Novel bispecific anti CD19-CD20 car-t constructs and uses thereof

Abstract

The present disclosure provides CD19 / CD20-binding domains and chimeric antigen receptors (CARs) based on the same, as well as corresponding nucleic acid molecules, vectors, cells, compositions, methods and uses, e.g., for the prevention and / or treatment of cancer and / or autoimmune disease.

Description

NOVEL BISPECIFIC ANTI CD19-CD20 CAR-T CONSTRUCTS AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure provides, inter alia, bispecific CD19 / CD20 binding domains and chimeric antigen receptors (CARs) comprising the same, as well as corresponding nucleic acid molecules, vectors, cells, compositions, methods and uses, which are particularly useful in the prevention and / or treatment of cancer and / or autoimmune disease.BACKGROUND

[0002] Chimeric antigen receptors (“CARs”) are engineered receptors designed to modulate T cell receptors (“TCRs”) response and generally include four main components: (i) an extracellular target antigen-binding domain, (ii) a hinge or spacer region, (iii) a transmembrane domain, and (iv) one or several intracellular signaling domains. (Ahmad et al. 2022). T-cells engineered to express the same are referred to as “CAR T-cells” and therapeutic methods using such cells are often referred to as a “CAR-T therapy.”

[0003] Further research has resulted in the creation of 4thgeneration CAR T-cells, which are further engineered to release a transgenic cytokine in the targeted tumor tissue thereby modulating the tumor microenvironment. These 4'1' generation CAR-T are also known as “armored” CAR-Ts or TRUCKs (“T- cells redirected for antigen-unrestricted cytokine-initiated killing”). (Chmielewski and Abken 2020).

[0004] Chimeric antigen receptor T-cell (“CAR-T”) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia and two anti-CD19 (Cluster of Differentiation 19, “CD19,” also known as B4 or Leu-12) CAR-T therapies were approved by the FDA in 2017, in particular for the treatment of patients with relapsed, refractory B cell lymphoid malignancies (Maude et al. 2018), such as patients with R / R DLBCL after failure on 2 lines of systemic therapy (Neelapu et al. 2017)), acute lymphoblastic leukemia (“ALL”), chronic lymphocytic leukemia (“CLL”) and / or large B-cell lymphomas (“DLBCL”). See (Ramos, Savoldo, and Dotti 2014). CD19-targeted CAR-T therapy has also been evaluated as a treatment for autoimmune diseases such as systemic lupus erythematosus (“SLE”). (Mackensen et al. 2022; Aghajanian, Rurik, and Epstein 2022).

[0005] CD20 is a transmembrane protein, encoded in humans by the MS4A1 gene, expressed on the surface of B lymphocytes during the development of a B lymphocyte from the early pre-B stage until terminal differentiation into plasmocytes, at which stage CD20 expression disappears.

[0006] CD20 is also expressed on malignant B cells, and prior studies have found it to be expressed on more than 90% of B cell non-Hodgkin's lymphoma (“NHL”) cells and over 95% of B-type Chronic Lymphocytic Leukemia (“B-CLL”) cells. (Casan et al. 2018).

[0007] Consequently, antibodies directed against CD20 have been used for the treatment of B-cell derived leukemia and lymphomas. To date, the most widely prescribed anti-CD20 antibody is rituximab, which is listed on the World Health Organization's List of Essential Medicines. See (Ang et al. 2023). Specifically, rituximab (Rituxan) is a genetically engineered chimeric murine / human monoclonal antibody directed against CD20. See (Casan et al. 2018). Rituximab is currently approved for the treatment of relapsed orrefractory follicular lymphoma. Reports indicate that weekly infusions with rituximab resulted in overall response rates of 48%. (Casan et al. 2018). However, many patients do not respond to rituximab treatment. In addition, responding patients often develop resistance to rituximab and eventually relapse.

[0008] Fully human monoclonal antibodies against CD20 have also been developed, for example Ofatumumab (CAS No. 679818-59-8) which has however received a black box warning due to side effects. See (Clinical Review Report: Ofatumumab (Kesimpta): (Novartis Pharmaceuticals Canada Inc.): Indication: Multiple Sclerosis, Relapsing-Remitting 202 f).

[0009] Similarly, CD20 may also be linked to autoimmune diseases, in particular type 2 rheumatoid arthritis, vasculitis, lupus erythematosus (Casan et al. 2018)) and / or multiple sclerosis (Hauser et al. 2023)).SUMMARY

[0010] T cell immunotherapies (e.g., CAR-T therapy) have shown great potential as a precision medicine, however significant limitations remain, such as antigen escape, on-target off-tumor effects, cell trafficking and tumor infiltration, Immunosuppressive microenvironment, and CAR-T cell-associated toxicities. See (Sterner and Sterner 2021). Therefore, one strategy to avoid antigen loss after CAR-T cell therapy is to target two or more epitopes of a single antigen, or multiple antigens, simultaneously. See (Trabolsi, Arumov, and Schatz 2024; Wang et al. 2024). However, CAR-T cell activation and activation kinetics are influenced by several factors, including but not limited to the level of tumor antigen expressed on malignant cells, tumor burden, antigen binding domain’s affinity to its target epitope, and / or the CAR’s costimulatory elements. Careful consideration of several components of the CAR’s modular structure is therefore necessary to optimize therapeutic efficacy and limit toxicity. (Sterner and Sterner 2021).

[0011] Despite the clinical success of various CAR T-cell therapies, the therapeutic index of these therapies remains high due to immunogenicity issues, toxicities associated with the infusion of the CAR T- cells, and relapse of the tumor. Accordingly, there is an urgent need in the art for novel approaches that can solve or mitigate the harmful side effects of CAR T-cell therapies and allow for more effective, safe, and efficient adoptive immunotherapy. In particular, there is a need for new CAR which is not associated with the disadvantages of prior constructs, e.g., to provide new treatment and method of treatment of cancer and / or autoimmune diseases.

[0012] Herein are provided, inter alia, bispecific CD19 / CD20 binding domains and chimeric antigen receptors (CARs) comprising the same, as well as corresponding nucleic acid molecules, vectors, cells, compositions, methods and uses, which are particularly useful in the prevention and / or treatment of cancer and / or autoimmune disease. In particular, herein are provided bispecific CD19 / CD20 CAR constructs operably linked to interleukin- 18 (CD19 / CD20-IL18 constructs), CAR T-cells comprising these CD19 / CD20-IL18 constructs, and methods of using these IL-18 “armored” CD19 / 20 CAR T-cells. In some aspects, these T-cells may show improved expansion, persistence, potency, and / or reduced toxicity.

[0013] In a first general aspect, the disclosure provides a nucleic acid molecule, comprising a polynucleotide sequence encoding a CAR comprising an anti-CD19 binding domain and an anti-CD20 binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain.

[0014] In some aspects, the disclosure provides a nucleic acid molecule comprising a polynucleotide sequence encoding a CAR comprising an anti-CD19 binding domain and an anti-CD20 binding domain, a transmembrane domain, a costimulatory, and an intracellular signaling domain; and further comprising a second polynucleotide sequence encoding a polypeptide that enhances an immune cell function, or a functional derivative thereof, wherein the polypeptide that enhances an immune cell function, or a functional derivative thereof is selected from the group consisting of a cytokine, an interferon, a chemokine, an antibody or antibody fragment, a checkpoint inhibitor antagonist, a dominant negative receptor, a switch receptor, and a combination thereof. In a particular embodiment, the polypeptide that enhances an immune cell function, or a functional derivative thereof is: (a) a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, or a combination thereof; (b) a cytokine selected from the group consisting of Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-7 receptor (IL-7R), Interleukin- 11 (IL-11), Interleukin- 12 (IL-12), Interleukin- 15 (IL-15), Interleukin- 15 receptor (IL- 15R), Interleukin- 18 (IL-18), Interleukin- 18 receptor (IL-18R), Interleukin-21 (IL-21), granulocyte macrophage colony stimulating factor, alpha, beta or gamma interferon, erythropoietin, and a combination thereof; or (c) a chemokine selected from CCL21, CCL19, or a combination thereof. In a more particular embodiment, the polynucleotide sequence encoding a polypeptide that enhances an immune cell function, or a functional derivative thereof, is an IL-18 encoding polynucleotide sequence. In some aspects, the IL- 18 encoding polynucleotide sequence encodes a polypeptide sequence that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 47.

[0015] In some aspects, herein is provided inter alia a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a chimeric antigen receptor (CAR), where the CAR comprises an anti-CD19 binding domain and an anti- CD20 binding domain, a transmembrane domain, a costimulatory, and an intracellular signaling domain.

[0016] In some aspects, the disclosure provides a vector comprising: (a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a nucleic acid sequence encoding a CAR comprising an anti-CD19 binding domain and an anti-CD20 binding domain, a transmembrane domain, a costimulatory, and an intracellular signaling domain; and (b) a second polynucleotide sequence comprising a nucleic acid sequence encoding a polypeptide that enhances an immune cell function, or a functional derivative thereof. In a particular embodiment, the polypeptide that enhances an immune cell function, or a functional derivative thereof is: (a) a chemokine, a chemokine receptor, a cytokine, a cytokine receptor, or a combination thereof; (b) a cytokine selected from the group consisting of Interleukin-2 (IL- 2), Interleukin-3 (IL-3), Interleukin-6 (IL-6), Interleukin-7 (IL-7), Interleukin-7 receptor (IL-7R), Interleukin- 11 (IL-11), Interleukin- 12 (IL-12), Interleukin- 15 (IL-15), Interleukin- 15 receptor (IL-15R), Interleukin- 18 (IL-18), Interleukin- 18 receptor (IL-18R), Interleukin-21 (IL-21), granulocyte macrophage colony stimulating factor, alpha, beta or gamma interferon, erythropoietin, and a combination thereof; or (c) a chemokine selected from CCL21, CCL19, or a combination thereof. In a more particular embodiment,the nucleic acid sequence encoding a polypeptide that enhances an immune cell function, or a functional derivative thereof, is an IL- 18 encoding nucleic acid sequence.

[0017] In some aspects, the first polynucleotide sequence and the second polynucleotide sequence are separated by an intervening polynucleotide sequence encoding one or more linker sequences (e.g., allowing for the expression of a polypeptide construct wherein the CAR is operably linked to the second polypeptide via a linker peptide). In a particular embodiment, the linker peptide is selected from F2A, E2A, P2A, T2A, Furin-(SG)2-T2A (F-SG2-T2A), Furin-(SG)2-T2A-Furin-, (F-SG2-T2A-F), Furin-(SG)2-P2A (F-SG2- P2A), Furin-(SG)2-P2A-Furin (F-SG2-P2A-F). In a more particular embodiment, the linker is P2A, F-SG2- P2A, or F-SG2-P2A-F. In a more particular aspect, the linker peptide is selected from SEQ ID NO: 46, SEQ ID NO: 83, SEQ ID NO: 84.

[0018] In some aspects, the polypeptide that enhances an immune cell function, or a functional derivative thereof further comprises a signal peptide selected from the group consisting of a CD59 signal peptide, an IL-2 signal peptide, an IL-12 signal peptide, an antibody kappa light chain signal peptide, an antibody lambda light chain signal peptide, a CD8 signal peptide, CD4 signal peptide, granulocyte-macrophage colony -stimulating factor receptor alpha, tPA signal peptide or any equivalent thereof. In a particular embodiment, the leader sequence is a CD59 signal peptide.

[0019] In some aspects, the polypeptide that enhances an immune cell function, or a functional derivative thereof comprises an IL- 18 polypeptide or a polypeptide having an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 47. In a particular embodiment, the polypeptide that enhances an immune cell function, or a functional derivative thereof comprises an IL- 18 polypeptide or a polypeptide having the amino acid sequence of SEQ ID NO: 47.

[0020] In some aspects, the polypeptide that enhances an immune cell function, or a functional derivative thereof comprises an IL- 18 polypeptide or a polypeptide encoded by a nucleic acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 48. In a particular embodiment, the polypeptide that enhances an immune cell function, or a functional derivative thereof comprises an IL- 18 polypeptide or a polypeptide encoded by a nucleic acid sequence according to SEQ ID NO: 48.

[0021] In some aspects, the IL-18 polypeptide further comprises a CD59 leader sequence, or the amino acid sequence of SEQ ID NO: 34 or an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 35.

[0022] In some aspects, the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, an adenoviral vector, or a retroviral vector.

[0023] In some aspects, the vector is an in vitro transcribed vector.

[0024] In some aspects of the vectors disclosed herein, the promoter (e.g. constitutive promoter) comprises a promoter selected from the group consisting of an EF-lalpha promoter, a truncated EF-lalpha promoter, a PGK-1 promoter, a truncated PGK-1 promoter, an UBC promoter, a CMV promoter, a CAGGpromoter, and an SV40 promoter. In a particular embodiment, the promoter is a constitutive promoter. In a particular embodiment, the constitutive promoter is a truncated EF-1 promoter. In a more particular embodiment, the constitutive promoter comprises the sequence of SEQ ID NO: 49.

[0025] In some aspects, the vector further comprises a rev response element (RRE), a poly(A) tail, a 3' UTR, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and / or a cPPT sequence. In a particular embodiment, the WPRE comprises the sequence of SEQ ID NO: 86, 87, 88, 89, 98, 99, or 100.

[0026] In some aspects, the anti-CD19 binding domain comprises: (a) a light chain variable domain including a light chain complementary determining region 1 (CD 19 LC CDR1) of SEQ ID NO: 1, a light chain complementary determining region 2 (CD 19 LC CDR2) of SEQ ID NO: 2, and a light chain complementary determining region 3 (CD19 LC CDR3) of SEQ ID NO: 3; and / or a heavy chain variable domain including a heavy chain complementary determining region 1 (CD19 HC CDR1) of SEQ ID NO: 4, a heavy chain complementary determining region 2 (CD19 HC CDR2) of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (CD 19 HC CDR3) of SEQ ID NO: 6.

[0027] In some aspects, the anti-CD19 binding domain comprises: (a) a light chain variable domain including a light chain complementary determining region 1 (CD 19 LC CDR1) of SEQ ID NO: 1, a light chain complementary determining region 2 (CD 19 LC CDR2) of SEQ ID NO: 2, and a light chain complementary determining region 3 (CD 19 LC CDR3) of SEQ ID NO: 3; and a heavy chain variable domain including a heavy chain complementary determining region 1 (CD19 HC CDR1) of SEQ ID NO: 4, a heavy chain complementary determining region 2 (CD19 HC CDR2) of SEQ ID NO: 5, and a heavy chain complementary determining region 3 (CD 19 HC CDR3) of SEQ ID NO: 6.

[0028] In some aspects, the anti-CD19 binding domain light chain variable region (CD19 VL) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 7. In a more particular embodiment, the anti-CD19 binding domain light chain variable region (CD 19 VL) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 7, wherein the CD 19 VL comprises the CD 19 LC CDR1 of SEQ ID NO: 1, the CD 19 LC CDR2 of SEQ ID NO: 2, and the CD 19 LC CDR3 of SEQ ID NO. 3. In a particular embodiment, the anti-CD19 binding domain light chain variable region (CD 19 VL) comprises the amino acid sequence of SEQ ID NO: 7. In a more particular embodiment, the amino acid sequence of the anti-CD19 binding domain light chain variable region (CD 19 VL) is according to SEQ ID NO: 7.

[0029] In some aspects, the anti-CD19 binding domain light chain variable region (CD19 VL) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 7. In a particular embodiment, the anti-CD 19 binding domain light chain variable region (CD19 VL) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 7, wherein the CD 19 VL comprises the CD 19 LC CDR1 of SEQ ID NO: 1, the CD 19 LC CDR2 of SEQ ID NO: 2, and the CD 19 LC CDR3 ofSEQ ID NO. 3. In other words, in this embodiment, the amino acid substitutions, insertions, or deletions compared to SEQ ID NO:7 are not within the CDRs. In a particular embodiment, the anti-CD19 binding domain light chain variable region (CD19 VL) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 7. In a particular embodiment, the anti-CD19 binding domain light chain variable region (CD19 VL) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 7, wherein the CD 19 VL comprises the CD 19 LC CDR1 of SEQ ID NO: 1, the CD19 LC CDR2 of SEQ ID NO: 2, and the CD19 LC CDR3 of SEQ ID NO. 3.

[0030] In some aspects, the anti-CD 19 binding domain heavy chain variable region (CD 19 VH) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 8. In a particular embodiment, the anti-CD 19 binding domain heavy chain variable region (CD 19 VH) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 8, wherein the CD 19 VH comprises the CD19 HC CDR1 of SEQ ID NO: 4, the CD19 HC CDR2 of SEQ ID NO: 5, and the CD19 HC CDR3 of SEQ ID NO. 6. In a particular embodiment, the anti-CD 19 binding domain heavy chain variable region (CD 19 VH) comprises the amino acid sequence of SEQ ID NO: 8. In a more particular embodiment, the amino acid sequence of the anti -CD 19 binding domain heavy chain variable region (CD 19 VH) is according to SEQ ID NO: 8.

[0031] In some aspects, the anti-CD19 binding domain light chain variable region (CD19 VH) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 8. In a particular embodiment, the anti-CD 19 binding domain heavy chain variable region (CD19 VH) comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, insertions, or deletions compared to SEQ ID NO: 8, wherein the CD19 VH comprises the CD19 HC CDR1 of SEQ ID NO: 4, the CD19 HC CDR2 of SEQ ID NO: 5 and the CD19 HC CDR3 of SEQ ID NO. 6. In other words, in this embodiment, the amino acid substitutions, insertions, or deletions compared to SEQ ID NO:8 are not within the CDRs. In a particular embodiment, the anti-CD19 binding domain light chain variable region (CD19 VH) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 8. In a particular embodiment, the anti-CD19 binding domain light chain variable region (CD19 VH) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to SEQ ID NO: 8, wherein the CD 19 VH comprises the CD 19 HC CDR1 of SEQ ID NO: 4, the CD19 HC CDR2 of SEQ ID NO: 5 and the CD19 HC CDR3 of SEQ ID NO. 6.

[0032] In some aspects, the anti-CD19 binding domain light chain variable region (CD19 VL) comprises the amino acid sequence of SEQ ID NO: 7 and the anti-CD 19 binding domain heavy chain variable region (CD 19 VH) comprises the amino acid sequence of SEQ ID NO: 8.

[0033] In some aspects, the anti-CD 19 binding domain has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 9. In a particular embodiment, the anti-CD 19 binding domain has the amino acid sequence of SEQ ID NO: 9.

[0034] In some aspects, the anti -CD 19 binding domain has a nucleic acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 21. In a particular embodiment, the anti-CD19 binding domain has the nucleic acid sequence of SEQ ID NO: 21.

[0035] In some aspects, the CD 19 binding domain is an scFv.

[0036] In some aspects, the anti-CD20 binding domain comprises: (a) a light chain variable domain including a light chain complementary determining region 1 (CD20 LC CDR1) of SEQ ID NO: 10, a light chain complementary determining region 2 (CD20 LC CDR2) of SEQ ID NO: 11, and a light chain complementary determining region 3 (CD20 LC CDR3) of SEQ ID NO: 12; and / or a heavy chain variable domain including a heavy chain complementary determining region 1 (CD20 HC CDR1) of SEQ ID NO: 13, a heavy chain complementary determining region 2 (CD20 HC CDR2) of SEQ ID NO: 14, and a heavy chain complementary determining region 3 (CD20 HC CDR3) of SEQ ID NO: 15.

[0037] In some aspects, the anti-CD20 binding domain comprises: (a) a light chain variable domain including a light chain complementary determining region 1 (CD20 LC CDR1) of SEQ ID NO: 10, a light chain complementary determining region 2 (CD20 LC CDR2) of SEQ ID NO: 11, and a light chain complementary determining region 3 (CD20 LC CDR3) of SEQ ID NO: 12; and a heavy chain variable domain including a heavy chain complementary determining region 1 (CD20 HC CDR1) of SEQ ID NO: 13, a heavy chain complementary determining region 2 (CD20 HC CDR2) of SEQ ID NO: 14, and a heavy chain complementary determining region 3 (CD20 HC CDR3) of SEQ ID NO: 15.

[0038] In some aspects, the anti-CD20 binding domain light chain variable region (CD20 VL) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16. In a more particular embodiment, the anti-CD20 binding domain light chain variable region (CD20 VL) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 16, wherein the CD20 VL comprises the CD20 LC CDR1 of SEQ ID NO: 10, the CD20 LC CDR2 of SEQ ID NO: 11, and the CD20 LC CDR3 of SEQ ID NO. 12. In a particular embodiment, the anti-CD20 binding domain light chain variable region (CD20 VL) comprises the amino acid sequence of SEQ ID NO: 16. In a more particular embodiment, the amino acid sequence of the anti-CD20 binding domain light chain variable region (CD20 VL) is according to SEQ ID NO: 16.

[0039] In some aspects, the anti-CD20 binding domain heavy chain variable region (CD20 VH) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 17. In a more particular embodiment, the anti-CD20 binding domain heavy chain variable region (CD20 VH) comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 17, wherein the CD20 VH comprises the CD20 HC CDR1 of SEQ ID NO: 13, the CD20 HC CDR2 of SEQ ID NO: 14, and the CD20 HC CDR3 of SEQ ID NO. 15. In a particular embodiment, the anti-CD20 binding domain heavy chain variable region(CD20 VH) comprises the amino acid sequence of SEQ ID NO: 17. In a more particular embodiment, the amino acid sequence of the anti-CD20 binding domain heavy chain variable region (CD20 VH) is according to SEQ ID NO: 17.

[0040] In some aspects, the anti-CD20 binding domain light chain variable region (CD20 VL) comprises the amino acid sequence of SEQ ID NO: 16 and the anti-CD20 binding domain heavy chain variable region (CD20 VH) comprises the amino acid sequence of SEQ ID NO: 17.

[0041] In some aspects, the anti-CD20 binding domain has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 18. In a particular embodiment, the anti-CD20 binding domain has the amino acid sequence of SEQ ID NO: 18.

[0042] In some aspects, the anti-CD20 binding domain has a nucleic acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 22. In a particular embodiment, the anti-CD20 binding domain has the nucleic acid sequence of SEQ ID NO: 22.

[0043] In some aspects, the CD20 binding domain is a single-chain fragment variable (“scFv”).

[0044] In some aspects, the CD19 and CD20 binding domains are in tandem (TanCD19 / CD20 BD). In a particular embodiment, the TanCD19 / CD20 BD has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 39. In a particular embodiment, the TanCD19 / CD20 BD has the amino acid sequence of SEQ ID NO: 39.

[0045] In some aspects, the CD19 and CD20 binding domain is in tandem (TanCD19 / CD20 BD). In a particular embodiment, the TanCD19 / CD20 BD is encoded by a nucleic acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 24. In a particular embodiment, the TanCD19 / CD20 BD has the nucleic acid sequence of SEQ ID NO: 24.

[0046] In some aspects, the CD 19 and CD20 binding domain is a diabody (DiaCD19 / CD20 BD). In a particular embodiment, the DiaCD19 / CD20 BD has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 38. In a particular embodiment, the DiaCD19 / CD20 BD has the amino acid sequence of SEQ ID NO: 38.

[0047] In some aspects, the CD19 and CD20 binding domain is a diabody (DiaCD19 / CD20 BD). In particular embodiment, the DiaCD 19 / CD20 BD is encoded by a nucleic acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 23. In a particular embodiment, the DiaCD19 / CD20 BD has the nucleic acid sequence of SEQ ID NO: 23.

[0048] In some aspects, the transmembrane domain comprises a transmembrane domain (TMD) of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD2, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In a particular embodiment, the TMD is a CD8 transmembrane domain. In a particular embodiment, the TMD has an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 25. In a particular embodiment, the TMD has the amino acid sequence according to SEQ ID NO: 25.

[0049] In some aspects, the binding domain is connected to the transmembrane domain by a hinge region. In a particular embodiment, the hinge is a CD8 hinge region. In a particular embodiment, the hinge region has an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 27. In a particular embodiment, the hinge region has the amino acid sequence according to SEQ ID NO: 27.

[0050] In some aspects, the costimulatory domain of the CAR is a functional signaling domain of a protein selected from the group consisting of a TNFR superfamily member, 0X40 (CD 134), CD2, CD5, CD7, CD27, CD28, CD30, CD40, PD-1, CD8, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CDlla, CD18, ICOS (CD278), LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, DAP10, DAP12, Lek, Fas and 4-1BB (CD137). In a particular embodiment, the costimulatory domain is 4-1BB (CD137) costimulatory domain. In a particular embodiment, the costimulatory domain has an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 29. In a particular embodiment, the costimulatory domain has the amino acid sequence according to SEQ ID NO: 29.

[0051] In some aspects, the intracellular signaling domain of the CAR comprises a signaling domain of a protein selected from the group consisting of CD3 zeta, FcyRIII, FceRI, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.

[0052] In a particular embodiment, the intracellular signaling domain is CD3 zeta. In a particular embodiment, the intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 30. In a particular embodiment, the intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 30.

[0053] In a particular embodiment, the intracellular signaling domain is CD3 zeta. In a particular embodiment, the intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 31. In a particular embodiment, the intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 31.

[0054] In a particular embodiment, the intracellular signaling domain is CD3 zeta. In a particular embodiment, the intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQID NO: 97. In a particular embodiment, the intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 97.

[0055] In some aspects, the CAR comprises a functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain. In a particular embodiment, the functional signaling 4- 1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 36. In a particular embodiment, the functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 36.

[0056] In some aspects, the CAR comprises a functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain. In a particular embodiment, the functional signaling 4- 1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 37. In a particular embodiment, the functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 37.

[0057] In some aspects, the CAR comprises a functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain. In a particular embodiment, the functional signaling 4- 1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has an amino acid sequence having 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 85. In a particular embodiment, the functional signaling 4-1BB costimulatory domain and a functional CD3 zeta intracellular signaling domain has the amino acid sequence according to SEQ ID NO: 85.

[0058] In some aspects, the CAR comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 39. In a particular embodiment, the CAR comprises the amino acid sequence according to SEQ ID NO: 39.

[0059] In some aspects, the polynucleotide sequence encoding the CAR comprises a nucleic acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 43. In a particular embodiment, the polynucleotide sequence encoding the CAR comprises a nucleic acid sequence according to SEQ ID NO: 43.

[0060] In some aspects, the CAR comprises an amino acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 38. In a particular embodiment, the CAR comprises the amino acid sequence according to SEQ ID NO: 38.

[0061] In some aspects, the polynucleotide sequence encoding the CAR comprises a nucleic acid sequence having about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, or about 99% identity to SEQ ID NO: 42. In a particular embodiment, the polynucleotide sequence encoding the CAR comprises a nucleic acid sequence according to SEQ ID NO: 42.

[0062] In some aspects, the CAR further comprises a leader sequence. In a particular embodiment, the leader sequence is according SEQ ID NO: 34.

[0063] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, and an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta.

[0064] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD19 LC CDR2 of SEQ ID NO: 2, and CD19 LC CDR3; and a heavy chain variable domain having a CD19 HC CDR1 of SEQ ID NO: 4, CD19 HC CDR2 of SEQ ID NO: 5, and CD19 HC CDR3 of SEQ ID NO: 6, and an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta.

[0065] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO:17, a CD8 transmembrane domain,an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta.

[0066] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta.

[0067] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30.

[0068] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD19 LC CDR2 of SEQ ID NO: 2, and CD19 LC CDR3 of SEQ ID NO: 3; and a heavy chain variable domain having a CD 19 HC CDR1 of SEQ ID NO: 4, CD 19 HC CDR2 of SEQ ID NO: 5, and CD19 HC CDR3 of SEQ ID NO: 6, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, anda CD3-zeta intracellular signaling domain according to SEQ NO: 30.

[0069] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO:17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30.

[0070] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a constitutive promoter operably linked to a polynucleotide sequence encoding a bispecific antiCD 19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30.

[0071] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31.

[0072] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD 19 LC CDR2 of SEQ ID NO: 2, and CD 19 LC CDR3 of SEQ ID NO: 3, and a heavy chain variable domain having a CD 19 HC CDR1 of SEQ ID NO: 4, CD 19 HC CDR2 of SEQ ID NO: 5, and CD19 HC CDR3 of SEQ ID NO: 6,an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31.

[0073] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31.

[0074] In some aspects, the disclosure provides a vector comprising a polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / C D20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31.

[0075] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, and an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, andan intracellular signaling domain comprising of CD3-zeta; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL-18) polypeptide. In some aspects, the IL- 18 is according to SEQ ID NO: 47.

[0076] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD19 LC CDR2 of SEQ ID NO: 2, and CD19 LC CDR3; and a heavy chain variable domain having a CD19 HC CDR1 of SEQ ID NO: 4, CD19 HC CDR2 of SEQ ID NO: 5, and CD19 HC CDR3 of SEQ ID NO: 6, and an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2,CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL-18) polypeptide. In some aspects, the IL-18 is according to SEQ ID NO: 47.

[0077] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promote (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO:17, a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL-18) polypeptide. In some aspects, the IL-18 is according to SEQ ID NO: 47.

[0078] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain, an intracellular signaling domain of a protein selected from the group consisting of 0X40, CD27, CD2, CD28, ICOS, and 4-1BB, and an intracellular signaling domain comprising of CD3-zeta; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL-18) polypeptide. In some aspects, the IL-18 is according to SEQ ID NO: 47.

[0079] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0080] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD19 LC CDR2 of SEQ ID NO: 2, and CD19 LC CDR3 of SEQ ID NO: 3, a heavy chain variable domain having a CD19 HC CDR1 of SEQ ID NO: 4, CD19 HC CDR2 of SEQ ID NO: 5, and CD 19 HC CDR3 of SEQ ID NO: 6, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15;a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0081] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO:17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0082] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 30; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0083] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD 19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD 19 binding domain, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and aheavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0084] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: a light chain variable domain having a CD19 LC CDR1 of SEQ ID NO: 1, CD 19 LC CDR2 of SEQ ID NO: 2, and CD 19 LC CDR3 of SEQ ID NO: 3, and a heavy chain variable domain having a CD 19 HC CDR1 of SEQ ID NO: 4, CD 19 HC CDR2 of SEQ ID NO: 5, and CD19 HC CDR3 of SEQ ID NO: 6, an anti-CD20 binding domain comprising: a light chain variable domain having a CD20 LC CDR1 of SEQ ID NO: 10, CD20 LC CDR2 of SEQ ID NO: 11, and CD20 LC CDR3 of SEQ ID NO: 12; and a heavy chain variable domain having a CD20 HC CDR1 of SEQ ID NO: 13, CD20 HC CDR2 of SEQ ID NO: 14, and CD20 HC CDR3 of SEQ ID NO: 15; a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31; and(b) a second polynucleotide sequence encoding an Interleukin- 18 (IL- 18) polypeptide according to SEQ NO: 47.

[0085] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31; and(b) a second polynucleotide sequence encoding Interleukin- 18 (IL-18) according to SEQ NO: 47.

[0086] In some aspects, the disclosure provides a vector comprising:(a) a first polynucleotide sequence comprising a promoter (e.g. constitutive promoter) operably linked to a polynucleotide sequence encoding a bispecific anti-CD19 / CD20 chimeric antigen receptor (CAR), where the CAR comprises: an anti-CD19 binding domain comprising: CD19 VL of SEQ ID NO: 7, CD19 VH of SEQ ID NO: 8, an anti-CD20 binding domain comprising: CD20 VL of SEQ ID NO: 16, CD20 VH of SEQ ID NO: 17, a CD8 transmembrane domain according to SEQ ID NO:25, a 4-1BB intracellular signaling domain according to SEQ NO: 29, and a CD3-zeta intracellular signaling domain according to SEQ NO: 31; and(b) a second polynucleotide sequence encoding Interleukin- 18 (IL-18) according to SEQ NO: 47.

[0087] In some embodiments, the first polynucleotide is operably linked to the second polynucleotide via a nucleic acid sequence encoding a linker peptide selected from the group consisting of F2A, E2A, P2A, T2A, Furin-(SG)2-T2A (F-SG2-T2A), Furin-(SG)2-T2A-Furin-, (F-SG2-T2A-F), Furin-(SG)2-P2A (F- SG2-P2A), Furin-(SG)2-P2A-Furin (F-SG2-P2A-F). In a particular embodiment, the first polynucleotide is operably linked to the second polynucleotide via a nucleic acid sequence encoding a linker peptide, selected from SEQ ID NO: 46, 83, and 84.

[0088] Herein is also provided inter alia, a cell composition comprising a plurality of any of the cells provided herein. In some aspects, herein is also provided inter alia, a method for making any of the cells provided herein, comprising transducing or transfecting a cell with a nucleic acid molecule or a vector provided herein.

[0089] Herein is also provided inter alia, a method for making a cell composition, comprising transducing or transfecting ex vivo a plurality of cells obtained from a subject with nucleic acid molecule or a vector provided herein. In some aspects, the plurality of cells may be obtained from a sample from the subject such as a blood sample or a derivative thereof, e.g., a peripheral blood mononuclear cell (PBMC) sample.

[0090] Herein is also provided inter alia, a pharmaceutical composition comprising any binding domain, CAR, nucleic acid molecule, vector, cell, or cell composition provided herein, together with a pharmaceutically acceptable carrier, diluent or excipient.

[0091] The binding domain, CAR, nucleic acid molecule, vector, cell, or cell composition provided herein may therefore be used in the manufacture of a pharmaceutical composition for treating cancer and / or autoimmune disease.

[0092] The pharmaceutical compositions provided herein may be for use as a medicament.

[0093] Accordingly, herein is also provided inter alia, a method for treating cancer and / or autoimmune disease comprising administering an effective amount of a pharmaceutical composition, provided herein, to a subject in need thereof.

[0094] In some aspects, the method may comprise (i) transducing or transfecting ex vivo a plurality of cells obtained from a subject with the pharmaceutical composition, wherein the pharmaceutical composition comprises a nucleic acid molecule or a vector provided herein, and (ii) administering the transduced or transfected cells back into the subject.

[0095] The pharmaceutical compositions provided herein may be for use in treating cancer and / or autoimmune disease.

[0096] Other objects and advantages will become apparent to those skilled in the art from a consideration of the ensuing detailed description.DESCRIPTION OF THE FIGURES

[0097] FIG. 1. Schematic representation of bispecific CD19 / CD20 CARs. VH1 and VL1 are the VH and VL domains of the CD 19 binder, respectively. VH2 and VL2 are the VH and VL domains of the CD20 binder, respectively. CD8H, CD8a hinge. CD8TM, CD8a transmembrane domain. CD3z, CD3 zeta domain.

[0098] FIG. 2. Transduction efficiency of bispecific CD19 / CD20 CARs. CAR-T cells were stained by a PE-conjugated anti-G4S linker antibody. The CAR-negative and -positive T cell populations are in light gray and dark gray, respectively. Transduction efficiency (percent positivity rates) is shown above the histograms and in the table for D1920 (SEQ ID NO: 38), and D1920-IL18 (SEQ ID NO: 40).

[0099] FIG. 3. in vitro killing activity of bispecific CD19 / CD20 CARs D1920 (SEQ ID NO: 38), and D1920-IL18 (SEQ ID NO: 40). The experiment was performed in duplicate. Data plotted are mean ± standard deviation.

[0100] FIG. 4. in vitro IL-18 release by bispecific CD 19 / CD20 CARs for D1920 (SEQ ID NO: 38), and D1920-IL18 (SEQ ID NO: 40). The experiment was performed in duplicate. Data plotted are mean ± standard deviation.

[0101] FIG. 5. in vitro killing activity of bispecific CD19 / CD20 CARs in a repetitive challenge assay. The experiment was performed at an E:T ratio of 1: 1 with two different donors of T cells for DI 920 (SEQ ID NO: 107), T1920 (SEQ ID NO: 39), D1920-IL18 (SEQ ID NO: 40) and T1920-IL18 (SEQ ID NO: 41). Data plotted are mean ± standard deviation. NT, non-transduced. Stim, stimulation. **, P<0.01 (unpaired t test) for killing activity of D1920-IL18 in rounds 5 and 6 of stimulation compared to T2019-IL18.

[0102] FIG. 6. in vitro IFN-y release by bispecific CD19 / CD20 CARs in a repetitive challenge assay for D1920 (SEQ ID NO: 107), T1920 (SEQ ID NO: 39), D1920-IL18 (SEQ ID NO: 40) and T1920-IL18 (SEQ ID NO: 41). The experiment was performed at an E:T ratio of 1 : 1 with two different donors of T cells. Data plotted are mean ± standard deviation. NT, non-transduced. Stim, stimulation. ***, P0.001; **, PO.01; and *, P<0.05 (unpaired t test) for IFN-y release by D1920-IL18 compared to T2019-IL18. ns, nonsignificant.

[0103] FIG 7. In vivo anti-tumor activity of the IL- 18 armed CAR not expressing a bispecific binding domain (SEQ ID NO: 108) in mice. The CARs were tested at a low (1E6) and a high (3E6) dose, while the untransduced T cells were tested at a high dose (3E6) only as a negative control. Tumor burden (bioluminescence) is plotted for each animal in all the groups.

[0104] FIG. 8. / n vivo anti-tumor activity of IL-18-armed bispecific CD19 / CD20 CARs for D1920 (SEQ ID NO: 107), T1920 (SEQ ID NO: 39), D1920-IL18 (SEQ ID NO: 40) and T1920-IL18 (SEQ ID NO: 41). The CARs including the unarmed and the IL-18-only CARs were tested at a low (1E6) and a high (3E6)dose, while the untransduced T cells were tested at a high dose (3E6) only as a negative control. Tumor burden (bioluminescence) is plotted for each animal in all the groups. ***, PO.OOl; **, PO.Ol; and *, P<0.05 (Bonferroni test) for tumor control by the armed CARs compared to the unarmed counterparts.

[0105] FIG. 9. In vivo expansion of IL-18-armed bispecific CD19 / CD20 CARs at the high dose (3E6) D1920 (SEQ ID NO: 107), T1920 (SEQ ID NO: 39), D1920-IL18 (SEQ ID NO: 40) and T1920-IL18 (SEQ ID NO: 41). Blood was collected from each animal in all the groups for detection of CD3+ and CD45+ human T cells. Data are plotted with percentages of double positive human T cells for those animals which survived for 20 days post CAR-T cell infusion.

[0106] FIG. 10: Top: Localization of ofatumumab somatic hypermutations on the cryo-EM structure of the CD20-ofatumumab complex. The structure of the protein complex is represented, and the amino acid residues are annotated by using the molecular visualization system PyMOL (version 2.5.4) and the IMGT number scheme, respectively. PDB, Protein Data Bank.

[0107] FIG. 11. Expression and purification of ABDs and their monomers by SEC. Purity and intensity of proteins in each collected fraction were analyzed on SDS-PAGE and graphs were embedded into the SEC graphs. The molecular weight standards for SDS-PAGE are shown on the left of the graphs and the related fraction numbers from SEC are indicated on the top. The molecular weight standards and their elution volumes for SEC are shown in the table. The elution volumes for each peak of the samples are indicated on the top of the peaks in the graphs.

[0108] FIG. 12. Binding of ABDs to K562-CD19, K562-CD20, and K562 cells as measured by flow cytometry. Bound ABDs were detected by 1:500 diluted APC-conjugated anti -FLAG tag antibody.

[0109] FIG. 13. Statistical analysis of ABD binding to K562-CD19, K562-CD20, and K562 cells at concentrations of 62.5, 250, and 1000 nM, respectively. Statistical analysis was performed using two-way ANOVA. ****, PO.OOOl; ***, PO.OOl; **, PO.Ol; ns, not significant; MFI, median fluorescence intensity.DETAILED DESCRIPTION

[0110] Herein are provided inter alia, bispecific CD19 / CD20 binding domains and chimeric antigen receptors (CARs) thereof, as well as corresponding nucleic acid molecules, vectors, cells, compositions, methods and uses, which are particularly useful in the prevention and / or treatment of cancer and / or autoimmune disease. In particular, herein is provided bispecific CD19 / CD20 CARs operably linked to interleukin- 18 (CD19 / CD20-IL18) constructs, CAR T cells comprising the CD19 / CD20-IL18 constructs, and method of using the IL-18 armored CD19 CAR T cells. Such T cells may show improved expansion, persistence, potency, and / or reduced toxicity.Definitions[oni] The following terms are intended to have the meanings presented therewith below and are useful in understanding the description and intended scope of the aspects herein provided.

[0112] When describing aspects herein provided, which may include nucleic acid sequences, nucleic acid molecules, vectors, cells, compositions, pharmaceutical compositions, binding domains, CARs, methodsand uses, etc., the following terms, if present, have the following meanings unless otherwise indicated. It should also be understood that when described herein any of the moieties defined forth below may be substituted with a variety of substituents, and that the respective definitions are intended to include such substituted moieties within their scope as set out below.

[0113] The articles “a” and “an” may be used herein to refer to one or to more than one ( / .e. at least one) of the grammatical objects of the article.

[0114] The term “antibody” means monoclonal antibodies (mAb), including any isotype, such as, IgG, IgM, IgA, IgD and IgE. An IgG antibody is comprised of two identical heavy chains and two identical light chains that are joined by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. Each variable region contains three segments called “complementarity-determining regions” (“CDRs”) or “hypervariable regions”, which are primarily responsible for binding an epitope of an antigen. They are referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portions of the variable regions outside of the CDRs are called the “framework regions.” An “antibody fragment” means an Fv, scFv, dsFv, Fab, a F(ab')2 fragment, a F(ab') fragment, or other fragment, which contains at least one variable heavy or variable light chain, each containing CDRs and framework regions. The CDRs provided herein are defined using the IMGT numbering scheme. (Dondelinger et al. 2018)

[0115] “ VH” refers to the variable region of an immunoglobulin heavy chain of an antibody, or antibody fragment. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3.

[0116] “ VL” refers to the variable region of the immunoglobulin light chain of an antibody, or antibody fragment. The CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3.

[0117] The term “CD 19” refers to the protein known as CD 19 or cluster differentiation 19, having the following synonyms: B4, B-lymphocyte antigen CD19, B-lymphocyte surface antigen B4, CVID3, Differentiation antigen CD19, MGC12802, and T-cell surface antigen Leu-12. Human CD19 is assigned UniProt Accession No. P15391, and the canonical wild-type sequence is 556 amino acids in length and represented by SEQ ID NO: 55.

[0118] The term “CD20” refers to the protein known as CD20 or B-lymphocyte surface antigen Bl , having the following synonyms: MS4A1, Bl, Bp35, CD20, CVID5, LEU-16, MS4A2, S7, membrane spanning 4-domains Al, FMC7. Human CD20 is assigned UniProt Accession No. Pl 1836, and the canonical wild-type sequence is 297 amino acids in length and is represented by SEQ ID NO: 56.

[0119] The term “IL-18” refers to the interleukin 18 cytokine, also known as IGIF, or IL1F4. An example of IL- 18 is interleukin IL- 18 of human origin, such as the protein under UniProtKB-accession number Q14116, or a homologue thereof, such as interleukin IL-18 of non-human mammalian origin, for example rodent.

[0120] The terms “antigen-binding domain”, “binding domain” or “binder” are used interchangeably herein to refer to a protein domain (e.g., portion of an antibody, portion of an antibody fragment, or portion of a CAR) that recognizes an antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody. The binding domain may be a full-length antibodyor an antibody fragment. It may be a full-length antibody, an Fv, single chain antibody fragment (scFv), tandem scFv (ScFv2), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment, a single domain antibody (sdAb), a VHH / nanobody, a nanobody, a diabody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin / nanoantibody, a centyrin, an alphabody, a nanofitin or a D domain. In particular, the term refers to a full-length antibody, an Fv, single chain antibody fragment (scFv), ScFv2), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment. In a particular embodiment, it refers to a tandem scFv, or a diabody.

[0121] The antigen-binding domain may specifically recognize an antigen (monospecific), i.e. it may be a specifically bind the antigen (such as CD20 or CD 19), or bind two antigens (bispecific) such as CD20 and CD19 (herein referred to as CD19 / CD20). For example, “specifically binding” in the context of the present disclosure means binding with an affinity corresponding to a KD of about 10 M or less, such as about 10xM or less, such as about I O9M or less, about 1019M or less, or about 10 " M or even less when determined for instance by surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen- binding domain as the analyte. This term also may mean that the antigen-binding domain binds to the predetermined antigen / epitope with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., bovine serum albumin, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the KD of the antigen-binding domain, so that when the KD of the antigen-binding domain is very low (that is, the antigen-binding domain is very specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000-fold. The term “KD” as used herein, means the dissociation rate constant of a particular antigen-binding domain -antigen interaction.

[0122] The binding domain (e.g., scFv, ScFv2, diabody) may be part of a chimeric antigen receptor (CAR). It may provide the antigen targeting capability (in other words, the capability to bind to the desired antigen) of the CAR. CARs that comprise the CD19 / CD20-binding domains provided herein may be referred to as “CD19 / CD20-targetting CARs” due to their ability to bind CD19 / CD20 via the binder component of the CAR. CARs that comprise the CD19 / CD20-binding domains provided herein may also be referred to as CARs that specifically target both CD 19 and CD20 expressing cells, or CARs that specifically bind to CD 19 and CD20 expressing cells. Methods for determining specific binding are well known in the art, see for example the examples section below.

[0123] The term “diabody” refers to a noncovalent dimer of single-chain Fv (scFv) fragment that consists of the heavy chain variable (VH) and light chain variable (VL) regions connected by a small peptide linker, typically 5 to 8 amino acids. The peptide linker being shorter than in a typical scFv (10 to 25 amino acids), causes the dimerization into an into an amphipathic helix.

[0124] The term “CAR” refers to chimeric antigen receptor, which is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signallingdomain (endodomain). The binder is typically derived from antibody fragments (e.g., scFv, ScFv2, Fab, VHH, scFab), ligands of receptors (e.g., peptides) or from Dectins. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of IgGl. More compact spacers may suffice, e.g., the stalk from CD8a and even just the IgGl hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain. Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FceRl or CD3L Consequently, these first-generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3c results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 4-1BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

[0125] A CAR may have the general structure: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iv) an intracellular domain which comprises or associates with a signalling domain (see FIG. 1).

[0126] The nucleic acid molecules, vectors, binding domains, CARs, cells, and compositions provided herein provided herein may be in isolated form. The term “isolated” means a substance in a form and / or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any substance that does not occur naturally, (2) any substance including, but not limited to, any nucleic acid, protein, peptide, cell, which is at least partially removed of one or more or all of the constituents that occur naturally with which it is associated in nature; (3) any substance modified by man in relation to that substance found in nature; and / or (4) any substance modified by increasing the amount of the substance in relation to other components with which it is naturally associated (e.g., enriched).

[0127] “Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government or the corresponding agency in countries other than the United States, or that is listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly, in humans.

[0128] “Subject” includes humans. The terms “human”, “patient” and “subject” are used interchangeably herein.

[0129] “ Administered” or “administration” includes but is not limited to delivery of a drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route ormucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution, capsule or tablet. Preferably, the administration is by an injectable form.

[0130] “ Effective amount” means the amount of a composition provided herein that, when administered to a subject for treating a disease, is sufficient to effect such treatment for the disease. The “effective amount” can vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0131] The term “prophylaxis” is related to “prevention,” and refers to a measure or procedure the purpose of which is to prevent, rather than to treat or cure a disease. Non-limiting examples of prophylactic measures may include the administration of vaccines; the administration of low molecular weight heparin to hospital patients at risk for thrombosis due, for example, to immobilization; and the administration of an anti- malarial agent such as chloroquine, in advance of a visit to a geographical region where malaria is endemic or the risk of contracting malaria is high.

[0132] “Treating” or “treatment” of any disease or disorder refers, in one embodiment, to ameliorating the disease or disorder (i.e. arresting the disease or reducing the manifestation, extent or severity of at least one of the clinical symptoms thereof). In another embodiment ‘treating’ or ‘treatment’ refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In a further embodiment, “treating” or “treatment” relates to slowing the progression of the disease.

[0133] As used herein, the term “autoimmune disease” refers to diseases that result when the acquired immune system mistakenly attacks the body's own tissues. In particular, the term refers to rheumatoid arthritis (RA), COPD, asthma, bronchitis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis, dermatomyositis, Sjogren’s syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus, atopic dermatitis, thyroiditis, contact dermatitis, eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis, chronic active hepatitis, scleroderma, pemphigus, and Hashimoto's thyroiditis and amyotrophic lateral sclerosis. More particularly, the term refers to systemic lupus erythematosus (SLE).

[0134] As used herein the term “Cancer” refers to diseases caused by an uncontrolled division of abnormal cells in a part of the body. In particular, the term refers to metastatic tumour cell types (such as but not limited to, melanoma, lymphoma, leukaemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). More particularly, the term refers to haematological cancer.

[0135] The term “haematological cancer” includes blood-borne tumours and diseases or disorders involving abnormal cell growth and / or proliferation in tissues of hematopoietic origin, such as lymphomas, leukaemia’s, and myelomas. More particularly, the term refers to Non-Hodgkin’s lymphoma (NHL), acutemyeloid leukaemia (AML), and acute lymphoblastic leukaemia (ALL) and chronic lymphoblastic leukaemia (CLL).

[0136] Non-Hodgkin’s lymphoma (“NHL”) is a heterogeneous malignancy originating from lymphocytes. In the United States, the incidence is estimated at 65,000 / year with mortality of approximately 20,000 (American Cancer Society, 2006; and SEER Cancer Statistics Review). The disease can occur in all ages, the usual onset begins in adults over 40 years, with the incidence increasing with age. NHL is characterized by a clonal proliferation of lymphocytes that accumulate in the lymph nodes, blood, bone marrow and spleen, although any major organ may be involved. The current classification system used by pathologists and clinicians is the World Health Organization (WHO) Classification of Tumours, which organizes NHL into precursor and mature B-cell or T-cell neoplasms. The PDQ is currently dividing NHL as indolent or aggressive for entry into clinical trials. The indolent NHL group is comprised primarily of follicular subtypes, small lymphocytic lymphoma, MALT (mucosa-associated lymphoid tissue), and marginal zone; indolent encompasses approximately 50% of newly diagnosed B-cell NHL patients. Aggressive NHL includes patients with histologic diagnoses of primarily diffuse large B cell lymphoma (DLBL, “DLBCL”, or DLCL) (40% of all newly diagnosed patients have diffuse large cell lymphoma), Burkitt's, and mantle cell lymphoma (“MCL”). The most commonly used agents for combination chemotherapy include cyclophosphamide, vincristine and prednisone (CVP); or cyclophosphamide, adriamycin, vincristine, prednisone (CHOP). Approximately 70% to 80% of patients will respond to their initial chemotherapy, duration of remissions last about 2-3 years. Ultimately, the majority of patients relapse. The discovery and clinical use of the anti-CD20 antibody, rituximab, has provided significant improvements in response and survival rate. The current standard of care for most patients is rituximab + CHOP (R-CHOP) or rituximab + CVP (R-CVP). Rituximab therapy has been shown to be efficacious in several types of NHL and is currently approved as a first line treatment for both indolent (follicular lymphoma) and aggressive NHL (diffuse large B cell lymphoma). However, there are significant limitations of anti-CD20 monoclonal antibody (mAb), including primary resistance (50% response in relapsed indolent patients), acquired resistance (50% response rate upon re-treatment), rare complete response (2% complete response rate in relapsed population), and a continued pattern of relapse.

[0137] Chronic lymphocytic leukaemia (also known as "chronic lymphoid leukaemia" or "CLL"), is a type of adult leukaemia caused by an abnormal accumulation of B lymphocytes. In CLL, the malignant lymphocytes may look normal and mature, but they are not able to cope effectively with infection. CLL is the most common form of leukaemia in adults. Men are twice as likely to develop CLL as women. However, the key risk factor is age. Over 75% of new cases are diagnosed in patients over age 50. More than 10,000 cases are diagnosed every year and the mortality is almost 5,000 a year (American Cancer Society, 2006; and SEER Cancer Statistics Review). CLL is an incurable disease but progresses slowly in most cases. Many people with CLL lead normal and active lives for many years. Because of its slow onset, early-stage CLL is generally not treated since it is believed that early CLL intervention does not improve survival time or quality of life. Instead, the condition is monitored over time. Initial CLL treatments vary depending on the exact diagnosis and the progression of the disease. There are dozens of agents used for CLL therapy.Combination chemotherapy regimens such as FCR (fludarabine, cyclophosphamide and rituximab), and BR (Ibrutinib and rituximab) are effective in both newly -diagnosed and relapsed CLL. Allogeneic bone marrow (stem cell) transplantation is rarely used as a first-line treatment for CLL due to its risk.

[0138] Another type of leukaemia is Small lymphocytic lymphoma (“SLL”) that is considered a CLL variant that lacks the clonal lymphocytosis required for the CLL diagnosis, but otherwise shares pathological and immunophenotypic features (Campo et al. 2011). The definition of SLL requires the presence of lymphadenopathy and / or splenomegaly. Moreover, the number of B lymphocytes in the peripheral blood should not exceed 5E+09 / L. The incidence of SLL is approximately 25% of CLL in the US (Dores et al. 2007).

[0139] Another type of leukaemia is acute lymphoblastic leukaemia (ALL), also known as acute lymphocytic leukaemia. ALL is characterized by the overproduction and continuous multiplication of malignant and immature white blood cells (also known as lymphoblasts) in the bone marrow. 'Acute' refers to the undifferentiated, immature state of the circulating lymphocytes ("blasts"), and that the disease progresses rapidly with life expectancy of weeks to months if left untreated.

[0140] The terms “combination” or "pharmaceutical combination" refer to the administration of one therapy in addition to another therapy. As such, "in combination with" includes simultaneous (e.g., concurrent) and consecutive administration in any order.CD19-binding domains

[0141] In some aspects, the disclosure provides a CD19-binding domain comprising a heavy chain variable (CD19 VH) domain comprising the following complementarity determining regions (CDRs): CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4), CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), and / orCD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6).

[0142] In some aspects, a CD19-binding domain comprises a heavy chain variable (CD19 VH) domain comprising the following complementarity determining regions (CDRs):CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), andCD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6).

[0143] In some aspects, a CD19-binding domain comprises a light chain variable region (CD19 VL) having CDRs with the following sequences:CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),CD 19 LCDR2 - HTS (SEQ ID NO: 2), and / orCD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3).

[0144] In some aspects, a CD19-binding domain comprises a light chain variable region (CD19 VL) having CDRs with the following sequences:CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),CD 19 LCDR2 - HTS (SEQ ID NO: 2), andCD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3).

[0145] In some aspects, a CD19-binding domain comprises: a) a heavy chain variable (CD19 VH) domain comprising the following CDRs:- CD19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), and / or- CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and b) a light chain variable (CD 19 VL) domain comprising the following CDRs:- CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- CD19 LCDR2 - HTS (SEQ ID NO: 2), and / or- CD19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3).

[0146] In some aspects, a CD19-binding domain comprises: a) a heavy chain variable (CD19 VH) domain comprising the following CDRs:- CD19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5),- CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and b) a light chain variable (CD 19 VL) domain comprising the following CDRs:- CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- CD19 LCDR2 - HTS (SEQ ID NO: 2), and- CD19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3).

[0147] In some aspects, one or more mutations (substitutions, additions or deletions) may be introduced into one or more of the CDRs in the CD19-binding domains provided herein without negatively affecting CD19-binding activity of the CD19-binding domain. In one embodiment, one or more CDR within the CD19-binding domain may have one, two or three amino acid mutations. In a particular embodiment, one or more CDR within the CD19-binding domain may have one or two amino acid mutations only. In a more particular embodiment, one or more CDR within the CD19-binding domain may have one amino acid mutation only. In a most particular embodiment, each CDR has no mutation.

[0148] In some aspects, a CD19-binding domain comprising a heavy chain variable (CD19 VH) domain comprises the following CDRs: a CD19 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 4; a CD19 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 5; and a CD19 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 6.

[0149] In some aspects, a CD19-binding domain comprising a light chain variable (CD 19 VL) domain comprises the following CDRs: a CD19 LCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 1; a CD19 LCDR2 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 2; and a CD19 LCDR3 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 3.

[0150] In some aspects, a CD19-binding domain comprises any combination of the CD 19 VH and VL domains described herein.

[0151] In some aspects, a CD19-binding domain comprising a heavy chain variable (CD19 VH) domain comprises the following CDRs: a CD19 HCDR1 having a sequence with at least 80% sequence identity to GVSLPDYG (SEQ ID NO: 4); a CD19 HCDR2 having a sequence with at least 94% sequence identity to IWGSETT (SEQ ID NO: 5); and a CD19 HCDR3 having a sequence with at least 88% sequence identity to AKHYYYGGSYAMDY (SEQ ID NO: 6).

[0152] In some aspects, a CD19-binding domain comprises a light chain variable region (CD19 VL) having CDRs with the following sequences: a CD19 LCDR1 having a sequence with at least 92% sequence identity to QDISKY (SEQ ID NO: 1); a CD 19 LCDR2 having a sequence with at least 85% sequence identity to HTS (SEQ ID NO: 2); and a CD 19 LCDR3 having a sequence with at least 90% sequence identity to QQGNTLPYT (SEQ ID NO: 3).

[0153] In some aspects, a CD19-binding domain comprises: a) a heavy chain variable (CD 19 VH) domain comprising the following complementarity determining regions (CDRs): a CD19 HCDR1 having a sequence with at least 80% sequence identity to GVSLPDYG (SEQ ID NO: 4); a CD19 HCDR2 having a sequence with at least 94% sequence identity to IWGSETT (SEQ ID NO: 5); and a CD19 HCDR3 having a sequence with at least 88% sequence identity to AKHYYYGGSYAMDY (SEQ ID NO: 6); and b) a light chain variable (CD 19 VL) domain comprising the following CDRs: a CD19 LCDR1 having a sequence with at least 92% sequence identity to QDISKY (SEQ ID NO: 1); a CD 19 LCDR2 having a sequence with at least 85% sequence identity to HTS (SEQ ID NO: 2); anda CD 19 LCDR3 having a sequence with at least 90% sequence identity to QQGNTLPYT (SEQ ID NO: 3).

[0154] The CDRs may be in the format of any appropriate CD19-binding domain provided herein (e.g., the CD19 binding domain may be a full-length antibody, or an antibody fragment comprising the recited CDRs It may be a full-length antibody, an Fv, single chain antibody fragment (scFv), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment, a single domain antibody (sdAb), a CD 19 VHH / nanobody, a nanobody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin / nanoantibody, a centyrin, an alphabody, a nanofitin or a D domain. In particular, the term refers to a full-length antibody, an Fv, single chain antibody fragment (scFv), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment.

[0155] The binding domain may be non-human, chimeric, humanized or fully human.

[0156] The CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (CD19 VH) and light chain variable region (CD19 VL) of an antibody, typically connected with a short linker peptide of about 10 to about 25 amino acids. The scFv may be in the orientation CD19 VH-CD19 VL, i.e., the CD19 VH is N-terminal to the CD19 VL. Accordingly, the CDRs may be grafted into the framework of a scFv.

[0157] The CD 19 binding domain provided herein may comprise a variant of the sequence shown as SEQ ID NO: 7, 8, or 9, wherein the variant has at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 7, 8, or 9, provided that the variant sequence also retains the capacity to bind CD 19 (when in conjunction with a complementary CD 19 VL or CD 19 VH domain, if appropriate). The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0158] “Retaining capacity to bind CD 19” refers to the ability of a variant of a CD19-binding domain herein provided (e.g., comprising at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 7, 8 or 9 and / or comprising one or more mutations introduced into one or more of the CDRs in the CD 19- binding domains provided herein) to bind to a CD19 molecule. For example, a variant CD19-binding domain may have the same or similar binding properties as a non- variant CD19-binding domain herein provided (e.g., the binder of SEQ ID NO: 9). For example, a variant CD19-binding domain may bind with an affinity the same as or similar to a non-variant CD19-binding domain herein provided (e.g., the binder of SEQ ID NO: 9). For example, a variant CD19-binding domain may have a Ka and / or Kd the same as or similar to a non-variant CD19-binding domain herein provided (e.g., the binder of SEQ ID NO: 9).

[0159] In a particular embodiment, the CD 19 VH domain of the CD19-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 8. In one example, the CD19 VH domain of the CD19-binding domain provided herein comprises the sequence of SEQ ID NO: 8.

[0160] In a particular embodiment, the CD 19 VL domain of the CD19-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least98, at least 99, 100%) to the sequence of SEQ ID NO: 7. In one example, the CD 19 VL domain of the CD19-binding domain provided herein comprises the sequence of SEQ ID NO: 7.

[0161] In a particular embodiment, the CD 19 VH domain of the CD19-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 8 and the CD 19 VL domain of the CD19-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 7.

[0162] In one embodiment, the CD19 VL domain of the CD19-binding domain provided herein comprises the sequence of SEQ ID NO: 7 and the CD19 VH domain comprises the sequence of SEQ ID NO: 8.

[0163] In one embodiment, the CD19-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 9. In one example, the CD19-binding domain provided herein comprises the sequence of SEQ ID NO: 9.

[0164] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of the antigen-binding domains (e.g., antibodies or antibody fragments provided herein). “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[0165] The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.

[0166] Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0167] Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.CD20-binding domains

[0168] In some aspects, a CD20-binding domain comprises a heavy chain variable (CD20 VH) domain comprising the following CDRs:- CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and / or- CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0169] In some aspects, a CD20-binding domain comprises a heavy chain variable (CD20 VH) domain comprising the following CD Rs:- CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and- CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0170] In some aspects, a CD20-binding domain comprises a light chain variable region (CD20 VL) having CDRs with the following sequences:- CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- CD20 LCDR2 - DAS (SEQ ID NO: 11), and / or- CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0171] In some aspects, a CD20-binding domain comprises a light chain variable region (CD20 VL) having CDRs with the following sequences:- CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- CD20 LCDR2 - DAS (SEQ ID NO: 11), and- CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0172] In some aspects, a CD20-binding domain comprises: a) a heavy chain variable (CD20 VH) domain comprising the following CDRs:- CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and / or- CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15); and b) a light chain variable (CD20 VL) domain comprising the following CDRs:- CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- CD20 LCDR2 - DAS (SEQ ID NO: 11), and / or- CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0173] In some aspects, a CD20-binding domain comprises: a) a heavy chain variable (CD20 VH) domain comprising the following CDRs:- CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14),- CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15); and b) a light chain variable (CD20 VL) domain comprising the following CDRs:- CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- CD20 LCDR2 - DAS (SEQ ID NO: 11),- CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0174] In some aspects, one or more mutations (substitutions, additions or deletions) may be introduced into one or more of the CDRs in the CD20-binding domains provided herein without negatively affecting CD20-binding activity of the CD20-binding domain. In one embodiment, one or more CDR within the CD20-binding domain may have one, two or three amino acid mutations. In a particular embodiment, oneor more CDR within the CD20-binding domain may have one or two amino acid mutations only. In a more particular embodiment, one or more CDR within the CD20-binding domain may have one amino acid mutation only. In a most particular embodiment, each CDR has no mutation.

[0175] In some aspects, a CD20-binding domain comprising a heavy chain variable (CD20 VH) domain comprises the following CDRs: a CD20 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 13; a CD20 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 14; and a CD20 HCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 15.

[0176] In some aspects, a CD20-binding domain comprising a light chain variable (CD20 VL) domain comprises the following CDRs: a CD20 LCDR1 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 10; a CD20 LCDR2 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 11; and a CD20 LCDR3 having a sequence with at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 12.

[0177] In some aspects, a CD20-binding domain comprises any combination of the CD20 VH and VL domains described herein.

[0178] In some aspects, a CD20-binding domain comprises a heavy chain variable (CD20 VH) domain comprising the following CDRs: a CD20 HCDR1 having a sequence with at least 80% sequence identity to GFTFDDYA (SEQ ID NO:13); a CD20 HCDR2 having a sequence with at least 94% sequence identity to ISWNSGSI (SEQ ID NO:14); and a CD20 HCDR3 having a sequence with at least 88% sequence identity to AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0179] In some aspects, a CD20-binding domain comprises a light chain variable region (CD20 VL) having CDRs with the following sequences: a CD20 LCDR1 having a sequence with at least 92% sequence identity to QSVSSY (SEQ ID NO: 10); a CD20 LCDR2 having a sequence with at least 85% sequence identity to DAS (SEQ ID NO: 11); and a CD20 LCDR3 having a sequence with at least 90% sequence identity to QQRSNWPIT (SEQ ID NO: 12).

[0180] In some aspects, a CD20-binding domain comprises: a) a heavy chain variable (CD20 VH) domain comprising the following CD Rs: a CD20 HCDR1 having a sequence with at least 80% sequence identity to GFTFDDYA (SEQ ID NO:13); a CD20 HCDR2 having a sequence with at least 94% sequence identity to ISWNSGSI (SEQ ID NO:14); and a CD20 HCDR3 having a sequence with at least 88% sequence identity to AKDIQYGNYYYGMDV (SEQ ID NO: 15); and b) a light chain variable (CD20 VL) domain comprising the following CDRs: a CD20 LCDR1 having a sequence with at least 92% sequence identity to QSVSSY (SEQ ID NO: 10); a CD20 LCDR2 having a sequence with at least 85% sequence identity to DAS (SEQ ID NO: 11); and a CD20 LCDR3 having a sequence with at least 90% sequence identity to QQRSNWPIT (SEQ ID NO: 12).

[0181] The CDRs may be in the format of any appropriate CD20-binding domain provided herein (e.g., the CD20 binding domain may be a full-length antibody, or an antibody fragment comprising the recited CDRs It may be a full-length antibody, an Fv, single chain antibody fragment (scFv), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment, a single domain antibody (sdAb), a CD20 VHH / nanobody, a nanobody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin / nanoantibody, a centyrin, an alphabody, a nanofitin or a D domain. In particular, the term refers to a full-length antibody, an Fv, single chain antibody fragment (scFv), dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment.

[0182] The binding domain may be non-human, chimeric, humanized or fully human.

[0183] The CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (CD20 VH) and light chain variable region (CD20 VL) of an antibody, typically connected with a short linker peptide of about 10 to about 25 amino acids. The scFv may be in the orientation CD20 VH-CD20 VL, i.e. the CD20 VH is N-terminal to the CD20 VL. Accordingly, the CDRs may be grafted into the framework of a scFv.

[0184] The CD20 binding domain provided herein may comprise a variant of the sequence shown as SEQ ID NO: 16, 17, or 18, wherein the variant has at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 16, 17, or 18, provided that the variant sequence also retains the capacity to bind CD20 (when in conjunction with a complementary CD20 VL or CD20 VH domain, if appropriate). The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0185] “Retaining capacity to bind CD20” refers to the ability of a variant of a CD20-binding domain herein provided (e.g., comprising at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 18 and / or comprising one or more mutations introduced into one or more of the CDRs in the CD20-binding domains provided herein) to bind to a CD20 molecule. For example, a variant CD20-binding domain may have the same or similar binding properties as a non-variant CD20-binding domain herein provided(e.g., the binder of SEQ ID NO: 18). For example, a variant CD20-binding domain may bind with an affinity the same as or similar to a non-variant CD20-binding domain herein provided (e.g., the binder of SEQ ID NO: 18). For example, a variant CD20-binding domain may have a Ka and / or Kd the same as or similar to a non-variant CD20-binding domain herein provided (e.g., the binder of SEQ ID NO: 18).

[0186] In some aspects, the CD20 VH domain of the CD20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 17. In one example, the CD20 VH domain of the CD20-binding domain provided herein comprises the sequence of SEQ ID NO: 17.

[0187] In some aspects, the CD20 VL domain of the CD20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 16. In one example, the CD20 VL domain of the CD20-binding domain provided herein comprises the sequence of SEQ ID NO: 16.

[0188] In some aspects, the CD20 VH domain of the CD20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 17 and the CD20 VL domain of the CD20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 16.

[0189] In some aspects, the CD20 VH domain of the CD20-binding domain provided herein comprises the sequence of SEQ ID NO: 17 and the CD20 VL domain comprises the sequence of SEQ ID NO: 16.

[0190] In some aspects, the CD20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 18. In one example, the CD20-binding domain provided herein comprises the sequence of SEQ ID NO: 18.

[0191] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of the antigen-binding domains (e.g., antibodies or antibody fragments provided herein). “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[0192] The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.

[0193] Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformationalepitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0194] Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.Bispecific CD19 / CD20-binding domains

[0195] In some aspects, the disclosure provides a CD19 / CD20-binding domain comprising(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs:- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), and / or- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and / or(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following complementarity determining regions (CDRs):- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and / or- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0196] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs:- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), and- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and / or(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following complementarity determining regions (CDRs):- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0197] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following complementarity determining regions (CDRs):- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD19 HCDR2 - IWGSETT (SEQ ID NO: 5),- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following CDRs:- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0198] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following complementarity determining regions (CDRs): a CD 19 LCDR1 having a sequence with at least 92% sequence identity to QDISKY (SEQ ID NO: 1), a CD 19 LCDR2 having a sequence with at least 85% sequence identity to HTS (SEQ ID NO: 2), and / or a CD 19 LCDR3 having a sequence with at least 90% sequence identity to QQGNTLPYT (SEQ ID NO: 3). and / or(b) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs: a CD20 LCDR1 having a sequence with at least 92% sequence identity to QSVSSY (SEQ ID NO: 10), a CD20 LCDR2 having a sequence with at least 85% sequence identity to DAS (SEQ ID NO: 11), and / or a CD20 LCDR3 having a sequence with at least 90% sequence identity to QQRSNWPIT (SEQ ID NO: 12).

[0199] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD19 LCDR2 - HTS (SEQ ID NO: 2), and- a CD19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3). and / or(b) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11), and- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0200] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD19 LCDR2 - HTS (SEQ ID NO: 2),- a CD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3), and(b) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11), and- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0201] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs:- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), and / or- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and / or(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following CDRs:- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), and / or- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15), and / or(c) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD 19 LCDR2 - HTS (SEQ ID NO: 2), and / or- a CD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3), and / or(d) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11), and / or- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0202] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs:- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD19 HCDR2 - IWGSETT (SEQ ID NO: 5),- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6), and / or(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following CDRs:- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14),- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15), and / or(c) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD19 LCDR2 - HTS (SEQ ID NO: 2),- a CD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3), and / or(d) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11),- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs:- a CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),- a CD19 HCDR2 - IWGSETT (SEQ ID NO: 5),- a CD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6),(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following CDRs:- a CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),- a CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14),- a CD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15),(c) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD19 LCDR2 - HTS (SEQ ID NO: 2),- a CD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3), and(d) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11),- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0203] In some aspects, one or more mutations (substitutions, additions or deletions) may be introduced into one or more of the CDRs in the CD19 / CD20-binding domains provided herein without negatively affecting CD19 / 20-binding activity of the CD19 / 20-binding domain. In one embodiment, one or more CDR within the CD19 / 20-binding domain may have one, two or three amino acid mutations. In a particular embodiment, one or more CDR within the CD19 / 20-binding domain may have one or two amino acid mutations only. In a more particular embodiment, one or more CDR within the CD19 / 20-binding domain may have one amino acid mutation only. In a most particular embodiment, each CDR has no mutation.

[0204] In some aspects, a CD19 / CD20-binding domain comprises:(a) a CD 19 binding heavy chain variable (CD 19 VH) domain comprising the following CDRs: a CD 19 HCDR1 having a sequence with at least 80% sequence identity to GVSLPDYG (SEQ ID NO: 4), a CD19 HCDR2 having a sequence with at least 94% sequence identity to IWGSETT (SEQ ID NO: 5), and / or a CD19 HCDR3 having a sequence with at least 88% sequence identity to AKHYYYGGSYAMDY (SEQ ID NO: 6), and / or(b) a CD20 heavy chain variable (CD20 VH) domain comprising the following CDRs: a CD20 HCDR1 having a sequence with at least 80% sequence identity to GFTFDDYA (SEQ ID NO:13), a CD20 HCDR2 having a sequence with at least 94% sequence identity to ISWNSGSI (SEQ ID NO:14), and / or a CD20 HCDR3 having a sequence with at least 88% sequence identity to AKDIQYGNYYYGMDV (SEQ ID NO: 15).

[0205] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding light chain variable (CD 19 VL) domain comprising the following CDRs:- a CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),- a CD 19 LCDR2 - HTS (SEQ ID NO: 2), and / or- a CD19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3). and / or(b) a CD20 binding light chain variable (CD20 VL) domain comprising the following CDRs:- a CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),- a CD20 LCDR2 - DAS (SEQ ID NO: 11), and / or- a CD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

[0206] In some aspects, a CD19 / 20-binding domain comprises a light chain variable region (VL) having CDRs with the following sequences: a LCDR1 having a sequence with at least 92% sequence identity to QSVSSY (SEQ ID NO: 10); a LCDR2 having a sequence with at least 85% sequence identity to DAS (SEQ ID NO: 11); and a LCDR3 having a sequence with at least 90% sequence identity to QQRSNWPIT (SEQ ID NO: 12).

[0207] In some aspects, a CD19 / 20-binding domain comprises: a) a heavy chain variable (VH) domain comprising the following CDRs: an HCDR1 having a sequence with at least 80% sequence identity to GFTFDDYA (SEQ ID NO: 13); an HCDR2 having a sequence with at least 94% sequence identity to ISWNSGSI (SEQ ID NO: 14); and an HCDR3 having a sequence with at least 88% sequence identity to AKDIQYGNYYYGMDV (SEQ ID NO: 15); and b) a light chain variable (VL) domain comprising the following CDRs: a LCDR1 having a sequence with at least 92% sequence identity to QSVSSY (SEQ ID NO: 10); a LCDR2 having a sequence with at least 85% sequence identity to DAS (SEQ ID NO: 11); and a LCDR3 having a sequence with at least 90% sequence identity to QQRSNWPIT (SEQ ID NO: 12).

[0208] The CDRs may be in the format of any appropriate CD19 / 20-binding domain provided herein (e.g., the CD 19 / 20 binding domain may be a full-length antibody, or an antibody fragment comprising the recited CDRs It may be a full-length antibody, an Fv, single chain antibody fragment (scFv), scFv2, dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment, a single domain antibody (sdAb), a VHH / nanobody, a nanobody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin / nanoantibody, a centyrin, an alphabody, a nanofitin or a D domain. In particular, the term refers to a full-length antibody, an Fv, single chain antibody fragment (scFv), scFv2, dsFv, a F(ab) fragment, a F(ab')2 fragment, a F(ab') fragment.

[0209] The binding domain may be non-human, chimeric, humanized or fully human.

[0210] The CDRs may be in the format of a single-chain variable fragment (scFv), which is a fusion protein of the heavy variable region (VH) and light chain variable region (VL) of an antibody, typically connected with a short linker peptide of about 10 to about 25 amino acids. The scFv may be in the orientation VH-VL, i.e. the VH is N-terminal to the VL. Accordingly, the CDRs may be grafted into the framework of a scFv.

[0211] The CDRs may be in the format of a diabody of a single -chain variable fragment (scFv), which is a fusion protein of the heavy variable region (VH) and light chain variable region (VL) of an antibody, typically connected with a short linker peptide of 5 to 8 amino acids. The scFv may be in the orientation VH-VL, i.e. the VH is N-terminal to the VL. Accordingly, the CDRs may be grafted into the framework of a scFv.

[0212] In some aspects, a CD19 / CD20-binding domain comprises:(a) a CD 19 binding heavy chain variable (CD 19 VH) domain having an amino acid sequence according to SEQ ID NO: 8, and / or(b) a CD20 heavy chain variable (CD20 VH) domain having an amino acid sequence according to SEQ ID NO: 17, and / or(c) a CD 19 binding light chain variable (CD 19 VL) domain having an amino acid sequence according to SEQ ID NO: 7, and / or(d) a CD20 binding light chain variable (CD20 VL) domain having an amino acid sequence according to SEQ ID NO: 16.

[0213] In some aspects, a CD19 / CD20-binding domain comprises(a) a CD 19 binding heavy chain variable (CD 19 VH) domain having an amino acid sequence according to SEQ ID NO: 8,(b) a CD20 heavy chain variable (CD20 VH) domain having an amino acid sequence according to SEQ ID NO: 17,(c) a CD 19 binding light chain variable (CD 19 VL) domain having an amino acid sequence according to SEQ ID NO: 7, and(d) a CD20 binding light chain variable (CD20 VL) domain having an amino acid sequence according to SEQ ID NO: 16.

[0214] The CD 19 / 20 binding domain provided herein may comprise a variant of the sequence shown as SEQ ID NO: 19 or 20, wherein the variant has at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 19 or 20, provided that the variant sequence also retains the capacity to bind CD19 / 20 (when in conjunction with a complementary VL or VH domain, if appropriate). The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http: / / blast.ncbi.nlm.nih.gov.

[0215] “Retaining capacity to bind CD19 / 20” refers to the ability of a variant of a CD19 / 20-binding domain herein provided (e.g., comprising at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID NO: 19 or 20 and / or comprising one or more mutations introduced into one or more of the CDRs in the CD19 / 20-binding domains provided herein) to bind to a CD19 / 20 molecule. For example, a variant CD19 / 20-binding domain may have the same or similar binding properties as a non-variant CD 19 / 20- binding domain herein provided (e.g., the binder of SEQ ID NO: 19 or 20). For example, a variant CD19 / 20- binding domain may bind with an affinity the same as or similar to a non-variant CD19 / 20-binding domain herein provided (e.g., the binder of SEQ ID NO: 19 or 20). For example, a variant CD 19 / 20-binding domain may have a Ka and / or Kd the same as or similar to a non-variant CD 19 / 20-binding domain herein provided (e.g., the binder of SEQ ID NO: 19 or 20).

[0216] In some aspects, the CD 19 / 20-binding domain provided herein comprises a CD19 VH domain having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 8, and / or a CD20 VH domain a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 17. In another example, the CD 19 / 20-binding domain provided herein comprises a CD19 VH domain having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 8 and a CD20 VH domain a sequence having at least 95% sequence identity(i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 17. In another example, the CD19 / 20-binding domain provided herein comprises a CD19 VH domain having the sequence of SEQ ID NO: 8 and a CD20 VH domain having the sequence of SEQ ID NO: 17.

[0217] In some aspects, the CD19 / 20-binding domain provided herein comprises a CD 19 VL domain having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 7, and / or a CD20 VL domain a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO:16. In another example, the CD19 / 20-binding domain provided herein comprises a CD19 VL domain having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 7 and a CD20 VL domain a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 16. In another example, the CD19 / 20-binding domain provided herein comprises a CD19 VL domain having the sequence of SEQ ID NO: 7 and a CD20 VL domain having the sequence of SEQ ID NO: 16.

[0218] In some aspects, the CD19 / 20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 19. In one example, the CD19 / 20-binding domain provided herein comprises the sequence of SEQ ID NO: 19.

[0219] In some aspects, the CD19 / 20-binding domain provided herein comprises a sequence having at least 95% sequence identity (i.e. at least 95, at least 96, at least 97, at least 98, at least 99, 100%) to the sequence of SEQ ID NO: 20. In one example, the CD19 / 20-binding domain provided herein comprises the sequence of SEQ ID NO: 20.

[0220] The CDRs contribute to the formation of the antigen-binding, or more specifically, epitope binding site of the antigen-binding domains (e.g., antibodies or antibody fragments provided herein). “Epitope” refers to a determinant that interacts with a specific antigen binding site in the variable region of an antibody molecule known as a paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characteristics, as well as specific charge characteristics. A single antigen may have more than one epitope.

[0221] The epitope may comprise amino acid residues directly involved in the binding (also called immunodominant component of the epitope) and other amino acid residues, which are not directly involved in the binding, such as amino acid residues which are effectively blocked by the specifically antigen binding peptide; in other words, the amino acid residue is within the footprint of the specifically antigen binding peptide.

[0222] Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and non-conformational epitopes may be distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0223] Antibodies that recognize the same epitope can be verified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.Chimeric Antigen Receptors (CARs)

[0224] Chimeric antigen receptors (CARs), also known as chimeric T cell receptors, artificial T cell receptors and chimeric immunoreceptors, are engineered receptors, which graft a desired specificity onto an immune effector cell. In a classical CAR, the specificity of an antibody or antibody fragment is grafted onto a T cell. CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. In this way, many cancer-specific T cells can be generated for adoptive cell transfer. Phase I clinical studies of this approach show efficacy.

[0225] The target antigen-binding domain of a CAR is commonly fused via a spacer and transmembrane domain to an endodomain. The endodomain may comprise or associate with an intracellular T -cell signaling domain. When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.

[0226] Examples of CARs and variants thereof are provided herein. Typically, A CAR comprises an antigen-binding domain, a transmembrane domain and an endodomain. Target antigen-binding domains provided herein are discussed in detail above (see “CD20-binding domains”, “CD19-binding domains”, and “CD19 / CD20-binding domains”). Details of other component parts of a CAR provided herein are described below.

[0227] Suitably, the CAR may comprise a sequence having at least 80% sequence identity to a sequence shown in any one of SEQ ID NO: 9, 18, l- 9, 38, 39, 40, or 41, wherein the CAR retains its capacity to i) bind CD19 and / or CD20 and ii) induce T-cell signaling. “Retaining capacity to bind CD19 / CD20” is defined elsewhere herein. “Retaining capacity induce T cell signaling” refers to the ability of a variant of a CAR provided herein (e.g., comprising at least 80, 85, 90, 95, 98 or 99% sequence identity to any one of SEQ ID NO: 9, 18, 27-29, 38, 39, 40, or 41 and / or comprising one or more mutations introduced into one or more of the CDRs in the CD20-binding domains provided herein) to recognize antigens presented on antigen presenting cells, and lead to a cascade of signaling events resulting in T-cell effector function at the same concentration and / or in the same timescale as a non-variant CAR provided herein. For example, a similar or same amount of a variant CAR may induce the same or similar level of T-cell signaling as a nonvariant CAR provided herein. Suitable variant component parts for the CARs provided herein are described in more detail below.Transmembrane Domains

[0228] In one embodiment, there is provided a CAR comprising a transmembrane domain which spans the membrane. In a particular embodiment, the transmembrane comprises a hydrophobic alpha helix. In a more particular embodiment, the transmembrane domain is derived from CD28, or CD8. In a more particular embodiment, the transmembrane domain is the transmembrane domain of human CD28, or human CD8. In a yet further more particular embodiment, the transmembrane domain is derived from CD8.In a yet further more particular embodiment, the transmembrane domain is the transmembrane domain of human CD8. In a most particular embodiment, the transmembrane domain is according to SEQ ID NO: 25.Endodomains

[0229] The endodomain is the signal-transmission portion of the CAR. After antigen recognition, receptors cluster and a signal is transmitted to the cell. The most commonly used endodomain component is that of CD3c which contains 3 IT AMs. This transmits an activation signal to the T cell after an antigen is bound. C D3c may not provide a fully competent activation signal and additional co-stimulatory signaling may be needed. For example, endodomains from CD28, or 0X40 or 4-1BB can be used with CD3c to transmit a proliferative / survival signal, or all three can be used together.

[0230] Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FceRl or CD3L Consequently, these first generation receptors transmitted immunological signal 1, which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains were constructed. Fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3c resulted in second generation receptors which could transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used was that of CD28. This supplies the most potent co-stimulatory signal, namely immunological signal 2, which triggers T-cell proliferation. Some receptors were also described which included TNF receptor family endodomains such as 0X40 and 4- IBB which transmit survival signals. Finally, even more potent third generation CARs were described which had endodomains capable of transmitting activation, proliferation and survival signals.

[0231] In one embodiment, there is provided a CAR comprising combinations of one or more of the C D3c endodomain, the 4- IBB endodomain, the 0X40 endodomain and the CD28 endodomain. In a particular embodiment, the endodomain is a fusion of 4-1BB and CD3^, CD28 and CD3^, or CD28, 4-1BB and CD3^. In a more particular embodiment, the endodomain is a fusion of 4-1BB and CD3L In a most particular embodiment, the endodomain is a fusion of 4-1BB and CD3c wherein the 4-1BB is according to SEQ ID NO: 29 or a variant thereof having at least 80% identity, and the CD3c is according to SEQ ID NO: 30 or a variant thereof having at least 80% identity. In a most particular embodiment, the endodomain is a fusion of 4-1BB and CD3c wherein the 4-1BB is according to SEQ ID NO: 29, and the CD3c is according to SEQ ID NO: 30. In another most particular embodiment, the endodomain is a fusion of 4- IBB and CD3c wherein the 4- IBB is according to SEQ ID NO: 29 or a variant thereof having at least 80% identity, and the CD3c is according to SEQ ID NO: 31 or a variant thereof having at least 80% identity. In a most particular embodiment, the endodomain is a fusion of 4-1BB and CD3c wherein the 4-1BB is according to SEQ ID NO: 29, and the CD3c is according to SEQ ID NO: 31. In yet another most particular embodiment, the endodomain is a fusion of 4-1BB and CD3c wherein the 4-1BB is according to SEQ ID NO: 29 or a variant thereof having at least 80% identity, and the CD3c is according to SEQ ID NO: 97 or a variant thereof having at least 80% identity. In a most particular embodiment, the endodomain is a fusion of 4-1BB and CD3c wherein the 4-1BB is according to SEQ ID NO: 29, and the CD3^ is according to SEQ ID NO: 97.

[0232] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 36 provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.

[0233] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 37 provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.

[0234] A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 85 provided that the sequence provides an effective transmembrane domain / intracellular T cell signaling domain.Signal Peptides or Leader Peptides

[0235] The CAR herein provided may comprise a signal peptide (or leader peptide) so that when the CAR is expressed inside a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. The signal peptide may be at the amino terminus of the molecule.

[0236] In one embodiment, the CAR herein provided may have the general (N- terminal to C-terminal) configuration of: Signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain. In a particular embodiment, the CAR herein provided may have the general (N- terminal to C-terminal) configuration of: CD8a signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain.

[0237] In a further embodiment, the CAR herein provided may have the general (N- terminal to C- terminal) configuration of: Signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain - self cleaving peptide -signal peptide - IL 18 domain. In a particular embodiment, the CAR herein provided may have the general (N- terminal to C-terminal) configuration of: CD8a signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain - self cleaving peptide - CD59 signal peptide - IL 18 domain. In a more particular embodiment, the CAR herein provided may have the general (N- terminal to C- terminal) configuration of: CD8a signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain - P2A cleaving peptide - CD59 signal peptide- IL18 domain. In a more particular embodiment, the CAR herein provided may have the general (N- terminal to C-terminal) configuration of: CD8a signal peptide - CD20 / CD19 binding domain - spacer domain - transmembrane domain / intracellular T cell signaling domain - furin-P2A-furin cleaving peptide- CD59 signal peptide - IL18 domain.

[0238] In one embodiment, there is provided a CAR comprising a CD8a signal peptide according to the SEQ ID NO: 34 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions, or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR. In a particular embodiment, the CD8a signal peptide according to SEQ ID NO: 34. Methods for determining cell surface expression of a CAR are well known in the art and can be used to determine suitable signal peptide variants.

[0239] In one embodiment, there is provided a CAR comprising a CD59 signal peptide according to the SEQ ID NO: 35 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions, or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR. In a particular embodiment, the CD59 signal peptide according to SEQ ID NO: 35. Methods for determining cell surface expression of a CAR are well known in the art and can be used to determine suitable signal peptide variants.Spacers

[0240] The CAR herein provided may comprise a spacer sequence to connect the binding domain with the transmembrane domain and spatially separate the binding domain from the endodomain. A flexible spacer allows to the binding domain to orient in different directions to enable binding to the antigen(s).

[0241] The spacer sequence may, for example, comprise an IgGl Fc region, an IgGl hinge or a CD8 stalk, or a combination thereof. The spacer may alternatively comprise an alternative sequence which has similar length and / or domain spacing properties as an IgGl Fc region, an IgGl hinge or a CD8 stalk. A human IgGl spacer may be altered to remove Fc binding motifs.

[0242] In one embodiment, there is provided a CAR comprising a spacer or hinge. In a particular embodiment, the hinge is a CD8 stalk. In a more particular embodiment, the hinge is according to SEQ ID NO: 27 or a variant thereof having at least 80% identity. In a most particular embodiment, the hinge is according to SEQ ID NO: 27.Full constructs

[0243] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, b) A transmembrane domain, and c) An endodomain.

[0244] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, b) A hinge domain, c) A transmembrane domain, and d) An endodomain.

[0245] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain,b) A CD8a hinge domain, c) A CD8a transmembrane domain, and d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain.

[0246] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, and e) an IL-18 polypeptide

[0247] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a P2A linker, andI) an IL- 18 polypeptide

[0248] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain.

[0249] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a P2A linker, andI) an IL- 18 polypeptide

[0250] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 19, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain.

[0251] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 19, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a P2A linker, andI) an IL- 18 polypeptide

[0252] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 19, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a furin-P2A-furin linker, and1) an IL- 18 polypeptide

[0253] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 20, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain.

[0254] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 20, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a P2A linker, andI) an IL- 18 polypeptide

[0255] In one embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 20, b) A CD8a hinge domain, c) A CD8a transmembrane domain, d) An endodomain, comprising a 4-1BB endodomain and a CD3c endodomain, e) a furin-P2A-furin linker, andI) an IL- 18 polypeptide

[0256] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15,b) A CD8a hinge domain according to SEQ ID NO: 27 c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30.

[0257] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, b) A CD8a hinge domain according to SEQ ID NO: 27 c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30, e) a P2A linker according to SEQ ID NO: 46, andI) an IL-18 polypeptide according to SEQ ID NO: 47.

[0258] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 19, b) A CD8a hinge domain according to SEQ ID NO: 27 c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30.

[0259] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 19, b) A CD8a hinge domain according to SEQ ID NO: 27 c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30, e) a P2A linker according to SEQ ID NO: 46, andI) an IL-18 polypeptide according to SEQ ID NO: 47.

[0260] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 19, b) A CD8a hinge domain according to SEQ ID NO: 27 c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30, e) a furin-P2A-furin linker according to SEQ ID NO: 84, andI) an IL-18 polypeptide according to SEQ ID NO: 47.

[0261] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 20, b) A CD8a hinge domain according to SEQ ID NO: 27, c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30.

[0262] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 20, b) A CD8a hinge domain according to SEQ ID NO: 27, c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30, e) a P2A linker according to SEQ ID NO: 46, andI) an IL-18 polypeptide according to SEQ ID NO: 47.

[0263] In a particular embodiment, there is provided a CAR comprising a) A CD19 / CD20-binding domain, according to SEQ ID NO: 20, b) A CD8a hinge domain according to SEQ ID NO: 27, c) A CD8a transmembrane domain according to SEQ ID NO: 25, and d) An endodomain, comprising a 4-1BB endodomain according to SEQ ID NO: 29 and a CD3c endodomain according to SEQ ID NO: 30, e) a furin-P2A-furin linker according to SEQ ID NO: 84, andI) an IL-18 polypeptide according to SEQ ID NO: 47.

[0264] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 38.

[0265] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 39.

[0266] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 40.

[0267] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 41.

[0268] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 102.

[0269] In a particular embodiment, there is provided a CAR according to SEQ ID NO: 103.

[0270] Herein are provided inter alia, nucleic molecules that encode a CD19 / CD20-binding domain provided herein.

[0271] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 42.

[0272] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 43.

[0273] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 44.

[0274] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 45.

[0275] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 104.

[0276] In a particular embodiment, there is provided a nucleic acid molecule encoding a CD19 / CD20- binding domain having the amino acid sequence of SEQ ID NO: 105.Vectors

[0277] Herein are provided inter alia, vectors which comprise a nucleic acid sequence provided herein. Such a vector may be used to introduce the nucleic acid sequence into a host cell so that it expresses and produces a CD19 / CD20-binding domain or CAR herein provided.

[0278] The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector.

[0279] In one embodiment, herein is provided a vector comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), according to SEQ ID NO: 50, 86, 87, 88, 89, 98, 99, or 100. In particular embodiment, the vector comprises a WPRE according to SEQ ID NO: 88.

[0280] The vector may be capable of transfecting or transducing a cell, such as a cytolytic immune cell. In a particular example, the cell may be a T cell or a natural killer (NK) cell.

[0281] In a particular embodiment, there is provided a vector according to SEQ ID NO: 51.

[0282] In a particular embodiment, there is provided a vector according to SEQ ID NO: 52.

[0283] In a particular embodiment, there is provided a vector according to SEQ ID NO: 53.

[0284] In a particular embodiment, there is provided a vector according to SEQ ID NO: 54.Recombinant Cells

[0285] In some aspects, the disclosure provides a recombinant cell which comprises a nucleic acid molecule as herein provided. In some aspects, is provided a recombinant cell which expresses a CD19 / CD20-binding domain or CAR herein provided, at the cell surface. The recombinant cell may be a cytolytic immune cell, such as a T-cell or natural killer (NK) cell.

[0286] A recombinant cell capable of expressing a CAR herein provided may be made by transducing or transfecting a cell with CAR-encoding nucleic acid. The CAR-expressing recombinant cell herein provided may be generated ex vivo. The recombinant cell may be from a cell sample, such as a peripheral blood mononuclear cell (PBMC) sample from the patient or a donor, recombinant cells may be activated and / or expanded prior to being transduced with CAR-encoding nucleic acid, for example by treatment with an anti-CD3 monoclonal antibody.PHARMACEUTICAL COMPOSITIONS

[0287] In some aspects, the disclosure provides a pharmaceutical composition containing a CD19 / CD20- binding domain, CAR, nucleic acid molecule, vector, recombinant cell, or plurality of recombinant cells, herein provided together with a pharmaceutically acceptable carrier, diluent and / or excipient, and optionallyone or more further pharmaceutically active polypeptides and / or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

[0288] Such compositions can be prepared in a manner well known in the pharmaceutical art and generally, a composition herein provided is administered in a pharmaceutically effective amount. The amount of composition herein provided actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual composition herein provided administered, the age, weight, and response of the individual patient, the severity of the patient’s symptoms, and the like.

[0289] The compositions provided herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.

[0290] The compositions provided herein may be in any form suitable for the above modes of administration. For example, compositions comprising cells may in any form suitable for infusion. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension, or emulsion. Alternatively, the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions herein provided is well within the routine capabilities of a person of skill in the art.

[0291] The compositions for oral administration can take the form of bulk liquid solutions or suspensions, or bulk powders. More commonly, however, the compositions are presented in unit dosage forms to facilitate accurate dosing. The term ‘unit dosage forms’ refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient, vehicle or carrier.

[0292] Injectable compositions are typically based upon injectable sterile saline or phosphate -buffered saline, or other injectable carriers known in the art.METHODS OF TREATMENT

[0293] CAR-expressing cells herein provided may be capable of killing cancer cells, such as B-cell lymphoma cells. CAR-expressing cells, such as T-cells or NK cells, may either be created ex vivo either from a patient's own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Alternatively, CAR-expressing cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to cells such as T-cells. In these instances, CAR cells are generated by introducing DNA or RNA coding for the CAR by one of many means including transduction with a viral vector, transfection with DNA or RNA. T or NK cells expressing a CAR molecule herein provided may beused for the treatment of a cancerous disease, in particular a cancerous disease associated with CD 19 / CD20 expression.

[0294] A method for the treatment of disease relates to the therapeutic use of a cell or population of cells herein provided. In this respect, the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and / or to slow down, reduce or block the progression of the disease. The method herein provided may cause or promote cell mediated killing of CD19 / CD20-expressing cells, such as B cells.

[0295] In one embodiment, there is provided a method for treating and / or preventing a disease, which comprises the step of administering a pharmaceutical composition herein provided to a subject. In a particular embodiment, the disease is cancer. In a more particular embodiment, the cancer is selected from metastatic tumour cell types (such as but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). In a most particular embodiment, the cancer is hematological cancer. In yet a most particular embodiment, the hematological cancer is selected from lymphomas, leukemias, and myelomas. In a further most particular embodiment, the hematological cancer is selected from to non-Hodgkin’s lymphoma (NHL), acute myeloid leukaemia (AML), and acute lymphoblastic leukaemia (ALL) and chronic lymphoblastic leukaemia (CLL).

[0296] In one embodiment herein provided, there is provided a method for treating and / or preventing a disease, which comprises the step of administering a pharmaceutical composition herein provided, to a subject. In a particular embodiment, the disease is an autoimmune disease. In a more particular embodiment, the autoimmune disease is selected from rheumatoid arthritis (RA), COPD, asthma, bronchitis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis, dermatomyositis, Sjogren’s syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus, atopic dermatitis, thyroiditis, contact dermatitis, eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. In a more particular embodiment, the autoimmune disease is, the term refers to systemic lupus erythematosus (SLE).

[0297] In one embodiment, there is provided a method for treating and / or preventing a disease, the method comprising the following steps:(i) isolation of a cell-containing sample from a subject;(ii) transduction or transfection of the cells (as described elsewhere herein); and(iii) administering the cells obtained in (ii) to the subject.

[0298] In one embodiment, there is provided a pharmaceutical composition herein provided, for use in treating and / or preventing a disease. In a particular embodiment, the disease is cancer. In a more particular embodiment, the cancer is selected from metastatic tumour cell types (such as but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lungcancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). In a most particular embodiment, the cancer is hematological cancer. In yet a most particular embodiment, the hematological cancer is selected from lymphomas, leukemias, and myelomas. In a further most particular embodiment, the hematological cancer is selected from to non-Hodgkin’s lymphoma (NHL), acute myeloid leukaemia (AML), and acute lymphoblastic leukaemia (ALL) and chronic lymphoblastic leukaemia (CLL).

[0299] In one embodiment, there is provided a pharmaceutical composition herein provided, for use in treating and / or preventing a disease. In a particular embodiment, the disease is an autoimmune disease. In a more particular embodiment, the autoimmune disease is selected from rheumatoid arthritis (RA), COPD, asthma, bronchitis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis, dermatomyositis, Sjogren’s syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus, atopic dermatitis, thyroiditis, contact dermatitis, eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. In a more particular embodiment, the autoimmune disease is, the term refers to systemic lupus erythematosus (SLE).

[0300] In one embodiment, there is provided the use of a cell expressing a CAR herein provided, in the manufacture of a medicament for treating and / or preventing a disease. In a particular embodiment, the disease is cancer. In a more particular embodiment, the cancer is selected from metastatic tumour cell types (such as but not limited to, melanoma, lymphoma, leukemia, fibrosarcoma, rhabdomyosarcoma, and mastocytoma) and types of tissue carcinoma (such as but not limited to, colorectal cancer, prostate cancer, small cell lung cancer and non-small cell lung cancer, breast cancer, pancreatic cancer, bladder cancer, renal cancer, gastric cancer, glioblastoma, primary liver cancer, ovarian cancer, prostate cancer and uterine leiomyosarcoma). In a most particular embodiment, the cancer is hematological cancer. In yet a most particular embodiment, the hematological cancer is selected from lymphomas, leukaemia, and myelomas. In a further most particular embodiment, the hematological cancer is selected from to non-Hodgkin’s lymphoma (NHL), acute myeloid leukaemia (AML), and acute lymphoblastic leukaemia (ALL) and chronic lymphoblastic leukaemia (CLL).

[0301] In one embodiment, there is provided the use of a cell expressing a CAR herein provided, in the manufacture of a medicament for treating and / or preventing a disease. In a particular embodiment, the disease is an autoimmune disease. In a more particular embodiment, the autoimmune disease is selected from rheumatoid arthritis (RA), COPD, asthma, bronchitis, systemic lupus erythematosus (SLE), cutaneous lupus erythematosus (CLE), lupus nephritis, dermatomyositis, Sjogren’s syndrome, multiple sclerosis, psoriasis, dry eye disease, type I diabetes mellitus, atopic dermatitis, thyroiditis, contact dermatitis, eczematous dermatitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), atherosclerosis and amyotrophic lateral sclerosis. In a more particular embodiment, the autoimmune disease is, the term refers to systemic lupus erythematosus (SLE).

[0302] Injection dose levels range from about 0.1 mg / kg / h to at least 10 mg / kg / h, all for from about 1 to about 120 h and especially 24 to 96 h. A preloading bolus of from about 0.1 mg / kg to about 10 mg / kg ormore may also be administered to achieve adequate steady state levels. The maximum total dose is not expected to exceed about 1 g / day for a 40 to 80 kg human patient.

[0303] For the prophylaxis and / or treatment of long-term conditions, such as degenerative conditions, the regimen for treatment usually stretches over many months or years so oral dosing is preferred for patient convenience and tolerance. With oral dosing, one to four (1-4) regular doses daily, especially one to three (1-3) regular doses daily, typically one to two (1-2) regular doses daily, and most typically one (1) regular dose daily are representative regimens. Alternatively for long lasting effect drugs, with oral dosing, once every other week, once weekly, and once a day are representative regimens. In particular, dosage regimen can be every 1-14 days, more particularly 1-10 days, even more particularly 1-7 days, and most particularly 1-3 days.

[0304] When used to prevent the onset of a condition, a composition herein provided, will be administered to a patient at risk for developing the condition, typically on the advice and under the supervision of a physician, at the dosage levels described above. Patients at risk for developing a particular condition generally include those that have a family history of the condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the condition.

[0305] The compositions described herein may be administered as the sole active agent or it can be administered in combination with other therapeutic agents, including other compositions herein provided, that demonstrate the same or a similar therapeutic activity and that are determined to be safe and efficacious for such combined administration. In a specific embodiment, co-administration of two (or more) agents allows for significantly lower doses of each to be used, thereby reducing the side effects seen.

[0306] In some aspects, a composition herein provided, is administered as a medicament. In a specific embodiment, said composition additionally comprises a further active ingredient. In another embodiment, the composition herein provided, is co-administered with a further active ingredient for the same and / or another disease.

[0307] By co-administration is included any means of delivering two or more therapeutic agents to the patient as part of the same treatment regime, as will be apparent to the skilled person. Whilst the two or more agents may be administered simultaneously in a single formulation, i.e. as a single pharmaceutical composition, this is not essential. The agents may be administered in different formulations and at different times.

[0308] The aspects herein provided will now be further described by way of examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the aspects herein provided and are not intended in any way to limit the scope herein provided.EXAMPLESExample 1. Generation and Characterization of Bispecific CD19 / CD20 CARs1.1. Materials and Methods1.1.1. Cells, plasmids, and other reagentsHuman peripheral blood pan-T cells were purchased from Stemcell Technologies. Raji, Daudi, and K562 cell lines were purchased from ATCC (Table 1). The pLV-tEFla-8BBz and V0048 lentiviral transfer vectors were subsequently designed and generated. The PE-conjugated anti-G4S linker antibody for detection of CAR surface expression was a product of Cell Signaling Technology. Other major reagents and supplies are listed in Table 2.Table I. Cell lines.Table II. Major reagents and supplies.Table III. Definition of abbreviations1.2. Cloning of T2019 CAR (SEQ ID NO: 39) with CD8a hinge (CD8H), CD8a transmembrane domain (CD8TM), 4-1BB costimulatory domain, and CD3 zeta domain (CD3z)

[0309] The following primers (synthesized by GENEWIZ), were used:24-2CPF, 5’-GATCGCATGCCGCCAGGCCAGAGGTGCAGCTGGTGG-3’ (sense) SEQ ID NO: 57;63-2CPR, 5’-GCAGGGGTTGTTGTAGCGGCCGCGGAGCTCACGGTCACGC-3’ (antisense) SEQ ID NO: 58;48BBzF, 5’-ACAACAACCCCTGCTCCTCGG-3’ (sense) SEQ ID NO: 59; and48SalR, 5’-GATTGTCGACGGTATCGCG-3’ (antisense) SEQ ID NO: 60.

[0310] the gene fragment encoding the extracellular binding domains of the CAR comprising the anti- CD20 binder CD20, a polypeptide linker composed of three repeats of the G4S motif [(G4S)3], and the anti-CD19 binder FMC63, was synthesized by GENEWIZ.

[0311] For cloning of T2019 CAR, the gene fragment encoding the extracellular binding domains of the CAR comprising the anti-CD20 binder gene fragment was PCR-amplified with primers 24-2CPF and 63- 2CPR. The gene fragment encoding the CD8H, CD8TM, 4-1BB costimulatory domain, and CD3z was PCR-amplified with the V0048 vector as a template and primers 48BBzF and 48SalR. The full-length T2019 CAR gene fragment was assembled by overlapping PCR with the two gene fragments in the same molarities for 18 cycles in the presence of primers 24-2CPF and 48SalR. The overlapping PCR product was digested with SphI and Sall restriction enzymes and cloned into the V0048 vector.1.3. Cloning of D1920 CAR (SEQ ID NO: 38) with CD8H, CD8TM and 4-1BB costimulatory domain, and CD3zThe following primers (synthesized by GENEWIZ), were used:1133, 5’- ACTACTCTAGAGATATCCAGATGACTCAG-3’ (sense) SEQ ID NO: 61;2125, 5’-ACTACGCGGCCGCGGATGACACGGTCACAGA-3’ (antisense) SEQ ID NO: 62;1136, 5’- ACTACGGATCCAGGCCTAAGGCCGCC-3’ (sense) SEQ ID NO: 63;2135, 5’-CTGGATATCTGGCCTGGCGGCATG-3’ (antisense) SEQ ID NO: 64;1141, 5’- GCCAGGCCAGATATCCAGATGACT-3’ (sense) SEQ ID NO: 65;2130, 5’-TCCGGATTAACGCGGGGGCAGCGCCTG-3’ (antisense) SEQ ID NO: 66;1138, 5’- CCCCCGCGTTAATCCGGACTCAGATCT-3’ (sense) SEQ ID NO: 67;2131, 5’-ACTACGTCGACGGTATCGCGATAAGCT-3’ (antisense) SEQ ID NO: 68.

[0312] The gene fragment encoding the extracellular binding domains of the CAR comprising the anti- CD20 binder, a polypeptide linker composed of (G4S)3, and the anti -CD 19 binder FMC63, was synthesized by GENEWIZ.

[0313] For cloning of DI 920 CAR, the gene fragment encoding the extracellular binding domains of the CAR comprising the anti-CD20 binder was PCR-amplified with primers 1133 and 2125, digested with Xbal and Notl restriction enzymes, and cloned into the pLV-tEFla-8BBz vector with built-in CD8H, CD8TM, 4-1BB, and CD3z. Then, the D1920 gene fragment was amplified with primers 1141 and 2130. A gene fragment encoding the BamHI restriction site was PCR-amplified with primers 1136 and 2135. Anothergene fragment encoding the Sall restriction site was PCR-amplified with primers 1138 and 2131. The full- length D1920 CAR gene fragment with BamHI and Sall restriction sites was assembled by using overlapping PCR with the three gene fragments in the same molarities and primers 1136 and 2131. The overlapping PCR product was digested with BamHI and Sall restriction enzymes and cloned into the V0048 vector.1.4. Cloning of T2019-IL18 CAR (SEQ ID NO: 41) with CD8H, CD8 TM domain, 4-1BB costimulatory domain, and CD3z

[0314] The following additional primers (synthesized by GENEWIZ), were used:V48zR, 5’-TCTTGGAGGCAGGGCCTG-3’ (antisense) SEQ ID NO: 69;P2ALF, 5’-GCCCTGCCTCCAAGACGCGCGAAACGCAGCGGC-3’ (sense) SEQ ID NO: 70;P2ALR, 5’-CAAGCTTGCCAAAGTACTGCAGGCTGTGTCCCG-3’ (antisense) SEQ ID NO: 71;IL18F, 5’-TACTTTGGCAAGCTTGAATC-3’ (sense) SEQ ID NO: 72;IL18R, 5’-GTCTTCGTTTTGAACAGTG-3’ (antisense) SEQ ID NO: 73; and48SalF, 5’-GTTCAAAACGAAGACTAATCCGGACTCAGATCTCG-3’ (sense) SEQ ID NO: 74.

[0315] For cloning of T2019-IL18 CAR, the T2019 CAR gene fragment was PCR-amplified with the T2019 CAR vector as a template and primers 24-2CPF and V48zR. The gene fragment (P2A-Leader) encoding the P2A and CD59 leader peptide was PCR-amplified with a previously generated, P2A-Leader encoding plasmid as a template and primers P2ALF and P2ALR. The IL- 18 gene fragment was PCR- amplified by using a previously generated, IL- 18 encoding plasmid as a template and primers IL18F and IL18R. Another gene fragment (Sall) encoding the Sall restriction site was PCR-amplified with primers 48SalF and 48SalR. The P2A-Leader, IL- 18, and Sall gene fragments were fused to each other by using overlapping PCR for 18 cycles with the three gene fragments in the same molarities and primers P2ALF and 48SalR. The full-length T2019-IL18 CAR gene fragment was assembled by overlapping PCR with the T2019 and P2A-Leader-IL-18-SalI gene fragments in the same molarities for 18 cycles in the presence of primers 24-2CPF and 48SalR. The overlapping PCR product was digested with SphI and Sall restriction enzymes and cloned into the V0048 vector.1.5. Cloning of D1920-IL18 CAR (SEQ ID NO: 40) with CD8H, CD8TM, 4-1BB costimulatory domain, and CD3z

[0316] The following primers (synthesized by GENEWIZ), were used:1136, 5’- ACTACGGATCCAGGCCTAAGGCCGCC-3’ (sense) SEQ ID NO:75;2157, 5’- TTTCGCGCGACGCGGGGGCAGCGCCTG-3’ (antisense) SEQ ID NO: 76;1167, 5’- CCCCCGCGTCGCGCGAAACGCAGCGGC-3 ’ (sense) SEQ ID NO: 77;2158, 5’- GCCAAAGTACTGCAGGCTGTGTCCCGA-3’ (antisense) SEQ ID NO: 78;1168, 5’- AGCCTGCAGTACTTTGGCAAGCTTGAA-3’ (sense) SEQ ID NO: 79;2159, 5’- GAGTCCGGATTAGTCTTCGTTTTGAAC-3’ (antisense) SEQ ID NO 80:;1169, 5’- GAAGACTAATCCGGACTCAGATCTCGA-3’ (sense) SEQ ID NO: 81; and 2131, 5’-ACTACGTCGACGGTATCGCGATAAGCT-3’ (antisense) SEQ ID NO:82.

[0317] For cloning of D1920-IL18 CAR, the D1920 CAR fragment was PCR-amplified with the D1920 CAR vector as a template and primers 1136 and 2157. The gene fragment (P2A-Leader) encoding the P2A and CD59 leader peptide was PCR-amplified with a previously generated, P2A-Leader encoding plasmid as a template and primers 1167 and 2158. The IL-18 gene fragment was PCR-amplified by using a previously generated, IL-18 encoding plasmid as a template and primers 1168 and 2159. Another gene fragment (Sall) encoding the Sall restriction site was PCR-amplified with primers 1169 and 2131. The full- length D1920-IL18 CAR gene fragment was assembled by using overlapping PCR with the four gene fragments (D1920, P2A-Leader, IL-18, and Sall) in the same molarities and primers 1136 and 2131. The overlapping PCR product was digested with SphI and Sall restriction enzymes and cloned into the V0048 vector.1.6. Lentiviral production

[0318] Lentiviruses encoding the CARs were produced by using the Thermo Fisher Scientific LV- MAX™ lentiviral production system. Lentiviral titers were briefly evaluated by using Lenti-X™ GoStix™ Plus according to the manufacturer’s instructions or for production of CAR-T cells for in vivo studies by transduction of Jurkat E6.1 cells followed by flow cytometry to measure the percentage of CAR-positive live cells. / .7. CAR- T cell manufacturing for in vitro cytotoxicity and IL-18 release assaysHuman peripheral blood pan-T cells (Stemcell Technologies) were activated by using T Cell TransAct™ according to the manufacturer’s instruction. For transduction, 250 pl lentiviruses were first diluted in 250 pl TexMACS™ medium containing 20 pg mL'1protamine sulfate and 1000 lU / mL IL-7 and 400 lU / mL IL-15, and then added into 106activated T cells in 1 mL TexMACS™ medium supplemented with 10 pg mL'1(final concentration) protamine sulfate and 500 lU / mL (final concentration) IL-7 and 200 lU / mL (final concentration) IL-15 in 12-well plates. After incubation at 37°C for 24 h, 1 mL medium was gently removed from each well and 2 mL fresh TexMACS™ medium containing 500 lU / mL IL-7 and 200 lU / mL IL-15 was added into each well of the 12-well plates. Transduced T cells were passaged every 2-3 days at a seed density of 5 * 105cells / mL in TexMACS™ supplemented with 500 lU / mL IL-7 and 200 lU / mL IL-15. For detection of CAR expression on the surface of transduced T cells, 106cells were mixed with 1:100 diluted PE-conjugated anti-G4S linker antibody and 1:1000 diluted SYTOX™ red dead cell stain, and incubated on ice for 30 min. The cells were washed with 200 pl PBS (pH7.4) containing 0.5% BSA (PBSA), resuspended in 100 pl PBSA, and used for flow cytometry analysis.1.8. CAR-T cell manufacturing for in vitro repetitive antigen stimulation and IFN-y release assays

[0319] Human peripheral blood pan-T cells (Stemcell Technologies) were thawed in X-VIVO medium (Lonza) supplemented with 3% human male AB Plasma-derived serum (Sigma), rhIL-7 (500 lU / mL) and rhIL15 (200 lU / mL) (both Miltenyi), and 1% Penicillin / Streptomycin (Thermo Fisher), and activated using TransAct (Miltenyi) as per manufacturer’s recommendations. After 24 hours, cells were transduced with lentiviruses at an MOI of 2 and then expanded for 6 days. On day 4 post transduction, transduction efficiency (i.e., percentage of CAR-positive T cells) was measured by flow cytometry with a PE-conjugatedanti-G4S linker antibody. To produce non-transduced T cells as a control, activated T cells were mock transduced without the addition of lentiviruses. The lack of CAR expression was confirmed by flow cytometry. Both CAR-T and non-transduced T cells were then used fresh in downstream applications.1.9. CAR-T cell manufacturing for in vivo studies

[0320] CAR T cells were manufactured starting from CD4+ / CD8+ T cells enriched by MACS separation from healthy donor leukapheresis material, according to the manufacturer’s instructions. T cells were then cultured in complete X-VIVO TM 15 medium (Lonza) supplemented with 3% human male AB Plasma- derived serum (Access Biologicals), rhIL-7 (20-2000 lU / mL) and rhIL15 (20-200 lU / mL) (both CellGenix), and 1% Penicillin / Streptomycin (Thermo Fisher), and activated using TransAct (Miltenyi Biotec) as per manufacturer’s recommendations. After 24 hours, cells were transduced with lentiviruses and then expanded for 6 days. At the end of the production (day 7), transduction efficiency (i.e., percentage of CAR-positive T cells) was measured by flow cytometry with PE-conjugated anti-G4S linker antibody. For the production of non-transduced T cells as a control, activated CD4+ / CD8+ enriched T cells were mock transduced without the addition of lentiviruses on day 1. The lack of CAR expression was confirmed by flow cytometry. Both CAR-T and non-transduced T cells were then used fresh in downstream applications such as co-culture assays or cryopreserved in X-VIVO TM 15 / 10% human male AB plasma- derived serum / 10% DMSO (Sigma- Aldrich).1.10. Cytotoxicity assay

[0321] Target cell lines were transduced with lentiviruses encoding firefly luciferase, as described above for transduction of primary T cells, except that different medium was used and IL-7 and IL- 15 were not added. Transduced cells with stable expression of luciferase were selected by adding puromycin and zeocin at final concentrations of 1 and 0.5 pg mL1, respectively.

[0322] For cytotoxicity assay, 104target cells with luciferase expression were mixed with transduced primary T cells at different effector : target (E:T) ratios in 100 pl TexMACS™ medium in each well of 96- well plates. Background luminescence signal was generated by treating target cells with 1% Tween-20. After 96-h incubation, assays were developed by using Promega Steady-Gio® luciferase assay system according to the manufacturer’s instructions. Percent lysis was calculated by using the formula: [1- (Luminescence of target cells plus effector cells - luminescence of target cells treated with Tween- 20) / (luminescence of target cells only - luminescence of target cells treated with Tween-20)] x 100.1.11. Repetitive challenge assay

[0323] Target cells containing stable expression of luciferase were co-cultured with CAR-T cells at several E:T ratios (4, 2, 1, 0.5) in a 384-well plate. Every 2-3 days, 10,000 target cells were added to the CAR-T culture for a total of 6 rounds of serial antigen stimulations. At every re -stimulation, luminescence was measured and supernatants harvested for IFN-y measurement.1.12. In vitro IL-18 release assay

[0324] 50 j l supernatant was collected from each well of the 96-well plates in a separate cytotoxicity assay after 24-h or 72-h incubation with Raji cells. IL-18 levels were measured by using the Human Total IL-18 / IL-1F4 Quantikine ELISA Kit according to the manufacturer’s instructions.1.13. In vivo IL- 18 release assay

[0325] Human IL- 18 in mouse serum was measured by the Ella automated immunoassay system and Simple Plex Human IL-18 cartridge. Mouse serum samples were harvested on day 16 and day 30 after CAR-T cell infusion. Serum samples were centrifuged to remove debris before being transferred into a labeled 96-well plate for storage. On the day of measurement, the Simple Plex Runner program was initiated, and the kit barcode was scanned to load the cartridge. Mouse serum samples were thawed at room temperature. Diluted mouse serum samples (up to 30 per run) are pipetted into the sample inlets, along with the prepared low and high IL- 18 controls. The assay was validated by the human IL- 18 controls that produced readings falling within the acceptable range specified by the Certificate of Analysis (CoA).1.14IFN- release assay

[0326] IFN-y was measured by duoset sandwich elisa (R&D systems) in a 384-well plate. Luminescence values were transformed to concentrations (pg / mL) using graphpad Prism.1.14. In vivo efficacy study

[0327] The in vivo efficacy study were carried out at the Dutch Cancer Institute. Each male NSG mouse was injected with 0.5E6 Daudi cells, a Burkitt’s lymphoma cell model, engineered to express firefly luciferase. One week post injection, tumor growth was confirmed by IVIS luciferase imaging and mice were randomized into groups. Every week post injection, blood was collected to monitor T cell expansion via flow cytometry and IVIS imaging was performed to assess tumor burden. Animals were euthanized by CO2 asphyxiation when humane endpoints were met or at the end of the experiment. CARs were tested at both a low dose (1E6 CAR-T cells per mouse) and high dose (3E6 CAR-T cells per mouse). Statistical analyses were performed by using non-parametric pairwise testing.1.15. Results and Discussion1.15.1. Generation of bispecific CD19 / CD20 CARs with CD8H, CD8TM, 4-1BB costimulatory domain, and CD3z

[0328] Two second-generation, bispecific CD19 / CD20 CARs (D1920 (SEQ ID NO: 38) and T2019 (SEQ ID NO: 39)) were generated with FMC63 as an anti-CD19 antibody, CD20 as an anti-CD20 antibody, CD8H, CD8TM, 4-1BB costimulatory domain, and CD3z (FIG. 1). T2019 is a tandem CAR, in which FMC63 scFv is linked to the C terminus of CD20 scFv via a flexible (G4S)3 polypeptide linker as the extracellular antigen binding domain. Two recent studies demonstrated that bispecific CARs with a different arrangement of the VH and VL domains of two binders and use of different linker lengths, so called “loop CARs”, were more effective than tandem CARs in controlling tumor growth in vitro and invivo (1-3). To test if a bispecific CD19 / CD20 CAR in a different structure could be more effective than T2019, we generated DI 920, in which the extracellular antigen binding domains were rearranged and forced to form a bispecific diabody due to the use of short G4S linkers between the VH and VL domains of the two antibodies. Both CARs were cloned into the clinically validated lentiviral vector V0048, which uses a truncated version of human elongation factor-1 alpha (tEFla) as a promoter.1.15.2. Generation of IL-18-armed bispecific CD19 / CD20 CARs

[0329] Bispecific CD19 / CD20 CARs have shown excellent clinical efficacy and safety in patients with refractory or relapsed B-cell non-Hodgkin lymphoma (4). However, some patients still did not respond to the therapy. Some of those patients who achieved an initial complete response relapsed again in a longterm follow-up. IL-18 is an inflammatory cytokine and an important mediator of T cell responses. It has recently been demonstrated that IL-18 could not only promote CAR-T cell expansion and persistence, but also stimulate the endogenous immunity and modulate the immunosuppressive tumor microenvironment (5, 6). An IL-18-armed CD19 CAR was safe and elicited durable responses in CD19 CAR-T -pretreated refractory or relapsed lymphoma (7). To test whether IL-18 arming could have an impact on the fitness and in vitro and in vivo efficacy of bispecific CD 19 / 20 CARs, two fourth-generation, bispecific CD19 / CD20 CARs (D1920-IL18 (SEQ ID NO: 40) and T2019-IL18 (SEQ ID NO: 41)) were generated by fusing D1920 and T2019, respectively, into human IL-18 via the self-cleaving peptide P2A, and cloning into the V0048 lentiviral vector (FIG. 1).1.16. Cytotoxicity and IL-18 release of human primary T cells transduced with bispecific CARs

[0330] To test cytotoxicity of the CARs against cancer cells expressing CD 19 and / or CD20 and IL- 18 release, human primary T cells were transduced with lentiviruses encoding the CARs. D1920 showed high surface expression on day 6 post transduction (FIG. 2). Arming DI 920 with IL- 18 for constitutive expression decreased the surface expression of D1920-IL18. In a 24-h cytotoxicity assay with two double positive B cell lines (Raji and Daudi), a double negative cell line (K562), a CD 19 knockout Raji cell line (Raji CD19 KO), and a CD20 knockout Raji cell line (Raji CD20 KO), D1920 and D1920-IL18 showed comparable killing activity against the double positive and single knockout B cell lines, suggesting that both the CD19- and CD20-targeting arms of the CARs should be functional (FIG. 3). D1920-IL18 did not show superior efficacy to D1920 in this short-term assay. No dose -dependent obvious killing of the double negative K562 cells was observed, suggesting their cytotoxicity for Raji, Daudi, Raji CD19 KO, and Raji CD20 KO cells should be mediated by interaction with CD19 and / or CD20. IL-18 release was detected for D1920-IL18 after 24 and 72 h of incubation with Raji cells with the level at 72 h approximately three-fold higher than at 24 h. (FIG. 4).1.17. Persistent killing and IFN-y release of the bispecific CARs in a repetitive antigen stimulation assay

[0331] To explore killing persistency of the bispecific CARs against cancer cells and if IL- 18 secretion could contribute to the efficacy, a repetitive antigen stimulation assay was designed in which Raji cellswere added every 2-3 days to CAR-T cell cultures for a total of 6 rounds of stimulation. At an E:T ratio of 1:1, D1920-IL18 (SEQ ID NO: 40) showed most persistent and significantly (p<0.01 , unpaired t test) better killing activity than T2019-IL18 (SEQ ID NO: 41) in the last three rounds of coculturing. D1920-IL18 (SEQ ID NO: 40)was also more potent than the unarmed D1920 (SEQ ID NO: 38) in rounds 5 and 6 of stimulation, although the difference in killing activity is not statistically significant (p>0.05, unpaired / test). In contrast, no improved cytotoxicity was observed with T2019-IL18 (SEQ ID NO: 41) compared to the unarmed counterpart. In correlation with the persistent killing activity, D1920-IL18 (SEQ ID NO: 40) released significantly higher levels of IFN-y than T2019-IL18 (SEQ ID NO: 41) in all rounds of stimulation except round 4 (FIG. 6). Compared to the unarmed D1920 (SEQ ID NO: 38), D1920-IL18 (SEQ ID NO: 40) also produced, on average, more IFN-y.1.18. Results in vitro at E: T = 1:1

[0332] The results were analysed via unpaired t-test statistical method.Table IV. % lysis after repeated stimulation (FIG 5)Table V. In vitro IFN-y release (pg / mL) by bispecific CD19 / 20 CARs in repetitive challenge assays(FIG 6)t-test value *** pO.OOl; ** p< 0.01, * p<0.051.19. In vivo anti-tumor activity, expansion, and IL-18 release of the bispecific CARs

[0333] In the first in vivo study with Daudi Burkitt lymphoma model, an aggressive CD 19 expressing disseminated model, D1920 (SEQ ID NO: 38) showed slightly better tumor control than T2019 (SEQ ID NO: 39) at the low dose(FIG. 8). At the high dose, D1920 and T2019 had similar efficacy. In the second in vivo study, IL- 18 only CAR (no binder-SEQ ID NO: 108) did not significantly inhibit tumor growth compared to the untransduced T cell group (FIG. 7). In contrast, D1920-IL18 (SEQ ID NO: 40) at both the low and high doses and T2019-IL18 (SEQ ID NO: 41) at the high dose on day 20 post infusion showed better tumor control than the unarmed counterparts, suggesting that IL- 18 should play a role in enhancing the anti-tumor activity (FIG. 8). After 10 days of infusion, increasing percentages of CD3+ and CD45+ human T cells were detected in the circulation of animals in the IL- 18-armed groups infused with the high dose, suggesting rapid expansion of the CAR-T cells (FIG. 9). CAR expression was confirmed with anti- FMC63 staining. High levels of serum IL-18 were also detected with the armed CAR groups on day 16 post infusion. Notably, the majority of animals at the high dose of armed CARs died without tumor growth due to the acute graft versus host diseases (GVHD).1.19.1.1. ResultsTable VI. In vivo IL-18 release for illustrative CARs1.20. Comparative study for antigen binding domains.1.20.1. Overview

[0334] The binding domains of the invention have a CD20 binding domain differing from ofatumumab CD20 by 3 mutations (see FIG. 10). This study aims at comparing the binding of the CD 19 / 20 antigen binding domains of the invention in tandem (SEQ ID NO: 20, T2019 ABD) or diabody (SEQ ID NO: 19,D1920 ABD) configuration against the CD19 / CD20 ofatumumab derived tandem (SEQ ID NO: 115, T2019-ofa ABD) or diabody (SEQ ID NO: 111, D1920-ofa ABD).1.20.2. Cloning of the antigen binding domains (ABD) of D1920, T2019, D1920-ofa (D1920 with ofatumumab as a CD20 binder), and T2019-ofa (T2019 with ofatumumab as a CD20 binder)

[0335] For cloning of D1920 ABD, we used the following primers synthesized by GENEWIZ:1217, 5’-TAGGAAGCTTTTAAAACAGC-3’ (sense) (SEQ ID NO: 126);2204, 5’-TCCATAAGCTCCGGAGAT-3’ (antisense) (SEQ ID NO: 119);1232, 5’-TCCGGAGCTTATGGAGATATCCAGATGACTCAG-3’ (sense) (SEQ ID NO: 127); and 2213, 5’-GCTGTTTTAAAAGCTTCCTAGCCCTTATCGTCATCGTC-3’ (antisense) (SEQ ID NO: 128).

[0336] The gene fragment of D 1920 ABD was amplified with a D1920-encoding plasmid as a template and primers 1232 and 2213. The pABm3.2 vector was linearized by PCR amplification with the plasmid as a template and primers 1217 and 2204. The gene fragment of D1920 ABD was then assembled into the linearized pABm3.2 vector by using Gibson Assembly® according to the manufacturer’s instructions.

[0337] For cloning of T2019 ABD, we used the following primers synthesized by GENEWIZ:1217, 5’-TAGGAAGCTTTTAAAACAGC-3’ (sense) (SEQ ID NO: 126);2204, 5’-TCCATAAGCTCCGGAGAT-3’ (antisense) (SEQ ID NO: 119);1218, 5’-ATCTCCGGAGCTTATGGAGAGGTGCAGCTGGTGGAA-3’ (sense) (SEQ ID NO: 129); and 2213, 5’-GCTGTTTTAAAAGCTTCCTAGCCCTTATCGTCATCGTC-3’ (antisense) (SEQ ID NO: 128).

[0338] The gene fragment of T2019 ABD was amplified with a D1920-encoding plasmid as a template and primers 1218 and 2213. The pABm3.2 vector was linearized by PCR amplification with the plasmid as a template and primers 1217 and 2204. The gene fragment of T2019 ABD was then assembled into the linearized pABm3.2 vector by using Gibson Assembly® according to the manufacturer’s instructions.

[0339] For cloning of D1920-ofa ABD, we used the following primers synthesized by GENEWIZ:1250, 5’-GGCCAGGCCGGCCACCAT-3’ (sense) (SEQ ID NO: 118);2204, 5’-TCCATAAGCTCCGGAGAT-3’ (antisense) (SEQ ID NO: 119);1252, 5’-TCCGGAGCTTATGGAGATATCCAGATGACTCAG -3’ (sense) (SEQ ID NO: 120);2217, 5’-CTGCACTTCACTTCCGCCTCCGCCGGT-3’ (antisense) (SEQ ID NO: 121);1241, 5’-GGCGGAAGTGAAGTGCAGCTGGTGGAG-3’ (sense) (SEQ ID NO: 122);2218, 5’-ATACAGGCTTTTCTTGGCGTTGTCTCT-3’ (antisense) (SEQ ID NO: 123);1242, 5’-GCCAAGAAAAGCCTGTATCTGCAGATG-3’ (sense) (SEQ ID NO: 124); and2226, 5’-GTGGCCGGCCTGGCCGGATGACACGGTCACAGA-3’ (antisense) (SEQ ID NO: 125).

[0340] Two gene fragments of FMC63 were amplified with a D1920-encoding plasmid as a template and primer pairs 1252 / 2217 and 1241 / 2218, respectively. The gene fragment of ofatumumab was amplified with an ofatumumab-encoding plasmid and primers 1242 / 2226. Then, the full-length DI 920-ofa ABD gene fragment was amplified by overlapping PCR with the three gene fragments and primers 1252 / 2226. The pABm3.2 vector was linearized by PCR amplification with the plasmid as a template and primers 1250 and2204. The gene fragment of D1920-ofa ABD was then assembled into the linearized pABm3.2 vector by using Gibson Assembly® according to the manufacturer’s instructions.

[0341] For cloning of T2019-ofa ABD, we used the following primers synthesized by GENEWIZ:1250, 5’-GGCCAGGCCGGCCACCAT-3’ (sense) (SEQ ID NO: 118);2204, 5’-TCCATAAGCTCCGGAGAT-3’ (antisense) (SEQ ID NO: 119);1251, 5’-TCCGGAGCTTATGGAGAAGTGCAGCTGGTGGAG -3’ (sense) (SEQ ID NO: 130);2216, 5’-GTACAGGCTTTTCTTGGCGTTGTCTCT-3’ (antisense) (SEQ ID NO: 133);1239, 5’-GCCAAGAAAAGCCTGTACCTGCAGATG-3’ (sense) (SEQ ID NO: 131); and 2225, 5’-GTGGCCGGCCTGGCCGGAGCTCACGGTCACGCT-3’ (antisense) (SEQ ID NO: 132).

[0342] The gene fragment of FMC63 was amplified with a T2019-encoding plasmid as a template and primer 1239 and 2225. The gene fragment of ofatumumab was amplified with an ofatumumab-encoding plasmid and primers 1251 / 2216. Then, the full-length T2019-ofa ABD gene fragment was amplified by overlapping PCR with the two gene fragments and primers 1251 / 2225. The pABm3.2 vector was linearized by PCR amplification with the plasmid as a template and primers 1250 and 2204. The gene fragment of T2019-ofa ABD was then assembled into the linearized pABm3.2 vector by using Gibson Assembly® according to the manufacturer’s instructions.1.20.3. Expression and purification qfD1920ABD, T2019 ABD, D1920-ofa ABD, and T2019-ofa ABD

[0343] Expi293F cells were transfected with plasmids encoding D1920 ABD, T2019 ABD, D1920-ofa ABD, and T2019-ofa ABD with both hexahistidine and FLAG tags at the C termini by using the ExpiFectamin 293 kit according to the manufacturer’s instructions. After 6 days of incubation, the supernatant was harvested by centrifugation at 8000 g for 10 min, and then concentrated and dialyzed with PBS by using a protein concentrator with a cutoff of 30 kDa. After dialysis, the proteins were purified by using the Ni-NTA agarose resin according to the manufacturer’s instructions. Eluted proteins were dialyzed against PBS by using a protein concentrator with a cutoff of 30 kDa. Oligomerization states of the dialyzed proteins were analyzed, and monomers were isolated by size -exclusion chromatography (SEC) with a Superdex 200 column.1.20.4. Cell binding of D1920 ABD, T2019ABD, D1920-ofa ABD, and T2019-ofa ABD

[0344] Binding of D1920 ABD, T2019 ABD, D1920-ofa ABD, and T2019-ofa ABD to K562-CD19, K562-CD20, and K562 cells was measured by flow cytometry. Briefly, antibodies were four-fold serially diluted from 2000 to 0.122 nM in PBS containing 3% BSA (assay buffer). Cells were collected by centrifugation at 500 g for 10 min and resuspended in the assay buffer at a density of 4E6 cells / mL. 50 pL of the serially diluted antibodies was mixed with 50 pL of the cells leading to the final antibody concentrations serially diluted from 1000 to 0.061 nM. After incubation on ice for 30 min, cells were washed 3 times with 200 pL assay buffer by centrifuging at 500 g for 3 min in each wash. After washing, cells were resuspended in 100 pL of 1 :500 diluted secondary antibody (APC -conjugated anti-FLAG). After incubation on ice for 30 min, cells were washed 3 times with 200 pL assay buffer by centrifuging at 500 gfor 3 min in each wash. Cells were then resuspended in 100 pL assay buffer and used for flow cytometry analysis. Median fluorescence intensity (MFI) was calculated to quantify the cell binding affinities of each antibody.1.20.5. Results1.20.5.1. Cloning, expression, and purification ofD1920ABD, T2019 ABD, D1920-ofaABD, and T2019-ofaABD

[0345] The CD20 binder used in the generation of D1920 and T2019 is an engineered form of ofatumumab harboring three amino acid mutations, one (N35D) in the CDR1 and the other two (T55G and K85N) in the FRs. To evaluate the effects of the mutations and provide insights into the development potential of D1920 and T2019 as novel cell therapies, we generated not only D1920 ABD and T2019 ABD but also D1920-ofa ABD and T2019-ofa ABD, which use wild-type ofatumumab as a CD20 binder. All ABDs were cloned into a mammalian expression vector pABm3.2 by using Gibson Assembly®. They were expressed in the Expi293F cell cultures and purified from the supernatants with yields of 13.6, 11.2, 12.7, and 9.2 mg L-l for D1920 ABD, T2019 ABD, D1920-ofa ABD, and T2019-ofa ABD, respectively. The purified ABDs migrated on a reducing SDS-PAGE with an apparent molecular weight of approximately 55 kDa, comparable to their calculated molecular weights (53.9, 55.7, 53.9, and 55.7 kDa, respectively) including the His and FLAG tags (Fig. 11). The oligomerization states of the purified proteins were analyzed by using SEC (Fig. 11). Monomers were isolated, confirmed by reloading onto the SEC column, and used for cell binding measurement.1.20.5.2. Binding ofD1920 ABD, T2019 ABD, D1920-ofa ABD, and T2019-ofa ABD to CD19- and CD20-overexpressing K562 cells

[0346] Binding of the ABDs to CD19-positive K562 (K562-CD19), CD20-positive K562 (K562-CD20), and dual negative K562 cells was measured by flow cytometry (Fig. 12).Table VII. Cell binding affinities of ABDs measured by flow cytometry within K562-CD19 cell linesTable VIII. Cell binding affinities measured by flow cytometry with K562-CD20 cell lines

[0347] When tested with K562-CD19 cells, D1920 ABD and T2019 ABD had comparable EC50s with D1920-ofa ABD and T2019-ofa ABD, respectively. When tested with K562-CD20 cells, however, T2019 ABD (EC50, 146.7 nM) and D1920 ABD (EC50, 496 nM) showed significantly lower affinities than T2019-ofa ABD (EC50, 19.12 nM) and D1920-ofa ABD (EC50, 22.41 nM), respectively (Figs. 12 and 13).None of the ABDs showed detectable binding to the dual negative K562 cells, suggesting they are highly specific to CD 19 and CD20.Table IX. Cell binding affinity comparison between illustrative tandem CD20 / 19 antigen binding domain of the invention and tandem ofatumumab variants at 3 concentrationsTable X. Cell binding affinity comparison between illustrative diabody CD20 / 19 antigen binding domain of the invention and diabody ofatumumab variants at 3 concentrations

[0348] The decreased affinities of DI 920 ABD and T2019 ABD compared to their counterparts with ofatumumab may result in lower trogocytosis, (Joly and Hudrisier 2003) which has been identified as one of the mechanisms for tumor escape from CAR-T therapies by mediating antigen loss on cancer cells and fratricide of CAR-T cells (Olson et al. 2022).

[0349] CAR with a lower affinity may result in reduced trogocytosis compared to those with higher affinity, (Olson et al. 2022), higher efficacy and persistence (Ghorashian et al. 2019). Furthermore, CAR- T cells with higher affinity binders are more prone to exhaustion and activation-induced cell death (Mao, Kong, and He 2022). Therefore, Binder A-based CARs may exhibit reduced trogocytosis and exhaustion than the wild-type ofatumumab-based counterparts.REFERENCESAghajanian, Haig, Joel G. Rurik, and Jonathan A. Epstein. 2022. “CAR-Based Therapies: Opportunities for Immuno-Medicine beyond Cancer.” Nature Metabolism 4 (2): 163-69. https: / / doi.org / 10.1038 / s42255- 022-00537-5.Ahmad, Ubaid, Zafran Khan, Daniya Ualiyeva, Obed Boadi Amissah, Zohaib Noor, Asaf Khan, Nasib Zaman, Marwa Khan, Ayub Khan, and Babar Ali. 2022. “Chimeric Antigen Receptor T Cell Structure, Its Manufacturing, and Related Toxicities; A Comprehensive Review.” Advances in Cancer Biology - Metastasis 4 (July): 100035. https: / / doi.Org / 10.1016 / j.adcanc.2022.100035.Ang, Zhiwei, Luca Paruzzo, Katharina E. Hayer, Carolin Schmidt, Manuel Torres Diz, Feng Xu, Urvi Zankharia, et al. 2023. “Alternative Splicing of Its 5'-UTR Limits CD20 mRNA Translation and Enables Resistance to CD20-Directed Immunotherapies.” Blood 142 (20): 1724-39. https: / / doi.org / 10.1182 / blood.2023020400.Campo, Elias, Steven H. Swerdlow, Nancy L. Harris, Stefano Pileri, Harald Stein, and Elaine S. Jaffe. 2011. “The 2008 WHO Classification of Lymphoid Neoplasms and beyond: Evolving Concepts and Practical Applications.” Blood 117 (19): 5019-32. https: / / doi.org / 10.1182 / blood-2011-01-293050.Casan, J. M. L., J. Wong, M. J. Northcott, and S. Opat. 2018. “Anti-CD20 Monoclonal Antibodies: Reviewing a Revolution.” Human Vaccines & Immunotherapeutics 14 (12): 2820-41. https: / / doi.org / 10.1080 / 21645515.2018.1508624.Chmielewski, Markus, and Hinrich Abken. 2020. “TRUCKS, the Fourth-generation CAR T Cells: Current Developments and Clinical Translation.” Advances in Cell and Gene Therapy 3 (April). https: / / doi.org / 10.1002 / acg2.84.Clinical Review Report: Ofatumumab (Kesimpta): (Novartis Pharmaceuticals Canada Inc.): Indication: Multiple Sclerosis, Relapsing-Remitting. 2021. CADTH Common Drug Reviews. Ottawa (ON): Canadian Agency for Drugs and Technologies in Health. http: / / www.ncbi.nlm.nih.gov / books / NBK572484 / .Dondelinger, Mathieu, Patrice Filee, Eric Sauvage, Birgit Quinting, Serge Muyldermans, Moreno Galleni, and Marylene S. Vandevenne. 2018. “Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition.” Frontiers in Immunology 9 (October) :2278. https: / / doi.org / 10.3389 / fimmu.2018.02278.Dores, Graca M., William F. Anderson, Rochelle E. Curtis, Ola Landgren, Evgenia Ostroumova, Elizabeth C. Bluhm, Charles S. Rabkin, Susan S. Devesa, and Martha S. Linet. 2007. “Chronic Lymphocytic Leukaemia and Small Lymphocytic Lymphoma: Overview of the Descriptive Epidemiology.” British Journal of Haematology 139 (5): 809-19. https: / / doi.Org / 10.l ll l / j.1365-2141.2007.06856.x.Ghorashian, Sara, Anne Marijn Kramer, Shimobi Onuoha, Gary Wright, Jack Bartram, Rachel Richardson, Sarah J. Albon, et al. 2019. “Enhanced CAR T Cell Expansion and Prolonged Persistence in Pediatric Patients with ALL Treated with a Low-Affinity CD19 CAR.” Nature Medicine 25 (9): 1408-14. https: / / doi.org / 10.1038 / s41591-019-0549-5.Hauser, Stephen L., Ludwig Kappos, Amit Bar-Or, Heinz Wiendl, David Paling, Mitzi Williams, Ralf Gold, et al. 2023. “The Development of Ofatumumab, a Fully Human Anti-CD20 Monoclonal Antibody forPractical Use in Relapsing Multiple Sclerosis Treatment.” Neurology and Therapy 12 (5): 1491-1515. https: / / doi.org / 10.1007 / s40120-023-00518-0.Joly, Etienne, and Denis Hudrisier. 2003. “What Is Trogocytosis and What Is Its Purpose?” Nature Immunology 4 (9): 815-815. https: / / doi.org / 10.1038 / ni0903-815.Mackensen, Andreas, Fabian Muller, Dimitrios Mougiakakos, Sebastian Boltz, Artur Wilhelm, Michael Aigner, Simon Vblkl, et al. 2022. “Anti-CD19 CAR T Cell Therapy for Refractory Systemic Lupus Erythematosus.” Nature Medicine 28 (10): 2124-32. https: / / doi.org / 10.1038 / s41591-022-02017-5.Mao, Rui, Wanqing Kong, and Yukai He. 2022. “The Affinity of Antigen-Binding Domain on the Antitumor Efficacy of CAR T Cells: Moderate Is Better.” Frontiers in Immunology 13. https : / / www.frontiersin. org / article s / 10.3389 / fimmu.2022.1032403.Maude, Shannon L., Theodore W. Laetsch, Jochen Buechner, Susana Rives, Michael Boyer, Henrique Bittencourt, Peter Bader, et al. 2018. “Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia.” New England Journal of Medicine 378 (5): 439-48. https: / / doi.org / 10.1056 / NEJMoal709866.Neelapu, Sattva S., Frederick L. Locke, Nancy L. Bartlett, Lazaros J. Lekakis, David B. Miklos, Caron A. Jacobson, Ira Braunschweig, et al. 2017. “Axicabtagene Ciloleucel CAR T-Cell Therapy in Refractory Large B-Cell Lymphoma.” New England Journal of Medicine 377 (26): 2531-44. https: / / doi.org / 10.1056 / NEJMoal707447.Olson, Michael L., Erica R. Vander Mause, Sabarinath V. Radhakrishnan, Joshua D. Brody, Aaron P. Rapoport, Alana L. Welm, Djordje Atanackovic, and Tim Luetkens. 2022. “Low-Affinity CAR T Cells Exhibit Reduced Trogocytosis, Preventing Rapid Antigen Loss, and Increasing CAR T Cell Expansion.” Leukemia 6 (7): 1943-46. https: / / doi.org / 10.1038 / s41375-022-01585-2.Ramos, Carlos A., Barbara Savoldo, and Gianpietro Dotti. 2014. “CD19-CAR Trials.” Cancer Journal (Sudbury, Mass.) 20 (2): 112-18. https: / / doi.Org / 10.1097 / PPO.0000000000000031.Sterner, Robert C., and Rosalie M. Sterner. 2021. “CAR-T Cell Therapy: Current Limitations and Potential Strategies.” Blood Cancer Journal 11 (4): 1-11. https: / / doi.org / 10.1038 / s41408-021-00459-7.Trabolsi, Asaad, Artavazd Arumov, and Jonathan H. Schatz. 2024. “Bispecific Antibodies and CAR-T Cells: Dueling Immunotherapies for Large B-Cell Lymphomas.” Blood Cancer Journal 14 (1): 1-10. https: / / doi.org / 10.1038 / s41408-024-00997-w.Wang, Lixin, Chuling Fang, Qingzheng Kang, Wenfa Huang, Ziren Chen, Weiqiang Zhao, Lei Wang, et al. 2024. “Bispecific CAR-T Cells Targeting CD 19 / 20 in Patients with Relapsed or Refractory B Cell NonHodgkin Lymphoma: A Phase I / II Trial.” Blood Cancer Journal 14 (1): 1-13. https: / / doi.Org / 10.1038 / s41408-024-01105-8.SEQUENCE LISTINGNote: The CDR sequences in the Sequence Listing below (e.g., SEQ ID NOs: 1-6 and 10-15) are defined using the IMGT numbering scheme. It is understood that in other aspects, alternative CDR sequences may be defined based upon other numbering scheme known in the art (e.g., the Kabat, Chothia, and Martin numbering schemes) and constructs using one or more CDR sequences based on these alternative numbering schemes are also contemplated by the present disclosure.

Claims

CLAIMS1. A CD19 / CD20-binding domain comprising:(a) a CD 19 binding heavy chain variable (“CD 19 VH”) domain comprising one or more of the following complementarity determining regions (CDRs):CD 19 HCDR1 - GVSLPDYG (SEQ ID NO: 4),CD 19 HCDR2 - IWGSETT (SEQ ID NO: 5), andCD 19 HCDR3 - AKHYYYGGSYAMDY (SEQ ID NO: 6); and(b) a CD20 binding heavy chain variable (“CD20 VH”) domain comprising one or more of the following CDRs:CD20 HCDR1 - GFTFDDYA (SEQ ID NO: 13),CD20 HCDR2 - ISWNSGSI (SEQ ID NO: 14), andCD20 HCDR3 - AKDIQYGNYYYGMDV (SEQ ID NO: 15);(c) a CD19 binding light chain variable (“CD19 VL”) domain comprising one or more of the following CDRs:CD 19 LCDR1 - QDISKY (SEQ ID NO: 1),CD 19 LCDR2 - HTS (SEQ ID NO: 2), andCD 19 LCDR3 - QQGNTLPYT (SEQ ID NO: 3); and(d) a CD20 binding light chain variable (“CD20 VL”) domain comprising one or more of the following CDRs:CD20 LCDR1 - QSVSSY (SEQ ID NO: 10),CD20 LCDR2 - DAS (SEQ ID NO: 11), andCD20 LCDR3 - QQRSNWPIT (SEQ ID NO: 12).

2. The CD19 / CD20-binding domain according to claim 1, wherein the CD19 / CD20-binding domain is a tandem single-chain fragment variable (“scFv”).

3. The CD19 / CD20-binding domain according to claim 1, wherein the CD19 / CD20-binding domain is a diabody.

4. The CD19 / CD20-binding domain according to claim 1, wherein the CD19 / CD20-binding domain comprises a polypeptide sequence of: a) SEQ ID NO: 19 b) SEQ ID NO: 20; or c) SEQ ID NO: 19 and SEQ ID NO: 20.

5. A CD19 / CD20-targeting chimeric antigen receptor (CAR), comprising a CD19 / CD20-binding domain according to any one of claims 1 to 4, and a transmembrane domain.

6. The CD19 / CD20-targeting CAR of claim 5, wherein the CAR further comprises a spacer between the CD19 / CD20-binding domain and the transmembrane domain.

7. The CD19 / CD20-targeting CAR according to claim 6, wherein the spacer comprises an IgGl hinge or a CD8 stalk.

8. The CD19 / CD20-targeting CAR according to any one of claims 5 to 7, wherein the CAR further comprises an intracellular T-cell signaling domain.

9. The CD19 / CD20-targeting CAR according to claim 8, wherein the intracellular T-cell signaling domain comprises a 4-1BB endodomain and a CD3c endodomain.

10. The CD19 / CD20-targeting CAR according to claim 9, further comprising a P2A or a furin-P2A-furin linker, and an IL-18 polypeptide.

11. A nucleic acid molecule encoding the CD19 / CD20-binding domain according to any one of claims 1 to 4, or the CD19 / CD20-targeting CAR according to any one of claims 5 to 10.

12. A vector comprising a nucleic acid sequence encoding the CD19 / CD20-binding domain according to any one of claims 1 to 4, or the CD19 / CD20-targeting CAR according to any one of claims 5 to 10.

13. A recombinant cell comprising the CD19 / CD20-binding domain according to any one of claims 1 to 4, the CD19 / CD20-targeting CAR according to any one of claims 5 to 10, the nucleic acid molecule according to claim 11, or the vector according to claim 12.

14. A composition comprising a plurality of recombinant cells according to claim 13.

15. A method for making a recombinant cell according to claim 13, comprising transducing or transfecting a cell with a nucleic acid molecule according to claim 11 or a vector according to claim 12.

16. A method for making a composition according to claim 14, comprising transducing or transfecting ex vivo a plurality of cells obtained from a subject, with the nucleic acid molecule according to claim 11 or the vector according to claim 12.

17. A pharmaceutical composition, comprising: the CD19 / CD20-binding domain according to any one of claims 1 to 4, the CD19 / CD20-targeting CAR according to any one of claims 5 to 10, the nucleic acid molecule according to claim 11, the vector according to claim 12, the recombinant cell according to claim13, or the composition according to claim 14, together with a pharmaceutically acceptable carrier, diluent, or excipient.

18. The pharmaceutical composition according to claim 17 for use as a medicament.

19. The pharmaceutical composition according to claim 17 for use in the treatment of cancer and / or autoimmune disease.

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