DESC:INDUCIBLE CYTOKINESIGNALS AND METHODS FOR IMMUNOTHERAPY

Introduction
The present invention provides for a Dimerization Induced Chimeric Cytokine Response Element (DICCRE) with ligand activation that can be stably and efficiently expressed in human immune cells. The present invention also related to immune cells expressing DICCRE and composition containing the same. The present invention further provides for a method for the production of DICCRE expressing immune cells and the use of the same for enhancing the immune response towards cancer and pathogens. It also relates to cells comprising antigen-recognizing receptors (e.g., chimeric antigen receptors (CARs) or T cell receptors (TCRs)) that are either engineered to express DICCRE or co-administered with either cells expressing DICCRE or other cancer immunotherapy drugs (checkpoint inhibitors etc.).

Background

Majority of cancers including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin’s lymphoma are incurable despite currently available therapies. Adoptive therapy with genetically engineered autologous T cells has shown evidence of therapeutic efficacy in many types of cancers including melanoma and B cell malignancies. T cells may be modified to target tumor-associated antigens through the introduction of genes encoding artificial T-cell receptors, termed chimeric antigen receptors (CAR), specific to such antigens. Though adoptive cell therapy (ACT) with antigen-specific T cells has shown remarkable clinical success, but approaches to safety and augmentation of T cell function, especially in solid tumors as well as in many types of blood cancers, remains a great interest.

Other cancer immunotherapies, for example check point inhibitors, lead to resistance in patients due to poor immune response. Cytokine therapies including cytokines IL-2, IL-12, IL-15 and others are involved non-targeted and uncontrolled deliveryof cytokines in patients, thus leading to systemic toxicity.

Though, adaptive transfer of tumor specific T cells has been shown to elicit tumor regression in leukaemias and melanoma in patients (Corrigan-Curay J, Kiem HP et.al. T cell immunotherapy: looking forward. MolTher. 2014; 22:1564-1574),it was estimated that out of the patients undergoing regression post CAR T cells treatment, 30-50% of the patients have a disease relapse within 1 year of treatment(Shah, N. N., & Fry, T. J. (2019). Mechanisms of resistance to CAR T cell therapy. Nature Reviews Clinical Oncology, 1.). The mechanism for the regression of the disease was understood to be (1) Less persistence and exhaustion of CAR T cells in patients post treatment, (2) Loss of antigen or antigen modulations in tumor cells making an immune escape, (3) Presence of immunosuppressive microenvironment which inactivates anti-tumor T cells in solid tumors. These poses a great challenge to methods of treatment involving stimulation of an immune response, such as targeted T cell therapies.

The immune checkpoint inhibitors majorly target immunosuppressive molecules like PD-1 and CTLA-4 which inhibit the T cell response against tumors. The monoclonal antibody targeting these immunosuppressive molecules have shown success in cancers like melanoma, lung cancers, renal cancers and others. However, the clinical trials have identified patients with innate resistance to this therapy or the group of patients who initially respond to the treatment and show disease progression in later stages. The major factors for causing the resistance of this therapy are 1.insufficient generation of anti-tumor T cells, 2.exhaustion of tumor specific T cells and 3.impaired formation of memory T cells (Jenkins, R. W., Barbie, D. A., & Flaherty, K. T. (2018). Mechanisms of resistance to immune checkpoint inhibitors. British journal of cancer, 118(1), 9.). It is also quite challenging to be able to monitor or map the expression of the variety of inhibitory receptors in order to find the best combinations of drugs depending on the occurring changes at cancer type over time. Further, some cancers can protect themselves from this therapy by stimulating immune checkpoint targets.

Various modifications have been made toward improving the antitumor effect of CAR- or TCR-engineered T cells as well as immune checkpoint inhibitors. Adjuvant therapy, such as administration of supporting cytokines or factors that modulate tumor microenvironment are two central approach that have been explored in preclinical and clinical studies to enhance T-cell therapy (Klebanoff CA et al, IL-15 enhances the in vivo antitumor activity of tumor-reactive CD8+ T cells. ProcNatlAcad Sci. 2004;101:1969-1974 and Wallace A et al, Transforming growth factor-beta receptors blockade augments the effectiveness of adoptive T cell therapy of established solid cancer. Clin Cancer Res. 2008; 14:3966-3974).

Cytokines are known to directly stimulate immune effector cells and stromal cells at the tumor site and enhance tumor cell recognition by cytotoxic effector cells. Numerous animal tumor model studies have demonstrated that cytokines have broad anti-tumor activity and this has been translated into a number of cytokine-based approaches for cancer therapy. Recent years have seen a number of cytokines, including GM-CSF, IL-7, IL-12, IL-15, IL-18 and IL-21, enter clinical trials for patients with advanced cancer. However, supplying adjuvant drugs at the right time and site appears crucial, as systemically administered immunomodulators show toxicities (Leonard JP et al, Effects of single-dose interlukin-12 exposure on interleukin associated toxicity and interferon-gamma production. Blood, 1997;90 2541-2548). For example, Pegram et al. describes a murine model of CAR-engineered T cells that constitutively secrete interleukin 12 (IL-12) and showed increased cytotoxicity towards CD19+ tumor cells (Pegram et al., BLOOD, Vol. 119, No. 18, 2012). However, the secretion of IL-12 led to suppression of interleukin 2 (IL-2), an important cytokine that promotes the proliferation and anti-tumor effect of T and B lymphocytes. Dotti et al. discloses CAR-engineered T cells that constitutively secrete interleukin 15 (IL-15) and an inducible caspase-9 based suicide gene (iC9), which showed increase cytotoxicity towards CD19<+>tumor cells (US 20130071414 A1). Yet, this modified CAR-T cell demonstrated unchanged levels of IL-2 expression both in vivo and in vitro. Cytokine secreting T cells with safety switch have been described in Hoyos, V.,et al. (Engineering CD19-specific T lymphocytes with interleukin-15 and a suicide gene to enhance their anti-lymphoma/leukemia effects and safety. Leukemia, 24(6), 1160 (2010)). However, since the safety switch is activated through a drug, this mechanism not only eliminates cytokine response but also eliminates anti-tumor T cells, thus again causing a chance of disease relapse. Accordingly, novel therapeutic strategies for treating neoplasia are urgently required. Especially, approaches for inducible production of cytokines in patients which reduces toxicity associated with cytokine administration and effectively augment adoptive cell therapy was a need in the art.

Summary of the invention

Accordingly, in one aspect, the invention pertains to a recombinant nucleic acid molecule encoding a novel Dimerization Induced Chimeric Cytokine Response Element (DICCRE), wherein the DICCRE comprises:
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain
wherein said dimerizing domain comprises FK506 binding protein (FKBP) domain of SEQ ID No: 2, or fragments thereof, and wherein the dimerizing domain is present as an extracellular domain or as an intracellular domain.

In an embodiment, the nucleic acid molecule encodes linker sequence comprising an amino acid sequence of SEQ ID No. 3.

In an embodiment, the nucleic acid molecule encodestransmembrane domain comprising the amino acid sequence selected from SEQ ID No. 4, SEQ ID No.6, SEQ ID No. 8, SEQ ID No.10, SEQ ID No.18 and SEQ ID No. 19.

In an embodiment, the nucleic acid molecule encodes intracellular cytokine signaling domain comprising the amino acid sequence selected from SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 24.

In an embodiment, the nucleic acid molecule encodes a DICCRE comprising the amino acid sequences of SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27 or SEQ ID No: 28.

In an embodiment, the nucleic acid molecule comprises a nucleic acid sequences encoding the DICCRE, wherein the nucleic acid sequence encoding the DICCRE comprises the nucleotide sequence selected from SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No: 43 or nucleotide sequence with at least 95% identity thereof.

In an embodiment, the nucleic acid molecule encodesDICCRE further comprising a Chimeric Antigen Receptor (CAR) sequence.

In an embodiment, the nucleic acid molecule encodes CAR-DICCRE comprising amino acid sequences selected from SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41 or SEQ ID No: 42.

In an embodiment, the nucleic acid molecule comprises a nucleic acid sequences encoding the CAR-DICCRE, wherein the nucleic acid sequence encoding the CAR-DICCRE comprises the nucleotide sequence selected from SEQ ID No. 44 and SEQ ID No. 45, or nucleotide sequence with at least 95% identity thereof.

In an embodiment, the nucleic acid molecule encodes DICCRE further comprises a leader sequence.

In an embodiment, the nucleic acid molecule encodes a leader sequence comprising the amino acid sequences of SEQ ID No. 1.

In another aspect, the present invention pertains to a Dimerization Induced Chimeric Cytokine Response Element (DICCRE) polypeptide, which comprises,
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain
wherein said dimerizing domain comprises FK506 binding protein (FKBP) domain of SEQ ID No: 2, or fragments thereof, and wherein the dimerizing domain is present as an extracellular domain or as an intracellular domain.

In an embodiment, in the DICCRE, the dimerizing domain is attached to the cytokine signaling domain via a linker, comprising the amino acid of SEQ ID NO: 3.

In an embodiment, the DICCRE comprises a transmembrane domain comprising the amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 18 and SEQ ID No. 19.

In an embodiment, the DICCRE comprises an intracellular cytokine signaling domain comprising the amino acid sequence selected fromSEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 24.

In an embodiment, the DICCRE comprises an amino acid sequence selected from SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27 or SEQ ID No: 28.

In an embodiment, theDICCREisco-expressed with Chimeric Antigen Receptor (CAR) comprises an amino acid sequence of SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41 or SEQ ID No: 42.

In an embodiment, the DICCRE further contains a leader sequence comprising an amino acid sequence of SEQ ID No: 1.

In another aspect, the invention pertains to a vector comprising the nucleic acid molecule according to the present invention.

In an embodiment, the vector is lentiviral vector, comprising the nucleotide sequence selected from SEQ ID No. 46 and SEQ ID No 47.

In another aspect, the invention pertains to an immune cell comprising the vector according to any of claims 19 to 20.

In an embodiment, theimmune cell is a human T lymphocyte including but not restricted to CD8+ and CD4+ T lymphocyte and its possible subsets.

In another aspect, the invention pertains to a pharmaceutical composition comprising the nucleic acid molecule or the DICCRE or the vector or the cell according to present invention, with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds alone or in combination.

In another aspect, the invention pertains to a method of making a DICCRE according to the present invention, comprising, transducing a T cell with a vector according to claims 19 to 20.

In another aspect, the invention pertains to a method for preparing immune cells expressing a DICCRE according the present invention, comprising:
(i) Providing a population of immune cells including T cells, NK cells, macrophages, or any other cells;
(ii) Introducing into the immune cells a nucleic acid according to claims 1 to 11; and
(iii) Culturing the immune cells under conditions allowing for expression of the chimeric receptor.

In an embodiment, the population of immune cells is derived from peripheral blood mononuclear cells (PBMC).

In an embodiment, theimmune cells comprise of CD3+ and CD8+ and CD4+ T lymphocyte and its possible subsets or other immune cells, NK cells, macrophages and B cells.

In an embodiment, theimmune cells are derived from a human cancer patient.

In another aspect, the invention pertains to a method of treating a subject having a disease associated with malignancy, comprising administering to the subject an effective amount of an immune effector cell according to the present invention.

In an embodiment, thedisease associated with CD19 expression is selected from a proliferative disease, such as a cancer or malignancy or a precancerous conditions such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of CD19.

In an embodiment, the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma.

In an embodiment, the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma including other CD19 positive malignancies.

Description of Figures

Figure 1: Schematic representation of DICCRE subunit, composed of extracellular dimerizing domain, a linker, transmembrane domains and an intracellular cytokine signaling domains.

Figure 2: Mechanism of action of DICCRE approach without (A) and with (B) ligand addition to improve the anti-tumor response.

Figure 3: Possible mode of treatment of cancer using the DICCRE approach
A. DICCRE armored T cell
B. DICCRE armored CAR T cell
C. DICCRE armored T cells with CAR T cell
D. DICCRE armored T cells as an adjuvant with other therapy

Figure 4: Manufacture of DICCRE T cells and its determination of its cytotoxicity potential. (A)Schematic representation of manufacturing and expansion of DICCRE T cells. (B)Flowcytometry analysis for determination of co-expression of DICCRE and CAR on T cells. (C)Percentage of T cells expressing CAR1-15 and CAR1-15A DICCRE T constructs after genetically modified using lentiviral vector. (D)Cytotoxicity potential assay of DICCRE T cells co-cultured with CD19 expressing Nalm-6 as determined by flowcytometry.

Figure 5: DICCRE T cells showed enhanced persistence and proliferation upon multiple antigen encounter. (A) Schematic representation of multiple antigen stimulation assay of DICCRE T cells. CD19 expressing Nalm-6 cells were used as target tumor cells. (B) T cells counts depicting the increased proliferation and persistence of DICCRE T cells upon tumor interaction in presence of AP20187 dimerizing ligand.

Detailed description of the invention

Though Immune therapy including cellular therapies, cytokine therapy and immune-checkpoint inhibitors may be effective in cancer treatment, their potential for expansion, damage of normal organs, and malignant transformation is a source of concern. The ability to conditionally eliminate aberrant cells in vivo would ameliorate these concerns and broaden the application of cellular therapy. Accordingly, the present inventors have devised a Dimerization Induced Chimeric Cytokine Response Element (DICCRE) with ligand activation that can be stably and efficiently expressed in human immune cells without impairing phenotype, function, or antigen specificity. This system is based on the fusion of modified human FK506-binding protein (FKBP), allowing conditional dimerization using a small- molecule drug. When exposed to a synthetic dimerizing drug, the inducible cytokine response element becomes activated by dimerization to induce cytokine specific signaling domain used in individual DICCRE construct for rapid immune-cell activation. (Figure 2). Therefore, the activation of the chimeric cytokine response element does not require extracellular cytokine for it dimerization. Hence the present invention provides for a safe small molecule induced dimerization of the cytokine responsive elements that not only eliminate cytokine toxicity but also augments anti-tumor effect of the immune cells without affecting bystander cell populations. The DICCRE is also found to enhance the anti-tumor activity, persistence and longevity of in adaptive immunotherapy, such as CAR T cell therapy.

The present invention therefore provides a novel Dimerization Induced Chimeric Cytokine Response Element (DICCRE) consisting of typically two receptor chains that may be the same or different. In each single subunit receptor, the subunits fulfil the dual role of binding to dimerizer and signaling. A single subunit comprises of
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain.

The dimerizing domain of the DICCRE is derived from subunit of protein involved in the dimerization of the bipartite subunits. The dimerizing domain described here in derived from FK506 binding proteins (FKBP) with additional modifications at particular amino acid and nucleotide level. FK506 is an immunosuppressive natural product that is known to heterodimerizes the proteins FKBP12 and calcineurin, potently inhibiting calcineurin (Liu et al., 1991). Calcineurin inhibition leads to impaired T-cell receptor signaling and consequent immunosuppression. The present inventors have devised a safety switch for immune cells that exploits the dimerization property of the cytokine receptor molecule, using the FK506 binding protein in order to control the cytokine signaling. Under physiological conditions, cytokine receptors are activated by the release of cytokines in the blood stream either from activated immune cells or through controlled intravenous administration, which activates cytokine production majorly through JAK-STAT pathway, with help from the PIP and Ras MAP Kinase pathways. The DICCRE according to the present invention comprising the FKBP as the dimerizingdomain is activated by the presence of FKBP dimerization ligand for production of cytokine and its downstream signaling and is not dependent on blood cytokine levels. The dimerization domains can also be chosen from drug-protein combinations in the literature for example combination of FKBP-FRB domain in presence of rapamycinanalogs and others plant derived combinations of Gibberellins receptor GID1 and others.(Choi, J., Chen, J., Schreiber, S. L., & Clardy, J. (1996).Structure of the FKBP12-rapamycin complex interacting with binding domain of human FRAP. Science, 273(5272), 239-242.)(Murase, K., Hirano, Y., Sun, T. P., &Hakoshima, T. (2008). Gibberellin-induced DELLA recognition by the gibberellin receptor GID1. Nature, 456(7221), 459.).In one embodiment the dimerizing domain is placed extracellular. In another embodiment the dimerizing domain is placed intracellular (see Figure 3).

The optimized inducible dimerizing domain comprises an FKBP12-F36V domain linked, via a flexible (Ser-Gly-Gly-Gly-Ser)n linker, to cytokine receptor, which is receptor without its physiological dimerization domain. The function of the FKBP domain is to dimerize the bipartite receptor upon addition of FKBP dimerization ligands like AP1903 and/or AP20187. This dimerization of the domain helps in the dimerization of the two subunits. In one embodiment, the dimerizing domain described herein is composed of amino acid sequence represented by SEQ ID NO. 2. In another embodiment, the linker is composed of amino acid sequence represented by SEQ ID NO. 3.

The transmembrane domain helps for membrane anchorage of the DICCRE subunits to immune cells. The transmembrane domain is the connecting link between the extracellular domain and the intracellular domain of the DICCRE. The transmembrane domain is derived from the cytokines or the receptors thereof which confer the cytokine specificity to the DICCRE.

In one embodiment the transmembrane domain is derived from human IL-15R???In a preferred embodiment, the IL-15R??transmembrane domain comprises of the amino acid sequence presented by SEQ ID NO. 4.

In another embodiment the transmembrane domain is derived from human IL-7R???In a preferred embodiment, the IL-7R?transmembrane domain comprises of amino acid sequence presented by SEQ ID NO. 6.

In another embodiment the transmembrane domain is derived from human IL-21R.?in a preferred embodiment, the IL-21R transmembrane domain comprises of amino acid sequence presented by SEQ ID NO. 8.

In another embodiment the transmembrane domain is derived from human gamma c receptor. In a preferred embodiment, the gamma c receptor transmembrane domain comprises of amino acid sequence presented by SEQ ID NO. 10.

In another embodiment the transmembrane domain is derived from human IL-27R??In a preferred embodiment, the IL-27R transmembrane domain comprises of amino acid sequence presented by SEQ ID NO. 18.

In another embodiment the transmembrane domain is derived from human IL-6R???In a preferred embodiment, the IL-6R?transmembrane domain comprises of amino acid sequence presented by SEQ ID NO. 19.

The intracellular cytokine signaling domain plays a major role in providing the cytokine signaling to the immune cells, thus improving its persistence and longevity. The receptor dimerization specifically of the intracellular domains, help in activation of signaling cascade which in this case helps in improving T cell longevity and anti-tumor efficacy. The cytokine signaling occurs majorly through JAK-STAT pathway, with help from the PIP and Ras MAP Kinase pathways (Figure 2). This pathways converging into certain genes, for example in case of IL-15 signaling, STAT5 is phosphorylated and genes encoding anti-apoptotic proteins like Bcl-2 are activated which enhances the T cell survival.

In one embodiment the intracellular cytokine signaling domain is derived from human IL-15R???In a preferred embodiment, the IL-15R?intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO.5 or SEQ ID NO. 24.

In another embodiment the intracellular cytokine signaling domain is derived from human IL-7R???In a preferred embodiment, the IL-7R??intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO. 7.

In another embodiment the intracellular cytokine signaling domain is derived from human IL-21R.?in a preferred embodiment, the IL-21R intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO. 9.

In another embodiment the intracellular cytokine signaling domain is derived from human gamma c receptor. In a preferred embodiment, the gamma c receptor intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO.11.

In another embodiment the intracellular cytokine signaling domain is derived from human IL-27Rb.??n a preferred embodiment, the IL-27R intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO. 20

In another embodiment the intracellular cytokine signaling domain is derived from human IL-6R?. In a preferred embodiment, the human IL-6R? intracellular cytokine signaling domain comprises of amino acid sequence presented by SEQ ID NO.21.

The DICCRE polypeptide described herein comprise a leader sequence at the amino terminal (N-ter). The leader sequence may comprise the amino acid sequence of SEQ ID NO. 1.

The DICCRE according to the present invention is composed of a polypeptide including an amino acid sequence selected from SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27 or SEQ ID NO.28. The DICCRE described herein are encoded by nucleotide sequence selected from SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO.43 or nucleotide sequence with at least 95% identity thereofand has been optimized for human codon usage to enhance the expression, efficacy and persistence of T cells.

The DICCRE according to the present invention can be combined with CAR expression and is composed of a polypeptide including an amino acid sequence selected from SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41 or SEQ ID NO.42. The DICCRE described herein are encoded by nucleotide sequence selected from SEQ ID NO:44 or SEQ ID NO.45 or nucleotide sequence with at least 95% identity thereof and has been optimized for human codon usage to enhance the expression, efficacy and persistence of CAR T cells.

The DICCRE according to the present invention can be provided in various embodiments as described below:

A. DICCRE armored T cell

Since DICCRE has low dimerizer-independent basal activity, it can be stably expressed in human immune cells without impairing their phenotype, function, and antigen specificity. The present invention therefore provides for an immune cells expressing DICCRE. The immune cells include but are not limited to T cells, NK cells, hematopoietic stem cells, etc. In this embodiment, cells are engineered to express DICCRE protein delivered via gene delivery vehicle / vector.

B. DICCRE armored CAR T cell

Adoptive cell therapy predominantly Chimeric Antigen Receptor (CAR) cell immunotherapy - the new ‘intent to cure’ strategy which involves generation of anti-tumor immune effector T cells against a particular cancer antigen is revolutionizing cancer treatment globally. Based on the higher remission rates in clinical trials involving patients with haematological malignancies, two CAR T cells products have been approved by the US FDA in 2017 against CD19 positive malignancies.

With newer CAR T been developed and increasing multi-centre trails for the available CAR T cells , more and more patients have been recruited and long-term follow up data of these clinical trials have generated some insightful observations. The conventional CAR T cells possess target antigen recognizing extracellular domain called the scFv and an intracellular domain encoding a CD3z signaling domain required for the T cell activation and costimulatory domain (CD28, 4-1BB , ICOS and others) required for the stimulation required for T cell proliferation. However, efficient T cells activation require an additional signal coming from immune-stimulatory cytokines and other gamma c cytokines which play a role a determining the anti-tumor efficacy.

For instance, cytokines IL-7 and IL-15 have shown to be involved in generation and maintenance of less differentiated T stem cell like memory population (Tscm) which is the known most potent anti-tumor T cells subsets. In the absence of these regulatory cytokines the CAR T cells tend to skew towards terminally differentiated state thereby leading to exhaustion and poor persistence of CAR T cells. However, translation of cytokines along with CAR T cells still remains a challenge. Direct administration of cytokines like IL-2, IL-7, IL-15 and IL-12 into the patients as an adjuvant treatment has shown higher toxicities due to uncontrolled and untargeted actions of these cytokines. To mitigate these toxicities, the cytokine secreting CAR T cells have designed and have shown success at the preclinical scale. This approaches though have reduced toxicities of this cytokines, they do not provide a complete solution to the uncontrolled and untargeted actions of this cytokines.

Accordingly, the present invention provides for a DICCRE armored CAR T cells which possess an antigen recognizing chimeric receptor along with the armor coding for cytokine response element. A major advantage of the DICCRE armored CAR T cells approach over the other available strategies is the induction of cytokine response in a controlled, targeted manner. The DICCRE armored CAR T cells when administered to patient for the treatment of cancer can function independent of CAR activity on cancer cells. In the event of relapse or disease progression where adjuvant therapy would be essential, the DICCRE could be activated using small moleculeligands like AP1903 and/or AP20187, which produces required level of cytokines at the tumor site. The DICCRE approach also has an improved clinical translatability due to use of pharmaceutically tolerable small molecules involvement and lack of any recombinant cytokines.

C. DICCRE armored T cells with CAR T cell

In patients with innate resistance to CAR T cell therapy or patients who initially respond to the treatment and show disease progression in later stages due to resistance of CAR T cell therapy or exhaustion of tumor specific T cells, a combination of DICCRE armored T cell with CAR T cell would prove effective in controlling disease progression.

Accordingly, the present invention provides for a combination of DICCRE armored T cell with CAR T cell for the treatment of cancer.

D. DICCRE armored T cells as an adjuvant with other cancer therapy regimens

The immune checkpoint inhibitors majorly target immunosuppressive molecules like PD-1 and CTLA-4 which inhibit the T cell response against tumors. However, the patients show innate resistance to this therapy or the group of patients who initially respond to the treatment and show disease progression in later stages. The major factors for causing the resistance of this therapy are 1.insufficient generation of anti-tumor T cells, 2.exhaustion of tumor specific T cells and 3.impaired formation of memory T cells.

Accordingly, the present invention provides DICCRE armored T cell for the treatment of cancer in combination with immune checkpoint inhibitors, to improve the T cell response and hence therapeutic outcome of the therapy.

Accordingly, the present invention provides DICCRE armored T cells for the treatment of cancer in combination with cancer therapies like chemotherapy, immune modulators and/or any other anticancer biologics including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant scFv and other anti-cancer proteins, wherein additional immune cell activation is required for the anti-tumor response.

Accordingly, the present invention provides for DICCRE armored T cells as an adjuvant with other therapy. This helps in utilization of the full anticancer potential of the cytokines at the same time limiting the toxicity.

The invention also encompasses a process forthe preparation of the DICCRE, composition containing the DICCRE, vectors containing the polynucleotide encoding the DICCRE and cells expressing the DICCRE at their surface, in particular for their use in immunotherapy.

In certain aspects, the invention pertains to provide immune response to the patient affected with cancer using DICCRE approaches as described above. In one aspect, the DICCRE polypeptide is expressed by T cells, which provide the anti-tumor response. The invention includes methods of generation DICCRE expressing T cells by using a vector comprising DNA encoding the DICCRE subunits, its expansion into clinically meaningful numbers to be used as a therapy.

In another aspect, the invention pertains to method of transducing T cells with the vector encoding DICCRE subunits. A vector is a molecule which contains nucleotide sequence encoding DICCRE subunits. In a preferred embodiment of the invention, the vector used is a lentiviral vector. Lentiviral vectors are known in art to facilitate long term stable gene transfer in the host cell and allowing its propagation in the daughter cells. The self-inactivating lentiviral vectors are more advantageous due to their capacity to accommodate larger gene of interest, can transduce non proliferating cells and possess low immunogenicity. The strategies for packaging of desired nucleic acid into viral vectors preferably lentiviral vectors are known in the art. The lentiviral particles expressing the gene of interest can be harvested and used for transducing the cells ex vivo.

The nucleotide sequence of the CAR construct is cloned into an expression vector suitable for integration and stable expression in mammalian cells with techniques known in art. The DICCRE subunits gene can also be synthesized chemically and added to the expression vector. The expression of DICCRE subunits is under the control of a constitutive promoter usually derived from a mammalian cell. A promoter is a DNA sequence usually present upstream of the gene of interest which drives the expression of the target gene in the cells.

In one embodiment, the promoter chosen for expression of DICCRE comprises of EF-1a promoter, comprising the nucleotide sequence of SEQ ID No. 48.

Along with promoter, the vector comprising DICCRE genes also comprises of the termination sequences for transcription and translation and other non-coding sequences which regulate the expression of desired gene of interest. The non-coding sequences usually called introns also a play in enhancing the gene expression in synergy with the promoter such as the EF-1a intron A.

In one embodiment, the EF-1a promoter chosen for expression of DICCRE comprises of EF-1a intron A.

The invention pertains to treatment of cancers preferably using cells expressing the DICCRE subunits. The cells expressing DICCRE subunits are immune cells which include but are not limited to T cells, NK cells and hematopoietic stem cells.

In one aspect, the T cells transduced for expressing DICCRE subunits are isolated from peripheral blood or apheresis product. The source of T cells can be ‘autologous’ or ‘allogenic’. In one aspect, the T cells to be used as a treatment for cancers are autologous.

In one aspect, the T cells transduced for expressing DICCRE subunits can be tumor infiltrating T cells. In one aspect, the T cells transduced for expressing DICCRE subunits can be genetically engineered T cells expressing CAR and/or TCR.

Examples:
Example 1: Design and synthesis of Dimerization Induced Chimeric Cytokine Response Element (DICCRE)
In the present invention Dimerization Induced Chimeric Cytokine Response Element (DICCRE) is a chimeric cytokine signaling receptor. DICCRE consists of typically two receptor chains that may be the same or different. In each single subunit receptor, the subunits fulfil the dual role of binding to dimerizer and signaling. A single subunit comprises of
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain.
In the present invention, the dimerizing domains have been derived from the part of human peptidyl-prolylcis-trans isomerase FKBP1A (UniProtKB - P62942 and PDB 1BL4). The transmembrane domain and intracellular signaling domain was adapted from IL-15 receptor subunit beta (UniProtKB - P14784), IL-7 receptor subunit alpha (UniProtKB - P16871), IL-21 receptor (UniProtKB - Q9HBE5), IL-27 receptor (UniProtKB - Q6UWB1), IL-6 receptor subunit beta (UniProtKB - P40189) and Cytokine receptor common subunit gamma (UniProtKB - P31785).

These DICCRE constructs were designed and chemically synthesized by GeneArt (Life Technologies) after codon optimization for human codon usage and expression.
The codon usage was adapted to the codon bias of Homo sapiens genes.
In addition, regions of very high (> 80 %) or very low (< 30 %) GC content have been avoided where possible.
During the optimization process the following cis-acting sequence motifs were avoided where applicable:
• internal TATA-boxes, chi-sites and ribosomal entry sites
• AT-rich or GC-rich sequence stretches
• RNA instability motifs
• repeat sequences and RNA secondary structures
• (cryptic) splice donor and acceptor sites in higher eukaryotes
The chemically synthesized DICCRE construct was further cloned into E1-T plasmid of the 3rd generation lentiviral system. In the present invention, the DICCRE constructs were expressed under EF-1 alpha promoter either alone or along with chimeric antigen receptor (CAR) against CD19.

Example 2: Manufacture of DICCRE T cells
T cells were isolated from the whole blood of healthy individuals using EasySep™ Direct Human T Cell Isolation Kit (Catalog #19661). The T cells were activated for 36-48hr by anti-CD3/anti-CD28 magnetic beads (Life Technologies, USA) in 1:1 cell to bead ratio in 24well plate (Corning, USA) with cell density 1million cells per 2ml of T cell media (AIM-V with 5% heat inactivated FBS; Gibco) supplemented with 50ng/ml recombinant human IL-2(rhIL-2)(Life Technologies). 3rdgeneration lentiviral vector system was used to generate the genetically modified DICCRE T cells. According to the literature 3rdgeneration lentivirus system is safer in clinic compared to second generation lentiviral vector system and retroviral vector system.

The activated T cells were spinoculated with lentiviral vector encoding DICCRE construct at 32°C for 2 hrs at 1000g. The mixture was further incubated for 24hrs followed by a second round of spinoculation. Next day, lentiviral vector was removed and cells were expanded as required by maintaining the cell density (0.3 million per ml) supplemented with 10ng/ml rhIL-2.

Flowcytometry analysis was performed to determine the expression of DICCRE and CAR on the surface of T cells using anti-FKBP antibody (Thermo,Catalog # PA1-026A) and Protein L (Genscript, Cat. No. M00097) staining respectively. The flowcytometry data showed that the cells successfully co-expressed DICCRE and CAR in 20-25% of T cells. This DICCRE T cells were used for further functional analysis.

Example 3: Efficacy study of DICCRE T cells
Cytotoxicity Assay:
The DICCRE T cells were supplemented with ligand AP20187 at a concentration of 10nM in presence of low rhIL-2 (5ng/ml) for 4 days. This cells were subjected to co-culture cytotoxicity assay with CD19 expressing Nalm-6 cells (target tumor cells) at different effector: target (E:T) (0.5:1, 1:1 and 2:1) for 16-24 hrs. The cells were subjected to flowcytometry analysis to determine the cytotoxicity potential of this cells. The DICCRE T cells showed 90% killing potential against the tumor cells in 2:1 ratio within 24 hrs.

T cell persistence and exhaustion assay:
The 7 day expanded DICCRE T cells were harvested and washed to remove residual rhIL-2 from the culture. This DICCRE T cells were co-culture with CD19 expressing Nalm-6 cells with and without supplementation of AP20187 (40nM) at 1:1 (E:T) ratio. The co-culture assay was devoid of any external cytokine supplementation. The cells were re-stimulated with target tumor cells at 3-day intervals and monitored for proliferation. Interestingly, the DICCRE T cells supplemented with AP20187 showed enhanced proliferation and persistence upon antigen encounter compared to the cells with no ligand supplementation.
This data suggests that, the DICCRE T cells might enhance the persistence of anti-tumor T cells like the CAR-T cells upon external ligand supplementation like AP20187 and/or AP1903.
,CLAIMS:We claim:

1. A recombinant nucleic acid molecule encoding anovel Dimerization Induced Chimeric Cytokine Response Element (DICCRE), wherein the DICCRE comprises:
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain
wherein said dimerizingdomain comprises FK506 binding protein (FKBP) domain of SEQ ID No: 2, or fragments thereof, and whereinthe dimerizing domain is present as an extracellulardomain or as an intracellular domain.

2. The nucleic acid molecule as claimed claim 1, wherein the encoded linker sequence comprises an amino acid sequence of SEQ ID No. 3.

3. The nucleic acid molecule as claimed in claim 1, wherein the encoded transmembrane domain comprises the amino acid sequence selected from SEQ ID No. 4, SEQ ID No.6, SEQ ID No. 8, SEQ ID No.10, SEQ ID No.18 and SEQ ID No. 19.

4. The nucleic acid molecule as claimed in claim 1, wherein the encoded intracellular cytokine signaling domain comprises the amino acid sequence selected from SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 24.

5. The nucleic acid molecule as claimed in any of the preceding claims, which encodes a DICCRE comprising the amino acid sequences of SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27 or SEQ ID No: 28.

6. The nucleic acid molecule as claimed in any of the preceding claims, which comprises a nucleic acid sequences encoding the DICCRE, wherein the nucleic acid sequence encoding the DICCRE comprises the nucleotide sequence optimized for human codon usage and selected from SEQ ID No. 16, SEQ ID No. 17 and SEQ ID No: 43 or nucleotide sequence with at least 95% identity thereof.

7. The nucleic acid molecule as claimed in any of the preceding claims, wherein the encoded DICCRE further comprises a Chimeric Antigen Receptor (CAR) sequence.

8. The nucleic acid molecule as claimed in claim 7, which encodes CAR-DICCRE comprising amino acid sequences selected from SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41 or SEQ ID No: 42.

9. The nucleic acid molecule as claimed in claim 7, which comprises a nucleic acid sequences encoding the CAR-DICCRE, wherein the nucleic acid sequence encoding the CAR-DICCRE comprises the nucleotide sequence optimized for human codon usage and selected from SEQ ID No. 44 and SEQ ID No. 45, or nucleotide sequence with at least 95% identity thereof.

10. The nucleic acid molecule as claimed in precedingclaims, wherein the encoded DICCRE further comprises a leader sequence.

11. The nucleic acid molecule as claimed in claim 10, wherein the leader sequence comprise the amino acid sequences of SEQ ID No. 1.

12. A Dimerization Induced Chimeric Cytokine Response Element (DICCRE) polypeptide, which comprises,
(a) an dimerizing domain,
(b) a linker sequence,
(c) a transmembrane domain, and
(d) an intracellular cytokine signaling domain
wherein said dimerizing domain comprises FK506 binding protein (FKBP) domain of SEQ ID No: 2, or fragments thereof, and wherein the dimerizing domain is present as an extracellular domain or as an intracellular domain.

13. The DICCRE according to claim 12, wherein the dimerizing domain is attached to the cytokine signaling domain via a linker, comprising the amino acid of SEQ ID NO: 3.

14. The DICCRE according to claim 12, wherein transmembrane domain comprises the amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 18 and SEQ ID No. 19.

15. The DICCRE according to claim 12, wherein the intracellular cytokine signaling domain comprises the amino acid sequence selected from SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 20, SEQ ID No. 21 and SEQ ID No. 24.

16. The DICCRE according to any of claims 12 to 15, wherein the DICCRE comprises an amino acid sequence selected from SEQ ID No: 12, SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15, SEQ ID No: 22, SEQ ID No: 23, SEQ ID No: 25, SEQ ID No: 26, SEQ ID No: 27 or SEQ ID No: 28.

17. The DICCRE according to claims 12 to 16, co-expressed with Chimeric Antigen Receptor (CAR).

18. The DICCRE according to claim 17, wherein the CAR comprises an amino acid sequence of SEQ ID No: 29, SEQ ID No: 30, SEQ ID No: 31, SEQ ID No: 32, SEQ ID No: 33, SEQ ID No: 34, SEQ ID No: 35, SEQ ID No: 36, SEQ ID No: 37, SEQ ID No: 38, SEQ ID No: 39, SEQ ID No: 40, SEQ ID No: 41 or SEQ ID No: 42.

19. The DICCRE according to claim 12 and 17, wherein it further contains a leader sequence comprising an amino acid sequence of SEQ ID No: 1.

20. A vector comprising the nucleic acid molecule as claimed in any of claims 1 to 11.

21. The vector as claimed in claim 20, wherein the vector is lentiviral vector and comprises the nucleotide sequences selected from SEQ ID No. 46 and SEQ ID No. 47.

22. An immune cell comprising the vector according to any of claims 20 to 21.

23. The immune cell as claimed in claim 22, wherein the immune cell is a human T lymphocyte including but not restricted to CD8+ and CD4+ T lymphocyte and its possible subsets.

24. A pharmaceutical composition comprising the nucleic acid molecule according to any of claims 1 to 11 or the DICCRE according to any of claims 12 to 19 or the vector according to any of claims 20 to 21 or the cell according to any of claims 22 to 23, with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds alone or in combination.

25. A method of making a DICCRE according to any of claims 12 to 19, comprising, transducing a T cell with a vector according to claims 20 to 21.

26. A method for preparing immune cells expressing a DICCRE according to any of claims 12 to 19, comprising:
(i) Providing a population of immune cells;
(ii) Introducing into the immune cells a nucleic acid according to claims 1 to 11; and
(iii) Culturing the immune cells under conditions allowing for expression of the chimeric receptor.

27. The method as claimed in claim 26, wherein the population of immune cells is derived from peripheral blood mononuclear cells (PBMC).

28. The method as claimed in claim 26 or 27, wherein the immune cells comprise of CD3+ and CD8+ and CD4+ T lymphocyte and its possible subsets or other immune cells, NK cells, macrophages and B cells.

29. The method as claimed in any of claims 25 to 28, wherein the immune cells are derived from a human cancer patient.

30. A method of treating a subject having a disease associated with malignancy, comprising administering to the subject an effective amount of an immune effector cell according to claims 22 to 23.

31. The method as claimed in claim 30, wherein the disease associated with CD19 expression is selected from a proliferative disease, such as a cancer or malignancy or a precancerous conditions such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of CD19.

32. The method as claimed in claim 31, wherein the cancer is selected from the group consisting of a cancer of B-cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyo sarcoma, leukemia, and Hodgkin's lymphoma.

33. The method of claim 32, wherein the cancer of B-cell origin is selected from the group consisting of B-lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, and B-cell non-Hodgkin's lymphoma including other CD19 positive malignancies.