Antigen binding molecules

Abstract

The disclosure relates to ROR1-specific binding molecules and chimeric antigen receptors (CARs), as well as immune effector cells, and their use in the treatment of cancer.

Description

[0001] ANTIGEN BINDING MOLECULES

[0002] Field of the Invention

[0003] The present invention relates to ROR1 -specific binding molecules and chimeric antigen receptors (CARs), and their use in the treatment of cancer.

[0004] Background of Invention

[0005] Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane receptor, consisting of an extracellular immunoglobin-like domain, a Frizzled domain (also called a cysteine-rich domain), a Kringle domain adjacent to the membrane, an intracellular tyrosine kinase domain and two serine / threonine-rich domains flanking a proline-rich domain. ROR1 plays a key role in embryonic and foetal development, but is expressed at low levels in healthy adults. Interestingly, ROR1 has been found to be overexpressed in many hematologic and solid tumours (Balakrishnan, A et al. 2017), and the high ROR1 expression in tumor cells is associated with adverse clinical prognosis. Furthermore, it has been shown that ROR1 signaling is crucial for promoting tumor growth and metastasis via the activation of multiple signaling pathways. ROR1 therefore represents a promising target for cancer therapy.

[0006] Chimeric antigen receptors (CARs) are synthetic receptors comprising an extracellular domain, often derived from an antibody single-chain variable fragment (scFv), and intracellular signalling and costimulatory domains derived from T cell receptors. Genetic insertion of CARs into immune cells can be used to develop cell-based approaches for treating cancer, including CAR-T cell (CAR-T) therapy. Anti-CD19 CAR T cells led to a paradigm change in cancer immunotherapy, based on their response rates in adult patients with recurrent / refractory diffuse large B cell lymphoma (DLBCL) or paediatric refractory B cell acute lymphoblastic leukaemia (B-ALL). Two CAR-T cell products specific for the B-cell marker CD 19, Kymriah (Novartis) and Yescarta (Kite Pharma), became the first therapeutic products registered by the FDA comprising a genetic engineering element for the treatment of B-ALL and DLBCL. However, despite the positive clinical outcomes of CAR-T therapy in the treatment of hematologic cancers, it remains a challenge to target solid tumours due to multiple reasons, including immunosuppressive tumour microenvironment and tumour cell heterogeneity, as well asthe poor persistence of CAR-T cells in vivo. Furthermore, CAR-T cell is often associated with severe toxicities, due to expression of antigens targeted by the CAR on normal healthy cells. Accordingly, there is still a need in the clinic for novel therapeutic strategies to target both hematologic and solid tumours with improved efficacy.

[0007] In immunoglobulin G (IgG) based CARs, the CH2 domain of IgG interacts with the Fcg receptor (FcgR) on monocytes, macrophages, and NK cells. FcgR binding to the IgG-based CAR T cells leads to off-tumour site and cancer antigen independent activation of CAR T cells that may lead to unwarranted toxicities, and reduced CAR T cell activity. (Almasbak et al 2015, Hornbach et al 2010, Hudecek et al 2015). The unwanted interactions with off-target cells and the conceivable side effects must be avoided to achieve functional therapeutic CAR T cells. In an attempt to address this problem, CARs comprising an inert and modifiable spacer were designed (WO2022 / 129692 Al, Koski J. et al 2022). The spacer was based on Ig-like Cl domains of signal -regulatory protein alpha (SIRP-alpha), resulting in a novel CAR backbone. These novel CARs, with inert SIRP-alpha spacer, evaded off-target binding by Fcg receptor (FcgR) expressing cells. However, the effect of the SIRP-alpha based spacers on clinical outcomes following CAR-T therapy has not been tested. Furthermore, the SIRP-alpha based spacers have yet to be tested in CAR constructs which specifically bind to ROR1.

[0008] Summary of Invention

[0009] It is an object of the invention to develop anti-RORl antigen-binding molecules, including ROR1 -specific CARs and immune effector cells engineered to expressed anti-RORl CARs, for treating cancer. Furthermore, most suitable ROR1 binding domains were combined with SIRP-alpha based spacers to study enhanced functionality of CAR T-cells targeting ROR1.

[0010] The inventors have generated antibodies and fragments thereof that bind to ROR1 but not to ROR2. Biophysical and functional characterization studies were performed to find antibodies and scFvs. Following epitope mapping studies, the selected scFvs were found to bind to specific amino acid residues in the human ROR1 Frizzled domain, which represent novel discontinuous epitopes. Chimeric antigen receptor (CAR) constructs were designed based on the scFv sequences identified, as well as known ROR1 scFvs, and theresulting CAR-encoding constructs were cloned into lentiviral vectors. CAR-T cells expressing the CARs of interest were generated by transducing T cells with the lentiviral vectors which encode the CARs. The inventors studied a large number of CARs containing different antigen-binding domains to identify antigen-binding domains which confer the CAR-T cells with optimal characteristics, such as viability, CAR expression, vector copy number, cell killing efficiency, effector cytokine release (for example IFN-gamma). The inventors then studied a number of different CAR structures with different functional elements and showed that particular CAR structures have optimal properties. For example, R0R1 -specific CAR structures which included a SIRP-alpha based spacer were shown to increase the relative proportion of CD4+ T cells by at least 10% in the transduced T cell population compared to non-transduced cells. The inventors also found that a number of the ROR1 -specific CAR T cells identified showed improved anti -tumor efficacy in vivo, including in solid tumors. In particular, a number of the ROR1 -specific CAR T cells identified were shown to reduce tumor volume. Furthermore, a number of the ROR1-specific CAR T cells identified displayed improved capability to expand, and maintained viability during expansion, freezing and thawing. The ROR1 -specific CAR T cells enabled the killing of ROR1 -expressing cells in target specific manner but not causing high activity without antigen binding.

[0011] Accordingly, the invention provides a ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising an anti-RORl antigen-binding domain; (b) an extracellular spacer which comprises at least one Ig-like Cl domain of signal -regulatory protein alpha (SIRPa) or a fragment or variant thereof; (c) a transmembrane domain; and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

[0012] The invention also provides a ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising a ROR1 -specific antigen-binding domain comprising three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within the HCVR / LCVR pair of SEQ ID NOs: 73 / 74; (b) an extracellular spacer which comprises at least one Ig-like Cl domain of signal-regulatory protein alpha (SIRPa) or a fragment or variant thereof; (c) a transmembranedomain; and (d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

[0013] In one embodiment, the Ig-like Cl domain of SIRPa is selected from (i) type 1 domain according to SEQ ID NO: 163; or (ii) type 2 domain according to SEQ ID NO: 164. In one embodiment, the extracellular spacer comprises Ig-like Cl type 1 domain and Ig-like Cl type 2 domain of SIRPa. In one embodiment, the extracellular spacer further comprises at least one multimerization domain. In one embodiment, the IgG hinge region is an IgG4 hinge region according to SEQ ID NO: 170 or a fragment or variant thereof. In one embodiment, the extracellular spacer is located between a transmembrane domain and the antigen binding domain and connects them. In one embodiment, the transmembrane domain comprises transmembrane domain of CD28 according to SEQ ID NO: 171. In one embodiment, the intracellular signaling domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof. In another embodiment, the co-stimulatory domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof. In one embodiment, the intracellular signaling domain comprises the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof. In another embodiment, the co-stimulatory domain comprises the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof. In a further embodiment, the intracellular signaling domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof and the co-stimulatory domain comprises the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof.

[0014] The invention also provides a ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising an anti-RORl antigen-binding domain, which binds to an epitope of the human ROR1 Frizzled domain comprising at least one amino acid residue selected from E26, M30, E33, Q37, D82, Ml 11, R112, KI 14, LI 15 and D120; (b) a transmembrane domain; and (c) a cytoplasmic domain comprising a costimulatory domain and a signaling domain. In one embodiment, the anti-RORl antigen-binding domain binds to an epitope of the human ROR1 Frizzled domain comprising the amino acid residues M30 and Ml 11. In apreferred embodiment, the anti-RORl antigen-binding domain binds to an epitope of the human R0R1 Frizzled domain comprising the amino acid residues M30, Q37 and Ml 11.

[0015] In one embodiment, the R0R1 -specific CAR disclosed herein does not bind to R0R2.

[0016] In one embodiment, the anti-RORl antigen-binding domain is a single chain variable fragment (scFv) comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR). In one embodiment, the anti-RORl antigen-binding domain comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within a HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162. In a further embodiment, the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 43-44-45, 51-52-53, 59-60-61, 67-68-69, 75-76-77, 83-84-85, 91-92-93, 99-100-101, 107-108-109, 115-116-117, 123-124-125, 131-132-133, 139-140-141, 147-148-149 or 155-156-157, and the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 46-47-48, 54-55-56, 62-63-64, 70-71-72, 78-79-80, 86-87-88, 94-95-96, 102-103-104, 110-111-112, 118-119-120, 126-127-128, 134-135-136, 142-143-144, 150-151-152 or 158-159-160. In one embodiment, the antigen-binding domain comprises an HCVR / LCVR pair having at least 95% identity to an HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162. In a further embodiment, the antigen-binding domain comprises or consists of an HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162.

[0017] The invention also provides a ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C-terminus: (a) an extracellular ligand-binding domain comprising an anti-RORl antigen-binding domain, which comprises comprising three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within the HCVR / LCVR pair of SEQ ID NOs: 73 / 74; (b) a transmembrane domain; and (c) a cytoplasmic domaincomprising a costimulatory domain and a signaling domain. In one embodiment, the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 67-68-69, and the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 70-71-72. In a further embodiment, the antigen-binding domain comprises a HCVR / LCVR pair having at least 95% identity to a HCVR / LCVR pair of SEQ ID NOs: 73 / 74. In a further embodiment, the antigen-binding domain comprises or consists of the HCVR / LCVR pair of SEQ ID NOs: 73 / 74.

[0018] The invention also provides a chimeric antigen receptor (CAR) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35.

[0019] The invention also provides an isolated nucleic acid molecule encoding the CAR disclosed herein. In one embodiment, the nucleic acid molecule comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

[0020] The invention also provides a vector comprising the nucleic acid molecule disclosed herein.

[0021] The invention also provides an engineered cell comprising a CAR disclosed herein, a nucleic acid molecule disclosed herein, or a vector disclosed herein. In one embodiment, the cell is a T cell, a natural killer cell, a natural killer T cell or a macrophage.

[0022] The invention also provides a pharmaceutical composition comprising the engineered cell disclosed herein and a pharmaceutically acceptable carrier.

[0023] The invention also provides an engineered cell disclosed herein or a pharmaceutical composition disclosed herein for use as a medicament. In one embodiment, the use is in the treatment of a ROR1 -expressing cancer. In one embodiment, the ROR1 -expressing cancer is a solid cancer. In one embodiment, the solid cancer is breast cancer, ovarian cancer, endometrial cancer, gastric cancer, non-small cell lung cancer, renal cell carcinoma or melanoma. In another embodiment, the ROR1 -expressing cancer is a liquid cancer. In one embodiment, the liquid cancer is chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL) or myeloid leukemia. In one embodiment, the myeloid leukemia is acute myeloid leukemia.The invention also provides a method of preparing an engineered cell, comprising introducing the nucleic acid disclosed herein, or the vector disclosed herein, into a cell. The invention also provides a R0R1 -specific antigen-binding molecule, which binds to an epitope of human R0R1 comprising the amino acid residues M30 and Ml 11. In a preferred embodiment, the antigen-binding molecule binds to an epitope of human R0R1 comprising the amino acid residues M30, Q37 and Ml 11. In one embodiment, the antigenbinding molecule does not bind to R0R2. In one embodiment, the antigen-binding molecule comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within the HCVR / LCVR pair of SEQ ID NOs: 73 / 74. In one embodiment, the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 67-68-69, and the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 70-71-72. In one embodiment, the antigenbinding molecule comprises an HCVR / LCVR pair having at least 95% identity to the HCVR / LCVR pair of SEQ ID NO: 73 / 74. In a preferred embodiment, the antigen-binding molecule comprises or consists of the HCVR / LCVR pair of SEQ ID NO: 73 / 74.

[0024] These and further aspects of the invention are described in more detail herein.

[0025] Brief Description of Figures

[0026] Figure 1: Cell surface expression of ROR1 on A549, NCI-H1975, PEO1, PANC-1, PEO-4, SK-MEL-5, HTC-116 cells was measured with flow cytometry.

[0027] Figure 2: ROR1 expression was studied on CD19+, CD3+ and CD19-CD3-immune cell populations of peripheral blood mononuclear cells (PBMCs) from CLL patients (Discovery Life Sciences) and on PBMCs of healthy controls. The stained cells were analysed by flow cytometry. ROR1 is highly expressed on CD 19+ B cells in CLL patient PBMC samples but not in healthy donor PBMC samples. ROR1 expression in CD3+ T cells is similar in CLL patient PBMCs and healthy PBMCs.

[0028] Figure 3: A) IHC against human ROR1 with rabbit monoclonal DB04C07 antibody in cell pellet samples processed to formalin-fixed paraffin-embedded (FFPE) samples. ROR1 expression is apparent in CHO cell expressing ROR1 (CHO-ROR1, arrows), while no staining / cross-reactivity is seen in ROR1 -negative CHO cells alone or CHO cellsexpressing R0R2. B) IHC against human R0R1 with DB04C07 in H1975 xenografts showing membrane-enriched expression and more cytoplasmic staining is present in MC38-ROR+ and TNBC xenografts. C) IHC against human R0R1 with DB04C07 and DB03B 11 antibodies in cell pellet samples processed to formalin-fixed paraffin-embedded (FFPE) samples. R0R1 expression is apparent in CHO cell expressing R0R1 (CH0-R0R1, arrows), while no staining / cross-reactivity is seen in R0R1 -negative CHO cells alone or CHO cells expressing R0R2.

[0029] Figure 4: Epitope mapping. A) Structural representation showing key amino acids Met-30, Gln-37, Met-111 in the R0R1 Frizzled domain epitope of binder DB04C07. B) Structural representation showing corresponding epitope of binder DB04C07 on the surface of the R0R1 Frizzled domain. C) Structural representation showing key amino acids Glu-26, Arg-112, Lys-114, Leu-115 in the R0R1 Frizzled domain epitopes of binder DB04D03.

[0030] D) Structural representation showing corresponding epitope of binder DB04D03 on the surface of the R0R1 Frizzled domain. E) Structural representation showing key amino acids Glu-26, Gln-37, Asp-82, Arg- 112, Lys-114, Leu- 115, Asp-120 intheRORl Frizzled domain epitopes of binder DB01D06. F) Structural representation showing corresponding epitope of binder DB01D06 on the surface of the R0R1 Frizzled domain. G) Structural representation showing key amino acids Glu-33, Gln-37, Lys-114 in the R0R1 Frizzled domain epitopes of binder DB01C11. H) Structural representation showing corresponding epitope of binder DB01C11 on the surface of the R0R1 Frizzled domain.

[0031] Figure 5: CAR expansion. The T cells were isolated and activated (days in vitro 0, DIVO) and subsequently transduced with lentiviral vectors carrying various CAR gene cassettes and were let to expand for 10 days (DIVIO). A) The cell amount, relative to the amount of T cells at DIV6 and DIVIO, was measured as well as the B) viability of the cells.

[0032] Figure 6: CAR expression. Lentiviral vector mediated CAR gene cassette transduction was measured for A) gene integration, vector copy number, with digital droplet PCR measuring integrated transgene in relation to housekeeping gene in the cell population and for B) CAR expression with flow cytometry using a fluorophore labelled ROR1.

[0033] Figure 7: Killing efficacy. CAR T (effector) and ROR1 positive cancer (target) cell were co-cultured with various effector to target ratios to determine the effectiveconcentration of killing at various timepoints. The target cells were labelled with fluorescence marker to study the killing efficacy.

[0034] Figure 8: IFN gamma release upon target engagement. After 24 hours co-culture of CAR T cells and R0R1 positive target cells, the supernatant was collected and measured for IFN-gamma (pg / ml).

[0035] Figure 9: Non-stimulated activity of CAR T cells. CAR T cells, after thaw, were incubated without activation in plain, non-supplemented, TexMACS medium for 24 hours and then supernatant was measured for INF-gamma. The INF-gamma amount was normalized to ORC-1035, a CAR structure having measurable non-stimulated residual activity.

[0036] Figure 10: Interferon gamma release against target cells representing cancer cells of various origin. CAR T cells and ROR1 -positive target cells were co-cultured for 24 hours after which supernatant was collected and measured for IFN-gamma.

[0037] Figure 11: Perforin, Granzyme B, Interleukin 2 and Interferon gamma release after 24 hour co-culture. CAR T cells and A549, a ROR1 expressing cancer cell line, were cocultured at 1 to 1 ratio of cells for 24 hours and subsequently measured with multiplex for A) perforin, B) granzyme B, C) interleukin 2 and D) interferon gamma amounts from the culture supernatant.

[0038] Figure 12: Effect of extracellular ROR1 to CAR structures. T and CAR T cells were incubated with various concentrations of recombinant protein containing extracellular domains of ROR1. After 24 hour incubation, the supernatant was collected and measured for IFN gamma amount.

[0039] Figure 13: Soluble ROR1 in plasma. Samples representing various cancer types were studied for soluble ROR1 expression.

[0040] Figure 14: Soluble ROR1 effect on CAR T survival. T and CAR T cells were thawn and incubated in the presence of variable amounts of recombinant protein containing the extracellular domains of (s)RORl. At the mentioned timepoints post thaw, days in vitro (DIV), the viable cell amount was measured. The relative fold expansion is normalized to the cell density at the start of the experiment compared live cell density at the measured timepoint. In addition to CAR T cells and sRORl, the mixture was studied in co-culture with ROR1 expressing cell line NCI-H1975 for IFN-gamma release and target cell killing.Figure 15: CAR T killing of Jekol cells. Jekol (target) cells, expressing luciferase, and CAR T (effector) cells were co-cultured for 24 hours after which the cells were lysed and measured for luciferase to calculate cell killing relative to Jekol cells only.

[0041] Figure 16: CAR expression. CAR expression was studied with flow cytometry using a fluorophore labelled R0R1. Lentiviral vector mediated CAR gene cassette transduction was measured for gene integration, vector copy number (VCN), with digital droplet PCR measuring integrated transgene in relation to housekeeping gene in the cell population Figure 17: CAR T killing of R0R1 positive target cells. A) NCI-H1975 (H1975 cell for short) and B) breast cancer cells were plated on impedance measuring plates and after 1 day culturing, CAR T cells were added to the cells with varying amounts. Killing efficacy of the CAR T cells was measured from the impedance signal relative to A) target cells only or B) for the 50% effective concentration of effector cells for breast cancer cells, at the different time-points.

[0042] Figure 18: CAR T responses to target positive and negative cancer cells. CAR T and R0R1 expressing A549 cells and their R0R1 -knockout counterpart were co-cultured for 24 hours and supernatant was studied for IL-2, Granzyme B and Perforin.

[0043] Figure 19: Various R0R1 expression levels and their effect on CAR T response. Cancer cells expressing various levels of R0R1 were subjected to co-culture with CAR T cells and after 24 hour incubation, the supernatant was studied for IFN-gamma release from the CAR T cells.

[0044] Figure 20: CD4 and CD8 populations in CAR T expansions. Expanded CAR T cells were studied for CD4 and CD8 expression in CD3 positive population. Two donors A and B shown.

[0045] Figure 21: Phenotyping of CAR T cells. Expanded CAR T cells were studied for their HLA-DR (A), 4-1BB (B), CD69 (C), PD-1 (D) and LAG3 (E) populations with flow cytometry. Two donors are shown in the figure.

[0046] Figure 22: Individual Jeko-1 tumor burden curves for the mice treated with PBS, NT T cells, ORC-2025, ORC -2091, ORC, 2027 and ORC-1023. Tumor burden, expressed in total flux (photons / s), are shown as a function of time (days post treatment). Individual values are shown. n=9 mice per group.Figure 23: Average Jeko-1 tumor burden in mice groups treated with PBS, NT T cells, ORC-2025, ORC -2091, ORC, 2027 and ORC-1023. Average tumor burden, expressed in total flux (photons / s), are shown as a function of time (days post treatment). Data presented as Mean ±SEM. n=9 mice per group.

[0047] Figure 24: Cytokine levels in serum on day 10 post treatment in mice treated with PBS, cetuximab, NT T cells, ORC-2025, ORC-1054, ORC-1023 and ORC-1055.

[0048] Figure 25: Gene expression on day 10 post treatment in tumors from the mice treated with PBS, cetuximab, NT T cells, ORC-2025, ORC-1054, ORC-1023 and ORC-1055. Expression of selected genes IFNG, GZMB, PRF1, IL2, PDCD1, CTLA4, TIM3, LAG3 and TIGIT in different study groups is represented in the figure.

[0049] Figure 26: Differential gene expression analysis of NT T cells versus different CAR T cell treatments. Volcano plots display the log2 fold change in gene expression (x-axis) against the -loglO p-value (y-axis) for each comparison: ORC-2025, ORC-1054, ORC-1023 and ORC-1055.

[0050] Figure 27: Individual H1975 tumor growth curves for the mice treated with PBS, NT T cells, ORC-2025, ORC-1054, ORC-1023. Tumor volumes, expressed in mm3, are shown as a function of time (days post treatment). Individual values are shown. n=10 mice per group.

[0051] Figure 28: Average H1975 tumor growth in mice groups treated with PBS, NT T cells, ORC-2025, ORC-1054, ORC-1023. Average tumor volumes, expressed in mm3, are shown as a function of time (days post treatment). Data presented as Mean ±SEM. n=10 mice per group.

[0052] Figure 29: Absolute CD3, CD4 and CD8 T cell counts per ml of blood at the end of efficacy study in mice treated with NT T cells, ORC-2025, ORC-1054, ORC-1023. A Kruskal-Wallis test followed by a Dunn’s multiple comparisons test was used to compare the treated groups. * p < 0.05, *** p < 0.001

[0053] Figure 30: Individual H1975 tumor growth curves for the mice treated with PBS, NT T cells, ORC-10546xlOA6 CAR T cells, ORC-10543xlOA6 CAR T cells, ORC-1054 lxlOA6 CAR T cells, ORC-1023 6xlOA6 CAR T cells, ORC-1023 3xlOA6 CAR T cells, ORC-1023 lxlOA6 CAR T cells, ORC-2025 6xlOA6 CAR T cells, ORC-2025 3xlOA6 CAR T cells, ORC -2025 lx!0A6 CAR T cells. Tumor volumes, expressed in mm3, areshown as a function of time (days post treatment). Individual values are shown. n=9 mice per group

[0054] Figure 31: Average H1975 tumor growth in mice groups treated with PBS, NT T cells, ORC-10546xlOA6 CAR T cells, ORC-10543xlOA6 CAR T cells, ORC-1054 lxlOA6 CAR T cells, ORC-1023 6xlOA6 CAR T cells, ORC-1023 3xlOA6 CAR T cells, ORC-1023 lxlOA6 CAR T cells, ORC-2025 6xlOA6 CAR T cells, ORC-2025 3xlOA6 CAR T cells, ORC -2025 lxlOA6 CAR T cells. Average tumor volumes, expressed in mm3, are shown as a function of time (days post treatment). Data presented as Mean ±SEM. n=9 mice per group

[0055] Detailed Description

[0056] It is to be understood that different applications of the disclosed methods and products may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the disclosure only, and is not intended to be limiting.

[0057] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0058] General definitions

[0059] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure belongs.

[0060] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes two or more such cells, and the like.

[0061] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase a method is one “comprising” particular steps, should be interpreted to mean that the method includes those steps, but the method may comprise further steps. The terms “comprising” or “comprises” may be replaced by “consisting of’ or “consisting essentially of’, unless the context dictates otherwise.It is to be understood that the term “nucleic acid” and the term “polynucleotide” are used interchangeably herein.

[0062] For the purpose of this invention, in order to determine the percent identity of two sequences (such as two nucleic acid sequences or two amino acids sequences), the sequences are aligned for optimal comparison purposes (e.g. gaps can be introduced in a first sequence for optimal alignment with a second sequence). The nucleotide or amino acid residues at each position are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the nucleotides or amino acids are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions in the reference sequence x 100).

[0063] Typically, the sequence comparison is carried out over the length of the reference sequence. For example, if the user wished to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. To assess whether a sequence is at least 95% identical to SEQ ID NO: 1 (an example of a reference sequence), the skilled person would carry out an alignment over the length of SEQ ID NO: 1, and identify how many positions in the test sequence were identical to those of SEQ ID NO: 1. If at least 95% of the positions are identical, the test sequence is at least 95% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, the gaps or missing positions should be considered to be non-identical positions.

[0064] The skilled person is aware of different computer programs that are available to determine the homology or identity between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In an embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (1970) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.As used herein, the term “abou may be interpreted to mean a value within + / -10% of the recited value.

[0065] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0066] A “chimeric antigen receptor” or “CAR” is a non-naturally occurring receptor protein which binds to a specific antigen and participates in cell activation. CARs generally comprise an extracellular portion comprising an antigen binding domain (also known as a “ligand binding domain”) specific for an antigen, linked via a spacer domain and a transmembrane domain to an intracellular cytoplasmic signalling domain comprising one or more signalling moieties. A CAR may also comprise an intracellular co- stimulatory domain.

[0067] As used herein, an “extracellular spacer domain” (also known to as a “spacer” or “extracellular spacer”) is a portion of a CAR located between the transmembrane domain and the antigen binding domain and connects them. The spacer domain has a role in finetuning the signalling of the CAR.

[0068] As used herein, the transmembrane domain refers to the portion of a CAR which spans the cell membrane, for example the cell membrane of a eukaryotic cell, and serves to transmit activation signals to the cytoplasmic signalling domains following ligand binding of the extracellular antigen -binding domains (e.g., scFv).

[0069] As used herein, the term, “cytoplasmic domain” refers to the intracellular portion of a CAR which comprises the intracellular signaling domain and the costimulatory signaling domain. The “intracellular signaling domain” or “signaling domain” refers to the part of the chimeric antigen receptor protein that participates in transducing the message of effective CAR binding to a target antigen (z.e., ROR1) into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited with antigen binding to the extracellular CAR domain. The term “effector function” refers to a specialized function of the cell. Effector function of the T cell, for example, may be cytolytic activity or help or activity including the secretion of a cytokine. Thus, the terms “intracellular signaling domain” or “signaling domain” used interchangeably herein, refer to the portion of aprotein which transduces the effector function signal and that directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of the entire domain as long as it transduces the effector function signal. The term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transducing effector function signal. The intracellular signaling domain is also known as the “signal transduction domain” and is typically derived from portions of the human CD3 or FcRy chains. The intracellular signalling domain of a CAR of the invention may comprise any signalling domain known in the art.

[0070] As used herein, the term, "costimulatory signaling domain," or "costimulatory domain", refers to the portion of a CAR comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Examples of such co-stimulatory molecules include CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, B7-H2 and a ligand that specifically binds CD83. Accordingly, while the present disclosure provides exemplary costimulatory domains derived from CD28 and 4- IBB, other costimulatory domains are contemplated for use with the CARs described herein. The inclusion of one or more co-stimulatory signaling domains may enhance the efficacy and expansion of T cells expressing CAR receptors. The intracellular signaling and costimulatory signaling domains may be linked in any order in tandem to the carboxyl terminus of the transmembrane domain.

[0071] CARs are typically expressed in immune effector cells. Cells expressing a CAR are able to bind specifically to a predetermined antigen (e.g., R0R1) resulting in activation of the cells. CAR-expressing cells are preferably CD3 T cells, naive T cells, memory T cells or effector T cells.

[0072] The antigen binding domain of a CAR specifically binds to a predetermined antigen. In particular, the antigen binding domain of a CAR specifically binds to an epitope of said antigen. The antigen binding domain is also referred to herein as the ligand bindingdomain. The antigen recognised by the CARs of the invention is the R0R1 protein, i.e. the antigen binding domain of the CARs of the invention specifically binds to R0R1. The expression “R0R1”, as used herein, refers to receptor tyrosine kinase-like orphan receptor 1. R0R1 (also known as NTRKR1) is a transmembrane glycoprotein that plays a central role in embryonic and foetal development and is found to be expressed at very low levels in human adult tissues. As used herein, “R0R1” typically refers to a human R0R1 protein unless specified as being from a non-human species (e.g., “mouse R0R1”, “monkey R0R1”, etc.). A preferred human R0R1 protein has an amino acid sequence shown in SEQ ID NO: 175. The Frizzled domain of the above human R0R1 protein has an amino acid sequence shown in SEQ ID NO: 174.

[0073] The term "antibody", as used herein, means any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., R0R1). The term "antibody" includes immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). The term “antibody” also includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CHI, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0074] The terms "antigen -binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of anantibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Such DNA is known and / or is readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0075] By “specific” or “specifically binds”, it is meant that the relevant antigen-binding domain of a CAR or an antigen binding molecule of the invention binds to one or more antigenic determinants of the desired antigen (i.e., R0R1) and does not bind or does not bind significantly to other polypeptides. For example, a CAR that specifically binds R0R1 may bind to R0R1 but not to a different polypeptide such as bovine serum albumin. A CAR may specifically bind if it binds to R0R1 with a stronger affinity when compared to binding an antigen of a different polypeptide such as bovine serum albumin. Methods for measuring the affinity of binding are well known in the art. For example, binding affinity may be quantified by determining the dissociation equilibrium constant (KD), dissociation rate constant (Kd) and / or association rate constant (Ka) for an antigen-binding domain and its target. Similarly, the specificity of binding of an antigen-binding domain to its target may be defined in terms of the comparative binding affinity of the antigen-binding domain for its target as compared to binding affinity with respect to the antigen-binding domain and another, non-target molecule. The dissociation equilibrium constant (KD) (M) refers to the dissociation equilibrium constant of a particular antigen-binding protein: antigen interaction. In particular, the KD represents the ratio of the free concentrations of antigenbinding protein and antigen to the concentration of the antigen-binding protein: antigen complex (at equilibrium). There is an inverse relationship between KD and binding affinity; thus the smaller the KD, the higher, i.e., stronger, the affinity. The dissociation rate constant (Kd) (sec -1 or 1 / s) refers to the dissociation rate constant of a particular antigenbinding protein: antigen interaction, and may also be referred to as kofif The association rate constant (Ka) (M-l x sec -1 or 1 / M) refers to the association rate constant of aparticular antigen-binding protein: antigen interaction, and may also be referred to as kon. Methods for measuring the affinity of binding are well known in the art. The affinity of binding may be measured for example by surface plasmon resonance or bio-layer interferometry. For instance, binding affinity may correspond to a KD value of about 10'7M or less, such as about 10'8M or less, such as about 10'9M or less when determined by, for instance, surface plasmon resonance using the antigen as ligand and the antigenbinding protein as analyte.

[0076] An “epitope” refers to an antigenic determinant that interacts with a specific antigen binding site in the variable region of an antigen binding domain therefor, also known as a paratope. An antigen may have more than one epitope. Thus, different antigen-binding domains may bind to different areas on an antigen and may have different biological effects. 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. In certain circumstances, an epitope may include moieties of saccharides, phosphoryl groups, or sulfonyl groups on the antigen.

[0077] CARs

[0078] The invention relates to CARs which specifically bind to R0R1. In particular, a CAR of the invention comprises:

[0079] i) an antigen binding domain which specifically binds to R0R1;

[0080] ii) a transmembrane domain; and

[0081] iii) a cytoplasmic domain comprising a costimulatory domain and a signalling domain.

[0082] The antigen binding domain may be selected from any domain able to specifically bind a target antigen, i.e. R0R1. For example, the antigen binding domain may comprise one or more immunoglobulin variable domains. The antigen binding domain may comprise a heavy chain variable domain and / or a light chain variable domain, each comprising three complementarity determining regions (CDRs). The antigen binding domain may be a single chain variable fragment (scFv) (typically arranged VH-VL in an N- to C-terminal orientation), a bivalent scFv (sc(Fv)2), single chain antibody fragment (scAb), amonoclonal antibody (comprising 2 heavy chains and 2 light chains), a polyclonal antibody, a chimeric antibody, a bispecific antibody, a camelid antibody, a Fab, a Fab’, a F(ab’)2 fragment, a heavy chain variable domain (VH) or a nanobody (VHH). Preferably, the antigen binding domain of a CAR of the invention is a single chain variable fragment (scFv). An scFv comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) of an immunoglobulin, connected by a (typically short) linker peptide. The scFv may be derived from a human immunoglobulin. Whilst an scFv is typically arranged HCVR-LCVR in an N-terminal to C-terminal orientation, antigen binding regions arranged LCVR-HCVR in an N-terminal to C-terminal orientation are also encompassed according to the invention. A CAR may comprise more than one antigen binding domain, such as two antigen-binding domains or three extracellular antigen binding domains. The two or more extracellular antigen binding domains may bind to different antigens, i.e. the CAR may be bispecific or multispecific.

[0083] The transmembrane domain serves to transmit activation signals to the cytoplasmic signal transduction domains following binding of the antigen to the antigen-binding domain. The transmembrane domain may be derived from a naturally occurring transmembrane protein, such as a type-I transmembrane protein. The transmembrane domain is typically the transmembrane domain of CD28 or CD8a. The transmembrane domain may be a transmembrane domain of the a, P, 5 or y subunits of the T-cell receptor, CD3s, CD3< CD4, CD6, CD8a, CD28, CD86, OX-40, 4-1BB or CD40L (CD154).

[0084] The cytoplasmic domain comprises a costimulatory domain and a signalling domain to activate the immune effector cell following engagement of the antigen binding domain.

[0085] The CAR may further comprise a signal peptide (otherwise referred to herein as a leader sequence). A nucleic acid molecule encoding the CAR typically encodes a signal peptide. A signal peptide targets the CAR to the endoplasmic reticulum during translation, and is typically co-translationally cleaved at a signal peptidase cleavage site. This enables cell surface expression of the CAR. Any suitable signal peptide may be used in the CAR (e.g. the signal peptide may have the amino acid sequence of SEQ ID NO: 177 or 178). Typically, the CAR referred to herein is mature and does not comprise the sequence cleaved by signal peptidase.Antigen binding domains

[0086] The inventors have identified a number of preferred antigen binding domains for R0R1 and characterised their amino acid sequences and nucleic acid sequences encoding them. They have also characterised preferred combinations of the preferred antigen binding domains with other functional domains for use in a CAR.

[0087] Accordingly, the antigen binding domain of a CAR of the invention may specifically bind to an epitope of human R0R1 which comprises at least one amino acid residue selected from E26, M30, E33, Q37, D82, Ml 11, R112, KI 14, LI 15 and DI 20, or amino acid residue(s) corresponding thereto, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. The antigen binding domain of the CAR may specifically bind to an epitope of human ROR1 which comprises two, three, four, five, six or seven of these residues. In one embodiment, the antigen binding domain of the CAR of the invention specifically binds to an epitope of human ROR1 which comprises amino acid residues E26, R112, KI 14 and LI 15, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigen binding domain of the CAR of the invention specifically binds to an epitope of human ROR1 which comprises amino acid residues E26, Q37, D82, R112, KI 14, LI 15 and D120, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigen binding domain of the CAR of the invention specifically binds to an epitope of human ROR1 which comprises amino acid residues E33, Q37 and KI 14, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigen binding domain of the CAR of the invention specifically binds to an epitope of human ROR1 which comprises the amino acid residues of M30 and Ml 11, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. In a preferred embodiment, the antigen binding domain of the CAR of the invention specifically binds to an epitope of human ROR1 which comprises amino acid residues M30, Q37 and Ml 11, wherein the amino acid numbering is based on the human ROR1 Frizzled domain, as shown in SEQ ID NO: 174. An antigen binding domain of a CAR of the invention may bind to an epitope comprisingcorresponding amino acid residues to those specified above in any human R0R1 amino acid sequence. Various techniques known in the art may be used to determine specific binding of the antigen binding domain to particular amino acid residues, such as crossblocking assays, alanine scanning mutational analysis or hydrogen / deuterium exchange detected by mass spectrometry.

[0088] The invention also provides a CAR comprising an antigen binding domain (specific for R0R1) comprising three heavy chain CDRs (HCDRs) contained with a HCVR selected from any of SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153 and 161, and the three light chain CDRs (LCDRs) contained within a LCVR selected from any of SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, 146, 154 and 162. Typically, the heavy chain CDRs and light chain CDRs are selected from those contained within the HCVR / LCVR pairs represented by SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154, and 161 / 162. The CDRs contained within a HCVR / LCVR may be identified according to the Kabat definition, the Chothia definition or the IMGT definition (see, for example, Kabat, Elvin Abraham 1991; Lefranc, Marie-Paule, et al. 2003)

[0089] The invention also relates to a CAR comprising an antigen binding domain (specific for ROR1) comprising an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination selected from the following combinations of CDR sequences: SEQ ID NOs: 43-48, SEQ ID NOs: 51-56, SEQ ID NOs: 59-64, SEQ ID NOs: 67-72, SEQ ID NOs: 75-80, SEQ ID NOs: 83-88, SEQ ID NOs: 91-96, SEQ ID NOs: 99-104, SEQ ID NOs: 107-112, SEQ ID NOs: 115-120, SEQ ID NOs: 123-128, SEQ ID NOs: 131-136, SEQ ID NOs: 139-144, SEQ ID NOs: 147-152 and SEQ ID NOs: 155-160. Preferred HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combinations are SEQ ID NOs: 43-48, SEQ ID NOs: 51-56, SEQ ID NOs: 59-64, SEQ ID NOs: 67-72, SEQ ID NOs: 75-80, SEQ ID NOs: 83-88, SEQ ID NOs: 91-96, SEQ ID NOs: 99-104, SEQ ID NOs: 107-112, SEQ ID NOs: 115-120, SEQ ID NOs: 123-128, SEQ ID NOs: 131-136 and SEQ ID NOs: 139-144. A particularly preferred HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination is SEQ ID NOs: 67-72.

[0090] The antigen binding domain may comprise a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination as disclosed above andcomprise an HCVR and LCVR having at least 95% sequence identity to the HCVR and LCVR from which the CDRs are derived. Thus, for example, the antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 43-48 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 49 and an LCVR having at least 95% sequence identity to SEQ ID NO: 50. The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 51-56 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 57 and an LCVR having at least 95% sequence identity to SEQ ID NO: 58.

[0091] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 59-64 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 65 and an LCVR having at least 95% sequence identity to SEQ ID NO: 66.

[0092] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 67-72 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 73 and an LCVR having at least 95% sequence identity to SEQ ID NO: 74.

[0093] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 75-80 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 81 and an LCVR having at least 95% sequence identity to SEQ ID NO: 82.

[0094] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 83-88 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 89 and an LCVR having at least 95% sequence identity to SEQ ID NO: 90.

[0095] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 91-96 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 97 and an LCVR having at least 95% sequence identity to SEQ ID NO: 98.

[0096] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 99-104and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 105 and an LCVR having at least 95% sequence identity to SEQ ID NO: 106.

[0097] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 107-112 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 113 and an LCVR having at least 95% sequence identity to SEQ ID NO: 114.

[0098] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 115-120 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 121 and an LCVR having at least 95% sequence identity to SEQ ID NO: 122.

[0099] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 123-128 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 129 and an LCVR having at least 95% sequence identity to SEQ ID NO: 130.

[0100] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 131-136 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 137 and an LCVR having at least 95% sequence identity to SEQ ID NO: 138.

[0101] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 139-144 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 145 and an LCVR having at least 95% sequence identity to SEQ ID NO: 146.

[0102] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 147-152 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 153 and an LCVR having at least 95% sequence identity to SEQ ID NO: 154.

[0103] The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 155-160 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 161 and an LCVR having at least 95% sequence identity to SEQ ID NO: 162.The invention further relates to a CAR comprising an antigen binding domain comprising an HCVR / LCVR pair selected from the following HCVR / LCVR pairs: SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154, and 161 / 162. Preferred HCVR / LCVR pairs for the antigen binding domain include SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146. A particularly preferred HCVR / LCVR pair for the antigen binding domain is SEQ ID NO: 73 / 74.

[0104] Any of the above described CARs comprising an antigen binding domain comprising a specified combination of CDRs or specified HCVR / LCVR may have one or more of the functional properties described above. For example, the CARs of the invention typically do not bind to ROR2. The CARs of the invention may be capable of increasing the relative proportion of CD4+ T cells in the population of T cells which is transduced, compared to non-transduced T cells. In some embodiments, the CARs are capable of increasing the relative proportion of CD4+ T cells by at least 10% compared to nontransduced T cells.

[0105] Extracellular spacer

[0106] Preferred CARs of the invention include an extracellular spacer domain located between and connecting the transmembrane domain and the antigen binding domain. It has been reported in literature that the spacer has a role in fine-tuning the signalling of the CAR. The spacer of a CAR of the invention can be selected from any of the extracellular spacer domains disclosed in WO 2022 / 129692 Al, which is hereby incorporated by reference in its entirety. As explained in WO 2022 / 129692 Al, the length of the spacer is adjustable by using different domains and their combinations in the spacer.

[0107] The extracellular spacer may be derived from an immunoglobulin Fc region or includes fragments from immunoglobulin Fc region. The immunoglobulin Fc region may be derived from IgG, IgM, IgA or IgE. The Fc region of IgG may be derived from IgGl, IgG2, IgG3 or IgG4. The IgG based spacer domain comprises CH2 and CH3 domains from IgG Fc region. An IgG based spacer domain having IgG constant regions CH2 and CH3 may be as described for example in Hornbach et al. (2010). Further spacer structures are described for example in Cappell, K. M. and Kochenderfer, J. N. (2023).The spacer domain of the CARs of the invention may comprise at least one Ig-like Cl domain of signal -regulatory protein alpha. Signal regulatory protein alpha is abbreviated SIRP-alpha throughout the specification. A SIRP-alpha Ig-like Cl domain may be selected from type 1 domain (SEQ ID NO: 163) and / or type 2 domain (SEQ ID NO: 164). In one embodiment a spacer comprises a SIRP-alpha Ig-like Cl-type 1 domain. In another embodiment a spacer comprises a SIRP-alpha Ig-like Cl-type 2 domain. In another embodiment a spacer comprises a SIRP-alpha Ig-like Cl-type 1 domain and SIRP-alpha Ig-like Cl-type 2 domain. The spacer may comprise multiple SIRP-alpha Ig-like Cl-type 1 domains and / or SIRP-alpha Ig-like Cl-type 2 domains.

[0108] In some embodiments, the spacer domains do not interact with FcgR resulting in functional effects. T cells with CARs comprising the aforementioned spacer domains do not affect CAR T cell activation caused by off-target binding, activation of FcgR-expressing cells, non CAR mediated activation and sequestration of CAR T cells in the lungs, activation induced cell death (AICD) and overall reduction of CAR T cell activity.

[0109] The CAR of the invention may be a monomer. However, in some embodiments, the spacer may further comprise a multimerization domain. A multimerization domain promotes multimerization of CAR monomers. In aspects relating to multimerization, CARs may form dimers, trimers, quadram ers, pentamers or multimers from CAR monomers. Preferably the CAR is a monomer or a dimer formed from two CAR monomers. The multimerization domain is typically capable of forming linkages between monomers of CARs. Preferably the linkages between the monomers are disulfide bridges. Preferably the multimerization domain forms at least one, two, or three disulfide bridges between the monomers. In some embodiments of the invention the multimerization domain of the spacer is selected from the following: IgGl hinge region, IgG2 hinge region, IgG3 hinge region, IgG4 hinge region, extracellular / intracellular CD28 domain or a fragment or variant of any thereof. A fragment or variant of a multimerization domain disclosed herein is defined as a truncated or modified portion of that domain which is capable of forming linkages between monomers of CARs. The IgGl hinge region may comprise or consist of an amino acid sequence according to SEQ ID NO: 167. The IgG2 hinge region may comprise or consist of an amino acid sequence according to SEQ ID NO: 168. The IgG3 hinge region may comprise or consist of an amino acid sequence according to SEQ ID NO:169. The IgG4 hinge region may comprise or consist of an amino acid sequence according to SEQ ID NO: 170. In a preferred embodiment the spacer of the CAR of the invention comprises a multimerization domain comprising the IgG4 hinge region. The IgG4 hinge region may comprise or consist of an amino acid sequence according to SEQ ID NO: 170. The multimerization domain or its fragment may be linked at one end to a SIRP-alpha Ig-like Cl type domain and at the other end to the antigen binding domain of the CAR of the invention. An additional linker sequence may be used between the spacer and the antigen binding domain.

[0110] In another embodiment the spacer may comprise a multimerization domain comprising an extracellular CD28 domain or a fragment thereof. The extracellular CD28 domain may comprise an amino acid sequence according to SEQ ID NO: 165. The extracellular CD28 domain or fragment thereof may be linked at one end to SIRP-alpha Ig-like Cl type domain and at the other end to the transmembrane domain, for example to the transmembrane domain of CD28 (SEQ ID NO: 171). An additional linker sequence may be used between the spacer and the transmembrane domain. The spacer may comprise multiple multimerization domains. The spacer may comprise multiple different multimerization domains. In some embodiments the spacer comprises both an IgGl hinge region and an extracellular CD28 domain. In some embodiments the spacer comprises both an IgG4 hinge region and an extracellular CD28 domain.

[0111] In some embodiments the domains in the spacer may be selected from an Ig-like Cl type 1 domain of SIRP-alpha, Ig-like Cl type 2 domain of SIRP-alpha, extracellular CD28 domain, IgG hinge region and / or a fragment or variant of any thereof.

[0112] Transmembrane domain

[0113] Preferably, the transmembrane domain of the CAR of the invention is a CD28 transmembrane domain. The CD28 transmembrane domain of a CAR of the invention may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 171, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 171.

[0114] Cytoplasmic domainThe cytoplasmic domain of a CAR of the invention may comprise a CD3zeta signalling domain, e.g. as in first generation CARs known in the art. The CD3zeta signalling domain of a CAR of the invention may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 173, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 173.

[0115] The cytoplasmic domain of a CAR of the invention may comprise a CD28 costimulatory domain and / or a 4- IBB costimulatory domain. Preferably, the cytoplasmic domain of a CAR of the invention comprises a CD28 costimulatory domain. The CD28 costimulatory domain of a CAR of the invention may comprise or consist of an amino acid sequence having at least 70% identity to SEQ ID NO: 172, such at least 80%, at least 90%, at least 95%, at least 99% or 100% identity to SEQ ID NO: 172.

[0116] The cytoplasmic domain of a CAR of the invention may comprise a CD3zeta signalling domain and a CD28 costimulatory domain, which may also be referred to as a CD28z domain, e.g. as in second generation CARs known in the art.

[0117] The cytoplasmic domain of a CAR of the invention may comprise a 4-1BB costimulatory domain and a CD3zeta signalling domain, which may also be referred to as a 4-lBBz domain, e.g. as in third generation CARs in the art.

[0118] The cytoplasmic domain of a CAR of the invention may comprise a 4-lBBz and a CD28z domain, which comprises CD3zeta, CD28 and the 4-1BB signalling domains.

[0119] The cytoplasmic domain of a CAR of the invention may comprise the CD3zeta signalling domain alone or in combination with a CD28, CD27, OX-40 (CD 134) and / or 4- 1BB (CD137) domain.

[0120] Other activation domains include IL-15Ra, CD2, CDS, ICAM-1, LTA-1 and ICOS and may be used in combination with the signalling domains described above.

[0121] Preferred CARs

[0122] As such, the inventors have identified a number of preferred CARs which specifically bind to ROR1 and characterised their sequences. A CAR of the invention may comprise any one of the antigen binding domains disclosed herein. In particular, a CAR of the invention comprises:

[0123] i) an antigen binding domain disclosed herein;ii) an extracellular spacer domain which comprises at least one Ig-like Cl domain of SIRPalpha or a fragment or variant thereof;

[0124] iii) a CD28 transmembrane domain; and

[0125] iv) a cytoplasmic domain comprising a CD28 or 4- IBB costimulatory domain and a CD3zeta signalling domain.

[0126] The invention provides a CAR comprising or consisting of the amino acid sequence selected from any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41. Preferably, the invention provides a CAR comprising or consisting of the amino acid sequence selected from any of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35. Most preferably, the invention provides a CAR comprising or consisting of the amino acid sequence of SEQ ID NO: 7, 27, 29, 31, 33 and 35.

[0127] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 1.

[0128] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 3.

[0129] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 5.

[0130] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 7.

[0131] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 9.

[0132] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 11.

[0133] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 13.

[0134] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 15.

[0135] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 17.

[0136] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 19.

[0137] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 21.

[0138] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 23.The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 25.

[0139] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 27.

[0140] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29.

[0141] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 31.

[0142] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33.

[0143] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35.

[0144] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37.

[0145] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 39.

[0146] The CAR may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 41.

[0147] The CAR may comprise any one of the amino acid sequences identified in Table 14. The CAR may be encoded by any one of the nucleic acid sequences identified in Table 14. The antigen-binding domain of the CAR may comprise any of the binding regions (e.g. CDRs or variable domains) from the amino acid sequences identified in Table 15. The CAR may include any one of the elements disclosed identified in Table 16.

[0148] The CAR may further comprise a signal peptide at the N-terminus. Any suitable signal peptide may be used in the CAR. The signal peptide may comprise or consist of an amino acid sequence of SEQ ID NO: 177 or 178. The CARs identified by the amino acid sequences in Table 14 do not include the signal peptide. The signal peptide having an amino acid sequence of SEQ ID NO: 177 may be employed with CARs having an amino acid sequence of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 39 or 41. The signal peptide having an amino acid sequence of SEQ ID NO: 178 may be employed with CARs having an amino acid sequence of SEQ ID NOs: 33, 35 or 37.

[0149] Antigen binding molecules

[0150] An antigen-binding molecule of the invention typically comprises a polypeptide, e.g. any antigen binding domain as described herein, which specifically binds to ROR1. Typically, an antigen-binding molecule is any protein that comprises an antigen-binding site or domain. ROR1 -specific antigen-binding molecules of the invention include antibodies and antigen-binding fragments thereof that specifically bind to ROR1. A ROR1 -specific antigen-binding molecule of the invention may comprise any one or more of the sequences identified in Table 15.

[0151] An anti-RORl antigen-binding molecule of the invention may specifically bind to an epitope of human ROR1 which comprises at least one amino acid residue selected from E26, M30, E33, Q37, D82, Ml 11, R112, KI 14, LI 15 and D 120, or amino acid residue(s)corresponding thereto, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. The antigen-binding molecule may specifically bind to an epitope of human R0R1 which comprises two, three, four, five, six, or seven of these residues. In one embodiment, the antigen-binding molecule of the invention specifically binds to an epitope of human R0R1 which comprises amino acid residues E26, R112, KI 14 and LI 15, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigen-binding molecule of the invention specifically binds to an epitope of human R0R1 which comprises amino acid residues E26, Q37, D82, R112, KI 14, LI 15 and D120, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigen-binding molecule of the invention specifically binds to an epitope of human R0R1 which comprises amino acid residues E33, Q37 and KI 14, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. In one embodiment, the antigenbinding molecule of the invention specifically binds to an epitope of human R0R1 which comprises amino acid residues M30 and Ml 11, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. In a preferred embodiment, the antigen-binding molecule of the invention specifically binds to an epitope of human R0R1 which comprises amino acid residues M30, Q37 and Ml 11, wherein the amino acid numbering is based on the human R0R1 Frizzled domain, as shown in SEQ ID NO: 174. The antigen-binding molecule of the invention may bind to an epitope comprising corresponding amino acid residues to those specified above in any human R0R1 amino acid sequence. Various techniques known in the art may be used to determine specific binding of the antigen-binding molecule to particular amino acid residues, such as cross-blocking assays, alanine scanning mutational analysis or hydrogen / deuterium exchange detected by mass spectrometry.

[0152] The invention provides an anti-RORl antigen-binding molecule (specific for R0R1) comprising three heavy chain CDRs (HCDRs) contained with a HCVR selected from any of SEQ IDNOs: 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, and 145, and the three light chain CDRs (LCDRs) contained within a LCVR selected from any of SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, and 146. Typically, the heavychain CDRs and light chain CDRs are selected from those contained within the HCVR / LCVR pairs represented by SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, and 145 / 146. The CDRs contained within a HCVR / LCVR may be identified according to the Kabat definition, the Chothia definition or the IMGT definition (see, for example, Kabat, Elvin Abraham 1991; Lefranc, Marie-Paule, et al. 2003).

[0153] The invention also relates to an antigen-binding molecule (specific for R0R1) comprising an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination selected from the following combinations of CDR sequences: SEQ ID NOs: 43-48, SEQ ID NOs: 51-56, SEQ ID NOs: 59-64, SEQ ID NOs: 67-72, SEQ ID NOs: 75-80, SEQ ID NOs: 83-88, SEQ ID NOs: 91-96, SEQ ID NOs: 99-104, SEQ ID NOs: 107-112, SEQ ID NOs: 115-120, SEQ ID NOs: 123-128, SEQ ID NOs: 131-136 and SEQ ID NOs: 139-144. A particularly preferred HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination is SEQ ID NOs: 67-72.

[0154] The antigen-binding molecule may comprise a HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination as disclosed above and comprise an HCVR and LCVR having at least 95% sequence identity to the HCVR and LCVR from which the CDRs are derived. Thus, for example, the antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 43-48 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 49 and an LCVR having at least 95% sequence identity to SEQ ID NO: 50. The antigen binding domain may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 51-56 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 57 and an LCVR having at least 95% sequence identity to SEQ ID NO: 58.

[0155] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 59-64 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 65 and an LCVR having at least 95% sequence identity to SEQ ID NO: 66.

[0156] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 67-72and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 73 and an LCVR having at least 95% sequence identity to SEQ ID NO: 74.

[0157] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 75-80 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 81 and an LCVR having at least 95% sequence identity to SEQ ID NO: 82.

[0158] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 83-88 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 89 and an LCVR having at least 95% sequence identity to SEQ ID NO: 90.

[0159] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 91-96 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 97 and an LCVR having at least 95% sequence identity to SEQ ID NO: 98.

[0160] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 99-104 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 105 and an LCVR having at least 95% sequence identity to SEQ ID NO: 106.

[0161] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 107-112 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 113 and an LCVR having at least 95% sequence identity to SEQ ID NO: 114.

[0162] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 115-120 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 121 and an LCVR having at least 95% sequence identity to SEQ ID NO: 122.

[0163] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 123-128 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 129 and an LCVR having at least 95% sequence identity to SEQ ID NO: 130.The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 131-136 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 137 and an LCVR having at least 95% sequence identity to SEQ ID NO: 138.

[0164] The antigen-binding molecule may comprise the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 139-144 and comprise an HCVR having at least 95% sequence identity to SEQ ID NO: 145 and an LCVR having at least 95% sequence identity to SEQ ID NO: 146.

[0165] The invention further relates to an antigen-binding molecule comprising an HCVR / LCVR pair selected from the following HCVR / LCVR pairs: SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, and 145 / 146. A particularly preferred HCVR / LCVR pair for the antigen-binding molecule is SEQ ID NO: 73 / 74.

[0166] Any of the above described antigen-binding molecules comprising a specified combination of CDRs or specified HCVR / LCVR may have one or more of the functional properties described above. For example, typically the antigen-binding molecule does not bind to ROR2.

[0167] Preferably, an antigen-binding molecule of the invention is an scFv. An ROR1-specific antigen-binding molecule of the invention may also be an antigen-binding fragments of a full anti-RORl antibody molecule, or a full anti-RORl antibody molecule.

[0168] The antigen-binding molecule may be an antibody-drug conjugate. The antigenbinding molecule may be used in an antibody -based therapy, for example, in a method of metabolite radiotherapy. The antigen-binding molecule may be used in a method of treatment of a cancer as described herein.

[0169] Nucleic acids

[0170] The invention also provides one or more isolated nucleic acids or polynucleotides encoding an anti-RORl CAR or anti-RORl antigen-binding molecule described herein. Nucleic acids of the invention can be isolated, purified, recombinantly produced or synthesized by any methods well known to those skilled in the art. The nucleic acids may encode for one or more particular amino acid sequences described herein. The nucleicacids may comprise any degenerate nucleic acid sequences capable of encoding for one or more particular amino acid sequences described herein.

[0171] The nucleic acids described herein may comprise a DNA sequence, or an RNA, such as mRNA sequence. The nucleic acids may be single stranded or double stranded. The nucleic acids may be comprised in one or more vectors. A vector may be a viral vector. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art.

[0172] The vectors may be cloning vectors or expression vectors. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

[0173] The vector is preferably an RNA vector. Suitable RNA vectors include the RNA vectors described in Schutsky, Keith, et al. 2015 and Beatty, Gregory L., et al. 2018.

[0174] General methods by which the vectors may be constructed, transfection methods and culture methods are well known to those skilled in the art. In this respect, reference is made to “Current Protocols in Molecular Biology”, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis Manual produced by Cold Spring Harbor Publishing.

[0175] A nucleic acid may be provided in the form of an expression cassette, which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the CAR of the invention in vivo. Hence, also provided is one or more expression cassettes encoding the one or more nucleic acids that encode a CAR described herein. These expression cassettes, in turn, are typically provided within vectors (e.g. plasmids or recombinant viral vectors). Hence, also provided is a vector encoding a CAR described herein. Further provided are vectors which collectively encode a CAR described herein.

[0176] The vector may be a human artificial chromosome. Human artificial chromosomes are described in e.g. Kazuki et al. 2011 and Kouprina et al. 2014.The vector may be a non-viral delivery system, such as a DNA plasmid, naked nucleic acid (e.g. naked RNA), and nucleic acid complexed with a delivery vehicle, such as a liposome or a nanoparticle.

[0177] The nucleic acids, expression cassettes or vectors described herein may be introduced into a host cell, e.g. by transfection. Hence, also provided is a host cell comprising the one or more nucleic acids, expression cassettes or vectors of the invention. The nucleic acids, expression cassettes or vectors described herein may be introduced transiently or permanently into the host cell, allowing expression of an antibody from the one or more nucleic acids, expression cassettes or vectors. Such host cells include transient, or preferably stable higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast, or prokaryotic cells, such as bacteria cells. Particular examples of cells include mammalian HEK293, such as HEK293F, HEK293T, HEK293S or HEK Expi293F, CHO, HeLa, NSO and COS cells, or any other cell line used herein. Preferred host cells are the immune effector cells described herein. In particular, preferred host cells include T cells, natural killer cells, natural killer T cells and macrophages. Preferably, the nucleic acids, expression cassettes or vectors described herein are introduced transiently into the host cell.

[0178] Also provided is a kit suitable for transforming and / or transfecting an immune effector cell to generate an engineered immune effector cell of the invention. The kit comprises a nucleic acid or vector described herein. The kit may comprise further agents such as those discussed herein that improve transfection or transformation efficacy.

[0179] Also described are one or more isolated nucleic acid molecules encoding the specific antigen-binding molecules or CARs, as described herein. The isolated nucleic acid molecule may encode a CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39 and 41. Preferably, the isolated nucleic acid molecule encodes a CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35. Most preferably, the isolated nucleic acid molecule encodes a CAR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 27, 29, 31, 33 and 35. For example, the isolated nucleic acid molecule may comprise a nucleotide sequence selected from the group consisting of SEQID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42. Preferably, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36. Most preferably, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 28, 30, 32, 34 and 36. Whilst SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40 and 42 are provided as a DNA sequence, the corresponding RNA sequence (replacing ‘T’ with ‘U’) is also encompassed.

[0180] Also described are nucleic acids encoding the antigen-binding molecule of the invention, including the scFv polypeptides disclosed herein. For example, the nucleic acid molecule may encode an antigen-binding molecule comprising the three heavy chain CDRs contained within a HCVR selected from any of SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, and 145, and the three light chain CDRs (LCDRs) contained within a LCVR selected from any of SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, and 146. The nucleic acid molecule may encode an antigen-binding molecule comprising an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination selected from the following combinations of CDR sequences: SEQ ID NOs: 43-48, SEQ ID NOs: 51-56, SEQ ID NOs: 59-64, SEQ ID NOs: 67-72, SEQ ID NOs: 75-80, SEQ ID NOs: 83-88, SEQ ID NOs: 91-96, SEQ ID NOs: 99-104, SEQ ID NOs: 107-112, SEQ ID NOs: 115-120, SEQ ID NOs: 123-128, SEQ ID NOs: 131-136, and SEQ ID NOs: 139-144. Preferably, the nucleic acid molecule of the invention encodes an antigen-binding molecule comprising an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 combination of SEQ ID NOs: 67-72.

[0181] The nucleic acid molecule may encode an antigen-binding molecule comprising an HCVR / LCVR pair selected from the following: SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, and 145 / 146. In a preferred embodiment, the nucleic acid molecule may encode an antigen-binding molecule comprising the HCVR / LCVR pair of SEQ ID NO: 73 / 74.

[0182] Also included is a pair of nucleic acid molecules, each of which encodes a variable region of the antigen-binding molecule or antigen-binding domain disclosed herein. For example, a first nucleic acid molecule may encode a HCVR comprising the three heavychain CDRs contained within a HCVR selected from any of SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, and 145, and a second nucleic acid molecule may encode a LCVR comprising the three light chain CDRs (LCDRs) contained within a LCVR selected from any of SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, and 146.

[0183] The first nucleic acid molecule may encode a HCVR comprising an HCDR1 / HCDR2 / HCDR3 combination selected from the following combinations of CDR sequences: SEQ ID NOs: 43-45, SEQ ID NOs: 51-53, SEQ ID NOs: 59-61, SEQ ID NOs: 67-69, SEQ ID NOs: 75-77, SEQ ID NOs: 83-85, SEQ ID NOs: 91-93, SEQ ID NOs: 99-101, SEQ ID NOs: 107-109, SEQ ID NOs: 115-117, SEQ ID NOs: 123-125, SEQ ID NOs: 131-133, and SEQ ID NOs: 139-141, and the second nucleic acid molecule may encode a LCVR comprising an LCDR1 / LCDR2 / LCDR3 combination selected from the following combinations of CDR sequences: SEQ ID NOs: 46-48, SEQ ID NOs: 54-56, SEQ ID NOs: 62-64, SEQ ID NOs: 70-72, SEQ ID NOs: 78-80, SEQ ID NOs: 86-88, SEQ ID NOs: 94-96, SEQ ID NOs: 102-104, SEQ ID NOs: 110-112, SEQ ID NOs: 118-120, SEQ ID NOs: 126-128, SEQ ID NOs: 134-136, and SEQ ID NOs: 142-144.

[0184] The first nucleic acid molecule may encode a HCVR selected from: SEQ ID NOs: 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137 and 145, and the second nucleic acid molecule may encode a LCVR selected from: SEQ ID NOs: 50, 58, 66, 74, 82, 90, 98, 106, 114, 122, 130, 138, and 146. In a preferred embodiment, the first nucleic acid molecule encodes a HCVR and the second nucleic acid molecule encodes a LCVR, selected from the following HCVR / LCVR pairs: SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, and 145 / 146. In a particularly preferred embodiment, the first nucleic acid molecule encodes a HCVR comprising SEQ ID NO: 73 and the second nucleic acid molecule encodes a LCVR comprising SEQ ID NO: 74.

[0185] The nucleic acid molecules described herein encode for the mature CARs and antigen-binding molecules described herein. The nucleic acid molecules described herein typically include nucleotides encoding for the signal peptide. The signal peptide is typically co-translationally cleaved at a signal peptidase cleavage site, in order to generate the mature protein. The signal peptide may have an amino acid sequence of SEQ ID NO:177 or 178. The signal peptide having an amino acid sequence of SEQ ID NO: 177 may be employed with CARs having an amino acid sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 39 or 41. The signal peptide having an amino acid sequence of SEQ ID NO: 178 may be employed with CARs having an amino acid sequence of SEQ ID NOs: 33, 35 or 37. In some embodiments, the nucleic acid molecule does not include nucleotides encoding for the signal peptide.

[0186] Engineered immune effector cells

[0187] An engineered cell or engineered immune effector cell of the invention comprises a CAR of the invention. The engineered immune effector cell may express a CAR of the invention. The engineered immune effector cell may be a cell capable of cell-mediated cytotoxicity against a target cell expressing R0R1.

[0188] An engineered immune effector cell of the invention may display improved capacity to expand in cell culture. An engineered cell of the invention may display maintained viability during expansion, freezing and / or thawing.

[0189] An engineered immune effector cell of the invention may be a T cell, a y5 T cell, a natural killer (NK) cell, an NKT cell, an induced pluripotent stem cell (iPSC) derived NK cell (iPSC-NK), a phagocyte, or a macrophage. The engineered immune effector cell is preferably a T cell. The T cell may be a CD8+ T cell, or cytotoxic T cell. The T cell may be a CD4-CD8+ T cell. The T cell may be a CD4+ T cell, or helper T cell (TH cell), such as a TH1, TH2, TH3, TH17, TH9, or TFH cells. The T cell may be a CD4+CD8+ T cells, CD4-CD8- T cells, regulatory T cell (Treg). The T cell may be a naive, effector, memory, effector memory, central memory, memory stem T cell. The T cell may be a peripheral lymphocyte.

[0190] The T cell may be expanded from peripheral blood mononuclear cells (PBMCs). The T cell may be autologous with respect to a subject into which it is to be administered. The T cell may be allogeneic with respect to a subject into which it is to be administered. The T cell may be partially HLA-mismatched with respect to a subject into which it is to be administered. The T cell may enable the killing of R0R1 -expressing cells in a targetspecific manner but not without causing high activity without antigen binding.The NK cell may be isolated from peripheral blood mononuclear cells (PBMCs) of the subject to be treated or of a healthy donor. The NK cell may be isolated from cord blood. The NK cell may be differentiated from a CD34+haematopoietic progenitor cell (HPC). The NK cell may be a cell of the NK92 cell line. The NK cell may enable the killing of R0R1 -expressing cells in a target-specific manner but not without causing high activity without antigen binding.

[0191] The gd T cell may be expanded from peripheral blood mononuclear cells (PBMCs). The gd T cell may be autologous with respect to a subject into which it is to be administered. The T gd cell may be allogeneic with respect to a subject into which it is to be administered.

[0192] The macrophage may be differentiated into the “Ml” phenotype. The Ml macrophage expresses pro-inflammatory cytokines and has strong anti-tumour activity. An undifferentiated macrophage expressing a CAR described herein may be induced to differentiate into the Ml phenotype by culturing in the presence of the glioma-associated antigen.

[0193] Sources of cells for use in accordance with the invention will be known to persons skilled in the art, illustrative examples of which include peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In an embodiment, the cells are derived from whole blood.

[0194] An engineered immune effector cell of the invention may be derived from an autologous cell. An engineered immune effector cell may be derived from an allogeneic cell. The term "autologous" refers to any material derived from the same individual to whom the material is later to be re-introduced to the individual. The term "allogeneic" refers to any material derived from a different individual of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic materials from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0195] The engineered immune effector cell may comprise a nucleic acid described herein. The engineered immune effector cell may comprise a vector described herein. Theengineered immune effector cell may comprise an RNA nucleic acid or RNA vector described herein. The engineered immune effector cell may transiently express the CAR. The engineered immune effector cell may be engineered to transiently express a CAR of the invention. This is to minimise on-target off-tumour toxicity. In some cases, the engineered immune effector cell may be transfected with mRNA encoding a CAR of the invention, for example by mRNA electroporation as demonstrated in Beatty et al, Gastroenterology 155.1 (2018): 29-32. (see supplementary materials 5) and Schutsky etal, Oncotarget 6.30 (2015): 28911.

[0196] The invention also relates to a method of preparing an engineered immune effector cell of the invention or a population of engineered immune effector cells of the invention. The method comprises introducing a nucleic acid or vector encoding a CAR of the invention into an engineered immune effector cell, e.g. by transformation (such as transfection or transduction).

[0197] The term “transduction” may be used to describe virus mediated nucleic acid transfer. A viral vector may be used to transduce the cell with the one or more constructs. Conventional viral based expression systems could include retroviral, alpha-retroviral, lentivirus, adenoviral, adeno-associated (AAV) and herpes simplex virus (HSV) vectors for gene transfer. Non-viral transduction vectors include transposon-based systems including PiggyBac and Sleeping Beauty systems. Methods for producing and purifying such vectors are known in the art. The vector is preferably a vector described herein. Immune effector cells may be transduced using any method known in the art. Transduction may be in vitro or ex vivo.

[0198] The term “transfection” may be used to describe non-virus-mediated nucleic acid transfer. The immune effector cells may be transfected using any method known in the art. Transfection may be in vitro or ex vivo. Any vector capable of transfecting immune effector cells may be used, such as conventional plasmid DNA or RNA transfection, preferably mRNA transfection. A human artificial chromosome and / or naked RNA may be used to transfect the cell with the nucleic acid sequence or nucleic acid construct.

[0199] Human artificial chromosomes are described in e.g. Kazuki et al., Mol. Ther. 19(9): 1591-1601 (2011), and Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014). Alternative non-viral delivery systems include DNA plasmids, naked nucleic acid, andnucleic acid complexed with a delivery vehicle, such as a liposome. Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipidmucleic acid conjugates, naked DNA, naked RNA, artificial virions, and agent-enhanced uptake of DNA.

[0200] Nanoparticle delivery systems may be used to transfect the engineered immune effector cell with the nucleic acid sequence. Such delivery systems include, but are not limited to, lipid-based systems, liposomes, micelles, microvesicles and exosomes. With regard to nanoparticles that can deliver RNA, see, e.g., Alabi et al. 2013; Zhang et al. 2013; Jiang et al. 2013; Karagiannis et al. 2012; Whitehead et al. 2012; Lee et al. 2012. Lipid Nanoparticles, Spherical Nucleic Acid (SNA™) constructs, nanoplexes and other nanoparticles (particularly gold nanoparticles) are also contemplated as a means for delivery of a nucleic acid or vector of the invention.

[0201] The engineered immune effector cell may be transfected by electroporation. The electroporation may be mRNA electroporation. This has the advantage of allowing transient expression of the CAR.

[0202] Uptake of nucleic acid constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectAmine, fugene and transfectam.

[0203] The invention also relates to a method, such as an ex vivo method, of preparing a population of engineered immune effector cells (e.g. for adoptive cell therapy), comprising culturing the engineered immune effector cell of the invention to produce a population of engineered immune effector cells. The invention also provides a population of engineered immune effector cells obtained or obtainable by any of the methods described herein. The invention also provides a population of engineered immune effector cells that express a CAR of the invention.

[0204] A population may comprise at least about IxlO6of the immune effector cells, such as at least about IxlO7, at least about IxlO8, at least about IxlO9or at least about IxlO10of the immune effector cells. A population may comprise at least about IxlO6to about IxlO12of the immune effector cells, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to aboutIxlO10of the immune effector cells. The population may comprise about IxlO6of the immune effector cells, such as about 5xl06, about IxlO7, about 5xl07, about IxlO8, about 5xl08, about IxlO9, about 5xl09, about IxlO10, about 5xlO10, about IxlO11, about 5xlOn, or about IxlO12of the immune effector cells.

[0205] Pharmaceutical compositions

[0206] Also provided is a composition comprising an engineered immune effector cell of the invention. Also provided is a composition comprising a population of engineered immune effector cells of the invention. The engineered immune effector cell or population of immune effector cells may be at least 50% of the total cells in the composition, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% of the total cells in the composition. The total cells in the composition may consist or consist essentially of the immune effector cell or population of immune effector cells of the invention, i.e. no other cells are detectable in the composition.

[0207] The composition may comprise at least about IxlO6to about IxlO12of the engineered immune effector cells of the invention, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to about IxlO10of the engineered immune effector cells. The composition may comprise about IxlO6of the engineered immune effector cells of the invention, such as about 5xl06, about IxlO7, about 5xl07, about IxlO8, about 5xl08, about IxlO9, about 5xl09, about IxlO10, about 5xl010, about IxlO11, about 5xl0n, or about IxlO12of the engineered immune effector cells. The composition may comprise a population of the engineered immune effector cells of the invention in the amounts described above.

[0208] The composition of engineered immune effector cells may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent or excipient. Briefly, pharmaceutical compositions of the present invention may comprise a population of engineered immune effector cells, such as CAR T cells, of the invention, in combination with one or more pharmaceutically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline, citrate and the like; saccharides orcarbohydrates such as glucose, mannose, sucrose or mannitol; a cryoprotective such as DMSO, dextran, trehalose or polyvinylpyrrolidone; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention may be formulated for intravenous administration.

[0209] The pharmaceutical composition may include one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects. Examples of such salts include acid addition salts and base addition salts.

[0210] The composition may comprise one or more additional therapeutic agent, such as a chemotherapeutic agent. The composition may comprise one or more preservative, such as an anti-fungal, anti-bacterial and / or anti-viral agent, aluminium salts, DMSO, sodium benzoate, sorbic acid, benzoic acid and the like.

[0211] Therapeutic uses and methods

[0212] Also provided herein is the use of a composition, engineered cell, antigen-binding molecule, nucleic acid(s), or vector(s) described herein in a method of treatment of the human or animal body by therapy.

[0213] Further provided is a method of treating a cancer or tumour in a subject, the method comprising administering to the subject an effective amount of a composition, engineered cell, antigen-binding molecule, nucleic acid(s), or vector(s) described herein. Also provided is a use of a composition, engineered cell, antigen-binding molecule, nucleic acid(s), or vector(s) described herein for the manufacture of a medicament for the treatment of a cancer or tumour.

[0214] The engineered cells described herein (e.g. therapeutic CAR T cells) may be used in immunotherapy. The immunotherapy may be targeted against solid tumours or hematological cancers. The engineered cells described herein may be autologous or allogeneic. Autologous cells are isolated from a subject, a polynucleotide encoding the CAR is introduced into the cells by a vector and cells expressing the CAR is administered back to the subject. Allogeneic cells are isolated from a different individual but aregenetically similar with cells of a subject. CAR expressing cells, preferably T cells, may be administered to a subject in a pharmaceutical composition. The pharmaceutical composition may comprise in addition to CAR expressing cells, other pharmaceutically active agents, preservatives and / or buffer substances, as described herein.

[0215] The subject is typically a mammal. Preferably, the mammal is a human.

[0216] The subject may be of any age. For example, the subject may be a juvenile. The subject may, for example, be an adult.

[0217] The cancer or tumour may be any cancer type which is positive for R0R1 (i.e. any cancer which expresses R0R1). The cancer or tumour may be a solid cancer or tumour. The solid cancer or tumour may be breast cancer, ovarian cancer, endometrial cancer, gastric cancer, non-small cell lung cancer, renal cell carcinoma or melanoma. The breast cancer may be triple negative breast cancer. The cancer may be a liquid cancer, such as a haematological cancer. The haematological cancer may be chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL) or myeloid leukemia, such as acute myeloid leukemia. The cancer or tumour may comprise R0R1+ cells. The cancer or tumour may be refractory to treatment with an antagonist of R0R1 (such as an antigen-binding protein specific for R0R1). The cancer or tumour may be refractory to treatment with an antibodydrug conjugate, BiTE, T cell engager or conditional agonist targeting R0R1.

[0218] The therapeutic methods and uses may comprise, prior to treatment with the immune effector cell, composition, antigen-binding molecule, or encoding nucleic acid(s) described herein, determining whether the cancer or tumour comprises R0R1+ cells.

[0219] The therapeutic methods and uses described herein may comprise inhibiting the disease state (i.e. the cancer or tumour), for example by arresting its development and / or causing regression of the disease state until a desired end point is reached. The therapeutic methods and uses of the invention may comprise achieving a partial response, a full response by the cancer or tumour. The therapeutic methods and uses of the invention may achieve remission of the cancer or tumour. The therapeutic methods and use of invention may promote bystander killing of cancer cells and / or long lasting immunological memory against cancer antigen.

[0220] The therapeutic methods and uses described herein may delay the growth of the cancer or tumour, arrest the growth of the cancer or tumour and / or reverse the growth ofthe cancer or tumour. The therapeutic methods and uses of the invention may reduce the size of the cancer or tumour by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or by 100%.

[0221] The therapeutic methods and uses described herein may comprise administering a therapeutically effective amount of the composition, engineered cell, antigen-binding molecule, nucleic acid(s), or vector(s) to the subject.

[0222] Typically, the therapeutic methods and uses are for a human subject in need thereof. However, non-human animals such as non -human mammals are also contemplated. The non-human mammals may be rats, rabbits, sheep, pigs, cows, cats or dogs.

[0223] The dose of the engineered immune effector cell may vary depending on the age and size of a subject, as well as on the disease, conditions and route of administration. The engineered immune effector cell may be administered at a dose of about IxlO6to about IxlO12cells, such as about IxlO6to about IxlO11, about IxlO6to about IxlO10, about IxlO6to about IxlO9, about IxlO7to about IxlO11, about IxlO8to about IxlO10cells. The composition or engineered immune effector cell may be administered at a dose of about IxlO6cells, such as about 5xl06cells, about IxlO7cells, about 5xl07cells, about IxlO8cells, about 5xl08cells, about IxlO9cells, about 5xl09cells, about IxlO10cells, about 5xl010cells, about IxlO11cells, about 5xl0ncells, or about IxlO12cells.

[0224] The composition or engineered immune effector cell may be administered at a dose of about IxlO5cells / kg to about IxlO11cells / kg, such as about IxlO5cells / kg to about IxlO10cells / kg, about IxlO5cells / kg to about IxlO9cells / kg, about IxlO5cells / kg to about IxlO8cells / kg, about IxlO6cells / kg to about IxlO11cells / kg, about IxlO6cells / kg to about IxlO10cells / kg, about IxlO6cells / kg to about IxlO9cells / kg, about IxlO7cells / kg to about IxlO11cells / kg, about IxlO7cells / kg to about IxlO10cells / kg, or about IxlO7cells / kg to about IxlO9cells / kg, The composition or engineered immune effector cell may be administered at a dose of about IxlO5cells / kg, such as about 5xl05cells / kg, IxlO6cells / kg, 5xl06cells / kg, IxlO7cells / kg, 5xl07cells / kg, IxlO8cells / kg, 5xl08cells / kg, IxlO9cells / kg, 5xl09cells / kg, IxlO10cells / kg, 5xl010cells / kg, or IxlO11cells / kg.

[0225] The composition or engineered immune effector cell may be administered as a single dose. The composition or engineered immune effector cell may be administered in amultiple dose regimen. For example, the initial dose may be followed by administration of a second or plurality of subsequent doses. The second and subsequent doses may be separated by an appropriate time. For example, the doses may be administered once about every week, once about every 2 weeks, once about every 3 weeks, once about every four weeks, or once about every month.

[0226] Typically, the composition or engineered immune effector cell is administered to a patient intravenously. The composition or engineered immune effector cell may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intracranially, intraventricularly, by intralymphatic injection, or intraperitoneally.

[0227] The composition or engineered immune effector cell may be administered with one or more additional therapy, such as one or more additional therapeutic agents. The additional therapeutic agent may be an anti-tumour agent. The additional therapeutic agent may be oncolytic viruses. The additional therapeutic agent may be a CAR-enhancing drug. The additional therapeutic agent may be an additional immune effector cell. The additional therapeutic agent may be a further immunotherapeutic agent, such as an immune checkpoint inhibitor.

[0228] Combined administration of the composition or engineered immune effector cell with the additional therapeutic agent may be achieved in a number of different ways. All the components may be administered together in a single composition. Each component may be administered separately as part of a combined therapy.

[0229] For example, the immune effector cell or the population of immune effector cells of the invention may be administered before, after or concurrently with the additional therapeutic agent.

[0230] The additional therapy may be chemotherapy, targeted therapy, radiotherapy and / or surgery.

[0231] Prior to administration of the immune effector cell or population of immune effector cells of the invention, the subject may undergo lymphodepletion.

[0232] Lymphodepletion may be achieved via administration to the subject with fluradabine, cyclophosphamide and / or bendamustine. Lymphodepletion may be carried out for at least about one day, such as about 2 days or about 3 days.The biological activity and / or therapeutic efficacy of the administered composition or engineered immune effector cell may be measured by known methods. For example, the method may comprise imaging, such as magnetic resonance imaging.

[0233] The following examples illustrate the invention.

[0234] Example 1 - ROR1 expression

[0235] R0R1 expression on different cancer cell lines

[0236] The R0R1 expression on the various cell lines was determined using microspheres (Quantum Simply Cellular, Bangs Laboratories) with various antibody binding capacity. The microspheres were prepared and used according to the manufactures’ protocol. In short, the cells in suspension and microspheres were stained with fluorochrome labelled anti-RORl antibody and studied with flow cytometry to determine the amount of R0R1 expression on the surface. Figure 1 summarizes the R0R1 expression on A549, NCI-111975, PEO1, PANC-1, PEO-4, SK-MEL-5, HTC-116 cells.

[0237] Flow cytometry analysis ofRORl expression on immune cell populations of peripheral blood mononuclear cell from CLL patients

[0238] R0R1 expression was studied on different immune cell populations of peripheral blood mononuclear cells (PBMCs) from CLL patient (Discovery Life Sciences) and healthy controls. The cells were stained for flow cytometry analysis with antibodies listed in Table 1.

[0239] Table 1. Antibodies used for flow cytometry staining

[0240]

[0241] R0R1 is highly expressed on CD 19+ B cells in CLL patient PBMC samples but not in healthy donor PBMC samples (Figure 2). R0R1 expression in CD3+ T cells is similar in CLL patient and healthy PBMCs (Figure 2).

[0242] Immunohistochemical analysis ofRORl expression on different cell lines Immunohistochemical analysis (IHC) for R0R1 was studied in samples with known R0R1 expression levels, including formalin-fixed paraffin-embedded (FFPE) pellets prepared from cell lines and xenograft tumors. The cell lines CHO and CH0-R0R2 are R0R1 -negative; triple-negative breast cancer (TNBC), chronic lymphocytic leukemia (CLL) and H1975 cell lines and xenograft tumors express low levels ofRORl; and CHO-R0R1 cell line and MC38-RORl+xenografts express high levels ofRORl.

[0243] R0R1 detection from tissues is known to be challenging based on literature and inhouse data. For anti-RORl Rabbit DB04C07 IHC, antigen retrieval was performed at pH6 using EnVision FLEX Target retrieval solution pH6 (Agilent) with DAKOLink48 autostainer (Agilent). The rabbit monoclonal primary antibody was applied at 20 ng / ul concentration, labelled polymer (EnVision+ / HRP anti-rabbit, Agilent) was used as a secondary antibody and EnVision FLEX HRP Magenta Substrate Chromogen System (Agilent) was used as a chromogen. Pictures of the staining are shown in Figure 3. CHO cells and CHO cells transfected with human ROR2 were negative and CHO cells transfected with human ROR1 were positive with DB04C07 based antibody (Figure 3A).

[0244] TNBC xenografts, MC38-ROR1+ xenografts and H1975 xenografts were positive with DB04C07 based rabbit antibody (Figure 3B).

[0245] ROR1 detection in immunohistochemical analysis with different ROR1 specific antibodies The monoclonal human IgG antibodies DB04C07 and DB03B11 stained ROR1-positive (CHO-ROR1) in formalin fixed paraffin-embedded (FFPE) cell pellet samples, while ROR1 -negative cells (CHO and CHO-ROR2) did not stain. IHC was performed as above, except for that Goat F(ab')2 Anti-Human IgG (Fab')2 (HRP) (Abeam) was used as a secondary antibody diluted 1:200 and DAB+ substrate (Agilent) was used as a chromogen. Table 2 lists the human monoclonal primary antibodies, antigen retrieval conditions and primary antibody concentrations applied in successful IHC analysis:Table 2: antibodies and conditions used for immunohistochemical analysis

[0246]

[0247] Pictures of the stains are shown in Figure 3C. CHO cells and CHO cells transfected with human R0R2 were negative and CHO cells transfected with human R0R1 were positive.

[0248] Example 2 - Binder screening and characterization and sequences

[0249] Antibody discovery

[0250] Table 15 sets forth the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of the selected anti-RORl binding molecules of the disclosure. To identify R0R1 binding antibodies, a humanized phage display library was used in in vitro selections. Selections were performed using the full HuRORl extracellular domain (ECD), as well as two different arms with selected HuRORl ECDs, combined with negative selection against HuR0R2 ECD. Following phage display selections, unique clones were re-arrayed, and scFvs were expressed. Filtered periplasmic extracts were then used in a primary biosensor screen with the HuRORl ECD, the HuRORl Frizzled domain (FZD), the HuRORl kringle domain (KRD), and the HuR0R2 ECD, by capturing them onto streptavidin sensor tips via biotinylated anti-V5-tag. Off-rates were analyzed based on the binding (association) and dissociation data of antigen analytes.

[0251] Unique binders to the primary target were further re-assayed using secondary flow cytometry cell binding screening. Binding analysis of unique scFv clones to high antigenexpressing cell lines was performed using Cytoflex flow cytometry with parental and Human R0R1 CH0-K1 cell lines. Cells were first incubated with scFv periplasmic extracts, then washed and labeled with secondary anti-myc APC antibody. The mean fold changes (MFI) of binding to R0R1 CHO cells over the parental CHO cell line geometricmeans were determined. The fold changes of the binders ranged from 2 to 96. Several binders with the best MFI and the desired target-binding domain were selected for further studies.

[0252] Protein production

[0253] For full biophysical characterization and binding studies, selected scFv hits were transiently produced in CHO cell lines and purified using a two-step method, combining a Protein A affinity column with analytical HPLC-SEC.

[0254] Basic QC

[0255] The Nanotemper Prometheus Panta instrument was used to measure the melting temperature (Tm) via nanoDSF and to determine the poly dispersity index (PDI) from DLS data. Antibody fragment (scFv or IgGl) samples were loaded by capillarity into standardgrade nanoDSF capillaries and placed in the Prometheus capillary holder. DLS data were collected at 25°C to determine the PDI, with a threshold value set below 0.2. Afterward, the samples were subjected to a temperature ramp of l°C / min from 20°C to 95°C. Tm values were obtained by monitoring intrinsic tryptophan and tyrosine fluorescence at emission wavelengths of 330 nm and 350 nm. To generate an unfolding curve, the fluorescence intensity ratio (F350 nm / F330 nm) was plotted against temperature. The thermal stability was characterized by the thermal unfolding transition midpoint (Tm), the temperature at which half of the protein population is unfolded. The Tm was determined as the inflection point of the unfolding curve, using its derivative (Table 4A and 4B for IgGl and Table 4C for scFv).

[0256] The molecular size distribution and protein aggregation of the scFv and IgGl solutions were analyzed using size-exclusion chromatography coupled with multi -angle light scattering (SEC-MALS). Analytical SEC was performed on selected fractions using Advance Bio SEC columns, connected sequentially to an HPLC system with a diode array detector, MALS, and a refractive index (RI) detector (Table 4A for IgGl and 4C for scFv).

[0257] Binding assaysBinding analysis of purified scFvs to antigen-expressing cell lines was performed using the NovoCyte Quanteon flow cytometer, with Human ROR1 CHO-KI, and Jeko-1 cell lines. Cells were incubated with 10 pg / ml of purified scFv, followed by washing and labelling with the secondary antibody, StrepMAB-Classic Conjugate DY-649. Table 3 shows that the selected ROR1 binding scFvs bind to ROR1 expressing CHO and Jeko-1 cells, but not to ROR2 expressing CHO cells. The values represent % of the cells that have bound the scFvs calculated relative to non-labelled and control cell line.

[0258] Table 3: Binding of different scFv fragments to ROR1 expressing cells and ROR2 expressing cells, % of positive cells

[0259]

[0260] Biophysical characterization, Surface Plasmon Resonance (SPR)

[0261] Kinetic analyses of antibody variants (scFv and IgGl) were conducted using SPR on a Biacore 8K+ (Cytiva) with a capture assay for affinity measurements. ScFv fragments as well as IgGl antibodies were captured anti-human IgG (Fc) antibody and IgGl with Human FcAvi -tagged R0R1 protein (Aero Biosystems) immobilized onto a CM5 sensor chip (Cytiva) (Table 4A and 4B for IgGl and Table 4C for scFv).Based on the combined biophysical and binding data of the produced scFv antibody fragments, the best hits were selected and incorporated as binding domains in CAR structures.

[0262] Table 4A: Biophysical characterisation of selected binders in antibody (IgGl) form

[0263]

[0264] Table 4B: Biophysical characterisation of selected binders in antibody (IgGl) form

[0265]

[0266] Table 4C: Biophysical characterization of selected binders in scFv form

[0267]

[0268]

[0269] Epitope mapping

[0270] Frizzled domain of human R0R1 was expressed on yeast cells and binding of selected scFvs DB04C07, DB04D03, DB01D06 and DB01C11 were studied. Deep Mutational Scanning library of the antigen (Frizzled domain) was generated to find mutants to which given scFv did not bind anymore. This library contained all the possible single mutants of the studied antigen. The mutants were sorted using FACS. The different pools of mutants were sequenced in order to have a precise mapping of the epitope to which of each scFv bound. The epitope was defined at the amino acid level. Figures 4A, C, E, G show key amino acids in R0R1 Frizzled domain epitopes recognised by binders DB04C07, DB04D03, DB01D06 and DB01C11, respectively. Figures 4B, D, F, H show corresponding epitopes on surface of R0R1 Frizzled domain. Key epitope positions for each scFv were: DB04D03: E26, R112, KI 14, LI 15; DB01D06: E26, Q37, D82, R112, K114, L115, D120; DB04C07: M30, Q37, Mill; DB01C11: E33, Q37, K114.

[0271] Example 3 - CAR structures and preparation of CAR expressing T cells

[0272] A gene encoding the chimeric antigen receptor includes a signaling domain, an antigen binding domain, spacer (with an optional hinge region), a transmembrane domain, co-stimulatory domains and stimulatory domains. CAR structures used in the examples and their sequences are summarized in Table 14. The CAR encoding gene was cloned to a lentiviral vector production suitable transfer plasmid, produced, and used for generation of 3rd generation self-inactivating lentiviral vector with VSV-G pseudotyping.

[0273] To generate T cells carrying the CAR gene, human CD4 and CD8 positive T-cells were isolated with magnetic sorting from peripheral blood mononuclear cells (PBMCs) of healthy donors. The cells were then activated with anti-CD3 and anti-CD28 antibodies (Transact, Miltenyi Biotec). The following day, the T cells were transduced with VSV-G pseudotyped 3rd generation self-inactivating lentiviral vectors carrying the CAR gene with a multiplicity of infection of approximately 2. 1-2 days post transduction, the cells were washed and expanded in suitable culture medium (TexMACS) with the presence ofrhInterleukin-7 (IL-7) and rhlnterleukin-15 (IL-15) at a cell density of 0,2-4 million cells / ml up to 9 days. Upon completion of expansion, cells were harvested and stored in vapor phase nitrogen (in Albunorm with 10% DMSO).

[0274] Example 4 - Cell killing efficacy with CAR structures having different antigen binding regions

[0275] Human CD4 and CD8 positive cells were isolated and prepared as in Example 3. During, and post the expansion period, the cells were counted with Nucleocounter (NC-100 and NC-202) to check for cell amounts to determine the expansion fold (Figure 5A) and for the viability of the cells (Figure 5B). At late stage of the expansion, some differences between CAR structures emerged with lower expansion matching lower viability, especially post thaw viability (Figure 5B). Vector copy number was determined by quantitative (q)PCR and / or digital droplet (dd)PCR. The genomic DNA was isolated from the cells and analyzed with q / ddPCR to determine the amount of transgene integrations in a cell pool based on reference gene. The functional part of the integrated transgene was verified with flow-cytometry with soluble extracellular part of ROR1 tagged with a fluorophore (Aero Biosystems). With these two analyses of, the study of transgene integration and the study of expression of functional protein, it was confirmed that sufficient and comparable level of integration and CAR expression had taken place for further analysis (Figure 6A and 6B).

[0276] To analyze cell killing efficacy of the T cells expressing different CARs, they were co-cultured with ROR1 -expressing target cells with various CAR-T cell to target cell ratios The target cells were imaged to quantify the target cell killing of them over time. Target cells without co-culture were used as a reference point. From imaging studies, the 50% effective killing efficacy (EC50) was measured from dilution series of CAR-T cells against target cells (Figure 7). To measure IFN-gamma release from the CAR T-cells upon target cell engagement, supernatant was collected after 24h of the co-culture and measured for IFN-gamma by ELISA or LUMIT based measurement techniques (Figure 8). For signs of tonic signaling, e.g. no-stimulated cytokine production, elevated or decreased cell expansion and reduced cellular viability, supernatant samples were collected for e.g. IFN-gamma measurement from high density cultured CAR T-cells, without exposure to ROR1expressing target cells, and the results referred to a structure producing some or no IFN-gamma release in the absence of R0R1+ cells (Figure 9). Cells were also studied for viability as previously described.

[0277] ORC -2025 showed highest killing efficacy and IFN-gamma release against the target cells, without loss of viability post thaw and no signs of tonic signaling. ORC-2027, -2057, 2087 and -2091 showed also killing efficacy and IFN-gamma release without loss of viability post thaw nor signs of tonic signaling (Figures 5B, 7, 8 and 9).

[0278] Based on these studies ORC -2025, -2027, -2057 and -2091 CAR constructs were chosen to be further studied. In addition to previously described studies, these CAR-T cells were co-cultured with target cell lines representing various cancer types, namely gastric carcinoma, breast cancer, lung cancer and melanoma (Figure 10). Moreover, a ROR1-negative, ROR2 -positive, K562 cell line was studied in co-culture with the CAR-T cells. To this cell line, ROR1 was knocked-in (KI) with an expression vector resulting in stable high ROR1 expression in a number of these cells. These cells were co-cultured with CAR-Ts and their IFN-gamma release was measured after 24 hours.

[0279] When the CAR expressing T cells were studied against target cells with various ROR1 expression levels, a clear response was seen for all studied constructs. ORC-2025 showed strongest response. As expected, none of the CAR-T cells responded to ROR1 negative, ROR2 positive K562 cell line (Figure 10). When ROR1 was introduced to the K562 cell line, clear engagement was seen through IFN-gamma secretion by the CAR-T cells. Responses of CARs with various scFvs were studied in detail. Target cells, namely the ROR1 expressing cell line (A549) were then co-cultured with (CAR-)T cells in 1 to 1 ratio. After 24h co-culture, supernatant was collected and analyzed for Perforin, Granzyme B, Interleukin 2 and Interferon gamma release (Figure 11). ORC -2025 showed highest response for all studied analytes in this co-culture experiment.

[0280] Soluble ROR1 can be detected in patients’ plasma with various cancers, and it is more prominently found in metastatic disease (Figure 13). When the CAR-T cells were incubated with plain medium supplemented with recombinant soluble extracellular domain part of ROR1 protein, CARs with DB04C07 antigen binding domain reacted more strongly to the antigen than anti-RORl Refl antigen binding domain; ORC-1054 / ORC-2025 vs. ORC-1026 and ORC-1061 vs. ORC-1023, more IFN-gamma was detected in the culturesupernatant after 24 hour culture (Figure 12). When the CAR-T cells were incubated with plain medium supplemented with recombinant soluble extracellular domain part of R0R1 protein (SEQ ID NO: 179), CARs with SIRP-alpha based spacer reacted more strongly to the antigen; ORC- 1026 vs ORC- 1023 and ORC-1054 / ORC-2025 vs ORC- 1061, more IFN-gamma was detected in the culture supernatant after 24 hour culture (Figure 12). The cytokine response was observed along with enhanced viability and survival for only in SIRP-alpha based CARs, that can benefit the long lasting anti -cancer effect of the CARs (Figure 14).

[0281] Dose dependency of cell killing efficacy of the CAR-T cells was studied with mantle cell lymphoma (Jekol) cells showing that all the CAR-T cells were able to kill the cells dose dependently (Figure 15). CAR-T cell co-culture with Jekol mantle cell lymphoma cell line, modified to express luciferase, was studied for killing efficacy at various effector to target ratios by lysing the cells with luciferin containing substrate and measured for luminescence. Target cell only result was used as a reference point to calculate the killing efficacy.

[0282] Example 5 - Cell killing efficacy with different CAR structures

[0283] Various CAR structures were introduced to T cells via transduction and cell were expanded. High CAR positivity with suitable vector copy number (VCN) were seen; with high CAR positivity with relatively low VCN for ORC-1054 and ORC-2025 (Figure 16).

[0284] This means, that the CAR structure is well expressed from the gene cassette, is stable structure and a good amount of functional protein is expressed even with relatively low integration amount. CAR-T cells with different CAR structures were studied in co-cultures with ROR1 expressing NCI-H1975 adherent cancer cells. The killing efficacies of the CAR-T cells were measured in co-cultures with imaging to determine effector cell to target cell ratios EC50 and with impedance-based co-culture assay to measure killing efficacy % (Figure 17A and 17B).

[0285] All ROR1 specific CAR-T cells were able to dose dependently kill the ROR1 expressing cancer cells. ORC-1054, ORC-1055 and ORC -2025 all outperformed ORC-1023 in killing performance in the two studied ROR1 positive cell linesWild type target cells and R0R1-K0 target cells were studied together in CAR-T co-culture. Interleukin 2 (IL-2), granzyme B and perforin secretion were studied and the response showed R0R1 -specificity (Figure 18). The cells with varying R0R1 expression, or knock-out of R0R1, were studied in co-culture with CAR-T cells. Number of R0R1 molecules expressed on R0R1 positive cells was for A54922879, NCI-H1975 13228, PEO1 8346, PANC-1 7388, PEO43989, SK-MEL-52558 and for HCT116 1625. Knockout (KO) cells were negative for R0R1 -expression. After 24 hours of co-culture, the medium was collected, and IFN-gamma was measured (Figure 19).

[0286] ORC- 1054, ORC- 1055 and ORC -2025 had higher IFN-gamma release in co-culture with ROR1 positive cancer cells compared to ORC-1023. ORC-1023 lost capacity to react to cancer cells with lower ROR1 expression, whereas ORC-1054, ORC-1055 and ORC-2025 did not. However, when ROR1 expression was knocked-out, none of the ROR1 specific CAR-T cells could mount an IFN-gamma response.

[0287] Example 6 - CD4+ and CD8+ cell populations of the CAR expressing T cells

[0288] To determine the portions of CD4 and CD8 population of the CAR T cells, CAR cells were thawed and stained for flow cytometry analysis. First CAR T cells were stained with the viability staining, which was followed by surface marker staining with the antibodies listed in Table 5. Samples were fixed before acquisition.

[0289] Table 5. Antibodies used in flow cytometry analysis

[0290]

[0291] Figure 20 shows the CD4 and CD8 portions of CD3 cells. The first donor (Donor A; Figure 20A) had slightly higher CD4 portions compared to the second donor (Donor B;

[0292] Figure 20B). In the first donor (Donor A; Figure 20A), SIRP-alpha including CAR structures ORC-2025, ORC-1054, and ORC-1055 had higher CD4 portions compared toCAR structures ORC- 1023 and ORC- 1062 without SIRP-alpha. Similarly, in the second donor (Donor B; Figure 20B), ORC -2025 and ORC-1054 had higher CD4 portions than ORC- 1023 and ORC- 1062 structures.

[0293] Example 7 - Phenotype

[0294] To determine the phenotype of the CAR T cells, CAR T cells were thawed and stained for flow cytometry analysis. First CAR T cells were stained with the viability staining, which was followed by surface marker staining with the antibodies listen in Table 6. Samples were fixed before acquisition.

[0295] For the measured markers, HLA-DR, 4-1BB, CD69, PD-1 and LAG3, some donor dependent variation was observed between two donors (Figure 21A-E). HLA-DR was not much affected with the various CARs. 4-1BB positive cell population was slightly increased, with the highest proportion of positive cells being in ORC-2025 cells. For CD69 and PD-1, some tendency for more positive cells for ORC -2025 and ORC-1055 were seen. The amount of LAG3 positive cells was increased in all CAR T cells with the largest increase seen for ORC -2025.

[0296] Table 6: Antibodies used in flow cytometry analysis.

[0297]

[0298] Example 8 - ROR1 specific CAR expressing T cells show improved anti-tumor efficacy in vivoR0R1 specific CAR expressing T cells have anti-tumor efficacy in vivo in Jeko-1 lymphoma model

[0299] In vivo efficacy was evaluated using the Jeko-1 tumor model in female NSG

[0300]

[0301] mice. Mice received an intravenous injection of 1 million Jeko-1 -luc-puro cells suspended in 100 pL of PBS via the tail vein to establish the tumor model.

[0302] Tumor burden was monitored weekly using bioluminescent imaging. Prior to imaging, mice were injected subcutaneously with D-luciferin (150 mg / kg) and imaged under isofhirane anaesthesia using the IVIS Spectrum in vivo imaging system. Body weight was measured twice a week to monitor overall health. Additionally, mice were monitored daily for signs of tumor progression and general health status.

[0303] One week post-inoculation, baseline tumor burden was assessed using the IVIS Spectrum imaging system. Mice were randomized based on bioluminescent signal intensities to receive either PBS (control group), ROR1 specific CAR-T cells (10 million cells), or non-transduced T cells (NT T cells). The NT T cells were administered intravenously in an amount equivalent to the total number of T cells administered in the CAR-T cell group. Table 7 summarizes the used agents, their doses and study groups.

[0304] Table 7: Summary of used agents and study groups used in the Jeko-1 in vivo efficacy study.

[0305]

[0306] Figure 22 shows tumor burden curves of the individual mice compared to PBS and NT treated mice. Already on day 6 post-treatment all treatment groups showed lowertumor burden when compared to PBS treated mice. Overall, all the CAR-T cell treatments slowed the tumor progression when compared to PBS treated mice.

[0307] When compared to non-transduced T cells (NT T cells), ORC-2025 and ORC-1023 showed improved antitumor efficacy. Even though, antitumor response was slower in ORC -2025 treated mice, it was able to stabilize tumor burden throughout the study and from day 33 post CAR-T cell treatment it had the lowest mean bioluminescence signal (total flux). Meanwhile ORC- 1023 showed fast anti -tumor response but tumor burden was comparable to ORC-2025 on day 33.

[0308] Mice treated with ORC -2091 and ORC- 1041 had comparable antitumor response to ORC- 1023 on day 6, but at the end of the study tumor burden in both treatment groups reached the same level as the NT T cell treated mice. ORC-1039 treated mice did not show improved anti-tumor response when compared to NT T cell treated mice. Figure 23 and Table 8 summarizes the average tumor burden in different study groups.

[0309] Table 8: Summary of the Jeko-1 tumor burden in mice on days 20 and 33.

[0310]

[0311] ROR1 CARs show in vivo antitumor efficacy in solid tumor.In vivo activity against solid tumors was evaluated in a mechanistic study using the H1975 tumor model in female NSG (NOD .Cg-PrkdcscldIl.2rgtmlWjl / SzJ') mice. H1975 is a human non-small cell lung cancer (NSCLC) cell line. Mice received a subcutaneous injection of 5 million H1975 cells suspended in 200 pL of PBS into the right flank to establish the tumor model.

[0312] Treatment was initiated when the tumor volume reached 52-87 mm3. Tumor volume was measured using calipers, and the volume was calculated using the formula: (1 X W2) X 0.5.

[0313] At the treatment initiation point, mice were randomized based on tumor volume to receive either PBS (control group), Cetuximab 30 mg / kg (model positive control), CAR-T cells (10 million cells), or non-transduced T cells (NT T cells) as a comparison group. The NT T cells were administered intravenously in an amount equivalent to the total number of T cells administered in the CAR-T cell group.

[0314] To evaluate CAR T response in vivo, blood, tumors, and spleens were collected 10 days post CAR-T treatment. The resulting serum, and tumors and spleen samples (1 / 2 snap frozen in RNAlater and 1 / 2 FFPE) were processed for ex vivo analysis. RNA was extracted from tumor and spleen tissues for Nanostring gene expression analysis.

[0315] Tumor volume was measured three times a week using calipers. Body weight was also measured three times a week to monitor overall health. Additionally, mice were monitored daily for humane endpoints based on their level of activity and behaviour.

[0316] Table 9: Summary of agents and study groups used in the H1975 in vivo mechanistic study.

[0317]

[0318]

[0319] Figure 24 shows the level of cytokines measured from the serums on day 10 post treatment. Cytokines TNF, IFN-gamma, IL-2, Granzyme B, perforin and IL- 10 are induced in the groups treated with T cells expressing CARs ORC -2025, ORC-1054 and ORC-1055, indicating activity in vivo. These cytokines are induced the most in mice treated with ORC- 1054. Surprisingly, cytokines were not induced in mice treated with ORC-1023, despite strong anti -turn or response in the efficacy group. Additionally, soluble ROR1, measured by human ROR1 ELISA kit (Abeam), was at the same level in all of the groups and only slightly elevated in ORC -2025, ORC-1054 and ORC-1055 treated mice.

[0320] Nanostring gene expression analysis with human immunology V2 panel was performed from the tumors and analysed via nSolver software (Figure 25). All CAR-T cell treatments induced genes in tumor that are associated with T cell response. GZMB, PRF1, IFNg and IL2 were elevated in CAR-T cell treated mice when compared to PBS, Cetuximab and NT T cell treated mice. Mice treated with ORC- 1023 expressing T cells showed increased expression of GZMB, PRF1, IFNg and IL2, when compared to mice treated with ORC-2025, ORC-1054 and ORC-1055 expressing T cells. ORC-1023 expressing T cells also promoted more exhaustion related genes PDCD1, CTLA4, TIMS, LAGS and TIGITm ' tumor, when compared to mice treated with ORC-2025, ORC- 1054 and ORC-1055 expressing T cells. Figure 26 shows the differential gene expression analysis of immunology-related genes of NT T cells versus different CAR T cell treatments using the Nanostring human immunology V2 panel. The overall gene expression profiles indicate a higher immune response in all CAR T treated groups compared to the NT T cell treatment.

[0321] In vivo efficacy against solid tumors was evaluated using the same H1975 tumor model with a second donor. 3 million H1975 cells suspended in 150 pL of PBS was inoculated subcutaneously into the right flank of female NSG mice to establish the tumor model. Treatment was initiated when the tumor volume reached 38-200 mm3. Tumorvolume was measured using calipers, and the volume was calculated using the formula: (1 X W2) X 0.5.

[0322] Tumor volume was measured two times a week using calipers. Body weight was also measured two times a week to monitor overall health. Additionally, mice were monitored daily for humane endpoints based on their level of activity and behaviour.

[0323] Table 10: Summary of agents and study groups used in the H1975 in vivo efficacy study.

[0324]

[0325] Figure 27 shows individual tumor growth curves of the mice in each group. Mice treated with NT T cells did not have anti-tumor response and tumor growth was similar to the PBS treated mice until day 21 post treatment. Mice treated with ORC-2025 showed anti -tumor response from day 16. All mice treated with ORC- 1054 showed strong antitumor response. 7 out of 10 mice treated with ORC- 1023 showed strong anti -turn or response. Figure 28 and Table 11 summarizes the anti-tumor activity in different study groups.

[0326] Table 11 Summary of anti-tumor activity in H1975 solid tumor model.

[0327]

[0328]

[0329] At the end of the study, blood samples were collected from the mice via cardiac puncture. Absolute T cell counts were measured from the blood by staining the whole blood with CD45-BV421 (BioLegend), CD3-BV785 (BioLegend), CD4-APC (BioLegend), and CD8-FITC (BioLegend) antibodies. After staining, the whole blood was lysed and fixed with lyse / fix buffer (BioLegend), and samples were analyzed via flow cytometer to acquire absolute T cell counts. Figure 29 shows absolute CD3, CD4, and CD8 counts per ml of blood. Average total CD3 cell amounts were significantly highest in ORC-1054 treated mice (66.3 x 10A6 cells / ml). Additionally, ORC-2025 treated mice had higher average CD3 counts (36.8 x 10A6 cells / ml) than ORC-1023 treated mice (11.7 x 10A6 cells / ml) and NT T cell treated mice (6.2 x 10A6 cells / ml).

[0330] In vivo dose response study was performed in same Hl 975 tumor model as described above. 3 million H1975 cells suspended in 150 pL of PBS was inoculated subcutaneously into the right flank of female NSG mice to establish the tumor model.

[0331] Treatment was initiated when the tumor volume reached 35-325 mm3. Tumor volume was measured using calipers, and the volume was calculated using the formula: (1 x w2) x 0.5. Table 12 summarizes the used agents, their doses and study groups.

[0332] Table 12: Summary of agents and study groups used in the H1975 dose response in vivo efficacy study.

[0333]

[0334]

[0335] Figure 30 shows individual tumor growth curves of the mice in each group. NT T cells showed no statistically significant effects on tumor size compared to vehicle indicating no therapeutic impact. ORC-1054 at all doses (6xlOA6, 3xlOA6 and lxlOA6) demonstrated highly significant reductions in in vivo tumor volume compared to vehicle and non-transduced control (p<0.001), highlighting their strong potential for tumor suppression. ORC-1023 exhibited dose dependent effects on tumor volume reduction, with significant reductions observed at the highest dose (6xlOA6, p<0.001), moderate effects at 3xlOA6 dose (p<0.01) and mild at lxlOA6 (p<0.05) compared to vehicle. However, when compared to non-transduced control, the tumor suppression effects of ORC- 1023 were less pronounced, with only highest dose showing a moderate reduction (6xlOA6, p<0.01), and no significant effects for CAR-T doses of 3xlOA6 and lxlOA6 cells. ORC -2025 at all doses (6xlOA6, 3xlOA6 and lxlOA6) demonstrated highly significant reductions in in vivo tumor volume compared to the vehicle and the non-targeting control (p<0.001), highlighting their strong potential for tumor suppression. Figure 31 summarizes the anti -turn or activity in different study groups.

[0336] Table 13: Summary of the dose response anti-tumor activity in H1975 solid tumor model.

[0337]

[0338]

[0339] Summary of amino acid sequences and nucleic acid sequences

[0340] Table 14

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368] Table 15

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376] Table 16

[0377]

[0378] Human R0R1 Frizzled domain (SEQ ID NO: 174):

[0379] EEDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCS QFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRL KLPNCEDLPQPESPEAANCIRI

[0380] Human ROR1 protein (SEQ ID NO: 175):

[0381] RARGCSARRRGTRPPLLALLAALLLAARGAAAQETELSVSAELVPTSSWNISSELN KDSYLTLDEPMNNITTSLGQTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSFRS TIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSSTGVLFVKFGPPPTASPGYSDEYE EDGFCQPYRGIACARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSHLSDKCSQ FAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILENVLCQTEYIFARSNPMILMRLK LPNCEDLPQPESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRGTVSVTKSGRQC QPWNSQYPHTHTFTALRFPELNGGHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCRNNQKSSSAPVQRQPKHV RGQNVEMSMLNAYKPKSKAKELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHA QLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNIVCLLGAVTQEQPVCMLFEYI NQGDLHEFLIMRSPHSD VGC S SDEDGTVKS SLDHGDFLHIAIQIAAGMEYLS SHFF VHKDLAARNILIGEQLHVKISDLGLSREIYSADYYRVQSKSLLPIRWMPPEAIMYG KFSSDSDIWSFGVVLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSEDCPPRMYS LMTECWNEIPSRRPRFKDIHVRLRSWEGLSSHTSSTTPSGGNATTQTTSLSASPVSN LSNPRYPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGYPIPPGYAAFPAAHYQPT GPPRVIQHCPPPKSRSPSSASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHPGGM GITVFGNKSQKPYKIDSKQASLLGDANIHGHTESMISAEL

[0382] Signal peptide 1 (SEQ ID NO: 177):

[0383] MEFGLSWLFLVAILKGVQCSR

[0384] Signal peptide 2 (SEQ ID NO: 178):

[0385] MLLLVTSLLLCELPHPAFLLIP

[0386] Soluble extracellular domain of ROR1 (SEQ ID NO: 179):

[0387] QETELSVSAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLGQTAELHCKVSGNPP PTIRWFKNDAPVVQEPRRLSFRSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSS TGVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIACARFIGNRTVYMESLHMQGEI ENQITAAFTMIGTSSHLSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRDECEILE NVLCQTEYIFARSNPMILMRLKLPNCEDLPQPESPEAANCIRIGIPMADPINKNHKC YNSTGVDYRGTVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNGGHSYCRNPGN QKEAPWCFTLDENFKSDLCDIPACDSKDSKEKNKMEILYReferences

[0388] Alabi et al. (2013) Proc Natl Acad Sci U S A 110(32): 12881-6.

[0389] Almasbak H et al (2015) Inclusion of an IgGl-Fc spacer abrogates efficacy of CD 19 CAR T cells in a xenograft mouse model. Gene Ther 22: 391-403.

[0390] Balakrishnan, A. et al. (2017) Analysis of ROR1 protein expression in human cancer and normal tissues. Clinical Cancer Research 23(12), 3061-3071.

[0391] Beatty, Gregory L., et al. (2018) Gastroenterology 155.1: 29-32.

[0392] Cappell, K. M. and Kochenderfer, J. N. (2023) Long-term outcomes following CAR T cell therapy: what we know so far. Nature Reviews 20, 359-371.

[0393] Hornbach A et al (2010) Adoptive immunotherapy with genetically engineered T cells: modification of the IgGl Fc ‘spacer’ domain in the extracellular moiety of chimeric antigen receptors avoids ‘off-target’ activation and unintended initiation of an innate immune response. Gene Ther 17: 1206

[0394] Hudecek M et al (2015) The Nonsignaling Extracellular Spacer Domain of Chimeric Antigen Receptors Is Decisive for In Vivo Antitumor Activity. Cancer Immunol Res 3 : 125— 135.

[0395] Jiang et al. (2013) Nano Lett. 13(3): 1059-64.

[0396] Kabat, Elvin Abraham (1991) Sequences of proteins of immunological interest. No. 91. US Department of Health and Human Services, Public Health Service, National Institutes of Health.

[0397] Karagiannis et al. (2012) ACS Nano. 6(10):8484-7.

[0398] Kazuki et al. (2011) Mol. Ther. 19(9): 1591-1601

[0399] Koski, J. et al. (2022) Novel modular chimeric antigen receptor spacer for T cells derived from signal regulatory protein alpha Ig-like domains. Frontiers in Molecular Medicine 2:1049580.

[0400] Kouprina et al., Expert Opinion on Drug Delivery 11(4): 517-535 (2014).

[0401] Lee et al. (2012) Nat Nanotechnol. 7(6):389-93.

[0402] Lefranc, Marie-Paule, et al. (2003) IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental & Comparative Immunology 27(1): 55-77.Schutsky, Keith, et al. (2015) Oncotarget 6.30: 28911. Whitehead et al. (2012) ACS Nano. 6(8):6922-9.

[0403] Zhang et al. (2013) Adv Mater. 25(33):4641-5.

Claims

CLAIMS1. A ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C -terminus:(a) an extracellular ligand-binding domain comprising an anti-RORl antigenbinding domain;(b) an extracellular spacer which comprises at least one Ig-like Cl domain of signal-regulatory protein alpha (SIRPa) or a fragment or variant thereof;(c) a transmembrane domain; and(d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

2. The CAR according to claim 1, wherein:(a) said Ig-like Cl domain of SIRPa is selected from (i) type 1 domain according to SEQ ID NO: 163; or (ii) type 2 domain according to SEQ ID NO: 164; and / or (b) the extracellular spacer comprises Ig-like Cl type 1 domain and Ig-like Cl type 2 domain of SIRPa.

3. The CAR according to claim 1 or 2, wherein the extracellular spacer further comprises at least one multimerization domain, optionally wherein the multimerization domain is an IgG hinge region, further optionally wherein the IgG hinge region is an IgG4 hinge region according to SEQ ID NO: 170 or a fragment or variant thereof.

4. The CAR according to any preceding claim, wherein the extracellular spacer is located between a transmembrane domain and the antigen binding domain and connects them.

5. The CAR according to any preceding claim, wherein the transmembrane domain comprises transmembrane domain of CD28 according to SEQ ID NO: 171.

6. The CAR according to any preceding claim, wherein the intracellular signaling domain and / or co-stimulatory domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof and / or the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof, optionally wherein the intracellular signaling domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof and the co-stimulatory domain comprises the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof.

7. A R0R1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C -terminus:(a) an extracellular ligand-binding domain comprising a R0R1 -specific antigen-binding domain comprising three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within the HCVR / LCVR pair of SEQ ID NOs: 73 / 74;(b) an extracellular spacer which comprises at least one Ig-like Cl domain of signal-regulatory protein alpha (SIRPa) or a fragment or variant thereof;(c) a transmembrane domain; and(d) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

8. The CAR according to claim 7, wherein the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 67-68-69, and wherein the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 70-71-72.

9. The CAR according to claim 7 or 8, wherein the antigen-binding domain comprises a HCVR / LCVR pair having at least 95% identity to the HCVR / LCVR pair of SEQ ID NO: 73 / 74, optionally comprising or consisting of the HCVR / LCVR pair of SEQ ID NO: 73 / 74.

10. The CAR according to any one of claims 7-9, wherein:(a) said Ig-like Cl domain of SIRPa is selected from (i) type 1 domain according to SEQ ID NO: 163; or (ii) type 2 domain according to SEQ ID NO: 164; and / or (b) the extracellular spacer comprises Ig-like Cl type 1 domain and Ig-like Cl type 2 domain of SIRPa.

11. The CAR according to any one of claims 7-10, wherein the extracellular spacer further comprises at least one multimerization domain, optionally wherein the multimerization domain is an IgG hinge region, further optionally wherein the IgG hinge region is an IgG4 hinge region according to SEQ ID NO: 170 or a fragment or variant thereof.

12. The CAR according to any one of claims 7-11, wherein the extracellular spacer is located between the transmembrane domain and the antigen binding domain and connects them.

13. The CAR according to any one of claims 7-12, wherein the transmembrane domain comprises transmembrane domain of CD28 according to SEQ ID NO: 171.

14. The CAR according to any one of claims 7-13, wherein the intracellular signaling domain and / or co-stimulatory domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof and / or the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof, optionally wherein the intracellular signaling domain comprises the intracellular domain of CD3zeta according to SEQ ID NO: 173 or a fragment thereof and the co-stimulatory domain comprises the intracellular domain of CD28 according to SEQ ID NO: 172 or a fragment thereof.

15. A ROR1 -specific chimeric antigen receptor (CAR) comprising, from N-terminus to C -terminus:(a) an extracellular ligand-binding domain comprising an anti-RORl antigenbinding domain, which binds to an epitope of the human ROR1 Frizzled domain comprising at least one amino acid residue selected from E26, M30, E33, Q37, D82,107Ml 11, R112, KI 14, LI 15 and D120, wherein the amino acid numbering is based on SEQ ID NO: 174;(b) a transmembrane domain; and(c) a cytoplasmic domain comprising a costimulatory domain and a signaling domain.

16. The CAR according to any preceding claim, wherein the anti-RORl antigenbinding domain binds to an epitope of the human R0R1 Frizzled domain comprising the amino acid residues M30 and Ml 11, optionally M30, Q37 and Ml 11, wherein the amino acid numbering is based on SEQ ID NO: 174.

17. The CAR according to any one of the preceding claims, which does not bind to R0R2.

18. The CAR according to any one of claims 1-6 or 15-17, wherein the anti-RORl antigen-binding domain is a single chain variable fragment (scFv) comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR).

19. The CAR according to any one of claims 1-6 or 15-18, wherein the anti-RORl antigen-binding domain comprises three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within a HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162, optionally wherein the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 43-44-45, 51-52-53, 59-60-61, 67-68-69, 75-76-77, 83-84-85, 91-92-93, 99-100-101, 107-108-109, 115-116-117, 123-124-125, 131-132-133, 139-140-141, 147-148-149 or 155-156-157, and wherein the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 46-47-48, 54-55-56, 62-63-64, 70-71-72, 78-79-80, 86-87-88, 94-95-96, 102-103-104, 110-111-112, 118-119-120, 126-127-128, 134-135-136, 142-143-144, 150-151-152 or 158-159-160.10820. The CAR according to any one of claims 1-6 or 15-19, wherein the antigen-binding domain comprises an HCVR / LCVR pair having at least 95% identity to an HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162, optionally wherein the antigen-binding domain comprises or consists of an HCVR / LCVR pair selected from SEQ ID NOs: 49 / 50, 57 / 58, 65 / 66, 73 / 74, 81 / 82, 89 / 90, 97 / 98, 105 / 106, 113 / 114, 121 / 122, 129 / 130, 137 / 138, 145 / 146, 153 / 154 and 161 / 162.

21. A chimeric antigen receptor (CAR) comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 and 35.

22. An isolated nucleic acid molecule encoding the CAR of any one of claims 1-21.

23. The nucleic acid molecule of claim 22, comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36.

24. A vector comprising the nucleic acid molecule of claim 22 or 23.

25. An engineered cell comprising a CAR according to any one of claims 1-21, a nucleic acid molecule of claim 22 or 23 or a vector of claim 24.

26. The engineered cell according to claim 25, wherein the cell is a T cell, a natural killer cell, a natural killer T cell or a macrophage.

27. A pharmaceutical composition comprising the engineered cell of claim 25 or 26 and a pharmaceutically acceptable carrier.10928. The engineered cell of claim 25 or 26 or the pharmaceutical composition of claim 27, for use as a medicament.

29. The engineered cell of claim 25 or 26 or the pharmaceutical composition of claim 27, for use in the treatment of a R0R1 -expressing cancer.

30. The engineered cell or pharmaceutical composition for use according to claim 29, wherein:(a) the ROR1 -expressing cancer is a solid cancer, optionally wherein the solid cancer is breast cancer, ovarian cancer, endometrial cancer, gastric cancer, non-small cell lung cancer, renal cell carcinoma or melanoma, or(b) the ROR1 -expressing cancer is a liquid cancer, optionally wherein the liquid cancer is chronic lymphocytic leukemia (CLL), Mantle cell lymphoma (MCL) or myeloid leukemia, such as acute myeloid leukemia.

31. A method of preparing an engineered cell, comprising introducing the nucleic acid of claim 22 or 23, or the vector of claim 24, into a cell.

32. A ROR1 -specific antigen-binding molecule, which binds to an epitope of human ROR1 comprising the amino acid residues M30 and Ml 11, optionally comprising the amino acid residues M30, Q37 and Ml 11, wherein the amino acid numbering is based on SEQ ID NO: 174.

33. The antigen-binding molecule of claim 32, which does not bind to ROR2.

34. The antigen-binding molecule according to claim 32 or 33, comprising three heavy chain complementarity determining regions (HCDRs) and three light chain complementarity determining regions (LCDRs) contained within the HCVR / LCVR pair of SEQ ID NOs: 73 / 74, optionally wherein the HCVR comprises HCDR1-HCDR2-HCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 67-68-69, and110wherein the LCVR comprises LCDR1-LCDR2-LCDR3 comprising the amino acid sequences, respectively, of SEQ ID NOs: 70-71-72.

35. The antigen-binding molecule according to any one of claims 32- 34, comprising an HCVR / LCVR pair having at least 95% identity to the HCVR / LCVR pair of SEQ ID NO: 73 / 74, optionally comprising or consisting of the HCVR / LCVR pair of SEQ ID NO: 73 / 74.Ill

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