Chimeric antigen receptors specific for antigen CLEC12a

Abstract

The present invention discloses a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A, b) a transmembrane domain, and, c) an intracellular signaling domain, wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:72 (VH) and SEQ ID NO:73 (VL) or is encoded by a nucleotide sequence comprising SEQ ID NO:67 (VH) and SEQ ID NO:68 (VL). Immune cells expressing said CARs for use in treatment of cancer cells expressing CLEC12A are also disclosed.

Description

[0001] MBG_178

[0002] Title

[0003] Chimeric antigen receptors specific for antigen CLEC12A

[0004] Field of the invention

[0005] The present invention generally relates to the field of immunotherapy using immune cells expressing a chimeric antigen receptor, in particular to the field of immunotherapy using immune cells expressing a chimeric antigen receptor specific for the antigen C-type lectin domain family 12 member A (CLEC12A).

[0006] Background of the invention

[0007] The use of chimeric antigen receptor (CAR)-expressing immune cells such as T cells re-directed to specifically recognize and eliminate target cells such as malignant cells, greatly increased the scope and potential of adoptive immunotherapy and is being assessed for new standard of care in certain human disorders such as malignancies. CARs are recombinant receptors that typically target surface molecules in a human leukocyte antigen (HLA)-independent manner. Generally, CARs comprise an extracellular antigen recognition moiety, often a single-domain antibody, a single-chain variable fragment (scFv) derived from antibodies or a Fab fragment, linked to an extracellular spacer, a transmembrane domain and intracellular co-stimulatory and signaling domains.

[0008] Current binders, i.e. the extracellular antigen recognition moiety of a CAR, used in clinically approved adoptive CAR T cell immunotherapies are either derived from murine or camelid origin. The identification of binders of human origin and their utilization in CAR constructs avoids the immune system’s recognition of sequences from other species origin. Thus, the application of human binders will contribute to higher efficiency of CAR T cell productions in therapeutic applications.

[0009] As an exemplary malignancy, acute myeloid leukemia (AML) is a type of cancer characterized by the patient’s bone marrow producing large numbers of abnormal blood cells. Several subtypes of AML have been described, and an increased number of immature myeloid blasts is common to them. In AML, but also other cancers, the antigen CLEC12A (CLL-l / CD371 / hMICL) has been described as a target antigen for CAR T cell therapy. The choice of the antigen is critical to prevent on-target, off-tumor toxicities. In this regard CLEC12A has been described a target antigen with distinctive expression on cancer cells compared to normal cellular subsets. The majority of AML blasts express CLEC12A at diagnosis (Bakker AB et al., Cancer Res. 2004 Nov 15;64(22):8443-50; Haubner S et al., 2019 Jan;33(l):64-74; Larsen H0 et al., MBG_178

[0010] Cytometry B Clin Cytom. 2012 Jan;82(l):3-8; Zhao X et al., Haematologica. 2010 Jan;95(l):71-8. Wang J et al., J Hematol Oncol. 2018 Jan 10; 1 l(l):7.)and expression was found to be independent of disease status (Haubner S et al., Leukemia. 2019 Jan;33(l):64-74; Larsen H0 et al., Cytometry B Clin Cytom. 2012 Jan;82(l):3-8; Coustan-Smith E et al., JCI Insight. 2018 May 3;3(9):e9856L), but similar across French-American-British (FAB) classification subtypes (Bakker AB et al., 2004 Nov 15;64(22):8443-50). In contrast, CLEC12A is absent on normal hematopoietic stem and progenitor cells (Bakker AB et al., Cancer Res. 2004 Nov 15;64(22):8443-50; Bill M et al., J Cell Mol Med. 2018 Apr;22(4):2311-2318; Haubner S et al., Leukemia. 2019 Jan;33(l):64-74; van Rhenen A et al., Leukemia. 2007 Aug;21(8): 1700-7,). Moreover, expression is not found on erythrocytes, basophils, thrombocytes, monocytes, natural killer cells as well as B- and T cells (Morsink LM et al., Blood Rev. 2019 Mar;34:26-33). However, CLEC12A was described on common myeloid progenitors, granulocyte progenitors and megakaryocyte-erythroid progenitors (Bill et al., J Cell Mol Med., 2018). To our knowledge, CLEC12A has been described as a safe target in CAR T cell treatments (Wang J et al., J Hematol Oncol. 2018 Jan 10; 11( 1): 7).

[0011] There is a need in the art for improved or alternative immunotherapies targeting CLEC12A expressing target cells.

[0012] Brief description of the invention

[0013] The inventors found novel sequences of antigen binding domains specific for the antigen CLEC12A for use as binders (antigen binding domains) in chimeric antigen receptors.

[0014] The invention comprises an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising

[0015] a) an antigen binding domain specific for the antigen CLEC12A,

[0016] b) a transmembrane domain, and

[0017] c) an intracellular signaling domain,

[0018] wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:72 VH) and SEQ ID NO:73 (VL), or is encoded by a nucleotide sequence comprising SEQ ID NO: 67 (VH) and SEQ ID NO: 68 (VL).

[0019] The use of said (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) is for directing immune cells such as T cells that comprise said nucleic acid molecule (and express the CAR encoded by said nucleic acid molecule) to target cells expressing CLEC12A, i.e. cancer or leukemia cells expressing CLEC12A, in a subject.

[0020] Said cancer or leukemia may be myelodysplastic syndrome (MDS), monoclonal gammopathy of undetermined significance (MGUS), myelofibrosis or acute myeloid leukemia (AML). MBG_178

[0021] Said cancer or leukemia preferentially may be acute myeloid leukemia (AML).

[0022] The present invention also comprises a CAR comprising

[0023] a) an antigen binding domain specific for the antigen CLEC12A,

[0024] b) a transmembrane domain, and

[0025] c) an intracellular signaling domain,

[0026] wherein said antigen binding domain comprises an amino acid sequence encoded by SEQ ID NO:72 (VH) and SEQ ID NO:73 VL), or by SEQ ID NO:67 (VH) and SEQ ID NO:68 (VL). The present invention also comprises a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A wherein the antigen binding domain comprises a nucleic acid molecule encoding SEQ ID NO:57 (VH) and SEQ ID NO:58 (VL), or encoding SEQ ID NO: 51 (VH) and SEQ ID NO: 52 (VL),

[0027] b) a transmembrane domain, and

[0028] c) an intracellular signaling domain.

[0029] The present invention also comprises a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A wherein the antigen binding domain comprises SEQ IDNO:57 (VH) and SEQ IDNO:58 (VL), or comprises SEQ IDNO:51 (VH) and SEQ ID NO: 52 (VL),

[0030] b) a transmembrane domain, and

[0031] c) an intracellular signaling domain.

[0032] The present invention also comprises immune cells such as T cells, NK cells or gammadelta T cells that comprise a nucleic acid molecule encoding the chimeric antigen receptor (CAR) as disclosed herein, or that comprise a CAR as disclosed herein. The present invention also discloses said immune cells for use in treatment of cancer, wherein the cancer cells express the antigen CLEC12A.

[0033] Brief description of the drawings

[0034] Figure 1: Unique binder sequences with reactivity on different hCLEC12A variants were identified via phage display. A Six different panning strategies were used to identify binder candidates using hCLEC12A (Sino) and hCLEC12A (LSBio) in Strategy SI - S4 as well as hCLEC12A ectodomain (Miltenyi Biotec) and hCLEC12A-Fc (Miltenyi Biotec) in Strategy S5 and S6. In all strategies hlgGl-Fc (amsbio) antigen was used for competition. B The used phage display antibody library revealed 1706 first hits for hCLEC12A in ELISA. The six different selection strategies lead to the discovery of scFvs that bind preferably to the target(s) that were used in the panning strategies. C Sequencing results confirm 428 unique sequences derived MBG_178

[0035] from 1443 processable sequences. D All panning strategies lead to the selection of 93 unique scFvs reactive against CLEC12A-Fc, 213 unique scFvs reactive against CLEC12A (Sino) and 122 unique scFvs cross-reactive against both targets

[0036] Figure 2: Flow cytometric screening revealed potential CAR T cell candidates in scFv-Fc format with high specificity for hCLEC12A, Candidates were produced in scFv-Fc format by transfection of HEK293 cells. OCI-AML-2 cells were used to test for specificity of the candidates for hCLEC12A. IxlO5cells were plated and stained with 1 pg scFv-Fc for 30 minutes at 4°C. An anti-IgG(Fc)-PE secondary antibody staining was performed for 10 minutes at 4°C. Flow cytometric measurements are displayed as % of stained cells. Selected candidates are marked in black, non-selected candidates and controls in grey. A Flow cytometric results of candidates in scFv-Fc format binding to hCLEC12A expressing OCI-AML-2 cells. B Flow cytometric results of candidates in scFv-Fc format binding to hCLEC12A non expressing SupTl wt cells.

[0037] Figure 3: CAR T cell screening reveals functional, specific anti-CLEC12A CAR T cell candidates. 2xl04GFP- and CLEC12A-expressing target cells (OCI-AML-2) were seeded and co-cultured with 5xl04anti-CLEC12A CAR T cells to measure antigen-dependent lysis of AML cells. After 92h of co-culture, 2xl04AML cells (OCI-AML-2) were added in addition, followed by 2xl04AML cells (OCI-AML-2) after 164h. Target cell lysis by the CAR T cell candidates was analyzed by flow cytometry. As negative controls, target cells (OCI-AML-2) were cultured without addition of CAR T cells or co-cultured with untransduced T cells. One positive control CAR T cell construct known to successfully induce CLEC12A specific lysis of target cells was included. CAR T cell expansion was assessed by flow cytometric quantification after 92h, 164h as well as 260h of co-culture, i.e. before each addition of target cells and at endpoint, respectively. A, B, C Lysis of AML cells (OCI-AML-2) expressing CLEC12A by 45 CAR T candidates of one donor identified to be specific for CLEC12A in the preciously performed flow-cytometric screening. D, E, F CAR T cell expansion of all candidates from one donor as CD3+LNGFR+ cells / well after 92h, 164h as well as 260h of co-culture.

[0038] Figure 4: Assessment of anti-CLEC12A CAR T cell functional candidates with (G4S)3 linker.

[0039] A, B, C After exchange of the YOL linker sequence in scFv for (G4S)s 5xl04anti-CLEC12A CAR T cells from a second donor were co-cultured with 2xlO4GFP- and CLEC12A-expressing target cells (OCI-AML-2). 2xl04AML cells (OCI-AML-2) were added after 92h, and the third MBG_178

[0040] round of co-culture was initiated after 164h by addition of 5xl04AML cells (OCI-AML-2). D, E IFN-y secretion was measured after 24h of addition of AML cancer cells in round 1 and 3, respectively. F, G, H Again, CAR T cell expansion was assessed by flow cytometric quantification after 92h, 164h as well as 260h of co-culture, i.e. before each addition of target cells and at end-point, respectively. I At the end-point CAR T cells of this donor as well as of a third, additional donor were analyzed for the expression of PD-1 and CD27 amongst CD3+transduced cells. Statistical analysis to identify reduced frequencies of PD-1' CD27+events for CAR constructs MB114-C03-G4S and MB117-C04-G4S in comparison to M26 control CAR was done by Bonferroni post-hoc analysis after 2-way ANOVA.

[0041] Figure 5: An vivo activity of CLEC12 A CAR T constructs in the HL60 xenograft model. A The CLEC12A CAR constructs are comprised of a fully human CLEC12A targeting scFv, a CD8hinge and transmembrane domain, 4-1BB co-stimulatory domain and a CD3(^ activation domain. B Primary T cells from a healthy donor were transduced with lentiviral vectors encoding CAR constructs. CAR surface expression was assayed by flow cytometry. UTD -untransduced control. C Schematic representation of the experimental design for CAR evaluation in vivo. D Percentage change in total body weight of the mice was recorded throughout the study, mean ± SEM. E Survival curves for CAR T cell - treated groups of mice and for control groups. F Images of mice representing tumor burden post treatment in CAR-treated and control groups, as detected by bioluminescence. G Quantitation of tumor growth kinetics per treatment group, as based on mouse whole body bioluminescence (radiance), n= 6, mean ± SEM.

[0042] Detailed description of the invention

[0043] In a first aspect the present invention provides an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A,

[0044] b) a transmembrane domain, and

[0045] c) an intracellular signaling domain,

[0046] wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:72 (VH) and SEQ ID NO:73 (VL) or is encoded by a nucleotide sequence comprising SEQ ID NO:67 (VH) and SEQ ID NO:68 (VL), (and wherein said said antigen binding domain is a scFv).

[0047] Said (isolated) nucleic acid molecule, wherein said antigen binding domain is a scFv. MBG_178

[0048] Said (isolated) nucleic acid molecule, wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:72 (VH) and SEQ ID NO:73 (VL), wherein the order in said CAR from the N-terminus to the C-terminus is SEQ ID NO:72 (VH) - SEQ ID NO:73 (VL), or is encoded by a nucleotide sequence comprising SEQ ID NO:67 (VH) and SEQ ID NO:68 (VL), wherein the order in said CAR from the N-terminus to the C-terminus is SEQ ID NO: 67 (VH) - SEQ ID NO: 68 (VL).

[0049] Said (isolated) nucleic acid molecule, wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:71 or SEQ ID NO:66.

[0050] Said (isolated) nucleic acid molecule, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs) such as the stimulatory domain of CD3zeta and / or one or more co-stimulatory domain(s) such as CD28 and / or 4-1BB.

[0051] Said (isolated) nucleic acid molecule, wherein said antigen CLEC12A is expressed on a target cell.

[0052] Said (isolated) nucleic acid molecule, wherein said target cell expressing CLEC12A is a cancer cell.

[0053] Said (isolated) nucleic acid molecule, wherein said cancer or leukemia may be myelodysplastic syndrome (MDS), monoclonal gammopathy of undetermined significance (MGUS), myelofibrosis or acute myeloid leukemia (AML).

[0054] Said (isolated) nucleic acid molecule, wherein said cancer cell is acute myeloid leukemia (AML).

[0055] In another aspect the present invention provides a CAR comprising

[0056] a) an antigen binding domain specific for the antigen CLEC12A,

[0057] b) a transmembrane domain, and

[0058] c) an intracellular signaling domain,

[0059] wherein said antigen binding domain comprises an amino acid sequence encoded by SEQ ID NO:72 (VH) and SEQ ID NO:73 VL), or by SEQ ID NO:67 (VH) and SEQ ID NO:68 (VL). Said CAR, wherein said antigen binding domain comprises an amino acid sequence encoded by SEQ ID NO:71 or SEQ ID NO:66.

[0060] In another aspect the present invention provides a chimeric antigen receptor (CAR) comprising MBG_178

[0061] a) an antigen binding domain specific for the antigen CLEC12A wherein the antigen binding domain comprises a nucleic acid molecule encoding SEQ ID NO:57 (VH) and SEQ ID NO:58 (VL), or encoding SEQ ID NO: 51 (VH) and SEQ ID NO: 52 (VL),

[0062] b) a transmembrane domain, and

[0063] c) an intracellular signaling domain.

[0064] Said CAR, wherein the antigen binding domain comprises a nucleic acid molecule encoding SEQ ID NO:56 or SEQ ID NO:50.

[0065] In another aspect the present invention provides a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A wherein the antigen binding domain comprises SEQ IDNO:57 (VH) and SEQ IDNO:58 (VL), or comprises SEQ IDNO:51 (VH) and SEQ ID NO: 52 (VL),

[0066] b) a transmembrane domain, and

[0067] c) an intracellular signaling domain.

[0068] Said CAR, wherein the antigen binding domain comprises SEQ ID NO:56 or SEQ ID NO:50. Said CAR, wherein said CAR comprises SEQ ID NO:59 or SEQ ID NO:53.

[0069] Said CAR, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (IT AMs) such as the stimulatory domain of CD3zeta and / or one or more co-stimulatory domain(s) such as CD28 and / or 4- IBB.

[0070] Said CAR, wherein said antigen CLEC12A is expressed on a target cell.

[0071] Said CAR, wherein said target cell expressing CLEC12A is a cancer cell.

[0072] Said CAR, wherein said cancer or leukemia may be myelodysplastic syndrome (MDS), monoclonal gammopathy of undetermined significance (MGUS), myelofibrosis or acute myeloid leukemia (AML).

[0073] Said CAR, wherein said cancer cell is acute myeloid leukemia (AML).

[0074] In another aspect the present invention provides an immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein.

[0075] Said immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein, wherein said immune cell does not comprise (does not express) additionally a transgenic TCR. MBG_178

[0076] Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

[0077] In another aspect the present invention provides an immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein for use in immunotherapy.

[0078] In another aspect the present invention provides an immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein for use in treatment of cancer, wherein the cancer cells express CLEC12A such as acute myeloid leukemia (AML).

[0079] Said immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein for use in treatment of cancer, wherein said immune cell does not comprise (does not express) additionally a transgenic TCR.

[0080] In another aspect the present invention provides an immune cell expressing a CAR as disclosed herein.

[0081] Said immune cell comprising a CAR as disclosed herein, wherein said immune cell does not comprise (does not express) additionally a transgenic TCR.

[0082] In another aspect the present invention provides an immune cell comprising a chimeric antigen receptor (CAR) as disclosed herein for use in immunotherapy.

[0083] In another aspect the present invention provides an immune cell comprising a chimeric antigen receptor (CAR) as disclosed herein for use in treatment of cancer, wherein the cancer cells express CLEC12A such as acute myeloid leukemia (AML).

[0084] Said immune cell comprising a CAR as disclosed herein for use in treatment of cancer, wherein said immune cell does not comprise (does not express) additionally a transgenic TCR.

[0085] In a further aspect the present invention provides a vector comprising a nucleic acid molecule encoding a CAR as disclosed herein.

[0086] A “vector” comprises a (isolated) nucleic acid molecule which can be used to deliver the (isolated) nucleic acid molecule to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or MBG_178

[0087] amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. Said vector may be preferentially a retroviral vector such as a lentiviral vector.

[0088] In a further aspect the present invention provides a pharmaceutical composition comprising an immune cell comprising an (isolated) nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein, and optionally a pharmaceutically acceptable carrier. In a further aspect the present invention provides a pharmaceutical composition comprising an immune cell comprising a CAR as disclosed herein, and optionally a pharmaceutically acceptable carrier.

[0089] Pharmaceutically acceptable carriers, diluents or excipients may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0090] In a further aspect the present invention provides an in-vivo method for treating a subject suffering from cancer, wherein the cancerous cells express CLEAC12A comprising administering to said subject an immune cell comprising an (isolated) nucleic acid molecule encoding a CAR as disclosed herein or administering to said subject an immune cell comprising a CAR as disclosed herein.

[0091] In one embodiment of the invention the immune cells comprising the nucleic acid molecule encoding a chimeric antigen receptor (CAR) as disclosed herein and / or comprising the CAR as disclosed herein are for use in treatment of a disease associated with a target cell of a subject suffering from said disease, wherein said target cell expresses CLEC12A and the disease may be a cancer such as AML. Immune cells, e.g. T cells orNK cells of a subject may be isolated. The subject may e.g. suffer from said cancer or may be a healthy subject. These cells are genetically modified e.g. in vitro to express the CAR as disclosed herein. These engineered cells may be activated and expanded in vitro. In a cellular therapy these engineered cells are infused to a recipient in need thereof. These cells may be a pharmaceutical composition (said MBG_178

[0092] cell plus pharmaceutical acceptable carrier). The infused cells may be e.g. able to kill (or at least stop growth of) cancerous cells in the recipient. The recipient may be the same subject from which the cells was obtained (autologous cell therapy) or may be from another subject of the same species (allogeneic cell therapy).

[0093] The immune cells, preferentially T cells or NK cells engineered to express the CAR as disclosed herein may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise a cell population of genetically modified cells (a plurality of immune cells) as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0094] Preferentially, the compositions are formulated for intravenous administration. The administration of cell compositions to the subject may be carried out in any convenient manner known in the art.

[0095] Pharmaceutical compositions may be administered in a manner appropriate to the disease to be treated. Appropriate dosages may be determined by clinical trials. But the quantity and frequency of administration will also be determined and influenced by such factors as the condition of the patient, and the type and severity of the patient's disease.

[0096] A pharmaceutical composition comprising the immune cells, preferentially T cells or NK cells as disclosed herein may be administered at a dosage of 104to 109cells / kg body weight, preferably 105to 106cells / kg body weight. The cell compositions may also be administered several times at these dosages. The compositions of cells may be injected e.g. directly into a tumor, lymph node, or site of infection.

[0097] The genetically engineered immune cells may be activated and expanded to therapeutic effective amounts using methods known in the art.

[0098] The immune cells of the invention may be used in combination with e.g. chemotherapy, radiation, immunosuppressive agents, antibodies or antibody therapies. MBG_178

[0099] Generation of of CAR T cells

[0100] Processes of generation of CAR immune cells such as CAR T cells are well known in the art. Exemplarily in the following methods of generation of immune cells expressing a CAR are disclosed.

[0101] The genetically modified immune cells expressing the CAR as disclosed herein, preferentially T cells, may be generated preferentially in an automated process in a closed system. A process for the generation of genetically modified cells, preferentially T cells, is disclosed e.g. in WO2015162211A1 and may comprise the e.g. steps:

[0102] a) providing a cell sample comprising immune cells (e.g. from a PBMC)

[0103] b) preparation of the cell sample by centrifugation

[0104] c) magnetic separation of the immune cells, preferentially T cells,

[0105] d) activation of the enriched immune cells, preferentially T cells, using modulatory agents e) genetically modifying the immune cells, preferentially T cells, to express the CAR as disclosed herein

[0106] f) expansion of the genetically modified immune cells, preferentially T cells, in a cultivation chamber

[0107] g) washing of the cultured immune cells, preferentially T cells.

[0108] All these steps may be performed automatically in a closed system, preferentially in a closed and sterile system.

[0109] The process is especially suited for preparing gene modified cells such as immune cells, preferentially T cells, wherein the enriched immune cells, preferentially T cells, are gene-modified by using viral and / or non-viral vectors, preferentially using a lentiviral vector.

[0110] In case of magnetically enrichment of T cells from PBMC or leukapheresis anti-CD4 and / or anti-CD8 antibodies or antigen binding fragments coupled to beads may be used.

[0111] The modulatory agents may be selected from agonistic antibodies such as anti-CD3 and / or anti-CD28 antibodies or antigen binding fragments thereof (especially in case of modifying T cells), and / or cytokines. MBG_178

[0112] The gene-modified immune cells, preferentially T cells, may be enriched by magnetic labelling of immune cells and magnetic separation before or after cultivation to obtain higher frequency of gene-modified immune cells, preferentially T cells, in the final cellular product.

[0113] The cultivation (expansion) may be over several day such as 8 to 12 days, or may be a shorter cultivation process without or with less cultivation / expansion as disclosed e.g. in WO2020239866A1. In case of a shorter in-vitro process of generation of immune cells such as T cells, the generated immune cells such as T cells may expand in-vivo after administration to a subject in need thereof to therapeutically effect amounts of immune cells expression the CAR as disclosed herein (see e.g. WO2020239866A1) . Such a short ex-vivo process may comprise e.g. (in a closed and sterile system for cell modification) the steps

[0114] a) providing a sample (e.g. from PBMC) comprising immune cells such as T cells

[0115] b) preparation of said sample by centrifugation

[0116] c) enrichment of the immune cells such as T cells of step b

[0117] d) activation of the enriched immune cells such as T cells using modulatory agents

[0118] e) genetic modification of the activated immune cells such as T cells by transduction e.g. with lentiviral vector particles

[0119] f) removal of said modulatory agents,

[0120] thereby generating a sample of genetically modified immune cells such as T cells, wherein said method is performed e.g. in equal or less than 3 days (72h).

[0121] As a closed and sterile system for cell modification, the fully automated cell processing device CliniMACS Prodigy® and associated tubing sets (Miltenyi Biotec GmbH, Germany) may be used (W02009 / 072003). This closed system meets the requirements of GMP -grade processing of almost any kind of cellular products and may allow reducing clean room requirements, improve technology transfer and harmonization of cell manufacturing processes.

[0122] Nucleotides, Expression, Vectors, and Host Cells

[0123] The nucleic acids (molecules) encoding a CAR as used herein may comprise a nucleotide sequence encoding any of the leader sequences, antigen binding domains, transmembrane domains, and / or intracellular T cell signaling domains described herein.

[0124] In some embodiments, the nucleotide sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleotide sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleotide sequence may involve substituting a native codon for another codon that encodes the same amino acid, but can be translated by tRNA that is more readily available within a cell, thus MBG_178

[0125] increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0126] "Nucleic acid" as used herein includes "polynucleotide", "oligonucleotide", "nucleic acid molecule" and “nucleic acid sequence” and generally means a polymer of DNA or RNA, which can be single-stranded or double-stranded, synthesized or obtained (e.g., isolated and / or purified) from natural sources, which can contain natural, non-natural or altered nucleotides, and which can contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

[0127] A recombinant nucleic acid may be one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. The nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art. For example, a nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed upon hybridization (e.g., phosphorothioate derivatives and acridine substituted nucleotides).

[0128] The nucleic acid can comprise any isolated or purified nucleotide sequence which encodes any of the CARs or functional portions or functional variants thereof. Alternatively, the nucleotide sequence can comprise a nucleotide sequence which is degenerate to any of the sequences or a combination of degenerate sequences.

[0129] Also provided is a nucleic acid comprising a nucleotide sequence that is at least about 70% or more, e.g., about 80%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any of the nucleic acids described herein.

[0130] In an embodiment, the nucleic acids can be incorporated into a recombinant expression vector. In this regard, an embodiment provides recombinant expression vectors comprising any of the nucleic acids.

[0131] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, MBG_178

[0132] polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors are not naturally-occurring as a whole.

[0133] However, parts of the vectors can be naturally-occurring. The recombinant expression vectors can comprise any type of nucleotides, including, but not limited to DNA and RNA, which can be single-stranded or double- stranded, synthesized or obtained in part from natural sources, and which can contain natural, non-natural or altered nucleotides. The recombinant expression vectors can comprise naturally occurring or non-naturally-occurring internucleotide linkages, or both types of linkages. Preferably, the non-naturally occurring or altered nucleotides or internucleotide linkages do not hinder the transcription or replication of the vector.

[0134] In an embodiment, the recombinant expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.

[0135] The recombinant expression vector may be a viral vector, e.g., a retroviral vector or a lentiviral vector. A lentiviral vector is a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector. Other examples of lentivirus vectors that may be used in the clinic, include, for example, and not by way of limitation, the LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica pic, the LENTIMAX.TM. vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0136] A number of transfection techniques are generally known in the art. Transfection methods include e.g. calcium phosphate co-precipitation, direct micro injection into cultured cells, electroporation, liposome mediated gene transfer, and lipid mediated transduction. If DNA or RNA is introduced into cells by using viral vector carriers, then the technique is called transduction.

[0137] Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell.

[0138] The recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based. The recombinant expression vector may comprise restriction sites to facilitate cloning. MBG_178

[0139] The recombinant expression vector can include one or more marker genes, which allow for selection of transformed or transfected host cells. Marker genes include biocide resistance, e.g., resistance to antibiotics, heavy metals, etc., complementation in an auxotrophic host to provide prototrophy, and the like. Suitable marker genes for the inventive expression vectors include, for instance, neomycin / G418 resistance genes, hygromycin resistance genes, histidinol resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0140] The recombinant expression vector can comprise a native or nonnative promoter operably linked to the nucleotide sequence encoding the CAR (including functional portions and functional variants thereof), or to the nucleotide sequence which is complementary to or which hybridizes to the nucleotide sequence encoding the CAR. The selection of promoters, e.g., strong, weak, inducible, tissue-specific and developmental-specific, is within the ordinary skill of the artisan. Similarly, the combining of a nucleotide sequence with a promoter is also within the skill of the artisan. The promoter can be a nonviral promoter or a viral promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, or a promoter found in the long-terminal repeat of the murine stem cell virus.

[0141] The recombinant expression vectors can be designed for either transient expression, for stable expression, or for both. Also, the recombinant expression vectors can be made for constitutive expression or for inducible expression.

[0142] Further, the recombinant expression vectors can be made to include a suicide gene. As used herein, the term "suicide gene" refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., a drug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes are known in the art and include, for example, the Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0143] An embodiment further provides a host cell comprising any of the recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive recombinant expression vector. The host cell can be a eukaryotic cell, e.g., plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human. The host cell can be an adherent cell or a suspended cell, i.e., a cell that grows in suspension. Suitable host cells are known in the art and include, for instance, DH5a E. coli cells, Chinese hamster ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For purposes of amplifying or replicating the recombinant expression vector, the host cell may MBG_178

[0144] be a prokaryotic cell, e.g., a DH5a cell. For purposes of producing a recombinant CAR, the host cell may be a mammalian cell. The host cell may be a human cell. While the host cell can be of any cell type, can originate from any type of tissue, and can be of any developmental stage, the host cell may be a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). The host cell may be a T cell. For purposes herein, the T cell can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. T cells can also be enriched for or purified. The T cell may be a human T cell. The T cell may be a T cell isolated from a human. The T cell can be any type of T cell and can be of any developmental stage, including but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, e.g., Thl and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, memory stem cells, i.e. Tscm, naive T cells, and the like. The T cell may be a CD8+ T cell or a CD4+ T cell.

[0145] In an embodiment, the CARs as described herein can be used in suitable non-T cells. Such cells are those with an immune-effector function, such as, for example, NK cells, and T-like cells generated from pluripotent stem cells.

[0146] General methods of treatment

[0147] It is contemplated that the CARs disclosed herein can be used in methods of treating or preventing a disease in a mammal. In this regard, an embodiment provides a method of treating cancer as disclosed herein in a mammal, comprising administering to the mammal (the subject) the CARs, the nucleic acids encoding the CARs, the recombinant expression vectors encoding the CARs, the immune cells expressing the CARs as disclosed herein in an amount effective to treat cancer in the mammal.

[0148] An embodiment, especially for the treatment of cancer, further comprises lymphodepleting the mammal prior to administering the CARs disclosed herein. Examples of lymphodepletion include, but may not be limited to, nonmyeloablative lymphodepleting chemotherapy, myeloablative lymphodepleting chemotherapy, total body irradiation, etc.

[0149] For purposes of the methods, wherein immune cells are administered, the cells can be cells that are allogeneic or autologous to the mammal. Preferably, the cells are autologous to the mammal. As used herein, allogeneic means any material derived from a different animal 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 material from individuals of the same species may be sufficiently unlike genetically to interact antigenically. As used herein, “autologous” means any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0150] The mammal referred to herein can be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). Preferably, the mammal is a human.

[0151] With respect to the methods, the cancer can be any cancer in that CLEC12A expressing target cells are involved, e.g. in AML.

[0152] The terms "treat," and "prevent" as well as words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods can provide any amount or any level of treatment of cancer or heart diseases in a mammal.

[0153] Furthermore, the treatment or prevention provided by the method can include treatment or prevention of one or more conditions or symptoms of the disease, e.g., cancer, being treated or prevented. Also, for purposes herein, "prevention" can encompass delaying the onset of the disease, or a symptom or condition thereof.

[0154] Any method of administration can be used for the disclosed therapeutic agents, including local and systemic administration. For example topical, oral, intravascular such as intravenous, intramuscular, intraperitoneal, intranasal, intradermal, intrathecal and subcutaneous administration can be used. The particular mode of administration and the dosage regimen will be selected by the attending clinician, taking into account the particulars of the case (for example the subject, the disease, the disease state involved, and whether the treatment is prophylactic). In cases in which more than one agent or composition is being administered, one or more routes of administration may be used; for example, a chemotherapeutic agent may be administered orally and a composition of immune cells expressing the CARs as disclosed herein may be administered intravenously.

[0155] Methods of administration include injection for which the CAR, CAR T cell or the compositions are provided in a nontoxic pharmaceutically acceptable carrier such as water, saline, Ringer's solution, dextrose solution, 5% human serum albumin, fixed oils, ethyl oleate, or liposomes. In some embodiments, local administration of the disclosed compounds or compositions (e.g. the cells expressing the CARs as disclosed herein) can be used, for instance by applying the compounds or compositions to a region of tissue from which a tumor has been removed, or a region suspected of being prone to tumor development. In some embodiments, sustained intra-tumoral (or near-tumoral) release of the pharmaceutical preparation that includes a therapeutically effective amount of the compounds or compositions may be beneficial. In other examples, the conjugate is applied as an eye drop topically to the cornea, or intravitreally into the eye.

[0156] The disclosed therapeutic agents can be formulated in unit dosage form suitable for individual administration of precise dosages. In addition, the disclosed therapeutic agents may be administered in a single dose or in a multiple dose schedule. A multiple dose schedule is one in which a primary course of treatment may be with more than one separate dose, for instance 1-10 doses, followed by other doses given at subsequent time intervals as needed to maintain or reinforce the action of the compositions.

[0157] Treatment can involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years. Thus, the dosage regime will also, at least in part, be determined based on the particular needs of the subject to be treated and will be dependent upon the judgment of the administering practitioner.

[0158] Treatment of cancer

[0159] In some embodiments, the disclosed methods include providing surgery, radiation therapy, and / or chemotherapeutics to the subject in combination with a disclosed CAR or T cell expressing a CAR (for example, sequentially, substantially simultaneously, or simultaneously). Methods and therapeutic dosages of such agents and treatments are known to those skilled in the art, and can be determined by a skilled clinician. Preparation and dosing schedules for the additional agent may be used according to manufacturer's instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described elsewhere.

[0160] In some embodiments, the combination therapy can include administration of a therapeutically effective amount of an additional cancer inhibitor to a subject. Non-limiting examples of additional therapeutic agents that can be used with the combination therapy include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, gene regulators, and angiogenesis inhibitors. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. For example, any suitable anticancer or anti-angiogenic agent can be administered in combination with the CARs or CAR- T cells disclosed herein. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

[0161] Additional chemotherapeutic agents include, but are not limited to alkylating agents, such as nitrogen mustards (for example, chlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan), nitrosoureas (for example, carmustine, fotemustine, lomustine, and streptozocin), platinum compounds (for example, carboplatin, cisplatin, oxaliplatin, and BBR3464), busulfan, dacarbazine, mechlorethamine, procarbazine, temozolomide, thiotepa, and uramustine; antimetabolites, such as folic acid (for example, methotrexate, pemetrexed, and raltitrexed), purine (for example, cladribine, clofarabine, fludarabine, mercaptopurine, and tioguanine), pyrimidine (for example, capecitabine), cytarabine, fluorouracil, and gemcitabine; plant alkaloids, such as podophyllum (for example, etoposide, and teniposide), taxane (for example, docetaxel and paclitaxel), vinca (for example, vinblastine, vincristine, vindesine, and vinorelbine); cytotoxic / antitumor antibiotics, such as anthracycline family members (for example, daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, and valrubicin), bleomycin, rifampicin, hydroxyurea, and mitomycin; topoisomerase inhibitors, such as topotecan and irinotecan; monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, gemtuzumab, rituximab, panitumumab, pertuzumab, and trastuzumab; photosensitizers, such as aminolevulinic acid, methyl aminolevulinate, porfimer sodium, and verteporfm; and other agents , such as alitretinoin, altretamine, amsacrine, anagrelide, arsenic trioxide, asparaginase, axitinib, bexarotene, bevacizumab, bortezomib, celecoxib, denileukin diftitox, erlotinib, estramustine, gefitinib, hydroxycarbamide, imatinib, lapatinib, pazopanib, pentostatin, masoprocol, mitotane, pegaspargase, tamoxifen, sorafenib, sunitinib, vemurafinib, vandetanib, and tretinoin. Selection and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.

[0162] The combination therapy may provide synergy and prove synergistic, that is, the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately.

[0163] In another aspect the present invention provides a pseudotyped retroviral vector particle comprising MBG_178

[0164] a) one envelope protein with antigen-binding activity, wherein said envelope protein is a recombinant protein and is fused at its ectodomain to a polypeptide (e.g. an antibody or antigen binding fragment thereof such as a scFv, a Fab or a single domain antibody) that specifically binds to an antigen expressed on the surface of a target cell, wherein said target cell is a CD4+ T cell and / or a CD8+ T cell, and wherein said antigen is CD4 and / or CD8, wherein said envelope protein is protein H of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the morbillivirus genus, or wherein said envelope protein is protein G of a virus of the Paramyxoviridae family, if said Paramyxoviridae virus is a virus of the Henipavirus genus,

[0165] b) one envelope protein with fusion activity (protein F) of a virus of the Paramyxoviridae family, wherein said Paramyxoviridae virus is a virus of the morbillivirus genus or the Henipavirus genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus,

[0166] c) a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising

[0167] i) an antigen binding domain comprising SEQ ID NO:57 and SEQ ID NO:58, or comprising SEQ ID NO:51 and SEQ ID NO:52,

[0168] ii) a transmembrane domain, and

[0169] iii) an intracellular signaling domain,

[0170] and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0171] Said pseudotyped retroviral vector particle, wherein said virus of the morbillivirus genus is canine distemper virus (CDV) or wherein said virus of the Henipavirus genus is Nipah Virus (NiV).

[0172] Said pseudotyped retroviral vector particle, wherein said protein H of CDV (CDV-H) is a modified protein CDV-H, wherein said modified protein CDV-H comprises a modified cytoplasmic tail and / or wherein said protein F of CDV (CDV-F) is a modified protein CDV-F, wherein said modified protein CDV-F comprises a modified cytoplasmic tail, when said virus of the morbillivirus genus is CDV , or

[0173] wherein said protein G of NiV (NiV-G) is a modified protein NiV-G, wherein said modified protein NiV-G comprises a modified cytoplasmic tail and / or wherein said protein F of NiV (NiV-F) is a modified protein NiV-F wherein said modified protein NiV-F comprises a modified cytoplasmic tail, when said virus of the Henipavirus genus is NiV. MBG_178

[0174] In-vivo gene therapy

[0175] Resting or non-dividing T cells such as CD+4 T cells and / or CD8+ T cells can also be efficiently transduced in-vivo using CD4-targeted and / or CD8-targeted viral vectors such as pseudotyoed retroviral vector particles that comprise a nucleic acid molecule encoding a transgenic polypeptide, herein regularly the CAR as disclosed herein. Such pseudotyped retroviral vectors are disclosed e.g. in WO2023015217A1, WO2022150731A1 and EP24159106.4.

[0176] Pseudo typed retroviral vector particles

[0177] Retroviridae is a virus family with a single-stranded, diploid, positive-sense RNA genome that is reverse-transcribed into a DNA intermediate that is then incorporated into the host cell genome. Relroviridae- v\ viruses are enveloped particles with a diameter of 80-120 nm. (Retro- / lenti- / gammaretro-) viral vectors are replication-deficient viral particles that are derived from the corresponding virus family. They contain Gag and Pol proteins, a singlestranded RNA genome and are usually pseudotyped with heterologous envelope proteins derived from other viruses. The RNA genome of said viral vectors do not contain any viral gene to produce viral progeny, but psi elements and LTRs that are required for efficient packing and reverse transcription into DNA. The DNA intermediate may contain a gene of interest under the control of a suitable promoter, for example, the CMV promoter and the gene of interest is expressed upon integration of said DNA into the genome of the host cell. The process of entering the host cell, delivering the RNA genome, integration and expression of the gene of interest is called transduction. The minimal requirements of a gammaretrovirus or lentivirus based viral vector has been well-described in the art.

[0178] Lentivirus is a genus of Retroviridae that cause chronic and deadly diseases characterized by long incubation periods, in the human and other mammalian species. The best-known lentivirus is the Human Immunodeficiency Virus (HIV), which can efficiently infect nondividing cells, so lentiviral derived retroviral vectors are one of the most efficient methods of gene delivery. Gammaretroviridae is a genus of the Retroviridae family. Representative species are the murine leukemia virus (MLV) and the feline leukemia virus (FLV).

[0179] Paramyxoviridae is a family of viruses in the order of Mononegavirales. There are currently 49 species in this family, divided among 7 genera. Diseases associated with this virus family include measles, mumps, and respiratory tract infections. Members of this virus family are enveloped viruses with a non-segmented, negative-strand RNA genome of about 16 kb. Two MBG_178

[0180] membrane proteins with two distinct functions appear as spikes on the virion surface. The H / HN / G proteins mediate binding to the receptor at the cell surface.

[0181] The “Nipah virus” (NiV) is a member of the family Paramyxoviridae, genus Henipavirus. Nipah virus is an enveloped virus with negative-stranded polarity and a non-segmented RNA genome encoding the main structural proteins: nucleopcapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), attachment glycoprotein (G) and RNA polymerase protein (L). Nipah virus enters the cell via binding of the G protein to its receptor ephrinB2 or ephrinB3, followed by pH-independent fusion of the virus with the cell membrane on the plasma membrane induced by the F protein. Of note, induction of fusion requires activation of the F protein by the G protein.

[0182] The “Canine Distemper virus” (CDV) is a member of the family Paramyxoviridae, genus Morbillivirus. CDV is an enveloped virus with negative-stranded polarity and a non-segmented RNA genome encoding the main structural proteins: nucleopcapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F), haemagglutinin protein (H) and the large protein (L). The non- structural protein (C) is encoded from the gene sequence of the P protein overlapping open reading frame. CDV enters the cell via binding of the H protein to its receptor Nectin-4 or SLAM, followed by pH-independent fusion of the virus with the cell membrane on the plasma membrane induced by the F protein. Of note, induction of fusion requires activation of the F protein by the G protein.

[0183] Thus, the term “(virus) envelope protein(s) that have antigen binding activity” as used herein refers to protein(s) on the viral envelope that are responsible for binding to complementary receptors or antigens on the cell membrane of a target cell. For Paramyxoviridae H, HN or G proteins are virus envelope protein(s) that have antigen binding activity.

[0184] Upon binding the H / HN / G proteins change their conformation that induces a process called fusion helper function, leading to subsequent conformational changes within the F protein that is mediating the fusion of the viral and cellular membrane. The capsid and viral genome may now enter and infect or transduce the host cell.

[0185] The term “(virus) envelope proteins(s) that have fusion activity” as used herein refers to protein(s) that initiate fusion of viral and cellular membrane. For Paramyxoviridae F proteins refer to virus envelope protein(s) that have fusion activity. MBG_178

[0186] The term "pseudotyping” or “pseudotyped" as used herein refers to a viral vector particle bearing envelope glycoproteins derived from other viruses having envelopes. The host range of the lentiviral vectors or viral vector particles of the present invention can thus be expanded or altered depending on the type of cell surface receptor used by the glycoprotein.

[0187] The terms “cytoplasmic domain”, "cytoplasmic portion", "cytoplasmic tail", "cytoplasmic region", “intracellular domain” or “endodomain”, as used in herein refer to the portion of the respective protein that is adjacent to the transmembrane domain of the protein and, if the protein is inserted into the membrane under physiological conditions, extends into the cytoplasm or in case of viral particles reaching into the intravirion side. Within Paramyxoviridae all envelope proteins with antigen-binding function are characterized to date as type II membrane proteins, meaning that the cytoplasmic domain is located at the N-terminus of the envelope protein. Within Paramyxoviridae all envelope proteins with fusion function are characterized to date as type I membrane proteins, meaning that the cytoplasmic domain is located at the C-terminus of the envelope protein.

[0188] The term “modified cytoplasmic tail”, as used herein refers to a cytoplasmic tail is truncated, mutated or replaced by a heterologous cytoplasmic tail (or part of a heterologous cytoplasmic tail) from a different virus.

[0189] The term "truncated", as used in the present invention, refers to a deletion of amino acid residues of the designated protein. It is clear to the skilled person that a protein is encoded by a nucleic acid. Thus, "truncated" also refers to the corresponding coding nucleic acids in a nucleic acid molecule that codes for a given "truncated" protein.

[0190] I. Retroviral vector particle pseudotyped with Nipah virus envelope proteins For selective retroviral vector particle pseudotyped with Nipah virus envelope proteins, the truncated protein G fused to the polypeptide comprising an antigen binding domain specific for CD4 or CD8 as disclosed herein may have mutations that reduce or ablate productive interactions with its native receptors ephrin-B2 and ephrin-B3. The potential receptor binding site of Nipah-G was described by Guillaume et al (2006; doi: 10.1128 / JVI.00190-06). They identified the mutation E533Q, E505A, W504A, Q530A, 531 A, A532K and N557A to abolish binding and fusion induction suggesting that these residues are implicated in receptor recognition. These residues were screened by Bender et al (2016; doi: 10.1371 / joumal.ppat.1005641) for ablation of receptor binding ability using Nipah-pseudotyped lentiviral vectors. Therefore, E501, W504, Q530, E533 were either evaluated as MBG_178

[0191] single mutation or in combination. The combined mutation of E501 A, W504A, Q530A, E533 A showed completely ablated receptor binding ability for both receptors ephrin-B2 and ephrin-B3. Mutation of amino acids in receptor binding domains of virus attachment proteins is a well-established method in the art to ablate receptor binding.

[0192] The person skilled in the art will readily be able to introduce mutations as, for example, additions and deletions, into a given nucleic acid or amino acid sequence.

[0193] II. Retroviral vector particle pseudotyped with CDV envelope proteins

[0194] For selective retroviral vector particle pseudotyped with CDV envelope proteins, the truncated protein H fused to the polypeptide comprising an antigen binding domain specific for CD4 or CD8 as disclosed herein may have mutations that reduce or ablate productive interactions with its native receptors SLAM and Nectin-4. A mutation that ablates interaction of canine distemper virus H protein with SLAM and Nectin 4 may be e.g. the point mutation at position D526, 1527, S528, R529; Y547 and T548, wherein amino these amino acids are replaced with another amino acid and this mutation prevents or assists in preventing interaction of the H protein with SLAM and Nectin-4 (Bah et al (2020), doi: 10.1158 / 1535-7163.MCT-20-0134; von Messing et al (2005), doi: 10.1128 / JVI.79.9.5857-5862).

[0195] The person skilled in the art will readily be able to introduce mutations as, for example, additions and deletions, into a given nucleic acid or amino acid sequence.

[0196] Generation of pseudotyped retroviral vector particles for administration

[0197] The quality and quantity of the pseudotyped retroviral vector should be sufficient to enable regulatory approval and safe treatment of larger patient cohorts.

[0198] In one embodiment the pseudotyped retroviral particles for clinical use are manufactured serum-free in suspension applying shaker flasks, bags or stirred bioreactors. In another embodiment HEK293 or HEK293T cells are used as packaging cell lines for the pseudotyped retroviral vectors described herein.

[0199] The pseudotyped retroviral vectors are generated by transient transfection, or stable producer cell lines (improving reproducibility), optionally including inducible expression systems to restrict the expression of pseudotyped retroviral vector components to the harvesting period only. MBG_178

[0200] Packaging cells are transiently transfected using magnetofection, electroporation or lipid- or non-lipid based transfection reagents established in the art, e.g. PEI, Calcium phospate, liposomes, LNPs.

[0201] The packaging cell line might be derived from a oligoclonal pool or a single clone that shows e.g. superior productivity, reproducibility, beneficial growth kinetics, cell media consumption or less impurities.

[0202] The packaging cell line might be genetically engineered for the expression of additional factors or for reducing or inhibiting the expression of specific factors.

[0203] In one embodiment the supernatant containing pseudotyped retroviral vectors is filtrated to remove cellular debris, enzymatically treated (e.g. DNAse), applied to tangential flow filtration, size exclusion chromatography, affinity chromatography, anion exchange chromatography and / or is sterile filtered.

[0204] In one embodiment the pseudotyped retroviral vector is formulated in pharmaceutically acceptable carrier, diluent or excipient compatible for administration in human.

[0205] In one application the pseudotyped retroviral vector is filled in vials or bags.

[0206] In another embodiment, the dose of filled pseudotyped retroviral vector is adjusted to the body weight.

[0207] In another embodiment, the pseudotyped retroviral vector is filled in the presence of a cryoprotectant.

[0208] In another embodiment, the pseudotyped retroviral vector is lyophilized and / or reconstituted before administration.

[0209] In another embodiment, the ratio of functional to non-functional pseudotyped retroviral vector particles is >1000:1, >100:1, >10:1, >1:1, >1:10, >1:100; >1:1000.

[0210] Methods of Treatment with pseudotyped retroviral vector particles

[0211] A pseudotyped retroviral vector particle as disclosed herein may be used to transduce T cells in-vivo at any effective dosage. In some embodiments, the viral particle is administered to a subject in-vivo by application to the tissue, the organ or to the blood circulation of a subject in need of therapy.

[0212] In some embodiments, the pseudotyped retroviral vector particle as disclosed herein may be administered via a route of parenteral, intravenous, intramuscular, subcutaneoustanous, intratumoral, intraperitoneal, or intralymphatic administration. In some embodiments, the viral particle may be administered multiple times. MBG_178

[0213] In one embodiment the pseudotyped retroviral vector particle as disclosed herein may be administered intratumorally to a subject and thereby activates and transduces the T cell portion of the tumor-infiltrating lymphocytes at the tumor site.

[0214] In one embodiment the pseudotyped retroviral vector particle as disclosed herein may be administered intravenously to a subject, and thereby activates and transduces the T cells in the circulatory blood system.

[0215] In one embodiment the pseudotyped retroviral vector particle as disclosed herein may be administered by intranodal (lymphnode) injection to a subject, and thereby transduces the T cells in the lymph node.

[0216] In one embodiment the pseudotyped retroviral vector particle as disclosed herein may be administered by intra splenic injection to a subject, and thereby activates and transduces the T cells in the spleen.

[0217] In one embodiment, administration of a total dose of the pseudotyped retroviral vector as disclosed herein is a unique administration of said pseudotyped retroviral vector to the subject. In one embodiment, administration of a total dose of the pseudotyped retroviral vector as disclosed herein includes administration of a total desired dose that includes at least two repeated doses that are each separately administered to the subject resulting in multiple administrations over a specified time period. In some embodiments, each repeated dose may be administered from a separate composition containing the pseudotyped retroviral vector as disclosed herein so that the total dose is provided as a plurality of compositions that are administered separately over a specified time period. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one month. In some embodiments, a first dose and second dose, and in some cases one or more additional doses, are administered over more than one day. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one week. In some embodiments, the repeated doses are administered over a period of no more than three days, such as once a day for two days (e.g. a first dose and a second dose) or once a day for three days (e.g. a first dose, a second dose, and a third dose).

[0218] In one embodiment the pharmaceutical composition comprises the pseudotyped retroviral vector specific for the antigen CD4 as disclosed herein and the pseudotyped retroviral vector specific for the antigen CD8 as disclosed herein. 1

[0219] MBG_178

[0220] In one embodiment, administration of a total dose of said pharmaceutical composition is a unique administration of said pharmaceutical composition to the subject.

[0221] In one embodiment, administration of a total dose of said pharmaceutical composition includes administration of a total desired dose that includes at least two repeated doses that are each separately administered to the subject resulting in multiple administrations over a specified time period. In some embodiments, each repeated dose may be administered separately over a specified time period. In some embodiments, the plurality of pharmaceutical compositions are administered over a time period that is no more than one month. In some embodiments, a first dose and second dose, and in some cases one or more additional doses, are administered over more than one day. In some embodiments, the plurality of compositions (e.g. providing a first dose and a second dose, and optionally one or more successive doses) are administered over a time period that is no more than one week. In some embodiments, the repeated doses are administered over a period of no more than three days, such as once a day for two days (e.g. a first dose and a second dose) or once a day for three days (e.g. a first dose, a second dose, and a third dose).

[0222] All definitions, characteristics and embodiments defined herein with regard to the first aspect of the invention as disclosed herein also apply mutatis mutandis in the context of the other aspects of the invention as disclosed herein.

[0223] Definitions

[0224] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0225] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein, “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1% from the specified value

[0226] In general, a CAR may comprise an extracellular domain (extracellular part) comprising the antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain MBG_178

[0227] (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker (or spacer or hinge region). The extracellular domain may also comprise a signal peptide.

[0228] A "signal peptide" refers to a peptide sequence that directs the transport and localization of the protein within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0229] Generally, an “antigen binding domain” refers to the region of the CAR that specifically binds to an antigen, e.g. to a tumor associated antigen (TAA) or tumor specific antigen (TSA). The CARs may comprise one or more antigen binding domains (e.g. a tandem CAR). Generally, the targeting regions on the CAR are extracellular. The antigen binding domain may comprise an antibody or an antigen binding fragment thereof. The antigen binding domain may comprise, for example, full length heavy chain, Fab fragments (Fab), single chain Fv (scFv) fragments, divalent single chain antibodies, nanobodies, single domain antibodies, VHH or diabodies. Any molecule that binds specifically to a given antigen such as affibodies or ligand binding domains from naturally occurring receptors may be used as an antigen binding domain. Often the antigen binding domain is a scFv or nanobody. Normally, in a scFv the variable regions of an immunoglobulin heavy chain and light chain are fused by a flexible linker to form a scFv. Such a linker may be for example the “(G4 / S)3-linker”.

[0230] In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will be used in. For example, when it is planned to use it therapeutically in humans, it may be beneficial for the antigen binding domain of the CAR to comprise a human or humanized antibody or antigen binding fragment thereof. Human or humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.

[0231] “Spacer” or “hinge” as used herein refers to the hydrophilic region which is between the antigen binding domain and the transmembrane domain. The CARs may comprise an extracellular spacer domain but is it also possible to leave out such a spacer. The spacer may include e.g. Fc fragments of antibodies or fragments thereof, hinge regions of antibodies or fragments thereof, CH2 or CH3 regions of antibodies, accessory proteins, artificial spacer sequences or combinations thereof. A prominent example of a spacer is the CD8alpha hinge.

[0232] The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such domain. When the source is natural the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in the MBG_178

[0233] CARs described herein may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8alpha, CD9, CD 16, CD22, CD28, mesothelin, CD33, CD37, CD64, CD80, CD83, CD86, CD134, CD137, CD154, TNFRSF16, or TNFRSF19. The transmembrane domain preferentially may be derived from CD8alpha or CD28. When the key signaling and antigen recognition modules (domains) are on two (or even more) polypeptides then the CAR may have two (or more) transmembrane domains. The splitting key signaling and antigen recognition modules enable for a small molecule-dependent, titratable and reversible control over CAR cell expression (e.g. WO2014127261A1) due to small molecule-dependent heterodimerizing domains in each polypeptide of the CAR.

[0234] The cytoplasmic signaling domain (the intracellular signaling domain or the activating endodomain) of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed, if the respective CAR is an activating CAR (normally, a CAR as described herein refers to an activating CAR, otherwise it is indicated explicitly as an inhibitory CAR (iCAR)). "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines. The intracellular signaling domain refers to the part of a protein which transduces the effector function signal and directs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain may include any complete, mutated or truncated part of the intracellular signaling domain of a given protein sufficient to transduce a signal which initiates or blocks immune cell effector functions. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0235] Prominent examples of intracellular signaling domains for use in the CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors that initiate signal transduction following antigen receptor engagement.

[0236] Generally, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences, firstly those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domain) and secondly those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences, co-stimulatory signaling domain). Therefore, an intracellular signaling domain of a CAR may comprise one or more MBG_178

[0237] primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0238] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs).

[0239] Examples of IT AM containing primary cytoplasmic signaling domains often used in CARs are that those derived from TCR^ (CD3Q, FcRgamma, FcRbeta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. Most prominent is sequence derived from CD3^.

[0240] The cytoplasmic domain of the CAR may be designed to comprise the CD3^ signaling domain by itself or combined with any other desired cytoplasmic domain(s). The cytoplasmic domain of the CAR can comprise a CD3^ chain portion and a co-stimulatory signaling region (domain). The co-stimulatory signaling region refers to a part of the CAR comprising the intracellular domain of a co-stimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples for a co-stimulatory molecule are CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3.

[0241] The cytoplasmic signaling sequences within the cytoplasmic signaling part of the CAR may be linked to each other with or without a linker in a random or specified order. A short oligo- or polypeptide linker, which is preferably between 2 and 10 amino acids in length, may form the linkage. A prominent linker is the glycine-serine doublet.

[0242] As an example, the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD28. In another example the cytoplasmic domain may comprise the signaling domain of CD3^ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3^, the signaling domain of CD28, and the signaling domain of CD137.

[0243] As aforementioned either the extracellular part or the transmembrane domain or the cytoplasmic domain of a CAR may also comprise a heterodimerizing domain for the aim of splitting key signaling and antigen recognition modules of the CAR.

[0244] The CAR may be further modified to include on the level of the nucleic acid encoding the CAR one or more operative elements to eliminate CAR expressing immune cells by virtue of a MBG_178

[0245] suicide switch. The suicide switch can include, for example, an apoptosis inducing signaling cascade or a drug that induces cell death. In one embodiment, the nucleic acid expressing and encoding the CAR can be further modified to express an enzyme such thymidine kinase (TK) or cytosine deaminase (CD). The CAR may also be part of a gene expression system that allows controlled expression of the CAR in the immune cell. Such a gene expression system may be an inducible gene expression system and wherein when an induction agent is administered to a cell being transduced with said inducible gene expression system, the gene expression system is induced and said CAR is expressed on the surface of said transduced cell.

[0246] In some embodiments the CAR may be a “SUPRA” (split, universal, and programmable) CAR, where a “zipCAR” domain may link an intra-cellular costimulatory domain and an extracellular leucine zipper (WO2017 / 091546). This zipper may be targeted with a complementary zipper fused e.g. to an scFv region to render the SUPRA CAR T cell tumor specific. This approach would be particularly useful for generating universal CAR T cells for various tumors; adapter molecules could be designed for tumor specificity and would provide options for altering specificity post-adoptive transfer, key for situations of selection pressure and antigen escape. The CARs as disclosed herein may be designed to comprise any portion or part of the above-mentioned domains as described herein in any order and / or combination resulting in a functional CAR, i.e. a CAR that mediated an immune effector response of the immune effector cell that expresses the CAR as disclosed herein.

[0247] The engineered cell expressing a CAR as disclosed herein may be further modified by genetic engineering using methods well known in the art e.g. Meganucleases, TALEN, CrisprCas, zink finger nucleases, shRNA and / or miRNA. Said cells may be modified e.g. to reduce or lack expression of a specific gene, which is normally expressed in the cell e.g. T cell receptor (TCR), MHC, co-inhibitory molecules like PD-1, CTLA-4, BTLA, TIGIT, Tim-3, CD244, LAIR, Lag-3, CD160, HVEM . Said cells may be modified to express additional transgenes such as therapeutic controls, cytokines and / or fragments, cytokine receptors and / or fragments, cytokine receptor fusion proteins, costimulatory receptors or armoring molecules such as, but not limited to, metalloproteases / ECM-degrading enzymes.

[0248] The term “CLEC12A” is the abbreviation for the gene / protein “C-type lectin domain family 12 member A”. Alternative names are e.g. CLL1, MICL, CD371, CLL-1, and DCAL-2. This gene encodes a member of the C-type lectin / C-type lectin-like domain (CTL / CTLD) superfamily. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune MBG_178

[0249] response. The protein encoded by this gene is a negative regulator of granulocyte and monocyte function.

[0250] The term "antibody" as used herein is used in the broadest sense to cover the various forms of antibody structures including but not being limited to monoclonal and polyclonal antibodies (including full length antibodies), multispecific antibodies (e.g. bispecific antibodies), antibody fragments, i.e. antigen binding fragments of an antibody, immunoadhesins and antibody -immunoadhesin chimeras, that specifically recognize (i.e. bind) an antigen. "Antigen binding fragments" comprise a portion of a full-length antibody, preferably the variable domain thereof, or at least the antigen binding site thereof (“an antigen binding fragment of an antibody”). Examples of antigen binding fragments include Fab (fragment antigen binding), scFv (single chain fragment variable), single domain antibodies (VHH and nanobodies), diabodies, dsFv, Fab’, F(ab')2, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0251] A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

[0252] A “fully human antibody” or “human antibody” is an antibody or antigen binding fragment thereof which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some embodiments, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals.

[0253] The term “CDR” denotes a complementarity determining region as defined by at least one manner of identification to one of skill in the art. The precise amino acid sequence boundaries MBG_178

[0254] of a given CDR or framework region (FR) can be readily determined using any of a number of well-known schemes, including the numbering system of Kabat.

[0255] As used herein, the term “antigen” is intended to include substances that bind to or evoke the production of one or more antibodies and may comprise, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextran, and combinations thereof, for example a glycosylated protein or a glycolipid. The term “antigen” as used herein refers to a molecular entity that may be expressed on the surface of a target cell and that can be recognized by means of the adaptive immune system including but not restricted to antibodies or TCRs, or engineered molecules including but not restricted to endogenous or transgenic TCRs, CARs, scFvs or multimers thereof, Fab-fragments or multimers thereof, antibodies or multimers thereof, single chain antibodies or multimers thereof, or any other molecule that can execute binding to a structure with high affinity.

[0256] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0257] As used herein, the term “subject” refers to an animal. Preferentially, the subject is a mammal such as mouse, rat, cow, pig, goat, chicken, dog, monkey or human. More preferentially, the individual is a human. The subject may be a subject suffering from a disease such as cancer. The terms “nucleic acid”, “nucleic acid sequence”, “nucleic acid molecule” or “polynucleotide” may be used interchangeably herein and refer to polymers of nucleotides. Polynucleotides, which can be hydrolyzed into monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, the term “polynucleotides” encompasses, but is not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0258] A recombinant protein is a biotechnologically generated protein that does not occur naturally in a eukaryotic and / or prokaryotic cell. Often it is composed of different domains from different proteins, e.g. as used herein, a viral envelope protein is fused (at its ectodomain) to a polypeptide that comprises an antigen binding domain specific for an antigen.

[0259] The term “transduction” means the transfer of genetic material from a viral agent such as a lentiviral vector particle into a eukaryotic cell such as a T cell. MBG_178

[0260] The terms “having specificity for”, “specifically binds” or “specific for” with respect to an antigen-binding domain of an antibody or a fragment thereof refer to an antigen-binding domain which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antigen-binding domain that binds specifically to an antigen from one species may bind also to that antigen from another species. This cross-species reactivity is not contrary to the definition of that antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may bind also to different allelic forms of the antigen (allelic variants, splice variants, isoforms etc.). This cross reactivity is not contrary to the definition of that antigen-binding domain as specific.

[0261] Immunotherapy is a medical term defined as the "treatment of disease by inducing, enhancing, or suppressing an immune response". Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Cancer immunotherapy as an activating immunotherapy attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses cell-based, preferentially T cell-based cytotoxic responses to attack cancer cells. T cells that have a natural or genetically engineered reactivity to a patient's cancer are generated in vitro and then transferred back into the cancer patient or are directly generated in-vivo. Then the immunotherapy is referred to as “CAR T cell immunotherapy”.

[0262] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0263] The term “(therapeutically) effective amount” as used herein means an amount of a pharmaceutical composition which is sufficient to significantly and positively modify the symptoms and / or conditions to be treated. The effective amount of an active ingredient such a a pseudotyped retroviral vector particle or a genetically modified immune cell for use in a pharmaceutical composition will vary with the particular condition being treated, the severity of the condition, the duration of treatment, the nature of concurrent therapy, the particular active ingredient(s) being employed, the particular pharmaceutically-acceptable carrier(s) utilized. The terms “engineered cell” and “(genetically) modified cell” as used herein can be used interchangeably. The terms mean containing and / or expressing a foreign gene or nucleic acid sequence which in turn modifies the genotype or phenotype of the cell or its progeny. Especially, the terms refer to the fact that cells, preferentially T cells can be manipulated by recombinant methods well known in the art to express stably or transiently peptides or proteins which are MBG_178

[0264] not expressed in these cells in the natural state. For example, T cells, preferentially human T cells are engineered to express an artificial construct such as a chimeric antigen receptor on their cell surface.

[0265] For enrichment, isolation or selection of specific immune cells, e.g. T cells such as CD4+ and / or CD8+ T cells, in principle any sorting technology can be used. This includes for example affinity chromatography or any other antibody-dependent separation technique known in the art. Any ligand-dependent separation technique known in the art may be used in conjunction with both positive and negative separation techniques that rely on the physical properties of the cells. An especially potent sorting technology is magnetic cell sorting. Methods to separate cells magnetically are commercially available e.g. from Invitrogen, Stem cell Technologies, in Cellpro, Seattle or Advanced Magnetics, Boston. For example, monoclonal antibodies can be directly coupled to magnetic polystyrene particles like Dynal M 450 or similar magnetic particles and used e.g. for cell separation. The Dynabeads technology is not column based, instead these magnetic beads with attached cells enjoy liquid phase kinetics in a sample tube, and the cells are isolated by placing the tube on a magnetic rack. However, in a preferred embodiment for enriching e.g. CD4+ and / or CD8+ T cells from a sample comprising T cells monoclonal antibodies or antigen binding fragments thereof are used in conjunction with colloidal superparamagnetic microparticles having an organic coating by e.g. polysaccharides (Magnetic-activated cell sorting (MACS) technology (Miltenyi Biotec B.V. & Co. KG, Germany)). These particles (nanobeads or MicroBeads) can be either directly conjugated to monoclonal antibodies or used in combination with anti-immunoglobulin, avidin or anti-hapten-specific MicroBeads. The MACS technology allows cells to be separated by incubating them with magnetic nanoparticles coated with antibodies directed against a particular surface antigen. This causes the cells expressing this antigen to attach to the magnetic nanoparticles. Afterwards the cell solution is transferred on a column placed in a strong magnetic field. In this step, the cells attach to the nanoparticles (expressing the antigen) and stay on the column, while other cells (not expressing the antigen) flow through. With this method, the cells can be separated positively or negatively with respect to the particular antigen(s) / marker(s).

[0266] In case of a positive selection the cells expressing the antigen(s) of interest, which attached to the magnetic column, are washed out to a separate vessel, after removing the column from the magnetic field.

[0267] In case of a negative selection the antibody used is directed against surface antigen(s) which are known to be present on cells that are not of interest. After application of the cells / magnetic MBG_178

[0268] nanoparticles solution onto the column the cells expressing these antigens bind to the column and the fraction that goes through is collected, as it contains the cells of interest. As these cells are non-labelled by an antibody coupled to nanoparticels, they are “untouched”.

[0269] As used herein “autologous” means that cells, a cell line, or population of cells used for treating subjects are originating from said subject.

[0270] As used herein “allogeneic” means that cells or population of cells used for treating subjects are not originating from said subject but from a donor.

[0271] The terms “immune cell” or “immune effector cell” may be used interchangeably and refer to a cell that may be part of the immune system and executes a particular effector function such as T cells, alpha-beta T cells, NK cells, NKT cells, B cells, innate lymphoid cells (ILC), cytokine induced killer (CIK) cells, lymphokine activated killer (LAK) cells, gamma-delta T cells, regulatory T cells (Treg), monocytes or macrophages. Preferentially these immune cells are human immune cells. Preferred immune cells are cells with cytotoxic effector function such as alpha-beta T cells, NK cells, NKT cells, ILC, CIK cells, LAK cells or gamma-delta T cells. Most preferred immune effector cells are T cells and / or NK cells. Tumor infiltrating lymphocytes (TILs) are T cells that have moved from the blood of a subject into a tumor. These TILs may be removed from a patient' s tumor by methods well known in the art, e.g. enzymatic and mechanic tumor disruption followed by density centrifugation and / or cell marker specific enrichment. TILs are genetically engineered as disclosed herein, and then given back to the patient. "Effector function" means a specialized function of a cell, e.g. in a T cell an effector function may be cytolytic activity or helper activity including the secretion of cytokines.

[0272] The term "isolated" is used herein to indicate that the polypeptide, nucleic acid or host cell exist in a physical milieu distinct from that in which it occurs in nature. For example, the isolated polypeptide may be substantially isolated (for example enriched or purified) with respect to the complex cellular milieu in which it naturally occurs, such as in a crude extract.

[0273] A transgene may be a gene that has been transferred by genetic engineering techniques into a host cell that normally does not bear this gene. The gene may be a naturally gene that occurs in other cells or may be a recombinant gene. The expressed transgene may also be referred to a heterologous protein or transgenic polypeptide. MBG_178

[0274] Examples

[0275] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

[0276] The anti-CLEC12A (CLL-1 / CD371) CAR T candidate selection process consisted of an initial phage display selection, followed by a flow cytometric screening to ensure specificity of the candidates to CLEC12A+OCI-AML-2 target cells compared to CLEC12A' SupTl wt cells. Subsequently, CAR T screening approaches and an in vivo experiment allowed to identify novel, functional and specific anti-CLEC12A CAR candidates.

[0277] Example 1 : Identification of antibody fragments

[0278] Two human naive scFv phage display libraries constructed at Miltenyi Biotec were used for the identification of novel binders against CLEC12A. These libraries differed in the variable domains of the light chain, they contained either the K or the X isotype. For panning, human CLEC12A (hCLEC12A) was purchased from LSBio and Sino Biologicals, respectively. Additionally human CLEC12A ectodomain (hCLEC12Ae) and human CLEC12A-hIgGlFc (hCLEC12A-Fc) produced at Miltenyi Biotec was used. Human IgGl-Fc (hlgGlFc) was purchased from amsbio. Six different panning strategies were used to identify binder candidates using hCLEC12A variants for positive selection. In all strategies hlgGl-Fc antigen was used for competition (Fig. 1 A). For panning, each library was incubated on antigens immobilized on Maxi Sorb ELISA Plates (Nunc). After washing, scFv phage particles were eluted using trypsin. Eluted phage particles were used to infect E. coli TGI and phage production was facilitated using M13K07 helper phage. After the third panning round eluted phage particles were used to infect E. coli TGI and single cell colonies were inoculated the next day for expression of scFvs.

[0279] Expressed scFvs were analyzed for their capability to bind hCLEC12A (Sino), hCLEC12A-Fc or hlgGlFc in ELISA. This lead to the identification of 1706 first hits that bind hCLEC12A and do not bind hlgGlFc. Separating the ELISA hits by panning strategy revealed, that binder selected in strategies without hCLEC12Ae and hCLEC12A-Fc in the selection process recognized preferably hCLEC12A (Sino). On the other hand panning strategy S7 and S8 with selection on hCLEC12Ae and hCLEC12A-Fc revealed binder binding preferably to hCLEC12A-Fc over hCLEC12A (Sino) (Fig. IB).

[0280] 1706 first hit scFv genes were sequenced resulting in 1443 processable sequences. Alignment of the scFv genes revealed that 428 out of 1443 clones contain unique scFv sequences (Fig. 1C). All panning strategies lead to the selection of 93 scFvs reactive against CLEC12A-Fc, 213 MBG_178

[0281] scFvs reactive against CLEC12A (Sino) and 122 scFvs cross-reactive against both targets (Fig. ID).

[0282] Identified unique candidates were inoculated again for expression of scFvs. After expression cells were pelleted at 4000 g for 10 min and resuspended in TE-Buffer to isolate the Periplasmic Protein (PPP). Cells were incubated at 37°C o.n. on an orbital shaker. For flow cytometric validation, non-target cells were labeled with CellTrace™ Violet (ThermoFisher) and IxlO5cells per cell line were seeded into a 96-well plate. Cells were pelleted at 1200 g for 2 min at 4°C and stained with 50 pL PPP. Staining was performed for 30 min at 4°C followed by two successive washing steps with 150 pL PBS / 2 mM EDTA / 0.5% BSA (referred to as PEB, 1200 g, 2 min). Next, 50 pl of secondary staining mixture comprising the mouse anti His-APC (Miltenyi Biotec) as well as 7-AAD as dead cell marker (Miltenyi Biotec) was added to the cells. Staining was performed for 10 min at 4°C followed by two successive washing steps with 150 pL PEB. Cells were resuspended in 50 pL PEB for subsequent flow cytometric analysis at the MACSQuant® Analyzer 10 (Miltenyi Biotec).

[0283] Cells were gated on size, singularity and viability before distinguishing between the human CLEC12A+ OCI-AML-2 cell line and the human CLEC12A' SupTl wt cell line via CellTrace. Staining of the respective CLEC12A positive target cell line with the anti-CLEC12A scFv candidate was displayed as frequency (%) of stained cells. 79 candidates with a signal to noise ratio of >5 were identified. After analysis of sequence liabilities 46 positive clones were identified and cloned as scFv-Fc fusion into mammalian expression vectors followed by expression and purification. Purified scFv-Fc proteins were used for an additional analysis by flow cytometry.

[0284] Example 2: Expression and purification of anti-CLEC12A scFv-Fc candidates

[0285] Potential anti-CLEC12A scFv-Fc candidates were manufactured via transient transfection of HEK293 cells. On the day of transfection, HEK293 cells were seeded at a density of 2xl06cells per well in a total volume of 800 pl expression medium in a 96 deep well plate. Next, transfection reagent was mixed with diluted plasmid DNA, encoding for the respective anti-CLEC12A scFv-Fc candidate and added to the cells after an incubation period of 10 minutes. Cells were incubated at 37°C and 8% CO2 on an orbital shaker. Supernatants were collected on day 4 after transfection and scFv-Fc candidates were purified using Protein A PhyTip® columns (Biotage) in combination with an automated workstation (Beckman Coulter). Purity was confirmed via SDS page and protein concentrations were determined using a microplate MBG_178

[0286] reader by measuring UV absorbance at 280 nm. Purified proteins were stored at -20°C until further processing.

[0287] Example 3: Flow cytometric screening of anti-FOLRl scFv-Fc candidates

[0288] Anti-FOLRl scFv-Fc candidates were manufactured as described in example 2 and protein concentrations were normalized. For flow cytometric validation, non-target cells were labeled with CellTrace™ Violet or Cell Trace™ FarRed (ThermoFisher) and IxlO5cells per cell line were seeded into a 96-well plate. Cells were pelleted at 1200 g for 2 min at 4°C and stained with 50 pL staining mixture containing 100 nM scFv-Fc. Staining was performed for 30 min at 4°C followed by two successive washing steps with 150 pL PBS / 2 mM EDTA / 0.5% BSA (referred to as PEB, 1200 g, 2 min). Next, 50 pl of secondary staining mixture comprising the AffmitPure F(ab')2 Fragment Goat Anti-Human IgG-PE (Jackson ImmunoResearch) as well as 7-AAD as dead cell marker (Miltenyi Biotec) was added to the cells. Staining was performed for 10 min at 4°C followed by two successive washing steps with 150 pL PEB. Cells were resuspended in 50 pL PEB for subsequent flow cytometric analysis at the MACSQuant® Analyzer 10 (Miltenyi Biotec).

[0289] Cells were gated on size, singularity and viability before distinguishing between the human CLEC12A+OCI- AML-2 cell line and the and human CLEC12A- SupTl wt cell line via CellTrace. Staining of the respective CLEC12A positive target cell line with the anti-CLEC12A scFv-Fc candidate was displayed as frequency (%) of stained cells. All 46 anti-CLEC12A scFv-Fc candidates, which have been successfully produced and purified, showed a positive result (frequency > 15%) on human CLEC12A+OCI- AML-2 cells in flow cytometric analysis (Fig.

[0290] 2A). None of the candidates showed unspecific staining (frequency > 5%) on human CLEC12A' Sup-Tl wt cells (Fig. 2B). Aiming for anti-CLEC12A scFv-Fc candidates that show specific binding for human CLEC12A+OCI-AML-2 cells, but no signal on human CLEC12A-Sup-Tl wt, in total 46 candidates (black bars) were selected for further analysis.

[0291] Example 4: Identification of chimeric antigen receptors (CARs) for CLEC12A-expressing target cell recognition by CAR-transgenic T cells.

[0292] Human naive single chain variable fragments (scFv) were cloned into lentiviral vector constructs with scFv - human CD8 alpha hinge (SEQ ID NO:76) - CD8 alpha transmembrane domain (SEQ ID NO:77) - human 41BB costimulatory domain (SEQ ID NO:78) - human CD3 zeta domain configuration (SEQ ID NO:79). The scFv contained a YOL linker domain (SEQ ID NO:80) or (G4S)s linker domain (SEQ ID NO:81). Lentiviral vectors contained a truncated MBG_178

[0293] human low-affinity nerve growth factor receptor (ALNGFR) expression cassette separated by a 2A element for co-expression as transduction marker. Lentiviral particles were used to transduce primary human T cells of one donor for CAR expression. Frequency and absolute numbers of CAR T cells were determined by quantification of CD3+LNGFR+events using flowcytometry.

[0294] First, 40 YOL linker domain-containing CAR T cell productions were cultured at an effector-to-target ratio of 2.5 to 1 with CLEC12A-expressing OCI-AML-2 cells, which were transduced to express GFP, and compared to (G4S)s linker domain-containing M26 CAR T cells. As controls, CAR T cells were cultured in absence of target cells, and OCI-AML-2 cells were cultured in absence of CAR T cells. Additionally, OCI-AML-2 cells were cultured with untransduced T cells. Cytotoxicity was measured by quantification of GFP+ events using flow cytometry at the end of each round of co-culture (Fig. 3A-C). After 92h CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 3 D). In addition, fresh OCI-AML-2 cells were added according to the experimental set-up to initiate the second round of co-culture. After 164 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 3E). The third round of co-culture was started by addition of fresh OCI-AML-2 cells according to the experimental set-up. 24h later supernatants were again analyzed for cytokine secretion. After 260 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 3F). The total duration of three rounds of co-culture was 11 days, and cytotoxicity towards OCI-AML-2 was determined at the end of each round by quantification of GFP+events. These three consecutive rounds of co-culture of CAR T cells with OCI-AML-2 target cells allowed to identify seven CAR T candidates (CD371_33_MB108-G02, CD371_36_MB114-A04, CD371_37_MB114-C03, CD371_4O_MB117-C04, CD371_43_MB119-G12, CD371 60 MB116-D01, CD371 61 MB117-F05), which were able to mediate killing of CLEC12A -expressing cells. Second, these 7 CARs were tested with the YOL linker domain replaced by the (G4S)s linker domain and compared to the M26 CAR as well as 3 CARs that were found to be not functional in three rounds of co-culture in the first experiment. After transduction of a second donor derived T cells, CAR T cells were cultured at an effector-to-target ratio of 2.5 to 1 with CLEC12A-expressing OCI-AML-2 cells, which were transduced to express GFP. As controls, CAR T cells were cultured in absence of target cells, and OCI-AML-2 cells were cultured in absence of CAR T cells. Additionally, OCI-AML-2 cells were cultured with un-transduced T cells or with T cells expressing one control M26 CAR. Cytotoxicity was assessed by quantification of GFP+target cells at the end of each round by flow cytometry (Fig. 4 A-C). After 24h cell-culture supernatants were analyzed for cytokine secretion, e.g. IFN-y (Fig. 4D). MBG_178

[0295] After 92h CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 4F). In addition, fresh OCI-AML-2 cells were added according to the experimental set-up to initiate the second round of co-culture. After 164 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 4G). The third round of co-culture was started by addition of fresh OCI-AML-2 cells according to the experimental set-up. 24h later supernatants were again analyzed for cytokine secretion (Fig. 4E). After 260 h, CAR T cells (CD3+LNGFR+) were quantified by flow cytometry (Fig. 4H). The total duration of three rounds of co-culture was 11 days, and cytotoxicity towards OCI-AML-2 was determined at the end of each round by quantification of GFP+events.

[0296] Three consecutive rounds of co-culture of CAR T cells with OCI-AML-2 target cells allowed to identify seven CAR T candidates (CD371_73_MB113-F10-G4S, CD371_74_MB114-A04-G4S, CD371 75 MB114-C03-G4S, CD371_77_MB117-C04-G4S, CD371_79_MB119-G12-G4S, CD371 76 MB116-D01-G4S, CD371_78_MB117-F05-G4S), which were able to mediate killing of CLEC12A-expressing cells. CAR T cell expansion was observed for four CAR constructs (CD371_74_MB114-A04-G4S, CD371_75_MB114-C03-G4S, CD371 77 MB117-C04-G4S, CD371_79_MB119-G12-G4S) in co-cultures with OCI-AML-2 cells at the end of the first, second and the third round, i.e. numbers were exceeding the initial amount of CD3+LNGFR+T cells plated at day 0 (Fig. 4F-H). Expression of these four functional CARs (CD371_74_MB114-A04-G4S, CD371_75_MB114-C03-G4S, CD371 77 MB117-C04-G4S, CD371_79_MB119-G12-G4S) by T cells was associated with a profound secretion of cytokines in comparison to M26 CAR as assessed in rounds 1 and 3 of co-culture with OCI-AML-2 cells (Fig. 4D, E). CAR T cells of this donor were also used to analyze expression of PD-1 and CD27 amongst CD3+transduced cells (Fig. 41). No decrease of these four candidates for the frequency of PD-1' CD27+events was observed compared to M26 CAR T cells, and the analysis of an additional, third donor’s T cells resulted in the detection of reduced frequencies of PD-1 CD27+events when T cells expressed CAR constructs MB114-C03-G4S and MB117-C04-G4S. In sum, in vitro co-cultures allowed to select for two CARs that equipped T cells with the ability to mediate repeated killing, superior cytokine production as well as expansion upon repeated target cell encounter with frequencies of transduced PD-1' CD27+T cells comparable to M26 CAR-expressing T cells.

[0297] Example 5: In vivo efficacy evaluation of fully human CLEC12A CARs

[0298] Two anti-CLEC12A (CLL-1 / CD371) binders, MB114-A04 and MB119-G12 (both with (G4S)3 linker), were incorporated into chimeric antigen receptor (CAR moiety, comprised of a MBG_178

[0299] scFv in frame to a CD8 hinge and transmembrane domain, a 41BB (CD137) costimulatory domain and CD3(^ signaling domain, and named CAR D0399 and D0400, respectively. The CAR of the construct D0399 is identical with SEQ ID NO:53. The CAR of the construct D0400 is identical with SEQ ID NO:59. A clinically-relevant CAR incorporating a targeting domain derived from mouse scFv M26, CAR D0398, was included as a control (Fig. 5 A). The binder of the CAR of D0398 is identical with SEQ ID NO:1. Primary human T cells from a healthy donor were transduced with lentiviral vector encoding CAR CLEC12A at MOI20. CAR positivity of the transduced T cells was determined by flow cytometry using His-tagged human CLEC12A recombinant protein followed by anti-His Allophycocyanin (APC) conjugated secondary antibody (Fig. 5B). CARs were tested in a panel of standard assays in vitro to confirm functionality against tumor target cells (not shown). After functionality of the novel CLEC12A CAR candidates was confirmed, they were evaluated for anti-tumor activity in vivo.

[0300] The in vivo anti -turn or efficacy of CLEC12A CARs D0398, D0399 and D0400 was evaluated in a disseminated HL-60 NSG xenograft model expressing the CLEC12A antigen (Fig. 5C). HL-60 tumor-bearing mice were treated with five million CAR-positive T cells or UTD (untransduced) control T cells from same donor. Tumor alone control group, receiving no T cells (TA, Untreated) was included as well. Animals were monitored for body weight change vs day 0, survival, and tumor burden by bioluminescence (BLI), until termination on day 42 after CAR T dosing (Fig. 5C). Mice’s body weights remained stable for 20 days on the study, then began to decline, first in TA and UTD groups, followed by CAR D0400 treatment group, but not in CAR D0398 and D0399 groups (Fig. 5D).

[0301] Consistent with body weight change profile, all mice treated with CAR D0398 or D0399 survived until end of study, while 3 and 2 mice died in CAR D0400 and UTD treatment groups, respectively. All untreated mice (tumor alone, TA group) succumbed to disease by study day 35 (Fig. 5E). Tumor growth kinetics BLI data demonstrated early CAR-specific tumor regressions starting at day 7, but also spontaneous tumor regression in the surviving mice in UTD control group at the end of the study, implicating graft vs host disease (GVHD) and potential graft vs leukemia effect (GVL, Fig 6F).

[0302] To avoid the potential GVL bias in data interpretation, only tumor regression data from the first half of the study (up to day 14) was included in the evaluation of efficacy of the novel CAR candidates (Fig. 5G). All CAR T treatments were similarly potent, and significantly reduced tumor burden as compared to TA and UTD control groups (p<0.001).

[0303] In conclusion, novel CLEC12A CAR D0399 and CAR D400 are suitable candidates for further evaluation. MBG_178

[0304]

[0305] SEQ ID NO:1: Clone name / LV_CAR vector name: M26 / CD371_01_M26

[0306] SEQ ID NO:2: Clone name / LV_CAR vector name: CD371-5 / CD371_03_5

[0307] SEQ ID NO:3: Clone name / LV_CAR vector name: CD371-23 / CD371_04_21

[0308] SEQ ID NO:4: Clone name / LV_CAR vector name: CD371-52 / CD371_05_52

[0309] SEQ ID NO:5: Clone name / LV_CAR vector name: CD371-75 / CD371_06_75

[0310] SEQ ID NO:6: Clone name / LV_CAR vector name: MB107-A03 / LV_LTG3026_CD371_32_MB107-A03 (identical clone MB122-G09)

[0311] SEQ ID NO: 7: Clone name / LV_CAR vector name: MB108-G02 /

[0312] LV LTG3026 CD371_33_MB 108-G02

[0313] SEQ ID NO:8: Clone name / LV_CAR vector name: MB110-D01 / LV_LTG3026_CD371_34_MBl 10-D01

[0314] SEQ ID NO: 9: Clone name / LV_CAR vector name: MB113-E12 / LV_LTG3026_CD371_35_MB113- E12

[0315] SEQ ID NO: 10: Clone name / LV_CAR vector name: MB114-A04 / LV_LTG3026_CD371_36_MBl 14-A04

[0316] SEQ ID NO: 11: Clone name / LV_CAR vector name: MB114-C03 / LV_LTG3026_CD371_37_MBl 14-C03

[0317] SEQ ID NO: 12: Clone name / LV_CAR vector name: MB116-E01 / LV_LTG3026_CD371_38_MBl 16-E01

[0318] SEQ ID NO: 13: Clone name / LV_CAR vector name: MB117-B04 / LV_LTG3026_CD371_39_MBl 17-B04

[0319] SEQ ID NO: 14: Clone name / LV_CAR vector name: MB117-C04 / LV_LTG3026_CD371_40_MBl 17-C04

[0320] SEQ ID NO: 15: Clone name / LV_CAR vector name: MB117-E05 / LV_LTG3026_CD371_41_MBl 17-E05

[0321] SEQ ID NO: 16: Clone name / LV_CAR vector name: MB117-F02 / LV_LTG3026_CD371_42_MBl 17-F02

[0322] SEQ ID NO: 17: Clone name / LV_CAR vector name: MB119-G12 / LV_LTG3026_CD371_43_MBl 19-G12

[0323] SEQ ID NO: 18: Clone name / LV_CAR vector name: MB120-A10 /

[0324] LV LTG3026_CD371_44_MB 120-A 10

[0325] SEQ ID NO: 19: Clone name / LV_CAR vector name: MB120-B12 /

[0326] LV_LTG3026_CD371_45_MB 120-B 12

[0327] SEQ ID NO:20: Clone name / LV_CAR vector name: MB120-G06 / LV_LTG3026_CD371_46_MB120-G06 (identical clone: MB121-A05)

[0328] SEQ ID NO:21: Clone name / LV_CAR vector name: MB120-H06 /

[0329] LV LTG3026 CD371_47_MB 120-H06

[0330] SEQ ID NO:22: Clone name / LV_CAR vector name: MB120-H08 / LV_LTG3026_CD371_48_MB120-H08

[0331] SEQ ID NO:23: Clone name / LV CAR vector name: MB121-A11 / LV_LTG3026_CD371_49_MB121-Al 1

[0332] SEQ ID NO:24: Clone name / LV_CAR vector name: MB121-D02 /

[0333] LV LTG3026 CD371 50 MB 121 -D02

[0334] SEQ ID NO:25: Clone name / LV_CAR vector name: MB121-D07 /

[0335] LV LTG3026 CD371 51_MB 121-D07

[0336] SEQ ID NO:26: Clone name / LV_CAR vector name: MB122-A07 /

[0337] LV LTG3026 CD371 52 MB122-A07

[0338] SEQ ID NO:27: Clone name / LV_CAR vector name: MB122-B01 / LV_LTG3026_CD371_53_MB122-B01

[0339] SEQ ID NO:28: Clone name / LV CAR vector name: MB122-B03 / LV_LTG3026_CD371_54_MB122-B03

[0340] SEQ ID NO:29: Clone name / LV_CAR vector name: MB122-B10 /

[0341] LV LTG3026 CD371 55 MB 122-B 10 MBG_178

[0342] SEQ ID NO:30: Clone name / LV CAR vector name: MB122-E09 / LV_LTG3026_CD371_56_MB122-E09

[0343] SEQ ID NO:31: Clone name / LV_CAR vector name: MB121-B05 /

[0344] LV LTG3026 CD371 57 MB 121 -B05

[0345] SEQ ID NO:32: Clone name / LV_CAR vector name: MB109-D11 / LV_LTG3026_CD371_58_MB109-Dl 1

[0346] SEQ ID NO: 33: Clone name / LV_CAR vector name: MB111-B12 /

[0347] LV LTG3026 CD371 59 MB111-B12

[0348] SEQ ID NO:34: Clone name / LV_CAR vector name: MB116-D01 / LV_LTG3026_CD371_60_MBl 16-D01

[0349] SEQ ID NO:35: Clone name / LV CAR vector name: MB117-F05 / LV_LTG3026_CD371_61_MBl 17-F05

[0350] SEQ ID NO:36: Clone name / LV_CAR vector name: MB120-A09 /

[0351] LV LTG3026 CD371_62_MB 120-A09

[0352] SEQ ID NO:37: Clone name / LV CAR vector name: MB122-E10 / LV_LTG3026_CD371_63_MB122-E10

[0353] SEQ ID NO:38: Clone name / LV_CAR vector name: MB123-C11 /

[0354] LV LTG3026 CD371 64 MB123-C11

[0355] SEQ ID NO: 39: Clone name / LV_CAR vector name: MB113-F10 / LV_LTG3026_CD371_65_MBl 13-F10

[0356] SEQ ID NO:40: Clone name / LV CAR vector name: MB122-F03 / LV_LTG3026_CD371_66_MB122-F03

[0357] SEQ ID NO:41: Clone name / LV_CAR vector name: MB122-G12 /

[0358] LV LTG3026_CD371_67_MB 122-G 12

[0359] SEQ ID NO:42: Clone name / LV CAR vector name: MB123-B07 / LV_LTG3026_CD371_68_MB123-B07

[0360] SEQ ID NO:43: Clone name / LV_CAR vector name: MB123-D09 /

[0361] LV LTG3026 CD371 69 MB123-D09

[0362] SEQ ID NO:44: Clone name / LV_CAR vector name: MB123-G09 / LV_LTG3026_CD371_70_MB123-G09

[0363] SEQ ID NO:45: Clone name / LV CAR vector name: MB123-H03 / LV_LTG3026_CD371_71_MB123-H03

[0364] SEQ ID NO:46: Clone name / LV_CAR vector name: MB122-G12 / CD371_80_MB122-G12-G4S SEQ ID NO:47: Clone name / LV_CAR vector name: MBII3-FI0 / CD371_73_MB113-F10-G4S SEQ ID NO:48: Clone name / LV CAR vector name: MB123-D09 / CD371 81 MB123-D09-G4S SEQ ID NO:49: Clone name / LV_CAR vector name: MB108-G02 / CD371_72_MB108-G02-G4S SEQ ID NO:50: Clone name / LV_CAR vector name: MB114-A04 / CD371_74_MB114-A04-G4S SEQ ID NO:51: MB114-A04 / VH

[0365] SEQ ID NO:52: MB114-A04 / VL

[0366] SEQ ID NO:53: Clone name / LV_CAR vector name: MB114-A04 / CD371_74_MB114-A04-G4S (full)

[0367] SEQ ID NO:54: Clone name / LV CAR vector name: MB114-C03 / CD371 75 MB114-C03-G4S SEQ ID NO:55: Clone name / LV_CAR vector name: MB117-C04 / CD371_77_MB117-C04-G4S SEQ ID NO:56: Clone name / LV_CAR vector name: MB119-G12 / CD371_79_MB119-G12-G4S SEQ ID NO:57: MB119-G12 / VH

[0368] SEQ ID NO:58: MB119-G12 / VL

[0369] SEQ ID NO:59: Clone name / LV_CAR vector name: MB119-G12 / CD371_79_MB119-G12-G4S (full)

[0370] SEQ ID NO:60: Clone name / LV_CAR vector name: MBII6-D0I / CD371_76_MB116-D01-G4S SEQ ID NO:61: Clone name / LV_CAR vector name: MB117-F05 / CD371_78_MB117-F05-G4S SEQ ID NO:62: Clone name / LV_CAR vector name: MB122-G12 / CD371_80_MB122-G12-G4S SEQ ID NO:63: Clone name / LV CAR vector name: MBII3-FI0 / CD371 73 MB113-F10-G4S SEQ ID NO:64: Clone name / LV_CAR vector name: MB123-D09 / CD371_81_MB123-D09-G4S SEQ ID NO:65: Clone name / LV_CAR vector name: MB108-G02 / CD371_72_MB108-G02-G4S SEQ ID NO:66: Clone name / LV CAR vector name: MB114-A04 / CD371_74_MB114-A04-G4S SEQ ID NO : 67 : MB 114-A04 / VH MBG_178

[0371] SEQ ID NO:68: MB114-A04 / VL

[0372] SEQ ID NO:69: Clone name / LV_CAR vector name: MB114-C03 / CD371_75_MB114-C03-G4S SEQ ID NO:70: Clone name / LV_CAR vector name: MB117-C04 / CD371_77_MB117-C04-G4S SEQ ID NO:71: Clone name / LV CAR vector name: MB119-G12 / CD371_79_MB119-G12-G4S SEQ ID NO:72: MB119-G12 / VH

[0373] SEQ ID NO:73: MB119-G12 / VL

[0374] SEQ ID NO:74: Clone name / LV_CAR vector name: MBII6-D0I / CD371_76_MB116-D01-G4S SEQ ID NO:75: Clone name / LV CAR vector name: MB117-F05 / CD371_78_MB117-F05-G4S SEQ ID NO: 76. amino acid sequence of CD 8 hinge domain

[0375] SEQ ID NO:77: amino acid sequence of CD8 transmembrane domain

[0376] SEQ ID NO:78: amino acid sequence of 4-1BB co-stimulatory domain

[0377] SEQ ID NO:79: amino acid sequence of CD3zeta

[0378] SEQ ID NO: 80: YOL linker domain (aa)

[0379] SEQ ID NO: 81 (aa): (648)3 linker domain

Claims

MBG_178Claims1) A nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CLEC12A,b) a transmembrane domain, andc) an intracellular signaling domain,wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:72 (VH) and SEQ ID NO:73 (VL) or is encoded by a nucleotide sequence comprising SEQ ID NO: 67 (VH) and SEQ ID NO: 68 (VL).2) The nucleic acid molecule of claim 1, wherein said antigen binding domain is encoded by a nucleotide sequence comprising SEQ ID NO:71 or SEQ ID NO:66.3) The nucleic acid molecule of claim 1 or 2, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (IT AMs) and / or one or more co-stimulatory domain(s).4) The nucleic acid molecule of claim 3, wherein said intracellular signaling domain said stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (IT AMs) is the costimulatory domain of CD3zeta, and wherein said one or more costimulatory domain(s) are CD28 and / or 4- IBB.5) The nucleic acid molecule of claims 1 to 4, wherein said antigen CLEC12A is expressed on a target cell, and wherein said target cell is a cancer cell.6) The nucleic acid molecule of claim 5, wherein said cancer cell is a cell of acute myeloid leukemia (AML).7) An immune cell comprising the nucleic acid molecule according to any one of claims 1 to 6.8) An immune cell comprising the nucleic acid molecule according to any one of claims 1 to 5 for use in treatment of cancer, wherein the cancer cells express CLEC12A.MBG_1789) The immune cell for use in treatment of cancer according to claim 8, wherein said cancer is acute myeloid leukemia (AML).10) The immune cell of claim 7 or the immune cell for use in treatment of cancer of claims 8 or 9, wherein said immune cell is a T cell or an NK cell.

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