Modified CD19-targeted chimeric antigen receptor (CAR)-t cell and uses thereof

Abstract

The present disclosure relates to chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically recognizes human cluster of differentiation 19 (CD 19) antigen. The present disclosure also relates to a polynucleotide encoding the CAR, vectors, genetically modified cells and uses thereof.

Description

[0001] MODIFIED CD19-TARGETED CHIMERIC ANTIGEN RECEPTOR (CAR)-T CELL AND USES THEREOF

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from Australian provisional application No. 2024901053 entitled “Modified CD19-targeted chimeric antigen receptor (CAR)-T cell and uses thereof’ filed 15 April 2024, the entire contents of which are hereby incorporated by reference.

[0004] SEQUENCE LISTING

[0005] The present application is filed together with a Sequence Listing in electronic form. The entire contents of the Sequence Listing are hereby incorporated by reference.

[0006] FIELD

[0007] The present disclosure relates to chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically recognizes human cluster of differentiation 19 (CD 19) antigen. The present disclosure also relates to a polynucleotide encoding the CAR, vectors, genetically modified cells and uses thereof.

[0008] BACKGROUND

[0009] The human immune system typically defends against foreign substances and pathogens from invading the body. B lymphocytes, or B-cells, are a crucial part of this defence, producing antibodies that bind to and mediate destruction of these foreign substances. However, when the immune system becomes dysregulated, it can lead to diseases marked by uncontrolled B-cell proliferation, such as cancer, autoimmune disorders, and inflammatory diseases.

[0010] Cancer is a leading cause of death worldwide, accounting for nearly one in six deaths. Over the last decade, immunotherapy has become a promising approach for the treatment of cancer. A rapidly developing area of immunotherapy has been adoptive immune cell therapy, whereby immune cells (commonly T cells) are genetically modified with chimeric antigen receptors (CAR) that target tumour associated antigens. The advantage of CAR-T technology compared with chemotherapy or other antibody-based therapies is that CARs provide a patient's own T cells with the ability to recognize and kill cancer cells directly. Autoimmune diseases, such as systemic lupus erythematosus (SLE) are characterised by the immune system mistakenly attacking the body's own healthy tissues, leading to inflammation, organ damage, and a wide range of symptoms that can fluctuate in severity. Currently, therapies for autoimmune disorders aim to manage symptoms and minimize organ damage, with treatments varying depending on the specific condition and individual needs, but there's no cure for these chronic conditions. Common approaches include medications like corticosteroids, immunosuppressants, and targeted therapies, alongside lifestyle modifications like diet and exercise. More recently, CAR- T technology has emerged as a promising approach for treating autoimmune diseases by targeting and eliminating the faulty immune cells driving the condition.

[0011] Cluster of differentiation 19 (CD 19) is a B-cell antigen which is expressed very early in B-cell differentiation and is only lost at terminal B-cell differentiation into plasma cells. Accordingly, CD 19 is expressed on all B-cell malignancies. It is not expressed on other haematopoietic populations or non-haematopoietic cells and therefore CD 19 has been an attractive target for immunotherapy.

[0012] To date six CAR-T cell products have been approved by the US Food and Drug Administration for treatment of several B-cell malignancies, including Breyanzi®, Kymriah™, Tecartus™, Yescarta™ targeting CD 19. However, there are a number of major limitations to CAR-T cell therapy that remain an issue, including life-threatening CAR-T cell-associated toxicities, cytokine release syndrome (CRS), limited efficacy against solid tumours, antigen escape (or tumour resistance), limited persistence, poor trafficking and tumour infiltration, and the immunosuppressive microenvironment. In addition, to date there are currently no approved CAR-T cell products approved for the treatment of autoimmune diseases.

[0013] The antigen binding domain is the portion of the CAR that confers target antigen specificity, whilst affinity of the antigen binding domain to the target ultimately determines CAR function. The CARs antigen binding affinity must be sufficiently high enough to recognise antigens on tumour cells, induce CAR signalling, and activate T cells, but not high enough to result in activation induced death of the CAR expressing T cell and trigger toxicities. Other factors, such as epitope location and target antigen density, can also differentially impact the binding of the CAR to its target antigen and the CAR-T cell function.

[0014] Accordingly, it will be apparent to the skilled person that there is a need to develop improved genetically modified immune cells with CARs for adoptive cell therapy. SUMMARY

[0015] The present disclosure is based on the inventors’ production of a modified single chain variable fragment (scFv) specific to CD 19, and subsequent production of a CD 19- targeting chimeric antigen receptor (CAR). Following modification of both the variable light and heavy chains, the inventors generated CD19-CAR-T cells and found that the transfection efficiency and intracellular expression of the CD19-CAR in transduced T cells was enhanced. In particular, the modified polynucleotide sequences identified by the inventors were shown to led to at least the improved transfection efficiency. In addition, the inventors found long-term stable CAR expression in transduced T cells. The inventors also found strong and specific CD19-CAR-T mediated killing of CD 19- expressing cancer cells, with reduced CRS, in a murine model of lymphoblastic leukemia. The inventors have also found specific strong and specific CD19-CAR-T mediated killing of CD19-expressing cells derived from a patient diagnosed with systemic lupus erythematosus (SLE), together with a reduction in pro-inflammatory cytokines associated with CRS.

[0016] Thus, the present disclosure is broadly directed to a CAR comprising an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen and polynucleotides encoding thereof.

[0017] The present disclosure provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the nucleotide sequence encoding the modified scFv comprises:

[0018] (i) a light chain variable region (VL) comprising a complementarity determining region (CDR) 1 comprising a sequence set forth in SEQ ID NO: 3; a CDR2 comprising a sequence set forth in SEQ ID NO: 4; and a CDR3 comprising a sequence set forth in SEQ ID NO: 5; and

[0019] (ii) a heavy chain variable region (VH) comprising: a CDR1 comprising a sequence set forth in SEQ ID NO: 6; a CDR2 comprising a sequence set forth in SEQ ID NO: 7; and a CDR3 comprising a sequence set forth in SEQ ID NO: 8.

[0020] The present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises:

[0021] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the nucleotide sequence encoding the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 3; a CDR2 comprising a sequence set forth in SEQ ID NO: 4; and a CDR3 comprising a sequence set forth in SEQ ID NO: 5; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 6; a CDR2 comprising a sequence set forth in SEQ ID NO: 7; and a CDR3 comprising a sequence set forth in SEQ ID NO: 8;

[0022] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0023] (iii)a nucleotide sequence encoding at least one co-stimulatory domain.

[0024] The present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises, in order from 5’ to 3’:

[0025] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 3; a CDR2 comprising a sequence set forth in SEQ ID NO: 4; and a CDR3 comprising a sequence set forth in SEQ ID NO: 5; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 6; a CDR2 comprising a sequence set forth in SEQ ID NO: 7; and a CDR3 comprising a sequence set forth in SEQ ID NO: 8;

[0026] (ii) a nucleotide sequence encoding a transmembrane domain; and

[0027] (iii)a nucleotide sequence encoding at least one co-stimulatory domain.

[0028] In one example:

[0029] (i) the transmembrane domain is a CD8a transmembrane domain; and / or

[0030] (ii)the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

[0031] In one example, the transmembrane domain is a CD8a transmembrane domain.

[0032] In one example, the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3^ signalling domain. For example, the at least one co-stimulatory domain comprises a 4-1BB signalling domain. In another example, the at least one co-stimulatory domain comprises a CD27 signalling domain. In a further example, the at least one co-stimulatory domain comprises an OX -40 signalling domain. In one example, the at least one co-stimulatory domain comprises a GITR signalling domain. In another example, the at least one costimulatory domain comprises a HVEM signalling domain. In a further example, the at least one co-stimulatory domain comprises a CD3^ signalling domain. In one example, the co-stimulatory domain comprises a 4-1BB signalling domain and a CD3^ signalling domain. In a further example, the co-stimulatory domain consists of a 4- IBB signalling domain and a CD3^ signalling domain.

[0033] In one example, the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region. For example, the hinge region is a CD8a hinge region.

[0034] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5’ to 3’:

[0035] (i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen of the disclosure;

[0036] (ii) a nucleotide sequence encoding a CD8a hinge region;

[0037] (iii)a nucleotide sequence encoding a CD8a transmembrane domain;

[0038] (iv)a nucleotide sequence encoding a 4- IBB signalling domain; and

[0039] (v) a nucleotide sequence encoding a CD3^ signalling domain.

[0040] In one example, the polynucleotide further comprises a signal peptide located 5’ to the antigen binding domain. For example, the signal peptide is a CD8a signal peptide.

[0041] In one example, the polynucleotide encoding the CAR of the present disclosure comprises from 5’ to 3’:

[0042] (i) a nucleotide sequence encoding a CD8a signal peptide;

[0043] (ii)a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen of the disclosure;

[0044] (iii)a nucleotide sequence encoding a CD8a hinge region;

[0045] (iv)a nucleotide sequence encoding a CD8a transmembrane domain;

[0046] (v) a nucleotide sequence encoding a 4- IBB signalling domain; and

[0047] (vi)a nucleotide sequence encoding a CD3^ signalling domain.

[0048] In one example, the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 1 and a VH comprising a sequence set forth in SEQ ID NO: 2.

[0049] In one example, the VL and the VH are covalently linked to each other via a linker.

[0050] In one example, the linker is a peptide linker comprising at least 2 amino acids in length. For example, the linker is a GS linker. In one example, the linker is a GS linker and is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3 linker, a (Gly)x linker, a (Gly)r, linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4. For example, the linker is a (Gly4Ser)3 linker. In one example, the (Gly4Ser)3 linker comprises a polypeptide encoded by a nucleotide sequence set forth in SEQ ID NO: 17.

[0051] In one example, the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 19.

[0052] In one example, the nucleotide sequence encoding the CD8a transmembrane domain comprises a sequence set forth in SEQ ID NO: 23; the nucleotide sequence encoding the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 25; and / or the nucleotide sequence encoding the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 27.

[0053] In one example, the nucleotide sequence encoding the CD8a transmembrane domain comprises a sequence set forth in SEQ ID NO: 23.

[0054] In one example, the nucleotide sequence encoding the 4- IBB signalling domain comprises a sequence set forth in SEQ ID NO: 25.

[0055] In one example, the nucleotide sequence encoding the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 27

[0056] In one example, the nucleotide sequence encoding the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 21.

[0057] In one example, the present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises, in order from 5’ to 3’:

[0058] (i) the nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the nucleotide sequence encoding the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 3; a CDR2 comprising a sequence set forth in SEQ ID NO: 4; and a CDR3 comprising a sequence set forth in SEQ ID NO: 5; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 6; a CDR2 comprising a sequence set forth in SEQ ID NO: 7; and a CDR3 comprising a sequence set forth in SEQ ID NO: 8;

[0059] (ii)the nucleotide sequence encoding a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 23; and

[0060] (iii)the nucleotide sequence encoding a co-stimulatory domain comprising: a.a nucleotide sequence encoding a 4- IBB signalling domain comprising a sequence set forth in SEQ ID NO: 25; and b. a nucleotide sequence encoding a CD3^ signalling domain comprising sequence set forth in SEQ ID NO: 27.

[0061] In one example, the present disclosure also provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises, in order from 5’ to 3’:

[0062] (i) the nucleotide sequence encoding an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 3; a CDR2 comprising a sequence set forth in SEQ ID NO: 4; and a CDR3 comprising a sequence set forth in SEQ ID NO: 5; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 6; a CDR2 comprising a sequence set forth in SEQ ID NO: 7; and a CDR3 comprising a sequence set forth in SEQ ID NO: 8;

[0063] (ii)the nucleotide sequence encoding a CD8a hinge region comprising a sequence set forth in SEQ ID NO: 21

[0064] (iii)the nucleotide sequence encoding a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 23; and

[0065] (iv)the nucleotide sequence encoding a co-stimulatory domain comprising: a.a nucleotide sequence encoding a 4-1BB signalling domain comprising sequence set forth in SEQ ID NO: 25; and b. a nucleotide sequence encoding a CD3^ signalling domain comprising a sequence set forth in SEQ ID NO: 27.

[0066] In one example, the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 29.

[0067] The present disclosure provides a CAR encoded by the polynucleotide of the disclosure. The present disclosure provides a CAR encoded by the polynucleotide sequence set forth in SEQ ID NO: 29.

[0068] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 35; a CDR2 comprising a sequence set forth in SEQ ID NO: 12 or 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 38; and b) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 41 or 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 44 or 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 47. The present disclosure also provides a CAR comprising:

[0069] (i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 35; a CDR2 comprising a sequence set forth in SEQ ID NO: 12 or 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 38; and b) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 41 or 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 44 or 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 47; or

[0070] (ii) a transmembrane domain; and

[0071] (iii) at least one co -stimulatory domain.

[0072] The present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0073] (i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 35; a CDR2 comprising a sequence set forth in SEQ ID NO: 12 or 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 38; and b) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 41 or 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 44 or 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 47; or

[0074] (ii) a transmembrane domain; and

[0075] (iii) at least one co -stimulatory domain.

[0076] In one example, the amino acid sequence of the VL CDR1 comprises a sequence set forth in SEQ ID NO: 35, wherein the amino acid sequence comprises Aspartic Acid (D) or Serine (S) at position 5 and / or Isoleucine (I) or Valine at position 6 and / or Lysine (K) or Serine (S) at position 8 and / or Asparagine (N) or Alanine (A) at position 11.

[0077] In one example, the amino acid sequence of the VL CDR1 comprises a sequence set forth in SEQ ID NO: 35, wherein the amino acid sequence comprises Aspartic Acid (D) at position 5 and Isoleucine (I) at position 6 and Lysine (K) at position 8 and Asparagine (N) at position 11.

[0078] In one example, the amino acid sequence of the VL CDR1 comprises a sequence set forth in SEQ ID NO: 35, wherein the amino acid sequence comprises Serine (S) at position 5 and Valine at position 6 and Alanine (A) at position 11. In one example, the amino acid sequence of the VL CDR3 comprises a sequence set forth in SEQ ID NO: 38, wherein the amino acid sequence comprises Glycine (G) or Tyrosine (Y) at position 3 and / or Lysine (K) or Asparagine (N) at position 4 and / or Threonine (T) or Asparagine (N) at position 5 and / or Phenylalanine (F) or Leucine (L) or Tryptophan (W) at position 6 and / or Leucine (L) or Tyrosine (Y) at position 8.

[0079] In one example, the amino acid sequence of the VL CDR3 comprises a sequence set forth in SEQ ID NO: 38, wherein the amino acid sequence comprises Glycine (G) at position 3 and Lysine (K) at position 4 and Threonine (T) at position 5 and Phenylalanine (F) at position 6 and Leucine (L) at position 8.

[0080] In one example, the amino acid sequence of the VL CDR3 comprises a sequence set forth in SEQ ID NO: 38, wherein the amino acid sequence comprises Glycine (G) at position 3 and Asparagine (N) at position 4 and Threonine (T) at position 5 and Leucine (L) at position 6 and Tyrosine (Y) at position 8.

[0081] In one example, the amino acid sequence of the VL CDR3 comprises a sequence set forth in SEQ ID NO: 38, wherein the amino acid sequence comprises Tyrosine (Y) at position 3 and Asparagine (N) at position 4 and Asparagine (N) at position 5 and Tryptophan (W) at position 6 and Leucine (L) at position 8.

[0082] In one example, the amino acid sequence of the VH CDR1 comprises a sequence set forth in SEQ ID NO: 41, wherein the amino acid sequence comprises Phenylalanine (F) or Valine (V) at position 2 and / or Glutamic acid (E) or Proline (P) at position 5.

[0083] In one example, the amino acid sequence of the VH CDR1 comprises a sequence set forth in SEQ ID NO: 41, wherein the amino acid sequence comprises Phenylalanine (F) at position 2 and Glutamic acid (E) at position 5.

[0084] In one example, the amino acid sequence of the VH CDR1 comprises a sequence set forth in SEQ ID NO: 41, wherein the amino acid sequence comprises Valine (V) at position 2 and Proline (P) at position 5.

[0085] In one example, the amino acid sequence of the VH CDR2 comprises a sequence set forth in SEQ ID NO: 44, wherein the amino acid sequence comprises Asparagine (N) or Serine (S) at position 11.

[0086] In one example, the amino acid sequence of the VH CDR2 comprises a sequence set forth in SEQ ID NO: 44, wherein the amino acid sequence comprises Asparagine (N) at position 11.

[0087] In one example, the amino acid sequence of the VH CDR2 comprises a sequence set forth in SEQ ID NO: 44, wherein the amino acid sequence comprises Serine (S) at position 11. In one example, the amino acid sequence of the VH CDR3 comprises a sequence set forth in SEQ ID NO: 47, wherein the amino acid sequence comprises Alanine (A) or Tyrosine (Y) at position 9 and / or Methionine (M) or Phenylalanine (F) at position 10 and / or Tyrosine (Y) or Tryptophan (W) at position 12.

[0088] In one example, the amino acid sequence of the VH CDR3 comprises a sequence set forth in SEQ ID NO: 47, wherein the amino acid sequence comprises Alanine (A) at position 9 and Methionine (M) at position 10 and Tyrosine (Y) at position 12.

[0089] In one example, the amino acid sequence of the VH CDR3 comprises a sequence set forth in SEQ ID NO: 47, wherein the amino acid sequence comprises Tyrosine (Y) at position 9 and Phenylalanine (F) at position 10 and Tryptophan (W) at position 12.

[0090] In one example, the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

[0091] In one example, the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 14; n. a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 16.

[0092] In one example, the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b) a VH comprising: i.a CDR1 comprising a sequence set forth in SEQ ID NO: 42; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 16.

[0093] In one example, the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 36; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b) a VH comprising: i.a CDR1 comprising a sequence set forth in SEQ ID NO: 43; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

[0094] The present disclosure also provides a CAR comprising:

[0095] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0096] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0097] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

[0098] (ii) a transmembrane domain; and

[0099] (iii) at least one co -stimulatory domain.

[0100] The present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0101] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0102] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0103] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0104] 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48; and

[0105] (ii) a transmembrane domain; and

[0106] (iii) at least one co -stimulatory domain.

[0107] The present disclosure also provides a CAR comprising:

[0108] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 14; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0109] (ii) a transmembrane domain; and

[0110] (iii) at least one co-stimulatory domain.

[0111] The present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0112] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0113] (ii) a transmembrane domain; and

[0114] (iii)at least one co-stimulatory domain.

[0115] The present disclosure also provides a CAR comprising:

[0116] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b) a VH comprising: iv. a CDR1 comprising a sequence set forth in SEQ ID NO: 42; v. a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and vi. a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0117] (ii) a transmembrane domain; and

[0118] (iii) at least one co-stimulatory domain.

[0119] The present disclosure also provides a CAR comprising, in order from N- to C- terminus: (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0120] (ii) a transmembrane domain; and

[0121] (iii)at least one co-stimulatory domain.

[0122] The present disclosure also provides a CAR comprising:

[0123] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 36; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b) a VH comprising: vii. a CDR1 comprising a sequence set forth in SEQ ID NO: 43; viii. a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and ix. a CDR3 comprising a sequence set forth in SEQ ID NO: 48;

[0124] (ii) a transmembrane domain; and

[0125] (iii) at least one co-stimulatory domain.

[0126] The present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0127] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48;

[0128] (ii) a transmembrane domain; and

[0129] (iii)at least one co-stimulatory domain.

[0130] In one example: (i) the transmembrane domain is a CD8a transmembrane domain; and / or

[0131] (ii)the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3^ signalling domain.

[0132] In one example, the transmembrane domain is a CD8a transmembrane domain.

[0133] In one example, the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3^ signalling domain. For example, the at least one co-stimulatory domain comprises a 4-1BB signalling domain. In another example, the at least one co-stimulatory domain comprises a CD27 signalling domain. In a further example, the at least one co-stimulatory domain comprises an OX -40 signalling domain. In one example, the at least one co-stimulatory domain comprises a GITR signalling domain. In another example, the at least one co- stimulatory domain comprises a HVEM signalling domain. In a further example, the at least one co-stimulatory domain comprises a CD3^ signalling domain. In one example, the co-stimulatory domain comprises a 4-1BB signalling domain and a CD3^ signalling domain. In a further example, the co-stimulatory domain consists of a 4- IBB signalling domain and a CD3^ signalling domain.

[0134] In one example, the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region. For example, the hinge region is a CD8a hinge region.

[0135] In one example, the CAR of the present disclosure comprises from N- to C- terminus:

[0136] (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen of the disclosure;

[0137] (ii)the CD8a hinge region;

[0138] (iii)the CD8a transmembrane domain;

[0139] (iv)the 4- IBB signalling domain; and

[0140] (v)the CD3^ signalling domain.

[0141] In one example, the CAR further comprises a signal peptide located at the N- terminus of the antigen binding domain. For example, the signal peptide is a CD8a signal peptide.

[0142] In one example, the CAR of the present disclosure comprises from N- to C- terminus:

[0143] (i) the CD8a signal peptide; (ii)the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen of the disclosure;

[0144] (iii)the CD8a hinge region;

[0145] (iv)the CD 8a transmembrane domain;

[0146] (v)the 4- IBB signalling domain; and

[0147] (vi)the CD3^ signalling domain.

[0148] In one example, the modified scFv comprises:

[0149] (i) a VL comprising a sequence set forth in SEQ ID NO: 9 and a VH comprising a sequence set forth in SEQ ID NO: 10; or

[0150] (ii) a VL comprising a sequence set forth in SEQ ID NO: 49 and a VH comprising a sequence set forth in SEQ ID NO: 51; or

[0151] (iii)a VL comprising a sequence set forth in SEQ ID NO: 50 and a VH comprising a sequence set forth in SEQ ID NO: 52.

[0152] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 9 and a VH comprising a sequence set forth in SEQ ID NO: 10.

[0153] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 49 and a VH comprising a sequence set forth in SEQ ID NO: 51.

[0154] In one example, the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 50 and a VH comprising a sequence set forth in SEQ ID NO: 52.

[0155] In one example, the VL and the VH are covalently linked to each other via a linker.

[0156] In one example, the linker is a peptide linker comprising at least 2 amino acids in length. For example, the linker is a GS linker. In one example, the linker is a GS linker and is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3 linker, a (Gly)s linker, a (Gly)r, linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4. For example, the linker is a (Gly4Ser)3 linker. In one example, the (Gly4Ser)3 linker comprises a sequence set forth in SEQ ID NO: 18.

[0157] In one example, the modified scFv comprises a sequence set forth in any one of SEQ ID NOs: 20, 53 or 54.

[0158] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO: 20.

[0159] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO:

[0160] 53.

[0161] In one example, the modified scFv comprises a sequence set forth in SEQ ID NO:

[0162] 54. In one example, the CD8a transmembrane domain comprises a sequence set forth in SEQ ID NO: 24; the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 26; and / or the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 28.

[0163] In one example, the CD8a transmembrane domain comprises a sequence set forth in SEQ ID NO: 24.

[0164] In one example, the 4- IBB signalling domain comprises a sequence set forth in SEQ ID NO: 26.

[0165] In one example, the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 28.

[0166] In one example, the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 22.

[0167] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0168] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a.a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0169] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b. a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c.a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0170] 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48; and

[0171] (ii) a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and (iii)a co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising a sequence set forth in SEQ ID NO:

[0172] 26; and b. a CD3^ signalling domain comprising sequence set forth in SEQ ID NO: 28.

[0173] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0174] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0175] (ii) a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0176] (iii)a co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising a sequence set forth in SEQ ID NO:

[0177] 26; and b. a CD3^ signalling domain comprising sequence set forth in SEQ ID NO: 28.

[0178] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0179] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0180] (ii) a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0181] (iii)a co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising a sequence set forth in SEQ ID NO:

[0182] 26; and b. a CD3^ signalling domain comprising sequence set forth in SEQ ID NO: 28.

[0183] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0184] (i) an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48;

[0185] (ii) a CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0186] (iii)a co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising a sequence set forth in SEQ ID NO:

[0187] 26; and b. a CD3^ signalling domain comprising sequence set forth in SEQ ID NO: 28.

[0188] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0189] (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a.a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO:

[0190] 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b. a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c.a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48; and

[0191] (ii)the CD8a hinge region comprising a sequence set forth in SEQ ID NO: 22

[0192] (iii)the CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0193] (iv)the co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising sequence set forth in SEQ ID NO:

[0194] 26; and b. a CD3^ signalling domain comprising a sequence set forth in SEQ ID NO: 28.

[0195] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0196] (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0197] (ii)the CD8a hinge region comprising a sequence set forth in SEQ ID NO: 22

[0198] (iii)the CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0199] (iv)the co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising sequence set forth in SEQ ID NO:

[0200] 26; and b. a CD3^ signalling domain comprising a sequence set forth in SEQ ID NO: 28. In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0201] (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 11 ; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16;

[0202] (ii)the CD8a hinge region comprising a sequence set forth in SEQ ID NO: 22

[0203] (iii)the CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0204] (iv)the co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising sequence set forth in SEQ ID NO:

[0205] 26; and b. a CD3^ signalling domain comprising a sequence set forth in SEQ ID NO: 28.

[0206] In one example, the present disclosure also provides a CAR comprising, in order from N- to C- terminus:

[0207] (i) the extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, wherein the modified scFv comprises: a. a VL comprising a CDR 1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b. a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48;

[0208] (ii)the CD8a hinge region comprising a sequence set forth in SEQ ID NO: 22

[0209] (iii)the CD8a transmembrane domain comprising a sequence set forth in SEQ ID NO: 24; and

[0210] (iv)the co-stimulatory domain comprising: a.a 4-1BB signalling domain comprising sequence set forth in SEQ ID NO:

[0211] 26; and b. a CD3^ signalling domain comprising a sequence set forth in SEQ ID NO: 28. In one example, the CAR comprises a sequence set forth in any one of SEQ ID NOs: 30, 55 or 56.

[0212] In one example, the CAR comprises a sequence set forth in SEQ ID NO: 30.

[0213] In one example, the CAR comprises a sequence set forth in SEQ ID NO: 55.

[0214] In one example, the CAR comprises a sequence set forth in SEQ ID NO: 56.

[0215] The present disclosure provides a polynucleotide encoding the CAR of the present disclosure. The present disclosure provides a polynucleotide encoding a CAR comprising a sequence set forth in any one of SEQ ID NOs: 30, 55 or 56.

[0216] The present disclosure provides a polynucleotide encoding a CAR comprising a sequence set forth in SEQ ID NO: 30.

[0217] The present disclosure provides a polynucleotide encoding a CAR comprising a sequence set forth in SEQ ID NO: 55.

[0218] The present disclosure provides a polynucleotide encoding a CAR comprising a sequence set forth in SEQ ID NO: 56.

[0219] The present disclosure also provides a vector comprising the polynucleotide of the disclosure.

[0220] In one example, the vector is a plasmid, a cosmid, a phage or a viral vector. For example, the vector is a plasmid. In another example, the vector is a cosmid. In a further example, the vector is a phage. In one example, the vector is a viral vector.

[0221] In one example, the viral vector is a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector. For example, the viral vector is a lentiviral vector. For example, the lentiviral vector is a self-inactivating lentiviral plasmid. In another example, the viral vector is an adeno- associated viral vector. In a further example, the viral vector is an adenoviral vector. In one example, the viral vector is a HSV vector. For example, a HSV type 1 vector. In another example, the viral vector is a retroviral vector.

[0222] The present disclosure also provides a genetically modified cell comprising the vector of the disclosure.

[0223] The present disclosure also provides a population of genetically modified cells comprising the vector of the disclosure.

[0224] The present disclosure further provides a method of producing a genetically modified cell for adoptive cell therapy, the method comprising introducing a vector comprising the polynucleotide of the disclosure into the cell, thereby producing the genetically modified cell.

[0225] In one example, the cell is an immune cell. For example, the immune cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

[0226] In one example, the cell is a T cell. For example, the T cell is a gamma delta (y5) T cell, a cytotoxic T cell, or a helper T cell. In another example, the cell is aNK cell.

[0227] The present disclosure provides a composition comprising a plurality of genetically modified cells (i.e., a population of genetically modified cells) of the disclosure for use as a medicament.

[0228] The present disclosure also provides a method of treating a CD19-expressing proliferative disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0229] The present disclosure further provides use of a population of genetically modified cells in the manufacture of a medicament for treating a CD19-expressing proliferative disease in a subject in need thereof.

[0230] The present disclosure provides use of a composition or a population of genetically modified cells for treating a CD19-expressing proliferative disease in a subject in need thereof.

[0231] In one example, the proliferative disease is selected from the group consisting of a malignant proliferative disease or autoimmune disease.

[0232] The present disclosure also provides a method of treating a CD19-expressing malignant proliferative disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0233] The present disclosure provides a method of treating a CD19-expressing autoimmune disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0234] The present disclosure further provides use of a population of genetically modified cells in the manufacture of a medicament for treating a CD19-expressing malignant proliferative disease in a subject in need thereof.

[0235] The present disclosure further provides use of a population of genetically modified cells in the manufacture of a medicament for treating a CD19-expressing autoimmune disease in a subject in need thereof.

[0236] The present disclosure provides use of a composition or a population of genetically modified cells for treating a CD19-expressing malignant proliferative disease in a subject in need thereof. The present disclosure provides use of a composition or a population of genetically modified cells for treating a CD19-expressing autoimmune disease in a subject in need thereof.

[0237] In one example, the CD19-expressing malignant proliferative disease is selected from the group consisting of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia. In one example, the CD19-expresssing disease is BALL. In another example, the CD19-expressing malignant proliferative disease is TALL. In a further example, the CD19-expressing malignant proliferative disease is CML. In one example, the CD19-expressing malignant proliferative disease is CLL. In another example, the CD19-expressing malignant proliferative disease is B cell prolymphocytic leukemia. In a further example, the CD19-expressing malignant proliferative disease is blastic plasmacytoid dendritic cell neoplasm. In one example, the CD19-expressing malignant proliferative disease is Burkitt's lymphoma. In another example, the CD19-expressing malignant proliferative disease is diffuse large B cell lymphoma. In a further example, the CD19-expressing malignant proliferative disease is follicular lymphoma. In one example, the CD19-expressing malignant proliferative disease is hairy cell leukemia. In another example, the CD19-expressing malignant proliferative disease is small cell- or large cell-follicular lymphoma. In a further example, the CD19-expressing malignant proliferative disease is malignant lymphoproliferative conditions. In one example, the CD19-expressing malignant proliferative disease is MALT lymphoma. In another example, the CD19-expressing malignant proliferative disease is mantle cell lymphoma. In a further example, the CD19-expressing malignant proliferative disease is Marginal zone lymphoma. In one example, the CD19-expressing malignant proliferative disease is multiple myeloma. In another example, the CD 19- expressing malignant proliferative disease is myelodysplasia and myelodysplastic syndrome. In a further example, the CD19-expressing malignant proliferative disease is non-Hodgkin's lymphoma. In one example, the CD19-expressing malignant proliferative disease is plasmablastic lymphoma. In another example, the CD19-expressing malignant proliferative disease is plasmacytoid dendritic cell neoplasm. In a further example, the CD 19-expressing malignant proliferative disease is Waldenstrom macroglobulinemia. In one example, the CD 19-expressing malignant proliferative disease is preleukemia.

[0238] In one example, the CD 19-expressing autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgG4-related disease (IgG4-RD), neuromyelitis optica spectrum disorder (NMOSD), generalized myasthenia gravis (gMG), idiopathic inflammatory myositis (IIM), systemic sclerosis (SSc) and Multiple Sclerosis (MS). In one example, the CD 19-expressing autoimmune disease is SLE. In another example, the CD 19-expressing autoimmune disease is RA. In a further example, the CD 19-expressing autoimmune disease is IgG4- RD. In one example, the CD 19-expressing autoimmune disease is gMG. In another example, the CD 19-expressing autoimmune disease is IIM. In a further example, the CD 19-expressing autoimmune disease is SSc. In one example, the CD 19-expressing autoimmune disease is MS. In one example, the subject is a human. In one example, the subject is an adult, for example over 18 years of age. In one example, the subject is a child, for example less than 18 years of age.

[0239] BRIEF DESCRIPTION OF THE DRAWINGS

[0240] Figure 1 is a series of graphical representations showing (A) BC-0041 (hCD19- CAR) and (B) control vector (BC-hCD19del).

[0241] Figure 2 is a series of graphical representations showing co-culture of BC-0041 (hCD 19-CAR) or control vector or PBMCs with (A) NALM-6-Luc or (B) Raji-Luc Cells expressing luciferase, at various effector to target ratios (E: T of 20: 1, 10: 1 and 5: 1) to evaluate cytotoxic potential.

[0242] Figure 3 is a series of graphical representations showing IFN-y levels of BC- hCD19 or BC-hCD19del vectors or untransduced controls co-cultured with (A) NALM- 6 or (B) Raji cells, and TNF-a levels of BC-hCD19 or BC-hCD19del vectors or untransduced controls co-cultured with (C) NALM-6 or (D) Raji cells.

[0243] Figure 4 is a series of graphical representations showing administration of modified CD19-scFv-CAR-T cells reduces tumour burden in a murine model of lymphoblastic leukemia. (A) Tumour burden over time as determined by total radiance (biolumine scent signal) at day 21. (B) Average animal body weight in the treatment groups. (C) Serum IFN-g levels in treated animals, n.s not significant; *P is <0.05; ***P<0.001

[0244] Figure 5 is a series of graphical representations showing transduction of human PBMCs with the modified BC-hCD19 in the presence of a transduction enhancer (A) increases percent CAR expression, (B) decreases levels of secreted IL-6 and (C) increases Raji tumour cell killing. *P is <0.05; **P<0.01

[0245] Figure 6 is a series of graphical representations showing (A) in vivo activity of BC-hCD19 CAR T cells transduced in the presence of a transduction enhancer reduces tumour burden in a murine model of lymphoblastic leukemia as determined by total radiance (biolumine scent signal) at day 21. (B) Serum IL-6 levels of treated mice, n.s not significant; *P is <0.05; ***P<0.001

[0246] Figure 7 is a graphical representation showing long-term stability of BC-hCD19 (BC-001) transduced PBMCs from healthy donors as assessed by flow cytometry.

[0247] Figure 8 is a series of graphical representations showing healthy donor PBMCs transduced with BC-001 (hCD19-CAR) or murine FMC63 or humanized C2146, or PBMCs alone, co-cultured with (A) Raji-Luc Cells or (B) NALM-6-Luc cells expressing luciferase, at various effector to target ratios (E: T of 20: 1, 10: 1 and 5: 1) to evaluate cytotoxic potential. *P is <0.05; **P<0.01

[0248] Figure 9 is a series of graphical representations showing Diffuse Large B-cell Lymphoma (DLBCL) patient derived PBMCs transduced with BC-001 (hCD19-CAR) or murine FMC63 or humanized C2146, or PBMCs alone, co-cultured with (A, C) NALM-6-Luc cells or (B,D) Raji-Luc cells expressing luciferase, at (A, B) MOI 5 or (C, D) MOI 2.5 and at various effector to target ratios (E: T of 20: 1, 10: 1 and 5: 1) to evaluate cytotoxic potential. *P is <0.05; **P<0.01; ***P<0.001

[0249] Figure 10 is a graphical representation showing CD 19 CAR expression in Diffuse Large B-cell Lymphoma (DLBCL) patient derived PBMCs transduced with BC-001 (hCD19-CAR) or murine FMC63 or humanized C2146 at MOI 2.5 or 5.

[0250] Figure 11 is a series of graphical representations showing (A) co-culture of SLE patient derived CD19+B cells with autologous SLE patient derived CD3+CD19‘ T cells transduced with BC-001 (hCD19-CAR) or murine FMC63 or humanized C2146 at MOI 5 at various effector to target ratios (E: T of 20: 1, 10: 1 and 5: 1) to evaluate cytotoxic potential; and cytokine levels in the supernatant of co-cultured cells including (B) TNF- a levels, (C) IFN-y levels, (D) IL-6 levels, (E) IL- 10 levels, (F) IL- 13 levels, (G) GM- CSF levels and (H) IL-2 levels. *P is <0.05; **P<0.01; ***P<0.001.

[0251] KEY TO SEQUENCE LISTING

[0252] SEQ ID NO: 1 CD 19 VL nucleotide sequence

[0253] SEQ ID NO: 2 CD 19 VH nucleotide sequence

[0254] SEQ ID NO: 3 CD 19 VL CDR1 nucleotide sequence

[0255] SEQ ID NO: 4 CD 19 VL CDR2 nucleotide sequence SEQ ID NO: 5 CD 19 VL CDR3 nucleotide sequence SEQ ID NO: 6 CD 19 VH CDR1 nucleotide sequence SEQ ID NO: 7 CD 19 VH CDR2 nucleotide sequence SEQ ID NO: 8 CD 19 VH CDR3 nucleotide sequence SEQ ID NO: 9 CD 19 VL amino acid sequence SEQ ID NO: 10 CD 19 VH amino acid sequence SEQ ID NO: 11 CD 19 VL CDR1 amino acid sequence SEQ ID NO: 12 CD 19 VL CDR2 amino acid sequence SEQ ID NO: 13 CD 19 VL CDR3 amino acid sequence SEQ ID NO: 14 CD 19 VH CDR1 amino acid sequence SEQ ID NO: 15 CD 19 VH CDR2 amino acid sequence SEQ ID NO: 16 CD 19 VH CDR3 amino acid sequence SEQ ID NO: 17 GS linker nucleotide sequence SEQ ID NO: 18 GS linker amino acid sequence SEQ ID NO: 19 scFv nucleotide sequence SEQ ID NO: 20 scFv amino acid sequence SEQ ID NO: 21 CD8 alpha hinge region nucleotide sequence SEQ ID NO: 22 CD8 alpha hinge region amino acid sequence SEQ ID NO: 23 CD8 alpha transmembrane domain nucleotide sequence SEQ ID NO: 24 CD8 alpha transmembrane domain amino acid sequence SEQ ID NO: 25 4- IBB signalling domain nucleotide sequence SEQ ID NO: 26 4- IBB signalling domain amino acid sequence SEQ ID NO: 27 CD3 zeta (CD3 signalling domain nucleotide sequence SEQ ID NO: 28 CD3 zeta (CD3 signalling domain amino acid sequence SEQ ID NO: 29 CD 19-scFv-CAR nucleotide sequence SEQ ID NO: 30 CD19-scFV-CAR amino acid sequence SEQ ID NO: 31 N-terminal CD8a signal peptide nucleotide sequence SEQ ID NO: 32 N-terminal CD8a signal peptide amino acid sequence SEQ ID NO: 33 scFv amino acid sequence SEQ ID NO: 34 scFv amino acid sequence SEQ ID NO: 35 CD 19 VL CDR1 amino acid consensus sequence SEQ ID NO: 36 CD 19 VL CDR1 amino acid sequence SEQ ID NO: 37 CD 19 VL CDR2 amino acid sequence SEQ ID NO: 38 CD 19 VL CDR3 amino acid consensus sequence SEQ ID NO: 39 CD 19 VL CDR3 amino acid sequence SEQ ID NO: 40 CD 19 VL CDR3 amino acid sequence SEQ ID NO: 41 CD 19 VH CDR1 amino acid consensus sequence

[0256] SEQ ID NO: 42 CD 19 VH CDR1 amino acid sequence

[0257] SEQ ID NO: 43 CD 19 VH CDR1 amino acid sequence

[0258] SEQ ID NO: 44 CD 19 VH CDR2 amino acid consensus sequence

[0259] SEQ ID NO: 45 CD 19 VH CDR2 amino acid sequence

[0260] SEQ ID NO: 46 CD 19 VH CDR2 amino acid sequence

[0261] SEQ ID NO: 47 CD 19 VH CDR3amino acid consensus sequence

[0262] SEQ ID NO: 48 CD 19 VH CDR3amino acid sequence

[0263] SEQ ID NO: 49 CD 19 VL amino acid sequence

[0264] SEQ ID NO: 50 CD 19 VL amino acid sequence

[0265] SEQ ID NO: 51 CD 19 VH amino acid sequence

[0266] SEQ ID NO: 52 CD 19 VH amino acid sequence

[0267] SEQ ID NO: 53 CD 19 scFv amino acid sequence

[0268] SEQ ID NO: 54 CD 19 scFv amino acid sequence

[0269] SEQ ID NO: 55 CD19-scFV-CAR amino acid sequence

[0270] SEQ ID NO: 56 CD19-scFV-CAR amino acid sequence

[0271] DESCRIPTION

[0272] General

[0273] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter.

[0274] Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0275] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0276] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0277] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0278] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0279] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Uaboratory Manual, Cold Spring Harbour Uaboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRE Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0280] The description and definitions of variable regions and parts thereof, antibodies and fragments thereof herein may be further clarified by the discussion in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991.

[0281] The term “EU numbering system of Kabat” will be understood to mean the numbering of an antibody heavy chain is according to the EU index as taught in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda. The EU index is based on the residue numbering of the human IgGl EU antibody.

[0282] The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0283] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0284] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0285] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0286] Selected Definitions

[0287] As used herein, a “Chimeric Antigen Receptor” or “CAR” refers to a protein or polypeptide comprising an extracellular domain capable of binding an antigen (i.e., an antigen binding domain linked to immune cell signalling domains. CARs are able to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, thus giving T cells expressing CARs the ability to recognize antigens independent of antigen processing. CARs do not dimerize with endogenous T cell receptor (TCR) a- and -chains. T cells expressing a CAR are referred to herein as CAR T cells, CAR-T cells or CAR modified T cells, and these terms are used interchangeably herein. A “CD19-CAR” refers to a CAR having an extracellular binding domain specific for CD 19.

[0288] Ther term “co-stimulatory domain” refers to any oligopeptide or polypeptide known to act as a domain that transmits signals to activate or inhibit biological processes within a cell.

[0289] As used herein, the term “transmembrane domain” refers to any three- dimensional structure formed by a sequence of amino acids which is thermodynamically stable in a biological membrane, e.g. a cell membrane.

[0290] The term “hinge region” refers to an amino acid sequence which provides for flexible linkage of the antigen-binding and transmembrane domains of the CAR.

[0291] As used herein, the terms “treating”, “treat” or “treatment” include any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition described herein, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0292] An “effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be a therapeutic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect treatment of a disease as herein described. In some examples of the present disclosure, the term “effective amount” is meant an amount necessary to effect a change associated with a disease as herein described. The effective amount may vary according to the disease to be treated or factor to be altered and also according to the weight, age, racial background, sex, health and / or physical condition and other factors relevant to the subject being treated. Typically, the effective amount will fall within a relatively broad range (e.g. a “dosage” range) that can be determined through routine trial and experimentation by a medical practitioner. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity, e.g., weight or number cells. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

[0293] A “therapeutically effective amount” is at least the minimum concentration required to effect a measurable improvement of a particular disease. A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the cells of the present disclosure to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.

[0294] As used herein, “adoptive cell therapy” (ACT) or “CAR-T cell therapy” refers to a treatment that involves the collection of immune cells from a subject, modifying the cells to express a CAR, growing or expanding these cells in vitro and reinfusing the modified cells back into the subject.

[0295] As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited to humans and non-human primates. For example, the subject is a human.

[0296] The term “protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex). For example, the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

[0297] The term “polypeptide” or “polypeptide chain” will be understood from the foregoing paragraph to mean a series of contiguous amino acids linked by peptide bonds.

[0298] As used herein, the term “nucleotide sequence” or “nucleic acid sequence” will be understood to mean a series of contiguous nucleotides (or bases) covalently linked to a phosphodiester backbone. By convention, sequences are presented from the 5' end to the 3' end, unless otherwise specified.

[0299] The term “recombinant” shall be understood to mean the product of artificial genetic recombination. A recombinant protein also encompasses a protein expressed by artificial recombinant means when it is within a cell, tissue or subject, e.g., in which it is expressed.

[0300] As used herein, the term “antigen binding domain” shall be taken to mean a structure formed by a protein that is capable of binding or specifically binding to an antigen. The antigen binding domain need not be a series of contiguous amino acids. For example, in a scFv the antigen binding domain is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always in the one or more of the CDRs in each variable region. In some examples, an antigen binding domain is a VH or a VL or a scFv.

[0301] As used herein, the term “Fv” shall be taken to mean any protein, whether comprised of multiple polypeptides or a single polypeptide, in which a VL and a VH associate and form a complex having an antigen binding site, i.e., capable of specifically binding to an antigen. A “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.

[0302] As used herein, “variable region" refers to the portions of the light and / or heavy chains of an antibody as defined herein that is capable of specifically binding to an antigen and includes amino acid sequences of complementarity determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). Exemplary variable regions comprise three or four FRs (e.g., FR1, FR2, FR3 and optionally FR4) together with three CDRs. VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. As used herein, the term "complementarity determining regions” (syn. CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of a variable region the presence of which are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3. The amino acid positions assigned to CDRs and FRs can be defined according to Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 or other numbering systems in the performance of this disclosure, e.g., the canonical numbering system of Chothia and Lesk J. Mol Biol. 196: 901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J Mol Biol 273: 927-948, 1997; the IMGT numbering system of Lefranc et al., Devel. And Compar. Immunol., 27: 55- 77, 2003; or the AHO numbering system of Honnegher and Pltikthun J. Mol. Biol., 309: 657-670, 2001. For example, according to the numbering system of Kabat, VH framework regions (FRs) and CDRs are positioned as follows: residues 1-30 (FR1 ), 31- 35 (CDR1), 36-49 (FR2), 50-65 (CDR2), 66-94 (FR3), 95-102 (CDR3) and 103- 113 (FR4). According to the numbering system of Kabat, VL FRS and CDRs are positioned as follows: residues 1-23 (FR1), 24-34 (CDR1), 35-49 (FR2), 50-56 (CDR2), 57-88 (FR3), 89-97 (CDR3) and 98-107 (FR4). The present disclosure is not limited to FRs and CDRs as defined by the Kabat numbering system, but includes all numbering systems, including those discussed above. In one example, reference herein to a CDR (or a FR) is in respect of those regions according to the Kabat numbering system.

[0303] "Framework regions" (FRs) are those variable region residues other than the CDR residues.

[0304] As used herein, the term “specifically binds” or “binds specifically” shall be taken to mean that the interaction between an antigen binding domain of the disclosure with an antigen is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen. In addition, an antigen binding domain of the disclosure reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular antigen or cell expressing same than it does with alternative antigens or cells. For example, a CAR comprising an antigen binding domain of the disclosure binds to CD 19 with materially greater affinity (e.g., 20 fold or 40 fold or 60 fold or 80 fold to 100 fold or 150 fold or 200 fold) than it does to other antigens. Reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term.

[0305] For the purposes of clarification and as will be apparent to the skilled artisan based on the exemplified subject matter herein, reference to “affinity” in this specification is a reference to KD of a protein or scFv. As used herein, the term “epitope” (syn. “antigenic determinant”) shall be understood to mean a region of CD 19 to which a protein comprising an antigen binding site of an antibody binds. This term is not necessarily limited to the specific residues or structure to which the protein makes contact. For example, this term includes the region spanning amino acids contacted by the protein and / or 5-10 or 2-5 or 1-3 amino acids outside of this region. The skilled artisan will also be aware that the term "epitope" is not limited to peptides or polypeptides. For example, the term “epitope” includes chemically active surface groupings of molecules such as sugar side chains, phosphoryl side chains, or sulfonyl side chains, and, in certain examples, may have specific three dimensional structural characteristics, and / or specific charge characteristics.

[0306] Chimeric Antigen Receptor (CAR)

[0307] The CAR of the present disclosure, or the polynucleotide encoding the CAR, comprises an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, a transmembrane domain and at least one co-stimulatory domain.

[0308] In one example, the CAR of the present disclosure comprises from N-terminus to C -terminus: a signal peptide, an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, a transmembrane domain and at last one co-stimulatory domain.

[0309] In one example, the CAR of the present disclosure comprises from N-terminus to C -terminus: a CD8a signal peptide, an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19 antigen, a CD8a hinge region, a CD8a transmembrane domain, a 4- IBB signalling domain and a CD3^ signalling domain.

[0310] In one example, the CAR of the present disclosure comprises a sequence set forth in any one of SEQ ID NOs: 30, 55 or 56.

[0311] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 30. In one example, the CAR of the present disclosure comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 29.

[0312] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 55.

[0313] In one example, the CAR of the present disclosure comprises a sequence set forth in SEQ ID NO: 56.

[0314] Polynucleotide sequences encoding the CARs described herein can be obtained using standard recombinant techniques and will be apparent to the skilled person. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizers or PCR techniques.

[0315] Extracellular antigen binding domain

[0316] The present disclosure provides a CAR, or polynucleotide encoding the CAR, comprising an extracellular antigen binding domain comprising a modified scFv that specifically binds human CD 19.

[0317] CD19 is a 556 amino acid Type I transmembrane glycoprotein that is encoded by the CD19 gene located on the short arm of chromosome 16. It contains at least fifteen exons, four that encode extracellular domain and nine that encode cytoplasmic domains.

[0318] The skilled person will be aware that scFvs comprise VH and VL regions in a single polypeptide chain and a polypeptide linker between the VH and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv).

[0319] In one example, the linker is a peptide linker. For example, a peptide linker comprising at least 2 amino acids in length. In one example, the peptide linker is between 2 and 35 amino acids in length. For example, the linker is about 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in length. In another example, the linker is between about 10 and 20 amino acids in length. For example, the peptide linker is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In one example, the peptide linker is 15 amino acids in length. In another example, the peptide linker is between about 20 and 35 amino acids in length. For example, the peptide linker is about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 amino acids in length.

[0320] Suitable linkers for use in the present disclosure for linking the VH and VL will be apparent to the skilled person and / or disclosed herein. Exemplary linkers include GS linkers. For example, the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3linker, a (Gly)s linker, a (Gly)r, linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4. In one example, the linker is a (Gl4Scr)3 linker and comprises a sequence set forth in SEQ ID NO: 18. In one example, the linker is a (Gly4Ser)3 linker and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 17.

[0321] It will be apparent from the disclosure herein that the scFv is a modified scFv.

[0322] In one example, the modified scFv is a humanised scFv. The term "humanized scFv” shall be understood to refer to a scFv having an antigen binding domain or variable region derived from an antibody from a non-human species (e.g., murine) and the remaining antibody structure based upon the structure and / or sequence of a human antibody. In a humanized scFv, the antigen-binding domain generally comprises the CDRs from the non-human antibody grafted onto appropriate FRs in the human variable regions and the antigen binding domains are modified by one or more amino acid substitutions. In some instances, FR residues of the human antibody are replaced by corresponding non-human residues.

[0323] Methods for modifying non-human antibodies or parts thereof (e.g., variable regions) are known in the art. Modification can be performed following the method of US5225539, or US5585089. Other methods for modifying an antibody are not excluded.

[0324] The present disclosure provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises:

[0325] (i) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or

[0326] (ii) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or

[0327] (iii)a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

[0328] The present disclosure provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 14; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 16.

[0329] The present disclosure provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 11; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 42; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 16.

[0330] The present disclosure provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 36; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 40; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in SEQ ID NO: 43; ii. a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and iii. a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

[0331] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in amino acid residues 24 to

[0332] 34 of SEQ ID NO: 9; ii. a CDR2 comprising a sequence set forth in amino acid residues 50 to 56 of SEQ ID NO: 9; and iii. a CDR3 comprising a sequence set forth in amino acid residues 89 to 97 of SEQ ID NO: 9; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in amino acid residues 26 to

[0333] 35 of SEQ ID NO: 10; ii. a CDR2 comprising a sequence set forth in amino acid residues 50 to 64 of SEQ ID NO: 10; and iii. a CDR3 comprising a sequence set forth in amino acid residues 98 to 109 of SEQ ID NO: 10.

[0334] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising: i. a CDR1 comprising a sequence set forth in amino acid residues 24 to 34 of SEQ ID NO: 20; ii. a CDR2 comprising a sequence set forth in amino acid residues 50 to 56 of SEQ ID NO: 20; and iii. a CDR3 comprising a sequence set forth in amino acid residues 89 to 97 of SEQ ID NO: 20; and b) a VH comprising: i. a CDR1 comprising a sequence set forth in amino acid residues 148 to 157 of SEQ ID NO: 20; ii. a CDR2 comprising a sequence set forth in amino acid residues 172 to 186 of SEQ ID NO: 20; and iii. a CDR3 comprising a sequence set forth in amino acid residues 220 to 231 of SEQ ID NO: 20.

[0335] The present disclosure further provides a polynucleotide encoding a CAR, wherein the polynucleotide comprises an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a light chain variable region (VL) comprising: i. a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 3; ii. a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 4; and iii. a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 5; and b) a heavy chain variable region (VH) comprising: i. a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 6; ii. a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 7; and iii. a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 8. In one example, the CAR further comprises a signal peptide located at the N- terminus of the extracellular antigen binding domain. For example, the signal peptide is a CD8a signal peptide. In one example, the signal peptide is a CD8a signal peptide and comprises a sequence set forth in SEQ ID NO: 32. In one example, the signal peptide is a CD8a signal peptide and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 31.

[0336] Transmembrane Domain

[0337] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen and a transmembrane domain.

[0338] It will be apparent to the skilled person that the transmembrane domain provides for anchoring the CAR to the cell membrane of a cell expressing a CAR, with the antigenbinding domain in the extracellular space and signalling domain (or co-stimulatory domain) inside the cell.

[0339] In one example, the transmembrane domain comprises or consist of a sequence of amino acids which forms a hydrophobic alpha helix or beta-barrel. The amino acid sequence of the transmembrane domain of the CAR of the present disclosure may be, or may be derived from, the amino acid sequence of a transmembrane domain of a protein comprising a transmembrane domain. Transmembrane domains are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as TMHMM (Krogh et al., J Mol Biol (2001) 305:567-580).

[0340] In one example, the transmembrane domain is from a CD8 molecule or a CD28 molecule. In one example, the transmembrane domain is from a CD8 molecule. For example, the transmembrane domain is a CD 8a. In another example, the transmembrane domain is a CD28 molecule.

[0341] In one example, the transmembrane domain is human. For example, the transmembrane domain is a human CD8a. In another example, the transmembrane domain is a human CD28.

[0342] An exemplary transmembrane domain of the present disclosure comprises a sequence set forth in SEQ ID NO: 24. In one example, the transmembrane domain comprises a polypeptide encoded by the nucleotide sequence of SEQ ID NO: 23.

[0343] In one example, the transmembrane domain that can be directly or indirectly connected to the extracellular antigen binding domain.

[0344] In one example, the transmembrane domain is directly connected to the extracellular antigen binding domain.

[0345] In one example, the transmembrane domain is indirectly connected to the extracellular antigen binding domain. For example, the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region. In one example, the CAR further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

[0346] It will be apparent to the skilled person that the hinge region not only provides separation between the antigen binding domain and the transmembrane domain but also allows the binding moiety to orient in different directions.

[0347] In one example, the hinge region is a CD8a hinge region. In one example, the hinge region is a CD8a hinge region and comprises a sequence set forth in SEQ ID NO: 22. In one example, the hinge region is a CD8a hinge region and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 21.

[0348] Co-stimulatory Domains

[0349] The present disclosure also provides a CAR comprising an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen and at least one costimulatory domain. It will be apparent to the skilled person that the co-stimulatory domain provides the co-stimulation signal necessary for enhancing immune cell activation and effector function.

[0350] In one example, the at least one co-stimulatory domain is at least one intracellular signalling domain. It will be apparent to the skilled person that the signalling domain comprises amino acid sequences required activation of immune cell function.

[0351] In one example, the signalling domain comprises ITAM-containing sequence. An ITAM-containing sequence comprises one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs comprise the amino acid sequence YXXL / I, wherein “X” denotes any amino acid. In ITAM-containing proteins, ITAM sequences are often separated by 6 to 8 amino acids. When phosphate groups are added to the tyrosine residue of an ITAM by tyrosine kinases, a signalling cascade is initiated within the cell.

[0352] Suitable co-stimulatory domains suitable for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, the at least one co- stimulatory domain is obtained or derived from a CD28 molecule, a CD3 zeta (Q molecule or modified versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27 molecule, an OX-40 molecule, a 4-1BB molecule, a glucocorticoid-induced TNFR-related (GITR) molecule and / or a herpesvirus entry mediator (HVEM) molecule.

[0353] In one example, the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a GITR signalling domain, a HVEM signalling domain and a CD3^ signalling domain. For example, the co-stimulatory domain comprises the 4- IBB signalling domain and the CD3^ signalling domain.

[0354] In one example, the at least one co-stimulatory domain is human. For example, the co-stimulatory domain comprises a human 4- IBB signalling domain and a human CD3^ signalling domain.

[0355] In one example, the co-stimulatory domain comprises a human 4- IBB signalling domain and comprises a sequence set forth in SEQ ID NO: 26. In one example, the co- stimulatory domain comprises a human 4-1BB signalling domain and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 25.

[0356] In one example, the co-stimulatory domain comprises a human CD3^ signalling domain and comprises a sequence set forth in SEQ ID NO: 28. In one example, the co- stimulatory domain comprises a human CD3^ signalling domain and comprises a polypeptide encoded by a nucleotide sequence of SEQ ID NO: 27.

[0357] Vectors The present disclosure also provides a vector comprising the polynucleotide sequences encoding the CAR described herein.

[0358] Suitable vectors and methods of preparing a vector will be apparent to the skilled person and / or described herein. For example, the vector is a plasmid, a cosmid, a phage or a viral vector. In one example, the viral vector is a lentiviral vector, an adeno- associated viral vector (AAV), an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector.

[0359] In one example, the viral vector is derived from the genome of an adenovirus (e.g. Ad5), an adeno-associated virus (e.g. AAV2, 3, 5, 6, 8 or 9), a herpes simplex virus (e.g. HSV, HSV1), a retrovirus (e.g. avian leukosis virus (ALV), mouse mammary tumour virus (MMTV), murine leukemia virus (MLV), human T-lymphotropic virus (HTLV), Walleye dermal sarcoma virus (WDSV) or murine stem cell virus (MSCV)), a spumavirus (e.g., a human foamy virus (HFV) or simian foamy virus (SFV)), a lentivirus (e.g. simian immunodeficiency viruses (SIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritis-encephalitis virus (CAEV), or the ovine visna-maedi virus (VMV)), an alphavirus (e.g. Semliki forest virus (SFV), Sindbis virus (SIN), Venezuelan equine encephalitis (VEE)), a flavivirus (e.g. Kunjin, West Nile, Dengue), a rhabdovirus (e.g. rabies), a measles virus, (e.g. MV-Edm), Newcastle disease virus, a poxvirus (e.g. Vaccinia virus) or a picomavirus (e.g. Coxsackievirus).

[0360] In one example, the viral vector is derived from the genome of a retrovirus. For example, the viral vector is a retroviral vector.

[0361] In one example, the viral vector is derived from the genome of a lentivirus. For example, the viral vector is a lentiviral vector. In one example, the lentiviral vector is a pseudotyped lentiviral vector. In another example, the lentiviral vector is a selfinactivating lentiviral vector.

[0362] In one example, the viral vector is derived from the genome of human immunodeficiency virus (HIV). For example, the viral vector is derived from the genome of HIV-1. It will be apparent to the skilled person that to increase safety, the viral vector only contains HIV genes which are necessary for infection and gene delivery, whilst the genes necessary for replication and virulence factors have been removed. For example, the envelope protein of HIV-1 is exchanged with that of another virus (e.g., VSV-G protein from Vesicular stomatitis Indiana virus (V SV)) to allow infection of a wide range of target cells.

[0363] Methods for the production of viruses, viral vectors and viral particles will be apparent to the skilled person and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48: 362-377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474- 486; and Rodrigues etal., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40. For example, the methods generally involve introducing plasmids encoding the polynucleotide of the disclosure together with packaging components encoding the structural proteins required for virion assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of the plasmids with inducible promoters, in which lentivirus production can be induced. Once virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells. The lentivirus (comprising the polynucleotide encoding the CAR of the disclosure introduced into its genome) is harvested from these producer cells and subsequently purified and concentrated for use in the downstream process, e.g., in the transduction of immune cells.

[0364] The skilled person will be aware of the cell types suitable for transfection with viral plasmids (e.g. producer cells, or ‘host’ cell). For example, mammalian cells are well known hosts for the production of viral vectors and are suitable for transfection with viral plasmids.

[0365] As used herein, “transfection”, “transformation”, or transduction” refer to the introduction of one or more exogenous polynucleotides into a cell by using physical or chemical methods.

[0366] The host or producer cell may be selected from any cell allowing production of e.g., the lentivirus. According to one example, the cell is selected from a human cell (HEK293T, HEK293FT, HEK293S, HEK29FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEKEBNA1-6E, HEK293MSR and HEK293A cells), a musteli cell (NIH-3T3), a mustelidae cell (Mpf), a canid cell (DI 7), and derivatives thereof. According to one example, the cell is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells, and derivatives thereof.

[0367] The skilled person will be aware that in addition to the polynucleotide sequences encoding the CAR, the vector additionally comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators and internal ribosome entry sites (IRES), that provide for the expression of the polynucleotide sequence in the host cell. Exemplary expression control sequences are known in the art and described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). Genetically Modified Cells

[0368] The present disclosure also provides a genetically modified cell comprising the vector of the disclosure.

[0369] As used herein, the term “cell” refers to any type of cell that can contain the expression vector. For example, the cell is modified to express an expression vector (e.g., a retroviral, lentiviral, adenoviral, adeno-associated viral vector). In one example, the cell is modified to express a CAR of the present disclosure.

[0370] Methods of genetically modifying cells with viral vectors will be apparent to the skilled person and / or described herein. For example, the lentivirus (comprising the polynucleotide encoding the CAR of the disclosure introduced into its genome) is harvested from the producer cells and applied to the cells to be modified, resulting in expression of the CAR of the disclosure by the modified cell.

[0371] In one example, the cell is a eukaryotic cell, for example, a plant, animal, fungi, or algae. In another example, the cell is a prokaryotic cell, for example bacteria or protozoa.

[0372] In one example, the cell is a cultured cell or a primary cell, i.e., isolated directly from an organism (e.g., a human).

[0373] In one example, the cell is an adherent cell or a suspended cell, i.e., a cell that grows in suspension.

[0374] Suitable cells for use in the present disclosure will be apparent to the skilled person and / or are disclosed herein.

[0375] In one example, the cell is an immune cell. For example, the cell is an immune effector cell. In one example, the immune effector cell expresses FcyRIII and performs antibody-dependent cellular cytotoxicity (ADCC) effector function.

[0376] In one example, the immune effector cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

[0377] In one example, the cell is of hematopoietic origin. For example, the cell is a neutrophil, an eosinophil, a basophil, a lymphocyte, or a monocyte. In one example, the lymphocyte is a T cell, a B cell, a NK cell or precursor thereof.

[0378] In one example, the cell expresses CD3 polypeptides. For example, the cell expresses a CD3y, a CD3a, a CD3^ or CD35 polypeptide.

[0379] In one example, the cell expresses a T cell receptor (TCR) polypeptide. For example, a TCRa or TCRp polypeptide.

[0380] In one example, the cell expresses CD27, CD28, CD4 and / or CD8. For example, the cell expresses CD4 and / or CD8. In one example, the cell is a T-cell. For example, the T-cell is a human T-cell. For example, isolated from a human. In one example, the T cells are autologous T cells. In one example, the T cells are allogeneic T cells.

[0381] In one example, the T-cell is of any developmental stage. Stages of T cell differentiation include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and terminal effector T cells, from least to most differentiated. Methods of identifying T cells at different stages of differentiation will be apparent to the skilled person and / or described herein. For example, naive T cells express the cell surface markers CCR7+, CD62L+, CD45RO-, CD95-, stem central memory T cells (Tscm) express CCR7+, CD62L+, CD45RO-, CD95+, central memory T cells (Tcm) express CCR7+, CD62L+, CD45RO+, CD95+, effector memory T cells (Tem) express CCR7-, CD62L-, CD45RO+, CD95+, and terminal effector T cells (Teff) express CCR7-, CD62L-, CD45RO-, CD95+.

[0382] In one example, the T cell is a gamma delta (y5) T cell, a cytotoxic T cell, or a helper T cell. In another example, the T cell is a CD3+ / CD8+ T cell. In another example, the T cell is a CD4+ / CD8+ double positive T-cell, a CD4+ helper T-cell, (e.g., Th and Th2 cells), a CD8+ T- cell (e.g., a cytotoxic T-cell), a tumour infiltrating cell, a memory T-cell, or a naive T-cell. In one embodiment, the T-cell is a CD8+ T-cell or a CD4+ T- cell. Suitable T-cell lines for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, suitable T cell lines are available from, e.g., the American Type Culture Collection (ATCC), and the German Collection of Microorganisms and Cell Cultures (DSMZ) and include, for example, Jurkat cells (ATCC TIB- 152), Sup-Tl cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC- 290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

[0383] In one example, the cell is a natural killer (NK) cell. In one example, the NK cell is a human NK cell. For example, the NK cell is isolated from a human. In one example, the NK cells are autologous NK cells. In one example, the NK cells are allogeneic NK cells. Exemplary NK cell lines suitable for use in the present disclosure are available from, e.g., the American Type Culture Collection (ATCC) and include, for example, NK- 92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), and derivatives thereof.

[0384] Methods of obtaining cells (e.g., T cells or NK cells) suitable for use in the present disclosure will be apparent to the skilled person and / or described herein.

[0385] In one example, the cell is a cultured cell, a primary cell, or a cell from a cultured cell line, or a cell obtained from a mammal. In one example, the cell is a cultured cell. In another example, the cell is a primary cell. In a further example, the cell is a cultured cell line. In one example, the cell is a cell obtained from a mammal (e.g., a human subject). In one example, the cell has been previously obtained from the subject (i.e., the human subject). In one example, the cells have been obtained from a tissue sample or biopsy.

[0386] It will be apparent to the skilled person that if obtained from a mammal, the cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. Cells for use in the present disclosure can also be enriched for or purified. Such methods may involve removing or substantially reducing the amount of, erythrocytes, platelets, serum and / or plasma in a sample. Methods disclosed herein may be performed on isolated immune cells, or a sample containing immune cells in addition to other cells.

[0387] In one example, the genetically modified cell of the disclosure is a CAR- expressing immune cell. In another example, the genetically modified cell of the disclosure is a CAR-T cell. In a further example, the genetically modified cell of the disclosure is a CAR-NK cell.

[0388] In one example, methods of producing a genetically modified cell of the disclosure comprises the stimulation and / or expansion of the immune cell. For example, stimulation and / or expansion of PBMCs or an immune cell population (e.g., T cells) from within a population of immune cells (e.g., PBMCs). In one example, a population of T- cells is expanded from within a population of PBMCs by stimulation of the T-cells within the population of PBMCs. In one example, stimulation and / or expansion of T cells involves stimulation of a population of PBMCs. In one example, a population of T-cells are expanded from within a population of tumor-infiltrating lymphocytes, by stimulation of the T-cells within the population of tumor-infiltrating lymphocytes. For example, in one example, stimulation and / or expansion of T cells involves stimulation of a population of tumor-infiltrating lymphocytes. In one example, a population of T-cells are expanded from within a population of T-cells (e.g. a population of T cells of heterogeneous specificity) obtained from a blood sample, a population of PBMCs, or from a population of tumor-infiltrating lymphocytes. In one example, the method involves the stimulation and / or expansion of T cells.

[0389] In one example, the cell is contacted with a transduction enhancer during a method of producing a genetically modified cell. For example, methods of producing a genetically modified cell of the disclosure comprise expanding and / or culturing the cell in the presence of a transduction enhancer. Transduction enhancers suitable for use in the present disclosure will be apparent to the skilled person and / or described herein. For example, transduction enhancers include, but are not limited to, cationic polymers (e.g. polybrene), fibronectin or fibronectin fragments (e.g. RetroNectin® or vitronectin), rapamycin and prostaglandin E2 (PGE2). In one example, the transduction enhancer is a human fibronectin fragment.

[0390] In one example, the transduction enhancer is RetroNectin®. The skilled person will understand that RetroNectin® is a recombinant human fibronectin (CH-296) fragment which contains three functional domains: (1) an N-terminal cell-binding domain, which binds cells via VLA-5 integrin (this domain contains an RGDS motif and comprises 3 fibronectin type III domains 8, 9 and 10), (2) a heparin-binding domain, comprising 3 fibronectin type III domains 12, 13 and 14, and (3) a C-terminal CS-1 sequence, which binds cells via VLA-4 integrin.

[0391] In one example, the transduction enhancer is vitronectin.

[0392] Additional transduction enhancers will be apparent to the skilled person and are described, for example, in WO2024023245, hereby incorporated by reference in its entirety.

[0393] Pharmaceutical Compositions

[0394] The present disclosure provides a composition comprising a population of genetically modified cells of the disclosure for use as a medicament. In one example, the present disclosure provides a composition for use in adoptive cell therapy. For example, adoptive T cell therapy.

[0395] Accordingly, the present disclosure provides a composition comprising a population of genetically modified cells of the disclosure and a pharmaceutically acceptable carrier.

[0396] It will be apparent to the skilled person that the population of genetically modified cells of the disclosure, e.g., population of genetically modified T cells, comprises a population of cells at different stages of differentiation.

[0397] Methods for preparing a compound into a suitable form for administration (e.g. a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990).

[0398] An appropriate pharmaceutical composition comprising a population of genetically modified cells to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives, or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. Exemplary carriers include water, saline, Ringer's solution, dextrose solution and <5% human serum albumin. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0399] The optimum concentration of the ingredient(s) in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

[0400] In one example, the composition additionally comprises human serum albumin. In one example, the composition comprises <5% human serum albumin. For example, the composition comprises 5% human serum albumin. In another example, the composition comprises 2.5% human serum albumin. In a further example, the composition comprises 0.25% human serum albumin.

[0401] In one example, the composition additionally comprises dimethylsulfoxide (DMSO). For example, the composition comprises <7.5% DMSO. In one example, the composition comprises 7.5% DMSO. In another example, the composition comprises 5% DMSO.

[0402] In one example, the composition additionally comprises sodium chloride.

[0403] In one example, the composition additionally comprises dextrose. For example, the composition comprises 5% dextrose.

[0404] Upon formulation, compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The dosage ranges for the administration of the composition of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an effective amount of the population of genetically modified cells. In one example, the composition comprises a therapeutically effective amount of the genetically modified cells.

[0405] The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication. In one example, the composition is formulated for injection or infusion, e.g., via intravenous infusion.

[0406] In one example, the CAR-T cells are formulated at a dose of about 1.0 x 105to 6 x 108cells / kg. In one example, the CAR-T cells are formulated at a dose of about 0.2 to 5.0 x 106cells / kg. For example, the CAR-T cells are formulated at a dose of about 2 x 106cells / kg. In another example, the CAR-T cells are formulated at a dose of about 0. 1 to 2.5 x io8cells / kg. In one example, the CAR-T cells are formulated at a dose of less than 1.0 x 108cells per subject. For example, the CAR-T cells are formulated at a dose of between 0.5-1.0 x 108cells per subject. In one example, the CAR-T cells are formulated at a dose of 0.6-6.0 x 108cells per subject. For example, the CAR-T cells are formulated at a dose of 2 x 108cells per subject.

[0407] Methods of Treatment

[0408] The present disclosure provides methods of using the composition or a population of genetically modified cells of the disclosure as a medicament. For example, in a method of treating a CD19-expressing proliferative disease in a subject in need thereof. In one example, the proliferative disease is selected from the group consisting of a malignant proliferative disease or autoimmune disease. For example, the present disclosure provides a method of treating a CD19-expressing malignant proliferative disease in a subject in need thereof. The present disclosure also provides a method of treating a CD19-expressing autoimmune disease in a subject in need thereof.

[0409] The present disclosure also provides a method of treating a CD19-expressing proliferative disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0410] The present disclosure also provides a method of treating a CD19-expressing malignant proliferative disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0411] In one example, the CD19-expressing malignant proliferative disease is a hematologic cancer. In one example, the hematologic cancer is selected from the group consisting of leukemia, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL) (e.g., relapsing and refractory ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and "preleukemia."

[0412] In one example, the CD19-expressing malignant proliferative disease is leukemia. For example, the leukemia is selected from the group consisting of B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL) (e.g., relapsing and refractory ALL), chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).

[0413] In one example, the CD19-expressing malignant proliferative disease is a B-cell expressing malignancy. In one example, the B-cell expressing malignancy is selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma (also called chronic lymphocytic leukaemia), mantle cell lymphoma, marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinaemia), hairy cell leukaemia and primary central nervous system lymphoma.

[0414] As used herein the term “preleukemia” is used to refer to a diverse collection of haematological conditions that shown ineffective production (or dysplasia) of myeloid blood cells.

[0415] In one example, the subject has an early or late-stage disease. The skilled person will be aware that the methods of producing the cells may be tailored for such different stages of cancer, such as by utilizing peptides for the APCs that are from antigens associated with early vs. late-stage cancer. In one example, the cancer is a primary, a metastatic, a recurrent, a refractory or a non-responsive cancer.

[0416] The present disclosure also provides a method of treating a CD19-expressing autoimmune disease in a subject in need thereof, the method comprising administering a composition or a population of genetically modified cells of the disclosure.

[0417] In one example, the CD19-expressing autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgG4-related disease (IgG4-RD), neuromyelitis optica spectrum disorder (NMOSD), generalized myasthenia gravis (gMG), idiopathic inflammatory myositis (IIM), systemic sclerosis (SSc) and multiple sclerosis (MS). In one example, the CD19-expressing autoimmune disease is SLE. In another example, the CD19-expressing autoimmune disease is RA. In a further example, the CD19-expressing autoimmune disease is IgG4- RD. In one example, the CD19-expressing autoimmune disease is gMG. In another example, the CD19-expressing autoimmune disease is IIM. In a further example, the CD19-expressing autoimmune disease is SSc. In one example, the CD19-expressing autoimmune disease is MS.

[0418] The term “refractory,” as used in connection to treatment with a particular treatment agent or medicament herein, refers to diseases or disease subjects that fail to respond to said treatment agent or medicament.

[0419] As used herein, the phrase “non-responsive disease” refers to either failure to achieve minimal response or development of progressive disease while on therapy.

[0420] In one example of any method described herein, a composition or population of genetically modified cells of the disclosure are for use in adoptive cell therapy.

[0421] Methods of adoptive cell therapy will be apparent to the skilled person and / or are described herein. For example, cells (e.g., immune cells) are obtained from a subject, e.g., by drawing a blood sample from which the cells are isolated. The cells are then genetically modified (e.g., with a CAR) and / or expanded, and then administered either to the same subject (in the case of adoptive therapy with autologous / autogeneic cells) or to a different subject (in the case of adoptive therapy of allogeneic cells).

[0422] Additional methods of adoptive cell therapy are described in Kalos and June, Immunity (2013) 39(l):49-60, and Davis etal., Cancer J. (2015) 21 (6) :486— 491, both of which are hereby incorporated by reference in their entirety.

[0423] In one example, the method comprises isolating an immune cell, or generating / expanding a population of immune cells. In one example, the method comprises modifying an immune cell to comprise / express a CAR according to the present disclosure. In one example, the method comprises modifying an immune cell to comprise / express nucleic acid encoding a CAR according to the present disclosure.

[0424] In one example, the method comprises administering to a subject an immune cell modified to express / comprise a CAR according to the present disclosure (or modified to express / comprise a nucleic acid encoding the CAR).

[0425] In one example, the subject from which the immune cells (e.g., T cells) are isolated is the same subject to which the composition or population of genetically modified cells of the disclosure are administered. For example, the adoptive cell therapy is autologous / autogeneic cell therapy.

[0426] In one example, the subject from which the immune cells (e.g., T cells) are isolated is a different subject to the subject to which the composition or population of genetically modified cells of the disclosure are administered. For example, the adoptive cell therapy is allogeneic cell therapy. In one example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance expression of the CAR and / or to induce / enhance proliferation or survival of immune cells comprising / expressing the CAR. In one example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance expression of the CAR. In another example, the method additionally comprises treating the genetically modified cells or the subject to induce / enhance proliferation or survival of immune cells comprising / expressing the CAR.

[0427] In one example of the methods described herein, administration of the composition or population of genetically modified cells disclosed herein reduces the development / progression of a CD19-expressing proliferative disease, alleviates the symptoms of a CD19-expressing proliferative disease, or reduces the pathology of a CD19-expressing proliferative disease. In one example of the methods described herein, administration of the composition or population of genetically modified cells disclosed herein reduces the development / progression of a CD19-expressing malignant proliferative disease, alleviates the symptoms of a CD19-expressing malignant proliferative disease, or reduces the pathology of a CD19-expressing malignant proliferative disease. In one example of the methods described herein, administration of the composition or population of genetically modified cells disclosed herein reduces the development / progression of a CD19-expressing autoimmune disease, alleviates the symptoms of a CD19-expressing autoimmune disease, or reduces the pathology of a CD19-expressing autoimmune disease. In one example, the methods prevent progression of the CD19-expressing malignant proliferative disease. For example, to prevent worsening of, or to slow the rate of development of, the CD19-expressing malignant proliferative disease. In one example, the methods lead to an improvement in the CD 19- expressing malignant proliferative disease. For example, a reduction in the severity of symptoms of the CD19-expressing malignant proliferative disease, or a reduction in some other correlate of the severity / activity of the CD19-expressing malignant proliferative disease. In one example, the methods prevent development of the CD19- expressing malignant proliferative disease to a later stage. For example, a chronic stage or metastasis. In one example, the methods prevent progression of the CD19-expressing autoimmune disease. For example, to prevent worsening of, or to slow the rate of development of, the CD 19-expressing autoimmune disease. In one example, the methods lead to an improvement in the CD 19-expressing autoimmune disease. For example, a reduction in the severity of symptoms of the CD 19-expressing autoimmune disease, or a reduction in some other correlate of the severity / activity of the CD 19-expressing autoimmune disease. In one example, the methods prevent development of the CD 19- expressing autoimmune disease to a later stage. For example, a chronic stage.

[0428] In one example, the subject has, or suffers from, a CD19-expressing proliferative disease. In one example, the subject has, or suffers from, a CD19-expressing malignant proliferative disease. In one example, the subject has, or suffers from, a CD 19-expressing autoimmune disease. For example, the subject is in need of treatment (i.e., in need thereof).

[0429] In one example, the subject has been diagnosed with a CD 19-expressing proliferative disease. In one example, the subject has been diagnosed with a CD 19- expressing malignant proliferative disease. In one example, the subject has been diagnosed with a CD 19-expressing autoimmune disease. Methods of diagnosing a CD 19- expressing proliferative disease will be apparent to the skilled person and / or described herein. Methods of diagnosing a CD 19-expressing malignant proliferative disease will be apparent to the skilled person and / or described herein. Methods of diagnosing a CD 19- expressing autoimmune disease will be apparent to the skilled person and / or described herein.

[0430] In one example, the CAR-T cells are administered at a dose of about 1.0 x 105to 6 x 108positive viable T cells / kg. In one example, the CAR-T cells are administered at a dose of about 0.2 to 5.0 x 106positive viable T cells / kg. For example, the CAR-T cells are administered at a dose of about 2 x 106positive viable T cells / kg. In another example, the CAR-T cells are administered at a dose of about 0.1 to 2.5 x 108positive viable T cells / kg. In one example, the CAR-T cells are administered at a dose of less than 1.0 x 108positive viable T cells per subject. For example, the CAR-T cells are administered at a dose of between 0.5-1.0 x 108positive viable T cells per subject. In one example, the CAR-T cells are administered at a dose of 0.6-6.0 x 108positive viable T cells per subject. For example, the CAR-T cells are administered at a dose of 2 x 108positive viable T cells per subject.

[0431] In one example, one or more administrations of the genetically modified cells of the disclosure are provided to a subject in need thereof. The length of time between each administration may be of any suitable duration, including for example, 1-7 days, 1-4 weeks, 1-12 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years. Multiple infusions within about a year may be employed, in some cases. In cases wherein more than one administration of cells is provided to the subject, the antigen to which the cells are targeted may or may not be the same antigen that was targeted with the cells utilized in earlier administration(s). Additional administration(s) may be required in cancers that become refractory, for example. Additional stimulations may be employed in conjunction with one or more other types of cancer treatments.

[0432] In one example, a composition of the present disclosure is administered in combination with an additional therapy useful for treating the proliferative disease, e.g., cancer or autoimmune disease, described herein. In one example, a composition of the present disclosure is administered in combination with an additional therapy useful for treating the malignant proliferative disease, e.g., cancer, described herein. In one example, a composition of the present disclosure is administered in combination with an additional therapy useful for treating the autoimmune disease, e.g., SLE, described herein.

[0433] In one example, the additional therapy may be a standard of care therapy for treating the proliferative disease, or for treating a complication associated with the proliferative disease. In one example, the additional therapy may be a standard of care therapy for treating the malignant proliferative disease, or for treating a complication associated with the malignant proliferative disease. In one example, the additional therapy may be a standard of care therapy for treating the autoimmune disease, or for treating a complication associated with the autoimmune disease.

[0434] In one example, the standard of care therapy is administered prior to, concurrently or simultaneously, or after treatment with a composition of the present disclosure.

[0435] In one example, the composition of the disclosure is administered before the additional therapy.

[0436] In one example, the composition of the disclosure is administered after the additional therapy.

[0437] In one example, the composition of the disclosure is administered concurrently or simultaneously with the additional therapy.

[0438] Standard of care therapies for treatment of the disease will be apparent to the skilled person and / or are described herein.

[0439] Exemplary additional therapies include, but are not limited to, chemotherapy (e.g., cyclophosphamide alone or in combination with fludarabine, bendamustine alone or in combination with fludarabine), radiation, surgery, tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib or ponatinib), bruton tyrosine kinase (BTK) inhibitors (e.g., ibrutinib), monoclonal antibodies (e.g., rituximab, daratumumab), antibody-drug conjugates (e.g., gemtuzumab, bretuxumab, inotuxumab), proteasome inhibitors (e.g., bortezomib, carfilzomib), immunomodulatory drugs (e.g., thalidomide, lenalidomide), hypomethylating drugs (e.g., azacitidine), immune checkpoint inhibitors (e.g., nivolumab, pembrolizumab), targeting cytokines (e.g., interleukin (IL)-7, IL-15) and haematopoietic stem cell transplantation (HSCT). Additional exemplary therapies include, but are not limited to corticosteroids (e.g., cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone) and immunosuppressants (e.g., cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil.

[0440] In one example, the subject has previously received a standard of care therapy for the CD19-expressing proliferative disease. In one example, the subject has previously received a standard of care therapy for the CD19-expressing malignant proliferative disease. For example, the subject has received a pre-conditioning treatment regimen for the CD19-expressing malignant proliferative disease prior to receiving treatment with a composition or population of genetically modified cells described herein. In one example, the pre-conditioning treatment regimen is chemotherapy. For example, the chemotherapy is lymphodepletion chemotherapy. In one example, the lymphodepletion chemotherapy comprises cyclophosphamide, cyclophosphamide in combination with fludarabine, bendamustine or bendamustine in combination with fludarabine. In one example, the subject has previously received a standard of care therapy for the CD 19- expressing autoimmune disease. In one example, the subject has received a preconditioning treatment regimen for the CD19-expressing autoimmune disease prior to receiving treatment with a composition or population of genetically modified cells described herein. In one example, the pre-conditioning treatment regimen is chemotherapy. For example, the chemotherapy is lymphodepletion chemotherapy. In one example, the lymphodepletion chemotherapy comprises cyclophosphamide, cyclophosphamide in combination with fludarabine, bendamustine or bendamustine in combination with fludarabine.

[0441] In one example, the standard of care is haematopoietic stem cell transplantation (HSCT). For example, an allogeneic HSCT.

[0442] In one example, the subject receives a HSCT after treatment with a composition of the disclosure. In one example, the subject receives allogeneic HSCT after treatment with a composition of the disclosure.

[0443] In one example, the subject receives a HSCT before treatment with a composition of the disclosure. In one example, the subject receives allogeneic HSCT before treatment with a composition of the disclosure.

[0444] In one example, the subject receives a HSCT concurrently with a composition of the disclosure. In one example, the subject receives allogeneic HSCT concurrently with a composition of the disclosure. In one example, the additional therapy is for the treatment of cytokine release syndrome (CRS). Exemplary therapies for the treatment of CRS include, but are not limited to, dexamethasone, tocilizumab, siltuximab, anakinra, corticosteroids and antiepileptic drugs.

[0445] In one example, the additional therapy is targeting cytokines. For example, the targeting cytokines are administered following administration of the composition of the disclosure.

[0446] In one example, the targeting cytokines are selected from the group consisting of IL-7, IL- 15, IL-2, IL- 18, IL- 12, IL-21 and combinations thereof.

[0447] Kits

[0448] Another example of the disclosure provides kits containing compositions useful for treating a CD19-expressing proliferative disease described herein.

[0449] In one example, the kit comprises (a) a container comprising a population of genetically modified cells as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a CD19-expressing proliferative disease in a subject in need thereof. In one example, the kit comprises (a) a container comprising a population of genetically modified cells as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a CD19-expressing malignant proliferative disease in a subject in need thereof. In one example, the kit comprises (a) a container comprising a population of genetically modified cells as described herein, optionally in a pharmaceutically acceptable carrier or diluent; and (b) a package insert with instructions for treating a CD19-expressing autoimmune disease in a subject in need thereof.

[0450] In accordance with this example of the disclosure, the package insert is on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, bags etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds or contains a composition that is effective for treating the CD19-expressing proliferative disease (i.e., malignant proliferative disease or autoimmune disease) and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the population of genetically modified cells (e.g., CAR-T cells). The label or package insert indicates that the composition is used for treating of a CD19-expressing proliferative disease in a subject eligible for treatment, with specific guidance regarding dosing amounts and intervals of compound and any other medicament being provided. The kit may further comprise an additional container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, fdters, needles, and syringes.

[0451] SEQUENCES OF THE DISCLOSURE

[0452] The present disclosure provides the following non-limiting examples.

[0453] EXAMPLES

[0454] Example 1: Generation of a modified CD19-scFv-CAR

[0455] The modified single-chain variable fragment (scFv) targeting the anti-CD19 binding domain was generated from the CD 19 binding sequences from the murine FMC63 monoclonal antibody.

[0456] Specifically, framework residues within the framework regions were substituted with the corresponding residues from a donor antibody using Sapiens deep-learning antibody humanization method, as detailed in Prihoda et al. MABS 14 (1) e2020203. In addition, the top ranked structure (prediction confidence level of >90%) were subjected to ClusPro docking simulation against the CD 19 receptor to generate clusters of candidate docking conformations. Binding interactions and parameters were determined to pinpoint crucial residues involved in the CD 19 antigen binding, focusing on both CDR and framework residues. Selection criteria also included Observed Antibody Space identity search (OASis) Identity and OASis Percentile, which provided providing insights into the “humaneness” of the selected scFv sequences. The template germline heavy / light chain and percent content retained in the final sequence was also generated using Biophi and is shown in Table 1.

[0457] Table 1: Modified scFv candidates with OASis and germline percentiles of VH / VL

[0458] Considering that binding affinity alone may not establish the superiority of an scFv in CAR applications, six candidate scFv’s spanning various free energy zones derived from Cluspro docking apart from OASis identity and percentile were selected. These candidate scFvs were cloned into a 2nd generation lentiviral vector comprising of CD8a leader, CD8 transmembrane domain 4-1BB and CD3zeta and assessed for their effectiveness at eliminating leukemic cells in vitro. As shown in Table 2, scFv BC-0041 demonstrated the highest potency in killing tumor cells compared to control lines FMC63 and C2146. Table 2 also describes modifications of the scFv relative to the FMC63 backbone sequence.

[0459] Table 2: In vitro cell kill evaluation of modified scFv candidates

[0460] NB: BB - backbone; A indicates a variation, or Am indicates site directed mutagenesis in sequence compared to FCM63.

[0461] BC-0041 (also known as BC-001) was selected for further characterisation. In addition, the intracellular 4-1BB and the CD3^ signaling domains were removed to generate a control vector (denoted as BC-hCD19del). A schematic of BC-0041 and the control vector are shown in Figure 1.

[0462] To generate modified CD19-scFv-CAR-T cells, peripheral blood mononuclear cells (PBMC’s) from healthy donors were activated with CD3 / CD28 cocktail and were transduced with lentiviral vectors encoding modified CD19-CAR (BC-hCD19), or modified CD 19 with a deletion in intracytoplasmic endo-domain (BC-hCD19 del), or left untransduced (i.e., PBMC Ctrl) and were cultured.

[0463] Example 2: Anti-tumour efficacy of modified CD19-scFv-CAR-T cells

[0464] To investigate the tumour cell kill efficiency of BC-hCD 19 CAR lentiviral vector, BC-hCD 19 CAR transduced T cells were incubated with Nalm-6 or Raji-cells expressing a luciferase reporter gene. Nalm-6 cells were isolated from the peripheral blood of a patient suffering from acute lymphoblastic leukemia while Raji were derived from a patient with Burkitt lymphoma. CAR-T-lymphocytes were co-cultured with either NALM-6-Luc or Raji-Luc Cells expressing luciferase, at various effector to target ratios (E: T of 20: 1, 10: 1 and 5: 1) to evaluate cytotoxic potential. The extent of cytolytic activity of CAR T-cells was obtained by specific cell lysis (a ratio of total cell kills to spontaneous cell kill) using BrightGlo™ luciferase activity measurements (Promega, USA).

[0465] As shown in Figure 2, cells transduced with modified CD 19 CAR T-cells (BC- hCD19) exhibited significant cell kill compared to the PBMC control at the various effector to target ratios whereas the cell kill exhibited by control vector BC-hCD19 was comparable to PBMC non-transduced controls, demonstrating functionality of BC- hCD19 CAR transduced T cells.

[0466] The levels of cytokines secreted from the supernatant of CAR-T cells (either BC- hCD19 or control) co-cultured with tumour cells (Nalm-6 or Raji) was measured using an automated ELISA (ELLA, Biotechne).

[0467] As shown in Figure 3, increased levels of IFN-y and TNF-alpha in BC-hCD19 CAR expressing T-cells could be detected in both Nalm6-Luc and Raji-Luc cell models but not in negative controls where activated T-cells were transduced with BC-hCD19del CAR T cells or non-transduced cells alone, thus demonstrating the potencies of BC- hCD 19 CAR T cells

[0468] Example 3: Administration of modified CD19-scFv-CAR-T cells reduces tumour burden in a murine model of lymphoblastic leukemia

[0469] The therapeutic effect of the modified CD19-scFv-CAR-T cells was assessed in a murine pre-clinical model of lymphoblastic leukemia. Briefly, six-eight week old nonobese diabetic severe combined immunodeficient (NOD scid ) gamma (NSG) mice were injected with NALM-6 Luc cells (a human pre-B acute lymphoblastic leukemia cell line; 1 x 106cells) 24 hours after sub-lethal irradiation and subsequently infused intravenously with either (i) lx PBS; 5 x 106human T-cells; 5 x 106BC-hCD 19del CAR- T cells (negative control construct without the co-stimulatory domain); or 5 x 106BC- hCD19 CAR-T cells. Bioluminescent signal was tracked for a period of 35 days by bioluminescence imaging using IVIS™ system and tumor burden was quantified.

[0470] As shown in Figure 4A, tumour burden at day 21 was significantly reduced in BC-hCD19 CAR-T cell treated animals.

[0471] Average body weight of all the animals was monitored throughout the treatment period as shown in Figure 4B there was no significant difference in the treatment groups. Serum was collected at different time intervals and was processed for measurement of IFN-g levels using ELISA. As shown in Figure 4C, a significant increase in serum IFN-g levels was detected in BC-hCD19 CAR-T cell treated animals.

[0472] Example 4: modified CD19-CAR-T cells transduced in the presence of a transduction enhancer

[0473] To assess whether a soluble transduction enhancer could enhance CD 19 CAR expression without affecting the effectiveness of the CAR at killing tumour cells, human PBMCs were activated by CD3 / CD28 (24h) and transduced, with lentiviral vector constructs encoding humanized CD 19 at a multiplicity of infection (MOI) of 5 or left untransduced. CD 19 CAR-T cells prepared in the presence or absence of a transduction enhancer (as previously described in WO2024023245) and co-cultured with Raji-Luc cells and the supernatants evaluated for IL-6 cytokine secretion analysis by an automated ELISA (ELLA, Biotechne, USA). The extent of CAR expression was evaluated based on FACS analysis of CD 19 receptor staining.

[0474] No impact on the abilities of the transduced CAR T cells to proliferate and expand was observed (data not shown).

[0475] The transduction enhancer enhanced the transduction efficiency by >15%, compared to control without enhancer and as shown in Figure 5, promoted CAR expression, down-regulated IL-6 secretion but did not affect the effectiveness of the CAR at killing tumour cells in Raji-Luc cells. Nalm6-luc cells transduced in a similar manner demonstrated the same results (data not shown).

[0476] The safety and functional efficacies of BC-hCD19 CAR T cells prepared using the transduction enhancer were evaluated in the animal model as described in Example 3 above.

[0477] CAR-T cell efficacy was evaluated by biolumine scent signal tracked over various time points and as shown in Figure 6 luciferase was not detectable in all animals treated with the CD 19 CAR-T cells transduced in the presence or absence of the transduction enhancer and serum IL-6 levels were decreased in mice treated with the CD 19 CAR-T cells transduced in the presence of the transduction enhancer compared to without.

[0478] These results demonstrate the efficacy and safety of the hCD19-CAR-T cells transduced in the presence or absence of a transduction enhancer.

[0479] Example 5: Long term stability of modified CD19-scFv-CAR-T cells

[0480] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were activated using CD3 / CD28 stimulation for 24 hours and subsequently transduced at a multiplicity of infection (MOI) of 5 with lentiviral vectors encoding BC-001 (BC- hCD19). Transduction efficiency and long-term CAR expression were assessed by flow cytometry at multiple time points (Day 7, Day 14, and Day 21) during in vitro expansion. As shown in Figure 7, analysis confirmed stable CAR expression in transduced T cells through Day 21.

[0481] Example 6: Healthy donor PBMCs transduced with modified CD19-scFv exhibit improved CAR T cell potencies

[0482] Activated human peripheral blood mononuclear cells (PBMCs) from healthy donor were transduced with lentiviral vectors encoding distinct single-chain variable fragments (scFvs) viz., BC-001 (humanized; BC-hCD19), FMC63 (murine), or C2146 (humanized), sharing the same backbone as BC-001. Transduction was carried out at a multiplicity of infection (MOI) of 5. Non-transduced T cells served as negative controls (NTC). The resulting CD19-targeted CAR-T lymphocytes were co-cultured with Raji cells (Burkitt lymphoma) or NALM6 cells (acute lymphoblastic leukemia), both engineered to express luciferase for cytotoxicity tracking. CAR-T cell-mediated cytolysis was quantified using the Bright-Glo™ cytotoxicity assay (PerkinElmer), and results were compared against non-transduced T-cell controls. Statistical significance was determined (*p < 0.05).

[0483] As shown in Figure 8, healthy donor PBMCs transduced with BC-001 (BC- hCD19) exhibit improved CAR-T cell potencies in both cell types.

[0484] Example 7: Lymphoma patient derived PBMCs transduced with modified CD19- scFv exhibit improved CAR T cell potencies

[0485] Peripheral blood mononuclear cells (PBMCs) were isolated from a donor diagnosed with DLBCL.

[0486] T-cells were activated for 24 hours with soluble CD3 / CD28 (Miltenyi Biotec) in either serum free medium or serum free medium supplemented with BC-TE (Transduction Enhancer) and IL-7 and IL-15. After activation, T-cells were transduced with lentiviral vectors encoding different single-chain variable fragments (scFv’s) viz., humanized BC-001 (BC-hCD19), murine FMC63, and humanized C2146 (structurally identical backbone to BC-001), at a multiplicity of infection (MOI) of 2.5 and 5. Transductions were performed by spinoculation (at 1000 x g for 45 minutes). Nontransduced T cells served as negative controls (NTC).

[0487] CAR-T cells were co-cultured with Raji cells (Burkitt lymphoma) or NALM6 cells (acute lymphoblastic leukemia) at various effector-to-target (E:T) ratio of 20: 1, 10: 1, 5: 1 in 96-well flat-botom plates for 24 hours. Cytolytic activity was quantified using the CyQUANT™ LDH Cytotoxicity Assay Kit (ThermoFisher Scientific), or Bright-Glow™ luciferase activity kit (Perkin Elmer) according to manufacturer’s instruction. Assays were performed in technical triplicates, normalized to spontaneous and maximum release controls, later cytotoxicity (%) was calculated using [% Cytotoxicity = [(Experimental Release - Spontaneous Release) / (Total Release - Spontaneous Release)] * 100],

[0488] As shown in Figure 9, lymphoma patient derived PBMCs transduced with BC- 001 (BC-hCD19) exhibit improved CAR-T cell potencies in both cell types at both MOI.

[0489] Transduction efficiency was assessed by flow cytometry at different time points post-transduction using anti -human IgG Fc or FITC-Labeled Human CD19-detecting reagents (Acrobio systems). Gating was applied on CD3+lymphocytes, and dead cells were excluded. Figure 10 shows the percentage of T cells expressing surface CARs at each MOI.

[0490] Example 8: SLE patient derived PBMCs transduced with modified CD19-scFv exhibit improved CAR T cell potencies

[0491] Peripheral blood samples were purchased from a patient diagnosed with systemic lupus erythematosus (SLE) (Stemcell Technologies). CD19+B cells were subsequently enriched from freshly isolated PBMCs using the human CD 19 MicroBeads, kit (Miltenyi Biotec) and separated on MACS® LS columns using a MACS® Separator, following the protocol provided by the manufacturer.

[0492] CD3 CD 19 T cells were obtained by magnetic depletion of CD19+cells followed by positive selection of CD3+cells using sequential MACS® enrichment.

[0493] T-cells were activated for 24 hours with soluble CD3 / CD28 (Miltenyi Biotec) in either serum free medium or serum free medium supplemented with BC-TE (Transduction Enhancer) and IL-7 and IL- 15 (5 ng / mL). After activation, T-cells were transduced in the presence or absence of a transduction enhancer with lentiviral vectors encoding different single-chain variable fragments (scFv’s) viz., humanized BC-001 (BC-hCD19), murine FMC63, and humanized C2146 (structurally identical backbone to BC-001), at a multiplicity of infection (MOI) of 5. Transductions were performed by spinoculation. Non-transduced T cells served as negative controls (NTC).

[0494] CAR-T cells were co-cultured with autologous CD19+B cells at various effector- to-target (E:T) ratio of 20: 1, 10: 1, 5: 1 in 96-well flat-botom plates for 24 hours. Cytolytic activity was quantified using the CyQUANT™ LDH Cytotoxicity Assay Kit (ThermoFisher Scientific), or Bright-Glow™ luciferase activity kit (Perkin Elmer) according to manufacturer’s instruction. Assays were performed in technical triplicates, normalized to spontaneous and maximum release controls, later cytotoxicity (%) was calculated using [% Cytotoxicity = [(Experimental Release - Spontaneous Release) / (Total Release - Spontaneous Release)] * 100],

[0495] As shown in Figure 11A, SLE patient derived T cells transduced with BC-001 (BC-hCD19) exhibit improved CAR-T cell toxicity against autologous B-cells.

[0496] Cell-free supernatants were harvested from co-cultures after 24 hours and analyzed using the Ella™ Simple Plex Immunoassay System (Bio-Techne). Cytokines including IFN-y, TNF-a, and IL-6 etc., were quantified using Simple Plex™ Cartridges compatible with the ELLA platform. The system uses microfluidic cartridges pre-loaded with capture antibodies and detection reagents, enabling automated, high-throughput cytokine profiling. Assays were conducted according to the user manual, and concentrations were calculated based on standard curves generated by the instrument’s internal calibration of reference standards.

[0497] As shown in Figure 11B-G, PBMCs from SLE patient transduced with BC-001 in the presence and / or absence of a transduction enhancer exhibited lower levels of pro- inflammatory cytokines compared to murine FMC63. In addition, IL-2 levels (which are typically reduced in patients with SLE) were increased in PBMCs from SLE patient transduced with BC-001 in the presence of a transduction enhancer (Figure 11H).

Claims

CLAIMS:

1. A polynucleotide encoding a chimeric antigen receptor (CAR), wherein the polynucleotide comprises:(i) a nucleotide sequence encoding an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the nucleotide sequence encoding the modified scFv comprises: a) a light chain variable region (VL) comprising: i. a complementarity determining region (CDR) 1 comprising a nucleotide sequence set forth in SEQ ID NO: 3; ii. a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 4; and iii. a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 5; and b) a heavy chain variable region (VH) comprising: iv. a CDR1 comprising a nucleotide sequence set forth in SEQ ID NO: 6; v. a CDR2 comprising a nucleotide sequence set forth in SEQ ID NO: 7; and vi. a CDR3 comprising a nucleotide sequence set forth in SEQ ID NO: 8;(ii) a nucleotide sequence encoding a transmembrane domain; and(iii)a nucleotide sequence encoding at least one co-stimulatory domain.

2. The polynucleotide of claim 1, wherein:(i) the transmembrane domain is a CD8a transmembrane domain; and / or(ii)the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

3. The polynucleotide of claim 2, wherein the co-stimulatory domain consists of the 4- IBB signalling domain and the CD3^ signalling domain.

4. The polynucleotide of any one of claims 1 to 3, wherein the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region.

5. The polynucleotide of claim 4, wherein the hinge region is a CD8a hinge region.

6. The polynucleotide of any one of claims 1 to 5, wherein the nucleotide sequence encoding the modified scFv comprises a VL comprising a sequence set forth in SEQ ID NO: 1 and a VH comprising a sequence set forth in SEQ ID NO: 2.

7. The polynucleotide of any one of claims 1 to 6, wherein the VL and the VH are covalently linked to each other via a linker.

8. The polynucleotide of claim 7, wherein the linker is a peptide linker comprising at least 2 amino acids in length.

9. The polynucleotide of claims 7 or 8, wherein the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSG linker, a (Gly4Ser)3 linker, a (Gly)x linker, a (Gly)r, linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4.

10. The polynucleotide of any one of claims 7 to 9, wherein the linker is a (Gly4Ser)3 linker and comprises a polypeptide encoded by a nucleotide sequence set forth in SEQ ID NO: 17.

11. The polynucleotide of any one of claims 1 to 10, wherein the nucleotide sequence encoding the modified scFv comprises a sequence set forth in SEQ ID NO: 19.

12. The polynucleotide of any one of claims 2 to 11, wherein:(i) the nucleotide sequence encoding the CD8a transmembrane domain comprises a sequence set forth in SEQ ID NO: 23;(ii)the nucleotide sequence encoding the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 25; and / or(iii)the nucleotide sequence encoding the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 27.

13. The polynucleotide of any one of claims 5 to 12, wherein the nucleotide sequence encoding the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 21.

14. The polynucleotide of any one of claims 1 to 13, wherein the polynucleotide encoding the CAR comprises a sequence set forth in SEQ ID NO: 29.

15. A chimeric antigen receptor (CAR) encoded by the polynucleotide of any one of claims 1 to 14.

16. A chimeric antigen receptor (CAR) comprising:(i) an extracellular antigen binding domain comprising a modified single chain variable fragment (scFv) that specifically binds human cluster of differentiation 19 (CD 19) antigen, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 35; a CDR2 comprising a sequence set forth in SEQ ID NO: 12 or 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 38; and b) a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 41 or 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 44 or 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 47; or(ii) a transmembrane domain; and(iii) at least one co -stimulatory domain.

17. The CAR of claim 16, wherein the modified scFv comprises: a) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 13; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 14; a CDR2 comprising a sequence set forth in SEQ ID NO: 15; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or b) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 11; a CDR2 comprising a sequence set forth in SEQ ID NO: 12; and a CDR3 comprising a sequence set forth in SEQ ID NO: 39; and a VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 42; a CDR2 comprising a sequence set forth in SEQ ID NO: 45; and a CDR3 comprising a sequence set forth in SEQ ID NO: 16; or c) a VL comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 36; a CDR2 comprising a sequence set forth in SEQ ID NO: 37; and a CDR3 comprising a sequence set forth in SEQ ID NO: 40; anda VH comprising a CDR1 comprising a sequence set forth in SEQ ID NO: 43; a CDR2 comprising a sequence set forth in SEQ ID NO: 46; and a CDR3 comprising a sequence set forth in SEQ ID NO: 48.

18. The CAR of claim 16 or 17, wherein:(i) the transmembrane domain is a CD8a transmembrane domain; and / or(ii)the at least one co-stimulatory domain is selected from the group consisting of a 4- IBB signalling domain, a CD27 signalling domain, an OX-40 signalling domain, a glucocorticoid-induced TNFR-related (GITR) signalling domain, a herpesvirus entry mediator (HVEM) signalling domain and a CD3 zeta (CD3Q signalling domain.

19. The CAR of claim 18, wherein the co-stimulatory domain comprises the 4-1BB signalling domain and the CD3^ signalling domain.

20. The CAR of any one of claims 16 to 19, wherein the extracellular antigen binding domain is operably linked to the transmembrane domain through a hinge region.

21. The CAR of claim 20, wherein the hinge region is a CD8a hinge region.

22. The CAR of any one of claims 16 to 21, wherein the modified scFv comprises: a) a VL comprising a sequence set forth in SEQ ID NO: 9 and a VH comprising a sequence set forth in SEQ ID NO: 10; or b) a VL comprising a sequence set forth in SEQ ID NO: 49 and a VH comprising a sequence set forth in SEQ ID NO: 51; or c) a VL comprising a sequence set forth in SEQ ID NO: 50 and a VH comprising a sequence set forth in SEQ ID NO: 52.

23. The CAR of any one of claims 16 to 22, wherein the VL and the VH are covalently linked to each other via a linker.

24. The CAR of claim 23, wherein the linker is a peptide linker comprising at least 2 amino acids in length.

25. The CAR of claims 23 or 24, wherein the linker is selected from the group consisting of a GS linker, a GSGGS linker, a GGSSG linker, a GGGGS linker, a GSGSGlinker, a (Gly4Ser)3 linker, a (Gly)s linker, a (Gly)r, linker and a (GGGS)nlinker, wherein n=l, 2, 3 or 4.

26. The CAR of any one of claims 22 to 25, wherein the linker is a (Gly4Ser)3 linker and comprises comprising a sequence set forth in SEQ ID NO: 18.

27. The CAR of any one of claims 16 to 25, wherein the modified scFv comprises a sequence set forth in SEQ ID NO: 20, SEQ ID NO: 53 or SEQ ID NO: 54.

28. The CAR of any one of claims 18 to 27, wherein:(i) the CD 8 a transmembrane domain comprises a sequence seq forth in SEQ ID NO: 24;(ii)the 4-1BB signalling domain comprises a sequence set forth in SEQ ID NO: 26; and / or(iii)the CD3^ signalling domain comprises a sequence set forth in SEQ ID NO: 28.

29. The CAR of any one of claims 21 to 28, wherein the CD8a hinge region comprises a sequence set forth in SEQ ID NO: 22.

30. The CAR of any one of claims 16 to 28, wherein the CAR comprises a sequence set forth in SEQ ID NO: 30, SEQ ID NO: 55 or SEQ ID NO: 56.

31. A polynucleotide encoding the CAR of any one of claims 16 to 30.

32. A vector comprising the polynucleotide of any one of claims 1 to 15 or 31.

33. The vector of claim 32, wherein the vector is a plasmid, a cosmid, a phage or a viral vector.

34. The vector of claim 33, wherein the viral vector is a lentiviral vector, an adeno- associated viral vector, an adenoviral vector, a herpes simplex viral (HSV) vector or a retroviral vector.

35. The vector of claim 34, wherein the HSV vector is a HSV type 1 vector or the lentiviral vector is a self-inactivating lentiviral plasmid.

36. A genetically modified cell comprising the vector of any one of claims 32 to 35.

37. A method of producing a genetically modified cell for adoptive cell therapy, the method comprising introducing a vector comprising the polynucleotide of any one of claims 1 to 15 or 31 into a cell, thereby producing the genetically modified cell.

38. The genetically modified cell of claim 36 or the method of claim 37, wherein the cell is an immune cell.

39. The genetically modified cell or the method of claim 38, wherein the immune cell is selected from the group consisting of peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, T cells, neutrophils, eosinophils and combinations thereof.

40. The genetically modified cell of claim 36 or 38 to 39, or the method of claim 37 to 39, wherein the cell is a T cell.

41. The genetically modified cell or the method of claims 39 or 40, wherein the T cell is a gamma delta (y5) T cell, a cytotoxic T cell, or a helper T cell.

42. A composition comprising a population of genetically modified cells of any one of claims 36 or 38 to 41, for use as a medicament.

43. A method of treating a CD 19-expressing proliferative disease in a subject in need thereof, the method comprising administering a composition of claim 41 or a population of genetically modified cells of any one of claims 36 or 38 to 41.

44. Use of a population of genetically modified cells of any one of claims 36 or 38 to 41 in the manufacture of a medicament for treating a CD 19-expressing proliferative disease in a subject in need thereof.

45. The method of claim 43, or the use of claim 44, wherein the CD 19-expressing proliferative disease is a CD- 19 malignant proliferative disease or a CD 19-expressing autoimmune disease.

46. The method or use of claim 45, wherein the CD19-expressing malignant proliferative disease is selected from the group consisting of B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and preleukemia.

47. The method or use of claim 45, wherein the CD-19-expressing autoimmune disease is selected from the group consisting of systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgG4-related disease (IgG4-RD), neuromyelitis optica spectrum disorder (NMOSD), generalized myasthenia gravis (gMG), idiopathic inflammatory myositis (IIM), systemic sclerosis (SSc) and multiple sclerosis (MS).

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