. Humanization is usually necessary for human use, since patients usually respond with an immune response against the non-human antibody, which can lead to ineffective treatment and, at worst, to a life-threatening situation. Humanized constructs can be derived from the sequences of antigen-binding fragments shown in SEQ ID NOs: 1 and 5, respectively. The CDR regions structurally define the antigen-binding site, i.e., the contact site between the antigen-binding fragment and the antigen. The remainder of the sequence encodes the framework regions that form the scaffold for the antigen-binding site. For the process of humanization (e.g., by in silico modeling), it is first compared with other antigen-binding sequences of human origin. Usually, the human sequence (receptor framework) with the highest similarity to the framework sequence shown in the mouse sequence is selected. The CDR regions are grafted onto the human receptor framework in order to remove amino acid sequences that may cause undesired human anti-mouse antibody (HAMA) immune responses. Substitutions at potentially important positions (e.g., amino acids involved in folding of the antigen-binding site or the VH-VL interface) are analyzed for possible back mutations. Even in CDR sequences, exceptional modifications of amino acids can be made to avoid immunogenicity, ensure correct folding of the antigen-binding site, and maintain antigen-specific binding.

[0020] In the process of humanizing an antibody, a sequence is preferably selected from among the human immune sequences that have the highest homology with the corresponding mouse sequence.Then, the position of CDR is determined. The determination of CDR is well known in the art. It should be noted that various measurement methods are known, which may result in slightly different CDR positions. In the process of the present invention, the CDR measurement according to Kabat is used, and the measurement according to IMGT (International Immunogene Ticks) is also used.

[0021] In a preferred embodiment of the invention, the humanization was performed according to the so-called "CDR grafting". Functional CDRs are determined, preferably according to the IMGT method, and these CDRs are transferred into the human framework regions with the highest sequence homology to the starting mouse antibody. Then, differences regarding single amino acids in the framework regions of the humanized antibody and the mouse antibody are determined with respect to biochemical properties such as size, polarity or charge. In a first step, similar amino acids are matched and successively different amino acids are changed to end up with a completely human framework.

[0022] Since the humanized versions disclosed herein either completely or to a great extent maintain the CDRs, the humanized variants have the same functionality of the murine antibody, although their affinities may differ somewhat from each other.

[0023] The sequences obtained in the humanization experiments are shown as SEQ ID NOs: 15-36.

[0024] The results of the humanization experiments are shown in Figures 1 to 6. Shown is always the nucleic acid sequence of the coding strand. Also shown are the amino acid sequences with the CDR regions shown in grey. These CDR sequences are preferred embodiments of the invention. In most cases, the CDRs of the heavy chain were found to be: CDR-H1: GFTFSDYY, CDR-H2: ISDGGYYT and also CDR-H3: TRGFPLLRHGAMDYWG.

[0025] For the light chain, it is preferred to use the following CDRs: CDR-L1: GNVDTN, CDR-L2: SAS, and CDR-L3: QQYDSYPYT. The preferred humanized CDRs were deduced from the mouse sequence but fitted into the appropriate human framework.

[0026] In a preferred form, the humanized antigen-binding fragment, such as preferably an scFv fragment, has at least three, preferably at least four, more preferably at least five, and especially preferably six CDRs, as described above.

[0027] For good maintenance of specific binding to PSMA-positive cells, it has been found that in particular the following humanized VH and VL chains should be used: human A5-VL1, human A5-VL4, human A5-VL5 and human A5-VL6. These sequences can preferably be combined with any of the humanized sequences human A5-VH1, human A5-VH2, human A5-VH3, human A5-VH4 and human A5-VH5. The use of human A5-VL2 and human A5-VL3 is less preferred.

[0028] In recent years, adoptive immune cell therapy has been introduced as a novel concept to treat different cancers by re-directing the immune system to eliminate tumor cells. One of the most successful concepts is based on the genetic engineering of T cells expressing chimeric antigen receptors (CARs) that bind tumor antigens or tumor-associated antigens in a human leukocyte antigen (HLA)-independent manner. CD19 targeting CAR T cells have been successfully used to treat B-cell acute lymphoblastic leukemia (B-ALL), leading to complete remission in more than 90% of patients in several clinical trials. Based on this success, more than 200 clinical trials have been initiated, mainly to treat hematological malignancies. However, in the case of solid tumors, the potential of CAR T cell therapy seems rather low so far. The main reason for this failure seems to be the tumor microenvironment (TME), the cellular environment in which the tumor resides. It induces various types of immune cells, fibroblasts, and extracellular matrix (ECM), as well as the surrounding blood vessels. Many mechanisms have been described that elucidate the restriction of cytotoxic T cell activity in the TME, including the activation of PD-1-based T cell immune checkpoint inhibition. Overcoming these limitations, in combination with T cell checkpoint antagonists, would help improve antitumor activity in the TME.

[0029] Eradication of tumors as used in the present invention requires proper survival and intratumoral activation of tumor antigen-specific immune cells, preferably T cells. To meet these requirements, T cells must be provided with appropriate activation signals upon antigen priming and stimulation. Thus, the chimeric antigen receptor of the present invention binds an antigen-binding fragment as part of the receptor on T cells. The antigen-binding fragment binds to the specific antigen (here PSMA) to which the T cells should bind. In addition, the receptor contains sequences from CD28 and 4-1BB, respectively, as costimulatory signal domains. The addition of CD28 sequences or other costimulatory signal domains to CD3ζ chain-based receptors has been shown to increase antigen-induced secretion of interleukin-2 and in vitro T cell expansion. In the present case, the signal domains consist of the CD3ζ domain and either the intracellular CD28 or 4-1BB domains.

[0030] In general, the design of CARs can vary. Meanwhile, several generations of CARs are known. The main components of the CAR system are the CD3ζ intracellular domain, the transmembrane domain, the hinge region, and the antigen-binding portion of the T cell receptor (TCR) complex. In the design of CARs, the antigen-binding domain is linked to the hinge region, also called the spacer region, the transmembrane domain, and the cytoplasmic domain. These portions are responsible for the location of the antigen-binding portion, its binding in the T cell membrane, and intracellular signaling. Besides this structural rule in CAR design, the morphological features of the hinge region (e.g., length and sequence, etc.) are important for efficient targeting. The intracellular domain acts as a signal transducer. The cytoplasmic segment of CD3ζ plays a major role, since its function is different in activated and resting T cells. However, this cytoplasmic portion cannot activate only resting T cells. Therefore, at least a secondary signal for the complete activation of T cells is required. In the present invention, the 4-1BB or CD28 costimulatory domain was preferably used. Other costimulatory domains, such as those derived from CD27, ICOS, and 0X40, can be substituted.

[0031] In a preferred embodiment, mutations are introduced into the human IgG1 Fc hinge region, thereby preventing LcK activation and avoiding the side effect of unintended initiation of an innate immune response. One of these mutations may prevent LcK binding, and another of the mutations may inhibit Treg cell binding to the construct. Such mutations may improve the biological activity of the construct.

[0032] To treat human patients, T cells must be enriched from the peripheral blood of the individual patient (autologous setting) or provided by a donor (allogeneic setting). This can be done, for example, by leukapheresis. The enriched T cells are then transduced or transduced ex vivo with a suitable vector containing the genetic information of the CAR.

[0033] The genetic information encoding the CAR is inserted into a suitable vector. Such vectors are preferably lentiviral or retroviral vectors. The gold standard for transduction of primary T cells is currently considered to be lentiviral vectors, which appear to be an effective alternative to the simpler retroviral vectors. As a further alternative to lentiviral vectors, the information can be introduced into T cells with the help of transposons or plasmids. An alternative to both viral and non-viral delivery is the recently described gene editing tools designed as CRISPR / Cas or other designer nucleases such as transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs). This technology platform offers the possibility to target virtually any genomic site in a targeted manner. In the case of CRISPR / Cas, the editing complex comprises a Cas nuclease and a guide RNA, which usually consists of a CRISPR RNA (crRNA) and a trans-acting crRNA. Upon hybridization of the guide RNA to the target sequence, Cas9 (or other Cas nucleases, such as Cpf1 / Cas 12a) produces a double-stranded break that can be repaired by non-homologous end joining (NHEJ), an event that can result in loss of function of the genomic locus. In the presence of a suitable donor DNA, the mechanism of homology-directed repair (HDR) can introduce an exogenous sequence (CAR sequence) into the target locus. This can be utilized to deliver a CAR expression cassette at a desired genomic locus that does not interfere with endogenous gene function, thereby minimizing the genotoxic effects experienced by integrating a viral vector. In a further preferred embodiment, genome editing is used to place the CAR-encoding sequence under the control of an endogenous promoter. Expression from an endogenous promoter, such as the promoter of the TRAC locus, can ensure optimal expression levels of the CAR construct and achieve its function. The nucleic acid sequence encoding the CAR of the present invention is preferably optimized for human codon usage.Particularly preferred embodiments are SEQ ID NO:37, which encodes the CAR28 construct (Figures 7A-D) and SEQ ID NO:38, which encodes the CAR41 construct (Figures 8A-D).

[0034] In another preferred embodiment of the present invention, the RNA encoding the CAR construct is introduced into target cells, such as T cells. The nucleic acid encoding the CAR construct can be introduced into target cells by physical procedures such as electroporation, or by fusion of the cell membrane with suitable vesicles. In this embodiment, the CAR construct is preferably expressed transiently in T cells. The advantage is that the population of transduced T cells is only present temporarily in the treated patient.

[0035] In other preferred forms, the CAR construct is introduced into natural killer cells (NK), invariant natural killer T cells (iNKT), diverse natural killer cells (dNKT), cytokine-induced killer cells (CIK), or gamma-delta T cells. Additionally, suitable allogeneic cells may be used.

[0036] In a further preferred embodiment, in addition to the CAR construct described herein, another transgene that regulates the immune system can be introduced into the immune cells, for example, a gene encoding a cytokine, a chemokine receptor, and / or a checkpoint inhibitor. In a further preferred embodiment, genome editing is used to disrupt the expression of a gene that regulates the immune system, such as a gene encoding a cytokine, a chemokine receptor, and / or a checkpoint inhibitor.

[0037] In another embodiment, the constructs according to the present invention and immune cells containing such constructs can be used for localized treatment by targeted tumor injection. This embodiment is preferably carried out using an automated device that applies CAR T cells to a specific location on the patient's body where the local tumor area is located. A biopsy needle is then used to withdraw the sample, which creates a small cavity. Transduced or transduced T cells are introduced into this cavity, and then the needle is withdrawn. This embodiment is particularly advantageous when there is a solid tumor that is very difficult to treat with conventional methods.

[0038] The chimeric antigen receptor disclosed herein can be used to treat diseases associated with the expression of PSMA. PSMA is expressed in tumor cells derived from prostate cancer. Although there are several stages of prostate cancer, PSMA appears to be one of the most suitable markers for the treatment of prostate cancer. The term "prostate cancer" includes any form of prostate cancer cell derived from primary tumor, metastatic tumor, or circulating tumor cell. In a particularly preferred embodiment, the immune cells engineered with the chimeric antigen receptor of the present invention are used against the angiogenesis of solid tumors expressing PSMA.

[0039] Thus, the chimeric antigen receptor or nucleic acid encoding according to the present invention can be used either as a nucleic acid alone or in a format embedded in a vector for use in the treatment of prostate cancer and prostate-derived tumors. The use of a vector with an embedded encoding nucleic acid is a preferred embodiment. The vector can be used in such a way that it is either already introduced into the patient in the appropriate immune cells, preferably T cells, or applied as a nucleic acid that can enter the appropriate cells of the patient.

[0040] Prostate cancer affects a high percentage of men and causes a very high percentage of cancer deaths. Although the initial stage of the cancer may be localized in the prostate, the disease often progresses to metastasis, which affects other organs of the human body, namely bones, liver, hepatic system, lungs, brain, and sometimes other organs. Prostate cancer metastasis can be treated by injecting T cells obtained from the patient that have been transformed with a suitable nucleic acid encoding a chimeric antigen receptor according to the present invention. Alternatively, such cells can be specifically inserted into metastatic hotspots in the patient's body, for example by injection. The distribution of where prostate cancer cells are localized can be performed by labeling the area with a suitable detection means, such as an antibody against PSMA labeled with a radionuclide sequence scan of the patient's body.

[0041] In a further embodiment of the present invention, the immune cells engineered with chimeric antigen receptors disclosed herein are used in combination with therapeutic agents, particularly cytotoxic agents. Cytotoxic agents used in the treatment of prostate cancer are known. Preferably, such agents include taxol derivatives, 5-fluorouracil, cyclophosphamide, mitoxantrone, docetaxel, cabazitaxel, and etoposide. The following drugs are approved for prostate cancer and are preferably used: abiraterone acetate, apalutamide, bicalutamide, cabazitaxel, casodex (bicalutamide), degarelix, docetaxel, eligard (leuprolide acetate), enzalutamide, erreada (apalutamide), firumagon (degarelix), flutamide, goserelin acetate, jebatana (cabazitaxel) ), leuprolide acetate, luprondep (leuprolide acetate), mitoxantrone hydrochloride, nilandrone (nilutamide), nilutamide, provenzi (sipulcel-T), radium 223 dichloride, sipuleucel-T, taxotere (docetaxel), zofigo (radium 223 dichloride), xandi (enzalutamide), zoladex (goserelin acetate), zytiga (abiraterone acetate). As will be appreciated, the chimeric antigen receptors of the present invention may also be used in combination with drugs used to treat hormone-sensitive forms of prostate cancer (e.g., leuprolide acetate).

[0042] Preferred embodiments of the present invention are further described and illustrated in the figures and examples of this application. All aspects disclosed in these figures or examples relate to the present invention unless expressly excluded. Single features of the present invention disclosed in the experimental section can be combined unless there is a technical reason to refer to such combination. Specifically, these figures show the experimental results as follows:

[0043] The following abbreviations have been used in the figures and experiments: JPEG0007625281000001.jpg193134JPEG0007625281000002.jpg55134

[0044] It should be mentioned that the sequences, figures and sequence protocols disclosed herein, especially the CDR-sequences, are preferred embodiments of the present invention. [Brief description of the drawings]

[0045] Figure 1-6: Humanized sfv sequences derived from A5

[0046] [Figure 1-3] Shown in Figures 1-3 are the sequences of five humanized variants of the variable domain of the heavy chain (VH) of A5, whereby the positions of CDR-H1, CDR-H2, and CDR-H3 are shown as grey boxes according to the determination of IMGT.

[0047] [Figure 4-6] Shown in Figures 4-6 are the sequences of six humanized variants of the variable domain of the light chain (VL) of A5, with their CDR-L1, CDR-L2, and CDR-L3 shown in grey boxes. As will be understood, the VH and VL domains can be combined with each other. For example, the VH sequence of variant 1 (human A5-VH1) can be combined with the VL sequence of variant 5 (human A5-VL5).

[0048] [Figure 7] Figures 7A-D show the complete sequence of a preferred embodiment of a construct comprising the scFv A5 antigen-binding region linked to the relevant portions of CD28 and CD3ζ.

[0049] [Figure 8] 8A-D show all constructs of the antigen-binding fragment scFv A5 linked to human 4-1BB and the relevant portion of CD3ζ.

[0050] [Figure 9] Comparison of the CAR constructs according to the invention with constructs of PSMA antigen-binding fragments already described in the prior art

[0051] (A) Schematic of the self-inactivating gamma-retroviral vector for expressing second generation anti-PSMA CARs. CAR expression is driven by the EFS promoter. Anti-PSMA CARs contain either single chain variable fragments (scFvs) 3D8 or J59 (both described in the prior art) or A5 (from mouse monoclonal antibody 3 / A12 against PSMA) fused to a hinge region from Fc IgG1, a transmembrane (tm) domain, a costimulatory domain from CD28, and an intracellular signaling domain derived from the CD3 ζ chain. The hinge region provides a physical spacer between the scFv and tm domains for optimal target recognition. The CD28 costimulatory domain contains amino acid exchanges that prevent LCK binding and enhance antitumor activity in the presence of inhibitory regulatory T cells (Tregs). All retroviral constructs produced have the same scaffold but differ only in the scFv fragments that allow for side-by-side comparison of CAR constructs in terms of activity and cytotoxicity.

[0052] (B) CAR expression in Jurkat cells was transduced with three different PSMA-CAR-encoding retroviral particles. Following viral transduction, Jurkat cells were grown for 16-21 days, and cells were harvested and stained with an anti-human IgG antibody (CAR) to assess transduction efficiency and CAR expression levels.

[0053] (C) Antigen-specific activation profile. Jurkat cells expressing CAR were stimulated with either PSMA-positive, PD-L1-negative C4-2 tumor cells (PSMA+ / PDL1-) or PSMA-positive, PD-L1-positive LNCaP tumor cells (PSMA+ / PDL1+) at a 1:1 effector-to-target ratio for 24 h. As a negative control, CAR cells were co-cultured with PSMA-negative DU145 tumor cells (PSMA-). After 24 h stimulation, cells were harvested and activation profiles were assessed by assessing the percentage of cells positive for the activation marker CD69. Statistical significance is indicated by **** (P<0.001).

[0054] (D) Characterization of the prostate cancer cell lines used. The degree of PSMA target antigen expression (left) and PD-L 1 (CD274) expression (right) was assessed for C4-2, LNCaP, and DU145 prostate cell lines. For flow cytometry analysis, cells were stained with 3 / F11 1 antibody (anti-PSMA) or anti-CD274 antibody. UT: untransduced T cells; PD-L1: programmed cell death ligand 1; PSMA: prostate-specific membrane antigen.

[0055] As shown in panel D (left), both C4-2 and LNCaP tumor cells express the PSMA antigen, while DU145 are negative for the PSMA antigen. Panel D (right) shows that the majority of LNCaP and DU145 cells express PD-L1, while C4-2 cells are negative for PD-L1.

[0056] The antigen-specific activation profile of A5-based CAR T Jurkat cells was compared side-by-side with J59 and 3D8-based CAR T Jurkat cells. As shown in panel C, A5-based CAR and J59-based CAR were able to mediate massive activation of Jurkat cells upon antigen-specific sensitization, as measured by upregulation of the activation marker CD69 in approximately 70% of the cells. Activation was not affected by the presence of the inhibitory ligand PD-L1. On the other hand, 3D8-based CAR T cells were only poorly activated (up to 20% of CD69-positive cells).

[0057] [Figure 10] Functional evaluation of PSMA-CAR T cells. (A) Cytotoxicity profile of CAR T cells on PSMA-positive C4-2 tumor cells. T cells were activated with anti-CD2 / 3 / 28 antibodies for 2–3 days prior to retroviral transduction. CAR T cells were generated by transducing activated T cells with a self-inactivating retroviral vector construct to express a second-generation anti-PSMA CAR (see Figure 9A). After 6–9 days of expansion without antigenic stimulation, CAR T cells were co-cultured with either antigen-positive C4-2 (PSMA+ / PDL1+) or LNCaP (PSMA+ / PDL1-) tumor cells, or PSMA-negative cells (DU145) at a given effector-to-target (E:T) ratio for 48 h. Cytotoxicity was measured by an XTT ELISA-based colorimetric assay (% cytotoxicity = 100%-% viability). Statistical significance is indicated by * (P<0.05) or ** (P<0.01). UT: untransduced cells; PSMA: prostate-specific membrane antigen.

[0058] The cytotoxicity profile of A5-based CAR T cells was compared side-by-side with that of J59-based CAR T cells. A5-based CAR T cells were significantly more cytotoxic against both tumor cell targets compared to J59-based CAR T cells. This is evidenced by the fact that both tumor cell lines can be eliminated at a lower effector-to-target ratio. In conclusion, A5-based CAR T cells outperformed CAR T cells based on prior art CARs in terms of activation and cytotoxicity.

[0059] [Figure 11] Proliferation and exhaustion of PSMA targeting CAR T cells. (A) CAR expression. After transduction of activated T cells with a self-inactivating retroviral vector construct to express a second generation anti-PSMA CAR (see Fig. 9A, 10A), CAR T cells were expanded for 6 days without antigenic stimulation (-Ag). In parallel, CAR T cells were expanded and exposed to irradiated PSMA-positive (C4-2) cells (+Ag). Expanded CAR T cells were harvested every 3 days and exposed to fresh irradiated antigen-positive cells at an effector-target ratio of 1:1 up to 4 times ((4X). CAR and CD3 expression at the indicated exposure times to irradiated PSMA-positive cells were visualized by flow cytometry after staining the cells with antibodies against human IgG (CAR) and CD3. UT: untransduced cells.

[0060] (B, C, D) CAR T cell proliferation. Cells were harvested every 3 days during CAR T cell proliferation and stained with anti-human IgG antibody (CAR) and CD3 to assess the fraction of CAR-positive cells (B), total cell number (C), and cell division number (D). Statistical significance is indicated by * (P<0.05). UT: untransduced cells.

[0061] As shown, multiple exposures to antigen-expressing cells lead to enrichment (>95%) of CAR-positive T cells in the cell population for both A5- and J59-based CAR T cells (Figure 11A-B). However, A5-based CAR T cells proliferate significantly better with multiple antigen-specific stimulation compared to J59-based CAR T cells. This is evidenced by an approximately 160-fold increase in total cell number at the end of the expansion period (Figure 11C) and a marked increase in the number of cell divisions (Figure 11D). This data suggests that A5-based CAR T cells are less exhausted than J59-based CAR T cells.

[0062] (E) Quantitative assessment of CAR T cell phenotype. CAR T cells were stimulated with PSMA-positive C4-2 tumor cells at a 1:1 effector-to-target (E:T) ratio before the T cell phenotype profile was assessed based on CD62L and CD45RA expression. The average percentages of different T cell subsets from three experiments are shown. Statistical significance is indicated by *** (P<0.001, n=3). Tn / Tscm: T cell naive or T stem cell memory; Tcm: T cell central memory; Tem: T cell effector memory; Teff: T cell effector; UT: untransduced cells.

[0063] (F) Quantitative assessment of CAR T cell exhaustion. CAR T cells were stimulated with PSMA-positive C4-2 tumor cells at a 1:1 effector-to-target (E:T) ratio before T cell exhaustion profiles were determined based on LAG-3 and PD-1 expression. The mean percentages of PD-1 and LAG3 double positive or double negative cells are shown. Statistical significance is indicated by *** (P<0.001, n=3). UT: untransduced cells.

[0064] The experimental set showed the CAR T cell phenotype and the exhaustion profile of CAR T cells upon antigen stimulation. As seen in panels 3E and 3F, A5-based and J59-based CAR T cells, respectively, differentiate towards effector cells and are exhausted to some extent after antigen exposure. However, the fraction of naive and memory CAR T cells is significantly higher for A5-CAR T cells compared to J59-based CAR T cells (Figure 11E). Also, A5-CAR T cells are exhausted less frequently upon antigen exposure than J59-based CAR T cells, as indicated by a very high proportion of PD1-negative / LAG3-negative CAR T cells (Figure 11F). Overall, the CAR T cell phenotype and the exhaustion profile analysis upon antigen exposure show a favorable phenotype of A5-CAR T cells over J59-CAR T cells, which correlates with the better proliferation of A5-CAR T cells compared to J59-CAR T cells.

[0065] [Figure 12] The sequence of mouse scFv A5 is provided (SEQ ID NOs: 1 and 5), which shows the regions of CDR-H1, CDR-H2, and CDR-H3 (SEQ ID NOs: 2 to 4), CDR-L1, CDR-L2, and CDR-L3 (SEQ ID NOs: 6 to 8) according to the Kabat method.

[0066] [Figure 13] Sequence Figure 13A-C shows the amino acid sequence of the CAR construct A5CAR28 containing the mouse-derived scFv A5 ("Frame 1"). Additionally, the coding nucleic acid sequence and its complementary strand are provided. The CDR H1-H3 and CDR L1-L3 nucleic acid and amino acid sequences are marked with grey arrows.

[0067] [Figure 14]Sequence Figure 14A-C shows the amino acid sequence ("frame") of the CAR construct A5CAR 41, which comprises the murine-derived scFv antigen-binding construct A5. Additionally, the coding nucleic acid sequence and its complementary strand are provided. The CDR H1-H3 and CDR L1-L3 nucleic acid and amino acid sequences are marked with grey arrows. The amino acid sequence of the scFv fragment of A5 is essential for the present invention insofar as it is the starting sequence for the humanization of the antigen-binding fragment. The humanized sequence has at least 80% homology with SEQ ID NO:1 or / and SEQ ID NO:5, respectively. In a more preferred embodiment, said sequence has at least 90%, more preferably at least 95%, even more preferably at least 98% homology with SEQ ID NO:1 and / or SEQ ID NO:5. It should be noted that the CDR regions shown in Figure 13 are conserved at a very high level. This means that the CDR regions as determined by the Kabat method have no more than three amino acid exchanges, preferably no more than one amino acid exchange, and preferably no amino acid exchanges. Figure 14A shows the sequence of the A5 CAR41 construct, and the preferred homology values ​​listed above also apply to that construct.

[0068] JPEG0007625281000003.jpg211134JPEG0007625281000004.jpg49134

[0069] The results of the experiments shown in the figures can be interpreted as follows.

[0070] Generally, CARs contain an extracellular domain containing an antigen-recognizing scFv, a hinge region, a transmembrane region, and one or more intracellular signal domains that activate T cells, including the CD3ζ chain. Second or third generation CARs usually contain costimulatory domains derived from CD28, 4-1BB, 0X40, CD27, and / or ICOS. Many attempts have utilized CARs targeting PSMA epitopes to treat prostate cancer, and some of these methods have already entered clinical trials (e.g., NCT01140373, NCT01929239). However, the efficacy of these CARs appears to be rather low both in vitro and in vivo. In particular, early studies based on first generation CARs based on anti-PSMA scFv 3D8 or J591 (known as Pzl) showed low efficacy of the resulting CAR T cells, as indicated by the need to use high effector-to-target (E:T) ratios of up to 100:1 to eliminate tumor cells in vitro. The use of second- or third-generation CARs based on scFvs derived from D2B or J591 improved in vitro potency, but required very high CART cell doses, sometimes up to 20x10 6 Despite the fact that multiple injections of CAR T cells or multiple injections were applied (PMID 16204083, PMID:18026115, PMID:19773745, PMID:25358763, PMID:23242161, PMID:4174378, PMID:25279468), the efficacy of these CAR T cells remained low in xenograft tumor mouse models, as shown by the fact that these PSMA-targeting CAR T cells were able to suppress tumor growth but failed to eliminate tumors in vivo. Given the mixed results with these rather inefficient PSMA-CAR T cells, we intended to produce and validate a novel, more efficient PSMA-targeting CAR based on scFv A5. For this purpose, we designed a second-generation PSMA-CAR that contains a costimulatory domain derived from CD28 (CAR28).

[0071] A5-based CAR T cells were compared side-by-side with J59- and 3D8-based CAR T cells (Figure 9). Antigen-specific activation profiles were compared in Jurkat cells transduced with expression vectors encoding A5-, J59-, and 3D8-based CARs. A5- and J59-based CARs were able to mediate extensive activation of Jurkat cells upon antigen-specific sensitization (Figure 9C), whereas cells containing 3D8-CAR were only weakly activated.

[0072] Furthermore, upon transduction of primary T cells, A5-based CAR T cells exhibited a superior cytotoxicity profile compared to J59-based CAR T cells in two PSMA-positive tumor cell lines, as evidenced by the fact that both tumor cell lines were eliminated at a lower effector-to-target ratio (Figure 10A). Also, in the presence of antigen-bearing tumor cells, the manufactured A5-CAR T cell product proliferated better (Figure 11C), was less exhausted (Figure 11F), and had a higher proportion of undifferentiated T cells, such as naive T cells and T stem cell memory cells, compared to J59-based CAR T cells (Figure 11E).

[0073] In summary, A5-based anti-PSMA CAR T cells were demonstrated to have unexpected properties superior to previously published PSMA-targeted CAR T cells, especially considering their superior in vitro cytotoxicity, as well as their T cell phenotype and their proliferation and exhaustion profile upon antigen-specific stimulation. Based on these results, A5-based CAR T cells are promising tools for the development of novel immunotherapies for the treatment of localized and advanced prostate cancer.

[0074] Thus, the present invention relates to a chimeric antigen receptor for T cells comprising an antigen-binding fragment that specifically binds to the PSMA antigen. The antigen-binding fragment is preferably a V-linked fragment linked with a suitable linker. H and V LThe chimeric antigen receptor preferably comprises a spacer element, a transmembrane fragment, and a CDR3ζ cytoplasmic domain. The chimeric antigen receptor preferably comprises a fragment from the CD28 and / or 4-1BB cytoplasmic domain.

[0075] The chimeric antigen receptor of the present invention preferably comprises at least three CDRs selected from the group consisting of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. The CDRs are indicated by grey arrows above the amino acid sequence and related nucleic acid sequence coding. In a preferred embodiment, the chimeric antigen receptor comprises at least three CDRs (CDR-H1, CDR-H3, CDR-L3), preferably four CDRs (CDR-H1, CDR-H3, CDR-L2, CDR-L3), more preferably at least five CDRs (CDR-H1, CDR-H3, CDR-L1, CDR-L2, CDR-L3).

[0076] Said chimeric antigen receptor is preferably present in a humanized format, which can be obtained by inserting at least three CDRs, preferably four CDRs, more preferably five or six CDRs, into a suitable human antigen-binding scaffold with high homology to the mouse scaffold as shown in Figures 1 to 6.

[0077] Example 1 Preparation of CAR encoding retroviral particles HEK293T cells were cultured in DMEM (Gibco, Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (Biochrom, Berlin, Germany), penicillin (100 U / ml), streptomycin (100 mg / L), and 10 mM HEPES (Sigma-Aldrich) at 37°C in a humidified incubator with 5% CO2. One day before transfection, cells were cultured at 5 × 10 6Cells were seeded in 10 cm dishes at a cell density of 1000 cells / dish. After 24 hours, cells were transduced using polyethyleneimine (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 plasmid encoding gag / pol, and 10 μg of a vector plasmid encoding each CAR construct (FIG. 9A) were used. After 48 and 72 hours of transduction, the supernatants containing the viral vectors were collected and concentrated using ultracentrifugation (WX Ultra Series; Thermo Scientific: 25,000 rpm, 4°C for 2 hours). The concentrated vectors were suspended in 100 μl of cold PBS and stored at -80°C until use.

[0078] The biological titer of the vector preparation was measured by transducing Jurkat T cells and then staining the transduced cells with anti-human IgG to measure the fraction of CAR-positive cells.

[0079] Example 2 Generation of PSMA-targeting CAR T cells Peripheral blood mononuclear cells (PBMCs) were isolated using phase separation (Ficoll, Sigma-Aldrich) according to the manufacturer's recommendations and then frozen in liquid nitrogen until use. To generate CAR T cells, PBMCs were thawed and allowed to recover for 24 hours 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)]. PBMCs were then activated with anti-CD2 / CD3 / CD28 antibodies (Immunocult, Stemcell Technologies), cultured for 2–3 days in RPMI complete medium supplemented with 100 U / ml IL-2, 25 U / ml IL-7, and 50 U / ml IL-15 (all from Miltenyi Biotech), and transduced with γ-retroviral constructs encoding either PSMA-targeted CAR T cells at doses ranging from 50 to 300 transducing units per cell. Transduced cells were cultured in poly-D-lysine (PDL, Sigma-Aldrich)-coated wells containing RPMI complete medium supplemented with 5 μg / ml protamine sulfate (Sigma-Aldrich) and 1000 U / ml IL-2, 25 U / ml IL-7, and 50 U / ml IL-15. After 1 day, the medium was changed and the cells were grown for an additional 8–9 days in RPMI complete medium supplemented with 100 U / ml IL-2, 25 U / ml IL-7, and 50 U / ml IL-15 and then frozen in liquid nitrogen until further use.

[0080] Example 3 Cell activation upon antigen stimulation The biological activity of preferred constructs according to the invention was compared to constructs in which the PSMA-binding fragment was derived from an antibody, and further compared to similar constructs in which the antigen-binding construct was derived from another antibody (3D8).

[0081] The constructs were introduced into Jurkat T cell line, and the CAR expression level was measured by flow cytometry after staining the cells with anti-human IgG. As shown in Figure 9B, all CAR constructs were properly expressed in Jurkat T cells. UT (untransduced cells) served as negative control for staining.

[0082] Antigen-specific activation of transduced Jurkat T cells was measured by monitoring the expression of the activation marker CD69 upon antigen stimulation (Figure 9C). In contrast to A5- and J59-based CARs, respectively, 3D8-based CARs mediated only weak activation of transduced Jurkat T cells upon exposure to antigen-positive cells. This was independent of the expression of the inhibitory ligand PD-L1. CAR constructs were not activated when they were stimulated with antigen-negative (DU145) cells.

[0083] Example 4 Expansion of PSMA-targeted CAR T cells CAR T cells were generated from PBMCs after retroviral transduction as described in Example 2. Six days after transduction, CAR T cells were co-cultured with irradiated PSMA-positive (C4-2) tumor-positive cells at an effector-to-target ratio of 1:1 in RPMI complete medium supplemented with 100 U / ml IL-2, 25 U / ml IL-7, and 50 U / ml IL-15 (all from Miltenyi Biotech) for 12 days. Every 3 days, cells were harvested, counted, and plated on fresh irradiated antigen-positive (C4-2) tumor-positive cells. Cells were stained with anti-human IgG-PE (Southern Biotech) to measure CAR expression levels and the fraction of CAR-positive cells by flow cytometry (FACS Canto II or Accuri, BD Biosciences).

[0084] The number of CAR T cells was counted at different time points using a NucleoCounter (NC-250, ChemoMetec). The number of cell divisions was calculated based on the absolute cell number.

[0085] Furthermore, the quality of CAR T cells was measured by flow cytometry analysis to confirm the phenotype and exhaustion pattern of CAR T cells.

[0086] Example 5 Quality assessment of expanded CAR T cells For quality assessment, CAR T cells were exposed to antigen-positive (C4-2) tumor cells, after which 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). T cell phenotype was measured based on CD62L and CD45RA expression. Cells were pre-gated on CD3+ / CAR- for untransduced (UT) T cells, or on CD3+ / CAR+ for both types of CAR T cells (not shown). As observed, A5-CAR T cells were less differentiated compared to J591 CAR T cells, as evidenced by a higher proportion of undifferentiated cells, naïve T cells (Tn) and T stem cell memory (Tscm), but a lower proportion of effector T cells (Teff).

[0087] Example 6 CAR T cell exhaustion upon antigen stimulation To monitor CAR T cell exhaustion upon antigen-specific stimulation, CAR T cells were exposed to PSMA-positive tumor cells (C4-2) and then 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). Exhaustion profiles were measured by flow cytometry (FACS Canto II) based on expression of CD279 (PD-1) and CD223 (LAG-3). Cells were pre-gated on CD3+ / CAR- for UT T cells or CD3+ / CAR+ for both types of CAR (not shown). Compared to J591-CAR T cells, A5-CAR T cells maintained significantly increased numbers of LAG-3 and PD-1 double negative cells upon antigen encounter, indicating less exhausted T cell phenotype.

[0088] Taken together, the data in Examples 4, 5, and 6 indicate that antigen exposure is a major determinant of CAR T cell phenotype, CAR T cell proliferation, and CAR T cell exhaustion. A5-CAR T cells outperformed J59-CAR T cells in all of these assays.

[0089] Example 7 In vitro cytotoxicity of produced PSMA-CAR T cells The cytotoxic potential of PSMA targeting CAR T cells was measured by assessing their ability to kill PSMA-positive tumor cells using a cell viability XTT assay. CAR T cells were co-cultured with either PSMA-positive (C4-2) or (LNCaP) tumor cells or antigen-negative tumor control cells (DU145) in 96-well plates at different effector-to-target ratios in a final volume of 200 μml / well of cytokine-free RPMI complete medium for 48 h. To measure cell viability as a function of metabolic activity, 100 μml / well of medium was removed and replaced with 100 μl / well of XTT solution (Sigma-Aldrich) and cells were incubated at 37 °C. Colorimetric changes were quantified using an ELISA reader (Infinite F50, Tecan) at 450 nm. Cytotoxicity was expressed as the percentage of dead cells, calculated as 100% minus the percentage of viable cells. Viability was calculated using the formula [OD E+T -OD Eのみ ] / [OD Tのみ -OD 培地のみ ] (E: effector cells = CAR T cells; T: target cells = tumor cells). A5-CAR T cells were able to eliminate tumor-positive cells at a significantly lower effector-to-target ratio compared to J59-CAR T cells. The results show that A5-CAR T cells outperformed J59-CAR T cells in in vitro cytotoxicity assays.

[0090] Usually, the genetic information encoding chimeric antigen receptor is introduced into target immune cells (e.g., T cells) with the help of suitable vector. Such vectors can be preferably lentiviral vectors, retroviral vectors, transposons, or plasmids. Alternatively, said genetic information can be introduced into the genome of T cells in a targeted manner with the help of designer nuclease technology, such as CRISPR / Cas technology or TALEN technology, as described herein.

[0091] In a further embodiment, the present invention relates to an in vitro method for providing T cells comprising the chimeric antigen receptor described herein. In a first step, T cells are isolated from a donor, preferably by leukapheresis. This results in a substantial enrichment of the T cells. The T cells are then genetically modified by transduction with a suitable vector or by transduction with a viral vector containing the genetic information of the chimeric antigen receptor. Such genetically modified T cells can then be isolated and expanded. Thereby, T cells not containing the desired genetic information can be separated from the modified T cells, or at least reduced. The genetically modified T cells can then be applied to the patient to be treated.

Claims

1. A chimeric antigen receptor comprising an antigen-binding fragment that specifically binds to a PSMA antigen, characterized in that the antigen receptor for PSMA is humanized and comprises a single-chain variable fragment A5 having SEQ ID NO:1 and SEQ ID NO:5, a transmembrane domain, and an intracellular signaling domain, and contains six CDRs having the amino acid sequences of GFTFSDYY, ISDGGYYT, TRGFPLLRHGAMDYWG, QNVDTN, SAS, and QQYDSYPYT.

2. The chimeric antigen receptor of claim 1, wherein the transmembrane domain is a CD3 zeta cytoplasmic domain and the intracellular signaling domain is a CD28 and / or 4-1BB cytoplasmic domain.

3. The chimeric antigen receptor according to claim 1 or 2, characterized in that it contains at least one amino acid sequence selected from SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO:

36.

4. A nucleic acid encoding a chimeric antigen receptor according to any one of claims 1 to 3, comprising a nucleic acid encoding the chimeric antigen receptor.

5. A vector encoding the chimeric antigen receptor according to any one of claims 1 to 3, comprising a nucleic acid encoding the chimeric antigen receptor according to claim 4.

6. 6. The vector according to claim 5, characterized in that it comprises SEQ ID NO: 37 or SEQ ID NO: 38 or a sequence which is at least 95% identical thereto.

7. An in vitro method for providing an immune cell comprising a chimeric antigen receptor according to any one of claims 1 to 3, comprising:

10. Transducing / transducing immune cells isolated from a donor by leukapheresis with the vector of claim 5 encoding a chimeric antigen receptor; Isolating and amplifying transduced immune cells An in vitro method comprising:

8. The in vitro method of claim 7 , wherein the immune cell is a T cell.

9. An immune cell, comprising genetic information encoding the chimeric antigen receptor according to any one of claims 1 to 3.

10. The immune cell of claim 9 , wherein the immune cell is a T cell.

11. A chimeric antigen receptor according to any one of claims 1 to 3, or a nucleic acid according to claim 4, or a vector according to claim 5 or 6, for use in the treatment of prostate cancer and / or prostate-derived tumors.

12. A chimeric antigen receptor according to any one of claims 1 to 3 and / or a nucleic acid according to claim 4 and / or a vector according to claim 5 or 6 for use in the treatment of a solid tumor expressing PSMA, wherein an agent having antitumor activity is introduced into the solid tumor.

13. A chimeric antigen receptor according to any one of claims 1 to 3 for use according to claim 11 or 12, characterized in that it is combined with the administration of a chemotherapeutic active agent selected from taxol derivatives, 5-fluorouracil, cyclophosphamide, mitoxantrone, docetaxel and / or cabazitaxel and / or with the administration of a biological agent comprising a protein, an antibody, a vaccine, blood, a blood component, an allergen, a recombinant therapeutic protein, a gene therapy agent, a somatic cell, tissue or cell therapy agent.

14. A chimeric antigen receptor according to any one of claims 1 to 3 for use according to claim 11 or 12, characterized in that it is combined with the administration of a chemotherapeutic active agent selected from taxol derivatives, 5-fluorouracil, cyclophosphamide, mitoxantrone, docetaxel and / or cabazitaxel, and / or with the administration of a biological agent including a protein kinase inhibitor, an enzyme inhibitor or an anti-genomic therapeutic agent.

Images