Enhancement of activity of cytolytic t cells via inhibition of EBAG9

Abstract

To provide alternative means or improved means for enhancing cytolytic activity of therapeutic T cells, in particular, those having an antigen specific targeting construct that directs the T cells to a specific tumor target.SOLUTION: The invention relates to a genetically modified cytotoxic T cell comprising one or more exogenous nucleic acid molecule encoding a transgenic construct targeting an antigen, where in the cells, the activity of the estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is inhibited. Further, the invention relates to a modified cytotoxic T cell of which antigen targeting construct is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Furthermore, the invention relates to modified T cells for use as a medicament in the treatment of proliferative diseases, and, in particular, to methods for treating hematologic malignancies.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of cellular therapeutics, in particular to a means of enhancing the cytotoxic activity of therapeutic T cells suitable for treating cancer.

[0002] The present invention relates to genetically modified cytotoxic T cells comprising one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct, in which the activity of estrogen receptor-binding fragment-associated antigen 9 (EBAG9) is inhibited. The present invention further relates to modified cytotoxic T cells, in which the antigen-targeting construct is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). The present invention further relates to modified T cells for use as a medicament in the treatment of proliferative diseases and corresponding treatment methods, in particular for the treatment of hematological malignancies. The present invention further relates to pharmaceutical compositions comprising the modified T cells, nucleic acid vectors encoding the antigen-targeting constructs, and means for inhibiting EBAG9, preferably using RNA interference, and in vitro methods for increasing the cytolytic activity of cytotoxic T cells. [Background technology]

[0003] Hematological tumors are heterogeneous and can be differentiated by aggressive and indolent courses. Standard treatment is often antibody / chemotherapy combinations in combination with autologous stem cell transplantation, immunomodulatory drugs, radiation, proteasome inhibitors, signaling pathway inhibitors, and allogeneic stem cell transplantation in a small minority of patients. In many B-NHL entities, the median age at diagnosis is >66-72 years, and comorbidities are also present, which precludes intensive and long-term chemotherapy or even allogeneic bone marrow transplantation.

[0004] Inhibitors of BCR signaling in mature B-cell lymphomas, especially ibrutinib, have led to a significant increase in remission rates. Despite the initial high sensitivity to this class of kinase inhibitors, it is uncertain whether tumor eradication can be achieved, and secondly, several studies have revealed that clonal evolution of lymphomas and leukemias leads to the emergence of resistance to BTK inhibition. Therefore, the rapid emergence of secondary resistance to targeted therapy calls for finding an acceptable salvage therapy solution applicable to patients who have received several other types of chemotherapy, which leads to a decline in clinical outcome (IPI score).

[0005] Adoptive chimeric antigen receptor (CAR)-T cell therapy targeting the CD19 antigen, which is widely expressed on leukemia and lymphoma B cells, has produced significant clinical benefits. CAR-T approaches based on antigen specificity for BCMA (Patent Document 1) and CXCR5 (Patent Document 2) as tumor-associated antigens have also been described.

[0006] However, resistance may emerge for anti-CD19 antibodies or CAR-T cell therapy directed against B-NHL due to antigen loss or reduced expression. Recent studies have shown that alternatively spliced ​​CD19 isoforms are selected during anti-CD19 CAR-T cell therapy, which results in the emergence of escape variants due to the loss of the cognate epitope of the CD19 CAR. Thus, in addition to existing therapeutic mAbs or CAR-T cell therapy, other CAR specificities emerge as alternative targets for immunotherapy of B cell lymphoma. Similarly, the BCMA antigen associated with multiple myeloma has been shown to be shedding from tumor cell membranes, which has resulted in a significant reduction in structures that can be targeted by BCMA CAR.

[0007] Effective adoptive T cell therapy (ATT) depends on the generation of tumor antigen-specific CD8+ and / or CD4+ T cells with high avidity and long-lived. Functional avidity depends not only on the structural avidity of TCR or CAR to its cognate antigen, but also on the cytolytic efficiency of T cells, which is influenced by the synthesis, transport, and storage of effector cytokines and cytolytic molecules. In addition, adoptively transferred T cells with either TCR or CAR are prone to develop resistance or dysfunctional states in the presence of tumor microenvironment. Therefore, T cell function is justified to break this dysfunctional state or prevent such a state.

[0008] The function of estrogen receptor-binding fragment-associated antigen 9 (EBAG9) has been evaluated in T lymphocytes and found to regulate cytotoxic activity. Non-Patent Document 1 teaches that EBAG9 negatively regulates the cytolytic ability of mouse CD8+ T cells. Deletion of EBAG9 resulted in increased CTL secretion of granzyme A. Furthermore, Non-Patent Document 2 teaches that EBAG9 controls the cytotoxic activity of mouse T lymphocytes, thereby regulating tumor growth and metastasis by negatively regulating adaptive immune responses. Despite these findings, there has been no proposal or attempt to incorporate EBAG9 inhibition into human cytotoxic T cell containing antigen-targeting transgenic constructs. To date, findings regarding EBAG9 have been discussed in terms of regulating EBAG9 expression in cancer cells. According to Miyazaki et al., EBAG9 expression is elevated in cancer cells, which then negatively regulates the host's endogenous immune response. However, there has been no proposal in the art to inhibit EBAG9 in order to increase the cytolytic capacity of therapeutic T cells with engineered antigen specificity that are introduced into a subject. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2017 / 211900 [Patent Document 2] International Publication No. 2019 / 038368 [Non-patent literature]

[0010] [Non-Patent Document 1] Rueder et al (J. Clin. Invest. 2009, 119:2184-2203) [Non-Patent Document 2] Miyazaki et al (Oncogenesis (2014) 3, e126) Summary of the Invention [Problem to be solved by the invention]

[0011] In view of the above drawbacks and inherent difficulties in the development of cellular therapeutics using T cells that exhibit high avidity and cytotoxicity against malignant cells, the art is in dire need of novel means to improve the activity and ultimately therapeutic efficacy of cytolytic T cells to overcome difficulties in, for example, tumor resistance or tumor antigen shedding.

[0012] In view of the prior art, the technical problem underlying the present invention was to provide alternative or improved means of enhancing the cytolytic activity of therapeutic T cells, particularly those bearing an antigen-specific targeting construct directing the T cells to a specific tumor target. A further object of the present invention was to provide means of improving CAR-T or TCR T cell therapy. Another object of the present invention was to provide means of facilitating the production of competent CAR T cells. [Means for solving the problem]

[0013] This problem is solved by the features of the independent claims. Preferred embodiments of the invention are provided by the dependent claims. The present invention seeks to solve the above-mentioned problem while avoiding the disadvantages of the prior art.

[0014] Thus, the present invention relates to genetically modified cytotoxic T cells comprising one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct, wherein estrogen receptor-binding fragment-associated antigen 9 (EBAG9) activity is inhibited in said cells.

[0015] To apply the stimulatory effect of EBAG9 deletion or knockdown on the release of cytolytic effector molecules to the therapeutic setting of ATT, we developed engineered T cells in which EBAG9 was inhibited.

[0016] As shown below, in preferred embodiments and examples, the inventors combined miRNA targeting of EBAG9 with various CAR constructs, in particular BCMA CAR, CD19 CAR, and CXCR5 CAR, which are described in more detail herein. Retroviruses were generated and human T cells were transduced. These T cells were endowed with significantly improved cytolytic activity, as evidenced by enhanced cytolytic killing in vitro. Xenograft experiments were performed in vivo in NSG mice using human multiple myeloma cells, followed by transplantation of small numbers of engineered CAR T cells.

[0017] CAR T cells with silenced EBAG9 expression are thereby endowed with high cytolytic efficiency, which in some examples is seen as complete tumor eradication. Thus, EBAG9 inhibition, preferably mediated by RNAi, causes a surprising effect in terms of the increased efficacy brought to CAR-T and similar approaches in cell therapy. This increase represents a surprising effect that could not be predicted by those skilled in the art.

[0018] The present invention, characterized by the inhibition of EBAG9 in CTL expressing antigen targeting constructs, is further characterized by the advantage that there is no increased risk of autoimmunity. Other cell biological approaches to enhance the cytolytic efficiency of T cells are standard cell culture procedures described in the prior art, including endowing T cells with a predefined maturation state toward either effector or memory phenotype. However, downregulation of other obstacles to T cell activation, such as deletion of PD-1 or Cbl-b, is associated with a significantly higher risk of autoimmunity. Surprisingly, no autoimmunity was observed in EBAG9-KO mice, and therefore, essentially, there is no or a significantly lower risk of developing such diseases after ATT using the cells of the present invention.

[0019] As an additional advantage of the present invention, EBAG9 targets a post-translational step in cellular processes, thus interfering with the canonical secretory pathway that is only active in T cells, but not in other somatic cells. Selective targeting of EBAG9 using an in vitro miRNA approach in mature T cells has a very low risk profile.

[0020] Moreover, as an added benefit, EBAG9 silencing is not pro-oncogenic but rather confers enhanced immunological competence to CD8+ T cells (but not NK cells).EBAG9 deletion is also associated with increased formation of antigen-specific memory, which is important for preventing tumor recurrence.

[0021] The biological principle of the approach of EBAG9 inhibition (preferably silencing) is novel, as described above, and is associated with unexpected advantages that are described in more detail below: For the first time, a post-translational transport step in CTL is targeted, which serves to enhance the supply of cytolytic effector molecules.

[0022] This EBAG9 inhibition leads to enhanced cytolytic capacity and higher efficiency in tumor eradication without exhaustion or dysfunction, and no obvious side effects were observed in preclinical models, in stark contrast to other approaches targeting signaling processes (PD-1, Cbl-b).

[0023] Thus, the present invention further allows for a more rapid production of engineered (preferably CD8+ and CD4+) T cells endowed with better antitumor activity in vitro and in vivo. To obtain an effective dose of CAR T cells, it usually takes a minimum of 12 days for the cells to be ready for administration. Now, using EBAG9 inhibition, this period can be significantly accelerated, and in some cases, the preparation period for producing an effective dose of CAR T cells is reduced to less than 10 days. In other words, to obtain the same effect, fewer CAR T cells are required, and therefore fewer expansion steps are required ex vivo and in vitro. This will result in significant cost savings in cell production and more rapid treatment. Thus, improvements are observed at the manufacturing level, the biological level, and the treatment level, which provide a series of advantages that could not be predicted from the prior art.

[0024] Furthermore, in vitro analysis by the present inventors revealed that the engineered T cells were not prone to an early exhaustion phase, indicating that application of EBAG9 engineered CAR T cells is a feasible strategy to improve ATT of several hematological malignancies. In some embodiments, the engineered CTLs of the present invention are characterized by a relatively slower exhaustion of cytolytic activity compared to unmodified control cells, such as CTLs in which EBAG9 is not inhibited.

[0025] High affinity and high avidity enable CAR-T and TCR-T cells to recognize, activate against, and kill target tumor cells with high and moderate surface expression of cognate antigens. However, CARs with avidity maturation have not yet been described.

[0026] Thus, the present invention represents a breakthrough concept of improving CTL activity that has not been disclosed or proposed in the art until now. To the best of the inventors' knowledge, there is currently no comparable biological principle in use, whereby an increase in ATT efficiency is achieved by improving the secretory pathway in T cells.

[0027] Despite the known negative regulatory function of EBAG9 on cytolytic activity, there has been no proposal or attempt in the prior art to incorporate EBAG9 inhibition into cytotoxic T cells containing antigen-targeting transgenic constructs. Prior art on EBAG9 shows that EBAG9 expression is elevated in cancer cells, which then negatively regulates the host's endogenous immune response. Inhibiting EBAG9 in therapeutic T cells with engineered antigen specificity to increase cytolytic capacity is a completely novel approach aimed at directly manipulating EBAG9 function in cells where enhanced cytolytic activity is required.

[0028] Thus, the present invention solves various problems compared to the prior art literature on EBAG9 and cytolytic activity: the present invention seeks to improve CAR-T or TCR-based cell therapy by not only enhancing the cytolytic activity of the cells, but also improving the method of producing the cells by reducing the number of cells required for therapeutic application. Thus, the present invention provides a unique solution to these problems that could not be derived from the state of the art.

[0029] Furthermore, the combination of antigen-targeting constructs and EBAG9 inhibition in cytotoxic T cells results in a synergistic effect: by directing the cytolytic activity of T cells to the target cells of interest, the enhanced cytolytic activity of EBAG9 inhibition can be exerted locally and effectively, resulting in a cytolytic effect that exceeds expectations and exceeds the sum of the effects obtained by incorporating the antigen-targeting constructs or EBAG9 inhibition alone.

[0030] In the present invention, EBAG9 inhibition can be achieved by multiple possible mechanisms and using various molecular or chemical tools. Thus, the embodiments of EBAG9 inhibition disclosed herein are exemplary and are not intended to be limiting.

[0031] In some embodiments, inhibition of EBAG9 is preferably determined in comparison to control cytotoxic T cells, i.e., cytotoxic T cells obtained from a comparable or identical source in which the production or activity of EBAG9 has not been altered, e.g., by means described herein.

[0032] In some embodiments, inhibition of EBAG9 activity is associated with an increase in the release of cytolytic granules and / or secretory lysosomes containing granzymes (preferably compared to control cytotoxic T cells). Suitable assays by which the release of cytolytic granules and / or secretory lysosomes containing granzymes can be assessed without undue effort in two comparative cell populations are presented below and are known to those skilled in the art.

[0033] In some embodiments, the measurable effect of EBAG9 inhibition is enhanced delivery of cytolytic granules. In some embodiments, the measurable effect of EBAG9 inhibition is increased target cell killing following recognition mediated by an antigen-specific CAR. Those skilled in the art can determine these functional effects using routine and established assays.

[0034] One preferred assay is an "in vitro cytotoxicity assay" in which either EBAG9-inhibited or uninhibited CTLs are compared for their effectiveness in killing target cells in vitro. Examples of such in vitro assays are shown in more detail in the Examples below. In vivo techniques for determining EBAG9 inhibition based on CTL effectiveness are also available and can be performed without undue effort by those skilled in the art.

[0035] In one embodiment, isolated T cells modified with an antigen-targeting transgenic construct in which EBAG9 is inhibited are characterized by an increase in cytotoxic activity of 5% or more, 10% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% or more when compared to cytolytic T cells in the absence of EBAG9 inhibition. Functional assays that quantitatively or semi-quantitatively determine the increase in cytolytic activity of T cells in which EBAG9 is inhibited are available to the skilled artisan, some examples of which are described herein. For example, in vitro assays such as those used to generate Figures 7 and 8 below or described in the Examples can be applied to determine the increase in cytolytic activity compared to "unmodified" T cells, i.e., T cells in which no specific modification has been performed to reduce / inhibit EBAG9 function.

[0036] In some embodiments, EBAG9 silencing is achieved by miRNA-based retroviruses used for T cell transduction. The inventors have demonstrated >90% silencing of EBAG9 transcripts and protein in several primary cell types of mouse and human origin.

[0037] In some embodiments, inhibition of EBAG9 achieves 10% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more inhibition compared to control CTL. In some instances, for example, where EBAG9 is deleted or expression is prevented, for example, by genomic modification, 100% inhibition of EBAG9 can be achieved. In some embodiments, the invention uses miRNAs targeting mouse or human EBAG9 incorporated into retroviral expression constructs with various TCR or CAR antigen specificities.

[0038] In preferred embodiments, the genetically modified CTLs described herein are CD4+ and / or CD8+ T cells, and preferably the CTLs are a mixture of CD4+ and CD8+ T cells. These T cell populations, and preferably compositions comprising both transformed CD4+ and CD8+ cells, exhibit particularly effective cytolytic activity against a variety of hematological malignancies, and preferably against the cells and / or associated medical conditions described herein.

[0039] In a preferred embodiment, the genetically modified CTLs are CD4+ and CD8+ T cells, preferably in a ratio of 1:10 to 10:1, more preferably 5:1 to 1:5, 2:1 to 1:2, or 1:1. Administration of modified CAR-T cells expressing a CAR in the ratios described, preferably a 1:1 CD4+ / CD8+ ratio, provides beneficial properties in the treatment of diseases described herein, e.g., these ratios provide improved therapeutic responses and reduced toxicity.

[0040] In one embodiment, the genetically modified T cells described herein are characterized in that the antigen-targeting transgenic construct is a chimeric antigen receptor (CAR).

[0041] In one embodiment, the genetically modified T cells described herein are characterized in that the antigen-targeting transgenic construct is a T cell receptor (TCR).

[0042] The specific type or form of antigen targeting construct is not intended to limit the features of the present invention. The concept of the present invention is based on the increase in cytolytic activity mediated by EBAG9 inhibition. Therefore, the specific mode of delivering CTL to any given target cell does not limit the aspects of the present invention. Therefore, the enhanced activity of the CTL of the present invention does not depend on the antigen targeting construct, which is simply considered as a means of bringing the CTL into close proximity to the target (e.g., tumor) cell. Therefore, the CAR and TCR constructs described in more detail below represent preferred embodiments in which the effect of EBAG9 inhibition can be effectively utilized.

[0043] In further embodiments, the CAR constructs used can be easily exchanged, thus allowing for modular construction of clinically applicable CARs. The antigen specificity of the CAR is variable and is not a limitation of the present invention.

[0044] In one embodiment, the genetically modified T cells described herein are characterized in that inhibition of EBAG9 activity is achieved by knockdown of EBAG9, preferably by RNA interference of EBAG9 expression, such as by small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA). Gene knockdown is a technique that reduces the expression of one or more genes. In some embodiments, reduction of EBAG9 expression can occur by genetic modification or by treatment with reagents, such as short DNA or RNA, or other nucleic acids, oligonucleotides, having sequences complementary to either the gene or mRNA transcript of EBAG9.

[0045] RNA interference approaches to EBAG9 inhibition (i.e., "silencing" or "knockdown") are preferred for a number of reasons due to their unique advantages in biological systems. RNAi technology has a superior safety profile compared to engineered genomes by not interfering with genome structure or integrity.

[0046] In some embodiments, the sequence used to target EBAG9 is as follows: H17 (SEQ ID NO: 1; AAATAACCGAAACTGGGTGAT) or H18 (SEQ ID NO: 2; TTAAATAACCGAAACTGGGTG).

[0047] Other sequences targeting alternative sites in EBAG9 are as follows: TAAATAACCGAAACTGGGTGA (SEQ ID NO:55); TAGGAATGAGAATACTGTTGC (SEQ ID NO:56); CTTAATTTCCGTCCTCTGCCA (SEQ ID NO:57); TTTGGTCTCCACTTAATTTCC (SEQ ID NO:58); TTCAACATCTGTCTGCTTAGG (SEQ ID NO:59); AAGTCCACTCTTCAACATCTG (SEQ ID NO:60); ATCCCAGGAAGTCCACTCTTC (SEQ ID NO:61); TACTAGAGAAACCTGTGCTCC (sequence number 62).

[0048] In some embodiments, the present invention relates to a nucleic acid molecule, either in isolated form or preferably in a vector for CTL transfection, selected from the group consisting of: a) a nucleic acid molecule comprising a nucleotide sequence comprising a complementary or antisense sequence directed to an RNA region of EBAG9, capable of interfering with the stability or function of EBAG9 RNA, or A nucleic acid molecule comprising a nucleotide sequence comprising or consisting of a sequence set forth in SEQ ID NO: 1, 2, or SEQ ID NO: 55 to 62; b) a nucleic acid molecule complementary to the nucleotide sequence according to a); c) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to a nucleotide sequence according to a) or b), preferably comprising at least 50%, 60%, 70%, 80%, 90%, or 95% sequence identity to a nucleotide sequence according to a) or b); d) Nucleic acid molecules according to the nucleotide sequences of a) to c) which have been modified by deletions, additions, substitutions, translocations, inversions and / or insertions and which are functionally similar / equivalent to the nucleotide sequences according to a) to c).

[0049] In some embodiments, RNAi sequences may be designed to target one or more of the EBAG9 transcripts described herein, preferably as set forth in SEQ ID NOs: 3-6.

[0050] Those skilled in the art can design effective sequences for targeting RNA based on target sequences and general knowledge in the art.Software for such methods is generally available, allowing those skilled in the art to design additional sequences to be used as shRNA for silencing EBAG9.For example, Thermo Fisher's program BLOCK-iT RNAi can be used.Alternative software can be identified and used.

[0051] In some embodiments, miRNA is used to generate miRNA that targets EBAG9.

[0052] miRNAs are similar to small interfering RNAs (siRNAs), except that miRNAs are derived from regions of RNA transcripts that fold back to form short hairpins, whereas the small interfering RNAs (siRNAs) of the RNA interference (RNAi) pathway are derived from longer regions of double-stranded RNA.

[0053] In some embodiments, an endogenous miRNA can be used, such as endogenous miRNA-155. A sequence that specifically targets the EBAG9 sequence, such as the antisense sequence of H17 and H18, can be inserted into the endogenous miRNA, i.e., within a hairpin structure containing 21 nucleotides.

[0054] In some embodiments, EBAG9 knockdown can be achieved by using an expression vector for miRNA. Since miRNA genes are usually transcribed by RNA polymerase II (Pol II), an appropriate promoter for the expression vector can be selected. The polymerase often binds to a promoter found near the DNA sequence that encodes what becomes the hairpin loop of the pre-miRNA. The resulting transcript is capped with specially modified nucleotides at the 5' end, polyadenylated with multiple adenosines, and optionally spliced. The mature miRNA then becomes part of an active RNA-induced silencing complex (RISC) that contains Dicer and a number of associated proteins. Gene silencing can then occur by degrading EBAG9 mRNA or preventing translation of EBAG9 mRNA.

[0055] In some embodiments, small interfering siRNA is used to generate miRNA that targets EBAG9.

[0056] As the siRNA, for example, a short (10 bp to 50 bp, preferably 20 bp to 25 bp) double-stranded or hairpin RNA molecule containing a sequence having complementarity to a region of the mRNA encoding EBAG9 is used.

[0057] In some embodiments, EBAG9 inhibition is achieved using siRNA or miRNA directed against a region of any one of SEQ ID NOs: 3, 4, 5, or 6. The siRNA or miRNA (i.e., the targeted region) can be of 10-50 nucleotides, preferably 15-40 nucleotides, more preferably 18-30 nucleotides, more preferably 20-25 nucleotides, and exhibits sufficient sequence complementarity to the transcript of SEQ ID NO: 3, 4, 5, or 6 to cause a reduction in EBAG9 expression compared to a relevant control. Complementarity between the short hairpin RNA and the target mRNA can be 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or preferably 100% in some embodiments. Complementarity is preferably calculated similarly to sequence identity, where the total number of complementary / identical nucleotides over the entire length of the sequence (e.g., the length of the small interfering RNA) is determined.

[0058] In some embodiments, EBAG9 knockdown can be achieved by using an expression vector for siRNA. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is a short hairpin RNA (shRNA) that can be processed into functional siRNA by Dicer in the normal manner. A typical transcription cassette expressing siRNA in T cells in which EBAG9 is to be knocked down uses an RNA polymerase III promoter (e.g., U6 or H1) that directs the transcription of small nuclear RNA (snRNA) (U6 is involved in gene splicing, and H1 is the RNase component of human RNase P).

[0059] Those skilled in the art can generate further RNA interference constructs that effectively reduce or inhibit the production of EBAG9 without undue effort.

[0060] In one embodiment, the genetically modified T cells described herein are characterized in that inhibition of EBAG9 activity is achieved by genetic modification of the T cell genome with an exogenous nucleic acid molecule, preferably comprising an antigen-targeting transgenic construct and a vector encoding an RNA interference sequence for knockdown of EBAG9.

[0061] In some embodiments, viral vectors are used, preferably retroviral and lentiviral transduction.

[0062] In some embodiments, transposons can be used as vectors encoding antigen-targeting transgenic constructs.

[0063] Viral vectors are associated with a variety of advantages, such as being accepted by regulatory authorities, exhibiting good technical advantages such as reliable modification of cells, and having an acceptable risk profile.

[0064] In some embodiments, the CTLs of the present invention preferably use MP71 vectors in combination with a gamma retroviral expression system.

[0065] In this combination, significantly higher T cell transduction rates can be achieved. The transduction system is versatile due to the modular design of the CAR construct and miRNA, meaning that lentiviruses and transposons can be used alternatively.

[0066] In some embodiments, the CTLs of the present invention utilize retroviral SIN vectors (available, for example, from BIONTECH, Idar-Oberstein), preferably in combination with a gamma retroviral expression system.

[0067] Thus, these embodiments of the invention are further advantageous since manipulation of recipient T cells in a single step, due to retroviral, lentiviral or transposon-mediated introduction of a TCR or CAR (of any antigen specificity), is sufficient to inhibit the activity or expression of EBAG9.

[0068] In one embodiment, the genetically modified T cells described herein are characterized in that inhibition of EBAG9 activity is obtained by genetic modification of the T cell genome to prevent expression of the EBAG9 gene and / or to disrupt its sequence. Deletions or partial deletions of EBAG9 resulting in loss of function are envisaged.

[0069] As described in more detail below, CRISPR / Cas9 or TALEN technology or other genetic engineering tools can be used. The examples of the present application demonstrate that various guide RNAs targeting exon 4 of EBAG9 can achieve good knockout efficiency of EBAG9 together with Cas9 protein. In one embodiment, but not limited to, CRISPR / Cas9-mediated EBA9 inhibition targets exon 4 of EBAG9 gene. This embodiment shows effective but non-limiting preferred targets within EBAG9 for CRISPR-mediated knockout. Alternative targets suitable for CRISPR-mediated genetic manipulation of EBAG9 gene are known to those skilled in the art or can be evaluated without undue effort.

[0070] In other embodiments, genetic engineering tools such as site-directed mutagenesis or recombination-based gene targeting are known to those skilled in the art and can be used. However, genetic (genomic) manipulation usually requires two or more rounds of transfection or transduction, which is generally less efficient than viral transduction.

[0071] In some embodiments, introduction of a CAR or TCR and in a second step introduction of genetic information targeting EBAG9 can be used to introduce the required genetic modification. In some embodiments, introduction of a genetic information targeting EBAG9 and in a second step introduction of a CAR or TCR can be used to introduce the required genetic modification. In some embodiments, the modifications are performed simultaneously, either on the same vector or exogenous nucleic acid molecule, or on separate vectors or exogenous nucleic acid molecules.

[0072] In some embodiments, the exogenous nucleic acid molecule encoding the antigen-targeting transgenic construct is positioned within, adjacent to, or linked to the EBAG9 gene in the T cell genome, thereby preventing expression of the EBAG9 gene and / or disrupting its sequence.

[0073] In some embodiments, the CAR sequence can be integrated directly into the EBAG9 locus, thereby preventing EBAG9 gene expression.

[0074] Various alternative strategies for disrupting the EBAG9 gene sequence or mRNA are available to those skilled in the art and can be established without undue effort. Thus, the preferred embodiments of EBAG9 inhibition disclosed herein are illustrative and not intended to be limiting.

[0075] Thus, in a further aspect, the present invention relates to a genetically modified T cell according to any one of the preceding claims for use as a medicament. Given the novel given T cells characterized by EBAG9 inhibition, any medical application is contemplated. The skilled artisan can identify medical conditions that can be treated using CAR-T cells or TCR T cells and can design the modified T cells accordingly. In a preferred embodiment, the medical application is a proliferative disease or an autoimmune disease.

[0076] In a further aspect, the invention relates to the genetically modified T cells described herein for use as a medicament in the treatment of a proliferative disease, in which the antigen targeted by the antigen-targeting transgenic construct is expressed in target cells undergoing and / or associated with pathological cell proliferation. The invention thus encompasses a corresponding method of treatment, employing the administration of a therapeutically effective amount of the CTLs described herein to a subject in need thereof.

[0077] In one embodiment, the proliferative disease is a hematological malignancy, wherein the antigen targeted by the antigen-targeting transgenic construct, e.g., a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA), is expressed in cancerous cells of said hematological malignancy.

[0078] Non-limiting examples of hematological malignancies are non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and / or multiple myeloma. Further examples are provided below.

[0079] In some embodiments, the antigen targeted by the antigen-specific construct is a tumor-associated antigen (TAA). TAA refers to antigens that are primarily or preferably expressed on tumor cells, although in many cases these antigens are not exclusively expressed on such cells. CD19 falls under this definition, as it can occur on both transformed and benign B cells. BCMA is also expressed on normal plasma cells and transformed cells, such as multiple myeloma cells.

[0080] In some embodiments, the hematological malignancy being treated is a B-cell cancer that expresses CD19, and where the antigen-targeting transgenic construct binds to CD19.

[0081] Antibodies directed against CD19 can be used to develop antigen targeting constructs for CAR constructs that target cells expressing CD19. In some embodiments, the antigen targeting transgenic construct comprises an amino acid sequence (antigen binding domain) derived from an antibody that binds to CD19. CD19 antibodies include, but are not limited to, Blinatumomab, Coltuximabravtansine, MOR208, MEDI-551, Denintuzumabmafodotin, B4, Merck's DI-B4, Tapirtumomab paptox, XmAb 5871, MDX-1342, or AFM11.

[0082] Various CAR constructs targeting CD19 have been described in the art. Silencing of EBAG9 can essentially be applied to any one or more of such CAR-T cell therapeutics. Known CD19 CAR-T cells in clinical studies include, but are not limited to, CARs that contain antigen binding domains with ScFvs of FMC63 or SJ25C1. As reviewed in Park et al. (Blood, 2016, 127:3312-3320), CAR constructs with either 4-1BB or CD28 co-stimulatory domains are known. CAR-T cells in clinical investigations include, but are not limited to, tisagenlecleucel from Novartis, axicabtageneciloreucel from Gilead Sciences, JCAR015 and JCAR017 from Juno Therapeutics, CAR CD19 from Ziopharm Oncology, UCART19 from Cellectis, ALLO-501 from Allogene, BPX-401 from Bellicum Pharmaceuticals, PCAR-019 from PersonGen Biomedicine Suzhou, PBCAR-0191 from Precision Biosciences, SENL-001 from Hebei Senlang Biotechnology, and UWC-19 from UWELL Biopharma.

[0083] In some embodiments, the hematological malignancy being treated is a B cell cancer that expresses BCMA, and where the antigen-targeting transgenic construct binds to BCMA.

[0084] Antibodies directed against BCMA can be used to develop antigen targeting constructs for CAR constructs that target cells expressing BCMA. In some embodiments, the antigen targeting transgenic construct comprises an amino acid sequence (antigen binding domain) derived from an antibody that binds BCMA. BCMA antibodies include, but are not limited to, those described herein in addition to the antibodies used in the antibody drug conjugates GSK2857916, HDP-101, or MEDI2228, or the bispecific antibodies EM801, Ab-957, AFM26, or TNB383B.

[0085] Various CAR constructs targeting BCMA have been described in the art. Silencing of EBAG9 can essentially be applied to any one or more of such CAR-T cell therapeutics. Known BCMA CAR-T cells in clinical investigation include, but are not limited to, Bluebird Bio's bb2121, Nanjing Legend Biotech's LCAR-B38M, Novartis' CART-BCMA, Kite Pharma's KITE-585 and Pfizer / Cellectis SA's BCMA CAR, Poseida Therapeutics' P-BCMA-101, Tenebrioas' FHVH74-CD828Z, FHVH32-CD828Z, FHVH33-CD828Z, FHVH93-CD828Z, Cartesian Therapeutics' Descartes-08, Poseida Therapeutics' P-BCMA-ALLO1, and Juno's EGFRt / BCMA-41BBz, as reviewed in Cho et al. (Front Immunol. 2018; 9: 1821).

[0086] In some embodiments, the hematological malignancy being treated is a cancer that expresses CXCR5, and where the antigen-targeting transgenic construct binds to CXCR5.

[0087] Further tumor-associated antigens that can be targeted by antigen-specific constructs, such as CAR or TCR constructs, may be selected from the following:

[0088] The CAR-T or TCR antigen may be selected from those for which CAR constructs have been developed and are in clinical trials or preclinical trials, including, but not limited to, TAAs associated with hematological malignancies, such as CD30, CD20, CD22, ROR1, CD138, CD70, LeY, CD123, CD16V, CD123, CD33, Igκ, Igλ, IL-1RAP, NKG2D ligand, Muc1, GPC3, EpCam, CD38, CD5, IL-13Rα2, CD133, or CS1 (CD319).

[0089] TAAs from solid malignancies can also be targeted, including, but not limited to, GPC3, HER2, GD2, EGFR variant III (EGFR vIII), EGFR, CEA, PSMA, FRα, EPCAM, MUC1, ROR1, MUCI16eto, VEGFR2, CD171, PSCA, and EphA2, FAP, CAIX, c-MET, CD171, L1-CAM, mesothelin, Muc1, PD-L1.

[0090] Other tumor-associated antigens (TAA) can also be selected as targets for TCR-T or CAR, including MART-1, MAGE-A1, MAGE-A3, MAGE-A4, gp100, CEA, TIL 1383I, P53, HPV-16 E6, HPV-16 E7, and HBV.

[0091] Detailed summaries of published clinical trials of chimeric antigen receptor T cells (CAR-T) and TCR-transduced T cells (TCR-T) are disclosed in Mo et al. (Journal of Cancer, 2017; 8(9): 1690-1703), Hartmann et al. (EMBO Molecular Medicine, 2017; 9 (9): 1183-1197), and Townsend et al. (Journal of Experimental & Clinical Cancer Research, 2018, 37:163).

[0092] Although hematological cancers (blood-based malignancies) appear to be more amenable to T-cell therapy, solid tumors may also be treated, as evidenced by multiple ongoing clinical trials investigating the cytolytic effects of CAR-T or TCR-T cells in solid tumor disease.

[0093] For example, in lung cancer, candidate TAAs may include mesothelin, EGFR, MUC1, RORI, CEA, WT1, NY-ESO-1, or MAGE-A3 / 4.

[0094] A combinatorial approach based on antigen targeting constructs directed against two or more of the above antigens can also be used, for example, by using one or more targeting constructs targeting CD19 and CD20.

[0095] Furthermore, adoptive tandem-CAR-T therapy of glioblastoma appears to be effective by targeting human epidermal growth factor receptor 2 (HER2) and IL13Rα2, antigens that are heterogeneously expressed on glioblastoma.

[0096] Tumor type-specific and diversified TAAs are not only expressed in one type of cancer to be targeted, but one TAA may also be expressed in multiple types of cancer. These TAAs can also be targeted. For example, NY-ESO-1 is highly expressed in melanoma, multiple myeloma, NSCLC, synovial sarcoma, breast cancer, renal cell carcinoma, hepatocellular carcinoma, esophageal cancer, ovarian cancer, and bladder cancer. Similarly, mesothelin is highly expressed in mesothelioma and breast cancer, cervical cancer, pancreatic cancer, ovarian cancer, lung cancer, and endometrial cancer.

[0097] Abbreviations for the above antigens: PD-1: programmed cell death 1 receptor; NSCLC: non-small cell lung cancer; HLA: histocompatibility leukocyte antigen; ROR1: tyrosine kinase-like orphan receptor 1; BCMA: B-cell maturation antigen; LeY: Lewis (Le)-Y; GPC3: glypican-3; HER2: human epidermal growth factor receptor-2; GD2: tumor-associated ganglioside GD2; EGFR: epidermal growth factor receptor; CEA: carcinoembryonic antigen; PSMA: prostate-specific membrane antigen; FRα: folate receptor-α; EPCAM: epithelial cell adhesion molecule; MUC1: mucin 1; VEGFR2: vascular endothelial growth factor receptor-2; PSCA: prostate stem cell antigen; EphA2: erythropoietin-producing hepatocellular carcinoma A2; HPV: human papilloma virus; MAGE: genes encoding melanoma-associated antigens; TNF-α: tumor necrosis factor-α.

[0098] The specific TAAs described herein are of an exemplary nature and represent preferred, non-limiting embodiments of the present invention. The inventive concept of EBAG9 inhibition can be applied to any given engineered T cell, regardless of the antigen specificity of the T cell.

[0099] In a further aspect, the present invention relates to a pharmaceutical composition comprising the genetically modified T cells described herein suitable for the treatment of a proliferative disease, additionally comprising a pharma- ceutically acceptable carrier.

[0100] In a further aspect, the present invention relates to a nucleic acid vector or a combination of nucleic acid vectors comprising an antigen targeting construct and a sequence encoding an RNA interference sequence for knockdown of estrogen receptor binding fragment associated antigen 9 (EBAG9).

[0101] In a further aspect, the present invention relates to an in vitro method of increasing the cytolytic activity of genetically modified cytotoxic T cells, said T cells comprising one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct, the method comprising inhibiting the activity of estrogen receptor-binding fragment-associated antigen 9 (EBAG9) in said T cells.

[0102] In a further embodiment, the invention relates to the product of the in vitro method, namely a set of isolated T cells modified with an antigen-targeting transgenic construct in which EBAG9 is inhibited.

[0103] In one embodiment, the EBAG9 inhibited isolated T cells engineered with antigen-targeting transgenic constructs produced by the methods of the invention are characterized by 10% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more inhibition of EBAG9 compared to control CTLs. In some instances, for example, where EBAG9 is deleted or expression is prevented, for example, by genomic modification, 100% inhibition of EBAG9 may be achieved.

[0104] In one embodiment, isolated T cells modified with an antigen-targeting transgenic construct in which EBAG9 is inhibited, produced by the method of the invention, are characterized by an increase in cytotoxic activity of 5% or more, 10% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 300% or more, when compared to cytolytic T cells in the absence of EBAG9 inhibition. Functional assays that quantitatively or semi-quantitatively determine the increase in cytolytic activity of T cells in which EBAG9 is inhibited are available to the skilled artisan, some examples of which are described herein. For example, in vitro assays such as those used to generate Figures 7 and 8 below or described in the Examples can be applied to determine the increase in cytolytic activity compared to "unmodified" T cells, i.e., T cells in which a specific modification that reduces / inhibits EBAG9 function has not been performed. In some embodiments, this assay can be used to determine whether the method of the invention has been performed, i.e., to determine any reduction in EBAG9 function.

[0105] The in vitro method of increasing the cytolytic activity of cells represents an important aspect of the present invention, since T cell therapeutics usually require in vitro treatment before administration to patients. In a typical adoptive T cell therapy (ATT), T cells are isolated from a patient and then genetically modified to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). During this preparation process, CTLs can be further modified to increase their cytolytic activity.

[0106] Thus, the present invention allows for a method to enhance the efficacy of cellular therapeutics without adding additional burden or difficulty during the cell preparation process. Thus, in some embodiments, T cells intended for therapeutic applications are not placed under undue stress during preparation, since EBAG9 inhibition can be achieved in the same genetic modification step in which CTLs are modified with antigen targeting constructs.

[0107] In some embodiments, the in vitro methods described herein are characterized in that inhibiting EBAG9 activity preferably comprises RNA interference of EBAG9 expression, e.g., knockdown of EBAG9 by small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA).

[0108] In some embodiments, the in vitro methods described herein are characterized in that inhibiting EBAG9 activity comprises genetic modification of the T cell genome, preferably by CRISPR / Cas9 or TALEN to prevent expression of the EBAG9 gene and / or disrupt its sequence.

[0109] In further embodiments of the present invention, CRISPR / Cas and TALEN-mediated insertion of nucleic acids encoding CAR or TCR may be used. CRISPR / Cas, known to those skilled in the art and adapted from a process naturally occurring in bacteria, may be used to precisely and efficiently edit DNA to insert the appropriate coding sequence into immune cells of interest, preferably T cells. Cas9, a protein that acts as a pair of molecular scissors, is guided to a specific DNA sequence by an accompanying RNA molecule (guide RNA). Once Cas9 reaches the target location on the DNA, it promotes local changes in the genetic code, affecting the function of that gene. CRISPR / Cas9 can deliver the CAR or TCR gene to a very specific site in the T cell genome, which may reduce the risk of gene insertion at an incorrect or undesired location.

[0110] As mentioned above, the examples of the present application demonstrate that various guide RNAs targeting exon 4 of EBAG9 can achieve good knockout efficiency of EBAG9 together with Cas9 protein. In one embodiment of the method for enhancing the efficacy of cell therapy drugs, CRISPR / Cas9-mediated EBA9 inhibition is used, which preferably targets exon 4 of EBAG9 gene. This embodiment shows effective but non-limiting preferred targets within EBAG9 for CRISPR-mediated knockout. Alternative targets suitable for CRISPR-mediated genetic manipulation of EBAG9 gene are known to those skilled in the art or can be evaluated without undue effort.

[0111] The invention further relates to methods of treating the medical conditions described herein, which typically involve administration of a therapeutically effective amount of the modified CTLs to a patient in need of such treatment.

[0112] In some embodiments, the treatments described herein may be performed in new or unique patient populations due to the increased activity of the CTLs. In preferred embodiments, for example, the CTLs described herein are applicable to the treatment of patients who are not suitable for other therapies. More specifically, embodiments of the present invention relate to the treatment of the following patient populations: i) Patients with multidrug resistance, ii) patients who are not suitable for allogeneic stem cell transplantation; iii) patients with comorbidities precluding further chemotherapy; iv) Elderly patients who cannot tolerate chemotherapy; v) CTLs are applicable as salvage therapy even after disease progression and failure of multiple other standard therapies; vi) CTLs are applicable even when antigen density on the target tumor cells is low, where antibodies may fail, and / or vii) CTLs can be applied as a monotherapy where antibodies are not applicable.

[0113] In addition, the ability to specifically target B cells and / or plasma cells is highly effective in treating autoimmune diseases. Mild autoimmune diseases are usually initially treated with nonsteroidal anti-inflammatory drugs (NSAIDs) or disease-modifying antirheumatic drugs (DMARDs). More severe systemic lupus erythematosus (SLE), with active disease leading to organ failure, is usually treated with steroids in combination with strong immunosuppressants such as cyclophosphamide, a cytotoxic agent that targets circulating cells.

[0114] Recently, CAR-T cells have also been explored as a targeted approach to treat autoantibody-mediated diseases (Ellebrecht et al. (2016) Science 353:179-184). Long-lived, resident plasma cells in the bone marrow survival niche are often resistant to traditional immunosuppressive and cytotoxic drugs as well as therapies targeting B cells and their activation. This therapeutic challenge may be addressed by using CAR-T cell constructs, especially those with EBAG9 inhibition.

[0115] Thus, the present invention further relates to the genetically modified T cells described herein for use as a medicament in treating an autoimmune disease.

[0116] Thus, in a further aspect, the present invention relates to a genetically modified T cell as described herein for use as a medicament in the treatment of autoantibody-dependent autoimmune diseases, wherein the antigen targeted by the antigen-targeting transgenic construct is expressed in a target cell associated with the production of autoantibodies, preferably the medical disorder is systemic lupus erythematosus (SLE) or rheumatoid arthritis. Further embodiments of autoimmune-related diseases are provided below. The skilled artisan will be able to select a suitable target antigen for the antigen-targeting construct of the T cells of the present invention. One relevant antigen for targeting plasma cells producing autoantibodies is BCMA.

[0117] The technical guidance for treating cancer cells provided herein and by the examples herein is also applied to enable the treatment of autoimmune diseases. By selecting the appropriate antigen to be targeted by the antigen-specific targeting construct of engineered T cells, autoreactive B cells can be effectively targeted. The expression of EBAG9 in engineered T cells is of greatest interest, and the expression / activity of EBAG9 is reduced / inhibited, enhancing the cytotoxicity of T cells, regardless of EBAG9 expression in target cells. Thus, any given target cell can be attacked by engineered T cells with enhanced cytotoxicity due to EBAG9 inhibition.

[0118] Related embodiments of the features relating to the engineered T cells, their use as medicaments, vectors, pharmaceutical compositions, and in vitro methods are united by the novel concept of engineered cytolytic T cells comprising an antigen-specific targeting construct in which EBAG9 is inhibited. Related embodiments of the features disclosed herein relating to any one aspect of the engineered T cells, their use as medicaments, vectors, pharmaceutical compositions, and in vitro methods are considered to be disclosed in the context of other aspects of the invention such that the features of the cells may also be used, for example, to describe a method or composition disclosed herein, and vice versa.

[0119] Related embodiments of chimeric antigen receptor constructs and CAR T cells In one embodiment, the invention relates to a CTL as described herein comprising a chimeric antigen receptor polypeptide (CAR) comprising: an extracellular antigen-binding domain comprising an antibody or antibody fragment which binds to a target antigen, said antibody or antibody fragment comprising the VH and VL domains of a single chain antibody fragment, preferably wherein a linker polypeptide is disposed between the VH and VL domains, said linker being preferably configured so as not to interfere with antibody fragment-antigen interaction; a spacer polypeptide (also referred to as a hinge) disposed between the extracellular antigen-binding domain and the transmembrane domain, said spacer polypeptide preferably configured such that it does not interfere with antibody fragment-antigen interactions and / or T cell activation when the CAR is expressed in a T cell expressing the CAR; a transmembrane domain, wherein said transmembrane domain is preferably configured so as not to interfere with antibody fragment-antigen interaction and / or so as not to interfere with T cell activation when the CAR is expressed in a T cell expressing the CAR; and an intracellular domain, said intracellular domain comprising a costimulatory domain and a signaling domain, preferably configured to, upon binding to a target antigen, generate a signal to stimulate T cell activation, e.g., by increasing cytokine production and / or promoting T cell replication, thereby resulting in a cytotoxic effect.

[0120] Thus, the CAR of the present invention may use a variety of formats, including optionally different protein sequences for each of the functional domains described herein. Those skilled in the art may select and test the desired function of the CAR, for example, based on the experimental procedures shown in the following examples. Thus, the selection of any given specific protein sequence used in the CAR of the present invention in any of the functional domains discussed herein may be evaluated for functional effectiveness by those skilled in the art using routine methods. For example, different linker polypeptide sequences may be used between the VH and VL domains, different spacer polypeptide sequences (also called hinges) may be used between the extracellular antigen binding domain and the transmembrane domain, different transmembrane domains, and different intracellular domains, preferably including costimulatory and signal transduction domains. Different costimulatory domains may be used in series (e.g., CD28+4-1BB).

[0121] In embodiments of the invention, the CARs and each element or domain referred to herein are configured so as not to adversely interfere with antibody fragment antigen interactions, so as not to adversely interfere with T cell activation when the CAR is expressed in a T cell expressing said CAR, and so as not to adversely interfere with the CAR generating a signal upon binding to a target to stimulate T cell activation. In preferred embodiments, the elements of the CAR are selected so as not to interfere with EBAG9 inhibition and the associated increase in cytolytic activity.

[0122] Embodiments of the antigen-binding domain of the CAR As non-limiting examples, the antigen binding domain of the CAR can be directed against BCMA, CXCR5, or CD19. In other embodiments, the antigen binding fragment can be selected based on any given desired antigen that is considered a target of malignant cells. Some specific non-limiting embodiments of CXCR5 and BCMA CARs are described below. These have been evaluated by the inventors and shown to exhibit enhanced cytolytic activity when present on T cells in combination with EBAG9 inhibition.

[0123] CXCR5-specific antigen-binding domain In one embodiment, the CTL of the present invention comprises a CXCR5-specific chimeric antigen receptor (CAR) polypeptide described herein, wherein the antigen binding domain comprises: H-CDR1 according to SEQ ID NO: 7 (GFTFSTSG), H-CDR2 according to SEQ ID NO: 8 (ISSSSGFV), H-CDR3 according to SEQ ID NO: 9 (ARSEAAF), L-CDR1 according to SEQ ID NO: 10 (KSRLSRMGITP), L-CDR2 with a sequence comprising or consisting of RMS, and L-CDR3 according to SEQ ID NO: 11 (AQFLEYPPT).

[0124] In one embodiment, the CXCR5-specific CAR polypeptide comprises: A VH domain having at least 80% sequence identity to SEQ ID NO: 12 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSTSGMNWFRQAPGKGLEWVSYISSSSGFVYADSVKGRFTISRDNAQNSLYLQMNSLRAEDTAVYYCARSEAAFWGQGTLVTVSS), or a VH domain having at least 80% sequence identity to SEQ ID NO: 13 (EVQLVESGGGLVQPGKSLKLSCSASGFTFSTSGMHWFRQAPGKGLDWVAYISSSSGFVYADAVKGRFTISRDNAQNTLYLQLNSLKSEDTAIYYCARSEAAFWGQGTLVTVSS); and a VL domain having at least 80% sequence identity to SEQ ID NO: 14 (DIVLTQSPRSLPVTPGEPASISCRSSKSRLSRMGITPLNWYLQKPGQSPQLLIYRMSNRASGVPDRFSGSGSGTDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK), Or a VL domain having at least 80% sequence identity to SEQ ID NO: 15 (DIVLTQAPRSVSVTPGESASISCRSNKSRLSRMGITPLNWYLQKPGKSPQLLIYRMSNLASGVPDRFSGSGSETDFTLKISKVETEDVGVYYCAQFLEYPPTFGSGTKLEIK).

[0125] BCMA-specific antigen-binding domain In one embodiment, the CTL of the invention comprises a BCMA-specific chimeric antigen receptor (CAR) polypeptide described herein, wherein the antigen binding domain comprises: H-CDR1: GFTFSRYW (SEQ ID NO: 16), H-CDR2: INPSSSTI (SEQ ID NO: 17), H-CDR3: ASLYYDYGDAYDY (SEQ ID NO: 18), L-CDR1: QSVESN (SEQ ID NO: 19), L-CDR2:SAS, and L-CDR3: QQYNNYPLT (SEQ ID NO: 20), Or the antigen binding domain comprises: H-CDR1: RYWFS (SEQ ID NO: 21), H-CDR2: EINPSSSTINYAPSLKDK (SEQ ID NO: 22), H-CDR3: SLYYDYGDAYDYW (SEQ ID NO: 23), L-CDR1: KASQSVESNVA (SEQ ID NO: 24), L-CDR2: SASLRFS (SEQ ID NO: 25), and L-CDR3: QQYNNYPLTFG (sequence number 26).

[0126] In one embodiment, the BCMA-specific CAR polypeptide comprises: A VH domain having at least 80% sequence identity to SEQ ID NO: 27 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWFSWVRQAPGKGLVWVGEINPSSSTINYAPSLKDKFTISRDNAKNTLYLQMNSLRAEDTAVYYCASLYYDYGDAYDYWGQGTLVTVSS); and a VL domain having at least 80% sequence identity to SEQ ID NO: 28 (EIVMTQSPATLSVSPGERATLSCKASQSVESNVAWYQQKPGQAPRALIYSASLRFSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNYPLTFGAGTKLELK).

[0127] Linker, Spacer, Transmembrane and Signaling Domain Embodiments For the particular non-antigen-specific CAR domains used in the examples, several embodiments are shown below.

[0128] In a further embodiment, the present invention relates to a CTL comprising a chimeric antigen receptor (CAR) polypeptide as described herein, comprising one or more linkers, spacers, transmembrane domains, and signaling domains.

[0129] In one embodiment, the CAR comprises an intracellular domain that comprises one or two costimulatory domains and a signaling (activation) domain.

[0130] In one embodiment, the CAR comprises an extracellular antigen binding domain with a linker polypeptide disposed between the VH and VL domains, wherein said linker is preferably selected from: Whitlow (SEQ ID NO: 29; GSTSGSGKPGSGEGSTKG), or Gly-Ser (SEQ ID NO: 30; SSGGGGGSGGGGSGGGGS) linker, or A linker having at least 80% sequence identity to SEQ ID NO: 29 or 30.

[0131] In one embodiment, the CAR additionally comprises a spacer polypeptide positioned between the extracellular antigen binding domain and the transmembrane domain, wherein said spacer is selected from the following: IgG1 spacer (SEQ ID NO:31; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPK), IgG1Δspacer (SEQ ID NO:32; PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSSLSPGKK), IgG4(Hi-CH2-CH3) spacer (SEQ ID NO:33; ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), IgG4(Hi-CH3) spacer (SEQ ID NO:34; ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK), IgG4(Hi) spacer (SEQ ID NO: 35; ESKYGPPCPPCP), or A spacer having at least 80% sequence identity to any one of SEQ ID NOs: 31 to 35.

[0132] In one embodiment, the transmembrane domain is selected from the following: CD8α domain (SEQ ID NO: 36; IYIWAPLAGTCGVLLLSLVITLYC), or CD28 domain (SEQ ID NO: 37; FWVLVVVGGVLACYSLLVTVAFIIFWV), or A transmembrane domain having at least 80% sequence identity to SEQ ID NO: 36 or 37.

[0133] In one embodiment, the intracellular domain comprises: 4-1BB costimulatory domain (SEQ ID NO: 38; KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL), and / or CD28 costimulatory domain (SEQ ID NO: 39; RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSL), or A costimulatory domain that includes both a 4-1BB costimulatory domain (SEQ ID NO: 38) and a CD28 costimulatory domain (SEQ ID NO: 39) arranged adjacently; or A costimulatory domain having at least 80% sequence identity to SEQ ID NO: 38 or 39; A costimulatory domain selected from:

[0134] In one embodiment, the CAR additionally comprises a signaling domain (also known as an activation domain), wherein the signaling domain is: CD3zeta (4-1BB or CD28) signaling domain (SEQ ID NO: 40; LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR), or A signaling domain having at least 80% sequence identity to SEQ ID NO:40.

[0135] Furthermore, the present invention relates to an (isolated) nucleic acid molecule for use in the CTL of the present invention, preferably in the form of a vector such as a viral vector or a transposon vector, preferably in the form of a gamma retroviral vector, selected from the group consisting of: a) A nucleic acid molecule comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) polypeptide according to any embodiment of the CAR described herein; or A nucleic acid molecule comprising a nucleotide sequence encoding an extracellular antigen-binding domain, a leader, a transmembrane domain, a spacer, a linker, or an intracellular domain, preferably as set forth in SEQ ID NOs: 7 to 41, or a nucleic acid molecule comprising a nucleotide sequence encoding a leader, a transmembrane domain, a spacer, a linker, or an intracellular domain, the nucleic acid sequence comprising or consisting of one or more of SEQ ID NOs: 43-54; b) a nucleic acid molecule complementary to the nucleotide sequence according to a); c) a nucleic acid molecule comprising a nucleotide sequence having sufficient sequence identity to be functionally similar / equivalent to a nucleotide sequence according to a) or b), including at least 80% sequence identity to the nucleotide sequence according to a) or b), preferably 90% or 95% sequence identity to the nucleotide sequence according to a) or b), wherein the functional similarity is relevant to form a CAR construct, and wherein when the corresponding T cell expresses said construct, the product of the CAR-T cell confers cytotoxic activity to the T cell; and / or d) A nucleic acid molecule that is degenerate, due to the genetic code, to the nucleotide sequences in a) through c).

[0136] The exchange of signaling domains caters to either a potent and rapid effector phase (CD28 costimulatory domain) or a sustained relapse control ensured by T memory cell populations (4-1BB signaling domain). Different signaling domains can be exchanged in multiple configurations, providing CARs with flexibility in design without losing advantageous binding properties.

[0137] In a preferred embodiment, in combination with MP71 vectors and gamma-retrovirus expression systems, significantly higher transduction rates in human T cells can be achieved. The transduction system is flexible due to the modular design of the TCR or CAR constructs, meaning that lentiviruses and transposons can be used, depending on the needs and the preferences of the skilled artisan in carrying out the present invention. The introduction of genetic information / nucleic acid molecules for TCR, CAR, and EBAG9 inhibition also includes insertion into CTLs mediated by CRISPR / Cas and TALEN.

[0138] All suitable methods for introducing genetic information / nucleic acid molecules for TCR, CAR, and EBAG9 inhibition into cells expressing the CAR are encompassed by the present invention, and suitable methods can be selected by those skilled in the art when carrying out the present invention. For example, multiple methods of transforming T cells are known in the art, including any given virus-based gene transfer method, such as those based on modified retroviridae, and non-viral methods, such as DNA-based transposons, episomal cDNA vectors, and direct introduction of mRNA by electroporation.

[0139] In addition, the signaling components of the CAR construct can be exchanged in a simple three-step cloning procedure, allowing modular organization and tailor-made construction of clinically applicable CARs by those skilled in the art.

[0140] A further aspect of the present invention relates to a vector, preferably a viral vector, more preferably a gamma retroviral vector, comprising the nucleic acid molecule described herein.In another aspect of the present invention, the present invention relates to a transposon vector, preferably a sleeping beauty vector, capable of encoding and preferably expressing the TCR, CAR and / or EBAG9 inhibitors of the present invention.

[0141] In a preferred embodiment, the CTLs intended for administration in the treatment of the diseases described herein are genetically modified as described herein with nucleic acids encoding and expressing the TCR, CAR, and EBAG9 silencing molecules, e.g., miRNAs, described herein, using the "Sleeping beauty" transposon system, particularly the sleeping beauty transposase. The Sleeping Beauty transposon system, in the context of the present invention, is a synthetic DNA transposon designed to precisely introduce a defined DNA sequence into a vertebrate chromosome, for modifying immune cells to express the CAR or TCR described herein. The sleeping beauty transposon combines the advantages of viruses and naked DNA. Viruses have been evolutionarily selected based on their ability to infect and replicate in novel host cells. At the same time, cells have developed major molecular defense mechanisms to protect themselves against viral infection. Avoiding the use of viruses is also important for social and regulatory reasons. Thus, the use of non-viral vectors, such as the sleeping beauty system, avoids many, but not all, of the defenses that cells use against vectors. For this reason, the sleeping beauty system allows for a particularly effective and safe genetic modification of immune cells administered to patients.

[0142] Table A Preferred amino acid and nucleotide sequences of the present invention: SEQ ID NO: Sequence Description H17 EBAG9 targeting sequence 1 H18 EBAG9 targeting sequence 2 EBAG9 transcript variant 1 EBAG9 transcript variant 2 EBAG9 transcript variant 3 EBAG9 transcript variant X1 CXCR5 humanized VH CXCR5 Rat VH CXCR5 humanized VL CXCR5 Rat VL Whitlow Linker Gly-Ser linker IgG1 spacer IgG1ΔSpacer IgG4 (HI-CH2-CH3) spacer IgG4 (HI-CH3) spacer IgG4 (HI) spacer Transmembrane domain CD8α Transmembrane domain CD28 Costimulatory domain 4-1BB Costimulatory domain CD28 Activation domain CD3ζ(4-1BB) or (CD28) Lκ Leader Humanized Whitlow Rat Whitlow Humanized IgG1 Spacer Rat IgG1 Spacer Humanized IgG1ΔSpacer Rat IgG1Δspacer Transmembrane domain CD8α Transmembrane domain CD28 Costimulatory domain 4-1BB Costimulatory domain CD28 Activation domain CD3ζ(4-1BB) Activation domain CD3ζ (CD28) EBAG9 targeting sequence 3 EBAG9 targeting sequence 4 EBAG9 targeting sequence 5 EBAG9 targeting sequence 6 EBAG9 targeting sequence 7 EBAG9 targeting sequence 8 EBAG9 targeting sequence 9 EBAG9 targeting sequence 10 [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Brief description of the drawings]

[0143] [Figure 1] FIG. 1. Construction of retrovirus MP71 encoding EBAG9-targeting miRNA and GFP. [Diagram 2] FIG. 10. Reduction of human EBAG9 expression in Jurkat cells after transduction with retroviral vectors encoding different EBAG9-targeting miRNAs. [Diagram 3] FIG. 1: Experimental timeline of retroviral transduction of human primary T cells prior to in vitro functional assays. [Figure 4] FIG. 1 shows that EBAG9 downregulation promotes antigen-independent release of granzyme A from activated human CD8+ T cells. [Diagram 5] FIG. 1 shows that the MP71 vector is suitable for co-expression of miRNA targeting EBAG9 and CAR. [Figure 6-1] FIG. BCMA and CD19 CAR expression in transduced human primary T cells. [Figure 6-2] Same as above [Figure 7] FIG. 1 shows that antigen-specific cytolytic activity of BCMA CAR T cells can be increased by downregulation of EBAG9. [Figure 8] FIG. 1 shows that increased cytolytic activity of CAR T cells by silencing EBAG9 is a universally applicable cell biological mechanism. [Figure 9-1] FIG. 1 shows that in vivo engineered BCMA CAR T cells with silenced EBAG9 more efficiently eradicate multiple myeloma cells. [Figure 9-2] Same as above [Figure 10] FIG. 14. Target site validation of CRISPR-mediated EBAG9 knockout. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0144] All documents cited and their U.S. counterparts are hereby incorporated by reference.

[0145] The present invention relates to genetically engineered T cells that are modified by inhibiting EBAG function, thereby enhancing the cytolytic activity of the cells. Enhancement of the functional avidity of either TCR-T cells or T cells equipped with CAR will result in higher cytolytic capacity, thereby resulting in higher efficacy against tumors. As described herein, there are specific advantages associated with modified T cells (genetically engineered T cells) endowed with higher cytolytic capacity.

[0146] Tumor patients often undergo several previous treatment regimens, which causes strong myelosuppression and subsequent inefficient mobilization of peripheral lymphocytes as starting material for ex vivo culture and transduction. This process is called autologous transplantation. Since engineered T cells function much stronger on a single cell basis, endowing a small number of T cells with enhanced cytolytic ability helps to avoid quantity problems. In other words, genetic engineering of T cells improves and facilitates the manufacturing process.

[0147] Another aspect of the manufacturing process is the duration of the ex vivo expansion step. If the T cells are endowed with a higher cytolytic capacity, the in vitro expansion can be terminated sooner, resulting in lower production costs, earlier access to therapies, and a more undifferentiated T cell population. The latter aspect is important because the more advanced the differentiation state of T cells from in vitro cultures, the shorter their persistence in vivo.

[0148] In allogeneic bone marrow transplantation, a significant risk factor is the development of graft-versus-host disease (GvHD). This disease or syndrome is strongly correlated with the number of T cells transplanted. By endowing T cells with improved cytolytic capabilities, the need for large numbers of T cells is alleviated, and thus a smaller number of genetically engineered T cells provides the same therapeutic effect without mediating GvHD.

[0149] The threshold level for activation and effector function of engineered T cells is low, so that even weak tumor antigens, including neoantigens, self antigens, and non-major histocompatibility antigens, as well as the few antigens presented, can already trigger the release of effector molecules from either engineered T cells bearing only a TCR or those equipped with a CAR.

[0150] To prevent tumor recurrence, it is highly advantageous to generate memory T cells after the first tumor eradication. However, some tumor cells may escape the attack of T cells and eventually begin to proliferate thereafter. However, the inventors have shown that cytolytic ability correlates with the formation of antigen-specific memory T cells. The improved antitumor effect of ATT does not cause the release of more cytokines, which may be harmful to cause an inflammatory state.

[0151] In view of the above, the present inventors have presented a novel means of improving the cytolytic activity of T cells based on EBAG9 inhibition, which is described in more detail below. The following terms and definitions are provided for clarity. Unless otherwise defined, all terms retain their general meaning as understood by those skilled in the art.

[0152] Cytotoxic T cells and cytolytic activity Cytotoxic T cells (also known as TCs, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, T cells, or killer T cells, and may be used interchangeably herein) are T cells (a type of white blood cell) that have cytolytic activity, for example against cancer cells. In some embodiments, the cytolytic activity may be associated with CD8+ and / or CD4+ T cells. Both T cell subpopulations can produce and release lytic granule components that are cytolytic enzymes such as granzymes.

[0153] The term "cytolytic" refers to the ability of T cells to kill target cells through the release of lytic granule components, also called secretory lysosomes.

[0154] As a central component of the adaptive immune response, T cells can eliminate infected and transformed tumor cells. CD8 + T cells can mature into cytotoxic T lymphocytes (CTLs) that are primarily responsible for the destruction of infected or transformed cells by releasing cytolytic granules into the immune synapse. These granules contain Ca 2+ These proteins contain perforin and granzymes that are released via a regulated secretory pathway dependent on the IL-1 receptor and induce apoptosis in target cells.

[0155] As soon as CTLs recognize and bind to their target cells, secretory lysosomes migrate and cluster around the microtubule-organizing center. After membrane fusion, perforin and granzymes are released into the immune synapse. Perforin is a pore-forming molecule that can permeabilize the membrane, which is important for the entry of granzymes into the cytosol of target cells. Granzymes initiate a programmed cell death pathway in the target cells. Under some activation conditions, CD4+ T cells equipped with CAR receptors can also acquire cytolytic properties by releasing granzymes. Thus, they can be engineered by silencing EBAG9 in the same way as CD8+ cytolytic T cells.

[0156] Granzyme A induces caspase-independent apoptosis, characterized by the generation of single-stranded DNA nicks. The action of granzyme A causes a loss of mitochondrial inner membrane potential, which leads to the release of reactive oxygen species (ROS). As a result, single-stranded DNA nicks are induced. In contrast, granzyme B, in addition to activating the caspase-independent cell death program, can induce caspase-dependent apoptosis by cleaving and activating caspases 3, 7, 8, and 10 and some of their downstream substrates. In addition, granzyme B induces ROS generation and the release of cytochrome c from mitochondria.

[0157] EBAG9 and its role in regulated secretion of effector molecules Protein transport from the trans-Golgi network (TGN) to secretory lysosomes is highly regulated. We have demonstrated that the effector molecule Ca 2+ We have demonstrated the presence of regulatory proteins such as estrogen receptor-binding fragment-associated antigen 9 (EBAG9), which is a negative regulator of estrogen-dependent regulated secretion.

[0158] Human EBAG9 contains 213 amino acids and exhibits a domain structure. Due to the C-terminal coiled-coil structure, human EBAG9 forms homo-oligomers with the transmembrane domain located at the N-terminus. EBAG9 is an estrogen-inducible protein expressed in most tissues.

[0159] We show that deletion of EBAG9 inhibits CD8 + We demonstrated that promoting the release of secretory lysosomes containing granzyme A from T cells enhances the cytolytic activity of CTLs in vivo.

[0160] Mechanistically, EBAG9 interacts with the γ2-subunit of AP-1 and inhibits AP-1 activity, clathrin-coated vesicle formation. EBAG9 was also identified as an interacting partner of snapin and BLOS2, subunits of the lysosome-associated organelle complex-1 (BLOC-1). In the secretory pathway, BLOC-1 regulates protein sorting from endosomes to secretory lysosomes. Thus, EBAG9 is an attractive target to negatively regulate vesicle trafficking from the TGN to secretory lysosomes and increase the cytolytic activity of adoptively transferred T cells.

[0161] The net effect of trafficking regulation imposed by EBAG9 is inhibition of secretory lysosomal maturation, including the transport and fusion process of carriers containing precursor forms of proteolytic enzymes. Alternatively, reuptake of secreted lytic granule components may be inhibited by EBAG9, resulting in reduced availability of recycled cytolytically active granzymes.

[0162] EBAG9 was identified as an estrogen-responsive gene. Regulation of transcription by estrogen is mediated by estrogen receptors binding to estrogen-responsive elements found in the 5' flanking region of the gene. The encoded protein is a tumor-associated antigen that is frequently expressed in a variety of cancers. Alternative splicing results in multiple transcript variants. A pseudogene for this gene has been localized on chromosome 10.

[0163] In some embodiments, the present invention relates to the estrogen receptor binding site-associated antigen 9 (EBAG9) of Homo sapiens (human), preferably according to the gene number 9166 in the NCBI database. This gene is located on chromosome 8. EBAG9 may also be known as EB9 or PDAF.

[0164] Thus, to inhibit EBAG9 using RNAi, transcript variants according to SEQ ID NO: 3-6 can be targeted. Those skilled in the art can design appropriate means, i.e. antisense, siRNA, miRNA, or shRNA, based on the target sequence of interest. Exemplary EBAG9 transcripts are selected from, but not limited to: NM_004215.5 (EBAG9), transcript variant 1, mRNA, SEQ ID NO: 3; NM_198120.2 (EBAG9), transcript variant 2, mRNA, SEQ ID NO: 4; NM_001278938.1 (EBAG9), transcript variant 3, mRNA, SEQ ID NO: 5; XM_017013960.1 (EBAG9), transcript variant X1, mRNA, SEQ ID NO: 6.

[0165] Preferred methods for assessing EBAG9 inhibition are related to those disclosed herein, e.g., CD8 + Methods include assessing the cytolytic activity of CTLs either in vivo or in vitro by quantitatively or semi-quantitatively assessing the release of secretory lysosomes containing granzyme A from T cells. Suitable protocols are disclosed below as applied in the in vitro examples of the present application.

[0166] RNA interference (RNAi) RNAi is a post-transcriptional gene silencing mechanism triggered by double-stranded RNA (dsRNA) that results in sequence-specific translational repression or mRNA degradation. Historically, RNAi has been known by other names, including co-suppression, post-transcriptional gene silencing (PTGS), and quelling.

[0167] MicroRNA (miRNA) genes are transcribed into pri-miRNAs of 500-3000 nucleotides in the nucleus by the action of RNA polymerase II. These pri-miRNAs are capped and polyadenylated. In addition, pri-miRNAs contain one or more stem-loop sequences and are cleaved into double-stranded pre-miRNA hairpin structures of 60-100 nucleotides by the Drosha-DGCR8 complex. Ran GTPase and Exportin-5 mediate the export of pre-miRNAs from the nucleus to the cytoplasm. There, they are further processed into incomplete duplex structures of 22 nucleotides by an RNase III enzyme called Dicer. One of the strands resembles the mature miRNA that binds to Argonaute (Ago) proteins and is incorporated into the RNA-induced silencing complex (RISC). RISC binding results in mRNA degradation or inhibition of protein translation. The fate of the target mRNA molecule depends on the degree of complementarity between the target mRNA molecule and the miRNA, but is also influenced by the incorporated Ago protein: while incorporation of Ago 2 leads to direct cleavage of the target mRNA, other Ago proteins adversely affect mRNA stability or attenuate translation.

[0168] For knockdown of specific target by genetic manipulation, several dsRNA molecules can be used that participate in the RNAi pathway at different steps. Transfection with small interfering RNA (siRNA) molecules that participate in the RNAi pathway in the cytosol only results in a transient protein knockdown. For long-term manipulation of gene expression, it is necessary to deliver dsRNA molecules by incorporating gene transfer vectors. Therefore, short hairpin RNA (shRNA) or miRNA molecules can be applied.

[0169] Both participate in the RNAi pathway in the nucleus and are then processed into siRNA-like molecules. shRNAs mimic the stem-loop structure of pre-miRNA. Their expression is driven by a strong RNA polymerase III promoter that results in high-level expression and stable gene knockdown. However, overexpression of shRNAs has been shown to mediate toxicity due to saturation of the miRNA processing pathway.

[0170] Another option is the application of artificial miRNAs to mediate stable knockdown in primary T cells. These artificial miRNAs are similar to pri-miRNAs and are therefore a further step towards mimicking natural miRNA biology. This has several advantages as an option for clinical applications. Most importantly, the use of endogenous miRNA processing machinery does not activate cellular self-defense mechanisms such as interferon induction.

[0171] In addition, artificial miRNAs are transcribed by RNA polymerase II promoters, as most natural miRNAs are. These promoters mediate regulated and tissue-specific expression, and also allow for simultaneous expression of selectable or therapeutic transgenes. Furthermore, multiple miRNAs that target the same or different regions of mRNA can be integrated into one expression cassette, resulting in additive effects of target downregulation. Such target downregulation is referred to herein as, for example, knockdown, silencing, or RNA interference.

[0172] CRISPR technology In some embodiments, inhibition of EBAG9 comprises genetic modification of the T cell genome by disrupting the expression of the EBAG9 gene and / or its sequence with CRISPR. In further embodiments of the invention, CRISPR-mediated insertion of nucleic acid encoding a CAR or TCR may be used.

[0173] CRISPR, an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, is a family of bacterial DNA sequences. The sequences contain DNA fragments from viruses that have attacked the bacteria. These fragments are used by the bacteria to detect and destroy DNA from further attacks by similar viruses. These sequences play a key role in the bacterial defense system and form the basis of a technology known as CRISPR / Cas to effectively and specifically alter genes within an organism.

[0174] Sequences in the CRISPR locus are transcribed and processed into CRISPR RNA (crRNA), which, together with the transactivating crRNA (tracrRNA), forms a complex with CRISPR-associated (Cas) proteins and determines the specificity of DNA cleavage by Cas nucleases through Watson-Crick base pairing between the nucleic acids (Wiedenheft, B et al (2012). Nature 482: 331-338; Horvath, P et al (2010). Science 327: 167-170; Fineran, PC et a. (2012). Virology 434: 202-209).

[0175] It has been shown that the three components required for the type II CRISPR nuclease system are the Cas9 protein, the mature crRNA, and the tracrRNA, which can be reduced to two components by fusing the crRNA and the tracrRNA to a single guide RNA (sgRNA), and that retargeting of the Cas9 / sgRNA complex to new sites can be achieved by altering the sequence of a short portion of the gRNA (Garneau, JE et al (2010). Nature 468: 67-71; Deltcheva, E et al. (2011). Nature 471: 602-607; Jinek, M et al (2012) Science 337: 816-821).

[0176] The CRISPR-Cas system is an RNA-guided adaptive immune system in bacteria and archaea that provides sequence-specific resistance to viruses or other invading genetic material. This immune-like response has been classified into two classes based on the structure of the effector module responsible for target recognition and cleavage of the invading nucleic acid (Makarova KS et al. Nat Rev Microbiol. 2015 Nov; 13(11):722-36). Class 1 contains multisubunit Cas protein effectors, while class 2 consists of a single large effector protein. Both classes 1 and 2 use CRISPR RNA (crRNA) to guide the Cas nuclease component to its target site, where it cleaves the invading nucleic acid. Due to its simplicity, class 2 CRISPR-Cas systems have been the most studied and have been widely applied in genome editing. The most widely used CRISPR-Cas system is CRISPR-Cas9. It has been demonstrated that the CRISPR / Cas9 system can be engineered for effective gene modification in mammalian cells.

[0177] In some embodiments of the present invention, RNA-guided DNA endonucleases are used. In the context of the present invention, the term "RNA-guided DNA endonucleases" refers to DNA endonucleases that interact with at least one RNA molecule. DNA endonucleases are enzymes that cleave phosphodiester bonds in DNA polynucleotide chains. In the case of RNA-guided DNA endonucleases, the interacting RNA molecule can guide the RNA-guided DNA endonuclease to the site or position of DNA where the endonuclease becomes active. In particular, the term RNA-guided DNA endonuclease refers to naturally occurring or genetically engineered Cas nuclease components or CRISPR-Cas systems, including but not limited to the multi-subunit Cas protein effector of class 1 CRISPR-Cas systems and the single large effector Cas protein of class 2 systems.

[0178] The details of the technical application of the CRISPR / Cas system and suitable RNA-guided endonucleases are known to those skilled in the art and are detailed in the literature, for example, Barrangou R et al. (Nat Biotechnol. 2016 Sep 8;34(9):933-941), Maeder ML et al. (Mol Ther. 2016 Mar;24(3):430-46), and Cebrian-Serrano A et al. (Mamm Genome. 2017;28(7):247-261). The present invention is not limited to the use of a particular RNA-guided endonuclease and thus includes the use of any given RNA-guided endonuclease in the present invention suitable for use in the methods described herein.

[0179] Any RNA-guided DNA endonuclease known in the art may be used in accordance with the present invention. RNA-guided DNA endonucleases include, but are not limited to, Cas proteins of class 1 CRISPR-Cas systems, such as Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11, Csx10, and Csf1; Cas proteins of class 2 CRISPR-Cas systems, such as Cas9, Csn2, Cas4, Cpf1, C2c1, C2c3, and C2c2; corresponding orthologous enzymes / CRISPR effectors from various bacterial and archaeal species; and engineered CRISPR effectors with, for example, novel PAM specificities, increased fidelity (e.g., SpCas9-HF1 / eSpCas9), or altered function (e.g., nickases). Particularly preferred RNA-guided DNA endonucleases of the present invention are Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Neisseria meningitidis Cas9 (NmCas9), Francisella novicida Cas9 (FnCas9), Campylobacter jejuni Cas9 (CjCas9), Cas12a (Cpf1), and Cas13a (C2C2) (Makarova KS et al. (November 2015). Nature Reviews Microbiology. 13 (11): 722-36).

[0180] The definitions and explanations provided herein focus primarily on the SpCas9 Crispr / Cas system, however, those skilled in the art will know how to use alternative Crispr / Cas systems as well as tools and methods that provide or allow obtaining information regarding the details of such alternative systems.

[0181] According to the method of the present invention, the RNA-guided DNA endonuclease may be introduced as a protein, but alternatively, the RNA-guided DNA endonuclease may also be introduced in the form of a nucleic acid molecule that encodes the protein. It will be understood that the nucleic acid molecule encodes the RNA-guided DNA endonuclease in an expressible form, such that expression in a cell results in a functional RNA-guided DNA endonuclease protein, such as the Cas9 protein. Means and methods for ensuring the expression of a functional polypeptide are well known in the art. For example, the coding sequence of the endonuclease may be included in a vector, such as a plasmid, a cosmid, a virus, a bacteriophage, or another vector that is typically used, for example, in genetic engineering.

[0182] Furthermore, the method of the present invention comprises introducing at least one guide RNA into the cell. In the context of the present invention, "guide RNA" refers to an RNA molecule that interacts with an RNA-guided DNA endonuclease, thereby resulting in the recognition of a target sequence that is cleaved by the RNA-guided DNA endonuclease. Thus, in the present invention, the term "guide RNA" encompasses, but is not limited to, target sequence-specific CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and chimeric single-stranded guide RNA (sgRNA).

[0183] As described herein, genes encoding the elements of the CRISPR / Cas system, e.g., Cas9, tracrRNA, and crRNA, are typically organized into an operon(s). DR sequences that function with RNA-guided endonucleases, such as Cas9 proteins of other bacterial species, can be identified by bioinformatics analysis of the repeat sequences occurring in the respective Crispr / Cas operons, as well as by experimental binding studies of Cas9 proteins and tracrRNA in combination with putative DR sequences flanking the target sequence.

[0184] In some embodiments, chimeric single-stranded guide RNA sequences comprising such target sequence-specific crRNA and tracrRNA may be used. Such chimeric (ch)RNAs may be designed by fusing a target-specific sequence of 20 nucleotides (nt) or more with a part or whole of the DR sequence (defined as part of the crRNA) to the whole or part of the tracrRNA, as shown by Jinek et al. (Science 337:816-821). The segments of the DR and tracrRNA sequences in the chimeric RNA can hybridize complementary to each other and form a hairpin structure.

[0185] Furthermore, at least one guide RNA of the present invention may be encoded by a nucleic acid molecule that is introduced into a cell.The definitions and preferred embodiments described herein for the nucleic acid molecule that codes for an endonuclease also apply to the nucleic acid molecule that codes for these RNAs.The regulatory elements for expressing RNA, such as the U6 promoter, are known to those skilled in the art.

[0186] Cell Therapy or Adoptive Cell Transfer (ATT) The discovery of IL-2 therapy gave rise to the hypothesis that T lymphocytes could be extracted from patients, expanded in vitro, and readministered as a cancer treatment. This was achieved in humans in 1988, when researchers used an expanded lineage of tumor-infiltrating lymphocytes to produce regressions in patients with metastatic melanoma. ATT is done by excising a tumor specimen and digesting it into a suspension of single cells. In the development of these techniques, two new methods of ATT emerged, the TCR and chimeric antigen receptor (CAR) approaches, both of which are now well established.

[0187] In TCR therapy, normal circulating T cells are usually isolated from the patient's blood and genetically modified to express TCR against tumor antigens by transfection with retroviral vectors or transposons. Specific TCRs are collected from human patients or mice immunized against the TAA of interest. A limitation of TCR therapy is that the recombinant TCR still relies on MHC recognition to obtain cytotoxicity. Chimeric antigen receptor technology was developed to circumvent this problem and introduced in 2010. This method also utilizes transfection with viral vectors, but introduces antibody variable regions expressed on the membrane and linked to intracellular signaling domains into T cells. As described in more detail below, the initial CARs and methods linked to CD3ζ were later improved to include costimulatory receptors, such as CD28, OX40, etc.

[0188] exogenous nucleic acid "Exogenous nucleic acid", "exogenous genetic element", or "transgenic" nucleic acid or construct refers to any nucleic acid introduced into a cell that is not a component of the cell's "original" or "native" genome or the pool of nucleic acids "naturally" found in unmodified T cells. Exogenous nucleic acid may or may not be integrated into the genetic material of the target T cell, or exogenous nucleic acid refers to stably transduced nucleic acid. Delivery of exogenous nucleic acid may result in genetic modification of the original cell by permanent integration of the exogenous nucleic acid molecule in the original cell. However, delivery of exogenous nucleic acid may be transient, meaning that the delivered genetic material disappears from the cell after a certain time for the provision of one or more TFs. Delivery of nucleic acid molecule and optionally genetic modification of living cells (i.e. T cells) can be performed and determined by the skilled artisan using commonly available techniques. For example, to detect genetic modification, it is possible to sequence the genome of the cell or a part thereof, thereby identifying if the exogenous nucleic acid is present. Alternatively, other molecular biology techniques for identifying / amplifying the exogenous genetic material may be applied, such as polymerase chain reaction (PCR). The exogenous nucleic acid may be detected by the vector sequence or a portion of the vector sequence, e.g., that remains at the site of genetic modification. Even if the vector sequence (e.g., that flanking the therapeutic transgene) may be removed from the genome or does not remain after modification, e.g., by CRISPR technology, the addition of the transgene may be detected by attempting to sequence the sequence containing the exogenous sequence at a "non-native" location in the genome.

[0189] An embodiment of the present invention relates to genetically modified cytotoxic T cells comprising one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct. In an embodiment of the present invention, the exogenous nucleic acid represents a nucleic acid sequence that is not naturally found in T cells, for example, based on a comparison with an unmodified human genome sequence. In an embodiment of the present invention, the antigen-targeting transgenic construct is a nucleic acid sequence encoding an antigen-targeting construct whose coding sequence is not naturally found in T cells, for example, based on a comparison with an unmodified human genome sequence. In some embodiments, the sequence is present in a "non-natural location" of the genome. In some embodiments, the targeting construct comprises or consists of a non-natural sequence, i.e., a synthetic sequence designed and created using recombinant or other molecular biology techniques. According to the present invention, EBAG9 activity is inhibited (compared to control cytotoxic T cells). In some embodiments, the presence of an exogenous nucleic acid sequence can be complemented by a functional evaluation of EBAG9 to further demonstrate EBAG9 inhibition.

[0190] Antigen targeting constructs As used herein, the term "antigen targeting construct" or "targeting construct" refers to a transgenic molecule (encoded by an exogenous nucleic acid molecule) that can direct T cells to a specific antigen or group of antigens. Thus, the antigen targeting construct is preferably a chimeric antigen receptor (CAR) or a T cell receptor (TCR). Engineered T cells have emerged as a new stage in precision cancer therapy. By forced expression of these antigen targeting molecules, preferably on autologous or donor T cells, they provide recognition of specific tumor antigens and enhance treatment specificity and efficacy.

[0191] Chimeric Antigen Receptor According to the present invention, a chimeric antigen receptor polypeptide (CAR) comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a target antigen, a transmembrane domain, and an intracellular domain. CARs are generally described as comprising an extracellular ectodomain (antigen-binding domain) derived from an antibody and an endodomain comprising a signaling module derived from a T-cell signaling protein.

[0192] In a preferred embodiment, the ectodomain comprises variable regions made of immunoglobulin heavy and light chains, preferably arranged as a single chain variable fragment (scFv). The scFv is preferably linked to a hinge region that provides flexibility and transmits a signal through an anchored transmembrane to an intracellular signalling domain. The transmembrane domain is preferably derived from CD8α or CD28. In first generation CARs, the signalling domain consists of the zeta chain of the TCR complex. The term "generation" refers to the structure of the intracellular signalling domain. Second generation CARs are equipped with a single costimulatory domain derived from CD28 or 4-1BB. Third generation CARs already contain two costimulatory domains, e.g. CD28, 4-1BB, ICOS or OX40, CD3ζ. The present invention preferably relates to second generation CARs or third generation CARs.

[0193] In various embodiments, engineered receptors are provided that redirect the cytotoxicity of immune effector cells to B cells. These engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine antibody-based specificity for a desired antigen with a T cell receptor activation intracellular domain, resulting in chimeric proteins that exhibit antigen-specific cellular immune activity. As used herein, the term "chimeric" describes a composition of different protein or DNA parts from different sources.

[0194] The CARs discussed herein comprise an extracellular domain (also referred to as a binding domain or antigen-binding domain) that binds to a target antigen, a transmembrane domain, and an intracellular domain or an intracellular signaling domain. The antigen-binding domain of the CAR engages on the surface of the target cell, resulting in the clustering of the CAR and transmitting an activation stimulus to the cell containing the CAR. A key property of CARs is their ability to redirect the specificity of immune effector cells, triggering the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or cell death of target antigen-expressing cells independently of major histocompatibility complex (MHC), thus exploiting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific coreceptors.

[0195] In various embodiments, the CAR comprises an extracellular binding domain comprising a humanized antigen-specific binding domain, a transmembrane domain, and one or more intracellular signaling domains. In certain embodiments, the CAR comprises an extracellular binding domain comprising a humanized antigen-specific antibody or antigen-binding fragment thereof, one or more spacer domains, a transmembrane domain, and one or more intracellular signaling domains.

[0196] An "extracellular antigen binding domain" or "extracellular binding domain" are used interchangeably and confer to the CAR the ability to specifically bind to a target antigen of interest. The binding domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. Preferably, it is an scFv domain.

[0197] "Specific binding" should be interpreted as the skilled artisan clearly recognizes various experimental procedures that can be used to test binding and binding specificity. Methods for measuring equilibrium association constants or equilibrium dissociation constants are known in the art. Some cross-reactivity or background binding may be unavoidable in many protein-protein interactions, and this should not be deducted from the "specificity" of the binding between a CAR and an epitope. "Specific binding" describes the binding of an antibody or an antigen-binding fragment thereof (or a CAR comprising it) to a target antigen with a binding affinity greater than background binding. The term "directed against" can also be applied in the understanding of the interaction between an antibody and an epitope when considering the term "specificity".

[0198] "Antigen (Ag)" refers to a compound, composition, or substance capable of stimulating the production of antibodies or a T-cell response in an animal. In certain embodiments, the target antigen is an epitope of a desired polypeptide. "Epitope" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed both from contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein.

[0199] A "single chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain in either orientation (e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. In a preferred embodiment, the CAR contemplated herein comprises an antigen-specific binding domain that is an scFv and can be a mouse, human or humanized scFv. Single chain antibodies can be cloned from the V region genes of a hybridoma specific for the desired target. The scFv can also be obtained from a phage display library, thus obviating traditional hybridoma technology. In a particular embodiment, the antigen-specific binding domain is a humanized scFv that binds to a human target antigen polypeptide.

[0200] Illustrative examples of variable heavy chains suitable for constructing anti-CXCR5 CARs contemplated herein include, but are not limited to, the amino acid sequence set forth in SEQ ID NO: 13. Illustrative examples of variable light chains suitable for constructing anti-CXCR5 CARs contemplated herein include, but are not limited to, the amino acid sequence set forth in SEQ ID NO: 15.

[0201] Antibodies and antibody fragments The CAR comprises an extracellular antigen-binding domain comprising an antibody or antibody fragment that binds to a target polypeptide. Thus, the antibodies or antibody fragments of the present invention include, but are not limited to, polyclonal, monoclonal, bispecific, human, humanized, or chimeric antibodies, single chain fragments (scFv), single chain variable fragments (ssFv), single domain antibodies (e.g., VHH fragments derived from nanobodies), Fab fragments, F(ab')2 fragments, fragments generated by Fab expression libraries, anti-idiotypic antibodies, and epitope-binding fragments of any of the above or combinations thereof, provided that they retain the same degree of binding properties as the CARs described herein, preferably comprising the corresponding CDRs, or VH and VL regions, as described herein. Mini- and multivalent antibodies, such as diabodies, triabodies, tetravalent antibodies, and peptabodies, may also be used in the methods of the present invention. The immunoglobulin molecules of the present invention may be of any class (i.e., IgG, IgE, IgM, IgD, and IgA) or subclass of immunoglobulin molecules. Thus, as used herein, the term antibody encompasses antibodies and antibody fragments encompassed by the CAR of the present invention, either produced by the modification of a full-length antibody or synthesized de novo using recombinant DNA methodologies.

[0202] As used herein, an "antibody" generally refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. When the term "antibody" is used, it may also be considered to refer to the term "antibody fragment". Known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. The basic immunoglobulin (antibody) structural unit is known to comprise a tetramer or a dimer. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having one "light" (L) chain (about 25 kD) and one "heavy" (H) chain (about 50 kD-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" and "variable heavy chain" refer to these variable regions of the light and heavy chains, respectively. Optionally, antibodies or immunological portions of antibodies can be chemically conjugated to or expressed as fusion proteins with other proteins.

[0203] The CARs of the present invention are intended to bind to mammalian, and particularly human, protein targets. The protein names used may correspond to either the mouse or human version of the protein.

[0204] The affinity of the binding domain polypeptides and CAR proteins according to the present disclosure can be readily determined using conventional techniques, for example, by competitive ELISA (enzyme-linked immunosorbent assay) or by binding association assays using labeled ligands or using surface plasmon resonance devices such as Biacore, or by displacement assays.

[0205] A humanized antibody comprising one or more CDRs of or derived from an antibody of the invention can be produced using any method known in the art. For example, a monoclonal antibody can be humanized using four general steps: (1) determining the nucleotide and predicted amino acid sequence of the light and heavy chain variable domains of the starting antibody, (2) designing the humanized antibody, i.e., determining which antibody framework regions to use in the humanization process, (3) the actual humanization methodology / techniques, and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Patent Nos. 4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, 6,180,370, 5,225,539, and 6,548,640.

[0206] The term humanized antibody means that at least some of the framework regions of the immunoglobulin and optionally some of the CDR regions or other regions involved in binding are derived from or adapted to human immunoglobulin sequences. Humanized, chimeric or partially humanized versions of mouse monoclonal antibodies can be produced, for example, using recombinant DNA techniques, departing from mouse and / or human genomic DNA sequences encoding the H and L chains, or cDNA clones encoding the H and L chains. Humanized forms of mouse antibodies can be produced by linking the CDR regions of a non-human antibody to human constant regions by recombinant DNA techniques (Queen et al., 1989, WO 90 / 07861). Alternatively, the monoclonal antibodies used in the methods of the invention can be human monoclonal antibodies. Human antibodies can be obtained, for example, using phage display techniques (WO 91 / 17271, WO 92 / 01047).

[0207] As used herein, humanized antibodies also refer to forms of non-human (e.g., murine, camel, llama, shark) antibodies that are certain chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin.

[0208] As used herein, a human or humanized antibody or human antibody fragment refers to an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or made using any of the techniques for making human antibodies known in the art or disclosed herein. A human antibody or fragment thereof can be selected in competitive binding experiments or otherwise to have the same epitopic specificity as a particular mouse antibody. The humanized antibodies of the present invention surprisingly have useful functional properties in common to a large extent with mouse antibodies. Human polyclonal antibodies can also be obtained in the form of serum from humans immunized with an immunogenic agent. Optionally, such polyclonal antibodies can be enriched by affinity purification using amyloid fibril and / or non-fibrillar polypeptides or fragments thereof as affinity reagents. Monoclonal antibodies can be obtained from serum according to the techniques described in WO 99 / 60846.

[0209] Variable Regions and CDRs The variable region of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held in close proximity by the FRs and contribute to the formation of the antigen-binding site of the antibody with the CDRs of the other chain.

[0210] There are a number of techniques available for determining CDRs, including approaches based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)) and approaches based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948). Alternative approaches include the IMGT (international ImMunoGeneTics) information system (Marie-Paule Lefranc). The Kabat definition is based on sequence diversity and is the most commonly used method. The Chothia definition is based on the location of structural loop regions, where the AbM definition is a compromise between the two and is used by Oxford Molecular's AbM antibody modeling software (see www.bioinf.org.uk: Dr. Andrew CR Martin's group). As used herein, CDRs can refer to CDRs defined in one or more ways, or in a combination of these ways.

[0211] In some embodiments, the invention provides an antibody or fragment thereof to be incorporated into a CAR, the antibody or fragment thereof comprising at least one CDR, at least two, at least three or more CDRs substantially identical to at least one CDR, at least two, at least three or more CDRs of an antibody of the invention. Other embodiments include antibodies having at least two, three, four, five or six CDRs substantially identical to at least two, three, four, five or six CDRs of or derived from an antibody of the invention. In some embodiments, at least one, two, three, four, five or six CDRs are at least about 70%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98% or 99% identical to at least one, two or three CDRs of an antibody of the invention. For purposes of the present invention, it is understood that the degree of activity may differ (greater or less) as compared to the above-described antibodies, but binding specificity and / or overall activity will generally be retained.

[0212] Additional components of the CAR In certain embodiments, the CARs contemplated herein may include linker residues between the various domains that are added for proper spacing and conformation of the molecule, e.g., a linker comprising an amino acid sequence that connects the VH and VL domains and provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody comprising the same light and heavy chain variable regions. The CARs contemplated herein may include one, two, three, four, or five or more linkers. In certain embodiments, the length of the linker is about 1 amino acid to about 25 amino acids, about 5 amino acids to about 20 amino acids, or about 10 amino acids to about 20 amino acids, or any intermediate length of amino acids.

[0213] Illustrative linkers include glycine polymers, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, such as Whitlow linkers. Glycine and glycine-serine polymers are relatively amorphous and therefore may be able to function as neutral tethers between domains of fusion proteins such as the CARs described herein.

[0214] In certain embodiments, the binding domain of the CAR is followed by one or more "spacers" or "spacer polypeptides," which refer to regions that distance the antigen binding domain from the effector cell surface to allow proper cell-cell contact, antigen binding, and activation. In certain embodiments, the spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. In one embodiment, the spacer domain comprises the CH2 and CH3 domains of IgG1 or IgG4. In one embodiment, the Fc binding domain of such a spacer / hinge region is mutated to prevent binding of the CAR to Fc receptors expressed on macrophages and other innate immune cells.

[0215] In some embodiments, the binding domain of a CAR is followed by one or more "hinge domains," which are responsible for positioning the antigen binding domain away from the effector cell surface to allow for proper cell-cell contact, antigen binding, and activation. A CAR may include one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The hinge domain may comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or a modified immunoglobulin hinge region. Exemplary hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, PD1, CD 152, and CD7, which may be wild-type hinge regions from these molecules or may be modified. In another embodiment, the hinge domain comprises the hinge region of PD1, CD 152, or CD8α.

[0216] The "transmembrane domain" is the portion of the CAR that fuses the extracellular binding moiety and the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The TM domain can be derived from either natural, synthetic, semi-synthetic, or recombinant sources. The TM domain can be derived from the α, β, or ζ chains of the T cell receptors CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD 137, CD 152, CD 154, and PD1. In one embodiment, the CAR contemplated herein comprises a TM domain derived from CD8α or CD28.

[0217] In certain embodiments, the CARs contemplated herein comprise an intracellular signaling domain. The term "intracellular signaling domain" refers to a portion of a CAR that transmits information of effective binding to a human target polypeptide inside an immune effector cell and is involved in effector cell functions, such as activation, cytokine production, proliferation, and eliciting cytotoxic activity, including release of cytotoxic factors to a target cell bound to the CAR, or other cellular responses elicited by antigen binding to the extracellular domain of the CAR. The term "effector function" refers to a specialized function of an immune effector cell. The effector function of a T cell can be, for example, an auxiliary activity, including cytolytic activity or secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs a cell to perform a specialized function. The CARs contemplated herein comprise one or more costimulatory signaling domains that enhance the potency, proliferation, and / or memory formation of T cells expressing a CAR receptor. As used herein, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules, other than antigen receptors or Fc receptors, that, upon binding to antigen, provide a second signal required for efficient activation and function of T lymphocytes.

[0218] In one embodiment, the CAR comprises an intracellular domain that includes a costimulatory domain and a signaling (activation) domain. Thus, the CAR construct may comprise the intracellular signaling domain of the natural T cell receptor complex (CD3ζ) and one or more costimulatory domains that provide a second signal to stimulate full T cell activation. The costimulatory domain is believed to increase cytokine production of CAR T cells and promote T cell replication and T cell persistence. The costimulatory domain has also been shown to potentially prevent CAR T cell exhaustion, increase T cell antitumor activity, and enhance CAR T cell survival in patients. As a non-limiting example, CAR constructs with a 4-1BB costimulatory domain have been associated in preclinical studies with slow sustained proliferation and effector function, increased persistence, and enriched memory center cells (TCMs) in T cell subset composition. 4-1BB is an in vivo inducible glycoprotein receptor that belongs to the tumor necrosis factor (TNF) superfamily and is expressed primarily on antigen-activated CD4 and CD8 T cells. CD28, as a non-limiting example, belongs to the immunoglobulin (Ig) superfamily. It is constitutively expressed on resting and activated CD4 and CD8 T cells and plays a crucial role in T cell activation by stimulating the PI3K-AKT signaling pathway. In one embodiment, the intracellular domain contains both 4-1BB and CD28 costimulatory domains. Other costimulatory domains include ICOS and OX40, which can be combined with the CD3ζ signaling (activation) domain.

[0219] T cell receptor The T cell receptor or TCR is a molecule found on the surface of T cells or T lymphocytes that is usually responsible for the recognition of antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains. In humans, the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively) in 95% of T cells, whereas in 5% of T cells, the TCR consists of a gamma and a delta (γ / δ) chain (encoded by TRG and TRD, respectively). Each chain is composed of two extracellular domains, the variable (V) region and the constant (C) region, with both immunoglobulin superfamily (IgSF) domains forming an antiparallel β-sheet. The constant region is close to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex.

[0220] The variable domains of both the α and β chains of the TCR each have three hypervariable regions or complementarity determining regions (CDRs). There is also an additional hypervariable region on the β chain (HV4) that does not normally contact the antigen and is therefore not considered a CDR.

[0221] These variable domain residues are present in two regions of the TCR: the α-chain and β-chain interface, and the β-chain framework region that is thought to be in close proximity to the CD3 signaling complex. Although CDR3 is the main CDR responsible for recognition of processed antigen, CDR1 of the α-chain has also been shown to interact with the N-terminal portion of antigenic peptides, whereas CDR1 of the β-chain interacts with the C-terminal portion of peptides.

[0222] Recombinant TCRs have previously been transfected into therapeutic T cells for the treatment of proliferative diseases. For example, TCR-T cells are engineered by transducing preferably autologous αβ or γδ cells with a retroviral or lentiviral vector encoding a TCR (typically an α chain non-covalently linked to a β chain) that recognizes a peptide of interest and a CD3z gene. When engineered T cells recognize a peptide bound to major histocompatibility complexes (MHC) on the surface of antigen-presenting cells or tumor cells, they become activated and begin to proliferate. The first TCR-T cell therapy was used in clinical trials for metastatic melanoma, in which the TCR recognized an HLA-A2-restricted peptide derived from melanocyte differentiation antigen melanoma antigen recognized by T cells 1 (MART-1).

[0223] Polypeptides "Peptide," "polypeptide," "polypeptide fragment," and "protein" are used interchangeably, unless otherwise indicated, and are used according to their ordinary meaning, i.e., as a sequence of amino acids. Polypeptides are not limited to a particular length, e.g., they can include a full-length protein sequence or a fragment of a full-length protein, and can include post-translational modifications of the polypeptide, e.g., glycosylation, acetylation, phosphorylation, etc., as well as other modifications, both naturally occurring and non-naturally occurring, known in the art.

[0224] In various embodiments, a CAR polypeptide contemplated herein comprises a signal (or leader) sequence at the N-terminus of the protein that directs transport of the protein during or after translation. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically include the CARs of the present disclosure, or sequences having deletions from, additions to, and / or substitutions of one or more amino acids of the CARs disclosed herein.

[0225] As used herein, "isolated peptide" or "isolated polypeptide" and the like refer to the in vitro isolation and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., it is not significantly associated with in vivo materials. Similarly, an "isolated cell" refers to a cell obtained in vivo from a tissue or organ and is substantially free of extracellular matrix.

[0226] nucleic acid As used herein, the term "polynucleotide" or "nucleic acid molecule" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides of the invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, where the variants typically retain at least one biological activity of the reference sequence. In various exemplary embodiments, the invention contemplates polynucleotides, including, in part, expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising the same.

[0227] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Further exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are pClneo vector (Promega) for expression in mammalian cells; pLenti4 / V5-DEST™, pLenti6 / V5-DEST™, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of the chimeric proteins disclosed herein can be ligated into such expression vectors for expression of the chimeric proteins in mammalian cells. "Regulatory elements" or "control sequences" present in an expression vector are the untranslated regions of the vector, i.e., origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno or Kozak sequences), introns, polyadenylation sequences, 5' and 3' untranslated regions, which interact with host cell proteins to carry out transcription and translation. Such elements can differ in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0228] vector In certain embodiments, cells (e.g., immune effector cells such as T cells) are transduced with a retroviral vector, e.g., a lentiviral vector, encoding a CAR. For example, T cells are transduced with a vector encoding a CAR that includes a humanized antigen-specific domain, antibody, or antigen-binding fragment that binds to a target polypeptide, together with a transmembrane domain and an intracellular signaling domain, such that these transduced cells can elicit a CAR-mediated cytotoxic response.

[0229] Retroviruses are common tools for gene delivery. In certain embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once a virus is integrated into the host genome, it is called a "provirus". The provirus serves as a template for RNA polymerase II to direct the expression of RNA molecules that code for structural proteins and enzymes required to generate new viral particles.

[0230] Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV), and lentiviruses.

[0231] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2), Visna-Maedi virus (VMV), Caprine Arthritis Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In certain embodiments, lentiviruses are used to deliver polynucleotides, including CARs, to cells.

[0232] The term "vector" as used herein refers to a nucleic acid molecule capable of introducing or carrying another nucleic acid molecule. The nucleic acid to be introduced is generally linked, e.g., inserted, into the vector nucleic acid molecule. The vector may contain sequences directing autonomous replication in a cell or may contain sequences sufficient to allow integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids, DNA plasmids that allow episomal localization and persistence), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses. In further embodiments of the present invention, CRISPR / Cas and TALEN-mediated insertion of nucleic acids encoding CAR or TCR may be used. Suitable vectors for CRISPR / Cas and TALEN-mediated insertion are known to those skilled in the art.

[0233] As will be apparent to one of skill in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains virally derived nucleic acid elements that facilitate the introduction or integration of a nucleic acid molecule into the genome of a cell, or a viral particle that mediates the introduction of a nucleic acid. Viral particles typically contain, in addition to the nucleic acid(s), various viral and optionally host cell components.

[0234] The term viral vector can refer to either a virus or viral particle capable of introducing a nucleic acid into a cell or the introduced nucleic acid itself. Viral vectors and introduction plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or parts thereof that are primarily derived from a retrovirus.

[0235] Thus, in a preferred embodiment, the invention relates to a method of transfecting a cell with an expression vector encoding a CAR. For example, in some embodiments, the vector comprises additional sequences, such as sequences that facilitate expression of the CAR, such as a promoter, an enhancer, a polyA signal, and / or one or more introns. In a preferred embodiment, the sequence encoding the CAR is flanked by transposon sequences, such that the presence of a transposase allows the coding sequence to be integrated into the genome of the transfected cell.

[0236] In some embodiments, the genetically transformed cells are further transfected with a transposase that facilitates integration of the CAR-encoding sequence into the genome of the transfected cells. In some embodiments, the transposase is provided as a DNA expression vector. However, in preferred embodiments, the transposase is provided as an expressible RNA or protein such that long-term expression of the transposase does not occur in the transgenic cells. For example, in some embodiments, the transposase is provided as an mRNA (e.g., an mRNA including a cap and a polyA tail). Any transposase system can be used in accordance with embodiments of the present invention. However, in some embodiments, the transposase is a salmonid-type Tel-like transposase (SB). For example, the transposase can be the so-called "Sleeping beauty" transposase (see, e.g., U.S. Patent No. 6,489,458, which is incorporated herein by reference). In some embodiments, the transposase is an engineered enzyme with increased enzymatic activity. Some specific examples of transposases include, but are not limited to, SB 10, SB 11, or SB 100X transposase (see, e.g., Mates et al, 2009, Nat Genet. 41(6):753-61, or U.S. Patent No. 9,228,180, which are incorporated by reference herein). For example, the method can include electroporating a cell with mRNA encoding SB 10, SB 11, or SB 100X transposase.

[0237] Sequence variants Also included within the scope of the present invention are sequence variants of the claimed nucleic acids, proteins, antibodies, antibody fragments and / or CARs, defined, for example, by % sequence identity, that maintain similar binding properties of the present invention. Such variants that exhibit alternative sequences but maintain essentially the same binding properties, such as target specificity, as the particular sequence presented are known as functional analogs or functionally similar. Sequence identity relates to the percentage of identical nucleotides or amino acids when sequences are aligned.

[0238] As used herein, the description "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a comparison window.Therefore, "sequence identity percentage" can be calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical nucleobases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) are found in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, where a polypeptide variant typically retains at least one biological activity of the reference polypeptide.

[0239] As a result of the degeneracy of the genetic code, those skilled in the art will understand that there are many nucleotide sequences that code for the polypeptides described herein. Some of these polynucleotides bear minimal homology or sequence identity to the nucleotide sequence of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Deletions, substitutions and other changes in sequence that fall within the described sequence identity are also encompassed by the present invention.

[0240] Protein sequence modifications that may occur by substitution are also included within the scope of the present invention. Substitutions as defined herein are modifications made to the amino acid sequence of a protein, where one or more amino acids are replaced with the same number of (different) amino acids, resulting in a protein containing an amino acid sequence different from that of the primary protein. Substitutions may be made, preferably without significantly altering the function of the protein. As with additions, substitutions may be natural or artificial. It is known in the art that amino acid substitutions can be made without significantly altering the function of a protein. This is particularly true when the modification concerns "conservative" amino acid substitutions, which are the replacement of one amino acid with another of similar properties. Such "conservative" amino acids may be natural or synthetic amino acids that, due to size, charge, polarity and conformation, may be substituted without significantly affecting the structure and function of the protein. Many amino acids can often be substituted by conservative amino acids without adversely affecting the function of a protein.

[0241] In general, the non-polar amino acids Gly, Ala, Val, Ile and Leu, the non-polar aromatic amino acids Phe, Trp and Tyr, the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met, the positively charged amino acids Lys, Arg and His, the negatively charged amino acids Asp and Glu are conservative amino acid groups. This list is not exhaustive. For example, it is known that Ala, Gly, Ser and sometimes Cys can be substituted for each other despite belonging to different groups.

[0242] In substitution variants, at least one amino acid residue in an antibody molecule is removed and a different residue is inserted in its place. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR changes are also contemplated. If such substitutions result in changes in biological activity, larger changes may be introduced, as referred to as "exemplary substitutions" in the table immediately below, or as further described below in relation to amino acid classes, and the products are screened.

[0243] [Table B] Potential Amino Acid Substitutions: Original Residue Preferred Conservative Substitutions Exemplary Substitutions Norleucine

[0244] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone, e.g., as a sheet or helical structure, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chains.

[0245] Conservative amino acid substitution is not limited to natural amino acids, but also includes synthetic amino acids.Commonly used synthetic amino acids are omega amino acids of various chain lengths and cyclohexylalanine, which are neutral non-polar analogs, citrulline and methionine sulfoxide, which are neutral non-polar analogs, phenylglycine, which is aromatic neutral analog, cysteic acid, which is negatively charged analog, and ornithine, which is positively charged amino acid analog.As with natural amino acids, this list is not comprehensive, but merely illustrative of the substitutions known in the art.

[0246] Genetically modified cells and genetically modified T cells In certain embodiments, the present invention contemplates T cells genetically modified to express an antigen-specific targeting construct as contemplated herein for use in the treatment of cell proliferative disorders. The T cells of the present invention also include CD8+ and CD4+ T cells.

[0247] As used herein, the term "genetically engineered" or "genetically modified" refers to the addition of extra genetic material in the form of DNA or RNA to the total genetic material of a cell. The terms "genetically modified cell", "modified cell" and "redirected cell" are used interchangeably. As used herein, the term "gene therapy" refers to the permanent or transient introduction of extra genetic material in the form of DNA or RNA to restore, correct or modify the expression of a gene or to express a therapeutic polypeptide, e.g., a CAR. In certain embodiments, a TCR or CAR as contemplated herein is introduced and expressed in a CTL to redirect the specificity of the CTL to a target antigen of interest.

[0248] Immune effector cells (such as the CTLs of the present invention) can be autogeneic ("self") or non-autologous ("non-self", e.g., allogeneic, syngeneic, or xenogeneic). As used herein, "autogeneic" refers to cells from the same subject and is a preferred embodiment of the present invention. As used herein, "allogeneic" refers to cells of the same species that are genetically different from the cells being compared. As used herein, "syngeneic" refers to cells of a different subject that are genetically identical to the cells being compared. As used herein, "xenogeneic" refers to cells of a different species from the cells being compared. In a preferred embodiment, the cells of the present invention are autogeneic or allogeneic.

[0249] The term "T cells" or "T lymphocytes" is art-recognized and is intended to encompass thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, cytokine-induced killer cells (CIK cells), or activated T lymphocytes. Cytokine-induced killer (CIK) cells are typically CD3 and CD56 positive non-major histocompatibility complex (MHC)-restricted natural killer (NK)-like T lymphocytes. T cells can be T helper (Th; CD4+ T cells) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells. T cells can be cytotoxic T cells (CTL; ​​CD8+ T cells), CD4+ CD8+ T cells, CD4 CD8 T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in certain embodiments include naive T cells and memory T cells and stem cell-like memory cells (TSCM).

[0250] For example, when reintroduced into a patient after autologous cell transplantation, T cells modified with the inventive constructs described herein can recognize and kill tumor cells.

[0251] The present invention provides a method for producing CTLs expressing the intended constructs described herein. In one embodiment, the method comprises transfecting or transducing CTLs isolated from an individual so that the immune effector cells express one or more antigen-specific constructs (CAR or TCR) described herein. In a particular embodiment, CTLs are isolated from an individual and genetically modified (without further manipulation in vitro). Such cells can then be readministered directly to the individual. In a further embodiment, CTLs are first activated and stimulated to grow in vitro before being genetically modified to express CAR or TCR, optionally with an EBAG9 silencing agent. In this regard, CTLs can be cultured before and / or after genetic modification.

[0252] In certain embodiments, a cell source is obtained from a subject prior to the in vitro manipulation or genetic modification of immune effector cells as described herein. In certain embodiments, T cells can be obtained from a number of sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, induced pluripotent stem cells (iPSCs), tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood drawn from a subject using any number of techniques known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods, e.g., MACS™ separation (Miltenyi). In one embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells harvested by apheresis may be washed to remove the plasma fraction and to place the cells in a suitable buffer or medium for subsequent processing. The cells may be washed with PBS or another suitable solution lacking calcium, magnesium, and nearly all other divalent cations. As will be appreciated by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, for example, by using a semi-automated flow-through centrifuge, such as the Cobe 2991 cell processing machine, Baxter CytoMate, etc. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed directly in the culture medium in which the cells are resuspended.

[0253] In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and removing monocytes, for example, by PERCOLL™ gradient centrifugation. Specific subpopulations of T cells can be further isolated by positive or negative selection techniques. One method used herein is negative magnetic immunoadhesion or flow cytometric cell sorting and / or cell selection using a monoclonal antibody cocktail directed against cell surface markers present on the negatively selected cells.

[0254] PBMCs can be directly genetically modified to express CAR using the methods contemplated herein. In certain embodiments, T lymphocytes are further isolated after isolation of PBMCs, and in certain embodiments, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after genetic modification and / or expansion. CD8+ cells can be obtained by using standard methods. In some embodiments, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens associated with each of these types of CD8+ cells.

[0255] In some embodiments, the T cells described herein may be obtained from induced pluripotent stem cells (iPSCs) using methods known to those of skill in the art.

[0256] The accepted approach to generate CAR T cells relies on genetic modification and expansion of mature circulating T cells. Such a process utilizes autologous T cells and reduces the risk of graft-versus-host disease (GvHD) due to endogenous TCR expression by allogeneic T cells and rejection due to MHC mismatch. Alternatively, direct in vitro differentiation of pluripotent stem cells, e.g., induced pluripotent stem cells, into genetically engineered T cells provides an essentially unlimited source of cells that can be genetically modified to express the CAR of the present invention. In some embodiments, a so-called master iPSC line can be maintained that provides a renewable source for consistent and repeatable production of homogenous cell products. In some embodiments, transformation of the master iPSC cell line with a nucleic acid encoding a CAR prior to expansion and differentiation into the desired T cells is contemplated. For example, T lymphocytes can be generated from iPSCs so that the iPSCs can be expanded and differentiated into T cells for administration to patients after they have been modified with a nucleic acid encoding a CAR. Differentiation into appropriate T cells can be performed from the iPSCs prior to transformation with a nucleic acid encoding a CAR and expansion prior to administration. All possible combinations of iPSC expansion, genetic modification, and propagation to obtain suitable numbers of cells for administration are contemplated in the present invention.

[0257] T cells may be genetically modified after isolation using known methods, or T cells may be activated and expanded (or differentiated in the case of precursor cells) in vitro prior to genetic modification. In certain embodiments, T cells are genetically modified with a chimeric antigen receptor contemplated herein (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells may be genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells may be genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells may be genetically modified with a chimeric antigen receptor (e.g., transduced with a viral vector comprising a nucleic acid encoding a CAR) as described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869 ... Nos. 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041, and U.S. Patent Application Publication No. 20060121005, can be activated and expanded before or after being genetically modified to express a CAR.

[0258] In further embodiments, a mixture of, for example, 1, 2, 3, 4, 5 or more different expression vectors can be used to genetically modify a population of donor T cells, where each vector encodes a different antigen-targeting construct as contemplated herein.

[0259] In one embodiment, the present invention provides a method for preserving genetically modified T cells that exhibit EBAG9 inhibition, comprising cryopreserving T cells so that the cells remain viable when thawed. To provide a permanent source of such cells for future treatment of patients suffering from the condition to be treated, some immune effector cells can be cryopreserved by methods known in the art. When needed, the cryopreserved cells can be thawed and grown to increase into more such cells.

[0260] Compositions and Formulations Compositions contemplated herein may include one or more of the polypeptides, polynucleotides, vectors containing same, genetically modified T cells, etc. contemplated herein. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated into a pharma- ceutical or physiologically acceptable solution for administration to a cell or animal, alone or in combination with one or more other therapeutic modalities. It will also be understood that, if necessary, the compositions of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharma- ceutical active agents. There is virtually no limit to other components that may be included in the composition, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended treatment.

[0261] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0262] As used herein, a "pharmaceutically acceptable carrier, diluent, or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavoring, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent, or emulsifier that is approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. Exemplary pharma- ceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicones, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and any other compatible substances used in pharmaceutical formulations.

[0263] In certain embodiments, the compositions of the invention comprise an amount of T cells as contemplated herein. As used herein, the term "amount" refers to an "effective amount" or "effective amount" of genetically modified therapeutic cells, e.g., T cells, to achieve a beneficial or desired prophylactic or therapeutic outcome, including a clinical outcome.

[0264] "Prophylactically effective amount" refers to an amount of genetically modified therapeutic cells effective to achieve a desired prophylactic result. Because a prophylactic dose is used in subjects before or at an early stage of disease, the prophylactically effective amount is typically, but not necessarily, less than a therapeutically effective amount. The term prophylactic does not necessarily refer to the complete prevention or prevention of a particular medical disorder. The term prophylactic also refers to a reduction in the risk of developing a particular medical disorder or the risk of its symptoms worsening.

[0265] A "therapeutically effective amount" of genetically modified therapeutic cells may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the stem and progenitor cells to induce a desired response in the individual. A therapeutically effective amount is also one in which any toxic or adverse effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" encompasses an amount effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, and the condition of the patient (subject).

[0266] Generally, the pharmaceutical compositions comprising the T cells described herein are 2 Individual cells / kg body weight~10 10 cells / kg body weight, preferably 10 5 Individual cells / kg body weight~10 7 It can be stated that the composition may be administered in a dosage of 10 cells / kg body weight (including all integer values ​​within those ranges). The number of cells depends on the final use for which the composition is intended and the type of cells contained therein. For the uses provided herein, the cells are generally in a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. Thus, the desired cell density is typically 10 6 Higher than 10 cells / ml, generally 10 7 Higher than 10 cells / ml, generally 10 8 A clinically relevant number of cells is greater than 10 5 Individual cells, 10 6Individual cells, 10 7 Individual cells, 10 8 Individual cells, 10 9 Individual cells, 10 10 Individual cells, 10 11 individual cells, or 10 12 The amount of cells administered may be divided into multiple infusions equal to or greater than 100 cells. In some aspects of the invention, fewer cells may be administered, particularly if all of the infused cells are redirected to a specific target antigen. The cell composition may be administered multiple times at dosages within these ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient receiving the treatment.

[0267] In general, compositions comprising cells activated and expanded as described herein can be utilized in the treatment and prevention of diseases occurring in immunocompromised individuals. In particular, compositions comprising modified T cells as contemplated herein are used to treat hematological malignancies. The modified T cells of the present invention can be administered as pharmaceutical compositions alone or in combination with carriers, diluents, excipients, and / or other components, such as IL-2 or other cytokines or cell populations. In certain embodiments, pharmaceutical compositions as contemplated herein include a quantity of genetically modified T cells in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0268] Pharmaceutical compositions of the invention comprising T cells may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the invention are preferably formulated for parenteral administration, such as intravascular (intravenous or intraarterial), intraperitoneal, or intramuscular administration.

[0269] Liquid pharmaceutical compositions (whether they are in the form of a solution, suspension, etc.) may contain one or more of the following: sterile diluents, such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can act as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and isotonicity agents such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, multiple dose vials, or bags made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0270] In certain embodiments, the compositions contemplated herein comprise an effective amount of T cells, alone or in combination with one or more therapeutic agents. Thus, the T cell compositions may be administered alone or in combination with other known cancer treatments, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, and the like. The compositions may be administered in combination with antibiotics. Such therapeutic agents may be recognized in the art as standard treatments for certain conditions described herein, such as certain cancers. Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic drugs, radiation therapy, therapeutic antibodies, or other active and adjunctive agents.

[0271] The T cell product may be cryopreserved in dimethylsulfoxide (DMSO) / human serum albumin (10% / 90% vol / vol) in the vapor phase of liquid nitrogen until patient conditioning is performed. Such storage does not inhibit the viability and function of the T cell product.

[0272] Treatment method The genetically modified cells contemplated herein provide improved methods of adoptive immunotherapy for the treatment of medical disorders associated with the presence of unwanted cellular proliferation, preferably for use in hematological malignancies.

[0273] In certain embodiments, compositions comprising modified T cells as contemplated herein are used to treat hematological malignancies, including but not limited to, non-Hodgkin's lymphoma (NHL), e.g., B-cell malignancies with or without leukemic tumor cell seeding, such as B-cell NHL or T-cell non-Hodgkin's lymphoma.

[0274] In further embodiments, the method relates to the treatment of a hematological malignancy selected from non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and / or multiple myeloma.

[0275] Non-Hodgkin's lymphoma encompasses a large collection of cancers of lymphocytes (white blood cells). Non-Hodgkin's lymphoma can occur at any age and is often characterized by larger than normal lymph nodes, fever, and weight loss. Non-Hodgkin's lymphoma can also be present in extra-lymphatic sites, such as mucosal tissues, including the central nervous system, lungs, intestines, colon, and digestive tract. There are many different types of non-Hodgkin's lymphoma. For example, non-Hodgkin's lymphoma can be classified into aggressive (fast-growing) and indolent (slow-growing) types.

[0276] Non-Hodgkin's lymphoma can originate from B cells and T cells. As used herein, the term "non-Hodgkin's lymphoma" includes both "B cell" and "T cell" non-Hodgkin's lymphoma. B-cell non-Hodgkin's lymphoma (NHL) includes Burkitt's lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. Lymphomas that develop after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin's lymphoma.

[0277] T-cell lymphoma accounts for approximately 15 percent of all NHL in the United States. There are many different types of T-cell lymphoma, including angioimmunoblastic T-cell lymphoma (AITL), which is a mature T-cell lymphoma of immunoblasts in the blood or lymphatics. Additional types of T-cell lymphoma include cutaneous T-cell lymphoma and T-cell lymphoma with leukemic dissemination.

[0278] Chronic lymphocytic leukemia (CLL), which can also be treated with the CAR of the present invention, is an indolent (slow-growing) cancer that causes a slow increase in immature white blood cells (B lymphocytes). Cancer cells can spread through the blood and bone marrow and affect lymph nodes or other organs such as the liver and spleen. CLL eventually causes bone marrow failure. A different manifestation of the disease is called small lymphocytic lymphoma, which is mainly localized in secondary lymphoid organs, such as lymph nodes and the spleen.

[0279] Multiple myeloma is a B-cell malignancy with mature plasma cell morphology characterized by the malignant transformation of a single clone of plasma cells. These plasma cells proliferate in the BM and can invade adjacent bone and occasionally the blood. Atypical multiple myeloma includes overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, isolated plasmacytoma of bone, and extramedullary plasmacytoma.

[0280] Acute myeloid leukemia (AML) is a cancer of blood cells of the myeloid lineage, characterized by the rapid growth of abnormal cells that accumulate in the bone marrow and blood and interfere with normal blood cells. There appears to be a genetic risk for AML. The malignant cells in AML are myeloblasts. In normal hematopoiesis, myeloblasts are immature precursor cells of myeloid white blood cells. Normal myeloblasts gradually mature into mature white blood cells. In AML, a single myeloblast accumulates genetic changes that keep the cell in an immature state and prevent it from differentiating. Such mutations alone do not cause leukemia, but when combined with other mutations, they disrupt genes that control proliferation, leading to uncontrolled proliferation of immature clones of cells, causing AML.

[0281] Acute lymphoblastic leukemia (ALL) is a cancer of blood cells of the lymphocyte lineage, characterized by the development of large numbers of immature lymphocytes. In most cases, the cause is unknown. There may be genetic risk factors, and environmental risk factors may include radiation exposure or chemotherapy. The cancerous cells in ALL are lymphoblasts. Normal lymphoblasts develop into mature infection-fighting B or T cells, also called lymphocytes. In ALL, both the normal development of lymphocytes and the control of the number of lymphoid cells become defective.

[0282] Further hematological malignancies include essentially any other neoplasm of blood, blood cells, or precursor blood cells, such as any given leukemia, lymphoma, or myeloma.

[0283] The genetically modified cells contemplated herein also provide improved methods of adoptive immunotherapy for use in treating medical disorders associated with the presence of unwanted immune cell proliferation, preferably autoimmune diseases associated with the production of autoantibodies.

[0284] In one embodiment of the invention, the T cells described herein with silenced EBAG9 are intended for use in the treatment of autoimmune diseases, preferably autoantibody dependent autoimmune diseases, preferably autoimmune diseases with an inflammatory component, preferably Takayasu's arteritis, giant cell arteritis, familial Mediterranean fever, Kawasaki disease, polyarteritis nodosa, cutaneous polyarteritis nodosa, hepatitis associated arteritis, Behcet's syndrome, Wegener's granulomatosis, ANCA-vasculitis, Churg-Strauss syndrome, microscopic polyangiitis, vasculitis of connective tissue diseases, Henoch-Schönlein purpura, cryoglobulinemic vasculitis, cutaneous leukocytoclastic vasculitis, tropical aortitis, and / or other autoimmune diseases. aortitis), sarcoidosis, Cogan's syndrome, Wiskott-Aldrich syndrome, lepromatous arteritis, primary vasculitis of the CNS, thromboangiitis obliterans, paraneoplastic arteritis, urticaria, Degos' disease, myelodysplastic syndrome, erythema elevatum, hyperimmunoglobulin D, allergic rhinitis, bronchial asthma, chronic obstructive pulmonary disease, periodontitis, rheumatoid arthritis, atherosclerosis, amyloidosis, Crohn's disease (Morbus Chron), ulcerative colitis, autoimmune myositis, diabetes mellitus, Guillain-Barre syndrome, histiocytosis, osteoarthritis, atopic dermatitis, periodontitis, chronic sinusitis, psoriasis, rheumatoid arthritis, microscopic colitis, pulmonary fibrosis, glomerulonephritis, Whipple's disease, Still's disease, erythema nodosum, otitis, cryoglobulinemia, Sjogren's syndrome, lupus erythematosus, preferably systemic lupus erythematosus (SLE), aplastic anemia, myelofibrosis, chronic inflammatory demyelinating polyneuropathy, Kimura's disease, systemic sclerosis, chronic periaortitis, chronic prostatitis, idiopathic pulmonary fibrosis, chronic granulomatous disease, idiopathic amyloidosis, Chalasia, bleomycin-induced pulmonary inflammation, cytarabine-induced pulmonary inflammation, autoimmune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, autoimmune lymphopenia, Chagas' disease, chronic autoimmune thyroiditis, autoimmune hepatitis, Hashimoto's thyroiditis, atrophic thyroiditis, Graves' disease, autoimmune polyglandular syndrome, autoimmune Addison's syndrome, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, antiphospholipid syndrome, myasthenia gravis, stiff-man syndrome, Goodpasture's syndrome, sympathetic ophthalmia, folliculitis, Sharp's syndromesyndrome and / or Evans syndrome, in particular hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, most preferably SLE.

[0285] Systemic lupus erythematosus (SLE), also known as lupus, is an autoimmune disease in which the body's immune system attacks healthy tissue in various parts of the body. Symptoms vary from person to person and can range from mild to severe. Common symptoms include joint pain and swelling, fever, chest pain, hair loss, mouth ulcers, swollen lymph nodes, fatigue, and most commonly a red rash on the face.

[0286] As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits symptoms of a disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapeutics, and methods disclosed elsewhere herein. In a preferred embodiment, the subject includes any animal that exhibits symptoms of a hematopoietic disease, disorder, or condition, such as a B-cell malignancy, that can be treated with cell-based therapeutics and methods disclosed herein. Suitable subjects include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic animals or pets (e.g., cats or dogs). Non-human primates and preferably human patients are included. Exemplary subjects include human patients who have cancer, hematological malignancies, have been diagnosed with hematological malignancies, or are at risk of having hematological malignancies.

[0287] As used herein, "treatment" or "treating" includes any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even a slight decrease in one or more measurable markers of the disease or condition being treated. Treatment may optionally include a reduction or amelioration of the symptoms of a disease or condition, or a delay in the progression of a disease or condition. "Treatment" does not necessarily mean a complete eradication or cure of a disease or condition or its associated symptoms.

[0288] As used herein, "prevention" and similar terms, such as "prevented," "preventing," or "prophylactically," refer to an approach that prevents, inhibits, or reduces the likelihood of the onset or recurrence of a disease or condition. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar terms also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0289] figure The present invention is demonstrated by way of example only in the accompanying figures, which should be considered to provide further explanation of potentially preferred embodiments that strengthen the support of one or more non-limiting embodiments of the present invention.

[0290] Detailed Description of the Drawings Figure 1: Diagram of the generation of retroviral MP71 encoding EBAG9-targeting miRNA and GFP. EBAG9-targeting miRNA was generated by exchanging the hairpin antisense sequence of endogenous miRNA-155 with the predicted EBAG9 target site sequence. The resulting miRNA-encoding sequence was inserted at an intronic position into the retroviral MP71 vector encoding GFP (LTR: long terminal repeat, PRE: post-translational regulatory element, Amp R: ampicillin resistance, GFP: green fluorescent protein).

[0291] Figure 2: Diagram of reduction of human EBAG9 expression in Jurkat cells after transduction with gamma retroviral vectors encoding different EBAG9-targeting miRNAs. Retroviral transduction of human Jurkat cells with different GFP-encoding vectors expressing different miRNAs directed against human EBAG9. Positively transduced GFP+ cells were enriched by fluorescence-activated cell sorting (FACS) and analyzed by Western blot. Calnexin was used as a loading control. Lysates of 1x10e6 Jurkat cells were analyzed. UT: non-transduced.

[0292] Figure 3: Diagram of the experimental timeline for retroviral transduction of human primary T cells prior to in vitro functional assays.

[0293] Figure 4: EBAG9 downregulation promotes antigen-independent release of granzyme A from activated human CD8+ T cells. Activated human CD8+ T cells were transduced with vectors encoding SP6 or BCMA CAR together with miRNA H18 targeting EBAG9, or BCMA CAR alone. Enzyme activity in the supernatant was measured 15 days after CD8+ T cell activation. Granzyme A release was induced by restimulation of T cells with anti-human CD3 and anti-CD28 antibodies for 4 hours. Values ​​indicate release in percentage of total volume. Bars represent mean ± SEM of n=3 experiments with n=4 independent donors per group. * p<0.05, ** p<0.01, * p<0.001; ns: not significant. Paired t-test was performed.

[0294] Figure 5: MP71 vectors are suitable for co-expression of miRNAs targeting EBAG9 and CARs. Either SP6, BCMA, or CD19 CARs were introduced into the MP71 vectors containing miRNAs at the positions indicated using NotI and EcoRI restriction sites (LTR: long terminal repeat; PRE: post-translational regulatory element; Amp R: ampicillin resistance; CAR: chimeric antigen receptor).

[0295] Figure 6: BCMA and CD19 CAR expression in transduced human primary T cells. Human primary T cells were activated for 48 hours by stimulation with anti-human CD3 and anti-human CD28 antibodies. Transduction of human T cells with vectors encoding BCMA CAR (A or B) or CD19 CAR (C or D) together with miRNA H18 or H17 targeting EBAG9, respectively, was performed twice. Cells were cultured supplemented with IL-7 / IL-15. FACS was performed on day 6 of culture. One representative example per group is shown. Transduction rates are shown as percentages relative to the gate.

[0296] Figure 7: Antigen-specific cytolytic activity of BCMA CAR T cells can be increased by downregulation of EBAG9. (A or B) Human CD8+ T cells were activated for 48 h by stimulation with anti-human CD3 and anti-human CD28 antibodies. Transduction of human CD8+ T cells with vectors encoding either SP6 or BCMA CAR alone or together with miRNA H18 targeting EBAG9 was performed twice. Cells were cultured for 13 days supplemented with high concentration of IL-2. Before in vitro cytotoxicity assay, CAR T cells were cultured for 48 h supplemented with low concentration of IL-2. In vitro cytotoxicity assay was performed on day 15 of CAR T cell culture. Transduction rate was adjusted to 20%-30% by adding UT. [51Cr] Chromium-labeled MM cell lines (A) and B-NHL cell lines (B) were co-cultured with transduced human T cells at different effector-to-target ratios for 4 h. Data show mean ± SEM error bars. n = 5 experiments were performed in duplicate with 4-8 different donors per group. * p<0.05, ** p<0.01, *** p<0.001; ns: not significant. Mann-Whitney U test was used.

[0297] Figure 8: Increased cytolytic activity of CAR T cells by silencing EBAG9 is a universally applicable cell biological mechanism. Human CD8+ T cells were activated for 48 h by stimulation with anti-human CD3 and anti-human CD28 antibodies. Transduction of human CD8+ T cells with vectors encoding either SP6 CAR or CD19 CAR or with miRNA H17 targeting EBAG9 was performed twice. Cells were cultured for 13 days supplemented with high concentration of IL-2. Prior to in vitro cytotoxicity assay, CAR T cells were cultured for 48 h supplemented with low concentration of IL-2. In vitro cytotoxicity assay was performed on day 15 of CAR T cell culture. Transduction rate was adjusted to 15% by addition of UT. [51Cr] Chromium-labeled Jeko-1 cell line was co-cultured with transduced human T cells at different effector-to-target ratios for 4 h. Data show mean ± SEM error bars. n = 3 experiments with 3-6 different donors per group were performed in duplicate. * p<0.05, ** p<0.01, *** p<0.001; ns: not significant. Mann-Whitney U test was used.

[0298] Figure 9: In vivo engineered BCMA CAR T cells with silenced EBAG9 eradicate multiple myeloma cells more efficiently. (A) NSG mice were implanted with 1x10e7 MM.1S cells stably expressing GFP and firefly luciferase. On day 6, tumor inoculation was visualized by IVIS with 150 s illumination. One day later, 1x10e6 CAR+ cells (days 10-13 of culture supplemented with Il-7 / IL-15) were implanted and treatment efficiency was observed by IVIS with 60 s illumination. (B) Mean ± SEM of bioluminescence signal intensity obtained from regions of interest covering the entire body was plotted for each group and time point in one experiment. (C) On days 15 and 16, animals were sacrificed and CD138+GFP+ tumor cells in bone marrow were quantified by flow cytometry. Mean ± SEM of n=2 experiments is plotted. * p<0.05, ** p<0.01, *** p<0.001; ns: not significant. Mann-Whitney U test was used.

[0299] Figure 10: Target site validation of CRISPR-mediated EBAG9 knockout. (A) Schematic overview of the human EBAG9 gene consisting of seven exons. Six guide RNAs (E1-E6) targeting different regions of exon 4 were designed. (B and C) Human primary T cells were activated by stimulation with human CD3 / CD28 Dynabeads for 48 h before being electroporated with various guide RNAs (E1-E6) and Cas9 protein. At day 9 after activation, genomic DNA and protein samples were harvested for analysis of gene editing efficiency (B) or EBAG9 protein expression levels (C), respectively. A representative Western blot of one of three experiments is shown. EXAMPLES

[0300] The present invention is illustrated by the examples disclosed below, which provide technical support for the more detailed description of potentially preferred, non-limiting embodiments of the present invention.

[0301] Summary of the Examples 1. MiRNAs targeting mouse or human EBAG9 were cloned into retroviral expression vectors followed by transduction of human or mouse CD8+ T cells. As visualized by Western blot analysis, over 90% knockdown of EBAG9 was achieved in human and mouse CD138+ T cells. 2. Transduction of antigen-specific mouse T cells (polyclonal) was performed with a gamma-retrovirus encoding miRNA targeting EBAG9, followed by their adoptive transfer into RAG2-KO mice. These mice were exposed to splenocytes pulsed with SV40 large T antigen (peptide IV). In an in vivo killing assay, engineered T cells with EBAG9 knockdown were more effective at killing compared to unmodified or control T cells. 3. Human T cells were transduced with γ-retroviruses encoding miRNAs targeting EBAG9 in combination with various CARs. These cells were used in in vitro cytotoxicity assays, where target cells expressed the cognate antigen of the selected CAR. We achieved enhanced cytolysis of target cells expressing either BCMA or CD19 when the frequency of CAR-positive T cells was low (<30%). 4. Human T cells were transduced with a gamma-retrovirus encoding a miRNA targeting EBAG9 combined with an anti-BCMA CAR. NSG mice were implanted with the multiple myeloma cell line MM.1S and tumor development was measured by IVIS imaging. These mice were then treated with T cells expressing a control CAR, T cells expressing a standard BCMA CAR, and T cells expressing an engineered BCMA CAR co-expressing a miRNA targeting human EBAG9. Tumor progression was measured 14 days after CAR T cell administration. CAR T cells were kept in low numbers to better identify the performance of low-number T cells with enhanced cytolytic power. Anti-BCMA CAR T cells endowed with EBAG9-silenced miRNA were superior in tumor control, resulting in complete eradication of tumor cells from the bone marrow.

[0302] Retroviral Vector Design Cloning and miRNA sequences For silencing human EBAG9, different miRNAs were generated that target regions within the open reading frame of the human EBAG9 gene. Suitable target sites were identified using four different target site prediction programs for miRNA design that reflect the requirements of the endogenous RNAi machinery (WIsiRNA, BlockIT, siDESIGN, OligoWalk). The secondary structure of miRNAs is important for recognition and processing by the RNAi machinery. The miRNA structure is characterized by a highly base-paired hairpin that encodes a somewhat amorphous backbone and an antisense sequence.

[0303] To generate a miRNA targeting Ebag9, endogenous miRNA-155 was used. The 21 nucleotides containing the antisense sequence within the hairpin structure were replaced with the predicted EBAG9 target site sequence (Table 1).

[0304] Table 1: Hairpin sequences directed against the human EBAG9 gene. Antisense sequence targeting EBAG9 Sequence number [Table 1]

[0305] The resulting miRNA-encoding sequences were inserted into the GFP-encoding retroviral MP71 vector at an intronic position using MluI and NsiI restriction sites. The MP71 vector is known for its high transduction efficiency and stable expression of transgenes in primary T cells. Since the transcription of miRNAs is regulated by the polymerase II promoter, the highly active 5'LTR of MP71 can be used to drive the expression of miRNAs and transgenes.

[0306] Knockdown efficiency of miRNAs targeting EBAG9 The efficiency of miRNA-mediated EBAG9 downregulation at the protein level was tested in human acute T-cell leukemia cell line Jurkat J76. Compared with untransduced T cells (UT), about 80% EBAG9 knockdown efficiency could be detected in H16, H17, and H18. In contrast, H19 did not achieve EBAG9 protein downregulation. MP71-GFP vector without miRNA served as a control. The most effective miRNAs, H17 and H18, were selected for further analysis in human primary T cells.

[0307] Application Examples Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteer donors and CD8+ T cells were enriched by magnetic cell separation (negative selection). CD8+ T cells were activated for 48 h by stimulation with anti-human CD3 and anti-human CD28 antibodies. Activated human CD8+ T cells were then subjected to two rounds of retroviral transduction and cultured with high concentrations of IL-2 (100 IU / ml) and IL-15 (10 ng / ml) supplementation. 13 days after CD8+ T cell activation, the cytokine supplement was reduced to 10 IU / ml IL-2 and 1 ng / ml IL-15. Functional assays were performed 48 h later (Figure 3). To determine the activity of granzyme A, human CAR T cells (15 days after activation) were restimulated with plate-bound anti-human CD3 and anti-human CD28 antibodies for 4 h. Supernatants were analyzed for granzyme A activity by incubation with granzyme A substrate solution. Product concentration correlates with enzyme activity. Supernatants from CAR T cells transferred to uncoated plates were used as a control for basal granzyme A secretion.

[0308] To prove that EBAG9 silencing increases the release of effector molecules such as granzyme A from activated T cells, an in vitro release assay was performed. CD8+ T cells were isolated from healthy donors, transduced twice, and cultured for 13 days with high concentrations of IL-2 supplementation. IL-2 supplementation was reduced for 48 h before in vitro functional assays on day 15. On day 15 of culture, granzyme A release was induced by restimulating T cells with anti-human CD3 / CD28 antibodies for 4 h. The amount of granzyme A released from T cells transduced with BCMA CAR was comparable to that of UT. In contrast, T cells transduced with the H18-BCMA CAR construct released two-fold higher amounts of granzyme A. Similarly, H18 miRNA also endowed SP6 CAR T cells with enhanced secretion of cytolytic effector molecules (Figure 4).

[0309] Co-expression of EBAG9-targeting miRNA and CAR Cloning To generate a retroviral vector that allows simultaneous expression of EBAG9-specific miRNA and CAR, the GFP gene of the MP71-GFP vector encoding miRNA was replaced with a CAR cassette using the NotI and EcoRI restriction sites flanking GFP. Using this strategy, retroviral vectors encoding EBAG9-specific miRNA H18 and BCMA CAR and EBAG9-specific miRNA H17 and CD19 CAR were cloned (Figure 5). As a negative control for the functional assay, SP6 CAR without any naturally occurring ligand was combined with EBAG9-targeting miRNA H17 and H18.

[0310] CAR expression Retroviral transduction of anti-human CD3 / CD28-activated human primary T cells was performed twice using MP71 vectors encoding BCMA or CD19 CAR alone or in combination with miRNA H18 or H17 specific for EBAG9, respectively. Cells were cultured supplemented with IL-7 and IL-15. IgG hinge region staining was performed on day 6 to analyze CAR expression and determine transduction efficiency (Figure 6).

[0311] In vitro application example To examine the effect of EBAG9 downregulation on the antigen-specific cytolytic capacity of CAR T cells, an in vitro cytotoxicity assay was performed. In vitro cytolytic activity reports on the release of granzymes and perforin, a secretory process regulated by EBAG9.

[0312] CD8+ T cells were isolated from healthy donors, activated with anti-human CD3 and anti-CD28 antibodies for 48 h, transduced twice, and cultured for 13 days with high IL-2 supplementation. IL-2 supplementation was reduced for 48 h before in vitro cytotoxicity assays on day 15. On day 15, CAR T cells were co-cultured with target cell lines for 4 h. Assay supernatants were analyzed for [ 51Cr] chromium counts were measured. Maximal release of target cells was determined by direct counting of labeled cells. Spontaneous release was measured by incubating target cells alone.

[0313] BCMA high Expressing multiple myeloma (MM) cell lines OPM-2 and BCMA low The expressing B-cell non-Hodgkin's lymphoma (B-NHL) cell line DOHH-2 was used as target cells for BCMA CAR T cells. Prior to co-culture with CAR T cells on day 15 of culture, the target cells were [ 51 After 4 h of co-culture at different target-to-effector ratios, BCMA CAR-transduced CD8 + Cytolytic activity was observed in T cells, whereas no or little activity could be detected in UT or SP6 CAR T cells. Thus, nonspecific T cell activation due to EBAG9 downregulation did not occur. At the highest effector-to-target ratio of 80:1, target cell lysis of BCMA CAR T cells was about 30%. The combination of BCMA CAR and EBAG9 silencing in H18-BCMA CAR T cells resulted in a significant increase in cell lysis efficiency by CAR T cells in all cell lines tested. For example, in the MM cell line OPM-2, H18-BCMA CAR T cells had about 1.5-fold higher lysis rate compared to BCMA CAR alone. By different calculations, the maximum killing rate (E:T 80:1) of T cells transduced with BCMA CAR could be achieved by only 1 / 4-1 / 8 of EBAG9 knockdown BCMA CAR T cells. Thus, the effective dose level was significantly reduced.

[0314] To confirm the RNAi-mediated increase in CAR T cell cytotoxic activity, another miRNA sequence (H17) was used in the CD19 CAR. H17 targets the same region as H18 in the open reading frame of the EBAG9 gene. In vitro cytotoxicity assays were performed similarly to the BCMA CAR. The transduction rate of retrovirally transduced CD8+ T cells was adjusted to approximately 15% using UT. CD19 high The EBAG9-expressing B-NHL cell line JeKo-1 was used as target cells in a chromium release assay. After 4 h of coculture, barely any lytic activity could be detected in UT and control H17-SP6 CAR T cells. In Jeko-1 cells, T cells transduced with CD19 CAR achieved approximately 20% specific target cell lysis (E:T 80:1). Consistent with previous results, EBAG9 silencing endowed CAR T cells with a large increase in killing activity. RNAi-mediated genetic engineering of T cells resulted in a two-fold increase in cytotoxicity. Moreover, only 1 / 5-1 / 8 of H17-CD19 CAR T cells were required to achieve maximum lysis of JeKo-1 cells by CD19 CAR T cells (Figure 8).

[0315] In vivo applications To apply the findings of the in vitro functional assay to an in vivo model, a multiple myeloma xenograft model was established. A suitable animal model for xenografting of human multiple myeloma cell lines and human primary CAR T cells is the NOD / scid γ-chain deficient immunodeficient mouse (NSG) strain. These mice do not have mature T or B cells. In addition, NK cell differentiation is inhibited. NSG mice were inoculated with the BCMA-expressing multiple myeloma cell line MM.1S. Tumor progression was monitored by bioluminescence imaging of the MM.1S cell line stably expressing a firefly luciferase-eGFP construct. Tumor cell engraftment was detected by IVIS imaging 6 days after implantation. A single dose of 1×10e6 CAR+T cells on days 10-13 of culture supplemented with IL-7 / IL-15 was injected intravenously 1 day later. Tumor growth was followed by IVIS serial imaging until day 14 after implantation. Mice were sacrificed on days 15-16. Bone marrow was analyzed by flow cytometry for the number of remaining tumor cells and CAR T cells.

[0316] IVIS sequential imaging revealed rapid tumor growth between days 6 and 14. The strongest specific luciferase signal, which correlated with tumor activity, could be localized to the bone marrow. Treatment with non-targeted H18-SP6 CAR T cells failed to control tumor growth. The largest tumor burden was observed in this group of mice. Thus, antigen-independent T cell activation due to EBAG9 silencing and subsequent increased ability to release effector molecules did not occur. Mice treated with BCMA CAR T cells showed smaller tumor progression. However, clinical efficacy with this small number of effector CAR T cells was moderate. In contrast, mice administered with H18-BCMA CAR T cells showed almost no tumor signal (Figure 9A and Figure 9B). Correspondingly, when analyzing tumor cell numbers (GFP+CD138+) in bone marrow, the main niche for homing of myeloma cells, quantification of tumor cells in the H18-SP6 CAR control group showed a two-fold higher number compared to the BCMA CAR group. Notably, tumor cells were almost absent in mice treated with RNAi-mediated EBAG9 silencing in BCMA CAR T cells. Taken together, RNAi-mediated downregulation of EBAG9 led to a strong increase in antitumor efficiency even with low numbers of effector cells (Figure 9C).

[0317] CRISPR-mediated EBAG9 knockout In the above examples, EBAG9 downregulation was demonstrated by transducing cells with a retroviral vector that allows the expression of EBAG9-specific miRNA. Such vectors are also used to co-express EBAG9 miRNA with antigen-specific CAR constructs in transduced T cells. Enhancement of the cytolytic ability of CAR T cells by downregulated EBAG9 was demonstrated both in vitro and in vivo.

[0318] To complement the above examples, additional means of downregulating EBAG9 were evaluated. The following examples use CRISPR-mediated EBAG9 knockout to demonstrate that EBAG9 can be effectively removed or reduced from CRISPR-treated T cells, thereby enabling additional means of inhibiting EBAG9 in cytotoxic T cells.

[0319] To knock out EBAG9 in human primary T cells, we generated different guide RNAs (gRNAs) targeting exon 4 of the human EBAG9 gene (E1-E4). Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy volunteer donors and CD3 + T cells were activated by stimulation with human anti-CD3 / CD28 Dynabeads. + T cells were electroporated with gRNA and Cas9 protein and cultured under IL-2 supplementation. + Genomic DNA was isolated and protein lysates were made on day 9 after T cell activation. Analysis of gene editing efficiency at the EBAG9 locus by TIDE analysis (tracking of Indels by decomposition) and Western blot analysis of EBAG9 protein levels revealed a 50% knockout efficiency with gRNAs E2, E4, and E6. In contrast, gRNAs E1, E3, and E5 show only a minor reduction in endogenous EBAG9 expression levels. CD3 cells electroporated with non-targeting gRNAs + T cells served as a control (ctl). The results are shown in Figure 10. Thus, CRISPR-mediated gene editing can target the human EBAG9 locus and is a viable means of EBAG9 inhibition in cytotoxic T cells expressing antigen-targeting transgenic constructs.

[0320] In vitro cytotoxicity assays to examine the effect of CRISPR-mediated EBAG9 gene editing on the antigen-specific cytolytic capacity of CAR T cells are underway and are expected to demonstrate enhanced cytolytic activity of CRISPR-edited CAR T cells through the release of granzymes and perforin, as shown for miRNA-mediated downregulation of EBAG9 as described above. [Explanation of symbols]

[0321] Drawing translation Figure 1 Intron miR155 back bone miR155 back bone EBAG9-specific sequence Figure 2 Calnexin unspecific non-specific Figure 3 high IL-2 low IL-2 Day 0 CD8 stimulation + CD8 with anti-CD3 / 28 + Stimulation Day 2 Day 3 retroviral transduction Day 13 Day 15 functional in vitro assays Figure 4 Granzyme A secreted (% of total) Figure 5 Intron miR155 back bone miR155 back bone EBAG9-specific sequence Figure 6B Counts Figure 6D Counts Figure 7A Lysis (%) Lysis (%) Effector:Target Figure 7B Lysis (%) Lysis (%) Effector:Target Figure 8 Lysis (%) Lysis (%) Effector:Target Figure 9A H18-SP6 CAR T cells H18-SP6 CAR T cells BCMA CAR T cells BCMA CAR T cells H18-BCMA CAR T cells H18-BCMA CAR T cells Day 6 after tumor cells iv Days after T cell transfer Day -1 Day 6 Day 14 150 s exposure 60 s exposure Figure 9B Whole body tumor burden Avg Radiance [p / s / cm 2 / sr] Average luminance [p / s / cm 2 / sr] Time [d] Time [d] Figure 9C Tumor load bone marrow % of CD138 + GFP + Cells CD138 + GFP + % of cells Figure 10A EBAG9 potential sgRNA sites Translated region Figure 10B Genomic EBAG9 analysis Total gene editing efficiency Figure 10C EBAG9 protein level Different target sites Calnexin

Claims

1. A genetically modified cytotoxic T cell comprising one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct, wherein estrogen receptor-binding fragment-associated antigen 9 (EBAG9) activity is inhibited in said cell (compared to a control cytotoxic T cell).

2. The genetically modified T cell of claim 1, wherein inhibition of EBAG9 activity is associated with increased release of cytolytic granules and / or secretory lysosomes containing granzymes (compared to control cytotoxic T cells).

3. 3. The genetically modified T cell of claim 1 or 2, wherein the antigen-targeting transgenic construct is a chimeric antigen receptor (CAR).

4. The genetically modified T cell of any one of claims 1 to 3, wherein the antigen-targeting transgenic construct is a T cell receptor (TCR).

5. The genetically modified T cell of any one of claims 1 to 4, wherein inhibition of EBAG9 activity is achieved by knockdown of EBAG9 or by RNA interference of EBAG9 expression by small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA).

6. The genetically modified T cell of claim 5, wherein inhibition of EBAG9 activity is achieved by genetic modification of the T cell genome with one or more exogenous nucleic acid molecules, the exogenous nucleic acid molecules comprising an antigen-targeting transgenic construct and a vector encoding an RNA interference sequence for knockdown of EBAG9.

7. 7. The genetically modified T cell of any one of claims 1 to 6, wherein inhibition of EBAG9 activity is achieved by genetic modification of the T cell genome by preventing expression of the EBAG9 gene and / or disrupting its sequence, or wherein an exogenous nucleic acid molecule encoding an antigen-targeting transgenic construct is positioned within, adjacent to or linked to the EBAG9 gene in the T cell genome, thereby preventing expression of and / or disrupting the sequence of the EBAG9 gene.

8. The genetically modified T cell according to any one of claims 1 to 7, for use as a medicament.

9. 9. The genetically modified T cell of any one of claims 1 to 8, for use as a medicament in the treatment of a proliferative disease, wherein the antigen targeted by the antigen-targeting transgenic construct is expressed in target cells undergoing and / or associated with pathological cell proliferation.

10. 10. The genetically modified T cell for use according to claim 9, wherein the proliferative disease is a hematological malignancy or non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute myeloid leukemia, acute lymphoblastic leukemia, and / or multiple myeloma, and the antigen or tumor associated antigen (TAA) targeted by the antigen-targeting transgenic construct is expressed in cancerous cells of said hematological malignancy.

11. 11. The genetically modified T cell for use according to claim 10, wherein the hematological malignancy is a B cell cancer expressing CD19 and the antigen-targeting transgenic construct binds to CD19, or the hematological malignancy is a B cell cancer expressing BCMA and the antigen-targeting transgenic construct binds to BCMA, or the hematological malignancy is a cancer expressing CXCR5 and the antigen-targeting transgenic construct binds to CXCR5.

12. 10. The genetically modified T cell of claim 9, for use as a medicament in the treatment of an autoantibody-dependent autoimmune disease, wherein the antigen targeted by the antigen-targeting transgenic construct is expressed in a target cell associated with autoantibody production, or the medical disorder is systemic lupus erythematosus (SLE) or rheumatoid arthritis.

13. 13. A pharmaceutical composition comprising the genetically modified T cell of any one of claims 1 to 12 suitable for the treatment of a proliferative disease, additionally comprising a pharma- ceutically acceptable carrier.

14. A nucleic acid vector or a combination of nucleic acid vectors comprising an antigen targeting construct and a sequence encoding an RNA interference sequence for knockdown of estrogen receptor binding fragment associated antigen 9 (EBAG9).

15. 1. An in vitro method of increasing the cytolytic activity of genetically modified cytotoxic T cells, wherein the T cells comprise one or more exogenous nucleic acid molecules encoding an antigen-targeting transgenic construct, the method comprising inhibiting activity of estrogen receptor-binding fragment-associated antigen 9 (EBAG9) in the T cells, or wherein inhibiting EBAG9 activity is a. RNA interference of EBAG9 expression or knockdown of EBAG9 by small interfering RNA (siRNA), short hairpin RNA (shRNA), and / or microRNA (miRNA), or b. A method comprising genetic modification of the T cell genome by disrupting the expression of the EBAG9 gene and / or its sequence by CRISPR / Cas9 or TALEN.

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