Chimeric antigen receptors specific for CD318

Abstract

The present invention provides a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO:11, b) a transmembrane domain, and c) an intracellular signaling domain.

Description

[0001] MBG_183

[0002] Title

[0003] Chimeric antigen receptors specific for CD318

[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 CD318, wherein the antigen binding domain comprises a sequence of a single domain antibody.

[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-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. In some cases the antigen binding domain is derived from a single domain antibody (nanobody). CD318, also known as CUB domain-containing protein 1 (CDCP1), is a type I transmembrane glycoprotein that has gained attention for its role in cancer progression and metastasis1. Initially identified as a candidate biomarker for malignancies, CD318 has become increasingly recognized for its diverse roles in tumor biology and its potential as a therapeutic target2. CD318 is encoded by the CDCP1 gene and features extracellular CUB-like domains, a transmembrane domain, and an intracellular region rich in tyrosine phosphorylation sites3. While its expression is generally low or absent in most normal tissues, CD318 is upregulated in several cancers, including pancreatic cancer, non-small cell lung cancer, triple-negative breast cancer, renal cell carcinoma, and colorectal cancer4. This restricted expression pattern makes CD318 particularly attractive as a tumor marker for diagnostic and therapeutic purposes5. CD318 plays a significant role in tumorigenesis through its regulation of cell adhesion, motility, and survival4. It promotes metastasis by facilitating epithelial-to-mesenchymal transition, which enhances tumor cell detachment, migration, and invasion6. CD318 also contributes to resistance against apoptosis, particularly in conditions of cellular stress such as detachment from the extracellular matrix, a phenomenon known as anoikis resistance7. Additionally, CD318 supports tumor-stroma MBG_183 interactions that promote angiogenesis and immune evasion, further advancing tumor progression. These oncogenic effects are mediated through interactions with signaling molecules such as Src family kinases, PKC5, and integrins4.

[0008] CD318 has demonstrated significant potential as a diagnostic and prognostic marker1,3’5Elevated levels of CD318 in tumor tissues and patient plasma have been associated with poor prognosis in cancers like pancreatic and lung cancer4. Techniques such as immunohistochemistry and liquid biopsies can leverage CD318 for early detection or disease monitoring. Its expression correlates with advanced tumor stages, increased metastatic burden, and reduced overall survival, making it a valuable prognostic indicator in several malignancies. CD318’s location on the cell surface presents opportunities for targeted interventions8. Preclinical studies have explored various approaches, including monoclonal antibodies to block its function, antibody-drug conjugates for selective delivery of cytotoxic agents, and chimeric antigen receptor (CAR) T cells engineered to recognize CD318-expressing cancer cells5. These strategies highlight the promise of CD318 as a target in precision oncology.

[0009] There is a need in the art for improved or alternative immunotherapies targeting CD318.

[0010] Brief description of the invention

[0011] The inventors found novel sequences of antigen binding domains specific for the antigen CD318 for use as binders in chimeric antigen receptors. Regularly, these binder sequences are single domain antibodies (nanobodies).

[0012] The invention comprises a CAR comprising an antigen binding domain comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO: 11, that are specific for the antigen CD318, respectively. The use of the CARs as disclosed herein is for directing immune cells such as T cells to target cells, i.e. cancerous cells, expressing CD318 in a subject. Such cancers may be e.g. pancreatic cancer, bladder cancer, urothelial cancer, breast cancer, colon cancer, lung adenocarcinoma cancer, lung squamous cancer and prostate cancer.

[0013] Brief description of the drawings

[0014] Figure 1 : Density plots of CAR T-cells transduced with CD318-specific nanobody candidates, displaying CD318 binding and LNGFR expression as markers for CAR functionality and transduction efficiency, respectively. Each plot corresponds to an individual nanobody candidate (NB 1 to NB 16) from one representative donor, alongside non-transduced cells (NTC, negative control) and murine CD318 CAR (mCAR, positive control). Nanobody candidates MBG_183

[0015] NB1, NB2, NB3, NB4, NB5, NB6, NB11, NB13, and NB16 achieve binding levels exceeding 10%, illustrating CD318-specificity. In contrast, NB7, NB8, NB9, NB10, NB12, NB14, and NB 15 demonstrate transduction efficiencies above 20% but negligible or absent CD318 binding.

[0016] Figure 2: Kinetic results of repetitive killing assays performed with CAR T cells transduced with nanobody candidates against AsPCl (left) and BxPC3 (right) GFP+target cells. Negative control (NTC) and positive control (mCAR) are included for comparison and data displayed is derived from two representative donors. GFP signal confluence from tumor cells, normalized to a value of 1 at each time point of rechallenge, is illustrated over time. Tumor cell death is indicated by decreasing curves, while increasing curves reflect tumor cell growth. Two rechallenge time points, involving the addition of fresh tumor cells, are incorporated to evaluate the persistence and exhaustion of CAR activity over time. Nanobody candidates NB1, NB2, NB4, NB5, NB6, NB9, and NB11 (light grey) demonstrated sustained killing efficacy against both cell lines, maintaining activity even after two rechallenges. Conversely, NB3, NB7, NB8, NB10, NB12, NB13, NB14, NB15, and NB16 (dark grey) exhibited either no killing capacity or loss of persistence following initial activity during subsequent rechallenges.

[0017] Figure 3: Representative expression patterns of the 41BB early activation marker during coculture with AsPCl (top panel) or BxPC3 (bottom panel) target cells. Activation marker levels were assessed at distinct time points (Day 3, Day 7, and Day 9) following each rechallenge in the cytotoxicity assay for all 16 nanobody CAR candidates. Data are presented as mean ± s.e.m. (n = 4, two replicates per donor), with comparisons drawn against the negative control (NTC) and the positive control (mCAR). Nanobody CAR candidates NB1, NB2, NB4, NB5, NB6, NB9, and NB11 (in light grey) demonstrate robust and sustained 4 IBB expression, exceeding 20% in AsPCl and 10% in BxPC3 target cells, closely paralleling the activation levels observed in the positive control (mCAR) across all time points. In contrast, NB3, NB7, NB8, NB10, NB12, NB13, NB14, NB15, and NB16 exhibit negligible or undetectable 41BB activation, underscoring a lack of consistent early activation under these experimental conditions.

[0018] Figure 4: Representative profiles of GM-CSF and IFN-y cytokine release during co-culture of CAR T-cells with AsPCl (top panel) or BxPC3 (bottom panel) target cells. Cytokine levels were measured at three key time points (Day 3, Day 7, and Day 9) during successive rechallenges in the cytotoxicity assay, encompassing all 16 nanobody CAR candidates. Data are displayed as mean ± s.e.m. (n = 4, two replicates per donor) and benchmarked against both the negative control (NTC) and the positive control (mCAR). Nanobody CAR candidates NB1, NB2, NB4, NB5, NB6, NB9, and NB11 (in light grey) consistently exhibited robust cytokine MBG_183 release. GM-CSF levels surpassed 5000 ng / pl for AsPCl and 1000 ng / pl for BxPC3, while IFN-y levels exceeded 8000 ng / pl for AsPCl and 1000 ng / pl for BxPC3, matching or approaching the cytokine profiles of the mCAR positive control across all time points. Conversely, NB3, NB7, NB8, NB10, NB12, NB13, NB14, NB15, and NB16 exhibited either negligible or markedly attenuated GM-CSF and IFN-y release, underscoring their limited capacity for functional activation in this experimental framework.

[0019] Figure 5: Representative kinetic analysis of tumor cell killing by nanobody CAR candidates in AsPCl wild-type (WT), CD318 knockout (KO), and CD318 KO + murine chCDCPl knock-in (KI) target cells, evaluated in comparison to negative control (NTC) and positive control (mCAR). The X-axis represents time in hours, while the Y-axis shows the green area confluence, quantified from the GFP signal emitted by GFP+ tumor cells, with values normalized to 1 at the start of the experiment. A decrease in the curve indicates a reduction in GFP signal, correlating with tumor cell death, while an increase reflects tumor cell growth. The kinetics were assessed across two independent donor samples. The best-performing nanobody CAR candidates demonstrated high killing efficacy against the WT cell line, consistent with our previous findings. No killing was observed in the CD318 KO cells, confirming the specificity of the CAR candidates. One nanobody candidate showed inconsistent results, likely due to donor variability or cell inhibition, rather than actual killing. No killing was observed in the CD318 KO + murine chCDCPl KI cells, confirming a lack of cross-reactivity. Similar observations were made for the second donor, reinforcing the conclusions regarding specificity and cross-reactivity.

[0020] Figure 6: Representative expression patterns of the 4-1BB early activation marker for the most effective nanobody -based CAR (NB1) during co-culture with AsPCl cell lines corroborate the tendency observed during the cytotoxicity assay.

[0021] Detailed description of the invention

[0022] In a first aspect the present invention provides a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain. MBG_183

[0023] 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.

[0024] Said CAR, wherein said antigen CD318 is expressed on a target cell.

[0025] Said CAR, wherein said target cell expressing CD318 is a cancer cell.

[0026] Said CAR, wherein said target cell expressing CD318 may be pancreatic cancer, bladder cancer, urothelial cancer, breast cancer , colon cancer , lung adenocarcinoma cancer , lung squamous cancer or prostate cancer.

[0027] Said CAR, wherein said cancer cell expressing CD318 may be preferentially pancreatic cancer.

[0028] Said CAR, wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO: 1, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:2, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:2, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:4, the hinge domain of SEQ ID NO:23, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:5, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:6, the hinge domain of SEQ ID NO:24, the transmembrane domain of MBG_183

[0029] SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO:9, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27, or wherein said CAR comprises from the N-terminus to the C-terminus the antigen binding domain of SEQ ID NO: 11, the hinge domain of SEQ ID NO:24, the transmembrane domain of SEQ ID NO:25, the co-stimulatory domain of SEQ ID NO:26 and the stimulatory domain of SEQ ID NO:27.

[0030] In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0031] Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

[0032] In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) for use in immunotherapy, the CAR comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0033] Said immune cell expressing a chimeric antigen receptor (CAR) for use in immunotherapy, wherein the immunotherapy is for treatment of a (solid) cancer, wherein the cancerous cells express CD318 such as pancreatic cancer.

[0034] Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell. MBG_183

[0035] In another aspect the present invention provides an immune cell expressing a chimeric antigen receptor (CAR) for use in treatment of a (solid) cancer (in a subject), wherein the cancerous cells express CD318 such as pancreatic cancer, the CAR comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0036] Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

[0037] In another aspect the present invention provides a (isolated) nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0038] In another aspect the present invention provides an immune cell comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0039] In a further aspect the present invention provides a vector comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain. MBG_183

[0040] 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 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.

[0041] In a further aspect the present invention provides a vector for use in treatment of a (solid) cancer in a subject, wherein said cancer comprises cancerous cells expressing CD318 such as pancreatic cancer, wherein said vector is administered to the subject, the vector comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain, thereby treating said cancer in said subject.

[0042] Said vector for use in treatment of a (solid) cancer, wherein said vector is a pseudotyped vector targeting CD4+ and / or CD8+ T cells.

[0043] In a further aspect the present invention provides a composition comprising (a population of) immune cells expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0044] Said immune cells may preferentially be T cells, NK cells or a gammadelta T cells.

[0045] In a further aspect the present invention provides a composition comprising (a population of) immune cells expressing a chimeric antigen receptor (CAR) for use in a method of treating MBG_183 cancer in a subject, wherein the method comprises administering said composition to said subject, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain, wherein said cancer comprises cancer cells expressing CD318 such as pancreatic cancer. Said immune cells may preferentially be T cells, NK cells or a gammadelta T cells.

[0046] In a further aspect the present invention provides a pharmaceutical composition comprising i) immune cells expressing a chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain, and optionally ii) a pharmaceutically acceptable carrier.

[0047] 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.

[0048] In one embodiment of the invention the immune cells expressing 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 CD318 and the disease may be a solid cancer such as pancreatic cancer. Immune cells, e.g. T cells or NK cells of a subject may be isolated. The subject may e.g. suffer from said cancer or may be a healthy subject. These cells may be genetically modified 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 cell plus pharmaceutical acceptable carrier). The infused cells may be e.g. able to kill (or at least stop MBG_183 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).

[0049] 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.

[0050] Preferentially, the compositions of the present invention 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.

[0051] Pharmaceutical compositions of the present invention 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.

[0052] 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.

[0053] The genetically engineered immune cells may be activated and expanded to therapeutic effective amounts using methods known in the art.

[0054] The immune cells of the invention may be used in combination with e.g. chemotherapy, radiation, immunosuppressive agents, antibodies or antibody therapies.

[0055] In a further aspect the present invention provides in in-vivo method for treatment of a subject suffering from a (solid) cancer, wherein the cancerous cells express CD318 such as pancreatic MBG_183 cancer, the method comprising administering to said subject an immune cell expressing a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0056] Said immune cell may preferentially be a T cell, an NK cell or a gammadelta T cell.

[0057] In a further aspect the present invention provides in in-vivo method for treatment of a subject suffering from a (solid) cancer, wherein the cancerous cells express CD318 such as pancreatic cancer, the method comprising administering to said subject the vector comprising a nucleic acid molecule encoding a CAR, wherein said CAR comprises a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.

[0058] Said method, wherein said vector is a pseudotyped vector targeting CD4+ and / or CD8+ T cells.

[0059] In a further aspect the present invention provides a pseudotyped retroviral vector particle comprising 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 WIQ Paramyxoviridae family, if Paramyxoviridae virus is a virus of the Henipavirus genus, 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 MBG_183 genus, and wherein said envelope protein with antigen-binding activity and said envelope protein with fusion activity are from the same genus, c) a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising an antigen binding domain comprising SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9 or SEQ ID NO: 11, a transmembrane domain, and an intracellular signaling domain, and wherein said retroviral vector particle is a lentiviral or gammaretroviral vector particle.

[0060] 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).

[0061] 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 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.

[0062] Generation of of CAR immune cells such as CAR T cells

[0063] 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.

[0064] 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: a) providing a cell sample comprising immune cells (e.g. from a PBMC) b) preparation of the cell sample by centrifugation c) magnetic separation of the immune cells, preferentially T cells, d) activation of the enriched immune cells, preferentially T cells, using modulatory agents MBG_183 e) genetically modifying the immune cells, preferentially T cells, to express the CAR as disclosed herein f) expansion of the genetically modified immune cells, preferentially T cells, in a cultivation chamber g) washing of the cultured immune cells, preferentially T cells.

[0065] All these steps may be performed automatically in a closed system.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 expressing the CAR as disclosed herein (see e.g. WO2020239866A1) . Such a short ex-vivo process may comprise e.g. (in a closed system for cell modification) the steps a) providing a sample (e.g. from PBMC) comprising immune cells such as T cells b) preparation of said sample by centrifugation c) enrichment of the immune cells such as T cells of step b d) activation of the enriched immune cells such as T cells using modulatory agents e) genetic modification of the activated immune cells such as T cells by transduction e.g. with lentiviral vector particles f) removal of said modulatory agents, MBG_183 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).

[0071] As a closed system for cell modification, the fully automated cell processing device CliniMACS Prodigy® and associated tubing sets (Miltenyi Biotec B.V. & Co. KG, 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.

[0072] Nucleotides, Expression, and Vectors

[0073] The nucleic acids 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.

[0074] 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 increasing translation efficiency. Optimization of the nucleotide sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0075] "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.

[0076] 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.

[0077] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, 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 MBG_183 under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors are not naturally-occurring as a whole.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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 MBG_183 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.

[0085] 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.

[0086] 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.

[0087] Disclosure of general methods of treatment

[0088] 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 comprising cancerous cells expressing CD318 such as pancreatic cancer, 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 disclosed herein in an amount effective to treat cancer in the mammal.

[0089] 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.

[0090] 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 MBG_183 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.

[0091] With respect to the methods, the cancer can be any cancer in that CD318 expressing target cells are involved. The cancers may include pancreatic cancer, bladder cancer, urothelial cancer, breast cancer, colon cancer, lung adenocarcinoma cancer , lung squamous cancer or prostate cancer.

[0092] 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 in a mammal.

[0093] 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.

[0094] Another embodiment provides for the use of the CARs, nucleic acids, recombinant expression Vectors and immune cells for the treatment or prevention of disorder, e.g., cancer in a mammal. 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] In-vivo gene therapy

[0099] 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 transgene product, herein regularly the CAR as disclosed herein. Such pseudotyped retroviral vectors are disclosed e.g. in WO2023015217A1, WO2022150731A1 and EP24159106.4.

[0100] Pseudotvped retroviral vector particles

[0101] 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. Aetrow'rzt / ae-derived 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.

[0102] 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.

[0103] Gammaretroviridae is a genus of the Retroviridae family. Representative species are the murine leukemia virus (MLV) and the feline leukemia virus (FLV).

[0104] 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 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.

[0105] 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.

[0106] 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. MBG_183

[0107] 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 Par amyxoviridae H, HN or G proteins are virus envelope protein(s) that have antigen binding activity.

[0108] 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.

[0109] 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 Par amyxoviridae F proteins refer to virus envelope protein(s) that have fusion activity.

[0110] 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.

[0111] 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 Par amyxoviridae 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.

[0112] 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.

[0113] 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. MBG_183

[0114] I. Retroviral vector particle pseudotyped with Nipah virus envelope proteins

[0115] 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 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.

[0116] 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.

[0117] II. Retroviral vector particle pseudotyped with CDV envelope proteins

[0118] 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).

[0119] 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. MBG_183

[0120] Generation of pseudotyped retroviral vector particles for administration

[0121] The quality and quantity of the pseudotyped retroviral vector should be sufficient to enable regulatory approval and safe treatment of larger patient cohorts.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] The packaging cell line might be genetically engineered for the expression of additional factors or for reducing or inhibiting the expression of specific factors.

[0127] 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.

[0128] In one embodiment the pseudotyped retroviral vector is formulated in pharmaceutically acceptable carrier, diluent or excipient compatible for administration in human.

[0129] In one application the pseudotyped retroviral vector is filled in vials or bags.

[0130] In another embodiment, the dose of filled pseudotyped retroviral vector is adjusted to the body weight.

[0131] In another embodiment, the pseudotyped retroviral vector is filled in the presence of a cryoprotectant.

[0132] In another embodiment, the pseudotyped retroviral vector is lyophilized and / or reconstituted before administration.

[0133] 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. MBG 183

[0134] Disclosure of methods of treatment with pseudotyped retroviral vector particles

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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 MBG_183 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).

[0142] In one embodiment the pharmaceutical composition comprises the pseudotyped retroviral vector specific for the antigen CD4 as disclosed herein and / or the pseudotyped retroviral vector specific for the antigen CD8 as disclosed herein.

[0143] In one embodiment, administration of a total dose of said pharmaceutical composition is a unique administration of said pharmaceutical composition to the subject.

[0144] 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).

[0145] 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.

[0146] Definitions

[0147] 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. MBG_183

[0148] 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.

[0149] 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

[0150] In general, a CAR may comprise an extracellular domain (extracellular part) comprising the antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (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.

[0151] 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.

[0152] 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, single chain Fv (scFv) fragments, divalent single chain antibodies, nanobodies 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 a nanobody (a single domain antibody). 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”.

[0153] 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 MBG_183 humanized antibodies or antigen binding fragments thereof can be made by a variety of methods well known in the art.

[0154] “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.

[0155] 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. The transmembrane domain may be derived for example 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.

[0156] 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.

[0157] 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. 1

[0158] MBG_183

[0159] 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 primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0160] Primary cytoplasmic signaling domains that act in a stimulatory manner may contain ITAMs (immunoreceptor tyrosine-based activation motifs).

[0161] 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^.

[0162] 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.

[0163] 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.

[0164] 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. MBG_183

[0165] 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.

[0166] 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 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.

[0167] In some embodiments, the endodomain may contain a primary cytoplasmic signaling domains or a co-stimulatory region, but not both.

[0168] The CARs of the present disclosures 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.

[0169] The engineered cell expressing a CAR 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 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 .

[0170] 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.

[0171] The cluster of differentiation (CD) is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cells. In terms of physiology, CD molecules can act in numerous ways, often acting as receptors or ligands important to the cell. A signal cascade is usually initiated, altering the behavior of the cell. MBG_183

[0172] CD318 (also termed CUB domain-containing protein 1 (CDCP1)) is a 140 kD transmembrane glycoprotein with a large extracellular domain containing two CUB domains, and a smaller intracellular domain.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] Single domain antibodies (sdAbs), also known as nanobodies are derived from the heavychain antibodies found in Camelidae species (such as camel, llama, dromedary, alpaca and guanaco) using molecular biology techniques, which are also known as VhH fragments MBG_183

[0177] (herein also termed “VHH” or “VHH”). Similar antibody domains including Vnar fragments derived from heavy chain antibodies found in cartilaginous fish, such as sharks. sdAbs could also been generated from a heavy chain / light chain of conventional immunoglobulin G (IgGs) by engineering techniques followed by affinity maturations, or alternatively, from an immunized transgenic mouse or rat carrying the camelid heavy chain or humanized camelid gene loci.

[0178] Despite their small size (about lOx smaller than mAbs), sdAbs have comparable affinity and specificity, and quite stable under extreme pH, high temperature and proteolytic conditions that can be problematic for conventional antibodies and fragments thereof (e.g., Fab, scFv). Furthermore, VHHS could be expressed in simple prokaryotic and eukaryotic organisms (e.g., bacteria and yeast) in up to gram quantities and in properly folded / functional formats. The unique feature of a portion of VHH antibodies generated in Camelidae species is that they recognize cryptic or hidden epitopes such as enzyme active sites or cavities on virus surface proteins which are otherwise inaccessible to larger conventional antibodies and antibody fragments. This will place camelid sdAbs in a unique position for the development of enzyme inhibitor or viral neutralizer reagents.

[0179] 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 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.

[0180] 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.

[0181] 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. MBG_183

[0182] 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.

[0183] 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.

[0184] 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 well-known in the art.

[0185] In some embodiments, the nucleic acid sequence may be codon-modified. Without being bound to a particular theory, it is believed that codon optimization of the nucleic acid sequence increases the translation efficiency of the mRNA transcripts. Codon optimization of the nucleic acid 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 increasing translation efficiency. Optimization of the nucleic acid sequence may also reduce secondary mRNA structures that would interfere with translation, thus increasing translation efficiency.

[0186] 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.

[0187] 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.

[0188] 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 MBG_183 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.

[0189] 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”.

[0190] The term “treatment” as used herein means to reduce the frequency or severity of at least one sign or symptom of a disease.

[0191] 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.

[0192] 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 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. MBG_183

[0193] 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).

[0194] 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.

[0195] 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 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 nanoparticles, they are “untouched”. MBG_183

[0196] As used herein “autologous” means that cells, a cell line, or population of cells used for treating subjects are originating from said subject.

[0197] 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.

[0198] 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.

[0199] T cells or T lymphocytes are a type of lymphocyte that play a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells, each with a distinct function.

[0200] T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen- presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate a different type of immune response. Signaling from the APC directs T cells into particular subtypes. MBG_183

[0201] Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells.

[0202] Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0203] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

[0204] Two major classes of CD4+ Treg cells have been described — Foxp3+ Treg cells and Foxp3- Treg cells.

[0205] Natural killer T cells (NKT cells - not to be confused with natural killer cells of the innate immune system) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CDld. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic / cell killing molecules).

[0206] Natural killer cells (NK cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor-generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, where they then enter the circulation. NK cells differ from natural killer T cells (NKTs) phenotypically, by origin and by respective effector functions; often, NKT cell activity promotes NK cell activity by secreting fFNy. In contrast to NKT cells, NK cells do not express T cell antigen receptors (TCR) or pan T marker CD3 or surface immunoglobulins (Ig) B cell receptors, but they usually express the surface markers CD 16 (FcyRIII) and CD56 in humans, NK1.1 or NK1.2 in C57BL / 6 mice. Up to 80% of human NK cells also express CD8. Continuously growing NK cell lines can be established from cancer patients and common NK cell lines are for instance NK-92, NKL and YTS. MBG_183

[0207] 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.

[0208] 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. Normally the transgenes used in the present disclosures may be the chimeric antigen receptor specific for the antigen CD318 as disclosed herein. The expressed transgene may also be referred to as a transgene product, a heterologous protein or transgenic polypeptide.

[0209] Examples

[0210] The following examples are intended for a more detailed explanation of the invention but without restricting the invention to these examples.

[0211] Example 1 : Generation of single domain antibodies targeting CD318

[0212] A male Huacaya alpaca (Miltenyi Biotec, SMG Charlie Brown, AREU-025043, “Charlie”) was immunized with CD318 recombinant protein (Miltenyi Biotec, Uniprot Q9H5V8

[0213] AA30-666 fused with poly his and avi tag) according to a standard protocol (Pardon, E., Laeremans, T., Triest, S. et al. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc 9, 674-693 (2014)) with GERBU F adjuvant (GERBU Biotechnik GmbH, Cat. No. 3030) and using five boost immunizations over 6 weeks. 150 ml blood was collected afterwards. PBMCs were isolated using Human Pancoll solution (Pan Biotech, Cat. No. P04-60500) and stored in StemMACS TM Cryobrew (Miltenyi Biotec, Cat 130-109-558). Cells were lysed in Trizol (Thermo Scientific, Cat No. 15596018). Total RNA was extracted using Directzol RNA miniprep Plus Kit (Zymo Research, Cat No. R2070) and RNA quality was checked by the Agilent 2100 Bioanalyzer. cDNA was generated using the SuperScript IV first-Strand Synthesis System (Thermo Fischer, Cat. No. 18091050) using Oligo d(T). The VHH DNA was amplified using nested PCR. PCRI was run using Phusion polymerase using primers CALL001 and CALL002. The PCR product was separated on 1% agarose (v / v) gel. The generously cut band at -400 bp was extracted and purified using PCR Clean-up kit (Macherey -Nagel Cat. No. 740609.10). The second PCR (CloneAmp, Takara, Cat. 639298) was run using forward (VHH-PCRII-TypeIIS__F) and reverse primer (VHH-PCRII- MBG_183

[0214] TypeIIS__R) and purified using PCR Clean-up kit (Macherey -Nagel Cat. No. 740609.10). The insert was cloned into the Phagemid vector (Ml 3, Miltenyi Biotec) using Type IIS cloning and transformed into electrocompetent TGI cells (Lucigen Cat. 60522-19). After packaging of the library two (strategy 2) and three (strategy 1) rounds of panning selection were performed using absorbed CD318 (Miltenyi Biotec) on Nunc MaxiSorb ELISA plates (Thermo Scientific), respectively. Antigen binding phage were eluted using trypsin. TGI were infected with the phage. Two times 288 clones from both strategies were randomly picked using a automated colony picker (Molecular Devices). Bacterial culture supernatants of theses clones were screened using 384 well ELISA plates on CD318 (Miltenyi Biotec), CUB 1 -domain of CD318 (Sino Biological, 13262-H08H) and BSA with anti-c-myc antibody HRP conjugate (Miltenyi Biotec) and TMB substrate.

[0215] 379 initially positive clones were sent to sequencing, yielding 120 unique CUB1 and CUB2 / 3 binder sequences totally. The sequences clustered into 6 groups + 3 single sequences (CUB1) and 11 groups + 5 single sequences (CUB2 / 3), respectively. Sequences featuring stop codons, as well as Methionine and N-glyco sites in the CDR regions were excluded. Sixteen VHH sequences (7 CUB1, 9 CUB2 / 3) were selected for cloning into the CAR backbone and bacterial expression.

[0216] Primers used:

[0217] SEQ ID NO: 18: VHH-PCRII-TypeIIS_F

[0218] SEQ ID NO: 19: VHH-PCRII-TypeIIS_R

[0219] SEQ ID NO:20: CALL001

[0220] SEQ ID NO:21 : CALL002

[0221] Example 2: Construction and expression of CD318 targeting nanobody CARs in primary human T cells

[0222] CAR cloning

[0223] Commercial gene synthesis in combination with an optimization algorithm for codon usage in humans (IDT) was used to construct CD318 targeting nanobody CARs. The CD318-specific CARs comprised as a binding domain SEQ ID 1-16. To facilitate receptor trafficking to the plasma membrane, a human CD8a leader signaling peptide (SEQ ID NO:22) was added N- terminally to the respective nanobody sequence. The spacer region downstream of the nanobody domain encompassed the CD8a hinge (SEQ ID NO:23)) for nanobodies targeting CUB2 / 3 (NB3, NB4, NB7, NB8, NB10, NB12, NB13, NB15, NB16) and IgG4 hinge only (SEQ ID NO:24) for CUB1 binding nanobodies (NB1, NB2, NB5, NB6, NB9, NB11, NB14). MBG_183

[0224] All CARS have the hCD8 transmembrane domain (SEQ ID NO: 25), the costimulatory domain of 4-1BB (SEQ ID NO: 26) and the stimulatory domain of CD3zeta (SEQ ID NO: 27). The CAR genes were fused to a Furin-P2A sequence to include co-expression of the truncated low affinity nerve growth factor receptor (ALNGFR). Transgene expression was promoted by the PGK promoter located upstream of the CAR gene.

[0225] Lentiviral particle production

[0226] VSV-G pseudotype lentiviral particles encoding CARs that have SEQ IDs NO: 1 - 16 as binding domains were generated by transfection of HEK293 cells using a four or three plasmid system. 24h after transfection, 10 mM Sodium Butyrate (Sigma Aldrich) was added to the culture medium. Lentiviral particles were harvested 48h after transfection by passing the cell culture supernatant through a 0.45 pm filter and centrifugation over night at 4°C. Lentiviral particles were resuspended in TexMACS Medium (Miltenyi Biotec) and stored at -70 °C until transduction.

[0227] CAR T cell generation and cultivation

[0228] Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation from whole blood of healthy donors. Pan T cells were isolated from PBMC using the Pan T Cell Isolation Kit, human (Miltenyi Biotec). T cells were cultured at a density of 1E+06 cells per ml in TexMACS Medium (Miltenyi Biotec) supplement with 12.5 ng / ml IL-7 and IL-15 (Miltenyi Biotec) and activated with CD3 and CD28 agonists (T cell TransAct, Miltenyi Biotec). 24h after activation, T cells were transduced with VSV-G pseudotype lentiviral particles. Three days after isolation, the majority of TransAct was removed by replacing % of the media volume with fresh medium supplemented with IL-7 and IL-15. Transduction efficiency was determined via flow cytometry by staining with PE-labelled human CD318 protein (Miltenyi Biotec). CAR T cells were used for assays 10-14 days after isolation.

[0229] Flow cytometry analysis

[0230] Flow cytometry analysis was carried out in MACSQuant 10 analyzer. For cell staining, 0.5 million cells were harvested from culture and washed two times in cold AutoMACS buffer supplemented with 0.5% bovine serum albumin (Miltenyi Biotec). Non-transduced cells were used as negative controls. Dead cells in all studies were excluded by 7AAD staining (Miltenyi Biotec). Cells were washed twice and resuspended in 200 pl Staining Buffer before quantitative analysis by flow cytometry. Flow cytometric analysis and data plots were generated using MACSQuant software. MBG_183

[0231] Results

[0232] To characterize the CD318 specific CAR T cells, primary T cells were transduced. All CAR T cells showed comparable LNGFR expression at day 7 (Fig 1) indicating that the genetical CAR cassette was well expressed. Most of the CAR T cells showed good recognition of the human CD318 antigen (Figure 1) except NB 8 and NB 14 CARs. While the recombinant CD318 protein was not able to stain NB8 or NB14 CARs, it was still possible that CAR is on the surface, but not detectable due to steric reasons. Thus, all CARs were further evaluated in subsequent functional assays.

[0233] Example 3: Functional characterization of CD318 targeting nanobody CARs Cell lines and cell culture

[0234] For in vitro experiments, AsPCl and BxPC3 pancreatic cancer cell lines, were purchased from American Type Culture Collection (ATCC) and cultured according to ATCC recommendations. Cytotoxicity assay on PDAC cell lines

[0235] AsPCl and BxPC3 pancreatic cancer cell lines were stably transduced to express firefly luciferase (ffLuc) and green fluorescent protein (GFP). PDAC cell lines that underwent CRISPR mediated knock-out of CD318 were used as negative controls. Target cells were seeded in a density of 2E+04 cells per well in 96-well plates . Target cells were cocultured with CAR T cells at effector to target (E:T) ratios of 5: 1 (AsPCl) and 2: 1 (BxPC3). After 56h and 154h new 2E+04 target cells were added to the wells. Cytotoxicity was assessed by measuring confluency of target cells (green area confluence, GAC) with Incucyte®S3 Live-Cell Analysis Instrument (Sartorius) and analysing with the IncuCyte® S3 2019A software over the course of the co-culture. Data was normalized to GAC at start of the co-culture and after every new addition of target cells. T cell activation marker expression and cytokine secretion were assessed via flow cytometry at 48h of co-cultivation. For cytokine measurements 50 pl of supernatant were taken and stored at -20°C until analysis. Quantification of cytokines was performed using MACSplex Cytokine 12 Kit, human (Miltenyi Biotec) according to manufacturer’s instruction.

[0236] Results

[0237] To evaluate CD318 specific nanobody CAR T cell functionality, we assessed their cytotoxicity, activation marker expression and cytokine secretion in co-culture with target cell lines. Only 6 of the 16 CAR T cells performed target cell killing consistently through 3 rounds of cytotoxicity assays (Fig. 2) as reflected with decreasing GAC even in the 3rdround. Surprisingly, they acted better than the previously published murine CAR (mCAR) that comprise a scFv having SEQ MBG_183

[0238] ID NO: 17 followed by IgG4 hinge, hCD8 transmembrane domain, 4-1BB and CD3zeta (doi.org / 10.1038 / s41467-021-21774-4). The CAR T cells exhibiting killing for 3 rounds were the ones with clear upregulation of the activation marker 4 IBB (Fig. 3). NB7, 8 and 10 remained slight activated until the end, although no efficient killing was performed in the final coculture round, indicating a certain level of activation in which the activation threshold is not high enough to initiate target cell lysis. Cytokine release mirrored the results from the activation and killing, showing only cytokine release in the CAR groups that performed 3 rounds of killing (Fig. 4). CAR T cells performing 3 rounds of killing were subsequently cocultured with target cells not expressing the target through CRISPR mediated KO. No cytotoxic activity was observed on KO cells and no activation as shown by 41BB expression was observed (Fig. 5 and 6).

[0239] In conclusion, nanobody candidates NB1, NB2, NB4, NB5, NB6, NB9, and NB11 have emerged as exceptionally promising targets in the context of CAR-T cell therapies, exhibiting pronounced and sustained cytotoxic activity in vitro. Beyond their potent tumor-cell killing capabilities, these nanobodies robustly activate pivotal immune markers, such as 4 IBB, and drive the secretion of key cytokines, including GM-CSF and IFN-g, that are critical mediators of a durable and effective anti-tumor response. The combination of their high cytolytic efficiency, consistent functionality across rechallenges, and ability to engage immune effector pathways highlights their transformative potential in cancer immunotherapy. These findings lay a strong foundation for advancing these candidates into clinical development, offering new avenues for precision-targeted cancer treatments.

[0240] Description of the sequences of the sequence protocol

[0241] SEQ ID NO: 1 : NB1

[0242] SEQ ID NO:2: NB2

[0243] SEQ ID NO:3: NB3

[0244] SEQ ID NO:4: NB4

[0245] SEQ ID NO:5: NB5

[0246] SEQ ID NO:6: NB6

[0247] SEQ ID NO:7: NB7

[0248] SEQ ID NO:8: NB8

[0249] SEQ ID NO:9: NB9

[0250] SEQ ID NO: 10: NB10

[0251] SEQ ID NO: 11 : NB11

[0252] SEQ ID NO: 12: NB12

[0253] SEQ ID NO: 13: NB13

[0254] SEQ ID NO: 14: NB14

[0255] SEQ ID NO: 15: NB15

[0256] SEQ ID NO: 16: NB16

[0257] SEQ ID NO: 17: murineCAR (mCAR) scFv CD318 MBG_183

[0258] SEQ ID NO: 18 VHH-PCRII-TypeIIS_F

[0259] SEQ ID NO: 19 VHH-PCRII-TypeIIS_R

[0260] SEQ ID NO:20 CALL001

[0261] SEQ ID NO:21 CALL002

[0262] SEQ ID NO:22 CD8alpha leader

[0263] SEQ ID NO:23 CD8alpha hinge

[0264] SEQ ID NO:24 IgG4 hinge

[0265] SEQ ID NO:25 hCD8 transmembrane domain

[0266] SEQ ID NO:26 4-1BB

[0267] SEQ ID NO:27 CD3zeta

[0268] References:

[0269] 1. Awakura, Y. et al. Microarray -based identification of CUB-domain containing protein 1 as a potential prognostic marker in conventional renal cell carcinoma. J. cancer Res. Clin. Oncol. 134, 1363-1369 (2008).

[0270] 2. Uekita, T. & Sakai, R. Roles of CUB domain-containing protein 1 signaling in cancer invasion and metastasis. Cancer Sci. 102, 1943-1948 (2011).

[0271] 3. Wortmann, A. et al. The cell surface glycoprotein CDCP1 in cancer — Insights, opportunities, and challenges. IUBMB Life, 61 : 723-730 (2009).

[0272] 4. Khan, T, et al. The CDCP1 signaling hub : a target for cancer detection and therapeutic intervention. Cancer Res 2021;81 :2259-69 (2021).

[0273] 5. Schafer, D. et al. Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma. Nat. Com. 12: 1453 (2021).

[0274] 6. Celia-Terrassa T, Kang Y. How important is EMT for cancer metastasis? PLoS Biol 22(2): e3002487(2024).

[0275] 7. Adeshakin FO, et al. Mechanisms for Modulating Anoikis Resistance in Cancer and the Relevance of Metabolic Reprogramming. Front. Oncol. 11 :626577 (2021).

[0276] 8. Li, M., Li, S., Zhao, R. et al. CD318 is a target of chimeric antigen receptor T cells for the treatment of colorectal cancer. Clin Exp Med 23, 2409-2419 (2023).

Claims

MBG_183Claims1) A chimeric antigen receptor (CAR) comprising a) an antigen binding domain specific for the antigen CD318 wherein the antigen binding domain comprises SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO: 9 or SEQ ID NO: 11, b) a transmembrane domain, and c) an intracellular signaling domain.2) The CAR of claim 1, wherein said intracellular signaling domain comprises a stimulatory domain comprising one or more immunoreceptor tyrosine-based activation motifs (ITAMs) and / or one or more co- stimulatory domain(s).3) The CAR of claim 1 or 2, wherein said antigen CD318 is expressed on a target cell.4) The CAR of claim 3, wherein said target cell expressing CD318 is a cancer cell.5) The CAR of claim 4, wherein said cancer cell is pancreatic cancer, bladder cancer, urothelial cancer, breast cancer, colon cancer, lung adenocarcinoma cancer , lung squamous cancer or prostate cancer.6) An immune cell expressing a CAR according to any one of claims 1 to 5.7) The immune cell of claim 6 for use in immunotherapy.8) A nucleic acid molecule, such as a vector, encoding a CAR according to any one of claims 1 to 5.9) A vector for use in treatment of cancer in a subject, wherein said cancer comprises cancerous cells expressing CD318, such as pancreatic cancer, wherein said vector is administered to the subject, the vector comprising a nucleic acid molecule encoding a CAR, according to any one of claims 1 to 5, thereby treating said cancer in said subject.10) Said vector for use in treatment of cancer, wherein said vector is a pseudotyped vector targeting CD4+ and / or CD8+ T cells.

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