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Definitions

• the CAR of the present invention comprises:
• the CAR of the present invention may comprise one of the following amino acid sequences:
• the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http://blast.ncbi.nlm.nih.gov.
• the endodomain is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster and a signal is transmitted to the cell.
• the most commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.
• CD3-zeta may not provide a fully competent activation signal and additional co-stimulatory signaling may be needed.
• chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together (Pule et al, Molecular therapy, 2005: Volume 12; Issue 5; Pages 933-41).
• the CAR endodomain may also be derived from other signaling domains either individually or in combination, derived from signaling proteins found in nature or artificial ones constructed by those skilled in the art such that the CAR transmits a suitable signal to for an effective CAR therapeutic.
• the endodomain of the CAR of the present invention may comprise the CD28 endodomain and OX40 and CD3-Zeta endodomain.
• the transmembrane and intracellular T-cell signalling domain (endodomain) of the CAR of the present invention may comprise the sequence shown as SEQ ID No. 7 or a variant thereof having at least 80% sequence identity.
• FWVLWVGGVLACYSLLVTVAFI I FWVRSKRSRLLHSDYMN TPRRPGPTRKHYQPYAPPRDF AAYRSRDQRLPPDAHKPPGGGS FRTPI QEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 7, provided that the sequence provides an effective transmembrane domain and an effective intracellular T cell signaling domain.
• the CAR of the present invention may comprise a signal peptide so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
• the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
• the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
• At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
• Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases.
• the CAR of the invention may have the general formula: Signal peptide - BCMA-binding domain - spacer domain - transmembrane domain - intracellular T cell signaling domain.
• the signal peptide may comprise the SEQ ID No. 8 or 9 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.
• the signal peptide of SEQ ID No. 8 and SEQ ID No 9 is compact and highly efficient. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.
• the CAR of the present invention may comprise a spacer sequence to connect the BCMA-binding domain with the transmembrane domain and spatially separate the BCMA-binding domain from the endodomain.
• a flexible spacer allows to the BCMA- binding domain to orient in different directions to enable BCMA binding.
• a human lgG1 spacer may be altered to remove Fc binding motifs.
• amino acid sequences for these spacers are given below:
• SEQ ID No. 12 (human lgG1 hinge):
• the CAR of the first aspect of the invention comprises a domain which binds BCMA.
• BCMA also known as TNFRSF17,is a plasma cell specific surface antigen which is expressed exclusively on B-lineage haemopoietic cells or dendritic cells. It is a member of the TNF receptor family. BCMA is not expressed on naive B cells but is up-regulated during B-cell differentiation into plasmablasts, and is brightly expressed on memory B cells, plasmablasts and bone marrow plasma cells. BCMA is also expressed on the majority of primary myeloma cells. Unlike other CAR targets such as CD19, BCMA is expressed at low density ( Figure 2).
• BCMA functions within a network of interconnected ligands and receptors which is shown schematically in Figure 1.
• Two other TNF receptors share the ligands APRIL and BAFF with BCMA - TACI (TNFRSF 3B), which is found on activated T-cells and all B-cells and BAFF-R (TNFRSF13C) which is predominantly expressed on B- lymphocytes.
• BCMA - TACI TNFRSF 3B
• BAFF-R TNFRSF13C
• Multiple myeloma cells express TACI in some cases and BCMA in most cases, but never BAFF-R.
• the BCMA-binding domain of the CAR of the invention and comprises at least part of a proliferation-inducing ligand (APRIL).
• APRIL is also known as TNFSF13.
• APRIL APRIL sequence available at UNIPROT/075888 and is show below (SEQ ID No. 13). It is not a classical secreted protein in that it has no signal peptide. It has a furin cleavage site "KQKKQK” (underlined in SEQ ID No. 13). The amino terminus is involved in proteoglycan binding.
• the BCMA-binding domain may comprise the BCMA-binding site of APRIL.
• the BCMA-binding domain may comprise a fragment of APRIL which comprises the BCMA-binding site.
• the BCMA-binding domain may comprise a truncated APRIL, which lacks the amino terminal end of the molecule.
• the truncated APRIL may retain BCMA and TACI binding but lose proteoglycan binding.
• Truncated APRIL can be cleaved at or immediately after the furin cleavage site.
• Truncated APRIL may lack the amino terminal 116 amino acids from the wild-type APRIL molecule shown as SEQ ID No. 13.
• Truncated APRIL may comprise the sequence shown as SEQ ID No. 14 (which corresponds to the portion of SEQ ID No. 13 shown in bold) or a variant thereof. This corresponds to the portion of the molecule which is needed for BCMA and TACI binding.
• the CAR of the present invention may comprise a variant of the truncated APRIL molecule shown as SEQ ID No. 14 which has at least 80% amino acid sequence identity and which has the same or improved BCMA binding capabilities.
• the variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 14.
• the second aspect of the invention relates to a nucleic acid sequence which codes for a CAR of the first aspect of the invention.
• the nucleic acid sequence may be or comprise one of the following sequences:
• the nucleic acid sequence may encode the same amino acid sequence as that encoded by SEQ ID No. 15, 16, 17, 18 19 or 20 but may have a different nucleic acid sequence, due to the degeneracy of the genetic code.
• the nucleic acid sequence may have at least 80, 85, 90, 95, 98 or 99% identity to the sequence shown as SEQ ID No. 15, 16, 17, 18 19 or 20 provided that it encodes a CAR as defined in the first aspect of the invention.
• the present invention also provides a vector which comprises a nucleic acid sequence according to the present invention.
• a vector may be used to introduce the nucleic acid sequence into a host cell so that it expresses and produces a molecule according to the first aspect of the invention.
• the vector may, for example, be a plasmid or synthetic mRNA or a viral vector, such as a retroviral vector or a lentiviral vector.
• the vector may be capable of transfecting or transducing an effector cell.
• the invention also provides a host cell which comprises a nucleic acid according to the invention.
• the host cell may be capable of expressing a CAR according to the first aspect of the invention.
• the host cell may be human T cell or a human NK cell.
• a T-cell capable of expressing a CAR according to the invention may be made by transducing or transfecting a T cell with CAR-encoding nucleic acid.
• the T-cell may be an ex vivo T cell.
• the T cell may be from a peripheral blood mononuclear cell (PBMC) sample.
• PBMC peripheral blood mononuclear cell
• T cells may be activated and/or expanded prior to being transduced with CAR-encoding nucleic acid, for example by treatment with a anti-CD3 monoclonal antibody.
• the present invention also relates to a pharmaceutical composition containing a vector or a CAR-expressing T cell of the invention together with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds.
• a pharmaceutically acceptable carrier diluent or excipient
• Such a formulation may, for example, be in a form suitable for intravenous infusion).
• METHOD OF TREATMENT T cells expressing a CAR molecule of the present invention are capable of killing cancer cells, such as multiple myeloma cells.
• CAR- expressing T cells may either be created ex vivo either from a patient's own peripheral blood (1 st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2 nd party), or peripheral blood from an unconnected donor (3 rd party).
• CAR T-cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T-cells.
• CAR T-cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA.
• T cells expressing a CAR molecule of the present invention may be used for the treatment of a cancerous disease, in particular a plasma cell disorder or a B cell disorder which correlates with enhanced BCMA expression.
• Plasma cell disorders include plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullar ⁇ plasmacytoma, osteosclerotic myeloma (POEMS Syndrome) and heavy chain diseases as well as the clinically unclear monoclonal gammopathy of undetermined significance/smoldering multiple myeloma.
• Plasma cell disorders include plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullar ⁇ plasmacytoma, osteosclerotic myeloma (POEMS Syndrome) and heavy chain diseases as well as the clinically unclear monoclonal gammopathy of undetermined significance/smoldering multiple myeloma.
• POEMS Syndrome osteosclerotic myelo
• the disease may be multiple myeloma.
• B cell disorders which correlate with elevated BCMA expression levels are CLL (chronic lymphocytic leukemia) and non-Hodgkins lymphoma (NHL).
• CLL chronic lymphocytic leukemia
• NHL non-Hodgkins lymphoma
• the bispecific binding agents of the invention may also be used in the therapy of autoimmune diseases like Systemic Lupus Erythematosus (SLE), multiple sclerosis (MS) and rheumatoid arthritis (RA).
• SLE Systemic Lupus Erythematosus
• MS multiple sclerosis
• RA rheumatoid arthritis
• the method of the present invention may be for treating a cancerous disease, in particular a plasma cell disorder or a B cell disorder which correlates with enhanced BCMA expression.
• a method for the treatment of disease relates to the therapeutic use of a vector or T cell of the invention.
• the vector or T cell may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
• the method of the invention may cause or promote T-cell mediated killing of BCMA-expressing cells, such as plasma cells.
• APRIL in its natural form is a secreted type II protein.
• the use of APRIL as a BCMA binding domain for a CAR requires conversion of this type II secreted protein to a type I membrane bound protein and for this protein to be stable and to retain binding to BCMA in this form.
• the extreme amino-terminus of APRIL was deleted to remove binding to proteoglycans.
• a signal peptide was added to direct the nascent protein to the endoplasmic reticulum and hence the cell surface.
• an APRIL based CAR was generated comprising (i) a human lgG1 spacer altered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) the lgG1 hinge alone (cartoon in Figure 4 and amino acid sequences in Figure 5, and also amino acid sequences in figure 19 which differ from the sequences in figure 5 by having a different signal peptide and the V5 epitope tag).
• These CARs were expressed in a bicistronic retroviral vector ( Figure 6A) so that a marker protein - truncated CD34 could be co-expressed as a convenient marker gene.
• the aim of this study was to test whether the APRIL based CARs which had been constructed were expressed on the cell surface and whether APRIL had folded to form the native protein.
• T-cells were transduced with these different CAR constructs and stained using a commercially available anti-APRIL mAb, along with staining for the marker gene and analysed by flow-cytometry.
• the results of this experiment are shown in Figure 6B where APRIL binding is plotting against marker gene fluorescence.
• Recombinant BCMA and TACI were generated as fusions with mouse lgG2a- Fc. These recombinant proteins were incubated with the transduced T-cells. After this, the cells were washed and stained with an anti-mouse fluorophore conjugated antibody and an antibody to detect the marker gene conjugated to a different fluorophore. The cells were analysed by flow cytometry and the results are presented in Figure 6C. The different CARs were able to bind both BCMA and TACI. Surprisingly, the CARs were better able to bind BCMA than TACI. Also, surprisingly CARs with a CD8 stalk or lgG1 hinge spacer were better able to bind BCMA and TACI than CAR with an Fc spacer.
• Example 4 APRIL based chimeric antigen receptors are active against BCMA expressing cells
• T-cells from normal donors were transduced with the different APRIL CARs and tested against SupT1 cells either wild-type, or engineered to express BCMA and TACI.
• Assays were used to determine function.
• a classical chromium release assay was performed.
• the target cells (the SupT1 cells) were labelled with 51 Cr and mixed with effectors (the transduced T-cells) at different ratio. Lysis of target cells was determined by counting 51 Cr in the co-culture supernatant (Figure 6A shows the cumulative data, example data from a single assay with different effectontarget ratios is shown in figure 12).
• Example 5 - APRIL based CARs are able to kill primary myeloma cells
• Example 6 Secreted and truncated APRIL fused to an Fc spacer recognizes BCMA and TACI
• Example 7 - APRIL based chimeric antigen receptors are stably expressed on the surface of T-cells
• the CAR spacer domain can alter sensitivity and specificity.
• Three versions of an 0 APRIL-based CAR were generated with three spacer domains: (i) a human lgG1 spacer altered to remove Fc binding motifs; (ii) a CD8 stalk; and (iii) the lgG1 hinge alone ( Figure 10B).
• Primary human T-cells were transduced with these different CARs and stained using a commercially available anti-APRIL mAb ( Figure 1 1).
• Example 8 - APRIL based chimeric antigen receptors are active against cognate target expressing cells T-cells from normal donors were transduced with the different APRIL CARs and tested against SupT1 cells either wild-type, or engineered to express BCMA and TACI.
• Several different assays were used to determine function.
• a classical chromium release assay was performed.
• the target cells (the SupT1 cells) were labelled with 51 Cr and mixed with effectors (the transduced T-cells) at different ratio. Lysis of target cells was determined by counting 51 Cr in the co-culture supernatant ( Figure 12).
• T-cell expansion after one week of co-culture with SupT1 cells was also performed. T-cells were counted by flow-cytometry calibrated with counting beads. Initial data (not shown) appears to indicate that the CD8 stalk based construct results in more T-cell proliferation than the other constructs.
• MM1.s (ATCC CRL-2974) is a human myeloma cell line which expresses intermediate levels of BCMA. The inventors engineered this cell line to express firefly Luciferase to derive the cell-line MMls.FLuc.
• NOD scid gamma (NSG: NOD.Cg-Prkdc scid Il2rgtm1 wjl/Szj ) mice are profoundly immunosuppressed mice capable of engrafting several human cell lines and human peripheral blood lymphocytes.
• 5x10 6 APRIL-HNG-CD280XZ CAR T-cells were administered via tail vein injection.

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