Therapeutic Antibodies – Richard Morgan, Kevin Friedman, 2Seventy Bio Inc

Abstract for “BCMA chimeric antigen receptors”

“The invention provides improved compositions to adoptive T-cell therapies for B-cell related conditions.”

Background for “BCMA chimeric antigen receptors”

“Technical Field”

“The invention concerns improved compositions and methods to treat B cell-related conditions. The invention is primarily concerned with improved chimeric antigen receptors. These CARs are comprised of murine anti-BCMA antibodies and antigen binding fragments. Immune effector cells have been genetically modified to express these CARs. These compositions can be used to treat B cell-related conditions.

“Description of Related Art”

Many serious diseases are mediated by B lymphocytes (i.e. B cells). A disordered B cell physiology may lead to autoimmune diseases, including systemic lupus. Malignant transformation of B cell cells can lead to cancers, including lymphomas such as multiple myeloma or non-Hodgkins lymphoma.

“The majority of patients with B cell malignancies (including non-Hodgkin?s lymphoma and multiple myeloma) are significant contributors in cancer mortality. Mixed responses to different forms of treatment are common in B cell malignancies. Due to side effects toxic, traditional methods of treating B-cell malignancies such as radiotherapy and chemotherapy have limited utility. Anti-CD19, anti?CD20, anti?CD22, anti?CD23, anti?CD52, anti?CD80, anti?HLA-DR therapeutic antibody have not had much success. This is due to poor pharmacokinetic profiles and rapid elimination of antibodies via serum proteases. The glomerulus has limited penetration and low expression of target antigens on cancer cells. The success rate of genetically modified cells that express chimeric antigen receptors has been limited. The therapeutic efficacy for a particular antigen binding domain in a CAR can be unpredictable. If it binds too strongly, CAR T cells cause massive cytokine release, which could lead to a fatal immune reaction known as a “cytokine storm”. If the antigen binding domain is too weak, CAR T cells don’t have sufficient therapeutic efficacy to clear cancer cells.

“The invention provides improved vectors for the generation of T cell therapies and methods to use them.”

“Chimeric antigen receptors (CARs) are provided in various embodiments. They include an extracellular domain that contains a murine anti?BCMA (Bcell maturation antigen), antibody or antigen binding segment thereof that binds one to three epitopes of the human BCMA polypeptide. A transmembrane and intracellular co-stimulatory signals domains as well as a primary signaling area.

“In particular embodiments the murine anti-BCMA antibody/antigen binding fragment that binds to the human BCMA polypeptide can be selected from the following group: Camel Ig and Ig NAR, Camel Ig fragments, Fab fragments, Fv? fragments F(ab?2 fragments F(ab?3 fragments Fv, single-chain Fv antibody (??scFv) ), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (?dsFv? ), and single domain antibody (sdAb; Nanobody).

“In addition to these embodiments, the murine anti BCMA antibody or antigen-binding fragment that binds to the human BCMA protein is an scFv.”

“In certain embodiments, the murine anti BCMA antibody or antigen-binding fragment thereof includes one or more CDRs. These CDRs are set forth in any of SEQ ID Nos: 1-3.”

“In particular embodiments the murine anti-BCMA antibody/antigen binding fragment thereof includes one or more CDRs as described in any one SEQ ID NO: 4-6.”

“In some embodiments, the murine anti BCMA antibody or antigen-binding fragment of it comprises a variable light chains sequence as described in SEQ ID No: 7.

“In particular embodiments the variable light chain sequence includes the CDR sequences described in SEQ ID Nos: 1-3.”

“In other embodiments, either the murine anti-BCMA antibody (or antigen binding fragment thereof) comprises a variable heavy chains sequence as described in SEQ ID No: 8.

“In addition to the above embodiments, the variable chain sequence of heavy chains includes the CDR sequences set forth under SEQ ID NOs 4-6.”

“In further embodiments the transmembranedomain is made from a polypeptide selected out of the group consisting: alpha, beta, or zeta chains of the T-cell receptor CD3??,CD4?,CD5?,CD8?,CD9?,CD16?,CD22,CD27,CD28,CD33,CD37,CD45,CD64,CD64,CD80,CD86,CD134,CD137,CD152, and PD1

“In certain embodiments, the transmembranedomain is made from a polypeptide selected among the following: CD8? ; CD4, CD45 and PD1, respectively.

“In some embodiments, transmembrane domains are from CD8?”

“In particular embodiments the co-stimulatory signaling domains come from a costimulatory molecule selected among the following: CD2, CD7 and CD28, CD30, OX40, CD53, CARD11, OX40, CD83, and CD134 (OX40), as well as CD205 (SLAMF1)), CD152 (CTLA4)), CD225 (LAG3)), CD223 (LAG3)), CD270 (HVEM), PD-L2) CD274, CD274 (PD-1), CD274 (PD-1, PD-L1), TRIM, TRIM, NKD2C, TRIM, TRIM, TRIM, TRIM, TRIM and TRIM)

“In particular embodiments the one or more costimulatory signaling domains are selected from the following co-stimulatory molecules: CD28, CD134 and CD137.”

“In additional embodiments the one or more costimulatory signaling domains come from a costimulatory molecule selected among the group consisting: CD28, CD134 and CD137.”

“In additional embodiments the one or more costimulatory signaling domains are from CD28.”

“In particular embodiments the one or more costimulatory signaling domains are from CD134.”

“In other embodiments the one or more costimulatory signaling domains are from CD137.”

“In some embodiments, a CAR includes a hinge region polypeptide.”

“In further embodiments the hinge region polypeptide includes a CD8-like region.”

“In some embodiments, the CAR includes a spacer area.”

“In addition to the above embodiments, spacer region polypeptide includes CH2 and CH3 regions IgG1 and IgG4.”

“In particular embodiments, the CAR includes a signal protein.”

“In further embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8? sign polypeptide.

“In one embodiment, the CAR contains an amino acid sequence set forth in SEQ ID No: 9”

“In different embodiments, a polynucleotide that encodes a CAR contemplated in this invention is provided.”

“In various embodiments, a polynucleotide that encodes a CAR can be provided. The sequence of the polynucleotides is described in SEQ ID No: 10.

“In certain specific embodiments, a vector encoding a CAR as contemplated herein or set forth in SEQID NO: 10 is provided.”

“In some embodiments, the vector can be an expression vector.”

“In additional embodiments, a vector is an episomal Vector.”

“In particular embodiments the vector is a virus vector.”

“In further embodiments the vector is a retroviral Vector.”

“In other embodiments the vector is a vector that is lentiviral.”

“In one embodiment, the vector that encodes a BCMA CAR includes the polynucleotide sequence described in SEQ ID No: 36.”

“In additional embodiments, the 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) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).”

“In particular embodiments, the vector includes a left (5?). A retroviral LTR and a Psi are (?) Retroviral LTR, a Psi (?) retroviral LTR.”

“In other embodiments, the CAR includes a heterologous sequence of polyadenylation.”

“In certain embodiments, a CAR comprises a hepatitis B viral posttranscriptional regulatory elements (HPRE) and a woodchuck posttranscriptional regulatory component (WPRE).

“In some embodiments, the promoter for the 5? LTR can be replaced by a heterologous promoter.

“In further embodiments, a heterologous promoter can be a cytomegalovirus promoter (CMV), a Rous Sarcoma Virus promoter (RSV), or a Simian Virus 40 promoter (SV40).

“In particular embodiments the 5? LTR or 3? LTR is a lentivirus LTR.

“In particular embodiments the 3? LTR can include one or more modifications.

“In some embodiments the 3? LTR can include one or more deletions.

“In some embodiments, the 3? LTR is an LTR that self-inactivates (SIN).

“In some embodiments, polyadenylation is a bovine growthhormone polyadenylation or signal bunny?-globin sequence.”

“In addition embodiments, a polynucleotide that encodes a CAR contemplated in this invention includes an optimized Kozak sequence.”

“In further embodiments, a promoter operably linked with the polynucleotide encoded a CAR is selected from the following: a cytomegalovirus instant early gene promoters (CMV), a elongation factor1 alpha promoters (EF1-?) ), a promoter of phosphoglycerate-1 (PGK), an ubiquitin-1 alpha promoter(UBQ-C), and a polyoma enhancer/herpes simplux thymidinekinase (CAG), a promoter of beta actin (?-ACT), and a myeloproliferative virus enhancer, negative region deleted, d1587rev prime-binding site substituted promoter.

“In various embodiments, an immune cell that acts as an effector is provided with a vector contemplated in this invention.” The vector contemplated herein is used to transduce the immune effector cells in various embodiments.

“In further embodiments the immune effector cells are selected from the group consisting: a T lymphocyte, a natural killer (NK), cell.”

“In some embodiments, an immune effector cell is transduced using the vector of any of the embodiments above. The activated and stimulated immune effectors are maintained in the presence or absence of an inhibitor of PI3K pathway. This allows for the maintenance of proliferation of the transduced immune cells compared with the proliferation of activated and stimulated immune effectors cells.

“In particular embodiments, an immune effector cells activated or stimulated in the presence inhibitor of PI3K pathways has increased expression of i), one or more markers from the group consisting: CD62L and CD127, CD197 and CD38, or ii), all markers CD62L and CD127, CD197 and CD38, compared to an immune cell activated or stimulated in absence of inhibitor of PI3K.

“In one embodiment, ZSTK474 is the PI3K inhibitor.”

“In different embodiments, a composition includes an immune effector cells contemplated herein as well as a physiologically acceptable excipient.”

“In different embodiments, a method for generating an immune-effector cell comprising an AAR is contemplated. This involves injecting into an immune cell a vector containing a polynucleotide that encodes the CAR.”

“In addition, the method includes stimulating the immune effector and inducing the cells to proliferate through contact with antibodies that bind CD3 or antibodies that bind CD28 to the cell; thus, generating a population immune effector cells.”

“In certain embodiments, the immune effector cells are stimulated and inducible to proliferate prior to introducing the vector.”

“In some embodiments, immune effector cells include T lymphocytes.”

“In particular embodiments the immune effector cells include NK cells.”

“In particular embodiments, cells are activated or stimulated when there is an inhibitor of PI3K pathway. This allows for the maintenance of proliferation of transduced immune effector cell populations as opposed to proliferation of activated or stimulated immune cells in absence of inhibitor of PI3K pathway.”

“In certain embodiments, immune effector cell activation and stimulation in the presence inhibitor of PI3K pathways have increased expression of i), one or more markers from the group consisting: CD62L and CD127, CD197 and CD38, or ii), all of the markers CD62L and CD127, CD197 and CD38, compared to immune cells activated or stimulated in absence of inhibitor of PI3K path.”

“In one embodiment, ZSTK474 is the PI3K inhibitor.”

“In various embodiments, there is a method for treating a B-cell related condition in a subject who needs it. This involves administering to the subject a therapeutically effective amount of a compound comprising BCMA cells and optionally, a pharmaceutically acceptable adjuvant.

“In other embodiments, the B cell related condition is multiple myeloma, non-Hodgkin’s lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas’ disease, Grave’s disease, Wegener’s granulomatosis, poly-arteritis nodosa, Sjogren’s syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture’s disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy-chain disease, primary or immunocyte-associated amyloidosis, or monoclonal gammopathy of undetermined significance.”

“Another embodiment of the B-cell related condition is a malignancy.”

“In some embodiments, the B-cell malignancy is multiple myeloma or non-Hodgkin?s lymphoma.

“In some embodiments, the MM may be selected from the following: overt multiple meeloma or smoldering multimyeloma; plasma cell leukemia; plasma cell leukemia; non-secretory Myeloma; IgD myeloma. Osteosclerotic myeloma. Solitary plasmacytoma bone. Extramedullary Plasmacytoma.

“In certain embodiments, the NHL may be selected from the following: Burkitt lymphoma/small lymphocytic Leukemia (CLL/SLL), chronic lymphocytic lukemia/small lymphocytic Lymphoma (CLL/SLL), diffuse large B cell lymphoma and immunoblastic large-cell lymphoma.

“In particular embodiments, the plasma cell malignancy is the B-cell related condition.”

“In further embodiments the B-cell related condition is an auto immune disease.”

“Systemic lupus is another form of autoimmune disease.”

“In some embodiments, the B-cell related condition is rheumatoidarthritis.”

“In particular embodiments the B-cell related condition is idiopathic hemolytic anemia, myasthenia gravis or myasthenia purpura.

“BRIEF DESCRIPTION ABOUT CERTAIN VIEWS OF THE DRAWINGS.”

“FIG. 1. shows a schematic for murine B cell maturation antibody (muBCMA), CAR constructs.

“FIG. 2a displays the amount IFNg released by anti-BCMA02 CAR C cells, anti-BCMA10CART cells and CAR19? After co-culturing the cells for 24 hours with BCMA-expressing K562 cells, T cells were activated by BCMA.

“FIG. 2b is the amount IFNg released by anti-BCMA02 CART cell, anti-BCMA10CART cells and CAR19? After co-culture with K562 cells, T cells were exposed to IFNg from anti-BCMA02 CART cells and anti-BCMA10 CART cells.

“FIG. “FIG. T cells were stimulated for 10 days before the assay.”

“FIG. “FIG. T cells without antigen stimulation.

“FIG. 5. The expression of phenotypic markers for activation at anti-BCMA CAR t cell manufacturing. The expression of CD25 and HLA-DR was determined in anti-BCMA02 CART cell lines, anti-BCMA10CART cells, and anti-BCMA19 CART cells. T cells.”

“FIG. “FIG.

“FIG. “FIG.

“FIG. 8A illustrates the tumor volume in NOD Scid Gamma (NSG), mice with?100mm3 experimental subcutaneous human multiple myeloma RPMI-8226 tumors. The mice were given vehicle, 107 antiBCMA02 CART cells and 107 antiBCMA10 CAR-T cells or Bortezomib.

“FIG. 8B is the volume of a tumor in NOD scid Gamma (NSG), mice with?100mm3 experimental subcutaneous human multiple myeloma(RPMI-8226). The mice were given vehicle, 107 antiBCMA02 CART cells and 107 antiBCMA10 CAR-T cells or Bortezomib.

“FIG. “FIG. release, boxes). BCMA-negative (BCMA+), tumor cell lines: Myelogenous Leukemia (K562) and acute lymphoblastic Leukemia (NALM-6, NALM-16); Mantle Cell Lymphoma (REC-1); or Hodgkin?s lymphoma [HDLM-2) showed little to no IFN. release. 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, Ramos cells), Multiple myeloma (?RPMI-82226?) showed significant IFN. release.”

“FIG. 10A is an in vivo activity chart for vehicle, anti-CD19? In an NSG mouse model, CART cells, anti?CD19 CART cell and anti-BCMA (Daudi) cells to BCMA expressing Burkitt?s lymphoma cells in a CART cell-based system. CAR T cells are given to the mice 8 days after tumor induction.

“FIG. 10B is an in vivo activity chart for vehicle, anti-CD19? In an NSG mouse model, CART cells, anti CD19 CAR-T cells, and anti BCMA CAR-T cells to BCMA-expressing Burkitt’s Lymphoma cells (Daudi Cells) are given to CAR-T cells-treated mice 18 days after tumor induction.

“FIG. 11B shows the MFI of normalized anti-BCMA cells. It ranged between 885 and 1875, as determined by flow cytometry. FIG. FIG. 11.C: K562 cells and the K562-BCMA cell were co-cultured together with normalized anti?BCMA CAR cells at a 20/1 or 10/1 effector (E: T cell to target (T: 1:1 mix of K562 BCMA and K562 BCMA) ratio. They showed comparable cytolytic activities.”

“FIGS. 12A-D illustrate the reliability of anti-BCMA CAR-T cells manufacturing processes. FIG. FIG. 12A illustrates that anti-BCMA CAR T cells products made from PBMCs from 11 donors show similar levels of expansion to untransduced donor T cell cultures. FIG. 12B illustrates anti-BCMA CAR-T cell products made from 11 donors. They had comparable lentiviral transduction efficiency. FIG. FIG. 12C illustrates the frequency of anti-BCMA positive T cells as measured by flow cytometry. BCMA expression was comparable among all donors. FIG. FIG. 12.12D: Anti-BCMA CAR T cells products made from 11 donors had therapeutically relevant levels IFN? When exposed to BCMA-expressing K562 cell.”

“FIG. 13 shows Venn diagrams of co-expression of CD127 and CD197 in CD62L-positive anti-BCMA02T cells cultured for ten days in the presence or absence of IL-2. Anti-BCMA02 CAR-T cells treated with ZSTK474 showed a higher percentage of cells that co-express CD127, CD197, and CD38 than cells cultured in the absence of IL-2.

“FIG. 14 shows an increase in CD8 expressing anti BCMA02 CAR T cells when cultures were treated with IL-2 and ZSTK474 (n=7) compared with cultures that were treated with IL-2 only. CD8 expression was determined using a fluorescently-labeled anti-CD8 antibody and flow cytometry.”

“FIG. “FIG. Anti-BCMA02 CART cells of 14 donors were able to release IFN-? after being cultured with IL-2 only or with IL-2 plus ZSTK474. After the culture period ended, the same number of anti-BCMA02 T cells from 14 donors were re-cultured in media for 24 hours. How much IFN-? was released in 24 hours was determined by ELISA. ELISA was used to measure the amount of IFN-? released within 24 hours. The anti-BCMA02 CAR-T cell tonic cytokine production was not significantly increased in culture in ZSTK474 as compared to anti?BCMA02 T cells cultured only with IL-2.”

“FIG. “FIG. In 50% of mice that received the anti-BCMA02 CART cells with IL-2 or ZSTK474, complete tumor regression was observed.

“FIG. “FIG. The outgrowth of tumors was completely stopped by animals treated with IL-2 or IL-2 plus ZSTK474-cultured anti?BCMA02 CAR-T cells.

Summary for “BCMA chimeric antigen receptors”

“Technical Field”

“The invention concerns improved compositions and methods to treat B cell-related conditions. The invention is primarily concerned with improved chimeric antigen receptors. These CARs are comprised of murine anti-BCMA antibodies and antigen binding fragments. Immune effector cells have been genetically modified to express these CARs. These compositions can be used to treat B cell-related conditions.

“Description of Related Art”

Many serious diseases are mediated by B lymphocytes (i.e. B cells). A disordered B cell physiology may lead to autoimmune diseases, including systemic lupus. Malignant transformation of B cell cells can lead to cancers, including lymphomas such as multiple myeloma or non-Hodgkins lymphoma.

“The majority of patients with B cell malignancies (including non-Hodgkin?s lymphoma and multiple myeloma) are significant contributors in cancer mortality. Mixed responses to different forms of treatment are common in B cell malignancies. Due to side effects toxic, traditional methods of treating B-cell malignancies such as radiotherapy and chemotherapy have limited utility. Anti-CD19, anti?CD20, anti?CD22, anti?CD23, anti?CD52, anti?CD80, anti?HLA-DR therapeutic antibody have not had much success. This is due to poor pharmacokinetic profiles and rapid elimination of antibodies via serum proteases. The glomerulus has limited penetration and low expression of target antigens on cancer cells. The success rate of genetically modified cells that express chimeric antigen receptors has been limited. The therapeutic efficacy for a particular antigen binding domain in a CAR can be unpredictable. If it binds too strongly, CAR T cells cause massive cytokine release, which could lead to a fatal immune reaction known as a “cytokine storm”. If the antigen binding domain is too weak, CAR T cells don’t have sufficient therapeutic efficacy to clear cancer cells.

“The invention provides improved vectors for the generation of T cell therapies and methods to use them.”

“Chimeric antigen receptors (CARs) are provided in various embodiments. They include an extracellular domain that contains a murine anti?BCMA (Bcell maturation antigen), antibody or antigen binding segment thereof that binds one to three epitopes of the human BCMA polypeptide. A transmembrane and intracellular co-stimulatory signals domains as well as a primary signaling area.

“In particular embodiments the murine anti-BCMA antibody/antigen binding fragment that binds to the human BCMA polypeptide can be selected from the following group: Camel Ig and Ig NAR, Camel Ig fragments, Fab fragments, Fv? fragments F(ab?2 fragments F(ab?3 fragments Fv, single-chain Fv antibody (??scFv) ), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein (?dsFv? ), and single domain antibody (sdAb; Nanobody).

“In addition to these embodiments, the murine anti BCMA antibody or antigen-binding fragment that binds to the human BCMA protein is an scFv.”

“In certain embodiments, the murine anti BCMA antibody or antigen-binding fragment thereof includes one or more CDRs. These CDRs are set forth in any of SEQ ID Nos: 1-3.”

“In particular embodiments the murine anti-BCMA antibody/antigen binding fragment thereof includes one or more CDRs as described in any one SEQ ID NO: 4-6.”

“In some embodiments, the murine anti BCMA antibody or antigen-binding fragment of it comprises a variable light chains sequence as described in SEQ ID No: 7.

“In particular embodiments the variable light chain sequence includes the CDR sequences described in SEQ ID Nos: 1-3.”

“In other embodiments, either the murine anti-BCMA antibody (or antigen binding fragment thereof) comprises a variable heavy chains sequence as described in SEQ ID No: 8.

“In addition to the above embodiments, the variable chain sequence of heavy chains includes the CDR sequences set forth under SEQ ID NOs 4-6.”

“In further embodiments the transmembranedomain is made from a polypeptide selected out of the group consisting: alpha, beta, or zeta chains of the T-cell receptor CD3??,CD4?,CD5?,CD8?,CD9?,CD16?,CD22,CD27,CD28,CD33,CD37,CD45,CD64,CD64,CD80,CD86,CD134,CD137,CD152, and PD1

“In certain embodiments, the transmembranedomain is made from a polypeptide selected among the following: CD8? ; CD4, CD45 and PD1, respectively.

“In some embodiments, transmembrane domains are from CD8?”

“In particular embodiments the co-stimulatory signaling domains come from a costimulatory molecule selected among the following: CD2, CD7 and CD28, CD30, OX40, CD53, CARD11, OX40, CD83, and CD134 (OX40), as well as CD205 (SLAMF1)), CD152 (CTLA4)), CD225 (LAG3)), CD223 (LAG3)), CD270 (HVEM), PD-L2) CD274, CD274 (PD-1), CD274 (PD-1, PD-L1), TRIM, TRIM, NKD2C, TRIM, TRIM, TRIM, TRIM, TRIM and TRIM)

“In particular embodiments the one or more costimulatory signaling domains are selected from the following co-stimulatory molecules: CD28, CD134 and CD137.”

“In additional embodiments the one or more costimulatory signaling domains come from a costimulatory molecule selected among the group consisting: CD28, CD134 and CD137.”

“In additional embodiments the one or more costimulatory signaling domains are from CD28.”

“In particular embodiments the one or more costimulatory signaling domains are from CD134.”

“In other embodiments the one or more costimulatory signaling domains are from CD137.”

“In some embodiments, a CAR includes a hinge region polypeptide.”

“In further embodiments the hinge region polypeptide includes a CD8-like region.”

“In some embodiments, the CAR includes a spacer area.”

“In addition to the above embodiments, spacer region polypeptide includes CH2 and CH3 regions IgG1 and IgG4.”

“In particular embodiments, the CAR includes a signal protein.”

“In further embodiments, the signal peptide comprises an IgG1 heavy chain signal polypeptide, granulocyte-macrophage colony stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8? sign polypeptide.

“In one embodiment, the CAR contains an amino acid sequence set forth in SEQ ID No: 9”

“In different embodiments, a polynucleotide that encodes a CAR contemplated in this invention is provided.”

“In various embodiments, a polynucleotide that encodes a CAR can be provided. The sequence of the polynucleotides is described in SEQ ID No: 10.

“In certain specific embodiments, a vector encoding a CAR as contemplated herein or set forth in SEQID NO: 10 is provided.”

“In some embodiments, the vector can be an expression vector.”

“In additional embodiments, a vector is an episomal Vector.”

“In particular embodiments the vector is a virus vector.”

“In further embodiments the vector is a retroviral Vector.”

“In other embodiments the vector is a vector that is lentiviral.”

“In one embodiment, the vector that encodes a BCMA CAR includes the polynucleotide sequence described in SEQ ID No: 36.”

“In additional embodiments, the 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) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV).”

“In particular embodiments, the vector includes a left (5?). A retroviral LTR and a Psi are (?) Retroviral LTR, a Psi (?) retroviral LTR.”

“In other embodiments, the CAR includes a heterologous sequence of polyadenylation.”

“In certain embodiments, a CAR comprises a hepatitis B viral posttranscriptional regulatory elements (HPRE) and a woodchuck posttranscriptional regulatory component (WPRE).

“In some embodiments, the promoter for the 5? LTR can be replaced by a heterologous promoter.

“In further embodiments, a heterologous promoter can be a cytomegalovirus promoter (CMV), a Rous Sarcoma Virus promoter (RSV), or a Simian Virus 40 promoter (SV40).

“In particular embodiments the 5? LTR or 3? LTR is a lentivirus LTR.

“In particular embodiments the 3? LTR can include one or more modifications.

“In some embodiments the 3? LTR can include one or more deletions.

“In some embodiments, the 3? LTR is an LTR that self-inactivates (SIN).

“In some embodiments, polyadenylation is a bovine growthhormone polyadenylation or signal bunny?-globin sequence.”

“In addition embodiments, a polynucleotide that encodes a CAR contemplated in this invention includes an optimized Kozak sequence.”

“In further embodiments, a promoter operably linked with the polynucleotide encoded a CAR is selected from the following: a cytomegalovirus instant early gene promoters (CMV), a elongation factor1 alpha promoters (EF1-?) ), a promoter of phosphoglycerate-1 (PGK), an ubiquitin-1 alpha promoter(UBQ-C), and a polyoma enhancer/herpes simplux thymidinekinase (CAG), a promoter of beta actin (?-ACT), and a myeloproliferative virus enhancer, negative region deleted, d1587rev prime-binding site substituted promoter.

“In various embodiments, an immune cell that acts as an effector is provided with a vector contemplated in this invention.” The vector contemplated herein is used to transduce the immune effector cells in various embodiments.

“In further embodiments the immune effector cells are selected from the group consisting: a T lymphocyte, a natural killer (NK), cell.”

“In some embodiments, an immune effector cell is transduced using the vector of any of the embodiments above. The activated and stimulated immune effectors are maintained in the presence or absence of an inhibitor of PI3K pathway. This allows for the maintenance of proliferation of the transduced immune cells compared with the proliferation of activated and stimulated immune effectors cells.

“In particular embodiments, an immune effector cells activated or stimulated in the presence inhibitor of PI3K pathways has increased expression of i), one or more markers from the group consisting: CD62L and CD127, CD197 and CD38, or ii), all markers CD62L and CD127, CD197 and CD38, compared to an immune cell activated or stimulated in absence of inhibitor of PI3K.

“In one embodiment, ZSTK474 is the PI3K inhibitor.”

“In different embodiments, a composition includes an immune effector cells contemplated herein as well as a physiologically acceptable excipient.”

“In different embodiments, a method for generating an immune-effector cell comprising an AAR is contemplated. This involves injecting into an immune cell a vector containing a polynucleotide that encodes the CAR.”

“In addition, the method includes stimulating the immune effector and inducing the cells to proliferate through contact with antibodies that bind CD3 or antibodies that bind CD28 to the cell; thus, generating a population immune effector cells.”

“In certain embodiments, the immune effector cells are stimulated and inducible to proliferate prior to introducing the vector.”

“In some embodiments, immune effector cells include T lymphocytes.”

“In particular embodiments the immune effector cells include NK cells.”

“In particular embodiments, cells are activated or stimulated when there is an inhibitor of PI3K pathway. This allows for the maintenance of proliferation of transduced immune effector cell populations as opposed to proliferation of activated or stimulated immune cells in absence of inhibitor of PI3K pathway.”

“In certain embodiments, immune effector cell activation and stimulation in the presence inhibitor of PI3K pathways have increased expression of i), one or more markers from the group consisting: CD62L and CD127, CD197 and CD38, or ii), all of the markers CD62L and CD127, CD197 and CD38, compared to immune cells activated or stimulated in absence of inhibitor of PI3K path.”

“In one embodiment, ZSTK474 is the PI3K inhibitor.”

“In various embodiments, there is a method for treating a B-cell related condition in a subject who needs it. This involves administering to the subject a therapeutically effective amount of a compound comprising BCMA cells and optionally, a pharmaceutically acceptable adjuvant.

“In other embodiments, the B cell related condition is multiple myeloma, non-Hodgkin’s lymphoma, B cell proliferations of uncertain malignant potential, lymphomatoid granulomatosis, post-transplant lymphoproliferative disorder, an immunoregulatory disorder, rheumatoid arthritis, myasthenia gravis, idiopathic thrombocytopenia purpura, anti-phospholipid syndrome, Chagas’ disease, Grave’s disease, Wegener’s granulomatosis, poly-arteritis nodosa, Sjogren’s syndrome, pemphigus vulgaris, scleroderma, multiple sclerosis, anti-phospholipid syndrome, ANCA associated vasculitis, Goodpasture’s disease, Kawasaki disease, autoimmune hemolytic anemia, and rapidly progressive glomerulonephritis, heavy-chain disease, primary or immunocyte-associated amyloidosis, or monoclonal gammopathy of undetermined significance.”

“Another embodiment of the B-cell related condition is a malignancy.”

“In some embodiments, the B-cell malignancy is multiple myeloma or non-Hodgkin?s lymphoma.

“In some embodiments, the MM may be selected from the following: overt multiple meeloma or smoldering multimyeloma; plasma cell leukemia; plasma cell leukemia; non-secretory Myeloma; IgD myeloma. Osteosclerotic myeloma. Solitary plasmacytoma bone. Extramedullary Plasmacytoma.

“In certain embodiments, the NHL may be selected from the following: Burkitt lymphoma/small lymphocytic Leukemia (CLL/SLL), chronic lymphocytic lukemia/small lymphocytic Lymphoma (CLL/SLL), diffuse large B cell lymphoma and immunoblastic large-cell lymphoma.

“In particular embodiments, the plasma cell malignancy is the B-cell related condition.”

“In further embodiments the B-cell related condition is an auto immune disease.”

“Systemic lupus is another form of autoimmune disease.”

“In some embodiments, the B-cell related condition is rheumatoidarthritis.”

“In particular embodiments the B-cell related condition is idiopathic hemolytic anemia, myasthenia gravis or myasthenia purpura.

“BRIEF DESCRIPTION ABOUT CERTAIN VIEWS OF THE DRAWINGS.”

“FIG. 1. shows a schematic for murine B cell maturation antibody (muBCMA), CAR constructs.

“FIG. 2a displays the amount IFNg released by anti-BCMA02 CAR C cells, anti-BCMA10CART cells and CAR19? After co-culturing the cells for 24 hours with BCMA-expressing K562 cells, T cells were activated by BCMA.

“FIG. 2b is the amount IFNg released by anti-BCMA02 CART cell, anti-BCMA10CART cells and CAR19? After co-culture with K562 cells, T cells were exposed to IFNg from anti-BCMA02 CART cells and anti-BCMA10 CART cells.

“FIG. “FIG. T cells were stimulated for 10 days before the assay.”

“FIG. “FIG. T cells without antigen stimulation.

“FIG. 5. The expression of phenotypic markers for activation at anti-BCMA CAR t cell manufacturing. The expression of CD25 and HLA-DR was determined in anti-BCMA02 CART cell lines, anti-BCMA10CART cells, and anti-BCMA19 CART cells. T cells.”

“FIG. “FIG.

“FIG. “FIG.

“FIG. 8A illustrates the tumor volume in NOD Scid Gamma (NSG), mice with?100mm3 experimental subcutaneous human multiple myeloma RPMI-8226 tumors. The mice were given vehicle, 107 antiBCMA02 CART cells and 107 antiBCMA10 CAR-T cells or Bortezomib.

“FIG. 8B is the volume of a tumor in NOD scid Gamma (NSG), mice with?100mm3 experimental subcutaneous human multiple myeloma(RPMI-8226). The mice were given vehicle, 107 antiBCMA02 CART cells and 107 antiBCMA10 CAR-T cells or Bortezomib.

“FIG. “FIG. release, boxes). BCMA-negative (BCMA+), tumor cell lines: Myelogenous Leukemia (K562) and acute lymphoblastic Leukemia (NALM-6, NALM-16); Mantle Cell Lymphoma (REC-1); or Hodgkin?s lymphoma [HDLM-2) showed little to no IFN. release. 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, Ramos cells), Multiple myeloma (?RPMI-82226?) showed significant IFN. release.”

“FIG. 10A is an in vivo activity chart for vehicle, anti-CD19? In an NSG mouse model, CART cells, anti?CD19 CART cell and anti-BCMA (Daudi) cells to BCMA expressing Burkitt?s lymphoma cells in a CART cell-based system. CAR T cells are given to the mice 8 days after tumor induction.

“FIG. 10B is an in vivo activity chart for vehicle, anti-CD19? In an NSG mouse model, CART cells, anti CD19 CAR-T cells, and anti BCMA CAR-T cells to BCMA-expressing Burkitt’s Lymphoma cells (Daudi Cells) are given to CAR-T cells-treated mice 18 days after tumor induction.

“FIG. 11B shows the MFI of normalized anti-BCMA cells. It ranged between 885 and 1875, as determined by flow cytometry. FIG. FIG. 11.C: K562 cells and the K562-BCMA cell were co-cultured together with normalized anti?BCMA CAR cells at a 20/1 or 10/1 effector (E: T cell to target (T: 1:1 mix of K562 BCMA and K562 BCMA) ratio. They showed comparable cytolytic activities.”

“FIGS. 12A-D illustrate the reliability of anti-BCMA CAR-T cells manufacturing processes. FIG. FIG. 12A illustrates that anti-BCMA CAR T cells products made from PBMCs from 11 donors show similar levels of expansion to untransduced donor T cell cultures. FIG. 12B illustrates anti-BCMA CAR-T cell products made from 11 donors. They had comparable lentiviral transduction efficiency. FIG. FIG. 12C illustrates the frequency of anti-BCMA positive T cells as measured by flow cytometry. BCMA expression was comparable among all donors. FIG. FIG. 12.12D: Anti-BCMA CAR T cells products made from 11 donors had therapeutically relevant levels IFN? When exposed to BCMA-expressing K562 cell.”

“FIG. 13 shows Venn diagrams of co-expression of CD127 and CD197 in CD62L-positive anti-BCMA02T cells cultured for ten days in the presence or absence of IL-2. Anti-BCMA02 CAR-T cells treated with ZSTK474 showed a higher percentage of cells that co-express CD127, CD197, and CD38 than cells cultured in the absence of IL-2.

“FIG. 14 shows an increase in CD8 expressing anti BCMA02 CAR T cells when cultures were treated with IL-2 and ZSTK474 (n=7) compared with cultures that were treated with IL-2 only. CD8 expression was determined using a fluorescently-labeled anti-CD8 antibody and flow cytometry.”

“FIG. “FIG. Anti-BCMA02 CART cells of 14 donors were able to release IFN-? after being cultured with IL-2 only or with IL-2 plus ZSTK474. After the culture period ended, the same number of anti-BCMA02 T cells from 14 donors were re-cultured in media for 24 hours. How much IFN-? was released in 24 hours was determined by ELISA. ELISA was used to measure the amount of IFN-? released within 24 hours. The anti-BCMA02 CAR-T cell tonic cytokine production was not significantly increased in culture in ZSTK474 as compared to anti?BCMA02 T cells cultured only with IL-2.”

“FIG. “FIG. In 50% of mice that received the anti-BCMA02 CART cells with IL-2 or ZSTK474, complete tumor regression was observed.

“FIG. “FIG. The outgrowth of tumors was completely stopped by animals treated with IL-2 or IL-2 plus ZSTK474-cultured anti?BCMA02 CAR-T cells.

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