EA045264B1 - METHOD FOR TREATING HEMOBLASTOSIS USING IMMUNE EFFECTOR CELLS CONTAINING CHIMERIC ANTIGEN RECEPTORS FOR BCMA - Google Patents

Description

Cross references to related applications

This application claims priority under the Code of Federal Regulations

U.S. Title 35 §119(e) based on U.S. Provisional Patent Application No. 62/200505, filed Aug. 3, 2015, and U.S. Provisional Patent Application No. 62/091419, filed Dec. 12, 2014, each is incorporated herein by reference in its entirety.

Sequence listing statement

The sequence listing associated with this application is presented in a text file format in lieu of a paper copy, and is incorporated herein by reference. The specified text file containing a list of sequences is named: BLBD_043_02WO_ST25.txt. The size of the specified text file is 27 KB, it was created on December 4, 2015 and was submitted electronically via the EFS-Web system at the same time as the submission of this description.

State of the art Field of technology

The present invention relates to improved compositions and methods for treating B cell-related conditions. More specifically, the present invention relates to improved chimeric antigen receptors (CARs) containing a mouse anti-BCMA antibody or antigen-binding fragments thereof, immune effector cells genetically modified to express said CARs, and the use of said compositions for the effective treatment of B cell-associated conditions.

Description of the prior art

B lymphocytes, i.e. B cells, are involved in a number of significant diseases. Abnormal B cell physiology can also lead to the development of autoimmune diseases, including, but not limited to, systemic lupus erythematosus (SLE). Malignant transformation of B cells leads to the development of cancers, including, but not limited to, lymphomas such as multiple myeloma and non-Hodgkin's lymphoma.

The vast majority of patients with B-cell malignancies, including non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), contribute significantly to cancer-related mortality rates. The response to various forms of treatment for B-cell malignancies is mixed. Traditional treatments for B-cell malignancies, including chemotherapy and radiation therapy, are limited by toxic side effects. Immunotherapy with anti-CD19, anti-CD20, anti-CD22, anti-CD23, anti-CD52, anti-CD80, and anti-HLADR therapeutic antibodies has had limited success, in part due to unsatisfactory pharmacokinetic profiles, rapid clearance of antibodies by serum proteases and glomerular filtration, and limited site penetration tumor and target antigen expression levels on cancer cells. Attempts to use genetically modified cells expressing chimeric antigen receptors (CARs) have also had limited success. In addition, the therapeutic efficacy of a particular antigen binding domain used in a CAR is unpredictable: if the said antigen binding domain binds too strongly, the CAR-bearing T cells induce a massive release of cytokines, leading to a potentially lethal immune response called a cytokine storm; if the antigen binding domain is too weakly bound, the CAR T cells do not provide sufficient therapeutic efficacy to eliminate cancer cells.

Short description

The present invention generally provides improved vectors for T cell therapy and methods of using them.

Various embodiments provide a chimeric antigen receptor (CAR) comprising: an extracellular domain that contains a mouse anti-BCMA antibody (B-cell maturation antigen) or an antigen-binding fragment thereof that binds one or more epitopes of a human BCMA polypeptide; a transmembrane domain, one or more intracellular costimulatory signaling domains, and a primary signaling domain.

In specific embodiments, said mouse anti-BCMA antibody or said antigen-binding fragment that binds a human BCMA polypeptide is selected from the group consisting of: camel Ig, NAR Ig, Fab fragments, Fab' fragments, F(ab)' 2-fragment, F(ab)'3-fragment, Fv, single-chain Fv antibody (scFv), bi-scFv, (scFv)2, minibody, diabody, tribody, tetrabody, disulfide-stabilized Fv protein (dsFv) and single domain antibodies (sdAb, nanobodies).

In further embodiments, said mouse anti-BCMA antibody or said antigen-binding fragment that binds a human BCMA polypeptide is a scFv.

In some embodiments, the mouse anti-BCMA antibody or antigen binding fragment thereof comprises one or more CDR regions present in any of SEQ ID NOs: 1-3.

In specific embodiments, the mouse anti-BCMA antibody or antigen binding fragment thereof comprises one or more CDR regions present in any of the sequences of SEQ ID NOs: 4-6.

In some embodiments, the mouse anti-BCMA antibody or antigen binding fragment thereof comprises a light chain variable region sequence corresponding to SEQ ID NO:7.

In certain embodiments, said light chain variable region sequence comprises the CDR sequences set forth in SEQ ID NO: 13.

In other embodiments, the mouse anti-BCMA antibody or antigen binding fragment thereof comprises the heavy chain variable region sequence set forth in SEQ ID NO: 8.

In further embodiments, said heavy chain variable region sequence comprises the CDR sequences set forth in SEQ ID NOs: 4-6.

In further embodiments, said transmembrane domain is a polypeptide selected from the group consisting of: T cell receptor alpha, beta or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1.

In some embodiments, said transmembrane domain is a polypeptide selected from the group consisting of: CD8a; CD4, CD45, PD1 and CD154.

In some embodiments, said transmembrane domain is CD8a.

In specific embodiments, said one or more costimulatory signaling domains are represented by a costimulatory molecule selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 ( 4-1ВВ), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76 , TRIM and ZAP70.

In specific embodiments, said one or more costimulatory signaling domains are represented by a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

In further embodiments, said one or more costimulatory signaling domains are represented by a costimulatory molecule selected from the group consisting of: CD28, CD134, and CD137.

In further embodiments, said one or more co-stimulatory signaling domains are CD28.

In specific embodiments, said one or more co-stimulatory signaling domains are CD134.

In other embodiments, said one or more co-stimulatory signaling domains are CD137.

In some embodiments, the CAR comprises a hinge region polypeptide.

In further embodiments, the hinge region polypeptide comprises a CD8a hinge region.

In some embodiments, the CAR comprises a spacer region.

In further embodiments, said spacer region polypeptide comprises the CH2 and CH3 regions of IgG1 or IgG4.

In certain embodiments, the CAR comprises a signal peptide.

In further embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8a signal polypeptide.

In one embodiment, the CAR comprises an amino acid sequence corresponding to SEQ ID NO: 9.

According to various embodiments, a polynucleotide encoding a CAR provided by the present invention is provided.

According to various specific embodiments, a polynucleotide encoding a CAR is provided, wherein said polynucleotide sequence corresponds to the sequence of SEQ ID NO: 10.

According to various specific embodiments, there is provided a vector comprising a polynucleotide encoding a CAR provided by the present invention or represented by SEQ ID NO: 10.

In some embodiments, said vector is an expression vector

- 2 045264 vector.

In further embodiments, the vector is an episomal vector.

In certain embodiments, said vector is a viral vector.

In further embodiments, the vector is a retroviral vector.

In other embodiments, the vector is a lentiviral vector.

In one embodiment, the vector encoding the BCMA CAR comprises the polynucleotide sequence set forth in SEQ ID NO: 36.

In further embodiments, said lentiviral vector is selected from the group consisting essentially of: human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2), visna maedi virus (VMV); caprine arthritis-encephalitis virus (AEC); equine infectious anemia virus (EIAN); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

In specific embodiments, the vector comprises a left-handed (5') retroviral LTR sequence, a psi (Ψ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element; a promoter operably linked to a polynucleotide encoding a CAR as described herein; and the right (3') retroviral LTR sequence.

In other embodiments, the CAR comprises a heterologous polyadenylation sequence.

In some embodiments, the CAR comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck virus post-transcriptional regulatory element (WPRE).

In some embodiments, the 5' LTR sequence promoter is replaced with a heterologous promoter.

In further embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter.

In certain embodiments, the 5' LTR or 3' LTR sequence is a lentiviral LTR.

In certain embodiments, the 3' LTR sequence contains one or more modifications.

In some embodiments, the 3' LTR sequence contains one or more deletions.

In some embodiments, the 3' LTR sequence is a self-inactivating (SIN) LTR.

In some embodiments, the polyadenylation sequence is a bovine growth hormone polyadenylation sequence or a rabbit β-globin polyadenylation signal sequence.

In further embodiments, a CAR-encoding polynucleotide of the present invention comprises an optimized Kozak sequence.

In further embodiments, the promoter operably linked to the CAR-encoding polynucleotide of the present invention is selected from the group consisting of: cytomegalovirus (CMV) immediate-early gene promoter, elongation factor 1-alpha (EF1-a) promoter, phosphoglycerate kinase 1 (PGK) promoter ), ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer/chicken beta-actin promoter (CAG), polyoma virus enhancer/herpes simplex virus thymidine kinase promoter (MC1), beta-actin promoter (β-AST), simian virus promoter 40 (SV40) and a promoter containing the myeloproliferative sarcoma virus enhancer with the negative control region removed and replaced with the d1587rev primer binding site (MND promoter).

Various embodiments provide an immune effector cell containing a vector of the present invention. In various embodiments, said immune effector cell is transduced with a vector of the present invention.

In further embodiments, said immune effector cell is selected from the group consisting of: a T lymphocyte and a natural killer (NK) cell.

In some embodiments, said immune effector cell is transduced with a vector according to any one of claims 28-46, and is activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining the proliferation of the transduced immune effector cells compared to the proliferation of transduced immune effector cells that have been

- 3 045264 titrated and stimulated in the absence of the specified P13K pathway inhibitor.

In specific embodiments, said immune effector cell, activated and stimulated in the presence of said PI3K pathway inhibitor, is characterized by increased expression of: i) one or more markers selected from the group consisting of: CD62L, CD 127, CD197 and CD38 or ii) all listed markers: CD62L, CD127, CD197 and CD38, compared with an immune effector cell activated and stimulated in the absence of the specified P13K pathway inhibitor.

In one embodiment, said PI3K inhibitor is ZSTK474.

According to various embodiments, a composition is provided comprising an immune effector cell provided by the present invention and a physiologically acceptable excipient.

According to various embodiments, there is provided a method of producing an immune effector cell containing a CAR provided by the present invention, comprising introducing into the immune effector cell a vector containing a polynucleotide encoding the specified CAR.

In further embodiments, the method further comprises stimulating said immune effector cell and inducing proliferation of said cell by contacting said cell with antibodies that bind to CD3 and antibodies that bind to CD28; thereby obtaining a population of immune effector cells.

In specific embodiments, said immune effector cell is stimulated and induced to proliferate prior to administration of said vector.

In some embodiments, said immune effector cells include T lymphocytes.

In specific embodiments, said immune effector cells include NK cells.

In specific embodiments, said cells are activated and stimulated in the presence of a PI3K pathway inhibitor, thereby maintaining the proliferation of transduced immune effector cells relative to the proliferation of immune effector cells that are activated and stimulated in the absence of said PI3K pathway inhibitor.

In some embodiments, said immune effector cells, activated and stimulated in the presence of said P13K pathway inhibitor, have increased expression of: i) one or more markers selected from the group consisting of: CD62L, CD127, CD197, and CD38, or ii) all listed markers: CD62L, CD127, CD197 and CD38, compared with immune effector cells activated and stimulated in the absence of the specified PI3K pathway inhibitor.

In one embodiment, said PI3K inhibitor is ZSTK474.

In various embodiments, there is provided a method of treating a B cell-related condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising anti-BCMA CAR-bearing T cells of the present invention and, optionally, a pharmaceutically acceptable excipient.

In other embodiments, the B cell-associated condition is multiple myeloma, non-Hodgkin's lymphoma, B cell proliferation of undetermined malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas' disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's syndrome, Kawasaki disease, autoimmune hemolytic anemia and rapidly progressive glomerulonephritis, heavy chain disease, primary or immunocyte-associated amyloidosis, or monoclonal gammopathy of undetermined significance.

In further embodiments, the B cell-related condition is a B cell malignancy.

In some embodiments, the B cell malignancy is multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL).

In some embodiments, the MM is selected from the group consisting of: overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

In some embodiments, the NHL is selected from the group consisting of: Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell leukemia and mantle cell lymphoma.

In specific embodiments, the B cell-related condition is a plasma cell malignancy.

In further embodiments, the B cell-related condition is an autoimmune disease.

In additional embodiments, said autoimmune disease is systemic lupus erythematosus.

In some embodiments, the B cell-related condition is rheumatoid arthritis.

In specific embodiments, said B cell-related condition is idiopathic thrombocytopenic purpura, or myasthenia gravis, or autoimmune hemolytic anemia.

Brief description of graphic materials

In fig. 1 shows a schematic representation of CAR constructs directed against mouse B-cell maturation antigen (muBCMA).

In fig. 2a shows the amount of IFN-γ released by anti-BCMA02 CAR-bearing T cells, anti-BCMA10 CAR-bearing T cells, and CAR19A-bearing T cells after coculture of these cells for 24 hours with BCMA-expressing K562 cells.

In fig. 2b compares the amount of IFN-γ released by BCMA02 CAR-bearing T cells, BCMA10 CAR-bearing T cells, and CAR19A-bearing T cells after coculture of these cells for 24 hours with non-BCMA expressing K562 cells and with cells K562 expressing BCMA.

In fig. Figure 3 shows the amount of inflammatory cytokines in the growth media of untransduced control T cells, CAR-bearing BCMA02 T cells, CAR-bearing BCMA10 T cells, and CAR19A-bearing T cells stimulated 10 days before analysis.

In fig. Figure 4 shows the amount of inflammatory cytokines produced by BCMA02 CAR-bearing T cells, BCMA10 CAR-bearing T cells, and CAR19A-bearing T cells in the absence of antigenic stimulation.

In fig. Figure 5 shows the expression of phenotypic activation markers at the end of the process of generating anti-BCMA CAR-bearing T cells. HLA-DR and CD25 expression were measured in BCMA02 CAR-bearing T cells, BCMA10 CAR-bearing T cells, and CAR19A-bearing T cells.

In fig. Figure 6 shows the levels of activation of caspase-3, a necessary step for apoptosis, which is of great importance for AICD in CAR-BCMA10 and CAR-BCMA02-bearing T cells in the absence of antigenic stimulation.

In fig. Figure 7 shows the amount of inflammatory cytokines released in anti-BCMA02 CAR and anti-BCMA10 CAR T cells in media containing fetal bovine serum (FBS), human AB antigen serum (HABS), or 100 ng/ml soluble BCMA.

In fig. 8A shows the volume of tumors in NOD-scid-gamma (NSG) mice with experimental human subcutaneous multiple myeloma tumors (RPMI-8226) measuring ~100 mm ³ . Mice received vehicle, 10 ⁷ CAR-carrying anti-BCMA02 T cells, 10 ⁷ CAR-carrying anti-BCMA10 T cells, or bortezomib (Velcade).

In fig. 8B shows tumor volume in NOD-scid-gamma (NSG) mice bearing experimental subcutaneous human multiple myeloma tumors (RPMI-8226) measuring ~100 mm ³ . Mice received vehicle, 10 ⁷ CAR-carrying anti-BCMA02 T cells, 10 ⁷ CAR-carrying anti-BCMA10 T cells, or bortezomib (Velcade).

In fig. Figure 9 shows the level of BCMA expression on lymphoma and leukemia cell lines (circles) and the activity of BCMA CAR-carrying T cells in relation to each cell line (IFN-γ release, squares). BCMA-negative (BCMA-) tumor cell lines: myelogenous leukemia (K562), acute lymphoblastic leukemia (NALM-6 and NALM-16); mantle cell lymphoma (REC-1); or Hodgkin's lymphoma (HDLM-2) showed little or no release of IFN-γ. BCMA-positive (BCMA+) tumor cell lines: B-cell chronic lymphoblastic leukemia (MEC-1), mantle cell lymphoma (JeKo-1), Hodgkin's lymphoma (RPMI-6666), Burkitt's lymphoma (Daudi cells and Ramos cells) and multiple myelomas (RPMI8226) showed significant IFN-γ release.

In fig. 10A shows the in vivo activity of a backbone of anti-CD19A CAR-bearing T cells, anti-CD19 CAR-bearing T cells, and anti-BCMA CAR-bearing T cells against BCMA-expressing Burkitt's lymphoma cells (Daudi cells) in the NSG mouse model when administered to mice carrying CAR T cells 8 days after tumor induction.

In fig. 10B shows the in vivo activity of a backbone of anti-CD19A CAR T cells, anti-CD19 CAR T cells, and anti-BCMA CAR T T cells against BCMA-expressing Burkitt's lymphoma cells (Daudi cells) in the NSG mouse model when administered to mice. bearing CAR T cells 18 days after tumor induction.

In fig. Figure 11 shows the pronounced in vitro activity of anti-BCMA CAR-bearing T cells, observed with a 50% decrease in anti-BCMA CAR expression. (A) T cell populations were transduced with 4x108-5x107 transducing units of lentivirus encoding the anti-BCMA CAR molecule (multiplicity of infection (MOI): 5-40). The resulting T cell populations were normalized to contain 26±4% CAR anti-BCMA positive T cells. (B) MFI of normalized CAR-bearing anti-BCMA T cells ranged from 885 to 1875 as assessed using flow cytometric analysis. (C) For K562 cells and K562-BCMA cells when co-cultured with normalized CAR-carrying anti-BCMA T cells and the ratio of effector (E; T cells) to target (T; a mixture of K562 and K562 BCMA cells in a 1:1 ratio) cells at a ratio of 20:1 or 10:1, comparable cytolytic activity was observed.

Fig. 12 illustrates the robustness of the process for generating anti-BCMA CAR-bearing T cells. (A) Anti-BCMA CAR T cell products derived from PBMCs from 11 individual donors exhibited levels of expansion comparable to the corresponding culture of untransduced donor T cells. (B) Anti-BCMA CAR T cell products derived from 11 donors demonstrated comparable lentiviral transduction (VCN) efficiencies. (C) The abundance of anti-BCMA CAR positive T cells was measured by flow cytometry; all donors showed comparable expression of BCMA. (D) Anti-BCMA CAR T cell products derived from 11 donors exhibited therapeutically relevant levels of IFN-γ release when exposed to BCMA-expressing K562 cells.

In fig. 13 shows Venn diagrams depicting the co-expression of CD127, CD197 and CD38 in CD62L positive anti-BCMA02 T cells cultured in the presence of IL-2, or IL-2 and ZSTK474 for ten days. ZSTK474-treated cultures of anti-BCMA02 CAR T cells showed an increase in the percentage of cells coexpressing CD127, CD197, and CD38 compared with cultures of anti-BCMA02 CAR T cells cultured with IL-2 alone.

In fig. 14 shows an increase in the percentage of CD8 expressing anti-BCMA02 CAR T cells in cultures treated with IL-2 and ZSTK474 (n=7) compared to cultures treated with IL-2 alone. CD8 expression was determined using fluorescently labeled anti-CD8 antibody and flow cytometry.

In fig. 15 shows the amount of IFN-γ released by BCMA02 CAR-bearing T cells from 14 donors after culture with IL-2 alone, or with IL-2 and ZSTK474. At the end of the culture period, an equivalent number of anti-BCMA02 CAR-bearing T cells were recultured for 24 hours in unsupplemented medium. The amount of IFN-γ released over 24 hours was quantified using ELISA. Culture in ZSTK474 did not significantly increase the release of tonic cytokines by anti-BCMA02 CAR T cells compared with anti-BCMA02 CAR T cells cultured with IL-2 alone.

In fig. 16 shows the antitumor activity of anti-BCMA02 CAR-bearing T cells treated with IL-2, or IL-2 and ZSTK474, or carrying truncated anti-BCMA02 CAR (tBCMA02) T cells treated with IL-2 and ZSTK474 that do not provide full signaling in the model aggressive Daudi tumor. Complete regression of tumors was observed in 50% of mice injected with BCMA02 CAR-carrying T cells treated with IL-2 and ZSTK474.

In fig. 17 shows the antitumor activity of BCMA02 CAR-bearing T cells treated with IL-2, or IL-2 and ZSTK474, in a tumor model of multiple myeloma (RPMI-8226). In animals treated with IL-2-cultured, or IL-2- and ZSTK474-carrying CAR BCMA02 T cells, tumor proliferation was completely prevented.

Brief Description of Sequence Identifiers

SEQ ID NOs: 1-3 provide example amino acid sequences of the light chain CDR regions of the BCMA CAR receptors provided by the present invention.

SEQ ID NOs: 4-6 provide example amino acid sequences of the heavy chain CDR regions of the BCMA CAR receptors provided by the present invention.

SEQ ID NO: 7 provides examples of the amino acid sequence of the light chains of the BCMA CAR receptors provided by the present invention.

SEQ ID NO: 8 provides examples of heavy chain amino acid sequences of the BCMA CAR receptors provided by the present invention.

SEQ ID NO: 9 provides example amino acid sequences of the CAR to BCMA provided by the present invention.

SEQ ID NO: 10 provides polynucleotide sequences that encode examples of the BCMA CARs provided by the present invention.

SEQ ID NO: 11 provides the amino acid sequence of human BCMA.

SEQ ID NO: 12-22 shows the amino acid sequence of the various linkers.

SEQ ID NO: 23-35 provides the amino acid sequence of protease cleavage sites and cleavage sites of self-cleaving polypeptides.

- 6 045264

SEQ ID NO: 36 shows the polynucleotide sequence of the vector encoding CAR to

BCMA.

Detailed Description of the Invention

A. General information

The present invention relates generally to improved compositions and methods for treating B cell-related conditions. As used herein, the term B cell related conditions refers to conditions involving inappropriate B cell activity and B cell malignancies.

In specific embodiments, the present invention relates to improved adoptive cell therapy for B cell-related conditions using genetically modified immune effector cells. Genetic techniques can potentially be used to improve immune recognition and elimination of cancer cells. One promising strategy is to genetically engineer immune effector cells to express hybrid antigen receptors (CARs) that redirect cytotoxicity to cancer cells. However, current adoptive cell immunotherapy options for the treatment of B-cell disorders carry a significant risk of impairment of humoral immunity, since these cells target antigens expressed on all or most B cells. Thus, such therapy options are not clinically desirable and, accordingly, there remains a need in the art for more effective, humoral immune-friendly therapy options for B cell-related conditions.

The improved compositions and methods for adoptive cell therapy as described herein provide genetically modified immune effector cells that can be easily expanded, exhibit long-term persistence in vivo, and are less detrimental to humoral immunity by targeting B cells expressing maturation antigen B cells (BCMA, also known as CD269, or tumor necrosis factor superfamily member 17; TNFRSF17).

BCMA is a member of the tumor necrosis factor superfamily (see, for example, Thompson et al., J. Exp. Medicine, 192(1): 129-135, 2000, and Mackay et al., Annu. Rev. Immunol, 21: 231264, 2003. BCMA binds B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204: 43-54, 2005) BCMA expression has been described in non-malignant cells, mainly in plasma cells and subsets of mature B cells (see, for example, Laabi et al., EMBO J., 77(1): 3897-3904, 1992; Laabi et al. , Nucleic Acids Res., 22(7): 1147-1154, 1994; Kalled et al., 2005; O'Connor et al., J. Exp. Medicine, 199(1): 91-97, 2004; and Ng et al., J. Immunol., 73(2): 807-817, 2004. BCMA-deficient mice are healthy and contain normal numbers of B cells, but the survival of long-lived plasma cells is impaired (see, for example, O'Connor et al., 2004; Xu et al., Mol. Cell. Biol, 21(12): 4067-4074, 2001; and Schiemann et al., Science, 293(5537): 2111-2114, 2001). BCMA RNA has been found ubiquitously in cells of multiple myeloma and other lymphomas, and BCMA protein has been detected by a number of researchers on the surface of plasma cells of multiple myeloma patients (see, for example, Novak et al., Blood, 103(2): 689-694, 2004; Neri et al., Clinical Cancer Research, 73(19): 5903-5909, 2007; Bellucci et al., Blood, 105(10): 3945-3950, 2005; and Moreaux et al., Blood, 703( 8): 3148-3157, 2004.

In various embodiments, CARs containing a mouse antibody against BCMA sequences are highly potent compared to anti-BCMA CARs containing specific human antibody sequences; demonstrate robust reproduction in vivo; recognize human B cells expressing BMCA; demonstrate cytotoxic activity against BCMA-expressing B cells; and do not show signs of inducing cytokine storm, a potentially lethal condition in which cytokines released by activated T cells cause an acute systemic inflammatory response that initiates a non-infectious fever.

In one embodiment, a CAR is provided comprising a mouse anti-BCMA antibody or an antigen-binding fragment thereof, a transmembrane domain, and one or more intracellular signaling domains.

In one embodiment, an immune effector cell is provided that is genetically modified to express a CAR of the present invention. T cells expressing CAR are referred to herein as CAR T cells or CAR-modified T cells.

In various embodiments, the genetically modified immune effector cells of the present invention are administered to a patient suffering from a B cell-related condition, such as a B cell-related autoimmune disease or a B cell malignancy.

In the practice of the present invention, unless specifically stated otherwise, standard methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, corresponding to the state of the art, many of which are described, are used. below for illustrative purposes. Such techniques are described in detail in published sources. See, for example, the manuals: Sambrook, et al.. Molecular

Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A

Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory

Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and

Sons, updated. dated July 2008); Short Protocols in Molecular Biology: A Compendium of

Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and WileyInterscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford,

1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New

York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A

Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology

Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991);

Annual Review of Immunology · as well as monographs in journals such as Advances in Immunology.

B. Definitions

Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly known to those skilled in the art to which the present invention relates. Although any methods and materials similar or equivalent to those presented herein may be used in the practice or testing of the present invention, preferred embodiments of the compositions, methods and materials are described herein. The following are definitions of terms used for the purposes of the present invention.

Singular forms, including those preceded by the definition specified(s), are used herein to denote one or more than one (i.e., at least one, or one or more) grammatical units of the specified object. For example, element means one element, or one or more elements.

The indication of an alternative (for example, the use of or) should be understood as indicating one, both, or any combination of the specified alternatives.

The term and/or should be understood to mean one or both of these alternatives.

As used herein, the term approximately, or about, refers to an amount, level, magnitude, number, frequency, percentage, dimension, size, value, mass or length varying up to 15%, 10%, 9%, 8%, 7 %, 6%, 5%, 4%, 3%, 2% or 1% relative to the reference amount, level, magnitude, number, frequency, percentage, dimension, size, value, mass or length. In one embodiment, the term approximately, or about, refers to a range of amount, level, magnitude, number, frequency, percentage, dimension, size, value, mass, or length corresponding to ±15%, ±10%, ±9%, ±8% ,±7%,±6%,±5%,±4%,±3%,±2% or±1% relative to the reference quantity, level, magnitude, number, frequency, percentage, dimension, size, value, mass or length.

Throughout this specification, unless the context otherwise requires, the terms include, contains, and comprising are intended to include the designated step or element, or group of steps or elements, without excluding any other step or element, or group of steps or elements. By consisting of is meant inclusion limited to the elements listed after the expression consisting of. Accordingly, the expression consisting of indicates that the listed elements are necessary or mandatory, and other elements cannot be present. By essentially consisting of is meant the inclusion of any of the elements listed after the specified expression, and the limitation of other elements that do not affect or contribute to the activity or action described herein for the listed elements. Accordingly, the expression consisting essentially of indicates that the listed elements are necessary or mandatory, but there are no other elements capable of substantially affecting the activity or effect of the listed elements.

Throughout this specification, reference to one embodiment, embodiment, specific embodiment, related embodiment, specific embodiment, additional embodiment, or further embodiment, or combinations thereof, means that the particular feature, structure, or characteristic described in connection with the specified embodiment is included in at least one embodiment of the present invention. Accordingly, the above expressions used elsewhere in the text of this specification do not necessarily always refer to the same embodiment. In addition, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It should also be understood that express disclosure of a feature in one embodiment provides a basis for excluding said feature in a particular embodiment.

C. Chimeric antigen receptors

Various embodiments provide genetically engineered receptors that redirect the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). CARs are molecules that combine the specificity of an antibody for a desired antigen (eg, BCMA) with a T-cell receptor-activating intracellular domain, in the form of a fusion protein, that exhibits specific cellular immune activity directed against BCMA. As used herein, the term hybrid or chimeric refers to molecules consisting of parts of different proteins or DNA of different origins.

The CARs of the present invention contain an extracellular domain (also called a binding domain or antigen-specific binding domain) that binds to BCMA, a transmembrane domain, and an intracellular signaling domain. Activation of the anti-BCMA antigen-binding domain of a CAR by BCMA on the surface of a target cell results in CAR aggregation and delivery of the activating stimulus to the CAR-containing cell. A major characteristic of CAR receptors is their ability to redirect the specificity of immune effector cells, thereby triggering proliferation, cytokine production, phagocytosis, or the synthesis of molecules that can mediate cell death of a target antigen-expressing cell in a major histocompatibility complex (MHC)-independent manner, involving the ability to cell-specific targeting of monoclonal antibodies, soluble ligands or cell-specific coreceptors.

In various embodiments, the CAR comprises an extracellular binding domain that comprises a mouse anti-BCMA antibody specific binding domain; transmembrane domain; one or more intracellular costimulatory signaling domains; and primary signaling domain.

In specific embodiments, the CAR comprises an extracellular binding domain that comprises a mouse anti-BCMA antibody or an antigen-binding fragment thereof; one or more hinge domains or spacer domains; a transmembrane domain including one or more intracellular co-stimulatory signaling domains; and primary signaling domain.

1. Binding domain

In specific embodiments, the CARs of the present invention comprise an extracellular binding domain that includes a mouse anti-BCMA antibody or an antigen-binding fragment thereof that specifically binds to a human BCMA polypeptide expressed on a B cell. As used herein, the terms binding domain, extracellular domain, extracellular binding domain, antigen-specific binding domain, and extracellular antigen-specific binding domain are used interchangeably; this domain provides the ability of the CAR to specifically bind to a target antigen of interest, for example, BCMA. Said binding domain comes from a natural, synthetic, semi-synthetic or recombinant source.

The terms specific binding affinity, or specifically binds, or specifically bound, or specific binding, or specifically targets as used herein describe the binding of an anti-BCMA antibody or an antigen-binding fragment thereof (or a CAR containing it) to BCMA with a higher binding affinity. than background binding. A binding domain (either a CAR binding domain-containing fusion protein or a binding domain-containing fusion protein) specifically binds to BCMA if it binds or binds to BCMA with affinity or a _{K a} constant (i.e., the equilibrium binding constant for a particular binding interaction , expressed in units of 1/M), for example greater than or equal to approximately 10 ⁵ M ^-1 . In some embodiments, the binding domain (or a fusion protein containing it) binds to the target with a Ka constant greater than or equal to about 10 ⁶ M' ¹ , 10 ⁷ M' ¹ , 10 ⁸ M' ¹ , 10 ⁹ M' ¹ , 10 ¹⁰ M' ¹ , 10 ¹¹ M' ¹ , 10 ¹² M ^-1 or 10 ¹³ M' ¹ . The term high-affinity binding domains (or single-chain fusion proteins containing them) refers to binding domains characterized by _a K a constant of at least 10 ⁷ M' ¹ , at least 10 ⁸ M' ¹ , at least 10 ⁹ M' ¹ , at least 10 ¹⁰ M' ¹ , at least 10 ¹¹ M' ¹ , at least 10 ¹² M' ¹ , at least 10 ¹³ M ^-1 or more.

Alternatively, affinity can be defined as the equilibrium dissociation constant (Kd) of a particular binding interaction in units of M (eg, 10 ^-5 M to 10 ^-13 M, or less). Affinity of polypeptide binding domains and CAR proteins according to the present invention

- 9 045264 em can be easily determined using standard techniques, for example, a competitive ELISA (enzyme-linked immunosorbent assay), or through a binding assay, or a displacement assay using labeled ligands, or using a surface plasmon resonance device such as the Biacore T100, offered by Biacore, Inc., Piscataway, New Jersey, or optical biosensor technology, for example, the EPIC or EnSpire systems offered by Corning and Perkin Elmer, respectively (see also, for example, Scatchard et al. (1949) Ann. N.Y. Acad. Sci. 51:660; US Pat. No. 5283173; No. 5468614, and equivalents).

In one embodiment, the specific binding affinity is about 2 times background binding, about 5 times background binding, about 10 times background binding, about 20 times background binding, about 50 times background binding, about 100 times the background binding, or approximately 1000 times or more the background binding.

In certain embodiments, the extracellular binding domain of the CAR comprises an antibody or an antigen binding fragment thereof. The term antibody refers to a binding agent that is a polypeptide containing at least an immunoglobulin light chain or heavy chain variable region that specifically recognizes and binds an epitope of an antigen, such as a peptide, lipid, polysaccharide or nucleic acid, containing an antigenic determinant, such as a recognized immune cell.

Antigen (Ag) refers to a compound, composition or substance that can stimulate antibody synthesis or a T cell response in an animal, including compositions (for example, a composition comprising a specific cancer-associated protein) injectable or penetrating the animal's body. The antigen reacts with products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein. In specific embodiments, the target antigen is an epitope of a BCMA polypeptide.

The term epitope or antigenic determinant refers to the region of an antigen to which a binding agent binds. Epitopes can be formed by amino acids located either sequentially or non-sequentially, the proximity of which is ensured by the tertiary folding of the protein. Epitopes formed by sequential amino acids are typically retained when exposed to denaturing solvents, whereas epitopes formed by tertiary stacking are typically lost when exposed to denaturing solvents. An epitope typically comprises at least 3, usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

Antibodies include antigen-binding fragments such as camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain Fv proteins (scFv), bi- scFv, (scFv) ₂ , minibodies, diabodies, tribodies, tetrabodies, disulfide-stabilized Fv proteins (dsFv), single-domain antibodies (sdAb, nanobody) and fragments of full-length antibodies responsible for binding antigens. The term also includes genetically engineered forms such as hybrid antibodies (eg, humanized mouse antibodies), heteroconjugate antibodies (eg, bispecific antibodies) and antigen binding fragments thereof. See also: Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL; Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

As will be appreciated by one skilled in the art and as described herein, a complete antibody contains two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second and third constant regions, and each heavy chain consists of a variable region and a constant region. Mammalian heavy chains are classified into α, δ, ε, γ and μ chains. Mammalian light chains are classified into λ or k. Immunoglobulins containing heavy chains α, δ, ε, γ and μ are divided into immunoglobulin classes (Ig)A, IgD, IgE, IgG and IgM. A complete antibody is Y-shaped. The Y base consists of the second and third constant regions (and, in the case of IgE and IgM, the fourth constant region) of two heavy chains connected to each other; Disulfide bonds (interchain) are formed in the hinge. The γ, α, and δ heavy chains contain a constant region consisting of three tandem (located in a straight line) Ig domains and a hinge region that provides additional flexibility; the μ and ε heavy chains contain a constant region consisting of four immunoglobulin domains. The second and third constant regions are called the CH2 domain and the CH3 domain, respectively. Each Y arm includes a variable region and a first constant region of one heavy chain linked to variable and constant regions of one light chain. The variable regions of the light and heavy chains are responsible for binding antigens.

The variable regions of the light and heavy chains contain a framework region interspersed by three hypervariable regions, also called complementarity determining regions, or CDR regions. CDR regions can be defined or identified using

- 10 045264 we use standard methods, for example, based on the sequence according to the system of Kabat et al (Wu, T.T. and Kabat, E. A., J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat E. A., PNAS, 84: 2440-2443 (1987); (See Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991, incorporated herein by reference), or based on structure according to the system of Chothia et al. 883 (1989)).

The framework sequences of different light or heavy chains are relatively conserved within a single species, such as humans. The framework region of an antibody, that is, the combination of the framework regions of the light and heavy chains that make up the antibody, serves to position and align the CDR regions in three-dimensional space. The CDR regions are primarily responsible for binding to the epitope of the antigen. The CDR regions of each chain are generally referred to as CDR1, CDR2, and CDR3; numbering starts sequentially from the N-end; for a specific CDR region, as a rule, they also indicate the chain in which it is localized. Accordingly, CDR regions located in the variable domain of an antibody heavy chain are referred to as CDRH1, CDRH2, and CDRH3, and CDR regions located in the variable domain of an antibody light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., containing different combining sites for different antigens) contain different CDRs. Although antibodies differ in their CDR regions, only a limited number of amino acid positions within the CDR regions are directly involved in antigen binding. These positions within the CDR regions are called specificity determining residues (SDRs). Illustrative examples of light chain CDR regions that are suitable for the construction of humanized BCMA CARs of the present invention include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 1-3. Illustrative examples of heavy chain CDR regions that are suitable for the design of humanized BCMA CARs of the present invention include, but are not limited to, the CDR sequences set forth in SEQ ID NOs: 4-6.

The terms VH or VH refer to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, dsFv, Fab or other antibody fragment as described herein. The terms VL or VL refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as described herein.

A monoclonal antibody is an antibody produced by a single clone of B lymphocytes or a cell that has been transfected with the genes for the light and heavy chains of a single antibody. Monoclonal antibodies are produced using methods known to those skilled in the art, for example, by producing hybrid antibody-producing cells by fusing myeloma cells with spleen immune cells. Monoclonal antibodies include humanized monoclonal antibodies.

A fusion antibody contains framework residues of one species, such as human, and CDR regions (which typically mediate antigen binding) of another species, such as mouse. In specific preferred embodiments, the CAR of the present invention comprises an antigen-specific binding domain that is a fusion antibody or an antigen-binding fragment thereof.

A humanized antibody is an immunoglobulin comprising a human framework region and one or more CDRs of a non-human immunoglobulin (eg, mouse, rat, or synthetic immunoglobulin). The non-human immunoglobulin that is the source of the CDR regions is called a donor, and the human immunoglobulin that is the source of the framework is called an acceptor.

In specific embodiments, the mouse anti-BCMA antibody or antigen-binding fragment thereof includes, but is not limited to, camelid Ig (camelid (VHH) antibody), Ig NAR, Fab fragments, Fab' fragments, F(ab)' ₂ fragments, F (ab)'3-fragments, Fv, single chain Fv antibody (scFv), bis-scFv, (scFv)2, minibody, diabody, tribody, tetrabody, disulfide stabilized Fv protein (dsFv) and single domain antibody (sdAb, nanobody ).

Camelid Ig or camelid VHH as used herein refers to the smallest known heavy chain antibody antigen binding unit (Koch-Nolte, et al., FASEB J., 21: 34903498 (2007)). The term heavy chain antibody or camelid antibody refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94/04678; WO94/ 25591; US Patent No. 6005079).

IgNAR, immunoglobulin novel antigen receptor, belongs to a class of antibodies in the shark immune repertoire that consists of homodimers of one novel antigen receptor variable domain (VNAR) and five novel antigen receptor constant domains (CNARs). IgNAR receptors are among the smallest known immunoglobulin-based protein scaffolds, are highly stable and provide efficient binding. Their inherent stability can be explained by (i) a supporting Ig framework containing a significant amount of charged and

- 11,045,264 hydrophilic surface residues compared to standard VH and VL domains from mouse antibodies; and (ii) stabilization of the structural characteristics of the complementarity determining region (CDR) loops, including inter-loop disulfide bridges and intra-loop hydrogen bonding patterns.

When papain cleaves antibodies, two identical antigen-binding fragments are formed, called Fab fragments, each of which contains one antigen binding site, and a residual Fc fragment (fragment crystallizable), the name of which reflects its ability to readily crystallize. When treated with pepsin, an F(ab')2 fragment is formed, which contains two antigen combining sites and is also capable of cross-linking antigens.

Fv is the minimal antibody fragment that contains the complete antigen binding site. In one embodiment, the double-chain Fv variant consists of a dimer comprising one heavy chain variable domain and one light chain variable domain tightly linked by a non-covalent bond. In single-chain Fv species (scFv), one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can join to form a dimeric structure similar to that in double-chain Fv species. It is in this configuration that the interaction of the three hypervariable regions (HVRs) of each variable domain defines the antigen binding site on the surface of the VH-VL dimer. Collectively, the six HVRs provide the antigen-binding specificity of the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigen, although with less affinity than the full-length binding site.

The Fab fragment contains the variable domains of the heavy and light chains, and also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments are distinguished from Fab fragments by the presence of several additional residues at the carboxyl terminus of the heavy chain of the CH1 domain, including one or more antibody hinge cysteines. As used herein, Fab'-SH corresponds to Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab)2 antibody fragments were initially obtained in the form of pairs of Fab fragments, between which hinge cysteines are located. Other types of chemical binding of antibody fragments are also known.

The term diabodies refers to antibody fragments with two antigen binding sites containing a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in a single polypeptide chain (VH-VL). By using a linker that is too short to pair two domains on the same strand, the domains are forced to pair with complementary domains on the other strand to form two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are described in more detail, for example, in EP 404097; WO 1993/01161; in: Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

A single domain antibody, or sdAb, or nanobody refers to an antibody fragment that consists of an antibody heavy chain variable region (VH domain) or an antibody light chain variable region (VL domain) (Holt, L., et al., Trends in Biotechnology, 21 (11): 484-490).

Single-chain Fv or scFv antibody fragments contain antibody domains VH and VL, with these domains located on the same polypeptide chain in any orientation (eg, VLVH or VH-VL). Typically, the scFv polypeptide additionally contains a polypeptide linker between the VH and VL domains, which ensures the formation of the scFv structure required for antigen binding. General information about scFv can be found, for example, in the source: Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

In preferred embodiments, the CAR of the present invention comprises an antigen-specific binding domain that is a mouse scFv. Single chain antibodies can be cloned from hybridoma V region genes that are specific for the desired target. Obtaining such hybridomas is currently a routine process. A technique suitable for cloning the heavy chain variable region (VH) and the light chain variable region (VL) has been described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.

In specific embodiments, the antigen-specific binding domain is a mouse scFv that binds a human BCMA polypeptide. Illustrative examples of heavy chain variable regions that are suitable for the construction of the BCMA CAR receptors of the present invention include, but are not limited to, the amino acid sequences shown in SEQ ID NO: 8. Illustrative examples of light chain variable regions that are suitable for the construction of receptors The anti-BCMA CARs of the present invention include, but are not limited to, the amino acid sequences set forth in SEQ ID NO: 7.

The BCMA-specific binding domains of the present invention further comprise one, two, three, four, five or six CDR regions. Such CDRs may be non-human CDRs or modified non-human CDRs selected from the light chain CDRL1, CDRL2 and CDRL3, and the heavy chain CDRH1, CDRH2 and CDRH3. In some embodiments, the BCMA-specific binding domain comprises (a) a light chain variable region that includes light chain CDRL1, light chain CDRL2, and light chain CDRL3, and (b) a heavy chain variable region that includes heavy chain CDRH1, heavy chain CDRH2 and CDRH3 heavy chain.

2. Linkers

In some embodiments, the CAR receptors of the present invention may contain linker residues between different domains, for example, added to provide proper spatial separation and conformation of the molecule. In certain embodiments, said linker is a variable region connecting sequence. The variable region joining sequence is the amino acid sequence connecting the V _H and V L domains and performs a spacer function consistent with the interaction of the two binding subdomains such that the resulting polypeptide retains the specific binding affinity for the same target molecule as the antibody that contains the same variable regions of light and heavy chains. A CAR of the present invention may contain one, two, three, four, or five or more linkers. In specific embodiments, the linker length is from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any number of amino acids in between. In some embodiments, the length of said linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

Illustrative examples of linkers include glycine (G) _n polymers; glycine-serine polymers (G ₁ . ₅ S ₁ .5)n, where n is an integer and is at least 1, 2, 3, 4 or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and accordingly may be able to function as a neutral link between the domains of fusion proteins, such as the CAR receptors described herein. The values of Ф and Ψ for glycine are significantly higher even than those for alanine; glycine is much less sterically restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). One of ordinary skill in the art will appreciate that the CAR design of particular embodiments may include fully or partially flexible linkers, such that said linker may comprise a flexible linker as well as one or more moieties that reduce the flexibility of the structure to obtain the desired CAR structure .

Other examples of linkers include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 12); TGEKP (SEQ ID NO: 13) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 14) (Pomerantz et al. 1995, supra); (GGGGS)n, where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 15) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 16) ( Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 17) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 18); LRQRDGERP (SEQ ID NO: 19); LRQKDGGGSERP (SEQ ID NO: 20); LRQKD(GGGS) ₂ ERP (SEQ ID NO: 21). Alternatively, flexible linkers can be prepared using rational design in a computer program that allows you to model both DNA binding sites and the peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or using phage display methods. In one embodiment The implementation of the linker contains the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 22) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

3. Spacer domain

In certain embodiments, downstream of the CAR binding domain are one or more spacer domains, that is, regions that separate the antigen binding domain from the surface of the effector cell to ensure proper cell-cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6:412 -419). The hinge domain may be from a natural, synthetic, semi-synthetic or recombinant source. In some 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 include the amino acid sequence of the hinge region of a naturally occurring or modified immunoglobulin hinge region.

In one embodiment, the spacer domain comprises the CH2 and CH3 domains of an IgG1 or IgG4 immunoglobulin.

4. Hinge domain

The CAR binding domain is typically followed by one or more hinge domains that participate in the spatial separation of the antigen binding domain from the effector surface.

- 13 045264 cells to ensure proper cell-to-cell contact, antigen binding and activation. A CAR typically contains one or more hinge domains between the binding domain and the transmembrane (TM) domain. The hinge domain comes from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring or modified immunoglobulin hinge region.

The term altered hinge region refers to (a) a naturally occurring hinge region in which up to 30% of the amino acids are changed (e.g., up to 25%, 20%, 15%, 10%, or 5% of the amino acids are replaced or deleted), (b) parts a naturally occurring hinge region containing at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) in which up to 30% of the amino acids are changed (e.g., up to 25%, 20%, 15% are replaced or deleted , 10% or 5% amino acids), or (c) a portion of a naturally occurring hinge region containing a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 in length or 15, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids). In some embodiments, one or more cysteine residues in the naturally occurring immunoglobulin hinge region may be replaced by one or more other amino acid residues (eg, one or more serine residues). The altered immunoglobulin hinge region may alternatively or additionally contain a different amino acid residue (eg, a serine residue) instead of the proline residue of the wild-type immunoglobulin hinge region.

Other exemplary hinge domains suitable for use in CARs as described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins, such as CD8a, CD4, CD28, and CD7, which may be hinge regions of these wild-type molecules, or may be changed. In another embodiment, the hinge domain comprises a CD8a hinge region.

5. Transmembrane (TM) domain

The transmembrane domain is the portion of the CAR receptor that connects the extracellular binding portion and the intracellular signaling domain, and anchors the CAR to the plasma membrane of the immune effector cell. The TM domain is derived from a natural, synthetic, semi-synthetic or recombinant source. The TM domain may be derived from (i.e., contain at least transmembrane region(s)) T cell receptor alpha, beta, or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154 and PD1. In a particular embodiment, the TM domain is synthetic and contains primarily hydrophobic residues such as leucine and valine.

In one embodiment, the CARs of the present invention comprise a CD8a-derived TM domain. In another embodiment, the CAR of the present invention comprises a CD8a-derived TM domain and a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, connecting the indicated TM domain and intracellular signaling domain of the CAR receptor. In particular, a linker based on glycine and serine is suitable.

6. Intracellular signaling domain

In certain embodiments, the CARs of the present invention comprise an intracellular signaling domain. The term intracellular signaling domain refers to the portion of the CAR receptor that is involved in transmitting the signal resulting from effective binding of the anti-BCMA CAR to the human BCMA polypeptide into an immune effector cell to stimulate effector cell function, such as activation, cytokine synthesis, proliferation, and cytotoxicity. activity, including the release of cytotoxic factors into the bound CAR target cell, or other cellular responses caused by the binding of antigen to the extracellular domain of the CAR.

The term effector function refers to the specialized function of an immune effector cell. The effector function of a T cell, for example, can be represented by cytolytic activity or helper activity, including the secretion of cytokines. Accordingly, the term intracellular signaling domain refers to the part of the protein that transmits the signal for effector function and directs the cell to perform a specialized function. Although, in general, a full-length intracellular signaling domain can be used, in many cases the use of a full-length domain is not necessary. In the case of using a truncated portion of an intracellular signaling domain, such a truncated portion may be used in place of the full-length domain provided that it is capable of transmitting a signal for effector function. The term intracellular signaling domain means any truncated portion of an intracellular signaling domain sufficient to transmit a signal for effector function.

It is known that signals generated through the TCR are not sufficient by themselves to fully activate a T cell, and a secondary or co-stimulatory signal is also required. Accordingly, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling

- 14 045264 domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (eg, the TCR/CD3 complex), and co-stimulatory signaling domains that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. In preferred embodiments, the CAR of the present invention comprises an intracellular signaling domain that comprises one or more co-stimulatory signaling domains and a primary signaling domain.

Primary signaling domains regulate the primary activation of the TCR complex either by stimulation or inhibition. Primary signaling domains that act by stimulation may contain signaling motifs that are known as immunoreceptor tyrosine activation motifs, or ITAMs.

Illustrative examples of ITAMs containing primary signaling domains particularly suitable for use in the present invention include ITAMs derived from TCRZ, FcRy, FcRe, CD3y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b and CD66d. In a particular preferred embodiment, the CAR comprises a primary CD3Z signaling domain, and one or more co-stimulatory signaling domains. Intracellular primary signaling and co-stimulatory signaling domains can be sequentially attached to the carboxyl terminus of the transmembrane domain in any order.

The CAR receptors of the present invention contain one or more co-stimulatory signaling domains to enhance the efficiency and expansion of T cells expressing the CAR receptors. As used herein, the term costimulatory signaling domain, or costimulatory 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 provide a second signal necessary for efficient T cell activation and function upon antigen binding. Illustrative examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70. In one embodiment, the CAR comprises one or more co-stimulatory signaling domains selected from the group consisting of the primary signaling domains CD28, CD137, and CD134 and CD3Z.

In another embodiment, the CAR comprises the co-stimulatory signaling domains CD28 and CD137, and the primary signaling domain CD3Z.

In yet another embodiment, the CAR comprises the co-stimulatory signaling domains CD28 and CD134, and the primary signaling domain CD3Z.

In one embodiment, the CAR comprises the costimulatory signaling domains CD137 and CD134, and the primary signaling domain CD3Z.

In specific embodiments, the CARs of the present invention comprise a mouse anti-BCMA antibody or an antigen-binding fragment thereof that specifically binds to a BCMA polypeptide expressed on B cells.

In one embodiment, the CAR comprises a scFv fragment of a mouse anti-BCMA antibody that binds a BCMA polypeptide; transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8a, CD9, CD 16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD 154 and PD1; and one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB ), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and the primary signaling domain of TCRZ, FcRy, FcRe, CD3y, CD3δ, CD3ε, CD3Z, CD22, CD79a, CD79b and CD66d.

In one embodiment, the CAR comprises a scFv fragment of a mouse anti-BCMA antibody that binds a BCMA polypeptide; a hinge domain selected from the group consisting of: an IgG1 CH2/CH3 hinge, an IgG4 CH2/CH3 hinge, and a CD8a hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154 and PD1; and one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB ), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM and ZAP70; and primary signaling domain from TCRZ, FcRy, FcRe, CD3y, CD3δ, CD3ε, CD3Z CD22, CD79a, CD79b and CD66d.

In one embodiment, the CAR comprises a scFv fragment of a mouse anti-antibody.

- 15 045264

BCMA, which binds BCMA polypeptide; a hinge domain selected from the group consisting of: an IgG1 CH2/CH3 hinge, an IgG4 CH2/CH3 hinge, and a CD8a hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of: T cell receptor alpha, beta or zeta chain, CD3ε, CD3Z, CD4, CD5, CD8a, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD 154 and PD1; a short oligopeptide or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length, connecting the TM domain to the CAR intracellular signaling domain; and one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB ), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and primary signaling domain from TCRZ, FcRy, FcRe, CD3y, CD3δ, CD3ε, CD3Z CD22, CD79a, CD79b and CD66d.

In a specific embodiment, the CAR comprises a scFv fragment of a mouse anti-BCMA antibody that binds a BCMA polypeptide; a hinge domain comprising an IgG1 CH2/CH3 hinge polypeptide and a CD8a polypeptide; a CD8a transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids in length; CD137 intracellular co-stimulatory signaling domain; and CD3Z primary signaling domain

In a specific embodiment, the CAR comprises a scFv fragment of a mouse anti-BCMA antibody that binds a BCMA polypeptide; a hinge domain comprising a CD8a polypeptide; a CD8a transmembrane domain containing a polypeptide linker that is from about 3 to about 10 amino acids in length; intracellular co-stimulatory signaling domain CD134; and the primary signaling domain of CD3Z.

In a specific embodiment, the CAR comprises a scFv fragment of a mouse anti-BCMA antibody that binds a BCMA polypeptide; a hinge domain comprising a CD8a polypeptide; a CD8a transmembrane domain containing a polypeptide linker that is from about 3 to about 10 amino acids in length; CD28 intracellular co-stimulatory signaling domain; and the primary signaling domain of CD3Z.

In addition, the design of the CARs provided by the present invention provides improved expansion, long-term persistence and satisfactory cytotoxic properties of T cells expressing these CAR receptors, compared to unmodified T cells or T cells modified to express other CAR receptors.

D. Polypeptides

The present invention includes, but is not limited to, CAR polypeptides and fragments thereof, cells and compositions containing them, and vectors expressing the polypeptides. In preferred embodiments, there is provided a polypeptide comprising one or more CARs as set forth in SEQ ID NO: 9.

The terms polypeptide, polypeptide fragment, peptide, and protein are used interchangeably unless otherwise noted and correspond to the standard meaning, i.e., amino acid sequence. Polypeptides are not limited to a particular length; for example, they may contain a full-length protein sequence or a fragment of a full-length protein, and may include polypeptides with post-translational modifications such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art , both naturally occurring and not occurring in nature. In various embodiments, the CAR polypeptides of the present invention contain a signal (or leader) sequence at the N-terminus of the protein that co-translationally or post-translationally directs the transfer of the protein. Illustrative examples of suitable signal sequences suitable for use in CAR receptors as described herein include, but are not limited to, an IgG1 heavy chain signal sequence and a CD8a signal sequence. Polypeptides can be produced using any of a variety of well-known recombinant and/or synthetic techniques. Polypeptides provided by the present invention specifically include CAR receptors as described herein, or sequences comprising deletions, additions and/or substitutions of one or more CAR receptor amino acids as described herein.

The terms isolated peptide or isolated polypeptide, etc. as used herein, refers to a peptide or polypeptide molecule isolated and/or purified in vitro from the cellular environment and lacking association with other cellular components, i.e., not significantly associated with substances in vivo. Likewise, an isolated cell refers to cells that have been obtained from a tissue or organ in vivo and are substantially free of extracellular matrix.

Polypeptides include polypeptide variants. Variant polypeptides may differ from the naturally occurring polypeptide by one or more substitutions, deletions, additions and/or insertions. Such variants may occur naturally or may be produced synthetically.

- 16 045264 by, for example, modifying one or more of the above polypeptide sequences. For example, in particular embodiments, it may be desirable to increase the binding affinity and/or improve other biological properties of CAR receptors by introducing one or more substitutions, deletions, additions, and/or insertions into the binding domain, hinge, TM domain, costimulatory signaling domain or primary signaling domain of a CAR polypeptide. Preferably, the polypeptides of the present invention include polypeptides differing by at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity.

Polypeptides include fragments of polypeptides. Polypeptide fragments refer to a polypeptide that may be monomeric or multimeric, contains a deletion at the amino terminus, a deletion at the carboxyl terminus, and/or a deletion or substitution in the internal sequence of a naturally occurring or recombinantly produced polypeptide. In some embodiments, the polypeptide fragment may comprise a chain of amino acids that is from at least 5 to about 500 amino acids in length. It should be understood that in some embodiments the length of the fragments is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids. In particular, suitable polypeptide fragments include functional domains, including antigen binding domains or antibody fragments. In the case of a mouse anti-BCMA antibody, suitable fragments include, but are not limited to: the CDR region, the heavy or light chain CDR3 region; heavy or light chain variable region; a portion of an antibody chain or variable region including two CDR regions; and so on.

The polypeptide may also be linked in frame or conjugated to a linker or other sequence to facilitate the synthesis, purification or identification of the polypeptide (eg, poly-His), or to enhance the binding of the polypeptide to a solid support.

As noted above, the polypeptides of the present invention can be modified in various ways, including amino acid substitutions, amino acid deletions, truncations and insertions. Methods for performing such manipulations are well known in the art. For example, variants of the amino acid sequence of a reference polypeptide can be obtained by introducing mutations into the DNA. Methods for performing mutagenesis and altering nucleotide sequences are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), US patent No. 4873192, Watson, J.D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance on making suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington , D.C.)).

In some embodiments, the variant contains conservative substitutions. A conservative substitution is a substitution in which an amino acid is replaced with another amino acid having similar properties such that one skilled in the art of peptide chemistry can assume that the secondary structure and hydropathic characteristics of the polypeptide are essentially unchanged. The structure of the polynucleotides and polypeptides of the present invention may be subject to modifications, but they may be represented by a functional molecule that encodes a variant or derivative of the polypeptide with the desired characteristics. In cases where it is necessary to change the amino acid sequence of a polypeptide to obtain an equivalent, or to obtain an improved version of the polypeptide according to the present invention, a person skilled in the art, for example, can change one or more codons of the DNA coding sequence, for example, in accordance with table. 1.

Table 1

Amino acid codons

Amino acid you Single letter code Three letter code Codons Alanin A Ala G.C. A G.C. WITH G.C. G G.C.U.

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Cysteine WITH Cys U.G. C UGU Asparaginova D Asp GA GAU I'm acid C Glutamine E Glu GA GAG acid A Phenylalanine F Phe UU UUU c Glycine — GG GG GG GGU G Gly A C G G istidin — C.A. CAU N His C Isoleucine — AU AU AUU I ISO A C Lysine — A.A. AAG TO Lys A Leucine — UU UU CU CU CU CU L Leu A G A C G U Methionine M Met AUG Asparagine — A.A. AAU N Asn C Proline cc CC CC CCU R Pro A C G Glutamine C.A. CAG Q Gln A

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Arginine R Arg A.G. A A.G. G C.G. A C.G. C C.G. G C.G. U Serin S Ser A.G. C A.G. U U.C. A U.C. C U.C. G U.C. U Threonine T Thr A.C. A A.C. C A.C. G ACU Valin V Val G.U. A G.U. C G.U. G GUU Tryptophan W Trp UGG Tyrosine Y Tyr U.A. C UAU

When identifying amino acid residues that can be replaced, inserted or deleted without loss of biological activity, computer programs well known in the art, such as DNASTAR™ software, can be used. Preferably, amino acid changes in protein variants as described herein are conservative amino acid changes, ie, substitutions with similarly charged or uncharged amino acids. A conservative amino acid change involves a substitution with one amino acid from a family of side chain related amino acids. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes grouped together as aromatic amino acids. Suitable conservative amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art will appreciate that, in general, single amino acid substitutions in non-critical regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224). Examples of conservative substitutions are described in US Provisional Application No. 61/241,647, the contents of which are incorporated herein by reference.

When making such changes, the hydropathic index of amino acids can be taken into account. The importance of the amino acid hydropathic index for the interactive biological function of a protein is well known in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid was assigned a hydropathicity index value based on hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). Values indicated: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is known in the art that certain amino acids can be replaced by other amino acids with a similar index or hydropathicity index to obtain a protein having similar biological activity, i.e., to obtain a biologically functionally equivalent protein. When making such changes, it is preferable to replace them with amino acids whose hydropathic indices differ within ±2, preferably within ±1, even more preferably within ±0.5. It is also known in the art that efficient substitution of similar amino acids can be made on the basis of hydrophilicity.

According to the detailed description in US patent No. 4554101, the amino acid residues were assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (0.5±1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It should be understood that an amino acid can be replaced by another amino acid having a similar hydrophilicity index, while maintaining biological equivalence, in particular, the immunological equivalence of the protein.

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When making such changes, it is preferable to replace them with amino acids, the differences in the hydrophilicity index of which do not exceed ±2, preferably do not exceed ±1, even more preferably do not exceed ±0.5.

As described above, amino acid substitutions can be made based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like.

Polypeptide variants also include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (eg, PEGylated molecules). Covalent variants can be obtained by combining functional groups with groups located in the amino acid chain, or with an N-terminal or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants and muteins. Options also include truncation or deletion of regions that do not affect the functional activity of the proteins.

In one embodiment, if expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence as described herein. In another embodiment, two or more polypeptides may be expressed as a fusion protein that contains one or more self-cleaving polypeptide sequences.

The polypeptides of the present invention include hybrid polypeptides. In preferred embodiments, hybrid polypeptides and polynucleotides encoding hybrid polypeptides, such as CAR receptors, are provided. Fusion polypeptides and fusion proteins refer to a polypeptide containing at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. In hybrid polypeptides, typically the C-terminus is connected to the N-terminus, although C-terminus to the C-terminus, N-terminus to the N-terminus, or N-terminus to the C-terminus may also be connected. The fusion protein polypeptides may be arranged in any order or in a specific order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and cross-species homologs, as long as the desired transcriptional activity of the fusion polypeptide is retained. Hybrid polypeptides can be obtained using chemical synthesis methods or by chemical linking of two fragments; or may generally be obtained using other standard techniques. The ligated DNA sequences containing the fusion polypeptide are operably linked to suitable transcriptional or translational control elements as described herein.

In one embodiment, the hybridization partner contains a sequence that promotes greater expression of the protein (expression enhancer) compared to the expression of the natural recombinant protein. Other hybridization partners may be selected to increase the solubility of the protein, or to target the protein to desired intracellular compartments, or to facilitate transport of the fusion protein across the cell membrane.

Fusion polypeptides may further comprise a polypeptide cleavage signal between all of the polypeptide domains described herein. In addition, the polypeptide site can be placed in any linker peptide sequence. Examples of polypeptide cleavage signals include recognition sites for polypeptide cleavage, such as protease cleavage sites, nuclease cleavage sites (eg, rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5 (8); 616-26).

Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gen. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, cleavage sites for potyviral NIa proteases (e.g., tobacco etch virus protease), potyviral HC proteases, potyviral proteases P1 (P35), bioviral NIa proteases, bioviral RNA-2-encoded proteases, aphthoviral proteases L , enteroviral proteases 2A, rhinoviral proteases 2A, picornaviral proteases 3C, comoviral proteases 24K, non-poviral proteases 24K, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip yellow spot virus) 3C-like protease, hepar inom, thrombin, factor Ha and enterokinase. Due to the high stringency of the cleavage conditions, in one embodiment, TEV (tobacco etch virus) protease cleavage sites, e.g., EXXYXQ(G/S) (SEQ ID NO: 23), e.g., ENLYFQG (SEQ ID NO: 24) and ENLYFQS, are preferred (SEQ ID NO: 25), where X is any amino acid (TEV cleavage occurs between Q and G, or Q and S).

In a specific embodiment, the self-cleaving peptides include sequences of such polypeptides derived from potyviral and cardioviral 2A peptides, FMDV

- 20 045264 (enteroviral vesicular stomatitis virus), equine rhinitis A virus, Thosea asigna virus and porcine teschovirus.

In some embodiments, said self-cleaving polypeptide site includes a 2A site or a 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen.

Virol. 82:1027-1041).

Table 2 Sites 2A include sequences:

SEQ ID NO: 26 LLNFDLLKLAGDVESNPGP SEQ ID NO: 27 TLNFDLLKLAGDVESNPGP SEQ ID NO: 28 LLKLAGDVESNPGP SEQ ID NO: NFDLLKLAGDVESNPGP 29 SEQ ID NO: thirty QLLNFDLLKLAGDVESNPGP SEQ ID NO: 31 APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 32 VTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQT SEQ ID NO: 33 LNFDLLKLAGDVESNPGP SEQ ID NO: 34 LLAIHPTEARHKQKIV APVKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 35 EARHKQKIVAPVKQTLNFDLLKLAGDVESNPGP

In preferred embodiments, the polypeptide provided by the present invention includes a CAR polypeptide.

E. Polynucleotides

In preferred embodiments, a polynucleotide encoding one or more CAR polypeptides is provided, for example, SEQ ID NO: 10. As used herein, the terms polynucleotide or nucleic acid refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), plus strand RNA (RNA). (+)), minus-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 present invention include polynucleotides or variants that differ by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99% or 100% sequence identity to any reference sequences described herein (see, for example, the sequence listing); in this case, as a rule, the specified variant retains at least one type of biological activity of the reference sequence. According to various illustrative embodiments, the present invention includes, but is not limited to, polynucleotides containing expression vectors, viral vectors and transfer plasmids, as well as compositions and cells containing them.

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In specific embodiments, the present invention provides polynucleotides that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more consecutive amino acid residues of the polypeptide of the present invention, as well as all products of intermediate length. It is easy to understand that intermediate length products in the specified context means any length in the range between the specified values, for example, 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

As used herein, the terms polynucleotide variant, variant, and the like are used. refer to polynucleotides whose sequences are substantially identical to a reference polynucleotide sequence, or polynucleotides that hybridize to a reference sequence under the stringent conditions defined hereinafter herein. These terms include polynucleotides in which, compared to the reference polynucleotide, one or more nucleotides have been added or deleted, or replaced with different nucleotides. It is well known in the art that when certain changes are made to a reference polynucleotide, including mutations, additions, deletions and substitutions, the altered polynucleotide may retain the biological function or activity of the reference polynucleotide.

As used herein, the terms sequence identity or, for example, 50% identical sequence, refer to the degree of identity of the arrangement of nucleotides or amino acids in specified sequences within a comparison window. Accordingly, percent sequence identity can be calculated by comparing two sequences after optimal alignment, within a window of comparison, to determine the number of positions where both sequences have identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met), giving the number of matches positions, dividing the specified number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percent sequence identity. Included are nucleotides and polypeptides that differ by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity to any reference sequences described herein, generally retaining at least one biological activity of the reference polypeptide in the polypeptide variant.

Terms used to describe the sequence relationship of two or more polynucleotides or polypeptides include the terms reference sequence, window of comparison, sequence identity, percent sequence identity, and substantial identity. The length of the reference sequence is at least 12, but often 1518, and often at least 25 monomer units, including nucleotides and amino acid residues. Since each of the two polynucleotides may contain (1) a sequence similar to the two polynucleotides (i.e., only part of the complete polynucleotide sequence) and (2) a sequence that differs in the two polynucleotides, comparison of the sequences of two (or more) polynucleotides is performed by typically by comparing the sequences of two polynucleotides within a window of comparison to identify and compare regions of local sequence similarity. The term comparison window refers to a conventional segment comprising at least 6 contiguous positions, typically from about 50 to about 100, more commonly from about 100 to about 150 positions, within which the sequence is compared with a reference sequence containing the same number of contiguous positions, after optimal alignment of these two sequences. For optimal alignment of two sequences, the comparison window may contain approximately 20% or less additions or deletions (i.e., gaps) relative to the reference sequence (which contains no additions or deletions). Optimal sequence alignment in the comparison window can be achieved using computerized variants of the algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, version 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA) or by inspection, and selecting the best alignment (i.e., one that gives the maximum percentage of homology over the comparison window) obtained using any of a variety of methods. Mention may also be made of the BLAST family of programs, for example those described in: Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed description of sequence analysis can be found in section 19.3 in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15.

As used herein, the term isolated polynucleotide refers to a polynucleotide that has been isolated from sequences flanking it in a naturally occurring state, for example, a DNA fragment that has been separated from sequences typically adjacent to the fragment. An isolated polynucleotide also refers to complementary DNA (cDNA), recombinant DNA or other polynucleotide not existing in nature and derived from humans

- 22 045264 century.

Terms to describe the orientation of polynucleotides include: 5' (generally the end of the polynucleotide containing a free phosphate group) and 3' (generally the end of the polynucleotide containing a free hydroxyl (OH) group). Polynucleotide sequences can be written in the 5'^3', or 3'^5' direction. In the case of DNA and mRNA, the 5'^3' strand is called the sense strand, plus strand, or coding strand because its sequence is identical to that of pre-template RNA (pre-mRNA) [except for uracil (U) in RNA instead of thymine (T) in DNA ]. In the case of DNA and mRNA, the complementary 3'^5' strand transcribed by RNA polymerase is called the template, antisense strand, minus strand, or non-coding strand. As used herein, the term reverse orientation refers to a 5'^3' sequence written in the 3'^5' direction or a 3'^5' sequence written in the 5'^3' direction.

The terms complementary and complementarity refer to polynucleotides (i.e., sequences of nucleotides) interconnected through the principle of base pairing. For example, the complementary strand to the DNA sequence 5'AGTCATG3' is strand 3' T C A G T A C 5'. The latter sequence is often written as reverse complement, starting at the 5' end on the left and ending at the 3' end on the right: 5' C A T G A C T 3'. A sequence that is also its own reverse complement is called a palindromic sequence. Complementarity can be partial, in which case only some nucleic acid bases conform to the base pairing principle. Complete or total complementarity of nucleic acids can also be observed.

In addition, those skilled in the art will appreciate that, due to the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein, or a fragment or variant thereof. Some of these polynucleotides have a minimal degree of homology to the nucleotide sequence of a naturally occurring gene. However, the present invention in particular provides polynucleotides whose composition varies due to differences in codon usage, for example, polynucleotides that are codon optimized for use in humans and/or primates. In addition, alleles of genes containing the polynucleotide sequences of the present invention can also be used. Alleles are endogenous genes that have been altered by one or more mutations, such as nucleotide deletions, additions, and/or substitutions.

The term nucleic acid cassette as used herein refers to genetic sequences within a vector from which RNA and then protein can be expressed. The nucleic acid cassette contains a gene of interest, such as the CAR gene. The nucleic acid cassette is located and oriented in the vector sequence such that the nucleic acid from the cassette can be transcribed into RNA, and optionally translated into a protein or polypeptide, subject to appropriate post-translational modifications necessary to ensure activity in the transformed cell, and translocated into a suitable compartment to carry out biological activity by targeting to suitable intracellular compartments or secretion into extracellular compartments. Preferably, the 3' end and 5' end of the cassette are designed to allow easy insertion into the vector, for example having restriction endonuclease cleavage sites at both ends of the cassette. According to a preferred embodiment of the present invention, said nucleic acid cassette contains a chimeric antigen receptor sequence useful for the treatment of B cell malignancy. Said cassette can be isolated and integrated into a plasmid or viral vector as a single unit.

In certain embodiments, the polynucleotides include at least one polynucleotide of interest. As used herein, the term polynucleotide of interest refers to a polynucleotide encoding a polypeptide (ie, a polypeptide of interest) inserted into an expression vector to be expressed. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 polynucleotides of interest. In some embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in treating or preventing a disease or disorder. Polynucleotides of interest and the polypeptides they encode include both polynucleotides that encode wild-type polypeptides and functional variants and fragments thereof. In certain embodiments, the functional variant differs in at least 80%, at least 90%, at least 95%, or at least 99% identity to the corresponding wild-type polynucleotide or polypeptide reference sequence. In some embodiments, the functional variant or fragment differs in at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

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In one embodiment, the polynucleotide of interest does not encode a polypeptide, but serves as a template for transcription of a microRNA, miRNA or shRNA, ribozyme or other inhibitory RNA. In various other embodiments, the polynucleotide includes a polynucleotide of interest encoding a CAR and one or more additional polynucleotides of interest, including, but not limited to, an inhibitory nucleic acid sequence, including, but not limited to: siRNA, microRNA, shRNA and ribozyme.

As used herein, the terms miRNA, or small interfering RNA, refer to a short sequence of polynucleotides that mediates the process of sequence-specific post-transcriptional gene silencing, translational inhibition, transcriptional inhibition or epigenetic RNA interference in animals (Zamore et al., 2000, Cell, 101 , 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886). In some embodiments, the siRNA comprises a first strand and a second strand containing the same number of nucleosides; however, the first and second strands are spaced apart in such a way that the two terminal nucleosides on the first and second strands are not paired with a residue on the complementary strand. In some cases, two unpaired nucleosides are represented by thymidine residues. The siRNA must include a region sufficiently homologous to the target gene and contain a sufficient number of nucleotides so that the siRNA or fragment thereof can mediate down-regulation of the target gene. Accordingly, the siRNA includes a region that is at least partially complementary to the target RNA. Absolute complementarity between the siRNA and the target is not required, but the match must be sufficient to allow the siRNA or its cleavage product to direct sequence-specific silencing, for example, by cleaving the target RNA during RNA interference. Complementarity, or the degree of homology of the target strand, is most important for the antisense strand. Although absolute complementarity is often required, particularly on the antisense strand, some embodiments include one or more, but preferably 10, 8, 6, 5, 4, 3, 2, or fewer mismatches to the target RNA. These mismatches are tolerated primarily in terminal regions, and, if present, are preferably located in the terminal region or regions, for example within 6, 5, 4 or 3 5'-terminal and/or 3'-terminal nucleotides. It is only necessary that the sense strand be sufficiently complementary to the antisense strand to maintain the overall double-stranded structure of the molecule.

In addition, the siRNA may be modified or may include nucleoside analogues. Single-stranded regions of siRNA may be modified or may include nucleoside analogues, for example, an unpaired region or regions with a hairpin structure, for example, a region connecting two complementary regions may contain modifications or nucleoside analogues. Modifications to stabilize one or more of the 3' or 5' ends of the siRNA, for example to protect against exonucleases, or to promote entry of an antisense siRNA agent into RISC may also be useful. Modifications may include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, base-deleted spacers, triethylene glycol, hexaethylene glycol), specific biotin or fluorescein reagents in the form of phosphoramidites and having an additional DMT-protected hydroxyl group, which ensures repeated binding during RNA synthesis. The length of each siRNA strand may be 30, 25, 24, 23, 22, 21 or 20 nucleotides or less. Preferably, the length of said strand is at least 19 nucleotides. For example, the length of each strand can be from 21 to 25 nucleotides. Preferred siRNAs contain a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more 2-3 nucleotide overhangs, preferably one or two 2-3 3' overhangs - protruding nucleotides.

As used herein, the term microRNA refers to small non-coding RNAs of 20-22 nucleotides in length, typically excised from self-hybridizing pre-RNA structures of ~70 nucleotides, known as pre-microRNAs. miRNAs negatively regulate their targets in one of two ways depending on the degree of complementarity between the miRNA and the target. First, microRNAs that bind to protein-coding mRNA sequences with absolute or near-absolute complementarity induce the RNA-mediated interference (RNAi) pathway. microRNAs that exert regulatory effects by binding to incompletely complementary sites within the 3' untranslated regions (UTRs) of their target mRNAs suppress the expression of target genes post-transcriptionally, presumably at the translational level, involving the RISC complex, similar to or possibly identical to the RISC complex involved in the RNA interference pathway. According to the translational control version, microRNAs that use this mechanism reduce the levels of proteins corresponding to their target genes, but the mRNA levels of these genes change minimally. miRNAs include both naturally occurring miRNAs and artificially engineered miRNAs that can target any specific mRNA sequence.

- 24 045264

For example, in one embodiment, one skilled in the art can design short hairpin RNA constructs that are expressed as primary human microRNA transcripts (eg, miR-30 or miR-21). A Drosha processing site is added to the hairpin construct of this design, which has been shown to significantly improve knockdown efficiency (Pusch et al., 2004). The hairpin stem consists of 22 nucleotides of dsRNA (for example, an antisense sequence with absolute complementarity to the desired target) and a 15-19 nucleotide loop from human microRNA. The addition of a miRNA loop and miR30 flanking sequences on either side or both sides of the hairpin results in a more than 10-fold increase in the level of processing of expressed hairpins by Drosha and Dicer enzymes compared to standard shRNA constructs without miRNA. Increasing levels of processing by Drosha and Dicer enzymes leads to increased miRNA/microRNA synthesis and greater activity of expressed hairpins.

As used herein, the terms shRNA or short hairpin RNA refer to a double-stranded structure formed by a single self-complementary strand of RNA. shRNA constructs containing a nucleotide sequence identical to part of either the coding or non-coding sequence of the target gene are preferred for inhibition. RNA sequences containing insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Preferred are inhibitory RNA sequences that are more than 90% identical to part of the target gene, or even 100% identical. In certain preferred embodiments, the length of the duplex-forming portion of the shRNA is at least 20, 21, or 22 nucleotides, for example, the size of the RNA products synthesized by Dicer-dependent cleavage. In some embodiments, the length of the shRNA construct is at least 25, 50, 100, 200, 300, or 400 bases. In some embodiments, the length of the shRNA construct is 400-800 bases. The shRNA constructs are largely tolerant of variations in sequence and loop size.

As used herein, the term ribozyme refers to a catalytically active RNA molecule capable of site-specific cleavage of a target mRNA. Several subtypes of ribozymes have been described, such as hammerhead ribozymes and hairpin ribozymes. The catalytic activity and stability of the ribozyme can be increased by replacing deoxyribonucleotides with ribonucleotides at sites that do not have catalytic activity. Although it is possible to use ribozymes that cleave mRNA at sites of site-specific sequence recognition to destroy specific mRNAs, it is preferable to use hammerhead ribozymes. Hammerhead ribozymes cleave mRNA at positions defined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA contains the following two-base sequence: 5'-UG-3'. The design and production of hammerhead ribozymes is well known in the art.

A preferred method of delivering the polynucleotide of interest, which contains siRNA, microRNA, shRNA or ribozyme, includes one or more regulatory sequences, such as, for example, a strong constitutive pol III promoter, for example, human U6 snRNA promoter, mouse U6 snRNA promoter, H1 RNA promoter human and mouse, the human tRNA-val promoter or the strong constitutive pol II promoter as described herein.

The polynucleotides of the present invention, regardless of the length of the actual coding sequence, can be combined with other DNA sequences, such as promoters and/or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome binding sites (IRES), recombinase recognition sites (eg, LoxP, FRT, and Att sites), termination codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags described in various sections herein or known in the art. this field of technology; thus, their overall length can vary significantly. Accordingly, it is contemplated that virtually any length of polynucleotide fragment may be used, with the overall length preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.

Preparation of polynucleotides, manipulation of polynucleotides, and/or expression of polynucleotides can be accomplished using any of a variety of conventional techniques known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding said polypeptide can be inserted into a suitable vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional examples of vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-based artificial chromosome (PAC), bacteriophages such as phage

- 25 045264 lambda or phage M13, and animal viruses. Examples of categories of animal viruses suitable for use as vectors include, but are not limited to, retroviruses (including lentivirus), adenoviruses, adeno-associated viruses, herpesviruses (eg, herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (eg, SV40 ). Examples of expression vectors are pClneo vectors (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 fusion proteins as described herein can be ligated into such expression vectors to express the fusion protein in mammalian cells.

In one embodiment, the CAR encoding vector of the present invention comprises the polynucleotide sequence set forth in SEQ ID NO: 36.

In certain embodiments, the vector is an episomal vector or an extrachromosomally supported vector. As used herein, the term episomal refers to a vector capable of replicating without integration into the host chromosomal DNA and without gradual loss during host cell division, and also means that the vector replicates extrachromosomal or episomal. Said vector is constructed such that it contains a sequence encoding a DNA origin of replication, or ori, from a lymphotropic herpesvirus or gammaherpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, in particular, an origin of replication of a lymphotropic herpesvirus or gammaherpesvirus corresponding to oriP EBV. In a particular aspect, said lymphotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), herpes saimiri virus (HS), or Marek's disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gammaherpesviruses. Typically, the host cell contains a viral replication transactivator protein that activates replication.

Control elements (control elements) or regulatory sequences present in the expression vector are the untranslated regions of the vector - the origin of replication, selection cassettes, promoters, enhancers, translation initiation signals (Shine-Dalgarno sequence or Kozak sequence), introns, polyadenylation sequence, 5' and 3' untranslated regions that interact with host cell proteins during transcription and translation. The effectiveness and specificity of such elements may vary. Depending on the vector system and host used, any number of suitable transcription and translation elements may be used, including universal promoters and inducible promoters.

In certain embodiments, vectors for use in the practice of the present invention, including, but not limited to, expression vectors and viral vectors, contain exogenous, endogenous, or heterologous control sequences, such as promoters and/or enhancers. An endogenous control sequence is a control sequence naturally associated with a specific gene in the genome. An exogenous control sequence is a control sequence placed directly adjacent to a gene through genetic manipulation (ie, molecular biology techniques) such that transcription of the gene is directed by the attached enhancer/promoter. A heterologous control sequence is an exogenous sequence that is not of the same species as the cell being genetically manipulated.

The term promoter as used herein refers to the recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes polyline cleotides that are functionally linked to a promoter. In specific embodiments, promoters operating in mammalian cells comprise an AT-rich region located approximately 25-30 bases 5' from the transcription start site, and/or another sequence located 70-80 bases 5' -direction from the start of transcription, the CNCAAT region, where e N can be any nucleotide.

The term enhancer refers to a segment of DNA that contains sequences capable of promoting increased transcription and, in some cases, capable of functioning independently of their orientation relative to another control sequence. An enhancer may function in conjunction with, or complement the action of, promoters and/or other enhancer elements. The term promoter/enhancer refers to a segment of DNA that contains sequences capable of serving as both a promoter and an enhancer.

The term functionally coupled refers to the arrangement of the described components in close proximity, while being interconnected in such a way that their proper

- 26 045264 functioning. In one embodiment, the term refers to the functional relationship of a nucleic acid expression control sequence (such as a promoter and/or enhancer) and a second polynucleotide sequence, such as a polynucleotide of interest, wherein the expression control sequence directs transcription of a nucleic acid corresponding to the second sequence.

As used herein, the term constitutive expression control sequence refers to a promoter, enhancer, or promoter/enhancer that allows continuous or sequential transcription of an operably linked sequence. The constitutive expression control sequence may be either a universal promoter, enhancer, or promoter/enhancer allowing expression in a wide range of cell types and tissues, or a cell-specific, cell type-specific, cell line-specific, or tissue-specific promoter, enhancer, or promoter. /enhancer that allows expression to be limited to a specific range of cell types and tissues, respectively.

Exemplary universal expression control sequences suitable for use in particular embodiments of the present invention include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) promoter (e.g., early or late), leukemia virus LTR promoter Moloney mice (MoMLV), Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) promoter (thymidine kinase), vaccinia virus H5, P7.5 and P11 promoters, elongation factor 1-alpha (EF1a) promoter, early response protein growth factors 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock protein 5 70 kDa (HSPA5), protein 90 kDa heat shock protein-β family 1 (HSP90B1), 70 kDa heat shock protein (HSP70), β-kinesin (β-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 ( 2007)), ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin (CAG) promoter, β-actin promoter, and myeloproliferative sarcoma virus enhancer-containing promoter with the negative control region removed by replacement with d1587rev primer binding site (MND promoter) (Challita et al., J Virol. 69(2):748-55 (1995)).

In one embodiment, a vector for use in the present invention includes an MND promoter.

In one embodiment, a vector for use in the present invention includes an EF1a promoter containing the first intron of the human EF1a gene.

In one embodiment, the vector of the present invention includes an EF1a promoter that lacks the first intron of the human EF1a gene.

In a particular embodiment, it may be desirable to express a CAR-containing polynucleotide under the control of a specific T cell promoter.

As used herein, conditional expression may refer to any type of conditional expression, including, but not limited to, inducible expression; repressed expression; expression in cells or tissues distinguished by a particular physiological, biological or disease state, etc. This definition does not imply the exclusion of cell type or tissue specific expression. Certain embodiments of the present invention provide for conditional expression of a polynucleotide of interest, eg, controlled expression in a cell, tissue, organism, or the like. treatments or conditions that result in expression of the polynucleotide, or that result in increased or decreased expression of the polynucleotide encoded by the polynucleotide of interest.

Illustrative examples of inducible promoters/systems include, but are not limited to, steroid-inducible promoters such as promoters of genes encoding glucocorticoid or estrogen receptors (inducible by treatment/treatment with the appropriate hormone), metallothionein promoter (inducible by treatment/treatment with various heavy metals), promoter MX-1 (interferon-inducible), mifepristone-regulated GeneSwitch system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved using a site-specific DNA recombinase. In accordance with certain embodiments of the present invention, the vector contains at least one site (usually two sites) of recombination mediated by a site-specific recombinase. As used herein, the terms recombinase or site-specific recombinase include excision or integration proteins, enzymes, cofactors, or associated proteins that are involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten , 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutant forms thereof, derivatives ( for example, fusion proteins containing protein sequences

- 27 045264 recombinations or fragments thereof), fragments and variants. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, PS31, Cin, Tn3 resolvase,

TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1 and ParA.

Vectors may contain one or more recombination sites from any of a wide range of site-specific recombinases. It should be understood that the target site of the site-specific recombinase is present as an additional site, along with any site(s) necessary for integration of the vector, for example, a retroviral vector or a lentiviral vector. As used herein, the terms recombination sequence, recombination site, or site-specific recombination site refer to a specific nucleic acid sequence that a recombinase recognizes and binds.

For example, one of the sites of recombination by Cre recombinase is IoxP, which is a 34-bp sequence containing two 13-bp inverted repeats (which serve as recombinase binding sites) flanking an 8-bp core sequence (see FIG. 1 in: Sauer, W., Current Opinion in Biotechnology 5:521-527 (1994)). Other examples of loxP sites include, but are not limited to: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al. , 2002), lox71 (Albert et al., 1995) and lox66 (Alberts al, 1995).

Suitable FLP recombinase recognition sites include, but are not limited to: FRT (McLeod, et al., 1996), F1, _F2 , _F3 (Schlake and Bode, 1994), F4 _; F ₅ (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

Other examples of recognition sequences are the sequences attB, attP, attL and attR, recognized by the recombinase enzyme integrase λ, for example, phi-c31. The site-specific recombinase (SSR) fS31 mediates recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). The attB and attP sites, so named because they are phage integrase attachment sites in bacterial and phage genomes; Accordingly, both of these sites contain non-absolute inverted repeats, presumably bound by fS31 homodimers (Groth et al., 2000). The resulting sites, attL and attR, are virtually inert with respect to further PS31-mediated recombination (Belteki et al., 2003), making this reaction irreversible. Regarding catalytic insertions, attB-carrying DNA has been found to insert into the attP genomic site more easily than the attP site into the attB genomic site (Thyagarajan et al., 2001; Belteki et al., 2003). Accordingly, typical strategies involve placement at a specific locus through homologous recombination of an attP-carrying docking site, which then binds to an inserted attB-carrying sequence.

As used herein, the term internal ribosome entry site, or IRES, refers to an element that facilitates direct ribosome entry into the initiation codon of an internal cistron (protein coding region), such as an ATG, thereby allowing cap-independent gene translation. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In certain embodiments, the vectors of the present invention include one or more polynucleotides of interest that encode one or more polypeptides. In specific embodiments, to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

As used herein, the term Kozak sequence refers to a short sequence of nucleotides that greatly facilitates the initial binding of mRNA to the small ribosomal subunit and enhances translation. The Kozak consensus sequence is represented by the sequence (GCC)RCCATGG, where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20): 8125-48). In certain embodiments, the vectors of the present invention comprise polynucleotides comprising a Kozak consensus sequence and encoding a desired polypeptide, for example, a CAR.

In some embodiments, a suicide gene, including an inducible suicide gene, is included in the polynucleotide or in a cell carrying the polynucleotide to reduce the risk of immediate toxicity and/or uncontrolled proliferation. In certain aspects, the suicide gene is not immunogenic to the host harboring the polynucleotide or cell. A specific example of a suitable suicide gene is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a specific chemical dimerization inducer (CID).

In some embodiments, the vectors contain gene segments that sensitize immune effector cells of the present invention, such as T-28045264 cells, to negative selection in vivo. By negative selection is meant that the infused cell can be eliminated as a result of a change in the conditions to which the individual is exposed in vivo. A phenotype for negative selection can be obtained by inserting a gene that confers sensitivity to an administered agent, such as a compound. Genes for negative selection are known in the art and include the following: the herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977), which confers sensitivity to ganciclovir; cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, cellular adenine phosphoribosyltransferase (APRT) gene and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

In some embodiments, the genetically modified immune effector cells, such as T cells, comprise a polynucleotide further comprising a positive selection marker to allow selection from cells of a negative selection phenotype in vitro. The positive selection marker may be a gene that, when introduced into a host cell, expresses a dominant phenotype that provides positive selection for cells carrying the gene. Genes of this type are known in the art and include, but are not limited to, the hygromycin B phosphotransferase (hph) gene, which confers resistance to hygromycin B, the aminoglycoside phosphotransferase (neo or aph) gene from Tn5, which encodes resistance to the G418 antibiotic, the dihydrofolate reductase (DHFR), adenosine deaminase (ADA) gene and multidrug resistance (MDR) gene.

Preferably, the positive selection marker and the negative selection element are connected in such a way that the loss of the negative selection element is also inevitably accompanied by the loss of the positive selection marker. Even more preferably, the markers for positive and negative selection are combined in such a way that the loss of one inevitably leads to the loss of the other. An example of a hybrid polynucleotide, the expression product of which is a polypeptide that provides the properties necessary for both positive and negative selection described above, is the hybrid hygromycin phosphotransferase/thymidine kinase (HuTK) gene. Expression of this gene produces a polypeptide that provides resistance to hygromycin B for positive selection in vitro and sensitivity to ganciclovir for negative selection in vivo. see Lupton S. D., et al., Mol. and Cell. Biology 1 1:3374-3378, 1991. In addition, in preferred embodiments, the polynucleotides of the present invention encoding the fusion receptors are included in retroviral vectors containing a fusion gene, particularly one conferring resistance to hygromycin B, for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo, for example, in the HyTK retroviral vector described by Lupton, S. D. et al. (1991), supra. See also PCT publications US91/08442 and PCT/US94/05601, S. D. Lupton, which describe the use of bifunctional selectable hybrid genes obtained by combining dominant positive selection markers with negative selection markers.

Preferred markers for positive selection are derived from genes selected from the group consisting of hph, nco, and gpt, and preferred markers for negative selection are derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. In particular, preferred markers are bifunctional selectable fusion genes, wherein the positive selection marker is derived from hph or peo and the negative selection marker is derived from a cytosine deaminase or thymidine kinase (TK) gene, or is represented by inducible suicide genes.

F. Viral vectors

In certain embodiments, a cell (eg, an immune effector cell) is transduced with a retroviral vector, such as a lentiviral vector encoding a CAR. For example, an immune effector cell is transduced with a vector encoding a CAR that contains a mouse anti-BCMA antibody or an antigen-binding fragment thereof that binds a BCMA polypeptide with the intracellular signaling domain CD3Z, CD28, 4-1BB, Ox40, or any combination thereof. Accordingly, these transduced cells can trigger a CAR-mediated cytotoxic response.

Retroviruses are a standard tool for gene delivery (Miller, 2000, Nature. 357: 455-460). In specific embodiments, a retrovirus is used to deliver a polynucleotide encoding a chimeric antigen receptor (CAR) into a cell. As used herein, the term retrovirus refers to an RNA virus that reverse transcribes its own genomic RNA to form a linear double-stranded DNA copy and then covalently integrates its own genomic DNA into the host genome. Once integrated into the host genome, the virus is called a provirus. The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules encoding structural proteins and enzymes necessary for the formation of new viral particles.

Illustrative examples of retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney murine leukemia virus (MMuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mammary tumor virus murine leukemia virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Freud's murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV)) and lentivirus.

As used herein, the term lentivirus refers to a group (or genus) of complex retroviruses. Illustrative examples of 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 (AEC); equine infectious anemia virus (EIAN); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (ie, the sequence of HIV cis-acting elements) are preferred. In specific embodiments, a lentivirus is used to deliver a CAR-containing polynucleotide into a cell.

Retroviral vectors and, more specifically, lentiviral vectors can be used in the practice of specific embodiments of the present invention. Accordingly, the term retrovirus or retroviral vector as used herein is intended to include lentivirus and lentiviral vectors, respectively.

The term vector is used herein to refer to a nucleic acid molecule capable of carrying or transporting another nucleic acid molecule. The transferred nucleic acid is usually linked, for example, embedded in the specified vector nucleic acid molecule. The vector may include sequences that direct cell-autonomous replication, or may include sequences sufficient to allow integration into the DNA of the host cell. Suitable vectors include, for example, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Suitable viral vectors include, for example, retroviruses and replication-defective lentiviruses.

As will be apparent to one skilled in the art, the term viral vector is widely used to refer to either a nucleic acid molecule (eg, a transfer plasmid) comprising virally derived nucleic acid elements that typically facilitate the transfer of said nucleic acid molecule or its integration into the genome of a cell or viral particle that mediates the transfer of nucleic acid. Viral particles typically include various viral components and sometimes also host cell components in addition to nucleic acid(s).

The term viral vector can refer either to a virus or viral particle capable of transferring a nucleic acid into a cell, or to the nucleic acid itself being transferred. Viral vectors and transfer plasmids contain structural and/or functional genetic elements originally derived from the virus. The term retroviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or parts thereof, originally derived from a retrovirus. The term lentiviral vector refers to a viral vector or plasmid containing structural and functional genetic elements, or parts thereof, including LTR regions originally derived from a lentivirus. The term hybrid vector refers to a vector, LTR, or other nucleic acid containing both retroviral, eg, lentiviral, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a transfer vector or plasmid containing retroviral, eg, lentiviral sequences for reverse transcription, replication, integration, and/or packaging.

In specific embodiments, the terms lentiviral vector, lentiviral expression vector can be used to refer to lentiviral transfer plasmids and/or infectious lentiviral particles. It should be understood that when referenced herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, and the like, the sequences of such elements are intended to be present in the form of RNA in the lentiviral particles of the present invention and in the form of DNA in DNA plasmids according to the present invention.

At each end of the provirus are structures called long terminal repeats, or LTRs. The term long terminal repeat (LTR) refers to base-pair domains located at the ends of retroviral DNA that, under natural sequence conditions, are direct repeats and contain the U3, R, and U5 regions. LTR regions typically perform fundamental functions associated with retroviral gene expression (eg, such as stimulation, initiation, and polyadenylation of gene transcripts) and viral replication. The LTR contains numerous regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The viral LTR region is divided into three regions called U3, R and U5. Area U3 contains

- 30 045264 enhancer and promoter elements. The U5 region is represented by the sequence between the primer binding site and the R region, and contains the polyadenylation sequence. Region R (repeat region) is flanked by regions U3 and U5. The LTR region consists of the U3, R and U5 regions, and is present at both the 5' and 3' ends of the viral genome. Adjacent to the 5' LTR region are sequences necessary for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (psi site).

As used herein, the term packaging signal or packaging sequence refers to sequences within the retroviral genome necessary for the insertion of viral RNA into the viral capsid or viral particle, see, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101-2109. Some retroviral vectors involve a minimal packaging signal (also called the psi [Ψ] sequence) required to enclose the viral genome into a capsid. Accordingly, as used herein, the terms packaging sequence, packaging signal, psi, and the symbol Ψ are used to refer to the non-coding sequence required for capsid packaging of retroviral RNA strands during the formation of viral particles.

In various embodiments, the vectors contain modified 5' LTR and/or 3' LTR regions. Any or both LTR regions may contain one or more modifications, including, but not limited to, one or more deletions, insertions or substitutions. Often, modifications to the 3' region of the LTR are made to improve the safety of lentiviral or retroviral systems by producing replication-defective viruses. As used herein, the term replication defective refers to a virus that is incapable of fully efficient replication such that infectious virions are not produced (eg, replication defective lentiviral particles). The term replication competent refers to a wild-type or mutant virus that is capable of replication; As a result of such viral replication, infectious virions (for example, replication competent lentiviral particles) can be formed.

Self-inactivating (SIN) vectors are replication-defective vectors, such as retroviral or lentiviral vectors, in which the right (3') enhancer region of the LTR, known as the U3 region, has been modified (eg, by deletion or substitution) to prevent viral transcription after the first round of viral replication. This is because the U3 region of the right (3') LTR is used as a template for the U3 region of the left (5') LTR during viral replication and, accordingly, the viral transcript cannot be synthesized without the U3 enhancer-promoter region. According to a further embodiment of the present invention, the 3' LTR region is modified by replacing the U5 region, for example, with an ideal poly(A) sequence. It should be noted that such modifications of LTR regions, such as modifications of the 3' LTR, 5' LTR, or both 3' and 5 LTR regions, are also covered by the present invention.

An additional increase in safety is provided by replacing the U3 5' LTR region with a heterologous promoter that controls the transcription of the viral genome during the formation of viral particles. Examples of suitable heterologous promoters include, for example, viral promoters: simian virus 40 (SV40) (eg, early or late), cytomegalovirus (CMV) (eg, immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus ( RSV) and herpes simplex virus (HSV) (thymidine kinase promoter). Typical promoters are capable of stimulating high levels of transcription in a Tat-independent manner. The substitution reduces the likelihood of recombination to form a replication-competent virus due to the absence of the complete U3 sequence in the virus production system. In some embodiments, a heterologous promoter provides the additional benefit of controlling the manner in which the viral genome is transcribed. For example, said heterologous promoter may be inducible; in this case, transcription of the entire viral genome or part of it occurs only in the presence of induction factors. Induction factors include, but are not limited to, the presence of one or more chemical compounds or physiological conditions, such as temperature or pH, in which the host cells are cultured.

In some embodiments, viral vectors contain a TAR element. The term TAR refers to a genetic transactivation response element located in the R region of long terminal repeats (LTRs) of lentiviruses (eg, HIV). This element interacts with the lentiviral transactivating (tat) genetic element, enhancing viral replication. However, this element is not required in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.

The R region refers to the region within the retroviral LTR that begins at the beginning of the capping group (i.e., the transcription start point) and ends just before the start of the polyadenine (poly-A) tract. The R region is also defined as the region flanked by the U3 and U5 regions. The R region is involved in reverse transcription, ensuring the transfer of synthesized DNA from one end of the genome to the other.

- 31 045264

As used herein, the term FLAP element refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (cPPT and CTS) of a retrovirus, for example, HIV-1 or HIV-2. Suitable FLAP elements are described in US Pat. No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, central initiation of plus-strand DNA in the central polypurine tract (cPPT) and central termination in the central termination sequence (CTS) lead to the formation of a triple-stranded DNA structure: the HIV-1 central DNA flap. Without being limited by any theory, DNA flap may function as a cis-active determinant of nuclear import of the lentiviral genome and/or may increase viral titer. In certain embodiments, the backbones of retroviral or lentiviral vectors contain one or more FLAP elements upstream or downstream of heterologous genes of interest in the vectors. For example, in certain embodiments a, the transfer plasmid includes a FLAP element. According to one embodiment of the present invention, the vector includes a FLAP element isolated from HIV-1.

In one embodiment, retroviral or lentiviral transfer vectors contain one or more export elements. The term export element refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcript from the nucleus to the cytoplasm of the cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) Rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991 . Cell 58: 423), and a post-transcriptional regulatory element (HPRE) of the hepatitis B virus. Typically, the RNA export element is localized in the 3' UTR of the gene, and can be inserted in one or more copies.

In certain embodiments, the expression of heterologous sequences in viral vectors is enhanced by including post-transcriptional regulatory elements, effective polyadenylation sites, and, optionally, transcription termination signals in the vectors. Various posttranscriptional regulatory elements can increase the expression of heterologous nucleic acid at the protein level, for example, the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); hepatitis B virus post-transcriptional regulatory element (HPRE) (Huang et al., Mol. Cell. Biol, 5:3864); and so on. (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vectors of the present invention comprise a post-transcriptional regulatory element such as a WPRE or HPRE.

In certain embodiments, the vectors of the present invention do not include or contain a post-transcriptional regulatory element (PTE), such as WPRE or HPRE, because in some cases these elements increase the risk of cellular transformation and/or do not substantially or significantly increase mRNA transcript abundance or stability mRNA. Accordingly, in some embodiments, the vectors of the present invention do not include or contain PTE. In other embodiments, the vectors of the present invention, as an additional security measure, do not include or contain WPRE or HPRE.

Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are typically located 3' to the polyadenylation signal. In certain embodiments, the vectors contain a polyadenylation sequence in the 3' direction of the polynucleotide encoding the polypeptide to be expressed. The term poly-A site or poly-A sequence as used herein refers to a DNA sequence that directs both termination and polyadenylation of a nascent RNA transcript by RNA polymerase II. Polyadenylation of sequences may contribute to mRNA stability by adding a polyadenine tail to the 3' end of the coding sequence and consequently contribute to increased translational efficiency. Efficient polyadenylation of a recombinant transcript is desirable because transcripts without a polyadenine tail are unstable and rapidly degraded. Illustrative examples of polyadenylation signals that may be used in a vector of the present invention include an ideal poly-A sequence (e.g., AATAAAA, ATTAAA, AGTAAA), a bovine growth hormone poly-A sequence (BGHpA), a rabbit β-globin poly-A sequence (regpA) or other suitable heterologous or endogenous poly-A sequence known in the art.

In some embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements. Insulator elements may contribute to the protection of lentiviral expressed sequences, such as therapeutic polypeptides, from integration site effects that may be mediated by cis-acting elements present in genomic DNA, leading to deregulation of expression of transferred sequences (i.e., position effect; see ., for example, Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci, USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471). In some embodiments, the vectors

- 32 045264 for transfer contain one or more insulator elements within the 3' LTR, and after integration of the provirus into the host genome, said provirus contains one or more insulators at both ends of the 5' LTR or 3' LTR, due to duplication of the 3' LTR. Suitable insulators for use in the present invention include, but are not limited to, chicken β-globin insulator (see references: Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS 94:575; and Bell et al. al., 1999. Cell 98:387, incorporated by reference herein). Examples of insulator elements include, but are not limited to, the β-globin locus insulator, such as chicken HS4.

In accordance with certain specific embodiments of the present invention, most or all of the backbone sequences of the viral vectors are derived from a lentivirus, for example, HIV-1. However, it should be understood that many different sources of retroviral and/or lentiviral sequences, or combinations thereof, can be used, and that numerous substitutions and changes to specific lentiviral sequences can be implemented without impairing the ability of the transfer vector to perform the functions described herein. In addition, various lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, US patents No. 6013516 and No. 5994136, many of which can be adapted to produce a viral vector or plasmid for transfer according to the present invention.

In various embodiments, the vectors of the present invention comprise a promoter operably linked to a polynucleotide encoding a CAR polypeptide. These vectors may contain one or more LTR sequences, characterized in that any of the LTRs contains one or more modifications, such as one or more nucleotide substitutions, additions or deletions. These vectors may further contain one or more auxiliary elements to enhance transduction efficiency (e.g., cPPT/FLAP), viral packaging (e.g., psi (Ψ) packaging signal, RRE), and/or other elements that enhance therapeutic gene expression (e.g., poly(A) sequences), and may optionally contain a WPRE or HPRE sequence.

In a specific embodiment, the transfer vector of the present invention comprises a left (5') retroviral LTR sequence; central polypurine tract/DNA flap (cPPT/FLAP); retrovirus export element; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; and right (3') retroviral LTR sequence; and optionally WPRE or HPRE.

In a specific embodiment, the transfer vector of the present invention comprises a left (5') retroviral LTR sequence; retrovirus export element; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; right (3') retroviral LTR sequence; sequence poly(A); and optionally a WPRE or HPRE sequence. According to another specific embodiment, the present invention provides a lentiviral vector comprising: a left (5') LTR; cPPT/FLAP; RRE; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; right (3') LTR; and polyadenylation sequence; and optionally a WPRE or HPRE sequence.

According to a certain embodiment, the present invention provides a lentiviral vector comprising: a left (5') LTR sequence of HIV-1; psi packing signal (Ψ); cPPT/FLAP; RRE; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; right (3') self-inactivating (SIN) LTR of HIV-1; rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE sequence.

According to another embodiment, the present invention provides a vector comprising: at least one LTR sequence; central polypurine tract/DNA flap (cPPT/FLAP); retrovirus export element; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; and optionally a WPRE or HPRE sequence.

According to a specific embodiment, the present invention provides a vector comprising at least one LTR region; cPPT/FLAP; RRE; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide of the present invention; polyadenylation sequence; and optionally a WPRE or HPRE sequence.

According to a certain embodiment, the present invention provides: at least one HIV-1 SIN LTR region; psi packing signal (Ψ); cPPT/FLAP; RRE; a promoter active in a T cell operably linked to a polynucleotide encoding a CAR polypeptide provided by the present invention; rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE sequence.

In various embodiments, said vector is an integrating viral vector.

In various other embodiments, the vector is an episomal or non-integrating viral vector.

In various embodiments, the vectors of the present invention contain a non-integrating or integration-deficient retrovirus. In one embodiment, an integration-deficient retrovirus or lentivirus refers to a retrovirus or lentivirus containing an integrase that is unable to mediate integration of the viral genome into the genome of host cells. In various embodiments, the integrase protein is mutated to specifically reduce integrase activity. Integration-incapable lentiviral vectors are produced by modifying the pol gene encoding the integrase protein, resulting in a mutant pol gene encoding a defective integrase that cannot integrate. Such integration-incapable viral vectors were described in patent application WO 2006/010834, incorporated herein by reference in its entirety.

Illustrative examples of HIV-1 pol gene mutations useful for reducing integrase activity include, but are not limited to: H12N, H12C, H16C, H16V, S81 R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R166A, D167A, E170A, N171A , K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A and K264N.

Illustrative examples of HIV-1 pol gene mutations useful for reducing integrase activity include, but are not limited to: D64E, D64V, E92K, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A , K159E, K159A, W235F and W235E.

In a particular embodiment, the integrase contains a mutation in one or more of amino acids D64, D116, or E152. In one embodiment, the integrase contains a mutation in amino acids D64, D116, and E152. In a specific embodiment, the defective HIV-1 integrase contains the D64V mutation.

A host cell includes cells into which a recombinant vector or polynucleotide of the present invention has been introduced by electroporation, transfection, infection or transduction in vivo, ex vivo or in vitro. Host cells may include packaging cells, production cells, and cells infected with viral vectors. In specific embodiments, host cells infected with a viral vector of the present invention are administered to a subject in need of therapy. In some embodiments, the term target cell is used interchangeably with the term host cell; it refers to transfected, infected or transduced cells of the desired type. In preferred embodiments, said target cell is a T cell.

Large-scale production of virus particles is often necessary to ensure satisfactory virus titers. Viral particles are produced by transfecting packaging line cells with a transfer vector that contains viral structural and/or accessory genes, for example gag, pol, env, tat, rev, vif, vpr, vpu, vpx or nef genes, or other retroviral genes.

As used herein, the term packaging vector refers to an expression vector or viral vector that lacks a packaging signal and contains a polynucleotide encoding one, two, three, four or more viral structural and/or accessory genes. Typically, packaging vectors are included in a packaging cell; they are introduced into the specified cell by transfection, transduction or infection. Methods of transfection, transduction or infection are well known to those skilled in the art. The retroviral/lentiviral transfer vector of the present invention can be introduced into a packaging cell line by transfection, transduction or infection to produce a production cell or cell line. The packaging vectors of the present invention can be introduced into human cells or cell lines using standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, packaging vectors are introduced into cells together with a dominant selection marker, such as neomycin, hygromycin, puromycin, blasticidin, zeocin, thymidine kinase, dehydrofolate reductase (DHFR), glutamine synthase, or adenosine deaminase (ADA), followed by selection in the presence of a suitable drug and isolation of clones. The selectable marker gene may be physically linked to genes encoded by the packaging vector, for example, through IRES or self-cleaving viral peptides.

Viral envelope (env) proteins define the range of host cells that can ultimately be infected and transformed by recombinant retroviruses derived from cell lines. In the case of lentiviruses such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells according to the present

- 34 045264 of the invention, encodes a different vector, separate from the vector encoding the viral gag and pol genes as described above.

Illustrative examples of retrovirus-derived env genes that may be used in the present invention include, but are not limited to: murine leukemia virus (MLV) envelope genes, 10A1 envelope, BAEV, FeLV-B, RD114, sSaV, Ebola virus, Sendai virus, FPV (fowl distemper virus) and influenza virus shell. Likewise, genes encoding the envelopes of RNA viruses (for example, RNA viruses of the families Picornaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, Retroviridae) can be used. as well as DNA viruses (from the families Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae and Iridoviridae). Representative examples include feline leukemia virus (FeLV), Venezuelan equine encephalitis (VEE), HFVW virus, dermal sarcoma stizostedion virus (WDSV), Semliki Forest virus (SFV), rabies virus, avian leukemia virus (ALV), SIVRS, large leukemia virus bovine virus (BLV), Epstein-Barr virus (EBV), AEC, Sin Nombre virus (SNV), cherry curl virus (ChTLV), simian T-lymphotropic virus (STLV), Mason-Pfizer simian virus (MPMV), squirrel monkey retrovirus (SMRV), Rous-associated virus (RAV), Fujinami sarcoma virus (FuSV), MH2 virus, avian encephalomyelitis virus (AEV), alfalfa mosaic virus (AMV), CT10 virus and INAN.

In other embodiments, the virus pseudotyping envelope proteins of the present invention include, but are not limited to, the envelope proteins of any of the following viruses: influenza A virus, e.g., H1N1, H1N2, H3N2, and H5N1 (avian influenza), influenza B virus, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the norovirus group, enteric adenoviruses, parvovirus, dengue fever virus, monkeypox virus, viruses of the order Mononegavirales, lyssavirus such as rabies virus, Lake Lagos bat fever virus, Mokola virus, Duvenhage virus, European bat viruses 1 and 2 and Australian bat virus, Ephemerovirus, Vesiculovirus, vesicular stomatitis virus (VSV), herpesviruses such as herpes simplex virus types 1 and 2, virus varicella, cytomegalovirus, Epstein-Barr virus (EBV), human herpesviruses (HHV), human herpesviruses 6 and 8, human immunodeficiency virus (HIV), papillomavirus, murine gammaherpesvirus, arenaviruses such as Argentine hemorrhagic fever virus, virus Bolivian hemorrhagic fever, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphatic choriomeningitis virus (LCMV), Bunyaviridiae (bunyaviruses) such as Crimean-Congo hemorrhagic fever virus, hantavirus, hemorrhagic fever virus with renal syndrome, Rift Valley fever virus, Filoviridae (filoviruses), including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae, including Kyasanur forest disease virus, Omsk hemorrhagic fever virus, tick-borne viral encephalitis virus and viruses of the Paramixoviridae family such as Hendra virus and Nipah virus, smallpox major and smallpox (variola), alphaviruses such as Venezuelan horse fever virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-associated coronavirus (SARS-CoV), West Nile virus, any virus that causes the development of encephalitis.

In one embodiment, the present invention provides packaging cells that produce a recombinant retrovirus, for example, a VSV-G glycoprotein pseudotyped lentivirus.

The terms pseudotype or pseudotyping as used herein refer to a virus whose viral envelope proteins have been replaced by the envelope proteins of another virus having preferred characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus envelope G protein (VSV-G), allowing HIV to infect a wider range of cells because HIV envelope proteins (encoded by the env gene) typically target the virus to CD4+ presenting cells. In a preferred embodiment of the present invention, the lentiviral envelope proteins are pseudotyped with VSV-G. In one embodiment, the present invention provides packaging cells that produce a recombinant retrovirus, for example, a VSV-G envelope glycoprotein pseudotyped lentivirus.

As used herein, the term packaging cell lines refers to cell lines that do not contain a packaging signal but stably or transiently express viral structural proteins and replication enzymes (eg, gag, pol, and env) necessary for proper packaging of viral particles. Any suitable cell line can be used to produce packaging cells according to the present invention. Typically, said cells are mammalian cells. In a specific embodiment, the cells used to produce the packaging cell line are human cells. Suitable cell lines include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1/2 cells, FLY cells, Psi-2 cells, BOSC23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC cells 40,

- 35 045264 BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, cells

HeLa, W163 cells, 211 cells and 211A cells. In preferred embodiments, said packaging cells are 293 cells, 293T cells, or A549 cells. In another preferred embodiment, said cells are A549 cells.

As used herein, the term production cell line refers to a cell line capable of producing recombinant retroviral particles, including a packaging cell line and a vector for transferring a construct containing a packaging signal. Infectious viral particles and viral stock solutions can be prepared using standard techniques. Methods for obtaining initial viral solutions are known in the art and their illustrative examples are given, for example, in the sources: Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633, and N. R. Landau et al. (1992) J. Virol. 66:5110–5113. Infectious viral particles can be obtained from packaging cells using standard techniques. For example, infectious particles can be obtained by lysing cells or collecting cell culture supernatant, as is known in the art. If necessary, the collected viral particles may optionally be purified. Suitable cleansing techniques are well known to those skilled in the art.

Delivery of gene(s) or other polynucleotide sequence using a retroviral or lentiviral vector by viral infection rather than transfection is called transduction. In one embodiment, retroviral vectors are used to transduce a cell through infection and integration of a provirus. In some embodiments, a target cell, eg, a T cell, is transduced if it contains a gene or other polynucleotide sequence delivered to the cell by infection using a viral or retroviral vector. In certain embodiments, the transduced cell contains one or more genes or other polynucleotide sequences delivered into the cell genome by a retroviral or lentiviral vector.

In specific embodiments, host cells transduced with a viral vector of the present invention expressing one or more polypeptides are administered to a subject to treat and/or prevent a B cell malignancy. Other methods that involve the use of viral vectors in gene therapy and can be used in accordance with certain embodiments of the present invention can be found, for example, in the sources: Kay, M. A. (1997) Chest 111(6 Supp.): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11:179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7:1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, S. L. and Crystal, R. G. (1994) Ann. N.Y. Acad. Sci. 716:90-101; Strayer, D. S. (1999) Expert Opinion. Investig. Drugs 8:2159–2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3:43-49; and Lee, H. C et al. (2000) Nature 408:483-8.

G. Genetically modified cells

The present invention provides, in particular embodiments, cells genetically modified to express a CAR of the present invention for use in treating B cell-related conditions. As used herein, the term genetically engineered or genetically modified refers to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms genetically modified cells, engineered cells, and redirected cells are used interchangeably. As used herein, the term gene therapy refers to the introduction of additional genetic material in the form of DNA or RNA into the general genetic material in a cell that restores, corrects or modifies the expression of a gene, or its introduction to express a therapeutic polypeptide, for example, a CAR.

In specific embodiments, the CARs of the present invention are administered to and expressed in immune effector cells in such a way as to redirect their specificity to a target antigen of interest, for example, a BCMA polypeptide. An immune effector cell is any cell of the immune system that performs one or more effector functions (eg, cytotoxic cell killing activity, cytokine secretion, induction of ADCC and/or CDC).

Immune effector cells according to the present invention can be autologous/autogeneic (self) or non-autologous (non-self, for example, allogeneic, syngeneic or xenogeneic).

Autologous as used herein refers to cells from the same subject.

Allogeneic as used herein refers to cells of the same species that are genetically different from the cell being compared.

Syngeneic as used herein refers to cells from another subject that are genetically identical to the cell being compared.

Xenogeneic herein refers to cells of a species to which it does not belong.

- 36 045264 compared cell. In preferred embodiments, the cells of the present invention are allogeneic.

Exemplary immune effector cells used with the CARs of the present invention include T lymphocytes. The terms T cell or T lymphocyte are accepted in the art and are intended to refer to thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cell may be a helper T cell (Th), such as a helper T cell 1 (Th1) or a helper T cell 2 (Th2). The T cell may be a helper T cell (HTL; CD4+ T cell) CD4 ⁺ T cell, a cytotoxic T cell (CTL; CD8+ T cell), CD4 ⁺ CD8+ T cell, CD4-CD8 ^- T- cell or T cells of any other subgroup. Other illustrative examples of T cell populations suitable for use in particular embodiments include naïve T cells and memory T cells.

As one skilled in the art will appreciate, other cells may also be used as immune effector cells with CAR receptors as described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils and macrophages. Immune effector cells also include effector cell precursors, and such precursor cells can be induced to differentiate into immune effector cells in vivo or in vitro. Accordingly, in specific embodiments, the immune effector cell includes immune effector cell precursors, such as hematopoietic stem cells (HSCs) contained in a population of CD34+ cells derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which, when administered to a subject, differentiate into mature immune cells. effector cells, or their differentiation into mature immune effector cells can be induced in vitro.

As used herein, genetically engineered immune effector cells containing BCMA-specific CAR receptors may be referred to as BCMA-specific redirected immune effector cells.

The term CD34+ cell as used herein refers to a cell expressing CD34 protein on its surface. CD34 as used herein refers to a cell surface glycoprotein (eg, sialomucin protein) that often functions as an intercellular adhesion factor and is involved in T cell entry into lymph nodes. The CD34+ cell population includes hematopoietic stem cells (HSCs), which, when injected into a patient, differentiate and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and cells of the monocyte/macrophage lineage.

The present invention provides methods for producing immune effector cells that express the CAR of the present invention. In one embodiment, the method comprises transfecting or transducing immune effector cells isolated from an individual such that the immune effector cells express one or more CARs as described herein. In some embodiments, the immune effector cells are isolated from the individual and genetically modified without further in vitro manipulation. Such cells can then be directly reintroduced back to the individual. In further embodiments, said immune effector cells are first activated and stimulated to proliferate in vitro before being genetically modified to express the CAR. Thus, these immune effector cells can be cultured before and/or after genetic modification (ie, transduction or transfection to express the CAR provided by the present invention).

In specific embodiments, prior to in vitro manipulation or genetic modification of immune effector cells described herein, parent cells are obtained from a subject. In specific embodiments, the CAR-modified immune effector cells include T cells. 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, tissue from an infected site, ascites, pleural effusion, tissue spleen and tumors. In some embodiments, T cells can be obtained from a volume of blood collected from a subject using any number of techniques known to one skilled in the art, such as sedimentation, such as Ficoll™ separation. In one embodiment, circulating blood cells from an individual are obtained through apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing. These cells can be washed with PBS or other suitable solution that is free of calcium, magnesium, and most or all other divalent cations. As will be appreciated by those skilled in the art, the washing step can be carried out using methods known in the art.

- 37 045264 field of technology methods, for example, using a semi-automatic flow centrifuge, for example, a Cobe 2991 cell processor, a Baxter CytoMate or the like. After washing, the cells can be resuspended in various biocompatible buffers or other saline solution, with or without buffer. In some embodiments, unwanted components of the apheresis sample can be removed from the culture medium where the cells are directly resuspended.

In some embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by erythrocyte lysis and monocyte depletion, for example, by Percoll gradient centrifugation (PERCOLL™). Using positive or negative selection techniques, a specific subpopulation of T cells expressing one or more of the following markers can then be isolated: CD3, CD28, CD4, CD8, CD45RA and CD45RO. In one embodiment, a specific subpopulation of T cells expressing CD3, CD28, CD4, CD8, CD45RA, and CD45RO is further isolated using positive or negative selection techniques. For example, negatively selected enrichment of a T cell population can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is the sorting and/or selection of cells by negative magnetic immune adhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on cells selected using negative selection. For example, to enrich for CD4+ cells using negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR and CD8. Flow cytometry and sorting may also be used to isolate cell populations of interest for use in the present invention.

PBMCs can be directly genetically modified to express CAR receptors using the methods provided by the present invention. In some embodiments, after the PBMCs are isolated, T lymphocytes are further isolated; in some embodiments, both cytotoxic and helper T lymphocytes can be sorted into naïve, memory, and effector T cell subpopulations, either before or after genetic modification and/or expansion.

CD8+ cells can be obtained using standard methods. In some embodiments, CD8+ cells are further sorted into naive cells, central memory cells, and effector cells by identifying cell surface antigens associated with each of these CD8+ cell types.

In some embodiments, naive CD8+ T lymphocytes are characterized by the expression of naïve T cell phenotypic markers, including CD62L, CCR7, CD28, CD3, CD127, and CD45RA.

In specific embodiments, memory T cells are present in both CD62L+ and ^CD62L− subsets of peripheral blood CD8+ lymphocytes. PBMCs are sorted into CD62L-CD8+ and CD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62L antibodies. In some embodiments, the expression of phenotypic markers of the central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127, and they are granzyme B negative. In some embodiments, the central memory T cells are CD45RO+, CD62L+, CD8+ T cells.

In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

In some embodiments, the CD4+ T cells are further sorted into subpopulations. For example, CD4+ helper T cells can be sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained using standard methods. In some embodiments, the naïve CD4+ T lymphocytes are ^CD45RO- , CD45RA+, CD62L+ CD4+ T cells. In some embodiments, the central CD4+ memory cells are CD62L positive and CD45RO positive. In some embodiments, the effector CD4+ cells are CD62L negative and CD45RO negative.

Immune effector cells, such as T cells, can be genetically modified after isolation using known methods, or these immune effector cells can be activated and expanded (or, in the case of progenitors, differentiated) in vitro before genetic modification is carried out. In a specific embodiment, said immune effector cells, such as T cells, are genetically modified with chimeric antigen receptors of the present invention (eg, transduced with a viral vector containing a nucleic acid encoding a CAR) and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR using methods as described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681;

7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and US Patent Application Published No. 20060121005.

Typically, T cells are propagated by contacting a surface to which is attached an agent that stimulates a CD3-TCR complex-associated signal and a ligand that stimulates a costimulatory molecule on the surface of said T cells. T cell populations can be stimulated by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or a surface-immobilized anti-CD2 antibody, or by contact with a protein kinase C activator (eg, bryostatin) in combination with a calcium ionophore. Costimulation of accessory molecules on the surface of T cells is also included.

In specific embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and a co-stimulatory agent, e.g., anti-CD3 and anti-CD28 antibodies, typically attached to a bead or other surface, in a culture medium with suitable cytokines, such as IL-2 , IL-7 and/or IL-15. An anti-CD3 antibody and an anti-CD28 antibody are used to stimulate the proliferation of either CD4 T cells or CD8 T cells. Suitable anti-CD28 antibodies such as 9.3, B-T3, XR-CD28 (Diacione, Besançon, France), as well as other methods well known in the art (Berg et al., Transplant Proc. 30(8) :3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999 ). Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a surrogate antigen presenting cell (APC). In other embodiments, T cells can be activated and stimulated to proliferate with feeder cells and suitable antibodies and cytokines using methods such as those described in US6040177; US5827642; and WO2012129514.

In other embodiments, artificial APCs (iAPCs) are produced by engineering K562, U937, 721.221, T2, and C1R cells to ensure stable expression and secretion of various co-stimulatory molecules and cytokines. In a specific embodiment, the K32 or U32 iAPC is used to cause one or more antibody-based stimulatory molecules to be displayed on the cell surface of the iAPC. Expression of different gene combinations on iAPCs allows us to precisely define the conditions required for activation of human T cells, so iAPCs can be modified to optimally expand T cell subsets with specific growth requirements and distinct functions. iAPCs support ex vivo growth and long-term proliferation of functional human CD8 T cells, and, unlike the use of natural APCs, there is no need for the addition of exogenous cytokines. T cell populations can be expanded using iAPCs expressing various co-stimulatory molecules, including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Finally, these iAPCs provide an effective platform for the expansion of genetically modified T cells and maintenance of CD28 expression on CD8 T cells. The iAPCs proposed in WO 03/057171 and US2003/0147869 are incorporated herein by reference in their entirety.

In one embodiment, CD34+ cells are transduced with a nucleic acid-containing construct of the present invention. In some embodiments, the transduced CD34+ cells differentiate into mature immune effector cells in vivo after administration to the subject, typically the same subject from which they were originally isolated. In another embodiment, CD34+ cells can be stimulated in vitro, before exposure to or after genetic modification of the CAR receptor as described herein, with one or more of the following cytokines: Flt-3 ligand (FLT3), stem cell factor (SCF), growth factor and megakaryocyte differentiation (TPO), IL-3 and IL-6, according to previously described methods (Asheuer et al., 2004; Imren, et al., 2004).

The present invention provides a population of modified immune effector cells for the treatment of cancer, wherein said modified immune effector cells contain CARs as described herein. For example, a population of modified immune effector cells is derived from peripheral blood mononuclear cells (PBMCs) obtained from a patient diagnosed with the B cell malignancy described herein (autologous donors). These PBMC cells form a heterogeneous population of T lymphocytes, which can be CD4+, CD8+, or CD4+ and CD8+ T lymphocytes.

PBMC cells may also include other cytotoxic lymphocytes such as NK cells or NKT cells. An expression vector carrying a CAR coding sequence provided by the present invention can be introduced into a population of donor T cells, NK cells or NKT cells of a person. Successfully transduced T cells carrying said expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then further expanded to increase the number of said CAR protein expressing T cells, along with cell activation using antibodies against CD3, and/or antibodies against CD28 and IL-2, or any other methods known in the art, as described in the text of this document. Standard procedures are used to cryopreserve CAR protein-expressing T cells for storage and/or formulation for use in a human subject. In one embodiment, transduction, culture, and/or expansion of T cells in vitro is performed in the absence of non-human animal products, such as fetal bovine serum and fetal bovine serum. Once a heterogeneous population of PBMC cells has been genetically modified, the resulting transduced cells are a heterogeneous population of modified cells containing the BCMA-targeting CAR receptor of the present invention.

In a further embodiment, a mixture of, for example, one, two, three, four, five or more different expression vectors, each vector encoding a different chimeric antigen receptor protein of the present invention, can be used to genetically modify a donor population of immune effector cells. The resulting modified immune effector cells form a mixed population of modified cells containing a number of modified cells expressing more than one CAR protein.

In one embodiment, the present invention provides a method for storing genetically modified CAR protein-expressing murine, human, or humanized cells targeting the BCMA protein, comprising cryopreserving said immune effector cells such that the cells remain viable when thawed. A fraction of immune effector cells expressing CAR proteins can be cryopreserved using methods known in the art to provide a continuous source of such cells for future treatment of patients suffering from a B cell-related condition. If necessary, these cryopreserved transformed immune effector cells can be thawed, cultured and expanded to obtain more such cells.

As used herein, cryopreservation/cryoprotection refers to the preservation of cells by cooling to a temperature below zero, for example (typically) 77 K or -196°C (the boiling point of liquid nitrogen). Cryoprotective agents are often used at temperatures below freezing to prevent cell damage when frozen at low temperatures or warmed to room temperature. Cryoprotective agents and optimal cooling rates can protect cells from damage. Suitable cryoprotective agents include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature, 1959; 183: 1394-1395; AshwoodSmith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann N.Y. Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196: 48). The preferred cooling rate is 1° to 3°C/minute. After at least two hours, the T cells reach -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage vessel.

H. Methods for obtaining T cells

T cells obtained using the methods provided by the present invention provide improved compositions for adoptive immunotherapy. Without being limited by any particular theory, it is believed that T cell compositions produced using the methods of the present invention have improved characteristics, including increased survival, expansion while remaining relatively undifferentiated, and persistence in vivo. In one embodiment, a method of producing T cells includes contacting said cells with one or more agents that modulate the cellular P13K signaling pathway. In one embodiment, a method of producing T cells includes contacting the cells with one or more agents that modulate the PI3K/Akt/mTOR cellular signaling pathway. In various embodiments, said T cells can be obtained from any source and brought into contact with said agent during the activation and/or expansion phase of the production process. The resulting T cell compositions are enriched for differentiation potential T cells capable of proliferation and expression of one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In one embodiment, cell populations containing T cells that have been treated with one or more PI3K inhibitors are enriched for a population of CD8+ T cells coexpressing one or more or all of the following biomarkers: CD62L, CD127, CD197, and CD38.

In one embodiment, modified T cells are produced that are characterized by maintaining levels of proliferation and decreasing differentiation. According to specific option

- 40 045264 implementations T cells are obtained by stimulating T cells to activate and proliferate in the presence of one or more stimulatory signals and an agent that is an inhibitor

P13K cell signaling pathways.

T cells can then be modified to express anti-BCMA CAR receptors. In one embodiment, T cells are modified by transducing said T cells with an anti-BCMA CAR-containing viral vector of the present invention. In a certain embodiment, T cells are modified to be stimulated and activated in the presence of an inhibitor of the P13K cell signaling pathway. In another embodiment, T cells are modified after stimulation and activation in the presence of an inhibitor of the P13K cell signaling pathway. In a specific embodiment, the T cells are modified within a period of 12 hours, 24 hours, 36 hours, or 48 hours after stimulation and activation in the presence of a PI3K cell signaling pathway inhibitor.

Once the T cells are activated, the cells are cultured to allow proliferation. The T cell culture may be for at least 1, 2, 3, 4, 5, 6 or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5 or 6 months or more, and include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds of breeding.

In various embodiments, T cell compositions are prepared in the presence of one or more PI3K pathway inhibitors. These inhibitors may target one or more activities within a pathway, or a single activity. Without being limited to any particular theory, it is believed that treating or exposing T cells to one or more PI3K pathway inhibitors during the stimulation, activation and/or expansion phases of the production process will preferentially increase the content of young T cells, thereby producing improved therapeutic compositions with T cells.

According to a specific embodiment, a method is provided for increasing the proliferation of T cells expressing an engineered T cell receptor. Such methods may include, for example, taking parental T cells from a subject, stimulating and activating said T cells in the presence of one or more PI3K pathway inhibitors, modifying said T cells to express an anti-BCMA CAR, e.g., an anti-BCMA02 CAR, and expanding the indicated T cells in culture.

In one embodiment, a method is provided for producing T cell populations enriched for cells expressing one or more of the following biomarkers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In one embodiment, the young T cells contain one or more of the following biological markers: CD62L, CD127, CD197, and CD38; or all specified biomarkers. One embodiment provides young T cells that do not express CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3. As described in various sections herein, the levels of expression of biomarkers by young T cells are related to the levels of expression of such markers in populations of more differentiated T cells or immune effector cells.

In one embodiment, peripheral blood mononuclear cells (PBMCs) are used as a source of T cells in the T cell production methods of the present invention. PBMC cells form a heterogeneous population of T lymphocytes, which may include CD4 ⁺ , CD8 + , or CD4 + and CD8 + T lymphocytes, and which may include other mononuclear cells such as monocytes, B cells, NK cells, and NKT cells. An expression vector containing a polynucleotide encoding an engineered TCR or CAR provided by the present invention can be introduced into a population of donor T cells, NK cells or NKT cells of a person. Successfully transduced T cells carrying the specified expression vector can be sorted using flow cytometry to isolate CD3 positive T cells and then expanded to increase the number of modified T cells, along with cell activation using anti-CD3 antibodies and or antibodies against CD28, as well as IL-2, IL-7 and/or IL-15 or any other methods known in the art, as described herein.

The production methods provided by the present invention may further include cryopreservation of the modified T cells for storage and/or formulation for use in a human subject. T cells are cryopreserved so that they remain viable when thawed. If necessary, these cryopreserved transformed immune effector cells can be thawed, cultured and expanded to obtain large quantities of such cells. As used herein, cryopreservation/cryoprotection refers to the preservation of cells by cooling to a temperature below zero, for example (typically) 77 K or 196°C. (boiling point of liquid nitrogen). Cryoprotective agents are often used at temperatures below freezing to prevent cell damage when frozen at low temperatures or warmed to room temperature. Cryoprotective agents and optimal cooling rates can protect cells from damage. Suitable cryoprotective agents include, but are not limited to, dimethyl sulfoxide (DMSO) (Lovelock and Bishop, Nature,

- 41 045264

1959; 183: 1394-1395; Ashwood-Smith, Nature, 1961; 190: 1204-1205), glycerol, polyvinylpyrrolidine (Rinfret, Ann. N.Y. Acad. Sci., 1960; 85: 576) and polyethylene glycol (Sloviter and Ravdin, Nature, 1962; 196:

48). The preferred cooling rate is 1° to 3°C/minute. After at least two hours, the T cells reach -80°C and can be placed directly into liquid nitrogen (-196°C) for permanent storage, such as in a long-term cryogenic storage vessel.

1. T cells

The present invention provides for the production of improved compositions bearing CAR T cells. The T cells used to generate CAR T cells may be autologous/autogeneic (self) or non-autologous (non-self, e.g. allogeneic, syngeneic or xenogeneic). In preferred embodiments, said T cells are obtained from a mammalian subject. In a more preferred embodiment, said T cells are obtained from a primate subject. In a most preferred embodiment, said T cells are obtained from a human subject.

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, infected site tissue, ascites, pleural effusion, spleen tissue and tumors. In some embodiments, T cells can be obtained from a volume of blood collected from a subject using any number of techniques known to one skilled in the art, for example, precipitation such as Ficoll™ separation. In one embodiment, circulating blood cells from an individual are obtained using apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected using apheresis may be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing. Cells can be washed with PBS or other suitable solution free of calcium, magnesium, and most or all other divalent cations. As will be understood by those skilled in the art, the washing step can be performed using methods known in the art, for example, using a semi-automated flow centrifuge, such as a Cobe 2991 Cell Processor, Baxter CytoMate or the like. After washing, the cells can be resuspended in various biocompatible buffers or other saline solution, with or without buffer. In some embodiments, undesired components of the apheresis sample can be removed from the culture medium in which the cells are directly resuspended.

In certain embodiments, the production methods of the present invention utilize a population of cells comprising T cells, such as PBMC cells. In other embodiments, the production methods of the present invention utilize an isolated or purified population of T cells. Cells can be isolated from peripheral blood mononuclear cells (PBMCs) by erythrocyte lysis and monocyte depletion, for example, by Percoll gradient centrifugation (PERCOLL™). In some embodiments, after PBMC isolation, both cytotoxic and helper T lymphocytes can be sorted into subpopulations of naïve T cells, memory T cells, and effector T cells, either before or after activation, expansion, and/or genetic modification.

Additionally, a specific subpopulation of T cells expressing one or more of the following markers can be isolated using positive or negative selection techniques: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127 and HLA-DR. In one embodiment, using positive or negative selection techniques, a specific subpopulation of T cells expressing one or more markers selected from the group consisting of i) CD62L, CCR7, CD28, CD27, CD122, CD127, CD197 is further isolated; or ii) CD38 or CD62L, CD127, CD197 and CD38. In various embodiments, the resulting T Cell compositions do not express, or substantially do not express, one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, the expression of one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38 is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, by at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least at least 25-fold or more compared to expression in a population of T cells activated and expanded in the absence of a PI3K inhibitor.

In one embodiment, the expression of one or more markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3 is reduced by at least 1.5-fold, at least 2-fold, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, according to at least 10 times, at least 25 times or more compared to the T cell population,

- 42 045264 activated and propagated in the presence of a PI3K inhibitor.

In one embodiment, the production methods of the present invention provide an increase in the number of CAR-bearing T cells containing one or more markers of naïve or differentiation-potential T cells. Without limitation to any particular theory, the present inventors believe that treatment of a population of cells containing T cells with one or more PI3K inhibitors results in stimulation of the expansion of differentiated T cells and provides more reliable and effective adoptive immunotherapy with CAR T cells. compared to existing CAR T cell-carrying therapy options.

Illustrative examples of naive or differentiation potential T cell markers that are elevated in T cells obtained using the methods of the present invention include, but are not limited to, CD62L, CD127, CD197, and CD38. In specific embodiments, naive T cells do not express or substantially do not express one or more of the following markers: CD57, CD244, CD160, PD-1, BTLA, CD45RA, CTLA4, TIM3, and LAG3.

With respect to T cells, the T cell populations resulting from the various expansion techniques provided by the present invention may exhibit different specific phenotypic properties depending on the conditions involved. In various embodiments, the expanded T cell populations contain one or more of the following phenotypic markers: CD62L, CD127, CD197, CD38, and HLA-DR.

In one embodiment, such phenotypic markers include increased expression of one or more of CD62L, CD127, CD197, and CD38, or all of these markers. In specific embodiments, CD8 T lymphocytes are expanded that are characterized by the expression of phenotypic markers of naïve T cells, including CD62L, CD127, CD197, and CD38.

In specific embodiments, T cells are expanded that express phenotypic markers of central memory T cells, including CD45RO, CD62L, CD127, CD197, and CD38 positive and granzyme B negative cells. In some embodiments, said central memory T cells are CD45RO+, CD62L+, CD8+ T cells.

In some embodiments, CD4+ T lymphocytes are expanded that are characterized by the expression of phenotypic markers of naïve CD4+ cells, including those positive for CD62L and negative for CD45RA and/or CD45RO expression. In some embodiments, CD4+ cells characterized by the expression of phenotypic markers of central CD4+ memory cells include CD62L positive and CD45RO cells. In some embodiments, the effector CD4+ cells are CD62L positive and CD45RO negative.

In some embodiments, T cells are isolated from an individual and activated and stimulated to proliferate in vitro before being genetically modified to express an anti-BCMA CAR. Thus, culturing of T cells can be carried out before and/or after genetic modification (ie, transduction or transfection to express the anti-BCMA CAR provided by the present invention).

2. Activation and reproduction

To provide sufficient therapeutic doses of T cell compositions, one or more rounds of T cell stimulation, activation and/or expansion are often performed. T cells can be activated and expanded generally using methods as described, for example, in US patents 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; and 6867041, each of which is incorporated herein by reference in its entirety. T cells modified to express an anti-BCMA CAR may be activated and expanded before and/or after modification of said T cells. In addition, T cells can be brought into contact with one or more agents that modulate the cellular P13K signaling pathway before, during, and/or after activation and/or proliferation. In one embodiment, T cells produced using the methods of the present invention undergo one, two, three, four, or five or more rounds of activation and expansion, each of which may include one or more agents that modulate the cellular PI3K signaling pathway .

In one embodiment, a costimulatory ligand is exposed on an antigen presenting cell (e.g., iAPC, dendritic cell, B cell, etc.) that specifically binds a cognate costimulatory molecule on the T cell, thereby providing a signal that, along with the primary signal , provided, for example, by the binding of the TCR/CD3 complex, mediates the desired T-cell response. Suitable costimulatory ligands include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), molecule intercellular adhesion (ICAM), CD30L, CD40, CD70, CD83,

- 43 045264

HLA-G, MICA, MICB, HVEM, lymphotoxin-beta receptor, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor, and a ligand that specifically binds to B7-H3.

In a specific embodiment, the costimulatory ligand includes an antibody or antigen-binding fragment thereof that specifically binds to a costimulatory molecule present on a T cell, including, but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD -1, 1COS, lymphocyte functional antigen-1 (LFA-1), CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds CD83.

Suitable costimulatory ligands also include target antigens that can be presented in soluble form or expressed on APCs or iAPCs that bind engineered TCRs or CARs expressed on engineered T cells.

In various embodiments, a method for producing T cells of the present invention includes activating a population of cells containing T cells and expanding said population of T cells. Activation of T cells can be accomplished by providing a primary stimulatory signal through the T-cell TCR/CD3 complex or stimulation of the surface protein CD2, as well as by providing a secondary co-stimulatory signal through an accessory molecule, such as CD28.

Stimulation of the TCR/CD3 complex can be accomplished by contacting the T cell with a suitable CD3 binding agent, for example, a CD3 ligand or an anti-CD3 monoclonal antibody. Illustrative examples of anti-CD3 antibodies include, but are not limited to, OKT3, G19-4, BC3, and 64.1.

In another embodiment, a CD2 binding agent may be used to provide a primary stimulatory signal to T cells. Illustrative examples of CD2 binding agents include, but are not limited to, CD2 ligands and anti-CD2 antibodies, for example, antibody T11.3 in combination with antibody T11.1 or T11.2 (Meuer, S. C. et al. (1984) Cell 36 :897-906), and antibody 9.6 (which recognizes the same epitope as T11.1) in combination with antibody 9-1 (Yang, S. Y. et al. (1986) J. Immunol. 137:1097-1100). Other antibodies that bind to the same epitopes as any of the antibodies described above may also be used. Additional antibodies or combinations of antibodies can be generated and identified using standard techniques as described in various sections herein.

In addition to the primary stimulatory signal passing through the TCR/CD3 complex, or through CD2, a second, co-stimulatory signal is required to induce a T-cell response. In certain embodiments, a CD28 binding agent may be used to provide a co-stimulatory signal. Illustrative examples of CD28 binding agents include, but are not limited to: natural CD28 ligands, e.g., natural CD28 ligand (e.g., a member of the B7 protein family, such as B7-1 (CD80) and B7-2 (CD86); and anti-CD28 monoclonal antibody or its fragment capable of cross-linking the CD28 molecule, for example, monoclonal antibodies 9.3, B-T3, XR-CD28, KOLT-2, 15E8, 248.23.2 and EX5.3D10.

In one embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR/CD3 or CD2 complex, and the co-stimulatory molecule are attached to the same surface.

In some embodiments, the binding agents that provide stimulatory and co-stimulatory signals are located on the cell surface. This can be achieved by transfecting or transducing the cell with a nucleic acid encoding the binding agent in a form suitable for expression on the cell surface or, alternatively, by coupling the binding agent to the cell surface.

In another embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR/CD3 or CD2 complex, and the co-stimulatory molecule are displayed on antigen presenting cells.

In one embodiment, the molecule that provides the primary stimulatory signal, for example, the molecule that provides stimulation through the TCR/CD3 or CD2 complex, and the co-stimulatory molecule are located on separate surfaces.

In a particular embodiment, one of the binding agents that provide the stimulatory and co-stimulatory signals is soluble (present in solution) and the other agent(s) are located on one or more surfaces.

In a specific embodiment, the binding agents that provide the stimulatory and co-stimulatory signals are provided in a soluble form (in solution).

In various embodiments, the methods for producing T cells of the present invention include activating the T cells with anti-CD3 and anti-CD28 antibodies.

T cell compositions prepared using the methods of the present invention contain T cells activated and/or expanded in the presence of one or more agents that inhibit the cellular P13K signaling pathway. T cells modified to express anti-BCMA CARs can be activated and expanded before and/or after modification of said T cells. In specific embodiments, a population of T cells is activated, modified to express anti-BCMA CARs, and then cultured for expansion.

In one embodiment, T cells produced using the methods of the present invention include an increased number of T cells expressing markers indicative of high proliferative potential and self-renewal capacity, but not expressing or expressing substantially undetectable markers of T cell differentiation . These T cells can be repeatedly activated and expanded in a reliable manner, thereby obtaining an improved therapeutic T cell composition.

In one embodiment, the population of T cells activated and expanded in the presence of one or more agents that inhibit the cellular PI3K signaling pathway is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, according to at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, at least 1000 times or more compared to the T cell population, activated and propagated in the absence of a PI3K inhibitor.

In one embodiment, the population of T cells characterized by the expression of markers of young T cells activated and expanded in the presence of one or more agents that inhibit the cellular P13K signaling pathway is increased by at least 1.5-fold, at least 2-fold , at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, at least 1000 times or more compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

In one embodiment, expansion of T cells activated using the methods of the present invention further comprises culturing a population of cells containing the T cells for a period ranging from several hours (about 3 hours) to about 7 days to about 28 days, or corresponding to any integer number of hours in the range between the specified values. In another embodiment, said T cell composition can be cultured for 14 days. In a specific embodiment, the T cells are cultured for approximately 21 days. In another embodiment, the T cell compositions are cultured for approximately 2-3 days. Several additional stimulation/activation/propagation cycles may be required; thus, the duration of T cell culture may be 60 days or more.

In specific embodiments, conditions suitable for culturing T cells include a suitable medium (e.g., minimal essential medium or RPMI 1640 medium, or Xvivo 15 (Lonza)) and one or more factors necessary for proliferation and viability, including, but not limited to, serum (eg, fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, IL-21, GM-CSF, IL- 10, IL12, IL-15, TRvf and TNF-α, or any other additives known to those skilled in the art that are suitable for cell growth.

Additional illustrative examples of cell culture media include, but are not limited to, RPMI 1640, Clicks, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, amino acid supplemented, pyruvate sodium and vitamins, either without serum or with the addition of an appropriate amount of serum (or plasma) or a specific set of hormones and/or an amount of cytokine(s) sufficient for the growth and proliferation of T cells.

Illustrative examples of other T cell expansion additives include, but are not limited to, surfactants, plasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.

Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures for infusion into the subject. The target cells are maintained under conditions necessary to ensure growth, for example, at a suitable temperature (eg, 37°C) and in a suitable atmosphere (eg, air plus 5% CO ₂ ).

In specific embodiments, PBMCs or isolated T cells are contacted with a stimulatory agent and a co-stimulatory agent, e.g., anti-CD3 and anti-CD28 antibodies, typically attached to a bead or other surface, in a culture medium with suitable cytokines, such as IL-2 , IL-7 and/or IL-15.

In other embodiments, artificial APCs (iAPCs) are produced by engineering K562, U937, 721.221, T2, and C1R cells to direct the stable expression and secretion of various co-stimulatory molecules and cytokines. In a specific embodiment, a K32 or U32-based iAPC is used to target one or more stimulus

- 45,045,264 antibody-based molecules on the surface of iAPC cells. T cell populations can be expanded using iAPCs expressing various co-stimulatory molecules, including, but not limited to, CD137L (4-1BBL), CD134L (OX40L), and/or CD80 or CD86. Finally, iAPCs provide an effective platform for the expansion of genetically modified T cells and maintenance of CD28 expression on CD8 T cells. The iAPC cells proposed in WO 03/057171 and US2003/0147869 are incorporated herein by reference in their entirety.

3. Agents

In various embodiments, a method of producing T cells is provided that allows expansion of undifferentiated or differentiated T cells, comprising contacting the T cells with an agent that modulates the PI3K pathway in said cells. In various embodiments, a method of producing T cells is provided, characterized by the expansion of undifferentiated or differentiated T cells, comprising contacting the T cells with an agent that modulates the PI3K/AKT/mTOR pathway in said cells. Contact of said cells with the agent may occur before, during and/or after activation and proliferation. T cell compositions retain sufficient differentiation potential such that T cells can undergo multiple rounds of expansion without significantly increasing the degree of differentiation.

As used herein, the terms modulate, modulator or modulating agent, or similar terms refer to the ability of an agent to cause changes in a cellular signaling pathway. A modulator may increase or decrease the amount or activity of a pathway component, or increase or decrease the desired effect or outcome of a cellular signaling pathway. According to one embodiment, said modulator is an inhibitor. According to another embodiment, said modulator is an activator.

The term agent refers to a compound, small molecule, e.g., small organic molecule, nucleic acid, polypeptide, or fragment, isoform, variant, analog, or derivative thereof, used to modulate the PI3K/AKT/mTOR pathway.

The term small molecule refers to a composition whose molecular weight is less than about 5 kDa, less than about 4 kDa, less than about 3 kDa, less than about 2 kDa, less than about 1 kDa, or less than about 0.5 kDa. Small molecules may include nucleic acids, peptides, polypeptides, peptidomimetics, peptoids, carbohydrates, lipids, components thereof, or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, for example based on extracts of fungi, bacteria or algae, are known in the art; they can be screened using any of the assays of the present invention. Examples of methods for synthesizing molecular libraries can be found in: (Carell et al., 1994a; Carell et al., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994; Zuckermann et al. , 1994).

The term analogue refers to a small organic compound, nucleotide, protein or polypeptide having similar or identical activity or function(s) as the compound, nucleotide, protein or polypeptide, or a compound having the desired activity according to the present invention, but not necessarily containing the sequence or a structure similar or identical to the sequence or structure according to a preferred embodiment.

The term derivative refers to a compound, protein or polypeptide that contains the amino acid sequence of the parent protein or polypeptide that has been modified by substitutions, deletions or additions of amino acid residues, or a nucleic acid or nucleotide that has been modified a) by introducing substitutions or deletions, additions or mutations of nucleotides.

A derivative of a nucleic acid, nucleotide, protein, or polypeptide has a function similar or identical to the function of the parent polypeptide.

In various embodiments, the PI3K pathway modulating agent activates a component of the pathway. The term activator or agonist refers to an agent that promotes, enhances or induces one or more activities of a molecule in the PI3K/AKT/mTOR pathway, including, without limitation, a molecule that inhibits one or more activities of PI3K.

In various embodiments, the PI3K pathway modulating agent inhibits a component of the pathway. The term inhibitor or antagonist refers to an agent that inhibits, reduces or attenuates one or more activities of a molecule in the PI3K pathway, including, but not limited to, PI3K. In one embodiment, the inhibitor is a dual molecular inhibitor. In a specific embodiment, the inhibitor may inhibit a class of molecules having the same or substantially similar activities (universal inhibitor) or may specifically inhibit the activity of one molecule (selective or specific inhibitor). Inhibition may also be irreversible or reversible.

- 46 045264

In one embodiment, the IC50 of said inhibitor is at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 µM, at least 10 µM, at least 50 µM or at least 100 µM. The IC50 concentration can be determined using any standard techniques known in the art. For example, the IC50 can be determined by the change in activity of a particular enzyme in the presence of a certain range of concentrations of the inhibitor of interest. Then a graph of the dependence of the experimentally obtained enzyme activity values on the inhibitor concentrations used is plotted. The inhibitor concentration at which 50% of the enzyme activity is observed (compared to the activity in the absence of any inhibitor) is taken as the IC50 value. Likewise, other inhibitory concentrations can be determined by determining the activity in a suitable manner.

In various embodiments, T cells are contacted, treated, or cultured with one or more PI3K pathway modulators at a concentration of at least 1 nM, at least 2 nM, at least 5 nM, at least 10 nM, at least 50 nM, at least 100 nM, at least 200 nM, at least 500 nM, at least 1 µM, at least 10 µM, at least 50 µM, at least 100 µM or at least 1M.

In specific embodiments, T cells may be contacted, treated, or cultured with one or more PI3K pathway modulators for at least 12 hours, 18 hours, at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, 6 months or more, with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rounds reproduction.

A. PI3K/Akt/mTOR pathway

The phosphatidylinositol 3-kinase/Akt/target of rapamycin pathway in mammals serves as a conduit for the integration of growth factor-related signaling with cell proliferation, differentiation, metabolism, and survival. PI3K kinases are a family of highly conserved intracellular lipid kinases. Class IA PI3Ks are activated by growth factor receptors with tyrosine kinase activity (RTK), either directly or through interaction with adapter molecules of the insulin receptor substrate family. This activity leads to the synthesis of phosphatidylinositol 3,4,5-trisphosphate (PIP3), a regulator of the serine-threonine kinase Akt. mTOR acts through the classical P13K pathway and its action is mediated by 2 different complexes, each characterized by different binding partners that mediate different activities. mTORC1 (mTOR in complex with PRAS40, Raptor and mLST8/GbL) functions as a dependent (downstream) effector in the PI3K/Akt signaling pathway, linking growth factor signals to protein translation, cell growth, proliferation and survival. mTORC2 (mTOR in complex with Rictor, mSIN1, Protor and mLST8 proteins) functions as an upstream regulatory activator of Akt.

Growth factor receptor-mediated activation of PI3K recruits Akt to the membrane through the interaction of its pleckstrin homologous domain with PIP3, thereby exposing the activation loop and allowing phosphorylation at threonine 308 (Thr308) by constitutively active phosphoinositide-dependent protein kinase 1 (PDK1). For maximum activation, Akt is further phosphorylated by mTORC2 at serine 473 (Ser473) on the C-terminal hydrophobic motif. The importance of DNA-PK and HSP in regulating Akt activity has also been demonstrated. Akt activates mTORC1 by inhibitory phosphorylation of TSC2, along with TSC1 negatively regulating mTORC1 by inhibiting the GTPase Rheb, a positive regulator of mTORC1. mTORC1 has 2 well-defined substrates, p70S6K (hereafter referred to as S6K1) and 4E-BP1; both are critical for the regulation of protein synthesis. Accordingly, mTORC1 is an important downstream effector of PI3K linking growth factor signals to protein translation and cell proliferation.

b. PI3K inhibitors

As used herein, the term PI3K inhibitor refers to a nucleic acid, peptide, compound or small organic molecule that binds to PI3K and inhibits at least one activity of PI3K. PI3K proteins can be divided into three classes, PI3K class 1, PI3K class 2 and PI3K class 3. PI3K class 1 exist as heterodimers consisting of one of four p110 catalytic subunits (p110a, p110b, p110δ and p110y) and one subunit from two families of regulatory subunits. The PI3K inhibitor of the present invention preferably targets class 1 PI3K inhibitors. In one embodiment, the PI3K inhibitor exhibits selectivity for one or more class 1 PI3K inhibitor isoforms (i.e., selectivity for p110a, p110b, p110δ, and p110y, or one or more of p110a, p110b, p110δ and p110y). In another aspect, the PI3K inhibitor does not exhibit isoform selectivity and is considered a universal PI3K inhibitor. In one embodiment, the PI3K inhibitor konku

- 47 045264 is responsible for binding to ATP with the catalytic domain of PI3K.

In some embodiments, a PI3K inhibitor may, for example, target PI3K as well as additional proteins of the PI3K-AKT-mTOR pathway. In specific embodiments, a PI3K inhibitor that targets both mTOR and PI3K may be referred to as either an mTOR inhibitor or a PI3K inhibitor. A PI3K inhibitor that exclusively targets PI3K may be called a selective PI3K inhibitor. In one embodiment, a selective PI3K inhibitor can be defined as an agent exhibiting a 50% PI3K inhibitory concentration that is at least 10-fold less, at least 20-fold, at least 30-fold, at least 50-fold less. times, at least 100 times, at least 1000 times or more than the IC50 of the specified inhibitor against mTOR and/or other proteins of the specified pathway.

In a specific embodiment, examples of PI3K inhibitors inhibit PI3K with an IC50 (inhibition concentration of 50% activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, the PI3K inhibitor inhibits PI3K with an IC50 of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Illustrative examples of PI3K inhibitors suitable for use in the T cell production methods of the present invention include, but are not limited to, BKM120 (class 1 PI3K inhibitor, Novartis), XL147 (class 1 PI3K inhibitor, Exelixis), (universal PI3K inhibitor, GlaxoSmithKline) and PX-866 (class 1 PI3K inhibitor; isoforms p110a, p110e and p110y, Oncothyreon).

Other illustrative examples of selective PI3K inhibitors include, but are not limited to, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, ZSTK474, and IPI-145.

Additional illustrative examples of generic PI3K inhibitors include, but are not limited to, BEZ235, LY294002, GSK1059615, TG100713, and GDC-0941.

With. AKT inhibitors

As used herein, the term AKT inhibitor refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one AKT activity. AKT inhibitors can be grouped into several classes, including lipid-based inhibitors (eg, inhibitors targeting the pleckstrin homologous domain of AKT, which prevents AKT localization to plasma membranes), ATP-competitive inhibitors, and allosteric inhibitors. In one embodiment, AKT inhibitors act by binding to the AKT catalytic site. In a specific embodiment, Akt inhibitors act by inhibiting phosphorylation of AKT-dependent targets such as mTOR. In another embodiment, inhibition of AKT activity occurs by inhibiting Akt activation inputs, for example, by inhibiting DNA-PK activation of AKT, PDK-1 activation of AKT, and/or mTORC2 activation of Akt.

AKT inhibitors can target all three AKT isoforms, AKT1, AKT2, AKT3, or can be isoform selective and target only one or two of the AKT isoforms. In one embodiment, the AKT inhibitor can target AKT as well as additional proteins of the PI3K-AKT-mTOR pathway. An AKT inhibitor that targets only AKT may be called a selective AKT inhibitor. In one embodiment, a selective AKT inhibitor may be an agent exhibiting a 50% AKT inhibitory concentration of at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold lower. times, at least 100 times, at least 1000 times or more relative to the IC50 of the specified inhibitor in relation to other proteins of the specified pathway.

In a specific embodiment, exemplary AKT inhibitors inhibit AKT with an IC50 (inhibition concentration of 50% activity) of about 200 nM or less, preferably about 100 nM or less, even more preferably about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM or less. In one embodiment, the AKT inhibitor inhibits AKT with an IC50 of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

Illustrative examples of AKT inhibitors for use in combination with auristatin antibody-drug conjugates include, for example, perifosine (Keryx), MK2206 (Merck), VQD-002 (VioQuest), XL418 (Exelixis), GSK690693, GDC-0068, and PX316 (PROLX Pharmaceuticals).

An illustrative non-limiting example of a selective Akt1 inhibitor is A-674563.

An illustrative non-limiting example of a selective Akt2 inhibitor is

- 48 045264

SST128930.

In specific embodiments, the Akt inhibitor inhibits DNA-PK activation of Akt,

PDK-1 activation of Akt, mTORC2 activation of Akt, or HSP activation of Akt.

Illustrative examples of DNA-PK inhibitors include, but are not limited to,

NU7441, PI-103, NU7026, PIK-75 and PP-121.

d. mTOR inhibitors

The terms mTOR inhibitor, or agent that inhibits mTOR, refers to a nucleic acid, peptide, compound or small organic molecule that inhibits at least one activity of the mTOR protein, such as, for example, the activity of at least a serine-threonine protein kinase on one of the substrates (e.g. p70S6 kinase-1, 4E-BP1, AKT/PKB and eEF2). mTOR inhibitors are able to directly bind to mTORC1, mTORC2, or both mTORC1 and mTORC2 and inhibit their activity.

Inhibition of mTORC1 and/or mTORC2 activity can be determined by attenuation of signaling through the PI3K/Akt/mTOR pathway. A wide range of indicators can be used to establish a decrease in the output of such a signaling pathway. Some non-limiting examples of indicators include (1) decreased phosphorylation of Akt, including but not limited to residues 5473 and T308; (2) a decrease in Akt activation, based, for example, on the observed decrease in phosphorylation of Akt substrates, including, but not limited to, Fox01/O3a T24/32, GSK3a/e; S21/9 and TSC2 T1462; (3) decreased phosphorylation of mTOR-dependent signaling molecules, including, but not limited to, ribosomal protein S6 S240/244, 70S6K T389 and 4EBP1 T37/46; and (4) inhibition of cancer cell proliferation.

In one embodiment, the mTOR inhibitors are active site inhibitors. They are mTOR inhibitors that bind to the ATP binding site (also called the ATP binding pocket) of mTOR and inhibit the catalytic activity of both mTORC1 and mTORC2. One class of active site inhibitors suitable for use in the T cell derivation methods of the present invention are dual-specificity inhibitors that target and directly inhibit both PI3K and mTOR. Dual specificity inhibitors bind to the ATP binding sites of both mTOR and PI3K. Illustrative examples of such inhibitors include, but are not limited to: imidazoquinolines, wortmannin, LY294002, PI-103 (Cayman Chemical), SF1126 (Semafore), BGT226 (Novartis), XL765 (Exelixis) and NVP-BEZ235 (Novartis).

Another class of mTOR active site inhibitors suitable for use in the methods of the present invention selectively inhibit the activity of mTORC1 and mTORC2 towards one or more type I phosphatidylinositol 3-kinases, such as PI3-kinase α, β, γ or δ. These active site inhibitors bind to the active site of mTOR, but not PI3K. Illustrative examples of such inhibitors include, but are not limited to: pyrazolopyrimidines, Thorin 1 (Guertin and Sabatini), PP242 (2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-3-yl)- 1Hindol-5-ol), PP30, Ku-0063794, WAY-600 (Wyeth), WAY-687 (Wyeth), WAY-354 (Wyeth) and AZD8055 (Liu et al., Nature Review, 8, 627-644, 2009). I

In one embodiment, the selective mTOR inhibitor is an agent that exhibits a 50% inhibitory concentration (IC50) for mTORC1 and/or mTORC2 that is at least 10-fold, at least 20-fold, or at least 50-fold lower. times, at least 100 times, at least 1000 times or more relative to the IC50 of the specified inhibitor against one, two, three or more type I PI3-kinases or against all type I PI3-kinases.

Another class of mTOR inhibitors for use in the present invention are referred to herein as rapalogs. As used herein, the term rapalogues refers to compounds that specifically bind to the FRB domain of mTOR (FKBP-rapamycin-binding domain), are structurally related to rapamycin, and retain mTOR inhibitory properties. The term rapalogue excludes rapamycin. Rapalogues include rapamycin esters, ethers, oximes, hydrazones and hydroxylamines, as well as compounds in which the functional groups of the rapamycin core structure have been modified, for example by reduction or oxidation. Pharmaceutically acceptable salts of such compounds are also considered rapamycin derivatives. Illustrative examples of rapalogues suitable for use in the methods of the present invention include, but are not limited to, temsirolimus (CC1779), everolimus (RAD001), deforolimus (AP23573), AZD8055 (AstraZeneca) and OSI-027 (OSI).

In one embodiment, the agent is the mTOR inhibitor rapamycin (sirolimus).

In a specific embodiment, exemplary mTOR inhibitors for use in the present invention inhibit either mTORC1 or mTORC2, or both mTORC1 and mTORC2, with an IC50 (50% activity inhibition concentration) of about 200 nM or less, preferably about 100 nm or less, even more preferably about 60 nm or less, about 25 nm, about 10 nm, about 5 nm, about 1 nm, 100 μm, 50 μm, 25 μm, 10 μm, 1 μm or less. In one aspect, an mTOR inhibitor for use according to the present invention inhibits either mTORC1 or mTORC2, or both mTORC1 and mTORC2, with an IC50 of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM, even more preferably from about 2 nM to about 15 nM.

In one embodiment, exemplary mTOR inhibitors inhibit either RBC and mTORC1 or mTORC2, or both mTORC1 and mTORC2 and PI3K with an IC50 (50% activity inhibition concentration) of about 200 nM or less, preferably about 100 nM or less, and more preferably, about 60 nM or less, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 μM, 50 μM, 25 μM, 10 μM, 1 μM, or less. In one aspect, an mTOR inhibitor for use according to the present invention inhibits PI3K and mTORC1 or mTORC2, or both mTORC1 and mTORC2 and PI3K with an IC50 of from about 2 nM to about 100 nM, more preferably from about 2 nM to about 50 nM , even more preferably from about 2 nM to about 15 nM.

Additional illustrative examples of mTOR inhibitors suitable for use in particular embodiments provided herein include, but are not limited to, AZD8055, INK128, rapamycin, PF-04691502, and everolimus.

mTOR has been shown to exhibit robust, specific catalytic activity for the physiological substrate proteins p70 S6 ribosomal protein kinase I (p70S6K1) and eIF4E binding protein 1 (4EBP1) as assessed using Western blotting with phosphospecific antibodies.

In one embodiment, said PI3K/AKT/mTOR pathway inhibitor is an s6 kinase inhibitor selected from the group consisting of: BI-D1870, H89, PF-4708671, FMK, and AT7867.

I. Compositions and formulations

The compositions provided by the present invention may contain one or more polypeptides, polynucleotides, vectors containing them, genetically modified immune effector cells, and the like, provided by the present invention. The compositions include, but are not limited to, pharmaceutical compositions. The term pharmaceutical composition refers to compositions provided as pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or body of an animal, either alone or in combination with one or more other therapies. It should also be understood that, if necessary, the compositions of the present invention may also be administered in combination with other agents, such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or various other pharmaceuticals. active agents. There are virtually no restrictions on the other components that may be further included in said compositions, provided that said additional agents do not adversely affect the ability of said composition to provide the intended treatment.

The expression pharmaceutically acceptable as used herein refers to compounds, materials, compositions and/or dosage forms that, according to sound medical judgment, are suitable for use in contact with human and animal tissues without causing unnecessary toxicity, irritation, allergic response or other problems or complications. , corresponding to a reasonable balance of benefit and risk.

As used herein, a pharmaceutically acceptable carrier, diluent or excipient includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, pigment/colorant, flavor enhancer, surfactant, wetting agent, dispersant agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier approved by the United States Food and Drug Administration as acceptable for use in humans or domestic animals. Examples of pharmaceutically 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, paraffins, silicones, bentonites, 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethanol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations.

In certain embodiments, the compositions of the present invention comprise a number of CAR-expressing immune effector cells provided by the present invention. As used herein, the term amount refers to an effective amount of genetically modified therapeutic cells, such as T cells, or an amount effective to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

A prophylactically effective amount refers to the amount of genetically modified therapeutic cells effective to achieve the desired prophylactic result. Generally, it is not necessary that since the prophylactic dose is administered to subjects prior to the onset of the disease or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

The therapeutically effective amount of genetically modified therapeutic cells may vary depending on factors such as the disease status, age, sex and body weight of the individual, and the ability of the stem and progenitor cells to produce the desired response in the individual. A therapeutically effective amount is also an amount at which the beneficial therapeutic effects outweigh any toxic or detrimental effects of the virus or transduced therapeutic cells. The term therapeutically effective amount includes an amount effective to treat a subject (eg, a patient). When prescribing a therapeutic amount, the precise amount to administer the compositions of the present invention may be determined by the attending physician, taking into account individual differences in age, body weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). In general, it can be stated that a pharmaceutical composition containing T cells as described herein can be administered at a dosage corresponding to 10 ² -10 ¹⁰ cells/kg body weight, preferably 10 ⁵ - 10 ⁶ cells/kg body weight, including all integer variants within the specified ranges. The number of cells depends on the final purpose of use of the composition, as well as on the type of cells included in its composition. According to the applications proposed in the present invention, the cells are generally in a volume of 1 L or less, may be in a volume of 500 ml or less, or even 250 ml, or 100 ml or less. Thus, the cell density required is typically greater than 10 ⁶ cells/ml, typically greater than 10 ⁷ cells/ml, typically 10 ⁸ cells/ml or more. A clinically relevant number of immune cells can be divided into multiple infusions totaling or exceeding 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. According to some aspects of the present invention, in particular, since all infused cells will be redirected to a specific target antigen (eg, K or λ light chain), smaller numbers of cells can be administered, in the range of 10 ⁶ /kg (106-1011 per patient). Compositions with CAR-expressing cells can be administered repeatedly at dosages within the specified ranges. The cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient receiving the therapy. If necessary, treatment may also include administration of mitogens (eg, PHA) or lymphokines, cytokines and/or chemokines (eg, IFN-γ, IL-2, IL-12, TNF-alpha, IL-18, and TNF-beta, GM -CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1a, etc.) as described herein to enhance the induction of an immune response.

In general, compositions containing cells activated and expanded as described herein can be used in the treatment and prevention of diseases occurring in individuals with immune deficiency. In particular, compositions containing CAR modified T cells provided by the present invention are used in the treatment of B cell malignancies. The CAR-modified T cells of the present invention can be administered either alone or as a pharmaceutical composition in combination with carriers, diluents, excipients and/or other components, such as IL-2 or other cytokines or cell populations. In specific embodiments, the pharmaceutical compositions of the present invention comprise a number of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

Pharmaceutical compositions of the present invention containing a population of CAR-expressing immune effector cells such as T cells may contain buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. Compositions according to the present invention

- 51 045264 are preferably presented in compositions for parenteral administration, for example intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

Liquid pharmaceutical compositions, which may be in solutions, suspensions or other similar forms, may include any 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, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose. The parenteral formulation may be contained in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

In a specific embodiment, the compositions of the present invention contain an effective amount of CAR-expressing immune effector cells, alone or in combination with one or more therapeutic agents. Accordingly, compositions containing CAR-expressing immune effector cells can be administered alone or used in combination with other known cancer treatment options, such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormonal therapy, photodynamic therapy, and the like. These compositions may also be administered in combination with antibiotics. Such therapeutic agents may be used in the course of standard treatment in the art for a particular disease state as described herein, for example, a particular cancer. Examples of therapeutic agents covered by the present invention include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapy agents, therapeutic antibodies, or other active and auxiliary agents.

In some embodiments, compositions comprising CAR-expressing immune effector cells as described herein may be administered in combination with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine group; mustargens, such as chlorambucil, chlornaphasine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, outramycin, azaserin, bleomycins, sactinomycin, calicheamicin, carabicin, carminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxoT-norleucine, doxorubicin, epirubicin, ezorubicin, idarubi icin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancytabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; adrenal suppressants such as aminoglutethimide, mitotane, trilostane; a folic acid replenishing agent such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisanthrene; edatrexate; defofamine; demecolcine; diaziquon; eflornithine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids, such as paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTER®, RhnePoulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine 52 045264 (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (aliretinoin); ONTAK™ (denileukin-diftitox); esperamycins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing agents. Also included in this definition are antihormonal agents whose effect is to regulate or inhibit the action of hormones on cancer, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibitory 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing agents.

A wide variety of other therapeutic agents may be used in combination with the compositions described herein. In one embodiment, a composition comprising CAR-expressing immune effector cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamathasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, ibuprofen , naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate.

Other examples of NSAIDs are selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib) and sialylates. Examples of analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Examples of glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone or prednisone. Examples of biological response modifiers include molecules targeting cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE) ®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Examples of DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofine) and intramuscular injection) and minocycline.

Illustrative examples of therapeutic antibodies suitable for combination with CAR-bearing modified T cells provided by the present invention include, but are not limited to, bavituximab, bevacizumab (Avastin), bivatuzumab, blinatumomab, conatumumab, daratumumab, duligotumab, dacetuzumab, dalotuzumab, elotuzumab (HuLuc63 ), gemtuzumab, ibritutomab, indatuximab, inotuzumab, lorvotuzumab, lucatumumab, milatuzumab, moxetumomab, ocaratuzumab, ofatumumab, rituximab, siltuximab, teprotumumab and ublituximab.

In some embodiments, the compositions described herein are administered in combination with a cytokine. By cytokine as used herein is meant a general term for proteins released by one population of cells and acting on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines and conventional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionylated human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); hepatocyte growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mullerian inhibitory factor; mouse gonadotropin-related peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factors I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon alpha, beta, and gamma; colony-stimulating factors (CSF), such as macrophage-CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF); and granulocyte CSF (GCSF); interleukins (IL) such as IL-1, IL-1-alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12; IL-15, IL-21, tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including leukemia inhibitory factor (LIF) and kit ligand (KL). As used herein, the term cytokine includes proteins derived from natural sources or from recombinant cell culture, as well as biologically active equivalents of natural cytokine sequences.

In specific embodiments, a composition may be further isolated using positive or negative selection techniques comprising CAR T cells of the present invention cultured in the presence of a PI3K inhibitor as described herein and expressing one or more of the following markers: CD3, CD4,

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CD8, CD28, CD45RA, CD45RO, CD62, CD127 and HLA-DR. In one embodiment, using positive or negative selection techniques, a composition is further isolated comprising a specific subpopulation of T cells expressing one or more markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; and CD38 or CD62L, CD127, CD197 and CD38. In various embodiments, the compositions do not express, or substantially do not express, one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

In one embodiment, the expression of one or more markers selected from the group consisting of CD62L, CD127, CD197, and CD38 is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, by at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least at least 25-fold or more compared to a population of T cells activated and expanded in the absence of a PI3K inhibitor.

In one embodiment, the expression of one or more markers selected from the group consisting of CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3 is reduced by at least 1.5-fold, at least 2-fold, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, according to at least 10-fold, at least 25-fold or more compared to a population of T cells activated and expanded in the presence of a PI3K inhibitor.

J. Methods of therapy

The genetically modified immune effector cells of the present invention provide improved adoptive immunotherapies for use in the treatment of B cell-related conditions, which include, but are not limited to, immune-related conditions and hematologic malignancies.

In certain embodiments, the specificity of a primary immune effector cell is redirected to B cells by genetically modifying the primary immune effector cell with a CAR receptor of the present invention. In various embodiments, a viral vector is used to genetically modify an immune effector cell with a specific polynucleotide encoding a CAR containing an antigen binding domain of a mouse anti-BCMA antibody that is bound by a BCMA polypeptide; hinge domain; transmembrane (TM) domain, a short oligopeptide or polypeptide linker connecting the TM domain and the intracellular signaling domain of CAR; one or more intracellular costimulatory signaling domains; and primary signaling domain.

In one embodiment, the present invention provides a type of cell therapy characterized in that the T cells are genetically modified to express a CAR that targets BCMA-expressing B cells. In another embodiment, anti-BCMA CAR-bearing T cells are cultured in the presence of IL-2 and a PI3K inhibitor to improve therapeutic properties and enhance persistence of the CAR-bearing T cells. The CAR-carrying T cells are then infused into the recipient in need. The infused cell is capable of killing disease-causing B cells in the recipient's body. Unlike antibody therapy options, when therapy is carried out by CAR T cells, they are capable of in vivo replication, allowing long-term persistence that can lead to prolonged results in cancer therapy.

In one embodiment, the CAR-bearing T Cells of the present invention can stably proliferate in vivo and persist for an extended period of time. In another embodiment, the CAR T cells of the present invention develop into specific memory T cells that can be reactivated to inhibit the formation of any additional tumors or any tumor growth.

In specific embodiments, immune effector cell compositions containing the CARs of the present invention are used in the treatment of conditions associated with abnormal B cell activity.

Illustrative examples of conditions that can be treated, prevented or alleviated by the use of immune effector cells containing the CARs provided herein include, but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antiphospholipid syndrome , Chagas disease, Graves' disease, Wegener's granulomatosis, polyarteritis nodosa, Sjögren's syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, antiphospholipid syndrome, ANCA-associated vasculitis, Goodpasture's syndrome, Kawasaki disease and rapidly progressive glomerulonephritis.

Modified immune effector cells may also find use in plasma cell disorders such as heavy chain disease, primary or immunocyte-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS).

As used herein, the term B-cell malignancy refers to the type

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In specific embodiments, compositions containing CAR-modified T cells provided by the present invention are used in the treatment of hematological malignancies, including, but not limited to, B-cell malignancies, such as, for example, multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL).

Multiple myeloma is a B-cell malignancy of morphologically mature plasma cells, characterized by neoplastic transformation of one clone of the specified cell types. These plasma cells proliferate in the bone marrow and can invade adjacent bones and sometimes the blood. Variant forms of multiple myeloma include overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma (see, for example, Braunwald, et al. (eds), Harrison's Principles of Internal Medicine, 15th Edition (McGraw-Hill 2001)).

Non-Hodgkin's lymphoma includes a large group of cancers of the lymphocytes (white blood cells). Non-Hodgkin's lymphoma can occur at any age and is often characterized by enlarged lymph nodes, fever, and weight loss. There are many types of non-Hodgkin lymphoma. For example, non-Hodgkin's lymphoma can be divided into aggressive (fast-growing) and indolent (slow-growing) types. Although non-Hodgkin's lymphomas can originate from B cells and T cells, the terms non-Hodgkin's lymphoma and B-cell non-Hodgkin's lymphoma are used interchangeably herein. B-cell non-Hodgkin's lymphomas (NHL) include Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, B-lymphoblastic progenitor cell leukemia; and mantle cell lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are usually B-cell non-Hodgkin lymphomas.

Chronic lymphocytic leukemia (CLL) is an indolent (slow-growing) cancer that results in a slow increase in the number of immature white blood cells called B lymphocytes, or B cells. Cancer cells spread through the blood and bone marrow, and may also affect lymph nodes or other organs such as the liver and spleen. CLL results in bone marrow failure. Sometimes in the later stages of the disease it is called small cell lymphocytic lymphoma.

In specific embodiments, methods are provided that include administering a therapeutically effective amount of CAR-expressing immune effector cells provided by the present invention, or a composition containing same, to a patient in need thereof, alone or in combination with one or more therapeutic agents. In some embodiments, the cells of the present invention are used in the treatment of patients who are at risk of developing a condition associated with abnormal B cell activity, or B cell malignancy. Accordingly, the present invention provides methods for treating or preventing a condition associated with abnormal B cell activity or a B cell malignancy comprising administering to a subject in need thereof a therapeutically effective amount of CAR modified cells provided by the present invention.

As used herein, the terms individual and subject are often used interchangeably and refer to any animal exhibiting a symptom of a disease, disorder, or condition that can be treated using gene therapy vectors, cell-based therapeutics, and methods described elsewhere in the text of this document. In preferred embodiments, the subject includes any animal that exhibits symptoms of a disease, disorder, or condition of the hematopoietic system, for example, Bcellular malignancy, which can be treated using gene therapy vectors, cell-based therapeutics, and methods described elsewhere in the text of this document. Suitable subjects (eg, patients) include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals, and domestic or companion animals (such as a cat or dog). Also included are non-human primate patients and, preferably, human patients. Exemplary subjects include human patients with a B-cell malignancy, diagnosed with, at risk of developing, or suffering from a B-cell malignancy.

As used herein, the term patient refers to a subject who has been diagnosed with a specific disease, disorder, or condition that can be treated using gene therapy vectors, cell-based therapeutics, and methods described elsewhere herein. document.

As used herein, the terms treat or treat include any beneficial or desired effect on the symptoms or pathology of a disease or condition, and may include even minimal reduction in the level of one or more measurable markers of the disease or condition being treated. Treatment may not necessarily involve either reducing or alleviating symptoms of a disease or condition, or delaying the progression of a disease or condition. Treatment does not necessarily indicate complete elimination or cure of the disease or condition or associated symptoms.

As used herein, the term prevent and similar terms such as prevented, preventative and the like refer to a method of preventing, inhibiting or reducing the likelihood of the occurrence 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, the term prevention and similar terms also include reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to the onset or recurrence of said disease or condition.

The terms enhance, promote, increase or increase generally mean the ability of a composition provided by the present invention, for example, a genetically modified T cell or a vector encoding a CAR, to provide, trigger or cause a greater physiological response (i.e. dependent effects) than with a response caused by either the base or the control molecule/composition. The measured physiological response, along with other responses known in the art and described herein, may include increased levels of proliferation, activation, persistence, and/or increased ability to kill cancer cells by T cells. An increase or increase in quantity is usually statistically significant and may include 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold multiple, 7x, 8x, 9x, 10x, 15x, 20x, 30x or greater (e.g. 500x, 1000x) (including all integer and decimal points from 1 and above, for example, 1.5, 1.6, 1.7, 1.8, etc.) increase relative to the response provided by the base or control composition.

The terms reduce, lower, decrease, reduce or attenuate generally mean the ability of a composition of the present invention to provide, trigger or cause a less pronounced physiological response (i.e. dependent effects) compared to the response caused by either the base or the control molecule. composition. The decrease or reduction in quantity is usually statistically significant and may include 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6 -x, 7x, 8x, 9x, 10x, 15x, 20x, 30x or greater (e.g. 500x, 1000x) (including all integer and decimal points from 1 and above eg, 1.5, 1.6, 1.7, 1.8, etc.) decrease relative to the response (reference response) provided by the vehicle, control composition or specific cell line.

The terms maintain, maintain, maintain, no change, no significant change, or no significant reduction generally refer to the ability of the composition provided by the present invention to provide, trigger, or cause a less pronounced physiological response (i.e., dependent effects) in a cell compared to the response induced by the substrate, control molecule/composition, or response in a particular cell line. A comparable effect is one that is not significantly or measurably different from the reference response.

In one embodiment, a method of treating a B cell-associated condition in a subject in need thereof comprises administering an effective amount, for example, a therapeutically effective amount of a composition comprising genetically modified immune effector cells provided by the present invention. The volume and frequency of administration will be determined by factors such as the patient's condition and the type and severity of disease in that patient, and appropriate dosages may be determined through clinical trials.

In one embodiment, the number of T cells in the composition administered to a subject is at least 0.1x105 cells, at least 0.5x105 cells, at least 1x105 cells, at least 5x105 cells, at least 1x106 cells, at least 0.5x107 cells, at least 1x107 cells, at least 0.5x108 cells, at least 1x108 cells, at least 0.5x109 cells, at least 1x109 cells, at least 2x109 cells, at least at least 3x109 cells, at least 4x109 cells, at least 5x109 cells, or at least ^1x1010 cells. In specific embodiments, the subject is administered from about 1x10 ⁷ CAR T cells to about 1x10 ⁹ CAR T cells, from about 2x10 ⁷ CAR T cells to about 0.9x10 9 CAR T cells, from about 3x10 ⁷ CAR T cells to approximately 0.8 x 109 CAR T cells, approximately 4 x 10 ⁷ CAR T cells to approximately 0.7 x 10 ⁹ CAR T cells, or approximately 5 x 10 ⁷ CAR T cells, or approximately 5x10 ⁷ CAR T cells to approximately 0.5x10 9 CAR T cells.

In one embodiment, the number of T cells in the composition administered to a subject is at least 0.1 x 10 ⁴ cells/kg body weight, at least 0.5 x 10 ⁴ cells/kg body weight, at least 1 x 10 ⁴ cells/kg body weight, at least 5x10 ⁴ cells/kg body weight, at least 1x10 ⁴ cells/kg body weight, at least 0.5x10 ⁶ cells/kg body weight, at least 1x10 ⁶ cells/kg body weight, according to at least 0.5x10 ⁷ cells/kg body weight, at least 1x10 ⁷ cells/kg body weight, at least 0.5x10 ⁸ cells/kg body weight, at least 1x10 ⁸ cells/kg body weight, at least 2x10 ⁸ cells/kg body weight, at least 3x10 ⁸ cells/kg body weight, at least 4x10 ⁸ cells/kg body weight, at least 5x10 ⁸ cells/kg body weight, or at least 1x10 ⁸ cells/kg body weight. In specific embodiments, the subject is administered from about 1x10 ⁶ CAR T cells/kg body weight to about 1x10 ⁸ CAR T cells/kg body weight, from about 2x10 ⁶ CAR T cells/kg body weight to about 0.9x10 ⁸ CAR T cells/kg body weight, from approximately 3 x 10 ⁶ CAR T cells/kg body weight to approximately 0.8 x 10 ⁸ CAR T cells/kg body weight, from approximately 4 x 10 ⁶ CAR T cells/kg body weight to approximately 0.7 x 10 ⁸ CAR T cells/kg body weight, from approximately 5 x 10 ⁶ CAR T cells/kg body weight to approximately 0.6 x 10 ⁸ CAR T cells/kg body weight, or approximately 5 x 10 ⁶ CAR T cells/kg body weight to approximately 0.5x10 CAR T cells/kg body weight.

One skilled in the art will appreciate that multiple administrations of the compositions of the present invention may be required to provide the desired therapy. For example, the composition may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more over a period of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

In some embodiments, it may be desirable to administer activated immune effector cells to a subject, followed by repeated blood collection (or apheresis), activation of immune effector cells in the collected blood in accordance with the present invention, and reinfusion of said activated and expanded immune effector cells into the patient. This process can be done repeatedly every few weeks. In some embodiments, immune effector cells can be activated in a volume of blood drawn ranging from 10 ^cc to 400 ^cc . In some embodiments, immune effector cells are activated in a volume of blood drawn that is 20 ^cc , 30 ^cc , 40 ^cc , 50 ^cc , 60 ^cc , 70 ^cc , 80 ^cc , 90 ^cc , 10θ ^cc , 150 cm ³ , 200 cm ³ , 250 cm ³ , 300 cm ³ , 350 cm ³ , or 400 cm ³ or more. Without being limited by theory, this multiple blood draw/reinfusion protocol may serve to eliminate certain populations of immune effector cells.

The compositions of the present invention may be administered in any convenient manner, including aerosol inhalation, injection, oral administration, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. The expressions parenteral administration and parenterally administered as used herein refer to modes of administration other than enteral and topical administration, typically administration by injection, and include, but are not limited to, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac , intradermal, intraperitoneal, transtracheal, subcutaneous, intradermal, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injections and infusions. In one embodiment, the compositions of the present invention are administered to a subject by injection directly into a tumor, lymph node, or site of infection.

In one embodiment, an effective amount of a composition is administered to a subject in need thereof to enhance a cellular immune response to a B cell related condition in the subject. Said immune response may include cellular immune responses mediated by cytotoxic T cells capable of killing infected cells, and cellular responses mediated by regulatory T cells and helper T cells. Humoral immune responses can also be induced, mediated primarily by helper T cells capable of activating B cells, which consequently leads to the production of antibodies. To analyze the types of immune responses induced by the compositions of the present invention, various techniques can be used, detailed descriptions of which are known in the art; see for example the manual: Current Protocols in Immunology, eds: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober (2001) John Wiley & Sons, NY, N.Y.

In T cell-mediated killing, the binding of a CAR to a ligand initiates a signal from the CAR to the T cell, which leads to the activation of various T cell signaling pathways, inducing the T cell to synthesize or release proteins capable of inducing apoptosis of target cells through various mechanisms. . These T cell-mediated mechanisms include (but are not limited to) the transfer of intracellular cytotoxic granules from the T cell to the target cell, the secretion of proinflammatory cytokines by T cells, which can directly (or indirectly, through the recruitment of other killer effector cells) , induce killing of target cells, and up-regulation of death receptor ligands (eg, FasL) on the surface of T cells, which induce apoptosis of target cells upon binding to a cognate death receptor (eg, Fas) on the target cell.

In one embodiment, the present invention provides a method of treating a subject who has been diagnosed with a B cell-related condition, comprising recovering immune effector cells from the body of a subject who has been diagnosed with a BCMA B cell-expressing condition, genetically modifying said immune effector cells. cells containing a CAR nucleic acid encoding vector according to the present invention, thereby obtaining a population of modified immune effector cells, and administering said population of modified immune effector cells to the same subject. In a preferred embodiment, said immune effector cells include T cells.

In some embodiments, the present invention also provides methods for stimulating an immune effector cell-mediated immunomodulatory response against a target cell population in a subject, comprising the steps of administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding a CAR molecule.

Methods of administering the cell-containing compositions described herein include any methods effective to provide either reintroduction of genetically modified ex vivo immune effector cells that directly express the CAR of the present invention into a subject, or reintroduction of genetically modified precursor immune effector cells that when administered to a subject, differentiate into mature immune effector cells expressing said CAR. One method involves transducing peripheral blood T cells ex vivo with a nucleic acid-containing construct of the present invention and reintroducing the transduced cells back into the subject.

All publications, patent applications, and issued patents referred to herein are incorporated herein by reference to the same extent as if each individual publication, patent application, or issued patent were specifically stated to be incorporated herein. document by reference.

Although the present invention has been described in some detail by way of illustrative material and examples for the sake of clarity of understanding, it will be apparent to one skilled in the art, in light of the teaching principles of the present invention, that certain changes and modifications may be made without departing from the spirit or scope of the appended claims. . The examples below are for illustrative purposes only and are not intended to be limiting. Those skilled in the art will readily be able to identify various secondary metrics that can be changed or modified to produce substantially similar results.

Examples

EXAMPLE 1

Construction of CAR receptors for BCMA

CAR receptors were constructed containing the mouse anti-BCMA scFv, containing the MND promoter operably linked to the anti-BCMA scFv, the hinge and transmembrane domain from CD8a, and the costimulatory domains of CD137, followed by the intracellular signaling domain of the CD3Z chain. See, for example, FIG. 1. Shown in FIG. 1 BCMA CAR contains the CD8a signal peptide (SP) sequence for surface expression on immune effector cells. The polynucleotide sequence for an example of the BCMA CAR receptor is shown in SEQ ID NO: 10; an example sequence of BCMA CAR receptor polypeptides is provided in SEQ ID NO: 9; a vector map for an example CAR receptor design is shown in FIG. 1. In table. Table 3 shows the characteristics, information from Genbank, source names and references for various nucleotide segments of the lentiviral vector carrying CAR to BCMA.

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Table 3

Nucleotides Characteristics Information in Gen Bank Original title Link 1-185 Plasmid backbone pUC 19 Accession number L09137.2, nucleotides 1185 pUC 19 New England Biolabs 185-222 Linker Not applicable Synthetic He applicable 223-800 CMV Not applicable pHCMV (1994) PNAS 91:9564-68 801-1136 R, U5, PBS and packaging sequences Accession number M19921.2, nucleotides 454-789 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 1137-1139 The Gag start codon (ATG) is replaced by a stop codon (TAG) Not applicable Synthetic He applicable 1140-1240 HIV-1 gag sequence Accession number M19921.2, nucleotides 793-893 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 1241-1243 HIV-1 gag sequence replaced with a second stop codon Not applicable Synthetic He applicable 1244-1595 HIV-1 gag sequence Accession number M19921.2, nucleotides 897-1248 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243

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1596-1992 Pol cPPT/CTS HIV- 1 Accession number M19921.2, nucleotides 4745-5125 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 1993-2517 HIV-1 NCVD Env region (RRE) isolated Accession number M14100L, nucleotides 1875-2399 PgTAT-CMV Malim, M. H. Nature (1988) 335:181–183 2518-2693 Sequences Env HIV-1 S/A Accession number M19921.2 nucleotides 8290-8470 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 2694-2708 Linker Not applicable Synthetic He applicable 2709-3096 MND Not applicable p ^cm3 1-s- MNDU3c-x2 Challita et al. (1995) J. Virol. 69: 748-755 3097-3124 Linker Not applicable Synthetic He applicable 3125-3187 Signal peptide Accession number NM_001768 Signal CD8a peptide He applicable

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3188-3934 scFv to BCMA02 (V _L 1 -l and n ker- V _H 0) Not applicable Synthetic Not applicable 3935-4141 Hinge and TM CD8a Accession number NM_001768 Hinge and TM CD8a Milone et al (2009) Mol Ther 17(8):1453-64 4144-4269 Signal domain CD137 (4-1BB) Accession number NM_001561 Signal domain CD 137 Milone et al (2009) Mol Ther 17(8):1453-64 4270-4606 Signal domain CD3- Accession number NM_000734 Signal CD3 domain Milone et al (2009) Mol Ther 17(8):1453-64 4607-4717 PRT and fragment 3’ izvich-1 Accession number M19921.2 nucleotides 9005-9110 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 4718-4834 HIV-1 U3 fragment (deletion of 399 i.i.) and R Accession number M19921.2, nucleotides 9511-9627 pNL4-3 Maldarelli et al. (1991) J Virol: 65(11):573243 4835-4858 Synthetic poly-A- subsequence Not applicable Synthetic Levitt, N. Genes & Dev (1989) 3:1019–1025 4859-4877 Linker Not applicable Synthetic He applicable 4878-7350 pUC19 backbone Accession number L09137.2, nucleotides 2636-2686 pUC 19 New England Biolabs

EXAMPLE 2

Evaluation of CAR to mouse BCMA

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Introduction

Adoptive transfer of genetically engineered T cells bearing chimeric antigen receptors (CARs) has been developed as a promising approach for the treatment of cancers. CAR is an artificial molecule consisting of an antigen-reactive single chain variable fragment (scFv) linked to T cell signaling domains via a transmembrane region. In this example, a CAR molecule specific for B-cell maturation antigen (BCMA) was evaluated. BCMA is expressed on cells of multiple myeloma, plasmacytoma and some lymphomas, although expression is normally restricted to plasma cells (Avery et al., 2003; Carpenito et al., 2009; Chiu et al., 2007).

The anti-BCMA02 CAR was constructed using the sequences of the mouse anti-BCMA antibody (C11D5.3). The BCMA10 CAR was constructed using modified sequences; it is a humanized version of the CAR to BCMA02. In a series of in vitro assays, both BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells demonstrated tumor specificity, high levels of CAR expression, and conferred high reactivity toward antigen-expressing targets. Anti-BCMA02 CAR-bearing T cells and anti-BCMA10 CAR-bearing T cells have been shown to have comparable reactivity against BCMA-expressing tumor cell lines. Although both anti-BCMA02 CAR-bearing T cells and anti-BCMA10 CAR-bearing T cells were able to mediate regression in a mouse tumor model, anti-BCMA10 CAR-bearing T cells exhibited antigen-independent secretion of inflammatory cytokines and, accordingly, could potentially cause clinical toxicity associated with high levels of cytokines.

results

Release of tonic inflammatory cytokines from BCMA10-targeted cells associated with apoptosis

BCMA protein is detected in the serum of patients suffering from multiple myeloma (Sanchez et al., 2012). The mean serum BCMA level in patients suffering from multiple myeloma was 10 ng/mL, but peak values reached levels of up to 100 ng/mL, and the effect of physiological levels of soluble BCMA on candidate anti-BCMA CAR-bearing T cells was assessed.

The release of IFN-γ by anti-BCMA02 CAR-bearing T cells, anti-BCMA10 CAR-bearing T cells, and CAR19A-bearing T cells was examined after 24 hours of culture with soluble BCMA (Fig. 2a). Anti-BCMA02 CAR-bearing T cells responded with minimal cytokine release after 24 hours of culture with BCMA at concentrations of up to 1 μg/ml. In contrast, BCMA10 CAR-bearing T cells responded by releasing increasing levels of IFN-γ proportional to the concentration of soluble BCMA added to the culture. At a concentration of 100 ng/ml BCMA, the maximum reported levels in patients with multiple myeloma secreted by BCMA10 CAR-bearing T cells were 82.1 ng/ml IFN-γ, relative to the secretion of 28.8 ng/ml IFN-γ by anti-CAR-carrying T cells. BCMA02 T cells. IFN-γ was detected even in several experiments where anti-BCMA10 CAR-bearing T cells were co-cultured with control cell lines that did not carry the BCMA antigen (Fig. 2b, co-culture with K562). These data indicate that BCMA10 CAR-carrying T cells are hypersensitive to stimulation with soluble BCMA and demonstrate the potential for an antigen-independent T-cell cytokine response.

The potential secretion of tonic cytokines by CAR-carrying BCMA02 T cells, CAR-carrying BCMA10 T cells (10 days after the start of culture) and CAR19A-carrying T cells was studied. After obtaining CAR-bearing T cells, the growth media in which BCMA02 CAR-bearing T cells, BCMA10CAR-bearing T cells, and CAR19A-bearing T cells were cultured were analyzed for the presence of inflammatory cytokines. Despite the lack of antigenic stimulation, cultures of anti-BCMA10 CAR-bearing T cells contained more than 10 ng/ml IFN-γ, whereas cultures of anti-BCMA02 CAR-bearing T cells contained less than 1 ng/ml IFN-γ (Fig. 3). . Cultures of CAR-carrying anti-BCMA10 T cells also contained a significantly (p<0.001) higher amount of TNF-α. To further quantify the amount of cytokines produced by BCMA10 CAR-bearing T cells without antigen stimulation, cytokine release from 5x10 ⁴ CAR-bearing T cells was measured during 24 hours of culture. BCMA10 CAR-carrying T cells produced significantly greater amounts of the inflammatory cytokines MIP1a, IFN-γ, GMCSF, MIP1e, IL-8, and TNF-α compared to BCMA02 CAR-carrying T cells (Fig. 4, p < 0.0001 ). Of all the cytokines studied, the maximum concentrations were observed for MIP1a and IFN-γ. Anti-BCMA10 CAR-carrying T cells produced 4.7 ng MIP1a/ ^5x104 cells/24 hours, 3.0 ng IFN-γ/ ^5x104 cells/24 hours, and ~1 ng/ ^5x104 cells/24 hours, or less amounts of other cytokines. No significant differences were detected in the concentrations of anti-inflammatory cytokines IL-10, IL-2 and IL-4.

At the end of the generation of anti-BCMA10 CAR-bearing T cells, the expression of phenotypic markers of T cell activation was measured to determine whether the secretion of tonic inflammation

- 62 045264 body cytokines on the hyperactive status in CAR-carrying BCMA10 T cells. HLA-DR and CD25 are surface markers whose expression levels typically peak 12 to 24 hours after T cell activation and then decline gradually. Using CAR-bearing T cells derived from cells from three healthy donors, it was demonstrated that on average 40±2% of CAR-carrying BCMA02 T cells expressed HLADR. The level of HLA-DR expression in these cells was comparable to that in nontransduced (43±2.3%) T cells and CAR19A (32±2.2%) control T cells. In contrast, 88 ± 1.2% of anti-BCMA10 CAR-bearing T cells expressed HLA-DR (Fig. 5). Expression of another marker, CD25 activation, was also higher on BCMA10 CAR-bearing T cells compared to BCMA02 CAR-bearing T cells (53±0.9% vs. 35±2.4%). Thus, BCMA10 CAR-bearing T cells exhibited the phenotypic characteristics of activated T cells in the absence of added antigens.

T cell hyperactivity is often associated with activation-induced cell death (AICD) through apoptosis. To determine whether hyperactivity of anti-BCMA10 CAR-bearing T cells could result in higher levels of apoptosis compared with those observed for anti-BCMA02 CAR-bearing T cells, levels of activated caspase-3 were measured. Active caspase-3 was contained in 48% of CAR-to-BCMA10 T cells from two donors, relative to 16% of CAR-to-BCMA02 T cells (Fig. 6). Accordingly, in the absence of added BCMA antigen, cultures of anti-BCMA10 CAR-bearing T cells contain more apoptotic cells, which is associated with increased activation and secretion of inflammatory cytokines compared to anti-BCMA02 CAR-bearing T cells.

BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells were assessed for the ability of CAR-bearing T cells to selectively respond to low levels of BCMA or to cross-react to an unrelated antigen in human serum used to culture the T cells. Both BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells were maintained in medium without human serum for two days and then transferred to medium containing fetal bovine serum (FBS), human serum (HABS), or HABS with or without the addition of 100ng/ml soluble BCMA (Fig. 7). IFN-γ release was analyzed 24 hours later using an ELISA. Both BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells responded to soluble BCMA. However, BCMA10 CAR-bearing T cells secreted 10 times more IFN-γ than BCMA02 CAR-bearing T cells. In the absence of BCMA, only BCMA10 CAR-bearing T cells released IFN-γ regardless of culture with fetal bovine serum (FBS) (p = 0.0002) or human AB antigen serum (HABS) (p = 0.0007). These data indicate that the secretion of inflammatory cytokines was characteristic of CAR-carrying BCMA10 T cells.

Less pronounced antitumor function of CAR-carrying BCMA10 T cells in a mouse model of multiple myeloma

Hyperactivation and increased apoptosis may negatively impact the persistence of CAR T cells in patients and ultimately clinical efficacy. The antitumor function of BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells was studied in a mouse tumor model. NOD scid gamma (NSG) mice bearing experimental human subcutaneous multiple myeloma tumors (RPMI-8226) measuring ~100 mm ³ were treated with 10 ⁷ CAR-carrying BCMA02 T cells, 10 ⁷ CAR-carrying BCMA10 T cells, or bortezomib (Velcade) . The growth of RPMI8226 was monitored using a caliper. In two independent experiments (Figs. 8a and 8b), bortezomib controlled tumor growth compared to vehicle-treated animals. In animals into which CAR-carrying BCMA02 T cells were adoptively transferred, rapid, prolonged tumor elimination was observed (the inset graphs show scaled data for early tumor regression). Adoptive transfer of CAR-carrying BCMA10 T cells also provided tumor regression, but with a delay in both experiments compared to CAR-carrying BCMA02 T cells.

conclusions

BCMA02 CAR-bearing T cells and BCMA10 CAR-bearing T cells demonstrated comparable antitumor functions in in vitro assays, but BCMA10 CAR-bearing T cells differed in characteristics that may compromise safety and efficacy in patient care.

BCMA10 CAR-bearing T cells responded with pronounced secretion of inflammatory cytokines when exposed to physiological levels of BCMA protein. Cytokine storm or cytokine release syndrome is a known cause of clinical toxicity with CAR T cell-bearing therapy options. Additional concern regarding cytokine release in response to BCMA was raised by the observed tonic activity of CAR-bearing BCMA10 T cells. Even in the absence of antigenic stimulation, BCMA10 CAR-bearing T cells released high levels of inflammatory cytokines. Sustained secretion of cytokines can potentially cause significant clinical toxicity and also negatively affect antitumor function. Thus, the inventors observed a higher content of apoptotic cells and a less pronounced antitumor function in cultures bearing CAR to BCMA10 T cells compared to cultures bearing CAR to BCMA02 T cells in a mouse model of multiple myeloma.

List of published sources

Avery et al., (2003). BAFF selectively enhances the survival of plasmablasts generated from human memory B cells. J Clin Invest, 112(2), 286-297.

Carpenito et al., (2009). Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD 137 domains. Proc Natl Acad Sci USA, 106(9), 3360-3365.

Chiu et al., (2007). Hodgkin lymphoma cells express TACI and BCMA receptors and generate survival and proliferation signals in response to BAFF and APRIL. Blood, 109(2), 729-739.

Sanchez et al. (2012). Serum B-cell maturation antigen is elevated in multiple myeloma and correlates with disease status and survival. Br J Haematol, 158(6), 727-738..

EXAMPLE 3

Minimal expression of BCMA in lymphomas activates anti-BCMA CAR-carrying T cells

The level of BCMA expression in lymphoma and leukemia cell lines (Daudi and Raji) was measured to determine whether expression is sufficient to activate anti-BCMA02 CAR-bearing T cells.

BCMA expression on lymphoma, leukemia, and multiple myeloma cells was quantified using flow cytometry. In this assay, the relative expression of BCMA on cells was assessed by relating the fluorescence intensity of expressed BCMA to the known amount of bound antibody (antibody binding capacity, ABC). Levels of BCMA expression in lymphoma cell lines were compared with levels of BCMA expression in a multiple myeloma cell line (RPMI-8226) known to activate CAR-bearing BCMA02 T cells. RPMI-8226 cells expressed 12590±1275 BCMA02 molecules on the surface. At the same time, Daudi cells expressed 1173 ± 234 BCMA02 molecules, and JeKo-1 cells (mantle cell lymphoma cell line) expressed only 222 ± 138 BCMA02 molecules (Fig. 9, circles).

In another series of experiments, the activity of CAR-carrying BCMA02 T cells was tested at low levels of BCMA observed in lymphoma and leukemia cells (Fig. 9, squares). Anti-BCMA02 CAR-bearing T cells were generated using standard methods; activity was assessed using an IFN-γ ELISA assay after co-cultivation with BCMA-positive and BCMA-negative tumor cell lines. The reactivity of CAR-bearing T cells to BCMA02 was correlated with the relative level of BCMA mRNA expression (above threshold) and/or BCMA receptor density on the surface of different tumor cell lines after co-culture (Fig. 9). When co-cultivating CAR-carrying T cells to BCMA and BCMA-negative (BCMA-) tumor cell lines: myelogenous leukemia (K562), acute lymphoblastic leukemia (NALM-6 and NALM-16); mantle cell lymphoma (REC-1); or Hodgkin's lymphoma (HDLM-2), little or no IFN-γ is released. Conversely, when co-cultivating BCMA02 CAR T cells with cells from BCMA-positive (BCMA+) tumor cell lines: B-cell chronic lymphoblastic leukemia (MEC-1), mantle cell lymphoma (JeKo-1), Hodgkin's lymphoma (RPMI-6666) , Burkitt's lymphoma (Daudi cells and Ramos cells) and multiple myeloma (RPMI-8226), significant amounts of IFN-γ were released.

Studies of the reactivity of CAR-bearing BCMA02 T cells against BCMA-expressing Burkitt's lymphoma cells (Daudi cells) continued in vivo in animals. Daudi cells also express CD19. The in vivo activity of anti-BCMA02 CAR-bearing T cells was compared with the in vivo activity of anti-CD19 CAR-bearing T cells. NOD scid gamma (NSG) mice were injected intravenously with 2 x 10 ⁶ Daudi cells and allowed to develop a significant systemic tumor load before treatment with CAR-bearing T cells. CAR-bearing T cells were administered 8 days and 18 days after tumor induction (Figures 10A and 10B, respectively). Scaffold and negative control (anti-CD19Δ CAR-bearing T cells) did not prevent tumor growth, as evidenced by prolongation of the logarithmic phase of bioluminescence, resulting in decreased body weight and death (Fig. 10A, two left mouse panels). Antibodies against CD19 and CAR-carrying BCMA02 T cells prevented tumor growth, promoting weight preservation and survival. Antibodies against CD19 and CAR-carrying anti-BCMA02 T cells were equally effective when administered on day 8 (Fig. 10A, two right panels of mice). CAR-carrying anti-BCMA02 T cells were also effective in reducing tumor burden

- 64 045264 when administered 18 days after tumor induction. Fig. 10V, right panel.

EXAMPLE 4

High in vitro activity of CAR-bearing anti-BCMA T cells

Pronounced in vitro activity of anti-BCMA02 CAR-bearing T cells was achieved with a 50% reduction in anti-BCMA02 CAR expression. T cell populations were transduced with 4x108 - ^5x107 transducing units of lentivirus encoding the CAR molecule against BCM02A. The resulting T cell populations showed reduced abundance of anti-BCMA02 CAR-bearing T cells (% defined as positive cells) and reduced expression of anti-BCMA02 CAR molecules (defined by mean fluorescence intensity: MFI).

The effect of reducing the expression of the CAR molecule on activity against BCMA02 was determined. The content of anti-BCMA CAR-positive T cells was normalized to non-transduced T cells to ensure a content of 26±4% BCMA-responsive T cells (Fig. 11A). The MFI for normalized anti-BCMA02 CAR-bearing T cells ranged from 885 to 1875 (Fig. 11B). K562 is a CML cell line that lacks BCMA expression. BCMA-expressing K562 cells were constructed and used in an in vitro cytolytic assay to evaluate the activity of anti-BCMA02 CAR-bearing T cells with varying levels of anti-BCMA CAR expression (Fig. 11C). K562 cells were labeled with Cell Trace Violet reagent; K562 cells stably expressing BCMA (K562-BCMA) were labeled with CFSE. T cells, K562 cells, and K562-BCMA cells were collected, washed, and resuspended in media without exogenous cytokines. Cells were cultured in a 20:1 or 10:1 ratio of effector cells (E; T cell) to target cells (T; 1:1 mixture of K562 and K562 BCMA cells) in an incubator for 4 hours at 37°C and 5 % CO ₂ . Cells were then stained with Live/Dead dye and analyzed by FACS. Cytotoxicity was determined based on differences in the relative abundance of K562 and K562-BCMA cells normalized to T-cell absence states.

EXAMPLE 5

Process for generating anti-BCMA CAR-carrying T cells

For the treatment of each patient, unique products containing CAR-carrying BCMA02 cells are obtained. The reliability of the process for generating anti-BCMA02 CAR-bearing T cell products was assessed by obtaining anti-BCMA02 CAR-bearing T cells from 11 individual healthy PBMC donors. The expansion of anti-BCMA02 CAR-bearing T cells from each donor was correlated with that of the corresponding untransduced parallel culture (Fig. 12A).

At the end of the culture period (day 10), T cell transduction efficiency was assessed by quantifying the number of integrated lentiviruses using qPCR and a set of specific lentiviral primers (vector copy number, VCN). Cultures bearing anti-BCMA02 CAR T cells from 11 donors showed comparable lentiviral transduction efficiency (Fig. 12B). The abundance of BCMA02 CAR positive T cells was measured by flow cytometry; comparable BCMA expression was found for all donors (Fig. 12C).

The activity of each product with anti-BCMA02 CAR-bearing T cells was assessed by IFN-γ release following co-culture with K562 cells engineered to express BCMA. All products with anti-BCMA02 CAR-bearing T cells demonstrated therapeutically relevant levels of IFN-γ release upon contact with BCMA-expressing K562 cells (Fig. 12D).

EXAMPLE 6

Expression of CD62L, CD127, CD197, and CD38 on CAR-bearing T cells treated with IL-2 or IL-2 and ZSTK474

CAR-bearing T cells cultured with IL-2 and ZSTK474 exhibit increased levels of CD62L expression compared to CAR-bearing T cells cultured with IL-2 alone. Expression analysis of 29 additional cell surface markers on BCMA02 CAR-bearing T cells cultured with IL-2 and ZSTK474 was performed using multiparameter mass cytometry (CyTOF) and compared with CAR-bearing T cells cultured with IL-2 alone . Three additional markers (CD127, CD197, and CD38) showed increased expression in IL-2 + ZSTK474-treated CAR T cells compared to CAR T cells treated with IL-2 alone. Accordingly, coexpression of CD62L, CD127, CD197, and CD38 was further stratified for ZSTK474-cultured CAR T cells. After culture in IL-2-containing medium, 7.44% of anti-BCMA02 CAR T cells coexpressed CD127, CD197, and CD38, compared with 24.5% of anti-BCMA02 CAR T cells cultured with IL-2 and ZSTK474 . Venn diagram in Fig. 13 illustrates the coexpression of CD127, CD197 and CD38 in CD62L positive anti-BCMA02 T cells.

EXAMPLE 7

ZSTK474 Treatment Increases CD8 T Cells

CD8 expression was quantified in anti-BCMA02 CAR-bearing T cells treated with

- 65 045264 IL-2 only, or IL-2 and ZSTK474. CD8 expression was determined using fluorescently labeled anti-CD8 antibody and flow cytometry. Anti-BCMA02 CAR-bearing T cells from seven healthy donors cultured with IL-2 and ZSTK474 showed significantly higher levels of CD8 expression compared with anti-BCMA02 CAR-bearing T cells cultured with IL-2 alone. Fig. 14.

EXAMPLE 8

Lack of antigen-independent activity in T cells treated with ZSTK474 carrying CAR against BCMA

Tonic activity of CAR-bearing T cells in the absence of antigen was associated with reduced biological activity. Tonic activity of anti-BCMA02 CAR T cells was assessed by quantifying interferon-γ (IFN-γ) release in the absence of antigen after culture in the presence of IL-2 and ZSTK474 and compared with that obtained under standard culture conditions with IL-2 alone. Cultures of anti-BCMA CAR-bearing T cells were generated using a system directly scalable to high-volume clinical manufacturing processes. Briefly, peripheral blood mononuclear cells (PBMCs) were cultured in stationary flasks in medium containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec). One day after the start of cultivation, 2x10 ⁸ transducing units of lentivirus encoding CAR receptors against BCMA were added.

Anti-BCMA02 CAR-bearing T cells were maintained in logarithmic growth phase by the addition of fresh medium containing IL-2 and an optimized dose of ZSTK474 for a total of 10 days of culture. At the end of the production process, an equivalent number of BCMA02 CAR-bearing T cells were recultured for 24 hours in unsupplemented medium. The amount of IFN-γ released over 24 hours was quantified using an ELISA. In this assay, IFN-γ levels below 200 pg/ml correspond to the absence of tonic activity. In fig. 15 shows that the amount of IFN-γ released by anti-BCMA02 CAR T cells from 14 donors is consistent with the absence of tonic activity, regardless of whether the CAR T cells were cultured with ZSTK474.

EXAMPLE 9

ZSTK474-Treated Anti-BCMA02 CAR-Carrying T Cells Show Therapeutic Activity in a Lymphoma Tumor Model

Daudi tumors were used to study the antitumor activity of anti-BCMA02 CAR-bearing T cells cultured with IL-2, or IL-2 and ZSTK474. Daudi cells express low levels of BCMA protein and provide a model of aggressive, difficult-to-treat lymphoma tumors.

2x10 ⁶ Daudi tumor cells were labeled with the firefly luciferase gene and administered to IL-2 receptor gamma chain knockout NOD scid (NSG) mice by intravenous injection. After tumor formation, tumor-bearing mice were injected with 1x10 ⁷ CAR T-cell-bearing cells. Mice were treated with i) anti-BCMA02 CAR T cells treated for 10 days with IL-2, or IL-2 and ZSTK474; or ii) containing truncated anti-BCMA02 signaling-deficient CAR T cells (tBCMA02) T cells treated for 10 days with IL-2 and ZSTK474. Tumor growth was monitored using bioluminescence using the Xenogen-IVIS imaging system.

Complete regression of tumors was observed in 50% of mice injected with BCMA02 CAR-carrying T cells treated with IL-2 and ZSTK474. Fig. 16.

EXAMPLE 10

ZSTK474-Treated CAR T Cells Show Therapeutic Activity in a Mouse Model of Human Myeloma

Animals bearing 100 mm ³ subcutaneous multiple myeloma tumors (RPMI-8226) were infused with equivalent doses of CAR-bearing T cells (1x10 ⁶ CAR BCMA02 positive T cells) or unmodified (nontransduced) T cells from a matched T cell donor. Anti-BCMA CAR-bearing T cells were treated with IL-2, or IL-2 and ZSTK474 as described in Example 8.

In animals that received CAR-to-BCMA02 T cells cultured with IL-2, or IL-2 and ZSTK474, complete prevention of tumor proliferation was observed. Fig. 17. In contrast, animals treated with nontransduced cells or scaffold failed to control tumor growth. Fig 17.

In general, the terms used in the appended claims are not to be construed as limiting the scope of the invention to the specific embodiments disclosed in the specification and claims; on the contrary, they include all possible embodiments, as well as all equivalents of such embodiments, included within the scope of the relevant claims. Thus, the claims of the present invention are not limited to the above description of the invention.

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Claims (20)

CLAIM 1. A method of treating a subject who has a hematologic malignancy, comprising administering immune effector cells containing a viral vector that expresses a chimeric antigen receptor (CAR) to B-cell maturation antigen (BCMA), wherein the CAR consists of the amino acid sequence represented by SEQ ID NO: 9. 2. The method of claim 1, wherein the hematological malignancy is a B-cell malignancy. 3. The method of claim 1 or 2, wherein the hematologic malignancy is a B-cell malignancy selected from the group consisting of multiple myeloma and non-Hodgkin's lymphoma. 4. Method according to any one of claims 1 to 3, wherein the hematological malignancy is multiple myeloma. 5. The method according to any one of claims 1 to 3, where the hematological malignancy is multiple myeloma selected from the group consisting of overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma bone and extramedullary plasmacytoma. 6. The method according to any one of claims 1 to 3, wherein the hematological malignancy is a non-Hodgkin's lymphoma selected from the group consisting of Burkitt's lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse B large cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, lymphoblastic lymphoma from B-cell precursors and lymphoma from mantle cells. 7. Method according to any one of claims 1 to 6, where the viral vector is a retroviral vector or a lentiviral vector. 8. The method according to claim 7, where the lentiviral vector is selected from the group essentially consisting of human immunodeficiency virus 1 (HIV-1); human immunodeficiency virus 2 (HIV-2); visna maedi virus (WMV); caprine arthritis-encephalitis virus (AEC); equine infectious anemia virus (EIAN); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV). 9. The method according to claim 7 or 8, where the lentiviral vector contains a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA flap (cPPT/FLAP), a retroviral export element, a promoter, operably linked with a polynucleotide encoding CAR; and right (3') retroviral LTR. 10. The method of claim 9, wherein the polynucleotide encoding the CAR contains the sequence set forth in SEQ ID NO: 10. 11. The method of claim 9, wherein the lentiviral vector further comprises a heterologous polyadenylation sequence. 12. The method of claim 11, wherein the heterologous polyadenylation sequence is a bovine growth hormone polyadenylation sequence or a rabbit β-globin polyadenylation signal sequence. 13. The method according to any one of claims 9 to 12, wherein the 5' retroviral LTR contains a retroviral promoter or a heterologous promoter. 14. The method of claim 13, wherein the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter, or a simian virus 40 (SV40) promoter. 15. The method according to any one of claims 9 to 14, wherein the 5' LTR or 3' LTR is a lentiviral LTR. 16. The method according to any one of claims 9 to 15, wherein the 3' LTR is a self-inactivating (SIN) LTR. 17. The method according to any one of claims 9 to 16, wherein the promoter operably linked to the polynucleotide encoding the CAR is selected from the group consisting of a cytomegalovirus (CMV) immediate-early gene promoter, an elongation factor 1-alpha (EF1-a) promoter , phosphoglycerate kinase-1 (PGK) promoter, ubiquitin-C promoter (UBQ-C), cytomegalovirus enhancer composite promoter/chicken betaactin promoter (CAG), polyoma virus enhancer composite promoter/herpes simplex virus thymidine kinase promoter (MC1), beta-actin promoter (β-AST), the simian virus 40 (SV40) promoter, and the myeloproliferative sarcoma virus enhancer-containing promoter, with the negative control region removed, with the primer binding site replaced with the sequence from d1587rev (MND). 18. The method according to any one of claims 1 to 17, wherein the cells are administered to the subject parenterally. 19. The method according to any one of claims 1 to 17, wherein the cells are administered intravascularly to the subject. 20. The method according to any one of claims 1 to 17, wherein the cells are administered to the subject intravenously. -