Small molecule-inducible recombinant receptors

Abstract

The present invention generally relates to recombinant receptor polypeptides and recombinant receptors, and cells expressing thereof, capable of activating cellular cytokine pathways by complexing small molecule ligands. The invention also relates to nucleic acid molecules encoding the recombinant cytokine receptors of the present invention.

Description

[0001] P39375-EP

[0002] Small molecule-inducible recombinant receptors

[0003] Technical Field

[0004] The present invention relates to recombinant receptor polypeptides and recombinant receptors, and cells expressing thereof, capable of activating cellular cytokine pathways by complexing small molecule ligands. After cooperative engagement of the antibody-based ligand binding domains to the small molecule, a complex is formed leading to intracellular signaling events. The invention also relates to nucleic acid molecules encoding the recombinant cytokine receptors of the present invention.

[0005] Background

[0006] Chimeric antigen receptor (CAR)-based cell therapy has become a clinically validated treatment option for haematologic malignancies. By contrast, CAR-T therapy has shown limited efficacy in solid cancer indications. Advanced solid tumors seem to be more difficult to treat with CAR-T cells. Possible reasons are tumor heterogeneity, immunosuppression in the tumor microenvironment (TME) resulting in CAR-T cell dysfunction, low in vivo persistence, and, most importantly, the lack of truly cancer-specific tumor associated antigens (TAAs) resulting in dose-limiting antigen mediated toxicities. Current research efforts are devoted to the engineering of next-generation CAR-T and TCR-T cells which can overcome these additional hurdles posed by solid tumors.

[0007] Cytokine pathways play a central role in T cell biology and their value in cancer immunotherapy is undisputed. However, most cytokines, naturally evolved to act locally and initiate potent self-amplifying signalling cascades at sites of infection or disease. As a consequence, cytokines are notoriously difficult to develop as therapeutic products. In cell therapy, a growing area of research is aiming to arm CAR- or TCR- engineered T cells with additional recombinant cytokine or cytokine receptor building blocks. A subset of these approaches are so-called orthogonal cytokine / receptor systems, i.e., synthetic cytokine receptors which bind synthetic ligands and do not cross-react with any endogenous ligands or receptors in humans.

[0008] For example, a mutant variant of lnterleukin-2 (IL2), termed orthogonal IL2 or ortho-IL2, has been engineered to bind to only a mutated variant of lnterleukin-2 receptor beta (IL2Rp) while still being able to engage with natural interleukin-2 receptor gamma (IL2Ry) (1). Ortho-IL2 and ortho-IL2Rp feature mutations in order to achieve mutually exclusive binding and induction of IL2 signaling, in engineered cells. Others have designed chimeric but not orthogonal cytokine receptors which can be triggered by small molecules. A prominent example is based on rapamycin (FK506), an approved immuno- suppressive drug which specifically engages two different protein domains, namely, FK506 binding protein (FRBP) and FKBP- rapamycin binding (FRB) domain of the rapamycin target (mTOR). The aforementioned rapamycin- controlled chimeric cytokine receptors feature FRBP and FRB as extracellular ligand binding domains (2). Finally, researchers have also investigated different constitutively active cytokine receptors that confer continuous cytokine signaling to engineered T cells (3,4).

[0009] There remains a need for improved cytokine / receptor systems which do not cross-react with the endogenous cytokine pathways. P39375-EP

[0010] Summary

[0011] The present invention generally relates to recombinant receptor polypeptides and recombinant receptors capable of activating cellular cytokine pathways by complexing small molecule ligands.

[0012] In a first aspect, the present disclosure provides a recombinant receptor polypeptide comprising:

[0013] (i) an extracellular portion comprising an antigen binding domain capable of binding to a small molecule;

[0014] (ii) an intracellular portion comprising the intracellular domains of a cytokine receptor; and

[0015] (iii) a transmembrane domain that joins the extracellular portion to the intracellular portion.

[0016] In some embodiments, the antigen binding domain of the extracellular portion comprises a light chain variable region (VL) or a heavy chain variable region (VH) of an antibody capable of specific binding to said small molecule.

[0017] In some embodiments, the small molecule is DOTAM or a DOTAM derivative.

[0018] In some embodiments, the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108.

[0019] In some embodiments, the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108.

[0020] In some embodiments, the VL comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111.

[0021] In some embodiments, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2, and wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0022] In some embodiments, the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:161 , SEQ ID NO:162, and SEQ ID NO:163, and wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0023] In some embodiments, the cytokine receptor is IL2R, IL7R, IL12R, IL18R or IL23R.

[0024] In a second aspect, provided is a recombinant receptor comprising a first recombinant receptor polypeptides as disclosed herein, e.g. in the first aspect, and a second recombinant polypeptide as disclosed herein, e.g. in the first aspect, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0025] In some embodiments, the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL. P39375-EP

[0026] In some embodiments, a) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 112, 1 14, 1 16, 1 17, 118, 119, and 120, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling; or b) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 128 and SEQ ID NO: 129, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling; or c) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 123, 125, and 126, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 124 and SEQ ID NO: 127, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL18 signaling; or d) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling and that is capable of IL12 signaling; or e) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 132, or an amino acid sequence having at least 80% sequence identity thereto thereto and that is capable of IL23 signaling; or f) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, the amino acid sequence of SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling. g) wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 158, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling and that is capable of IL12 signaling.

[0027] In a third aspect, provided is nucleic acid, or a plurality of nucleic acids, encoding a recombinant receptor polypeptide or a recombinant receptor disclosed herein, e.g. in the first and second aspects, and optionally further comprising a nucleic acid sequence encoding a chimeric antigen receptor P39375-EP

[0028] In a fourth aspect, provided is an expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids disclosed herein, e.g. in the third aspect.

[0029] In a fifth aspect, the provided is a cell comprising a recombinant receptor polypeptide or a recombinant receptor disclosed herein, e.g. in the first and second aspects, a nucleic acid or a plurality of nucleic acids disclosed herein, e.g. in the third aspect, or an expression vector or a plurality of expression vectors disclosed herein, e.g. in the fourth aspect.

[0030] In a sixth aspect, the present disclosure provides a pharmaceutical composition comprising the cell disclosed herein, e.g. in the fifth aspect, and optionally a pharmaceutically acceptable excipient.

[0031] In a seventh aspect, the present disclosure provides the recombinant receptor polypeptide disclosed herein, e.g. in the first aspect, the recombinant receptor disclosed herein, e.g. in the second aspect, the nucleic acid disclosed herein, e.g. in the third aspect, the vector disclosed herein, e.g. in the fourth aspect, the cell disclosed herein, e.g. in the fifth aspect, or pharmaceutical composition disclosed herein, e.g. in the sixth aspect for use in a method of medical treatment of a disease or prophylaxis of a disease. In some embodiments, the disease is a cancer, an autoimmune disease, or an infection.

[0032] In some embodiments, provided is a method of treating a disease in a subject, comprising administering to the subject the cell disclosed herein, e.g. in the fifth aspect.

[0033] In some embodiments, provided is the recombinant receptor polypeptide disclosed herein, e.g. in the first aspect, the recombinant receptor disclosed herein, e.g. in the second aspect, the nucleic acid disclosed herein, e.g. in the third aspect, the vector disclosed herein, e.g. in the fourth aspect, the cell disclosed herein, e.g. in the fifth aspect for the manufacture of a medicament for the treatment of a disease, in particular wherein the disease is a cancer, an autoimmune disease, or an infection.

[0034] Brief Description of the Figures

[0035] Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying figures.

[0036] Figure 1. Schematic representation of the DOTAM receptor concept. Wild-type (WT) cytokine receptors consist of extracellular cytokine binding domains, which heterodimerize upon engagement of specific cytokine ligands to form activated cytokine receptors. DOTAM receptors feature synthetic ligand binding domains, based on split-antibody variable domains VH and VL, replacing the natural cytokine binding domains. The antibody-derived variable domains ensure specific binding to the small molecule DOTAM. In the absence of DOTAM, the antibody variable domains do not interact and no signaling takes place. Upon engagement with DOTAM, the antibody variable domains assemble leading to receptor heterodimerization and downstream signaling events, e.g., via Janus kinases (JAKs) and signal transducers and activators of transcription (STATs). DOTAM and cytokines are not cross-reactive, making the receptors orthogonal to each other.

[0037] Figure 2. Schematic representations of different D2R formats. Comparison of wild type IL2R molecular architecture with D2Rv1 / D7Rv1 and D2Rv2 / D7Rv2 highlighting the difference between DOTAM receptors with and without the membrane proximal domains (D2 in the case of IL2R or IL7R). P39375-EP

[0038] Figure 3. Overview of gene expression cassettes of DOTAM receptors based on A) the heterodimeric IL2Rpy (D2Rs) and B) the heterodimeric IL7Ra-IL2Ry (D7Rs). C) Overview of gene expression cassettes of different D2R ICD variants.

[0039] Figure 4. Functional analysis of D2R or D7R in transiently transfected HEK Blue™ IL2 reporter cells. A) Schematic representation of HEK-Blue™ IL2 reporter cell function. B, C) Biological activity of D2R and D7R showing dose-dependent expression of phosphorylated pSTAT5-controlled SEAP reporter gene. D2Rv1 and D2Rv2 exhibit similar activity, same trend for D7Rv1 and D7Rv2. IL2 was used as a positive control. Depicted are technical average values from triplicates, error bars indicate standard deviation. D) Surface expression of D2R and D7R variants on HEK-Blue™ cells assessed using DOTAM-FITC or Fc(PGLALA)- DOTAM-Alexa-647, and RFP reporter gene, analyzed by flow cytometry.

[0040] Figure 5. Biological activity assay showing dose-dependent phosphorylation of STAT5 (pSTAT5) in primary T cells. A) Detection of D2Rv3 on engineered primary T cells. The expression of the engineered receptor is assessed indirectly by the expression of a reporter RFP or directly using DOTAM-biotin and Streptavidin- AF647 conjugate. B) Frequency of pSTAT5 positive cells in the RFP+ cell population (D2Rv3 T cells) upon stimulation with DOTAM, and D) in response to stimulation with IL2. C, E) Same data as in Fig. 5B, C but showing median fluorescence intensity (MFI) of pSTAT5.

[0041] Figure 6. Selective expansion and proliferation of primary T cells transduced with D2Rv3 A) Dosedependent specific expansion of a RFP+ D2Rv3+ T cell population after 6 days, when stimulated with DOTAM or IL2. Different starting cell densities of RFP+ cells (% RPF+) were used. IL2 was used as a control. B, C) Dose-dependent proliferation of D2Rv3-transduced T cells after stimulation with DOTAM or IL2. Different starting cell densities of RFP+ cells were used. Proliferation was assessed by flow cytometry by analyzing the dilution of CellTrace™ Violet dye in the dividing cell population.

[0042] Figure 7. Biological activity assay showing STAT5 phosphorylation of primary T cells transduced with DOTAM IL2R intracellular variants upon stimulation with DOTAM or IL2. A) RFP reporter expression of the D2R variants analyzed by flow cytometry. B, D) Frequency of pSTAT5+ cells in the RFP+ cell population in primary T cells transduced with different D2R variants and in mock transduced cells upon stimulation with DOTAM. C, E) Same data as in Fig. 7B, C but showing MFI of pSTAT5.

[0043] Figure 8. Proliferation of primary T cells transduced with different D2R variants after 6 days of incubation with DOTAM or IL2. A, B) Proliferation of RFP+ cells engineered with different D2R variants upon stimulation with DOTAM. Mock-transduced cells were used as a control. Proliferation was assessed by flow cytometry analyzing the decrease of CellTrace™ Violet dye in the dividing population. C, D) Same data as in Fig. 8A,B but showing RFP MFI.

[0044] Figure 9. Schematic representation of D12R constructs and concept. A) Depictions ofthe WT IL12 receptor in comparison to the DOTAM-inducible chimeric D12R receptor. In D12Rs, the extracellular domains of WT IL12Rs are replaced by the DOTAM antibody variable domains VH and VL. B) Overview of lentiviral expression cassettes of D12R constructs. D12Rv1 consists of genes encoding the two D12R chains followed by RFP. Alternatively, the individual D12R chains were also encoded on two separate plasmids D12Rv1 A and D12Rv1 B with downstream GFP / RFP reporter proteins. C) Overview of expression cassettes of a D12R as downstream gene element of a CAR construct. P39375-EP

[0045] Figure 10. Cell surface detection and ligand-induced internalization of D12Rs on engineered primary T cells. A) D12Rs stained in a 1 :1 ratio with DOTAM-FITC either directly or after previous blocking with DOTAM. B) D12Rs stained using DOTAM-biotin and a streptavidin-AF647 conjugate at 37 °C or 4 °C. C) D12R internalization assay showing rapid loss of D12R staining within the first 30 minutes after initial stimulation. D) Detection of cell surface expression of individual D12R chains (D12Rv1A or D12Rv1 B) by adding first, DOTAM-biotin together with the respective variable fragment counterpart as a purified protein, and second adding streptavidin-AF647.

[0046] Figure 11. Detection of D12R signaling in primary T cells via STAT4 phosphorylation and flow cytometry analysis. A) Histograms showing dose-dependent phosphorylation of STAT4 in response to stimulation with DOTAM or IL12 in D12R T cells and WT T cells which served as a negative control. B) Dose-response curves showing differences in EC50 and maximal STAT4 phosphorylation upon D12R signaling. C) Kinetic analysis of phosphorylation of STAT4 in T cells engineered with D12R and WT T cells after stimulation with DOTAM or IL12.

[0047] Figure 12. Functional analysis of T cells engineered with a D12R in response to DOTAM using flow cytometry or HTRF assay. A) Depiction of the D12Rv1 and downstream RFP reporter protein. B) RFP expression in D12Rv1 T cells. C) IL18Ra upregulation in response to stimulation with DOTAM or IL12. D) IL12Rp2 upregulation in response to stimulation with DOTAM. E) IFNy secretion in response to DOTAM or IL12 using the HTRF assay described above.

[0048] Figure 13. Functional analysis of T cells engineered with a P329G adaptor CAR and D12R using flow cytometry and HTRF assay. A) Depiction of the P329G adaptor CAR and D12Rv1 . B) Expression levels of P329G adaptor CAR in CAR-D12R-transduced T cells. C) IL18Ra upregulation in CAR-D12R T cells or CAR T cells in response to stimulation with DOTAM or IL12. D) IL12Rp2 upregulation in CAR-D12R T cells or CAR T cells in response to stimulation with DOTAM or IL12. E) IFNy secretion in CAR-D12R T cells or CAR T cells in response to DOTAM or IL12 using the HTRF assay.

[0049] Figure 14. Killing assay with T cells engineered with CAR-D12R and MKN45 cells using a CEACAM5- specific lgG1 P329G-CAR adaptor, with and without stimulation with DOTAM. A) Expression levels of P329G adaptor CAR in CAR-D12R-engineered T cells. B) Killing data obtained with CAR-D12R T cells at different effector to target cell (MKN45) ratios. Endpoint normalized killing is shown, highlighting the effect of DOTAM or IL12 at low E:T ratios. C,D) Killing kinetics of MKN-45 cell with CAR-D12R T cells at effectortarget ratios of 1 :8 and 1 :16, with or without addition of DOTAM or IL12.

[0050] Figure 15. Killing assay with CAR-D12R T cells and HPAF-II cells using a CEACAM5-specific IgG 1 P329G- CAR adaptor, with and without stimulation with DOTAM. A) Expression levels of P329G adaptor CAR in CAR-D12R-engineered T cells. B) Killing data obtained with CAR-D12R T cells at different effector to target cell (HPAF-II) ratios. Endpoint normalized killing is shown, highlighting the effect of DOTAM or IL12 at low E:T ratios. C,D) Killing kinetics of MKN-45 cell with CAR-D12R T cells at effector-target ratios of 1 :8 and 1 :16, with or without addition of DOTAM or IL12.

[0051] Figure 16. Schematic representation of D18R constructs and concepts. Comparison of wild type IL18R molecular architecture with D18R highlighting the difference between DOTAM receptors with (D18Rv1 , D18Rv2) and without (D18Rv3) the membrane proximal extracellular D3 domains. P39375-EP

[0052] Figure 17. Overview of lentiviral expression cassettes of D18R variants. A) D18Rv1 and D18Rv2 contain the membrane-proximal extracellular D3 domain but slightly differ with regard to their sequence composition. For D18Rv3, the DOTAM antibody variable domains were fused to the 3 extracellular amino acids of IL18Ra and IL18RAP. D18Rv1 -3 all feature a downstream RFP reporter protein. B) Overview of expression cassettes of a CAR-D18R featuring D18R as downstream gene element of a P329G adaptor CAR.

[0053] Figure 18. Functional analysis of D18R variants. A) Expression of downstream RFP analyzed by flow cytometry. B) Dose-dependent IFNy secretion in primary T cells engineered with different D18R variants. D18Rv1 and D18Rv2 show dose-dependent upregulation of IFNy with or without preconditioning with IL12, while D18Rv3 does not.

[0054] Figure 19. Functional analysis of P329G-CAR-D18R T cells. A) Flow cytometry histograms of CAR expression showing P329G-CAR expression in CAR-D18R and CAR-T cells. B) Phosphorylation Nf-KB analyzed by flow cytometry upon stimulation with DOTAM or IL18. C) IFNy secretion of CAR-D18R- and CAR-T cells upon stimulation with DOTAM or IL18.

[0055] Figure 20. Schematic representation of D23R constructs and concepts. A) Depictions of WT IL23R in comparison to the DOTAM inducible D23R. B) Overview of lentiviral expression cassettes of individual D23R genes (D23RA and D23RB) with a downstream RFP or GFP reporter.

[0056] Figure 21. Signaling analysis of D23R-engineered T cells. A) Schematic representation of D23R chains which were encoded on separate expression vectors. B) Analysis of D23R transduction via RFP / GFP expression in engineered T cells and detection of D23R in the GFP+RFP+ population. C) Phosphorylation of STAT3, D) STAT4 and E) STAT5 GFP+RFP+ D23R T cells. Baseline STAT3 and STAT4 phosphorylation stems from the expression of the D23RB chain.

[0057] Figure 22. Functional analysis of D23R-engineered T cells. DOTAM-dependent release of Granzyme B by D23R T cells. IL12 and WT T cells were used as a control.

[0058] Figure 23. Benchmarking different DOTAM binders for the use as extracellular domains of orthogonal IL12 receptors (D12R). A) Schematic representation of the construct design of two variants of the D12R with different VH frameworks. B) Flow cytometry histograms of D12R expression in T cells, assessed by RFR reporter signal and tabularized RFP MFI and frequency of positive cells. C) Dose response relationship of the upregulation of IL18Ra. D) Secretion of IFNg upon DOTAM or IL12 stimulation of D12R engineered and wild type T cells.

[0059] Figure 24. Functional analysis of D2R with different VH framework in transiently transfected HEK Blue™ IL2 reporter cells reporter assay. Biological activity of D2R variants showing dose-dependent expression of pSTAT5-controlled SEAP reporter gene. Depicted are technical average values from triplicates, error bars indicate standard deviation.

[0060] Detailed Description

[0061] The invention presented herein, relates to a novel split-antibody-based orthogonal chimeric cytokine receptor system that can be triggered by a small molecule. Antibody-based chimeric cytokine receptors, P39375-EP based on single-chain variable fragments (scFvs) have been described previously (5). However, our new orthogonal system differentiates itself from these early prototypes by the split-antibody receptor design. The centerpiece of the these new orthogonal receptors are hapten-binding split-antibody variable domains, which can serve as extracellular ligand binding domains of heterodimeric synthetic receptors. Importantly, the split-antibody variable domains only dimerize upon engagement of the small-molecule hapten. The variable domains can be genetically fused to different cytokine receptor chains. Hapten binding will then trigger a cytokine signaling pathway of choice specifically in cells which are genetically engineered to express the orthogonal split-antibody-based receptor. In absence of the small molecule ligand the receptors do not heterdimerize and are thus inactive. The use of split-antibody variable domains as the extracellular dimerization module is novel and comes with the advantage of a smaller gene size, making it easier to use in conjunction with CARs or other gene building blocks on the same transgene. Our new receptor system uses small molecule ligands, generally known as chelators of complex ions which are specifically recognized by cognate monoclonal antibodies. More specifically, we use DOTAM complex-ions as ligands, which are capable of complexing positively charged metal ions. DOTAM exhibits ideal properties for therapeutic application and have not been previously used in the context of small molecule-controlled chimeric receptors. Chelators like DOTAM, conjugated to tumor-targeting small molecules or antibodies, are used as targeted contrast agents for imaging in the clinic. These agents are typically well tolerated apart from radiation damage if certain radioisotopes are used, or if the tumor targeting agents exerts toxicity. For example, Pb-212-loaded, a somatostatin receptor-targeted DOTAM-derivative, termed 212Pb- DOTAMTATE, displayed a very favorable toxicity profile in the clinical setting (6). No known endogenous protein interaction partner has been discovered for DOTAM in humans so far. In our in vitro assays, DOTAM did not exhibit any toxic effect on T cells and, being a small molecule, likely has a low immunogenicity potential. Finally, being complex-ions, metal-DOTAM complexes are unlikely to penetrate cell membranes. Hence, we hypothesized that unmodified, non-radioactive DOTAM complexes are biologically inert and therefore ideal orthogonal ligands.

[0062] Previously, we reported the development of an antibody with extraordinarily high affinity and specificity for Me2+DOTAM complexes (WO2019201959). In the present work, we discovered that the variable domains of the anti-DOTAM antibody can be split into individual VH and VL domains while retaining their ability to bind DOTAM with sub-picomolar affinity. Notably, DOTAM displays no measurable binding to the individual VH or VL domains alone which would be consistent with a 2-to-1 binding mode. Importantly, the VH and VL domains have a very low or no measurable affinity to each other without DOTAM. We therefore postulated that the variable domains of the anti-DOTAM antibody may serve as extracellular domains of an orthogonal receptor system. Formation of the DOTAM-VH-VL complex indeed leads to heterodimerization and downstream signaling. To our surprise our novel DOTAM receptor system displayed broad applicability to different cytokine receptors. We were able to generate proof-of-concept data for pathways for which there are no previous reports in the context of orthogonal receptors. Specifically, we provide in vitro proof- of-concept data demonstrating the functionality of DOTAM receptors based on the signaling domains of lnterleukin-2 (IL2), lnterleukin-7 (IL7), Interleukin-12 (IL12), Interleukin-18 (IL18) and Interleukin-23 (IL23) receptors. Furthermore, we highlight that the DOTAM receptor technology can be used in conjunction with CARs in next-generation cell therapy products and that different cytokine signaling pathways may require slightly different DOTAM receptor designs. In summary, we developed a unique, modular synthetic receptor P39375-EP platform based on split-antibodies which enables the incorporation of orthogonal cytokine signaling into genetically engineered cells. Our system can be controlled by an otherwise inert small molecule ligand in a specific and dose-dependent manner.

[0063] The present invention relates to recombinant receptor polypeptides and recombinant receptors, and cells expressing thereof, capable of activating cellular cytokine pathways by complexing small molecule ligands. For example, the synthetic cytokine receptors consist of variable heavy or variable light antibody domains, which serve as extracellular ligand binding domains with affinity to DOTAM, and are connected to at least the transmembrane and intracellular domain of cytokine receptor chains.

[0064] After cooperative engagement of the antibody-based ligand binding domains to the small molecule, a complex is formed leading to intracellular signaling events akin to the corresponding natural cytokine pathways. The invention also relates to nucleic acid molecules encoding the recombinant (aka. Chimeric) cytokine receptors of the present invention.

[0065] In the following detailed description, the illustrative alternatives described in the detailed description and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0066] The present disclosure is based, inter alia, on recombinant receptor polypeptides which comprise an extracellular portion comprising an antigen binding domain capable of binding to a small molecule, an intracellular portion comprising the intracellular domains of a cytokine receptor, and a transmembrane domain that joins the extracellular portion to the intracellular portion. Immune cells expressing these recombinant receptors may be useful in the context of modulating immune cell activity. In some embodiments, the small molecule can be added exogenously and not be limited to production within the cell.

[0067] The present disclosure also relates to nucleic acid molecules encoding the recombinant cytokine receptors of the present invention, vectors, cells, pharmaceutical compositions, and uses and methods, as described herein, e.g. in the claims.

[0068] I. Definitions

[0069] Terms are used herein as generally used in the art, unless otherwise defined in the following.

[0070] As used herein, the terms “first”, “second” or “third” with respect to antigen binding domains etc., are used for convenience of distinguishing when there is more than one of each type of moiety. Use of these terms is not intended to confer a specific order or orientation of the moiety unless explicitly so stated.

[0071] “DOTAM” has the chemical name:

[0072] 1 ,4,7,10-Tetrakis(carbamoylmethyl)-1 ,4,7,10-tetraazacyclododecane, P39375-EP which is a compound of the following formula:

[0073] The present invention may in certain aspects and embodiments also make use of functional variants or derivatives of DOTAM incorporating a metal ion, e.g. Ca2+. Suitable variants / derivatives of DOTAM have a structure that differs to a certain limited extent from the structure of DOTAM and retain the ability to function (i.e. retains sufficient activity to be used for one or more of the purposes described herein). In such aspects and embodiments, the DOTAM or functional variant / derivative of DOTAM may be one of the active variants disclosed herein (e.g. in Figures 6, 7, 8 and 9) or disclosed in WO2019201959.

[0074] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0075] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab’-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g. scFv and scFab), single-domain antibodies, and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Hollinger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

[0076] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure.

[0077] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprised in the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, P39375-EP and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0078] An "isolated" antibody is one which has been separated from a component of its natural environment. In some aspects, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC, affinity chromatography, size exclusion chromatography) methods. For review of methods for assessment of antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007). In some aspects, the antibodies provided by the present invention are isolated antibodies.

[0079] The term “chimeric” antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0080] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human CDRs and amino acid residues from human FRs. In certain aspects, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. Such variable domains are referred to herein as “humanized variable region”. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. In some aspects, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity. A “humanized form” of an antibody, e.g. of a non-human antibody, refers to an antibody that has undergone humanization.

[0081] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. In certain aspects, a human antibody is derived from a non-human transgenic mammal, for example a mouse, a rat, or a rabbit. In certain aspects, a human antibody is derived from a hybridoma cell line. Antibodies or antibody fragments isolated from human antibody libraries are also considered human antibodies or human antibody fragments herein.

[0082] The term "antigen binding domain" refers to the part of an antibody that comprises the area which binds to and is complementary to part or all of an antigen. An antigen binding domain may be provided by, for example, one or more antibody variable domains (also called antibody variable regions). In preferred aspects, an antigen binding domain comprises an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).

[0083] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and complementarity determining regions (CDRs). See, e.g., Kindt P39375-EP et al., Kuby Immunology, 6thed., W.H. Freeman & Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 750:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0084] Glutamine or glutamate residues at the N-terminus of antibody heavy or light chains may be converted to pyro-glutamate spontaneously (see e.g. Liu et al., Journal of Pharmaceutical Sciences 97, 2426-2447 (2008), Rehder et al., Journal of Chromatography A 1 102, 164-175 (2006), Chelius et al., Anal Chem 78, 2370-2376 (2006)). Hence, variable regions or variable domains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus of an the antibody heavy or light chain, may comprise an N- terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Likewise, antibody heavy chains or light chains disclosed herein which comprise either a glutamine (Q) or a glutamate (E) amino acid residue at the N-terminus, may comprise an N terminal pyro-glutamate (pyroE) residue instead of the N-terminal Q or E residue. Accordingly, for each antibody heavy chain, light chain, or variable domain or region sequence disclosed herein that contains an N-terminal Q or E residue, the corresponding sequence with an N-terminal pyroE residue is also encompassed.

[0085] As used herein in connection with variable region sequences, "Kabat numbering" refers to the numbering system set forth by Kabat et al., Sequences of Proteins of Immunological Interest, 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0086] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), referred to as “numbering according to Kabat” or “Kabat numbering” herein. Specifically the Kabat numbering system (see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)) is used for the light chain constant domain CL of kappa and lambda isotype and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1 , hinge, CH2 and CH3), which is herein further clarified by referring to “numbering according to Kabat EU index” or “Kabat EU index numbering” in this case.

[0087] The term “hypervariable region” or “HVR”, as used herein, refers to each of the regions of an antibody variable domain which are hypervariable in sequence and which determine antigen binding specificity, for example “complementarity determining regions” (“CDRs”). Generally, antibodies comprise six CDRs; three in the VH (HCDR1 , HCDR2, HCDR3), and three in the VL (LCDR1 , LCDR2, LCDR3). Exemplary CDRs herein include:

[0088] (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987));

[0089] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and P39375-EP

[0090] (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H 1 ), 47- 58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)).

[0091] Unless otherwise indicated, the CDRs are determined according to Kabat et al., supra. One of skill in the art will understand that the CDR designations can also be determined according to Chothia, supra, McCallum, supra, or any other scientifically accepted nomenclature system.

[0092] "Framework" or "FR" refers to variable domain residues other than complementarity determining regions (CDRs). The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1 - HCDR1 (LCDR1 )-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0093] Unless otherwise indicated, CDR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0094] An “acceptor human framework” for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework “derived from” a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some aspects, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0095] A “human consensus framework” is a framework which represents the most commonly occurring amino acid residues in a selection of human immunoglobulin VL or VH framework sequences. Generally, the selection of human immunoglobulin VL or VH sequences is from a subgroup of variable domain sequences. Generally, the subgroup of sequences is a subgroup as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91 -3242, Bethesda MD (1991), vols. 1-3.

[0096] The term “immunoglobulin molecule” herein refers to a protein having the structure of a naturally occurring antibody. For example, immunoglobulins of the IgG class are heterotetra meric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable domain (VH), also called a variable heavy domain or a heavy chain variable region, followed by three constant domains (CH1 , CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable domain (VL), also called a variable light domain or a light chain variable region, followed by a constant light (CL) domain, also called a light chain constant region. The heavy chain of an immunoglobulin may be assigned to one of five types, called a (IgA), 5 (IgD), e (IgE), y (IgG), or p (IgM), some of which may be further divided into subtypes, e.g. yi (IgGi), Y2 (lgG2), ys (IgGs), Y4 (lgG4), cn (IgA-i) and c(2 (lgA2). The light chain of an immunoglobulin may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain. An immunoglobulin essentially consists of two Fab molecules and an Fc domain, linked via the immunoglobulin hinge region. P39375-EP

[0097] The “class” of an antibody or immunoglobulin refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, lgG2, IgGs, lgG4, IgAi , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, y, and p, respectively.

[0098] A “Fab molecule” refers to a protein consisting of the VH and CH1 domain of the heavy chain (the “Fab heavy chain”) and the VL and CL domain of the light chain (the “Fab light chain”) of an immunoglobulin.

[0099] By a “crossover” Fab molecule (also termed “Crosstab”) is meant a Fab molecule wherein the variable domains or the constant domains of the Fab heavy and light chain are exchanged (i.e. replaced by each other), i.e. the crossover Fab molecule comprises a peptide chain composed of the light chain variable domain VL and the heavy chain constant domain 1 CH1 (VL-CH1 , in N- to C-terminal direction), and a peptide chain composed of the heavy chain variable domain VH and the light chain constant domain CL (VH-CL, in N- to C-terminal direction). For clarity, in a crossover Fab molecule wherein the variable domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain constant domain 1 CH1 is referred to herein as the “heavy chain” of the (crossover) Fab molecule. Conversely, in a crossover Fab molecule wherein the constant domains of the Fab light chain and the Fab heavy chain are exchanged, the peptide chain comprising the heavy chain variable domain VH is referred to herein as the “heavy chain” of the (crossover) Fab molecule.

[0100] In contrast thereto, by a “conventional” Fab molecule is meant a Fab molecule in its natural format, i.e. comprising a heavy chain composed of the heavy chain variable and constant domains (VH-CH1 , in N- to C-terminal direction), and a light chain composed of the light chain variable and constant domains (VL-CL, in N- to C-terminal direction).

[0101] The term “Fc domain” or “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one aspect, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, antibodies produced by host cells may undergo post-translational cleavage of one or more, particularly one or two, amino acids from the C- terminus of the heavy chain. Therefore, an antibody produced by a host cell by expression of a specific nucleic acid molecule encoding a full-length heavy chain may include the full-length heavy chain, or it may include a cleaved variant of the full-length heavy chain. This may be the case where the final two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, numbering according to Kabat EU index). Therefore, the C-terminal lysine (Lys447), or the C-terminal glycine (Gly446) and lysine (Lys447), of the Fc region may or may not be present. Amino acid sequences of heavy chains including an Fc region (or a subunit of an Fc domain as defined herein) are denoted herein without C-terminal glycine-lysine dipeptide if not indicated otherwise. In one aspect, a heavy chain including an Fc region (subunit) as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine-lysine dipeptide (G446 and K447, numbering according to Kabat EU index). In one aspect, a heavy chain including an Fc region (subunit) as specified herein, comprised in an antibody according to the invention, comprises an additional C-terminal glycine residue (G446, numbering according to Kabat EU index). Unless otherwise specified herein, numbering of amino acid residues in the Fc region or heavy chain P39375-EP constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991 (see also above). A “subunit” of an Fc domain as used herein refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising C-terminal constant regions of an immunoglobulin heavy chain, capable of stable self-association. For example, a subunit of an IgG Fc domain comprises an IgG CH2 and an IgG CH3 constant domain.

[0102] By “fused” is meant that the components (e.g. a Fab molecule and an Fc domain subunit) are linked by peptide bonds, either directly or via one or more peptide linkers.

[0103] The term “multispecific” means that the antibody is able to specifically bind to at least two distinct antigenic determinants. A multispecific antibody can be, for example, a bispecific antibody. Typically, a bispecific antibody comprises two antigen binding sites, each of which is specific for a different antigenic determinant. In certain aspects the multispecific (e.g. bispecific) antibody is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells.

[0104] The term “valent” as used herein denotes the presence of a specified number of antigen binding sites in an antigen binding molecule. As such, the term “monovalent binding to an antigen” denotes the presence of one (and not more than one) antigen binding site specific for the antigen in the antigen binding molecule.

[0105] An “antigen binding site” refers to the site, i.e. one or more amino acid residues, of an antigen binding molecule which provides interaction with the antigen. For example, the antigen binding site of an antibody comprises amino acid residues from the complementarity determining regions (CDRs). A native immunoglobulin molecule typically has two antigen binding sites, a Fab molecule typically has a single antigen binding site.

[0106] As used herein, the term "antigenic determinant" or "antigen" refers to a site (e.g. a contiguous stretch of amino acids or a conformational configuration made up of different regions of non-contiguous amino acids) on a polypeptide macromolecule to which an antigen binding domain binds, forming an antigen binding domain-antigen complex. Useful antigenic determinants can be found, for example, on the surfaces of tumor cells, on the surfaces of virus-infected cells, on the surfaces of other diseased cells, on the surface of immune cells, free in blood serum, and / or in the extracellular matrix (ECM). In a preferred aspect, the antigen is a human protein.

[0107] “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice and rats), unless otherwise indicated. The term encompasses “full-length,” unprocessed CD3 as well as any form of CD3 that results from processing in the cell. The term also encompasses naturally occurring variants of CD3, e.g., splice variants or allelic variants. In one aspect, CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3e). The amino acid sequence of human CD3e is available at UniProt (www.uniprot.org) accession no. P07766 (version 209), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. In another aspect, CD3 is cynomolgus (Macaca fascicularis) CD3, particularly cynomolgus CD3e. The amino acid sequence of cynomolgus CD3e is available at NCBI GenBank no. BAB71849.1. In certain aspects the antibody of the invention binds to an epitope of CD3 that is conserved among the CD3 antigens from P39375-EP different species, particularly human and cynomolgus CD3. In preferred aspects, the antibody binds to human CD3.

[0108] “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by well- established methods known in the art, including those described herein. A preferred method for measuring affinity is Surface Plasmon Resonance (SPR).

[0109] An “affinity matured” antibody refers to an antibody with one or more alterations in one or more complementarity determining regions (CDRs), compared to a parent antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen.

[0110] “Reduced binding”, for example reduced binding to an Fc receptor, refers to a decrease in affinity for the respective interaction, as measured for example by SPR. For clarity, the term includes also reduction of the affinity to zero (or below the detection limit of the analytic method), i.e. complete abolishment of the interaction. Conversely, “increased binding” refers to an increase in binding affinity for the respective interaction.

[0111] “T cell activation” as used herein refers to one or more cellular response of a T lymphocyte, particularly a cytotoxic T lymphocyte, selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers. Suitable assays to measure T cell activation are known in the art and described herein.

[0112] A “modification promoting the association of the first and the second subunit of the Fc domain” is a manipulation of the peptide backbone or the post-translational modifications of an Fc domain subunit that reduces or prevents the association of a polypeptide comprising the Fc domain subunit with an identical polypeptide to form a homodimer. A modification promoting association as used herein preferably includes separate modifications made to each of the two Fc domain subunits desired to associate (i.e. the first and the second subunit of the Fc domain), wherein the modifications are complementary to each other so as to promote association of the two Fc domain subunits. For example, a modification promoting association may alter the structure or charge of one or both of the Fc domain subunits so as to make their association sterically or electrostatically favorable, respectively. Thus, (hetero)dimerization occurs between a polypeptide comprising the first Fc domain subunit and a polypeptide comprising the second Fc domain subunit, which may be non-identical in the sense that further components fused to each of the subunits (e.g. antigen binding domains) are not the same. In some aspects, the modification promoting the association of the first and the second subunit of the Fc domain comprises an amino acid mutation in the Fc domain, specifically an amino acid substitution. In a preferred aspect, the modification promoting the association of the first and the second subunit of the Fc domain comprises a separate amino acid mutation, specifically an amino acid substitution, in each of the two subunits of the Fc domain.

[0113] The term “effector functions” refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and P39375-EP complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B-cell receptor), and B-cell activation.

[0114] An “activating Fc receptor” is an Fc receptor that following engagement by an Fc domain of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Human activating Fc receptors include FcyRllla (CD16a), FcyRI (CD64), FcyRlla (CD32), and FcaRI (CD89).

[0115] Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immune mechanism leading to the lysis of antibody-coated target cells by immune effector cells. The target cells are cells to which antibodies or derivatives thereof comprising an Fc region specifically bind, generally via the protein part that is N-terminal to the Fc region. As used herein, the term “reduced ADCC” is defined as either a reduction in the number of target cells that are lysed in a given time, at a given concentration of antibody in the medium surrounding the target cells, by the mechanism of ADCC defined above, and / or an increase in the concentration of antibody in the medium surrounding the target cells, required to achieve the lysis of a given number of target cells in a given time, by the mechanism of ADCC. The reduction in ADCC is relative to the ADCC mediated by the same antibody produced by the same type of host cells, using the same standard production, purification, formulation and storage methods (which are known to those skilled in the art), but that has not been engineered. For example, the reduction in ADCC mediated by an antibody comprising in its Fc domain an amino acid substitution that reduces ADCC, is relative to the ADCC mediated by the same antibody without this amino acid substitution in the Fc domain. Suitable assays to measure ADCC are well known in the art (see e.g. PCT publication no. WO 2006 / 082515 or PCT publication no. WO 2012 / 130831).

[0116] As used herein, the terms “engineer, engineered, engineering”, are considered to include any manipulation of the peptide backbone or the post-translational modifications of a naturally occurring or recombinant polypeptide or fragment thereof. Engineering includes modifications of the amino acid sequence, of the glycosylation pattern, or of the side chain group of individual amino acids, as well as combinations of these approaches.

[0117] The term “amino acid mutation” as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor, or increased association with another peptide. Amino acid sequence deletions and insertions include amino- and / or carboxy-terminal deletions and insertions of amino acids. Preferred amino acid mutations are amino acid substitutions. For the purpose of altering e.g. the binding characteristics of an Fc region, non-conservative amino acid substitutions, i.e. replacing one amino acid with another amino acid having different structural and / or chemical properties, are particularly preferred. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids (e.g. 4-hydroxyproline, 3- methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, may also be useful. P39375-EP

[0118] Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution from proline at position 329 of the Fc domain to glycine can be indicated as 329G, G329, G329, P329G, or Pro329Gly.

[0119] “Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Alternatively, the percent identity values can be generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087 and is described in WO 2001 / 007611.

[0120] Unless otherwise indicated, for purposes herein, % amino acid sequence identity values are generated using the ggsearch program of the FASTA package version 36.3.8c or later with a BLOSUM50 comparison matrix. The FASTA program package was authored by W. R. Pearson and D. J. Lipman (“Improved Tools for Biological Sequence Analysis”, PNAS 85 (1988) 2444-2448), W. R. Pearson (“Effective protein sequence comparison” Meth. Enzymol. 266 (1996) 227- 258), and Pearson et. al. (Genomics 46 (1997) 24- 36) and is publicly available from www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi can be used to compare the sequences, using the ggsearch (global protein :protein) program and default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure a global, rather than local, alignment is performed. Percent amino acid identity is given in the output alignment header.

[0121] The term “polynucleotide” or “nucleic acid molecule” includes any compound and / or substance that comprises a polymer of nucleotides. Each nucleotide is composed of a base, specifically a purine- or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group. Often, the nucleic acid molecule is described by the sequence of bases, whereby said bases represent the primary structure (linear structure) of a nucleic acid molecule. The sequence of bases is typically represented from 5’ to 3’. Herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and mixed polymers comprising two or more of these molecules. The nucleic acid molecule may be linear or circular. In addition, the term nucleic acid molecule includes both, sense and antisense strands, as well as single stranded and double stranded forms. Moreover, the herein described nucleic acid molecule can contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring P39375-EP nucleotides include modified nucleotide bases with derivatized sugars or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules which are suitable as a vector for direct expression of an antibody of the invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or expression of the encoded molecule so that mRNA can be injected into a subject to generate the antibody in vivo (see e.g., Stadler et al. (2017) Nature Medicine 23:815-817, or EP 2 101 823 B1).

[0122] An “isolated” nucleic acid molecule refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0123] “Isolated polynucleotide (or nucleic acid) encoding an antibody” refers to one or more polynucleotide molecules encoding antibody heavy and light chains (or fragments thereof), including such polynucleotide molecule(s) in a single vector or separate vectors, and such polynucleotide molecule(s) present at one or more locations in a host cell.

[0124] The term “vector”, as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors”.

[0125] The terms "host cell", "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. A host cell is any type of cellular system that can be used to generate the antibodies of the present invention. Host cells include cultured cells, e.g. mammalian cultured cells, such as HEK cells, CHO cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, to name only a few, but also cells comprised within a transgenic animal, transgenic plant or cultured plant or animal tissue. In one aspect, the host cell of the invention is a eukaryotic cell, particularly a mammalian cell. In one aspect, the host cell is not a cell within a human body.

[0126] The term "pharmaceutical composition" or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. P39375-EP

[0127] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition or formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0128] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some aspects, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

[0129] An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). In certain aspects, the individual or subject is a human.

[0130] An “effective amount” of an agent, e.g., a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0131] The terms “administration” and “administering”, as used herein, refer to the delivery of a composition or formulation as disclosed herein by an administration route including, but not limited to, intravenous, intraarterial, intracranial, intramuscular, intraperitoneal, subcutaneous, intramuscular, or combinations thereof. The term includes, but is not limited to, administration by a medical professional and self-administration.

[0132] The terms “cell”, “cell culture”, and “cell line” refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell, cell culture, or cell line, without regard to the number of transfers or passages in culture. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutations (e.g., deliberate or inadvertent mutations) or environmental influences (e.g., methylation or other epigenetic modifications), such that progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the original cell, cell culture, or cell line.

[0133] The term “percent identity”, as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 80% sequence identity, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in P39375-EP length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J Mol Biol 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0134] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0135] All ranges disclosed herein also encompass any and all possible sub-ranges and combinations of subranges thereof. Any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, and so forth. As will also be understood by one skilled in the art all language such as “up to”, “at least”, “greater than”, “less than”, and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.

[0136] IL Therapeutic methods and compositions

[0137] In some aspects, the invention comprises a method for the treatment of a patient in need of therapy, characterized by administering to the patient a therapeutically effective amount of the combination therapy of a DOTAM or a DOTAM derivative as herein described with (cells expressing) a recombinant receptor polypeptide or a recombinant receptor as herein described.

[0138] Further provided is the use of DOTAM or a DOTAM derivative with (cells expressing) a recombinant receptor as herein described for the described combination therapy.

[0139] One preferred embodiment of the invention is the combination therapy of DOTAM or a DOTAM derivative with (cells expressing) a recombinant receptor as herein described for use in the treatment of cancer.

[0140] Thus one embodiment of the invention is DOTAM or a DOTAM derivative for use in the treatment of cancer in combination with (cells expressing) a recombinant receptor as herein described.

[0141] A further embodiment of the invention is (cells expressing) a recombinant receptor as herein described for use in the treatment of cancer in combination with DOTAM or a DOTAM derivative. P39375-EP

[0142] The treatment may be of a solid tumor. The treatment may be of a carcinoma. The cancer may be selected from the group consisting of colorectal cancer, head and neck cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer and gastric cancer. The cancer may be selected from the group consisting of lung cancer, colon cancer, gastric cancer, breast cancer, head and neck cancer, skin cancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer, brain cancer and cancer of the skeletal muscle.

[0143] The term “cancer” as used herein may be, for example, lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers. In one preferred embodiment such cancer is a breast cancer, colorectal cancer, melanoma, head and neck cancer, lung cancer or prostate cancer. In one preferred embodiment such cancer is a breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. In another preferred embodiment such cancer is breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric carcinoma cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myelomas.

[0144] An embodiment of the invention is DOTAM or a DOTAM derivative in combination with cells expressing a recombinant receptor as described herein for use in the treatment of any of the above described cancers or tumors. Another embodiment of the invention is cells expressing a recombinant receptor as described herein in combination with DOTAM or a DOTAM derivative for use in the treatment of any of the above described cancers or tumors.

[0145] The invention comprises the combination therapy with DOTAM or a DOTAM derivative with cells expressing a recombinant receptor as described herein for the treatment of cancer.

[0146] The invention comprises the combination therapy with DOTAM or a DOTAM derivative with cells expressing a recombinant receptor as described herein for the prevention or treatment of metastasis.

[0147] The invention comprises the combination therapy of DOTAM or a DOTAM derivative with cells expressing a recombinant receptor as described herein for use in stimulating an immune response or function, such as T cell activity. P39375-EP

[0148] The invention comprises a method for the treatment of cancer in a patient in need thereof, characterized by administering to the patient DOTAM or a DOTAM derivative and cells expressing a recombinant receptor as described herein.

[0149] The invention comprises a method for the prevention or treatment of metastasis in a patient in need thereof, characterized by administering to the patient DOTAM or a DOTAM derivative and cells expressing a recombinant receptor as described herein.

[0150] The invention comprises a method for stimulating an immune response or function, such as T cell activity, in a patient in need thereof, characterized by administering to the patient DOTAM or a DOTAM derivative and cells expressing a recombinant receptor as described herein.

[0151] The invention comprises DOTAM or a DOTAM derivative for use in the treatment of cancer in combination with cells expressing a recombinant receptor as described herein, or alternatively for the manufacture of a medicament for the treatment of cancer in combination with cells expressing a recombinant receptor as described herein.

[0152] The invention comprises DOTAM or a DOTAM derivative for use in the prevention or treatment of metastasis in combination with cells expressing a recombinant receptor as described herein, or alternatively for the manufacture of a medicament for the prevention or treatment of metastasis in combination with cells expressing a recombinant receptor as described herein.

[0153] The invention comprises DOTAM or a DOTAM derivative for use in stimulating an immune response or function, such as T cell activity, in combination with cells expressing a recombinant receptor as described herein, or alternatively for the manufacture of a medicament for use in stimulating an immune response or function, such as T cell activity, in combination with cells expressing a recombinant receptor as described herein.

[0154] The invention comprises cells expressing a recombinant receptor as described herein for use in the treatment of cancer in combination with DOTAM or a DOTAM derivative, or alternatively for the manufacture of a medicament for the treatment of cancer in combination with DOTAM or a DOTAM derivative.

[0155] In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing DOTAM or a DOTAM derivative formulated together with a pharmaceutically acceptable carrier.

[0156] In another aspect, the present invention provides a composition, e.g. a pharmaceutical composition, containing cells expressing a recombinant receptor as described herein formulated together with a pharmaceutically acceptable carrier.

[0157] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption / resorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.

[0158] A composition of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. P39375-EP

[0159] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. In addition to water, the carrier can be, for example, an isotonic buffered saline solution.

[0160] Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0161] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (effective amount). The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0162] In one aspect the invention provides a kit intended for the treatment of a disease, comprising in the same or in separate containers (a) DOTAM or a DOTAM derivative, and (b) cells expressing a recombinant receptor as described, and optionally further comprising (c) a package insert comprising printed instructions directing the use of the combined treatment as a method for treating the disease. Moreover, the kit may comprise (a) a first container with a composition contained therein, wherein the composition comprises cells expressing a recombinant receptor as described herein; (b) a second container with a composition contained therein, wherein the composition comprises DOTAM or a DOTAM derivative; and optionally (c) a third container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The kit in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the kit may further comprise a third (or fourth) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0163] In one aspect the invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising DOTAM or a DOTAM derivative, and (b) a package insert comprising instructions directing the use of the DOTAM or a DOTAM derivative in a combination therapy with cells expressing a recombinant receptor as described herein as a method for treating the disease.

[0164] In another aspect the invention provides a kit intended for the treatment of a disease, comprising (a) a container comprising cells expressing a recombinant receptor as described herein, and (b) a package insert comprising instructions directing the use of the (cells expressing) the MAB polypeptide in a combination therapy with DOTAM or a DOTAM derivative as a method for treating the disease. P39375-EP

[0165] In a further aspect the invention provides a medicament intended for the treatment of a disease, comprising DOTAM or a DOTAM derivative, wherein said medicament is for use in a combination therapy with cells expressing a recombinant receptor as described herein and optionally comprises a package insert comprising printed instructions directing the use of the combined treatment as a method for treating the disease.

[0166] The term “a method of treating” or its equivalent, when applied to, for example, cancer refers to a procedure or course of action that is designed to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of a cancer. “A method of treating” cancer or another proliferative disorder does not necessarily mean that the cancer cells or other disorder will, in fact, be eliminated, that the number of cells or disorder will, in fact, be reduced, or that the symptoms of a cancer or other disorder will, in fact, be alleviated. Often, a method of treating cancer will be performed even with a low likelihood of success, but which, given the medical history and estimated survival expectancy of a patient, is nevertheless deemed to induce an overall beneficial course of action.

[0167] The terms “administered in combination with” or “co-administration”, “co-administering”, “combination therapy" or “combination treatment” refer to the administration of the DOTAM or a DOTAM derivative and cells expressing the recombinant receptor e.g. as separate formulations / applications (or as one single formulation / application). The co-administration can be simultaneous or sequential in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities. Said active agents are co-administered either simultaneously or sequentially (e.g. intravenous (i.v.)) through a continuous infusion. When both therapeutic agents are co-administered sequentially the dose is administered either on the same day in two separate administrations, or one of the agents is administered on day 1 and the second is co-administered on day 2 to day 7, preferably on day 2 to 4. Thus in one embodiment the term “sequentially” means within 7 days after the dose of the first component, preferably within 4 days after the dose of the first component; and the term “simultaneously” means at the same time. The term “co-administration” with respect to the maintenance doses of DOTAM or a DOTAM derivative and / or cells expressing a recombinant receptor as herein described means that the maintenance doses can be either co-administered simultaneously, if the treatment cycle is appropriate for all drugs, e.g. every week. Or the maintenance doses are co-administered sequentially, for example, doses of DOTAM or a DOTAM derivative and cells expressing a recombinant receptor as herein described are given on alternate weeks.

[0168] It is self-evident that the recombinant DOTAM or a DOTAM derivative and cells expressing the recombinant receptor as herein described are administered to the patient in a “therapeutically effective amount” (or simply “effective amount”) which is the amount of the respective compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.

[0169] The amount of co-administration and the timing of co-administration will depend on the type (species, gender, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. Said DOTAM or a DOTAM derivative and / or cells expressing a recombinant receptor as herein described are suitably co-administered to the patient at one time or over a series of treatments e.g. on the same day or on the day after or at weekly intervals. P39375-EP

[0170] In addition to the recombinant DOTAM or a DOTAM derivative in combination with cells expressing a recombinant receptor as herein described, also a chemotherapeutic agent can be administered.

[0171] In one embodiment such additional chemotherapeutic agents, include, but are not limited to, anti- neoplastic agents including alkylating agents including: nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); Temodal ™ (temozolamide), ethylenimines / methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6- thioguamne, azathioprine, T-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; pipodophylotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycin C, and actinomycin; enzymes such as L- asparaginase; biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; miscellaneous agents including platinum coordination complexes such as oxaliplatin, cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o, p-DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; Gemzar ™ (gemcitabine), progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide. Therapies targeting epigenetic mechanism including, but not limited to, histone deacetylase inhibitors, demethylating agents (e.g., Vidaza) and release of transcriptional repression (ATRA) therapies can also be combined with the antigen binding proteins. In one embodiment the chemotherapeutic agent is selected from the group consisting of taxanes (like e.g. paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxel (e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar), and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine, and vinblastine. In one embodiment the chemotherapeutic agent is selected from the group consisting of taxanes (like e.g. taxol (paclitaxel), docetaxel (Taxotere), modified paclitaxel (e.g. Abraxane and Opaxio). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), leucovorin, irinotecan, or oxaliplatin. In one embodiment the chemotherapeutic agent is 5-fluorouracil, leucovorin and irinotecan (FOLFIRI). In one embodiment the chemotherapeutic agent is 5-fluorouracil, and oxaliplatin (FOLFOX).

[0172] Specific examples of combination therapies with additional chemotherapeutic agents include, for instance, therapies taxanes (e.g., docetaxel or paclitaxel) or a modified paclitaxel (e.g., Abraxane or P39375-EP

[0173] Opaxio), doxorubicin), capecitabine and / or bevacizumab (Avastin) for the treatment of breast cancer; therapies with carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin) for ovarian cancer, the therapies with a multi-kinase inhibitor, MKI, (Sutent, Nexavar, or 706) and / or doxorubicin for treatment of kidney cancer; therapies with oxaliplatin, cisplatin and / or radiation for the treatment of squamous cell carcinoma; therapies with taxol and / or carboplatin for the treatment of lung cancer.

[0174] Therefore, in one embodiment the additional chemotherapeutic agent is selected from the group of taxanes (docetaxel or paclitaxel or a modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine and / or bevacizumab for the treatment of breast cancer.

[0175] In one embodiment, the combination therapy of DOTAM or a DOTAM derivative with cells expressing a recombinant receptor as herein described is one in which no chemotherapeutic agents are administered.

[0176] The invention comprises also a method for the treatment of a patient suffering from such disease as described herein.

[0177] The invention further provides a method for the manufacture of a pharmaceutical composition comprising an effective amount of DOTAM or a DOTAM derivative according to the invention as described herein and cells expressing a recombinant receptor as described herein together with a pharmaceutically acceptable carrier and the use of the recombinant DOTAM or a DOTAM derivative and cells expressing a recombinant receptor as described herein for such a method.

[0178] The invention further provides the use of DOTAM or a DOTAM derivative according to the invention as described herein and cells expressing a recombinant receptor as described herein in an effective amount for the manufacture of a pharmaceutical agent, preferably together with a pharmaceutically acceptable carrier, for the treatment of a patient suffering from cancer.

[0179] Each of the components of the recombinant receptor (polypeptides) is explained in more detail herein below.

[0180] Extracellular portion comprising an antigen binding domain capable of binding to a small molecule

[0181] Herein provided are recombinant receptors comprising recombinant receptor polypeptides. In some aspects, the recombinant receptor polypeptides as herein disclosed comprise an extracellular portion comprising an antigen binding domain.

[0182] In some aspects, the antigen binding domain comprises an antigen-binding moiety.

[0183] As used herein, an ‘antigen-binding moiety’ refers to a moiety that binds to a given target antigen.

[0184] In preferred aspects, an antigen-binding moiety according to the present disclosure comprises, or consists of a variable fragment (Fv) moiety.

[0185] As used herein, ‘specific binding’ refers to binding which is selective for the target antigen (e.g. DOTAM or a DOTAM derivative), and which can be discriminated from non-specific binding to non-target antigen. An P39375-EP antigen-binding moiety that specifically binds to a given target antigen preferably binds the target antigen with greater affinity, and / or with greater duration than it binds to other, non-target antigens.

[0186] The ability of a given moiety to bind specifically to a given target antigen can be determined by analysis according to methods known in the art, such as by ELISA, Surface Plasmon Resonance (SPR; see e.g. Hearty et al., Methods Mol Biol (2012) 907:411 -442), Bio-Layer Interferometry (BLI; see e.g. Lad et al., (2015) J Biomol Screen 20(4): 498-507), flow cytometry, or by a radiolabeled antigen-binding assay (RIA) enzyme-linked immunosorbent assay. Through such analysis binding to a given target antigen can be measured and quantified. In some embodiments, the level of binding may be the response detected in a given assay.

[0187] An antigen-binding moiety that ‘does not bind’ or that ‘displays substantially no binding’ to a given antigen displays a level of binding to the given antigen which is similar to the level of binding to an antigen that the antigen-binding moiety is known not to bind, or known to not to bind specifically, e.g. a non-target antigen. In some embodiments, the level of binding of an antigen-binding moiety that does not bind, or that displays substantially no binding, to a given antigen is > 0.5 times and < 2 times, e.g. one of > 0.75 times and < 1 .5 times, > 0.8 times and < 1 .4 times, > 0.85 times and < 1 .3 times, > 0.9 times and < 1 .2 times, > 0.95 times and < 1.1 times the level of binding displayed by the antigen-binding moiety to an antigen that the antigenbinding moiety is known not to bind, or known to not to bind specifically, e.g. a non-target antigen.

[0188] An antigen-binding moiety according to the present disclosure may be, or may comprise, an antigen-binding peptide / polypeptide, or an antigen-binding peptide / polypeptide complex. An antigen-binding moiety may comprise more than one peptide / polypeptide that together form an antigen-binding domain.

[0189] In some embodiments, the antigen-binding moiety of the present disclosure comprises an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region of an antibody capable of binding to DOTAM or a DOTAM derivative. In some embodiments, the antigen-binding moiety comprises, or consists of, an Fv moiety formed by the VH region and a VL region of an antibody capable of binding to DOTAM or a DOTAM derivative.

[0190] Antigen-binding moieties of the present disclosure generally comprise six complementarity-determining regions CDRs; three in the heavy chain variable (VH) region: HC-CDR1 , HC-CDR2 and HC-CDR3, and three in the light chain variable (VL) region: LC-CDR1 , LC-CDR2, and LC-CDR3. The six CDRs together define the paratope of the antigen-binding moiety, which is the part of the moiety that binds to the target antigen.

[0191] The VH region and VL region comprise framework regions (FRs) either side of each CDR, which provide a scaffold for the CDRs. From N-terminus to C-terminus, VH regions comprise the following structure: N term- [HC-FR1]-[HC-CDR1]-[HC-FR2]-[HC-CDR2]-[HC-FR3]-[HC-CDR3]-[HC-FR4]-C term; and VL regions comprise the following structure: N term-[LC-FR1]-[LC-CDR1]-[LC-FR2]-[LC-CDR2]-[LC-FR3]-[LC-CDR3]- [LC-FR4]-C term.

[0192] There are several different conventions for defining antibody CDRs and FRs, such as (i) the Kabat system, described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991); (ii) the Chothia system, described in Chothia et al., J. Mol. Biol. 196:901-917 (1987); and (iii) the international IMGT (ImMunoGeneTics) information system P39375-EP

[0193] (LeFranc et al., Nucleic Acids Res. (2015) 43 (Database issue):D413-22), which uses the IMGT V-DOMAIN numbering rules as described in Lefranc et al., Dev. Comp. Immunol. (2003) 27:55-77.

[0194] The CDRs and FRs of the VH regions and VL regions of the antigen-binding moieties described herein are defined according to the Kabat system.

[0195] In some embodiments, the antigen-binding moiety comprises the CDRs of an antigen-binding moiety that binds to DOTAM or a DOTAM derivative. In some embodiments, the antigen-binding moiety comprises the FRs of an antigen-binding moiety that binds to DOTAM or a DOTAM derivative. In some embodiments, the antigen-binding moiety comprises the CDRs and the FRs of an antigen-binding moiety that binds to DOTAM or a DOTAM derivative. That is, in some embodiments, the antigen-binding moiety comprises the VH region and the VL region of an antigen-binding moiety that binds to DOTAM or a DOTAM derivative.

[0196] In some embodiments, the antigen-binding moiety comprises the CDRs, FRs and / or the VH and / or VL regions of an antigen-binding molecule described herein that binds to DOTAM or a DOTAM derivative, or comprises CDRs, FRs and / or VH and / or VL regions which are derived from those of an antigen-binding molecule described herein that binds to DOTAM or a DOTAM derivative.

[0197] In some embodiments, the antigen-binding moiety comprises a VH region incorporating the following CDRs:

[0198] HC-CDR1 having the amino acid sequence of SEQ ID NO:106

[0199] HC-CDR2 having the amino acid sequence of SEQ ID NO:107

[0200] HC-CDR3 having the amino acid sequence of SEQ ID NO:108, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0201] In some embodiments, the antigen-binding moiety comprises a VH region incorporating the following CDRs:

[0202] HC-CDR1 having the amino acid sequence of SEQ ID NQ:106

[0203] HC-CDR2 having the amino acid sequence of SEQ ID NO:164

[0204] HC-CDR3 having the amino acid sequence of SEQ ID NQ:108, or a variant thereof in which 1 or 2 or 3 amino acids in HC-CDR1 , and / or in which 1 or 2 or 3 amino acids in HC-CDR2, and / or in which 1 or 2 or 3 amino acids in HC-CDR3 are substituted with another amino acid.

[0205] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:2.

[0206] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO: 157. P39375-EP

[0207] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:160.

[0208] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:161.

[0209] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:162.

[0210] In some embodiments, the antigen-binding moiety comprises a VH region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:163.

[0211] In some embodiments, the antigen-binding moiety comprises a VL region according to (11) below: (11) a VL region incorporating the following CDRs:

[0212] LC-CDR1 having the amino acid sequence of SEQ ID NQ:109 LC-CDR2 having the amino acid sequence of SEQ ID NO:110 LC-CDR3 having the amino acid sequence of SEQ ID NO:111 , or a variant thereof in which 1 or 2 or 3 amino acids in LC-CDR1 , and / or in which 1 or 2 or 3 amino acids in LC-CDR2, and / or in which 1 or 2 or 3 amino acids in LC-CDR3 are substituted with another amino acid.

[0213] In some embodiments, the antigen-binding moiety comprises a VL region comprising an amino acid sequence having at least 70% sequence identity, more preferably one of at least >75%, >80%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100%, sequence identity, to the amino acid sequence of SEQ ID NO:8.

[0214] In some embodiments, a component of an antigen-binding moiety comprises or consists of an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NQ:10, 18 or 20. In some embodiments, a component of an antigen-binding moiety comprises or consists of an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:23. P39375-EP

[0215] It will be appreciated that where components of an antigen-binding moiety are provided in aspects and embodiments of the present disclosure, it is intended that the components provided are complementary, and capable of associating to form the (complete, functional) antigen-binding moiety.

[0216] Substitutions of amino acids in accordance with the present disclosure may be biochemically conservative. In some embodiments, where an amino acid to be substituted is provided in one of rows 1 to 5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid provided in the same row: By way of illustration, in some embodiments wherein substitution is of a Met residue, the replacement amino acid may be selected from Ala, Vai, Leu, lie, Trp, Tyr, Phe and Norleucine.

[0217] In some embodiments, a replacement amino acid in a substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, a replacement amino acid in a substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces: P39375-EP

[0218] That is, in some embodiments, a nonpolar amino acid is substituted with another, non-identical nonpolar amino acid. In some embodiments, a polar amino acid is substituted with another, non-identical polar amino acid. In some embodiments, an acidic polar amino acid is substituted with another, non-identical acidic polar amino acid. In some embodiments, a basic polar amino acid is substituted with another, non- identical basic polar amino acid. In some embodiments, a neutral amino acid is substituted with another, non-identical neutral amino acid. In some embodiments, a positive amino acid is substituted with another, non-identical positive amino acid. In some embodiments, a negative amino acid is substituted with another, non-identical negative amino acid.

[0219] In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g. target antigen binding) of the antigen-binding moiety comprising the substitution, as compared to the equivalent unsubstituted molecule.

[0220] In some embodiments, an antigen-binding moiety of the present disclosure comprises a VH as described herein. In some embodiments, an antigen-binding moiety comprises a VL as described herein. In some embodiments, an antigen-binding moiety comprises one or more antibody heavy chain constant regions (CH). In some embodiments, an antigen-binding moiety comprises one or more antibody light chain constant regions (CL). In some embodiments, an antigen-binding moiety comprises a CH1 , CH2 region and / or a CH3 region of an immunoglobulin (Ig). In some embodiments, an antigen-binding moiety comprises a linker sequence as described herein.

[0221] In some embodiments, the antigen-binding moiety of the present disclosure comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-CDR1 according to SEQ ID NO:106, HC-CDR2 according to SEQ ID NO:107, and HC-CDR3 according to SEQ ID NO:108, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NQ:109, LC-CDR2 according to SEQ ID NO:110, and LC- CDR3 according to SEQ ID NO:111 .

[0222] In some embodiments, the antigen-binding moiety of the present disclosure comprises a polypeptide or polypeptides comprising: (i) a VH region comprising HC-CDR1 according to SEQ ID NQ:106, HC-CDR2 according to SEQ ID NO:164, and HC-CDR3 according to SEQ ID NQ:108, and (ii) a VL region comprising LC-CDR1 according to SEQ ID NQ:109, LC-CDR2 according to SEQ ID NO:110, and LC- CDR3 according to SEQ ID NO:111 . P39375-EP

[0223] In some embodiments, an antigen-binding moiety of the present disclosure comprises an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:2. In some embodiments, an antigen-binding moiety of the present disclosure comprises an amino acid having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:8.

[0224] Intracellular portion comprising the intracellular domains of a cytokine receptor

[0225] In some aspects, the portion comprising the intracellular domains of a cytokine receptor comprises the intracellular domain of a wild-type cytokine receptor or a fragment thereof that retains signaling activity. The signaling activity can be determined; e.g., by enhanced cytokine release, as measured by ELISA (IFNy, TNFa, etc.), enhanced proliferative activity (as measured by enhanced cell numbers), or enhanced lytic activity as measured by LDH release assays.

[0226] In preferred aspect, the intracellular domains of a cytokine receptor comprises the intracellular domain of a wild-type cytokine receptor is selected from the group consisting of the IL2 receptor, the IL7 receptor, the IL12 receptor, the IL18 receptor, and the IL23 receptor.

[0227] In some embodiments, the intracellular domain of a cytokine receptor comprises the intracellular domain of a human cytokine receptor polypeptide selected from the group consisting SEQ ID NO: 146-156.

[0228] In some aspects, the cytokine receptor is a IL2 receptor (IL2R) or a fragment thereof that is capable of IL2 signaling.

[0229] In a particular aspect, the recombinant receptor comprises from the N-terminus to the C-terminus: Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a IL2 receptor chain (or a fragment thereof that is capable of IL2 signaling) - optional linker - optional marker.

[0230] In some aspects, the intracellular domain comprises an amino acid sequence selected from SEQ ID Nos: 30, 34, 35, 36, 37, 38 and 39, or an amino acid sequence having at least 80% sequence identity thereto.

[0231] In some aspects, the recombinant receptor comprises i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence selected from SEQ ID Nos: 30, 36, 37, 38 and 39, or an amino acid sequence having at least 80% sequence identity thereto; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence selected from SEQ ID Nos: 34 and 35, or an amino acid sequence having at least 80% sequence identity thereto.

[0232] In some aspects, the cytokine receptor is a IL7 receptor (IL7R) or a fragment thereof that is capable of IL7 signaling.

[0233] In a particular aspect, the recombinant receptor comprises from the N-terminus to the C-terminus: Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker P39375-EP

[0234] - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a IL7 receptor chain (or a fragment thereof that is capable of IL7 signaling) - optional linker - optional marker.

[0235] In some aspects, the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 34 and SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity thereto.

[0236] In some aspects, the recombinant receptor comprises i) a first recombinant receptor polypeptide comprising an intracellular domain comprising the amino acid sequence of SEQ ID Nos: 34, or an amino acid sequence having at least 80% sequence identity thereto; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity thereto

[0237] In some aspects, the cytokine receptor is an IL12 receptor (IL12R) or a fragment thereof that is capable of IL12 signaling.

[0238] In a particular aspect, the recombinant receptor comprises from the N-terminus to the C-terminus: Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker

[0239] - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a IL12 receptor chain (or a fragment thereof that is capable of IL18 signaling) - optional linker - optional marker.

[0240] In some aspects, the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 11 , or an amino acid sequence having at least 80% sequence identity thereto.

[0241] In some aspects, the recombinant receptor comprises i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity thereto; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 1 1 , or an amino acid sequence having at least 80% sequence identity thereto.

[0242] In some aspects, the cytokine receptor is a IL18 receptor (IL18R) or a fragment thereof that is capable of IL18 signaling.

[0243] In a particular aspect, the recombinant receptor comprises from the N-terminus to the C-terminus: Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker

[0244] - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a IL18 receptor chain (or a fragment thereof that is capable of IL18 signaling) - optional linker - optional marker.

[0245] In some aspects, the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 46 and SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity thereto.

[0246] In some aspects, the recombinant receptor comprises i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 46, or an amino acid sequence having at least 80% sequence identity thereto; and ii) a second recombinant receptor polypeptide P39375-EP comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity thereto.

[0247] In some aspects, the cytokine receptor is an IL23 receptor (IL23R) or a fragment thereof that is capable of IL23 signaling.

[0248] In a particular aspect, the recombinant receptor comprises from the N-terminus to the C-terminus: Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a IL23 receptor chain (or a fragment thereof that is capable of IL18 signaling) - optional linker - optional marker.

[0249] In some aspects, the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 52, or an amino acid sequence having at least 80% sequence identity thereto.

[0250] In some aspects, the recombinant receptor comprises i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity thereto; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 52, or an amino acid sequence having at least 80% sequence identity thereto.

[0251] Transmembrane domain that joins the extracellular portion to the intracellular portion

[0252] In some aspects, the transmembrane domain that joins the extracellular portion to the intracellular portion comprises part of a murine / mouse or preferably of a human transmembrane domain. Any protein having a transmembrane domain, as provided among others by the CD nomenclature, may be used as a transmembrane domain that joins the extracellular portion to the intracellular portion.

[0253] Human sequences might be beneficial, for example because (parts) of the transmembrane domain might be accessible from the extracellular space and hence to the immune system of a patient. In a preferred embodiment, the transmembrane domain comprises a human sequence.

[0254] In some embodiments, the transmembrane domain comprises the transmembrane domain of a human cytokine receptor polypeptide selected from the group consisting SEQ ID NO: 146-156.

[0255] In particular aspects, the transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:10, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 48, and SEQ ID NO:51 , or an amino acid sequence having at least 80% sequence identity thereto (and retaining the capability to anchor the recombinant receptor to the membrane).

[0256] Linkers

[0257] The polypeptides of the present disclosure (e.g. recombinant receptor polypeptides) may additionally comprise further amino acids or sequences of amino acids. P39375-EP

[0258] The polypeptides may comprise one or more linker sequences between sequences of amino acids. By way of example, a linker sequence may be provided between different domains of a recombinant receptor polypeptide (e.g. the extracellular portion and the transmembrane domain, or between the transmembrane domain and the intracellular portion).

[0259] Linker sequences are known to the skilled person, and are described, for example in Chen et al., Adv Drug Deliv Rev (2013) 65(10): 1357-1369, which is hereby incorporated by reference in its entirety. In some embodiments, a linker sequence may be a flexible linker sequence. Flexible linker sequences allow for relative movement of the amino acid sequences which are linked by the linker sequence. Flexible linkers are known to the skilled person, and several are identified in Chen etal., Adv Drug Deliv Rev (2013) 65(10): 1357-1369. Flexible linker sequences often comprise high proportions of glycine and / or serine residues.

[0260] In some embodiments, a linker sequence comprises at least one glycine residue and / or at least one serine residue. In some embodiments, the linker sequence comprises or consists of glycine and serine residues. In some embodiments, the linker sequence has the structure: (GxS)n or (GxS)nGm; wherein G = glycine, S = serine, x = 3 or 4, n = 2, 3, 4, 5 or 6, and m = 0, 1 , 2 or 3. In some embodiments, the linker sequence comprises one or more (e.g. 1 , 2, 3, 4, 5 or 6) copies (e.g. in tandem) of the sequence motif G4S. In some embodiments, a linker sequence comprises or consists of (648)3 or (648)4. In some embodiments, the linker sequence has a length of 1 -2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-30 amino acids. In some embodiments, the linker sequence is one or more repetitions of GGSG.

[0261] In some embodiments, the linker sequence comprises one or more copies of an amino acid sequence according to SEQ ID NO:3. In some embodiments, the linker sequence comprises at least 1 , 2, 3 or 4 copies of an amino acid sequence according to SEQ ID NO:20.

[0262] In some embodiments, the linker sequence comprises, or consists of, an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:18.

[0263] In some embodiments, the linker sequence comprises a cleavage site, e.g. a cleavage site as described hereinbelow.

[0264] Additional elements

[0265] The polypeptides of the present disclosure may comprise amino acid sequence(s) to facilitate expression, folding, trafficking, processing, purification or detection thereof. For example, recombinant receptor polypeptides of the present disclosure may additionally comprise a sequence of amino acids forming a detectable moiety, e.g. as described hereinbelow.

[0266] The polypeptides may additionally comprise a signal peptide (also known as a leader sequence or signal sequence). Signal peptides normally consist of a sequence of 5-30 hydrophobic amino acids, which form a single alpha helix. Secreted proteins and proteins expressed at the cell surface often comprise signal peptides. Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt and Ensembl, and / or can be identified / predicted e.g. using amino acid sequence analysis tools P39375-EP such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0267] The signal peptide may be present at the N-terminus of the recombinant receptor polypeptide, and may be present in the newly-synthesised polypeptide. The signal peptide provides for efficient trafficking of the recombinant receptor polypeptide. Signal peptides are often removed by cleavage, and thus are not comprised in the mature recombinant receptor polypeptide.

[0268] Signal peptides are known for many proteins, and are recorded in databases such as GenBank, UniProt, Swiss-Prot, TrEMBL, Protein Information Resource, Protein Data Bank, Ensembl, and InterPro, and / or can be identified / predicted e.g. using amino acid sequence analysis tools such as SignalP (Petersen et al., 2011 Nature Methods 8: 785-786) or Signal-BLAST (Frank and Sippl, 2008 Bioinformatics 24: 2172-2176).

[0269] In some embodiments, the signal peptide comprises, or consists of, an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:1 , SEQ ID NO:16, or SEQ ID NO:26.

[0270] In some embodiments, recombinant receptor polypeptides of the present disclosure comprise a detectable moiety, a.k.a. a marker. In some embodiments, a detectable moiety is provided at the N-terminus and / or C-terminus of the polypeptide.

[0271] In some embodiments, a detectable moiety is a fluorescent label, phosphorescent label, luminescent label, immuno-detectable label (e.g. an epitope tag), radiolabel, chemical, nucleic acid or enzymatic label. The recombinant receptor polypeptides may be covalently or non-covalently labelled with the detectable moiety.

[0272] Fluorescent labels include e.g. fluorescein, rhodamine, allophycocyanin, eosine and NDB, green fluorescent protein (GFP), enhanced GFP (eGFP), chelates of rare earths such as europium (Eu), terbium (Tb) and samarium (Sm), tetramethyl rhodamine, Texas Red, 4-methyl umbelliferone, 7-amino-4-methyl coumarin, Cy3, and Cy5. Radiolabels include radioisotopes such as Hydrogen3, Sulfur35, Carbon14, Phosphorus32, Iodine123, Iodine125, Iodine126, Iodine131, Iodine133, Bromine77, Technetium99m, Indium111, lndium113m, Gallium67, Gallium68, Ruthenium95, Ruthenium97, Ruthenium103, Ruthenium105, Mercury207, Mercury203, Rhenium99m, Rhenium101, Rhenium105, Scandium47, Tellurium121m, Tellurium122m, Tellurium125m, Thulium165, Thuliuml167, Thulium168, Copper67, Fluorine18, Yttrium90, Palladium100, Bismuth217and Antimony211. Luminescent labels include as radioluminescent, chemiluminescent (e.g. acridinium ester, luminol, isoluminol) and bioluminescent labels. Immuno-detectable labels include haptens, peptides / polypeptides, antibodies, receptors and ligands such as biotin, avidin, streptavidin or digoxigenin. Nucleic acid labels include aptamers.

[0273] In some embodiments, the recombinant receptor polypeptide comprises an epitope tag, e.g. a His, (e.g. 6XHis), FLAG, c-Myc, StrepTag, haemagglutinin, E, calmodulin-binding protein (CBP), glutathione-s- P39375-EP transferase (GST), maltose-binding protein (MBP), thioredoxin, S-peptide, T7 peptide, SH2 domain, avidin, streptavidin, and haptens (e.g. biotin, digoxigenin, dinitrophenol), optionally at the N- or C- terminus of the recombinant receptor polypeptide.

[0274] In some embodiments, the recombinant receptor polypeptide comprises a moiety having a detectable activity, e.g. an enzymatic moiety. Enzymatic moieties include e.g. luciferases, glucose oxidases, galactosidases (e.g. beta-galactosidase), glucorinidases, phosphatases (e.g. alkaline phosphatase), peroxidases (e.g. horseradish peroxidase) and cholinesterases.

[0275] In some embodiments, a polypeptide of the present disclosure comprises a fluorescent label. In some embodiments, the polypeptide comprises an eGFP moiety. In some embodiments, the polypeptide comprises an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:13 or SEQ ID NO: 55.

[0276] Recombinant receptor polypeptides of the present disclosure may also comprise one or more cleavage sites. A cleavage site refers to a sequence of amino acids that acts as a substrate for an enzyme capable of cleaving peptide bonds.

[0277] Many such cleavage sites are known to, and can be employed by, the person skilled in the art of molecular biology. In some embodiments, the cleavage sequence comprises an autocleavage site. Autocleavage sites include the 2A cleavage sequence from Picornavirus ‘NPGP’, which is cleaved at ’G / P’. Further autocleavage sites are described e.g. in Kim et al., PLoS ONE (201 1) 6: e18556 (hereby incorporated by reference in its entirety), and include e.g. T2A, P2A, E2A and F2A cleavage sites. The amino acid sequences of T2A and E2A cleavage sites are shown in SEQ ID NOs: 7 and 12, respectively.

[0278] A cleavage site may be included in a polypeptide according to the present disclosure to provide for removal of a moiety or domain. It might be desirable to remove a given moiety or domain so that it is not comprised in the polypeptide complex formed by the polypeptide. For example, in embodiments of recombinant receptor polypeptides of the present disclosure, a cleavage site (specifically, a T2A cleavage site) is provided upstream of an eGFP moiety, to provide for its removal such that the eGFP moiety is not included in the final recombinant receptor polypeptide. Accordingly, in some embodiments, a recombinant receptor polypeptides according to the present disclosure comprises a cleavage site adjacent to ( / .e. in the amino acid sequence of the polypeptide, e.g. immediately upstream or downstream of) a detectable moiety according to the present disclosure.

[0279] In some embodiments, a cleavage site according to the present disclosure is a 2A cleavage site, e.g. selected from a T2A, P2A, E2A and F2A cleavage site. In some embodiments, the cleavage site is a T2A cleavage site.

[0280] Particular exemplary recombinant receptor polypeptides P39375-EP

[0281] In some embodiments, a recombinant receptor polypeptide according to the present disclosure comprises or consists of one of the following structures:

[0282] Optional signal peptide - optional tag - optional linker - antigen binding domain (e.g. a VH or VL) - optional linker - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a wild-type cytokine receptor chain - optional linker - optional marker

[0283] Schematic representation of some of the recombinant receptor polypeptides of the present invention are provided in Figures 2, 9A, 16, 20A, and 23A.

[0284] In some embodiments, a recombinant receptor polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:112-120, 113, 115, 123-132.

[0285] In some embodiments, a recombinant receptor polypeptide according to the present disclosure comprises an amino acid sequence having at least 60%, preferably one of >70%, >75%, >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to SEQ ID NO:158, 165, 166, 167, or 168.

[0286] Nucleic acids and vectors

[0287] The present disclosure provides a nucleic acid, or a plurality of nucleic acids, encoding a recombinant receptor polypeptide according to the present disclosure. In some embodiments, the nucleic acid(s) comprise or consist of DNA and / or RNA.

[0288] A recombinant receptor polypeptide according to the present disclosure may be produced within a cell by translation of RNA encoding recombinant receptor polypeptide. A recombinant receptor polypeptide according to the present disclosure may be produced within a cell by transcription from nucleic acid encoding the recombinant receptor polypeptide, and subsequent translation of the transcribed RNA.

[0289] In some embodiments, the nucleic acid(s) may be, or may be comprised / contained in, a vector, or a plurality of vectors. A ‘vector’ as used herein is a nucleic acid molecule used as a vehicle to transfer exogenous nucleic acid into a cell.

[0290] Accordingly, the present disclosure also provides a vector, or plurality of vectors, comprising the nucleic acid or plurality of nucleic acids according to the present disclosure. The vector may facilitate delivery of the nucleic acid(s) encoding a recombinant receptor polypeptide according to the present disclosure to a cell. The vector may be an expression vector comprising elements required for expressing a recombinant receptor polypeptide, optionally together with or linked with a CAR molecule. The vector may comprise elements facilitating integration of the nucleic acid(s) into the genomic DNA of cell into which the vector is introduced. P39375-EP

[0291] Nucleic acids and vectors according to the present disclosure may be provided in purified or isolated form, i.e. from other nucleic acid, or naturally-occurring biological material.

[0292] A vector may be a vector for expression of the nucleic acid in the cell (i.e. an expression vector). Such vectors may include a promoter sequence operably linked to a nucleotide sequence encoding a recombinant receptor polypeptide according to the present disclosure. A vector may also include a termination codon (i.e. 3’ in the nucleotide sequence of the vector to the nucleotide sequence encoding the recombinant receptor polypeptide and expression enhancers. Any suitable vectors, promoters, enhancers and termination codons known in the art may be used to express a peptide or polypeptide from a vector according to the present disclosure.

[0293] The term ‘operably linked’ may include the situation where nucleic acid encoding a recombinant receptor polypeptide according to the present disclosure and regulatory nucleic acid sequence(s) (e.g. a promoter and / or enhancers) are covalently linked in such a way as to place the expression of the nucleic acid encoding a recombinant receptor polypeptide under the influence or control of the regulatory nucleic acid sequence(s) (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to the selected nucleic acid sequence if the regulatory sequence is capable of effecting transcription of the nucleic acid sequence. The resulting transcript(s) may then be translated into the desired polypeptide(s).

[0294] Vectors contemplated in connection with the present disclosure include DNA vectors, RNA vectors, plasmids (e.g. conjugative plasmids (e.g. F plasmids), non-conjugative plasmids, R plasmids, col plasmids, episomes), viral vectors (e.g. retroviral vectors, e.g. gammaretroviral vectors (e.g. murine Leukemia virus (MLV)-derived vectors, e.g. SFG vector), lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, vaccinia virus vectors and herpesvirus vectors), transposon-based vectors, and artificial chromosomes (e.g. yeast artificial chromosomes), e.g. as described in Maus et al., Annu Rev Immunol (2014) 32:189-225 and Morgan and Boyerinas, Biomedicines (2016) 4:9, which are both hereby incorporated by reference in their entirety. In some embodiments, a vector according to the present disclosure is a lentiviral vector.

[0295] In some embodiments, the vector may be a eukaryotic vector, i.e. a vector comprising the elements necessary for expression of protein from the vector in a eukaryotic cell. In some embodiments, the vector may be a mammalian vector, e.g. comprising a cytomegalovirus (CMV) or SV40 promoter to drive protein expression.

[0296] In some embodiments, a nucleic acid / plurality or vector / plurality according to the present disclosure comprises an EF1 a promoter.

[0297] In some embodiments, a nucleic acid / plurality or vector / plurality according to the present disclosure encodes a recombinant receptor polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to one of SEQ ID NOs: 82-105. P39375-EP

[0298] In some embodiments, a nucleic acid / plurality or vector / plurality according to the present disclosure encodes a recombinant receptor polypeptide comprising or consisting of an amino acid sequence having at least 70%, preferably one of >80%, >85%, >90%, >91 %, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99% or 100% amino acid sequence identity to one of SEQ ID NOs: 159, 169, 170, 171 , 172, and 173.

[0299] Constituent polypeptides of a recombinant receptor polypeptide according to the present disclosure may be encoded by different nucleic acids of a plurality of nucleic acids according to the present disclosure, or by different vectors of a plurality of nucleic acids according to the present disclosure.

[0300] In aspects and embodiments of the present disclosure, a nucleic acid, or a plurality of nucleic acids, according to the present disclosure encodes two or more (e.g. 2, 3, 4 or more) recombinant receptor polypeptides according to the present disclosure.

[0301] In some embodiments, wherein a nucleic acid / plurality or vector / plurality encodes two or more (e.g. 2, 3, 4 or more) recombinant receptor polypeptide according to the present disclosure, transcription of nucleic acid encoding the two or more recombinant receptor polypeptides is under the control of the same promoter.

[0302] In some embodiments, transcription of nucleic acid encoding the two or more recombinant receptor polypeptides is under the control of different promoters.

[0303] In some embodiments, the nucleic acid / plurality or vector / plurality is multicistronic (e.g. bicistronic, tricistronic, etc.). That is, in some embodiments the nucleic acid / plurality or vector / plurality vector comprises multiple polypeptide-encoding nucleotide sequences. In some embodiments, nucleic acid encoding two or recombinant receptor polypeptides is provided in different cistrons.

[0304] Cells comprisinq / expressinq the recombinant receptor polypeptides of the disclosure

[0305] The present disclosure also provides a cell comprising a recombinant receptor polypeptide according to the present disclosure, or a nucleic acid / plurality or vector / plurality according to the present disclosure.

[0306] It will be appreciated that where cells are referred to herein in the singular ( / .e. ‘a / the cell’), pluralities / populations of such cells are also contemplated.

[0307] The cell may be a eukaryotic cell, e.g. a mammalian cell. The mammal may be a primate (rhesus, cynomolgous, non-human primate or human) or a non-human mammal (e.g. rabbit, guinea pig, rat, mouse or other rodent (including any animal in the order Rodentia), cat, dog, pig, sheep, goat, cattle (including cows, e.g. dairy cows, or any animal in the order Bos), horse (including any animal in the order Equidae), donkey, and non-human primate). In preferred embodiments, the cell is a human cell. P39375-EP

[0308] In some embodiments, the cell is an immune cell. An immune cell may be a cell of hematopoietic origin, e.g. a neutrophil, eosinophil, basophil, dendritic cell, lymphocyte, or monocyte. A lymphocyte may be e.g. a T cell, B cell, NK cell, NKT cell or innate lymphoid cell (ILC), or a precursor thereof. The immune cell may express CD27, CD28, CD4 and / or CD8. In some embodiments, the immune cell is a T cell, e.g. a CD3+ T cell. In some embodiments, the T cell is a CD3+, CD4+ T cell. In some embodiments, the T cell is a CD3+, CD8+ T cell. In some embodiments, the T cell is a T helper cell (TH cell). In some embodiments, the T cell is a cytotoxic T cell (e.g. a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a tumor infiltrating lymphocyte (TIL), B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a Natural Killer T (NKT) cell, or other T cell.

[0309] Aspects and embodiments of the present disclosure relate particularly to T cells comprising / expressing recombinant receptor polypeptides according to the present disclosure.

[0310] In some aspects and embodiments, a cell according to the present disclosure expresses / presents a recombinant receptor polypeptide according to the present disclosure at the cell surface. That is, the recombinant receptor polypeptide may be present in or at the cell membrane. Cells can be evaluated for surface expression of recombinant receptor polypeptide, e.g. using antibody-based methods such as flow cytometry (e.g. as described in Examples of the present disclosure).

[0311] In aspects and embodiments of the present disclosure, a cell according to the present disclosure comprises or expresses a recombinant receptor polypeptide according to the present disclosure. In some aspects and embodiments, a cell according to the present disclosure comprises nucleic acid encoding a recombinant receptor polypeptide according to the present disclosure. In some aspects and embodiments, a cell according to the present disclosure comprises a nucleic acid / plurality or vector / plurality according to the present disclosure.

[0312] In aspects and embodiments of the present disclosure, a cell according to the present disclosure comprises or expresses a polypeptide complex according to the present disclosure that binds to a variant Fc domain as described herein.

[0313] Dose-dependent cytokine signalling mediated by exposure of the cells of the invention to the small molecule (e.g. DOTAM) can be investigated as described in the Examples herein, e.g. by detecting, qualifying and analysing the downstream molecules in the pathway, such as STAT3, STAT4, STAT5 and their phosphorylation status or level.

[0314] The level of recombinant receptor polypeptide -mediated signalling can also be analysed using reporterbased methods, e.g. methods quantifying the activity of a transcription factor or gene whose expression / activity is upregulated in response to the considered cytokine signalling pathway.

[0315] As used herein, ‘expression’ may be gene or protein expression. Gene expression encompasses transcription of DNA to RNA, and can be measured by various means known to those skilled in the art, for P39375-EP example by measuring levels of mRNA by quantitative real-time PCR (qRT-PCR), or using reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, e.g. antibody-based methods, for example by western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0316] An immune cell (e.g. a T cell) according to the present disclosure may display cytotoxicity to cells comprising / expressing a variant Fc domain according to the present disclosure. That is, an immune cell (e.g. a T cell) according to the present disclosure may posses the ability to kill cells comprising / expressing a variant Fc domain according to the present disclosure.

[0317] Cytotoxicity and cell killing can be investigated, for example, using any of the methods reviewed in Zaritskaya et al., Expert Rev Vaccines (201 1), 9(6):601 -616, hereby incorporated by reference in its entirety. Examples of in vitro assays of cytotoxicity / cell killing assays include release assays such as the51Cr release assay, the lactate dehydrogenase (LDH) release assay, the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide (MTT) release assay, and the calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing by a given test cell type (e.g. an immune cell (e.g. a T cell) according to the present disclosure) can be analysed e.g. by co-culturing the test cells with the given target cell type (e.g. a cell comprising a variant Fc domain according to the present disclosure), and measuring the number / proportion of viable ( / .e. nonlysed) / dead (e.g. lysed) target cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolytic in vitro potency assay described in Cerignoli etal., PLoS One. (2018) 13(3): e0193498 (hereby incorporated by reference in its entirety), and the Incucyte immune cell killing assay, which is employed in the experimental examples of the present disclosure.

[0318] The present disclosure also provides methods for producing a cell according to the present disclosure, and the cells obtained or obtainable by such methods.

[0319] Methods for producing cells comprising / expressing a polypeptide / polypeptide complex of interest are well known to the skilled person, and generally comprise introducing nucleic acid(s) / vector(s) encoding the polypeptide(s) of interest into the cells.

[0320] Such methods may comprise nucleic acid transfer for permanent ( / .e. stable) or transient expression of the transferred nucleic acid. In some embodiments, following introduction into a cell nucleic acid(s) encoding the polypeptide(s) of interest may be integrated into or form part of the genomic DNA of the cell. In some embodiments, following introduction into a cell nucleic acid(s) encoding the polypeptide(s) of interest may be maintained extrachromosomally.

[0321] Any suitable genetic engineering platform may be used, and include gammaretroviral vectors, lentiviral vectors, adenovirus vectors, DNA transfection, transposon-based gene delivery and RNA transfection, for example as described in Maus et al., Annu Rev Immunol (2014) 32:189-225, hereby incorporated by reference in its entirety. Methods also include those described e.g. in Wang and Riviere Mol Ther P39375-EP

[0322] Oncolytics. (2016) 3:16015, which is hereby incorporated by reference in its entirety. Suitable methods for introducing nucleic acid(s) / vector(s) into cells include transduction, transfection and electroporation.

[0323] Methods for generating / expanding populations of cells comprising / expressing polypeptide(s) of interest in vitro / ex vivo are well known to the skilled person. Suitable culture conditions ( / .e. cell culture media, additives, stimulations, temperature, gaseous atmosphere), cell numbers, culture periods and methods for introducing nucleic acid(s) / vector(s) encoding polypeptide(s) of interest into cells, etc. can be determined by reference e.g. to WO 2018 / 177966 A1 . In some embodiments, a cell / population of cells according to the present disclosure is prepared under GMP (good manufacturing practice; e.g. as described in the guidelines for good manufacturing practice published by the European Commission (Volume 4 of ‘The rules governing medicinal products in the European Union’ contains guidance for the interpretation of the principles and guidelines of good manufacturing practices for medicinal products for human and veterinary use laid down in Commission Directives 91 / 356 / EEC, as amended by Directive 2003 / 94 / EC, and 91 / 412 / EEC respectively) conditions.

[0324] Conveniently, cultures of cells according to the present disclosure may be maintained at 37°C in a humidified atmosphere containing 5% CO2. The cells of cell cultures can be established and / or maintained at any suitable density, as can readily be determined by the skilled person. Cultures can be performed in any vessel suitable for the volume of the culture, e.g. in wells of a cell culture plate, cell culture flasks, a bioreactor, etc. In some embodiments cells are cultured in a bioreactor, e.g. a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1 (8): 1435-1437, which is hereby incorporated by reference in its entirety. Immune cells (e.g. T cells) may be activated prior to introduction of nucleic acid(s) encoding the polypeptide(s) of interest. For example, T cells within a population of PBMCs may be non- specifically activated by stimulation in vitro with agonist anti-CD3 and agonist anti-CD28 antibodies, in the presence of IL-2.

[0325] Introducing nucleic acid(s) into a cell may comprise transduction, e.g. lentiviral transduction. Transduction of immune cells with viral vectors is described e.g. in Simmons and Alberola-lla, Methods Mol Biol. (2016) 1323:99-108, which is hereby incorporated by reference in its entirety.

[0326] Agents may be employed to enhance the efficiency of transduction. Hexadimethrine bromide (polybrene) is a cationic polymer which is commonly used to improve transduction, through neutralising charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include e.g. the poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech) and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs). In some embodiments the methods comprise centrifuging the cells into which it is desired to introduce nucleic acid encoding the polypeptide(s) of interest in the presence of cell culture medium comprising viral vector comprising the nucleic acid (referred to in the art as ‘spinfection’).

[0327] The methods generally comprise introducing a nucleic acid encoding polypeptide(s) of interest into a cell, and culturing the cell under conditions suitable for expression of the polypeptide(s) of interest by the cell. P39375-EP

[0328] In some embodiments, the methods comprise culturing immune cells into which nucleic acid encoding polypeptide(s) of interest has been introduced in order to expand their number.

[0329] In some embodiments, the methods comprise analysing the cells to confirm successful introduction of the nucleic acid into the cells. In some embodiments, the methods comprise analysing the cells to confirm expression of the polypeptide(s) of interest by the cells (e.g. via evaluation of a detectable entity).

[0330] In some embodiments the methods further comprise cells expressing the polypeptide(s) of interest, e.g. from other cells (e.g. cells which do not express the polypeptide(s) of interest). Methods for pu rifyi ng / isolati ng immune cells from heterogeneous populations of cells are well known in the art, and may employ e.g. FACS- or MACS-based methods for sorting populations of cells based on the expression of markers of the immune cells. In some embodiments the methods purifying / isolating cells of a particular type, e.g. CD8+ T cells or CTLs expressing the polypeptide(s) of interest.

[0331] Modification of a given target nucleic acid can be achieved in a variety of ways known to the skilled person, including modification of the target nucleic acid by homologous recombination, and target nucleic acid editing using site-specific nucleases (SSNs).

[0332] Suitable methods may employ targeting by homologous recombination, which is reviewed, for example, in Mortensen Curr Protoc Neurosci. (2007) Chapter 4:Unit 4.29 and Vasquez etal., PNAS 2001 , 98(15): 8403- 8410 both of which are hereby incorporated by reference in their entirety. Targeting by homologous recombination involves the exchange of nucleic acid sequence through crossover events guided by homologous sequences. Other suitable techniques include nucleic acid editing using SSNs. Gene editing using SSNs is reviewed e.g. in Eid and Mahfouz, Exp Mol Med. 2016 Oct; 48(10): e265, which is hereby incorporated by reference in its entirety. Enzymes capable of creating site-specific double strand breaks (DSBs) can be engineered to introduce DSBs to target nucleic acid sequence(s) of interest. DSBs may be repaired by either error-prone non-homologous end-joining (NHEJ), in which the two ends of the break are rejoined, often with insertion or deletion of nucleotides. Alternatively, DSBs may be repaired by homology- directed repair (HDR), a high-fidelity mechanism in which a DNA template with ends homologous to the break site is supplied and introduced at the site of the DSB.

[0333] SSNs capable of being engineered to generate target nucleic acid sequence-specific DSBs include zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced palindromic repeats / CRISPR-associated-9 (CRISPR / Cas9) systems. ZFN systems are reviewed e.g. in Umov et al., Nat Rev Genet. (2010) 11 (9):636-46, which is hereby incorporated by reference in its entirety. ZFNs comprise a programmable Zinc Finger DNA-binding domain and a DNA- cleaving domain (e.g. a Fok\ endonuclease domain). The DNA-binding domain may be identified by screening a Zinc Finger array capable of binding to the target nucleic acid sequence. TALEN systems are reviewed e.g. in Mahfouz et al., Plant Biotechnol J. (2014) 12(8):1006-14, which is hereby incorporated by reference in its entirety. TALENs comprise a programmable DNA-binding TALE domain and a DNA- cleaving domain (e.g. a Fok\ endonuclease domain). TALEs comprise repeat domains consisting of repeats P39375-EP of 33-39 amino acids, which are identical except for two residues at positions 12 and 13 of each repeat which are repeat variable di-residues (RVDs). Each RVD determines binding of the repeat to a nucleotide in the target DNA sequence according to the following relationship: ‘HD’ binds to C, ‘NT binds to A, ‘NG’ binds to T and ‘NN’ or ‘NK’ binds to G (Moscou and Bogdanove, Science (2009) 326(5959):1501 .). CRISPR / Cas9 and related systems e.g. CRISPR / Cpf1 , CRISPR / C2c1 , CRISPR / C2c2 and CRISPR / C2c3 are reviewed e.g. in Nakade et al., Bioengineered (2017) 8(3):265-273, which is hereby incorporated by reference in its entirety. These systems comprise an endonuclease (e.g. Cas9, Cpf1 etc.) and the singleguide RNA (sgRNA) molecule. The sgRNA can be engineered to target endonuclease activity to nucleic acid sequences of interest.

[0334] In some embodiments, modifying nucleic acid (e.g. endogenous nucleic acid) encoding the recombinant receptor polypeptide in accordance with the present disclosure employs a site-specific nuclease (SSN) system targeting nucleic acid encoding the recombinant receptor polypeptide. The SSN system may be a ZFN system, a TALEN system, CRISPR / Cas9 system, a CRISPR / Cpf1 system, a CRISPR / C2c1 system, a CRISPR / C2c2 system or a CRISPR / C2c3 system.

[0335] Compositions

[0336] The present disclosure also provides compositions, e.g. pharmaceutical compositions, comprising the polypeptides, nucleic acids, expression vectors and cells described herein.

[0337] The polypeptides, nucleic acids, expression vectors and cells described herein (and particularly the nucleic acids, expression vectors and cells described herein) may be formulated as pharmaceutical compositions or medicaments for clinical use and may comprise a pharmaceutically-acceptable carrier, diluent, excipient or adjuvant. In preferred aspects and embodiments, the present disclosure provides a pharmaceutical composition or medicament comprising a cell according to the present disclosure. Thus, the present disclosure also provides a pharmaceutical composition / medicament comprising a polypeptide, nucleic acid / plurality, expression vector / plurality or cell described herein. In preferred embodiments, a pharmaceutical composition / medicament according to the present disclosure comprises a nucleic acid / plurality, expression vector / plurality or cell described herein.

[0338] The pharmaceutical compositions / medicaments of the present disclosure may comprise one or more pharmaceutically-acceptable carriers (e.g. liposomes, micelles, microspheres, nanoparticles), diluents / excipients (e.g. starch, cellulose, a cellulose derivative, a polyol, dextrose, maltodextrin, magnesium stearate), adjuvants, fillers, buffers, preservatives (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium citrate, methyl paraben, propyl paraben), antioxidants (e.g. vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium), lubricants (e.g. magnesium stearate, talc, silica, stearic acid, vegetable stearin), binders (e.g. sucrose, lactose, starch, cellulose, gelatin, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), xylitol, sorbitol, mannitol), stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents or colouring agents (e.g. titanium oxide). P39375-EP

[0339] The term ‘pharmaceutically-acceptable’ as used herein pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g. a human subject) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, diluent, excipient, adjuvant, filler, buffer, preservative, anti-oxidant, lubricant, binder, stabiliser, solubiliser, surfactant, masking agent, colouring agent, flavouring agent or sweetening agent of a composition according to the present disclosure must also be ‘acceptable’ in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, binders, stabilisers, solubilisers, surfactants, masking agents, colouring agents, flavouring agents or sweetening agents can be found in standard pharmaceutical texts, for example, Remington’s ‘The Science and Practice of Pharmacy’ (Ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0340] Pharmaceutical compositions and medicaments of the present disclosure may be formulated for topical, parenteral, systemic, intracavitary, intravenous, intra-arterial, intramuscular, intrathecal, intraocular, intraconjunctival, intratumoral, subcutaneous, intradermal, intrathecal, oral or transdermal routes of administration. In some embodiments, a pharmaceutical composition / medicament may be formulated for administration by injection or infusion, or administration by ingestion.

[0341] Suitable formulations may comprise the cell provided in a sterile or isotonic medium. Medicaments and pharmaceutical compositions may be formulated in fluid, including gel, form. Fluid formulations may be formulated for administration by injection or infusion (e.g. via catheter) to a selected region of the human or animal body.

[0342] In some embodiments, the pharmaceutical compositions / medicament is formulated for injection or infusion, e.g. into a blood vessel, tissue / organ of interest, or a tumour.

[0343] The present disclosure also provides methods for the production of pharmaceutically useful compositions, such methods of production may comprise one or more steps selected from: producing a cell described herein; isolating / purifying a cell described herein; and / or mixing a cell described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent.

[0344] For example, a further aspect the present disclosure relates to a method of formulating or producing a medicament or pharmaceutical composition for use in the treatment of a disease / condition (e.g. a disease / condition described herein), the method comprising formulating a pharmaceutical composition or medicament by mixing a cell described herein with a pharmaceutically-acceptable carrier, adjuvant, excipient or diluent. P39375-EP

[0345] Therapeutic and prophylactic applications

[0346] The articles of the present disclosure find use in therapeutic and prophylactic methods. In particular, a cell according to the present disclosure, e.g. a cell comprising / expressing a recombinant receptor polypeptide according to the present disclosure, finds use in therapeutic and prophylactic methods. Similarly, a composition according to the present disclosure, e.g. a pharmaceutical composition comprising a cell according to the present disclosure, e.g. a cell comprising / expressing a recombinant receptor polypeptide according to the present disclosure finds use in such methods.

[0347] Accordingly, the present disclosure provides a cell or composition described herein for use in a method of medical treatment or prophylaxis. Also provided is a cell or composition described herein for use in a method of treating or preventing a disease or condition described herein. Also provided is the use of a cell or composition described herein in the manufacture of a medicament for treating or preventing a disease or condition described herein. Also provided is a method of treating or preventing a disease or condition described herein, comprising administering to a subject a therapeutically- or prophylactically- effective amount of a cell or composition described herein.

[0348] The intervention described in the preceding paragraph may be effective to reduce the development or progression of a disease / condition, alleviate the symptoms of a disease / condition or reduce the pathology of a disease / condition. The intervention may be effective to prevent progression of the disease / condition, e.g. to prevent worsening of, or to slow the rate of development of, the disease / condition. In some embodiments, the intervention may lead to an improvement in the disease / condition, e.g. a reduction in the symptoms of the disease / condition or reduction in some other correlate of the severity / activity of the disease / condition. In some embodiments, the intervention may prevent progression / development of the disease / condition a later stage (e.g. a chronic stage or metastasis).

[0349] Therapeutic or prophylactic intervention in accordance with the present disclosure generally comprises administering a cell or pharmaceutical composition according to the present disclosure to a subject to which an antigen-binding molecule comprising: (a) an antigen-binding domain that binds to the target antigen, and (b) a variant Fc domain according to the present disclosure, has been or is to be administered.

[0350] In particular, use of the cells and compositions according to the present disclosure in methods to treat / prevent diseases / conditions by adoptive cell transfer (ACT) is contemplated.

[0351] Adoptive cell transfer generally refers to a process by which cells (e.g. immune cells) are obtained from a subject, typically by drawing a blood sample from which the cells are isolated. The cells are then typically modified and / or expanded, and then administered either to the same subject (in the case of adoptive transfer of autologous / autogeneic cells) or to a different subject (in the case of adoptive transfer of allogeneic cells). The treatment is typically aimed at providing a population of cells with certain desired characteristics to a subject, or increasing the frequency of such cells with such characteristics in that P39375-EP subject. Adoptive transfer may be performed with the aim of introducing a cell or population of cells into a subject, and / or increasing the frequency of a cell or population of cells in a subject.

[0352] Adoptive transfer of immune cells is described, for example, in Kalos and June (2013), Immunity 39(1): 49- 60, and Davis et al. (2015), Cancer J. 21 (6): 486-491 , both of which are hereby incorporated by reference in their entirety. The skilled person is able to determine appropriate reagents and procedures for adoptive transfer of cells according to the present disclosure, for example by reference to Dai et al., 2016 J Nat Cancer Inst 108(7): djv439, which is incorporated by reference in its entirety.

[0353] The present disclosure provides methods comprising administering cells and compositions according to the present disclosure to a subject.

[0354] Administration of the articles of the present disclosure is preferably in a ‘therapeutically-effective’ or ‘prophylactically-effective’ amount, this being sufficient to show therapeutic or prophylactic benefit to the subject. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of the disease / condition and the particular article administered. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disease / disorder to be treated, the condition of the individual subject, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington’s ‘The Science and Practice of Pharmacy’ (ed. A. Adejare), 23rd Edition (2020), Academic Press.

[0355] Administration of the articles of the present disclosure may be parenteral, systemic, intravenous, intraarterial, intramuscular, intracavitary, intrathecal, intraocular, intravitreal, intraconjunctival, subretinal, suprachoroidal, subcutaneous, intradermal, intrathecal, oral, nasal, topical or transdermal. Administration may be by injection or infusion. Administration of the articles of the present disclosure may be intratumoral. In some cases, the articles of the present disclosure may be formulated for targeted delivery to specific cells, a tissue, an organ and / or a tumor.

[0356] Multiple doses of an article of the present disclosure may be provided. Multiple doses may be separated by a predetermined time interval, which may be selected to be one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or 1 , 2, 3, 4, 5, or 6 months.

[0357] Administration of a cell or composition according to the present disclosure with an antigen-binding molecule described herein to a subject in accordance with the therapeutic and prophylactic intervention described herein may be simultaneous or sequential.

[0358] Simultaneous administration refers to administration of (i) a cell or composition according to the present disclosure, and (ii) an antigen-binding molecule described herein together, for example as a pharmaceutical composition containing both agents ( / .e. a combined preparation), or immediately after one another, and optionally via the same route of administration, e.g. to the same artery, vein or other blood vessel. P39375-EP

[0359] Sequential administration refers to administration of one of (i) a cell or composition according to the present disclosure, and (ii) an antigen-binding molecule described herein, followed after a given time interval by separate administration of the other agent. It is not required that the two agents are administered by the same route, although this is the case in some embodiments. The time interval may be any time interval.

[0360] Subjects

[0361] A subject in accordance with the various aspects of the present disclosure may be any animal or human. Therapeutic and prophylactic applications may be in human or animals (veterinary use).

[0362] The subject to be administered with an article of the present disclosure (e.g. in accordance with therapeutic or prophylactic intervention) may be a subject in need of such intervention. The subject is preferably mammalian, more preferably human. The subject may be a non-human mammal, but is more preferably human. The subject may be male or female. The subject may be a patient.

[0363] A subject may have (e.g. may have been diagnosed with) a disease or condition described herein, may be suspected of having such a disease / condition, or may be at risk of developing / contracting such a disease / condition. In embodiments according to the present disclosure, a subject may be selected for treatment according to the methods based on characterisation for one or more markers of such a disease / condition.

[0364] In some embodiments, a subject may be selected for therapeutic or prophylactic intervention as described herein based on the detection of cells / tissue expressing a target antigen ( / .e. the target antigen of an antigen-binding molecule to be employed in conjunction with a cell or composition according to the present disclosure), or of cells / tissue overexpressing the target antigen, e.g. in a sample obtained from the subject.

[0365] A subject may be an allogeneic or non-autologous subject with respect to an intervention in accordance with the present disclosure. As used herein, where a subject is referred to herein as being ‘allogeneic’ or ‘non-autologous’ with respect to an intervention, the subject is a subject other than the subject from which the cell of the intervention ( / .e. the cell to be administered, or the cell of the pharmaceutical composition / medicament to be administered) is derived. A subject to be treated / prevented in accordance with the present disclosure may be genetically non-identical to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived. A subject to be treated / prevented in accordance with the present disclosure may comprise MHC / HLA genes encoding MHC / HLA molecules (e.g. MHC class I a and / or MHC class II molecules) that are non-identical to the MHC / HLA molecules (e.g. MHC class I a and / or MHC class II molecules) encoded by the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject. A subject to be treated / prevented in accordance with the present disclosure may be H LA-mismatched with respect to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived. P39375-EP

[0366] The subject to which cells are administered in accordance with the present disclosure may be allogeneic / non-autologous with respect to the source from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived. The subject to which cells are administered may be a different subject to the subject from which cells are / were obtained for the production of the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject. The subject to which the cell is administered may be genetically non-identical to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject cells are / were obtained for the production of the cells.

[0367] A subject may be an autogeneic / autologous subject with respect to an intervention in accordance with the present disclosure. As used herein, where a subject is referred to herein as being ‘autogeneic’ or ‘autologous’ with respect to an intervention, the subject is the same subject from which the cell of the intervention ( / .e. the cell to be administered, or the cell of the pharmaceutical composition / medicament to be administered) is derived. A subject to be treated / prevented in accordance with the present disclosure may be genetically identical to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived. A subject to be treated / prevented in accordance with the present disclosure may comprise MHC / HLA genes encoding MHC / HLA molecules (e.g. MHC class I a and / or MHC class II molecules) that are identical to the MHC / HLA molecules (e.g. MHC class I a and / or MHC class II molecules) encoded by the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject. A subject to be treated / prevented in accordance with the present disclosure may be HLA-matched with respect to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived.

[0368] The subject to which cells are administered in accordance with the present disclosure may be autogeneic / autologous with respect to the source from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject is derived. The subject to which cells are administered may be the same subject as the subject from which cells are / were obtained for the production of the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject. The subject to which the cell is administered may be genetically identical to the subject from which the cell (e.g. the cell of the pharmaceutical composition / medicament) to be administered to the subject cells are / were obtained for the production of the cells. g, J. 2005, Bioinformatics 21 , 951 -960).

[0369] It must be noted that, as used in the specification and the appended claims, the singular forms ‘a,’ ‘an,’ and ‘the’ include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from ‘about’ one particular value, and / or to ‘about’ another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent ‘about,’ it will be understood that the particular value forms another embodiment. P39375-EP

[0370] Where a nucleic acid sequence is disclosed herein, the reverse complement thereof is also expressly contemplated.

[0371] Methods described herein may preferably be performed in vitro. The term ‘in vitro’ is intended to encompass procedures performed with cells in culture whereas the term ‘in vivo’ is intended to encompass procedures with / on intact multi-cellular organisms.

[0372] II. Examples

[0373] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.

[0374] Example 1 : Materials and Methods

[0375] 1.1 Recombinant DNA / RNA Technique

[0376] Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturers’ instructions. General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5thed., NIH Publication No. 91 -3242.

[0377] 1 .2 DNA Sequencing

[0378] DNA sequences were determined by double-strand Sanger sequencing.

[0379] 1 .3 Gene Synthesis

[0380] Desired gene segments were either generated by PCR using appropriate templates or were synthesized by GeneArt AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. The gene segments flanked by singular restriction endonuclease cleavage sites were cloned into second generation lentiviral transgene vectors. The plasmid DNA was purified from transformed bacteria and concentration determined by UV spectroscopy. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene segments were designed with suitable restriction sites to allow sub-cloning into the respective expression vectors. All constructs were designed with a 5’- end DNA sequence coding for a signal peptide (SP) which targets proteins for secretion in eukaryotic cells. When more than one protein chain was expressed, the coding sequences were separated by DNA encoding P2A / T2A / E2A self-splicing peptides. E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; T2A is derived from the Asigna virus 2A.

[0381] 1 .4 T cell isolation from fresh healthy donor human blood

[0382] Bio-One LeucoSEP™ Polypropylene Tubes (Greiner, #10349081) were prepared with 15 mL Histopaque®- 1077 density gradient medium (Merck, #10771 -500ML) by centrifugation at 400 x G for 5 minutes until the P39375-EP liquid is under the filter-unit. Fresh blood from anonymized healthy donors was mixed in a 1 :1 ratio with Dulbecco’s phosphate buffered saline (DPBS, Merck #D8537-500ML) and transferred to the LeucoSEP™ tubes without disturbing the prepared solution. After centrifugation at 1200 x g for 20 minutes (Acceleration: 1 ; Break: 0), the buffy coat visible as a white layer was separated into a new 50 mL falcon tube using a 10 mL serological pipette. After washing three times with 40 mL DPBS and centrifugation at 780 >450 > 280 x g, the enriched cell population was further purified manually, according to the human Pan T cell isolation kit (Miltenyi, #130-094-535). In brief, cell pellets were labeled with Pan T cell Biotin-Antibody, incubated for 5 minutes at 4 °C and further labeled with Pan T cell magnetic MicroBead Cocktail. Following negative selection on a MACS® Manual Separator, the purified Pan T cell population was either cultured, or frozen as 5*106cells mL1aliquots in advanced RPMI 1640 (Fisher Scientific, #12633012) + 20 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 20 % DMSO (Sigma-Aldrich, #D2650- 100ML) in dedicated freezing containers at -80 °C for at least 24 hours, then transferred to liquid nitrogen.

[0383] 1.5 Production of Small molecules

[0384] DOTAM (2-[4,7,10-tris(2-amino-2-oxo-ethyl)-1 ,4,7,10-tetrazacyclododec-1-yl]acetamide) was synthesized from 1 ,4,7,10-tetraazacyclododecane, Kryptofix 11 aza and 2-bromoacetamide.

[0385] 1 ,4,7,10-tetraazacyclododecane, Kryptofix 11 aza (8 g, 46.4 mmol, Eq: 1) was dissolved in Ethanol (EtOH) (142 g, 180 ml, Eq: -) at room temperature. 2-bromoacetamide (28.8 g, 209 mmol, Eq: 4.50) and N,N- diisopropylethylamine (DIPEA) (30 g, 39.7 ml, 232 mmol, Eq: 5.0) was added to the colorless solution while stirring at room temperature. The reaction mixture was stirred for 4hr at reflux conditions. A white solid precipitate was formed at 44 °C. After 4hr at reflux Ion pair chromatography (IPC) by HPLC shows >99 % conversion. The white solid was filtered off, and washed and resuspended in EtOH. The white, incompletely dissolved suspension in EtOH was heated at reflux conditions. The volume of the hot reaction mixture was reduced by 30 % with a rotary evaporator at 45 °C for 1 h. The suspension was stirred at 0 °C for 30 min to allow crystal formation. The suspension was filtered and the crystals were dried at 50 °C and 5 mbar over night to reduce residual EtOH.

[0386] Ion-loading of DOTAM with Pb2+and Ca2+was done using Pb(C2H3O2)2 and Ca(C2H3O2)2, in H2O / MeOH (1 :9) at room temperature, respectively. The mixture was stirred for 2 h at reflux conditions (oil bath at 85 °C). MeOH was removed under reduced pressure and the remaining clear solution was dissolved again in 3x 10 ml EtOH and evaporated to leave a white powder. Working solution was dissolved in DMSO or PBS. Derivatives of the small molecule DOTAM (DOTAM-biotin, DOTAM-FITC) were produced by Macrocyclics Inc. using commercially available TCMC as a starting point. DOTAM-AF647 (AF647 = Alexa Fluor™ 647) was produced by Pharmaron Beijing Co Ltd. using commercially available TCMC and AF647 as starting points. In brief, an isothiocyanate acts as a warhead at slightly basic pH (9-11) to form a covalent bond with a terminal amine forming a benzoylthiourea conjugate. The molecular mass of all compounds was confirmed using LC-MS and where possible, structure was confirmed with NMR. All compounds were >95 % pure as measured by HPLC.

[0387] Unless otherwise specified, the different molecules are shortened in examples as followed:

[0388] DOTAM-biotin = [Pb2+]DOTAM-biotin P39375-EP

[0389] • DOTAM = [Ca2+]DOTAM

[0390] • DOTAM-FITC = [Pb2+]DOTAM-FITC

[0391] • DOTAM-AF647 = [Pb2+]DOTAM-AF647

[0392] 1.6 Production of IgG-like proteins in Expi293F cells

[0393] Antibodies and antibody-like proteins were generated by transient transfection of Expi293F cells. Cells were seeded in Expi293 media (Gibco, #1435101) at a density of 2.5 x 106mL-1. Expression vectors and ExpiFectamine (Gibco, ExpiFectamine transfection kit, #13385544) were separately mixed in OptiMEM (Gibco, #11520386). After 5 min, both solutions were combined, mixed by pipetting and incubated for 25 minutes at room temperature. Cells were added to the vector / ExpiFectamine solution and incubated for 24 hours at 37 °C in a shaking incubator with a 5 % CO2 atmosphere. One day post transfection, supplements (Enhancer 1 +2, ExpiFectamine transfection kit) were added. Cell supernatants were harvested after 4-5 days by centrifugation and subsequent filtration (0.2 pm filter), and proteins were purified from the harvested supernatant by standard methods as indicated below.

[0394] 1.7 Production of IgG-like proteins in CHO K1 cells

[0395] The antibodies and antibody-like proteins described herein were prepared by Evitria with conventional (non- PCR based) cloning techniques and using suspension-adapted CHO K1 cells (originally received from ATCC and adapted to serum-free growth in suspension culture at Evitria). For the production, animalcomponent free and serum-free media (eviGrow and eviMake2) were used. Supernatant was harvested by centrifugation and subsequent filtration (0.2 pm filter) and afterwards purified from the harvested supernatant by standard methods.

[0396] 1.8 Purification of IgG-like proteins

[0397] Proteins were purified from filtered cell culture supernatants referring to standard protocols. In brief, Fc containing proteins were purified from cell culture supernatants by Protein A-affinity chromatography (equilibration buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, pH 3.0). Elution was achieved at pH 3.0 followed by immediate pH neutralization of the sample. The protein was concentrated by centrifugation (Millipore Amicon® ULTRA-15, #UFC903096), and aggregated protein was separated from monomeric protein by size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, pH 6.0.

[0398] 1 .9 Analytics of IgG-like proteins

[0399] The concentrations of purified proteins were determined by measuring the absorption at 280 nm using the mass extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science, 1995, 4, 2411 -1423. Purity and molecular weight of the proteins were analyzed by CE- P39375-EP

[0400] SDS in the presence and absence of a reducing agent using a LabChipGXIl or LabChip GX Touch (Perkin Elmer). Determination of the aggregate content was performed by HPLC chromatography at 25 °C using analytical size-exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCI pH 6.2, 0.02 % NaN3).

[0401] 1.10 Modification of IqG-like proteins

[0402] Human IgG Fc proteins carrying the PGLALA mutations (P329G, L9A, L10A) were expressed and purified as explained above. The staining agent Fc(PGLALA)-AF647 was generated by labeling with commercially available AF647 NHS Ester (#A20106, Thermo Fisher Scientific) in a sodium carbonate solution (1 M NaHCO3, pH8.3) for 1 hour. Next, the buffer was exchanged for 20 mM Histidine, 140 mM NaCI, pH6.0 and the protein was purified using a desalting column (e.g., PD 10 Desalting Columns, #GE17-0851-01 , Sigma Aldrich). Fc(PGLALA)-DOTAM-AF647 was dual-labeled with p-SCN-Bn-TCMC (#B-1005, Macrocyclics) and AF647 NHS Ester (#A20106, Thermo Fisher Scientific) and purified as explained above.

[0403] 1.11 Transient transfection and HEK-Blue™ IL2 assays

[0404] HEK-Blue™ IL2 cells were modified with different DOTAM receptors by transient transfection using Lipofectamine LTX™ (#15338500, Invitrogen) according to the supplier’s manual, to assess STAT5 signaling.

[0405] 24 h after transfection of the cell line, 30000 cells were seeded per well of a 384-well transparent bottom plate in medium without antibiotics. Serial dilutions of the test compounds were added and cells were incubated for 24 h at 37 °C in a 5 % CO2 atmosphere. To monitor signaling (STAT5-induced SEAP), Quanti-Blue™ solution (InvivoGen, #rep-qbs) was added to the plate, followed by 10 minutes of incubation at 37 °C in a 5 % CO2 atmosphere. Optical density was then assessed at 620 nm using a Tecan Plate Reader.

[0406] 1.12 Preparation of virus-like particles

[0407] Lipofectamine LTX™ (#15338500, Invitrogen) based transfection of ~ 60 % confluent Lenti-X™ 293T cells (Takara, #632180) was performed with CAR / recombinant receptor encoding transfer vectors as well as packaging vectors pCAG-VSVG and psPAX2 at a 2:1 :2 molar ratio (Giry-Laterriere M, et al. Methods Mol Biol. 201 1 ;737:183-209, Myburgh R, et al. Mol Ther Nucleic Acids. 2014). As control for every experiment, mock virus-like particles (VLPs) using only the packaging vectors, but no transfer vector, were produced. After 48 hours, the supernatant was collected and centrifuged for 10 minutes at 500 x g to remove remaining cells and concentrated 10-fold (Lenti-x-Concentrator, Takara, #631231) by centrifugation and resuspension according to the manufacturer’s protocol.

[0408] 1.13 Transduction of healthy donor T cells P39375-EP

[0409] T cells were quickly thawed in a 37 °C water bath and washed with 10x (V:V) advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + Ix GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU*mL1IL2 (Proleukin, Novartis)) + 25 ng*mL1IL7 (Miltenyi, #130-095-364) + 50 ng*mL1Interleukin- 15 (I L15)(Miltenyi, #130-095-766). Cells were then seeded at 106cells*mb-1in a 24-well plate (Sigma Aldrich, # Z707791 -126EA) and activated using Immunocult CD3 / CD28 / CD2 T cell Activator cocktail (Stemcell Technologies, #10990) for 24 hours.

[0410] After brief incubation with 8 pg*ml-1Polybrene (Sigma Aldrich) and Lentiboost P (1 :100) (Sirion Biotech, #SB-P-LV-101-12), 100-500 pb of previously purified virus-like particles were added to the activated T cells. Following incubation for at least 72 hours, the transduction efficiency was assessed by flow cytometry. Transgenes encode different DOTAM chimeric cytokine receptors (DXR whereby X is any interleukin or interferon, e.g., Interleukin-2 (IL2) = D2R, lnterleukin-7 (IL7) = D7R, Interleukin-12 (IL12) = D12R, Interleukin-18 (IL18) = D18R, Interleukin-23 (RECOMBINANT RECEPTOR) = D23R) and / or a chimeric antigen receptor.

[0411] 1.14 Cultivation of T cells

[0412] Engineered or wild type T cells are cultured in G-Rex® 24 multi-well cell culture plates (Wilson Wolf, #80192M) at densities of 0.2*106mb1to 4*106mb1in advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU*mL1IL2 (Proleukin, Novartis) + 25 ng*mb1IL7 (Miltenyi, #130-095-364) + 50 ng*mb1IL15 (Miltenyi, #130-095-766). Before performing any analysis, T cells were cultured for a minimum of 7 days to avoid effects caused by pseudo transduction. Assays were usually performed between day 7 and day 15 post transduction.

[0413] 1.15 Flow cytometry surface staining

[0414] Transduction efficiency or cellular signaling events were assessed by flow cytometry. To control transduction efficiency and translation of DOTAM receptors, RFP or GFP were co-expressed downstream of a 2A self-cleaving peptide. The cell surface expression of DOTAM receptors was analyzed by staining with [Pb2+]DOTAM-FITC (DOTAM-FITC, Macrocyclics), Fc(PGbAbA)-DOTAM-Alexa-647 i.e., Fc PGbAbA dual-labeled with p-SCN-Bn-TCMC (#B-1005, Macrocyclics) and AF647 NHS Ester (#A20106, Thermo Fisher Scientific) or DOTAM-[Pb2+]-biotin (DOTAM-biotin, Macrocyclics) in conjunction with Streptavidin, AF647 conjugate (Fisher Scientific, #10308062). Chimeric antigen receptors were stained with fluorescently-labeled antigens (e.g. Fc PGbAbA).

[0415] The functionality of DOTAM receptors was assessed by flow cytometric analysis of cell surface receptors or by measuring cytokine release. 100,000 cells were prepared in a 96-well plate and stimulated with serial dilutions of cytokines. IL2 (Proleukin, Novartis), or IL7 (Miltenyi, #130-095-364), IL12 (R&D Systems #10018-IL-050), IL18 (R&D Systems, #9124-lb-050 / CF) or DOTAM-[Ca2+] (Fisher Scientific, #15407625).

[0416] After incubation for 16 hours at 37 °C in a 5 % CO2 atmosphere incubator, cells were washed with DPBS by subsequent centrifugation at 300 x g and stained with 50 pb of pre-diluted antibody solution (panels 1 - 5) at 4 °C for 30 min. Cells were washed two more times before being fixed with 4 % PFA solution (Fixation P39375-EP

[0417] Buffer, BD Biosciences, #554655). Cells were washed once more before final resuspension in FACS-buffer (PBS containing 2 % FBS, 10 % 0.5 M EDTA, pH 8 and 0.5 g*L1NaN3) and analyzed using a BD LSRFortessa™ Cell Analyzer. Panel 1

[0418] Panel 2 P39375-EP

[0419] Panel 3

[0420] Panel 4 P39375-EP

[0421] Panel 5 1.16 DOTAM internalization assay

[0422] 200,000 cells were collected and washed with cold DPBS. Next, the cells were stained for living cells with Live / Dead NIR stain (Fisher Scientific, # L10119) for 1 hour at 4 °C. The cells were washed with ice-cold DPBS before being resuspended with DOTAM-biotin (diluted in ice-cold PBS at 100 pM). The cells were then incubated with the compound for 1 hour at 4 °C. Cells were washed two times with ice-cold PBS before being resuspended in ice-cold 50 pL FACS buffer (PBS containing 2 % FBS, 10 % 0.5 M EDTA, pH 8 and 0.5 g*L1NaN3) per sample and condition in a 15 mL falcon tube. The tube containing the cells was placed in a water bath at 37 °C, while the control was kept at 4 °C and on ice to prevent D12R internalization. At each time point, triplicates of 50 pL cell suspension were taken into a 96W plate prepared with 150 pL ice- cold PBS. The 96W plate was stored on ice and in the fridge during the duration of the assay until further processing. After all samples were collected, cells were washed 2x with ice-cold PBS and stained with the secondary reagent Streptavidin-BV421 (Biolegend, # 405226) for 30 min. After washing 2x with ice-cold FACS Buffer, the cells were fixed with 4 % PFA solution (Fixation Buffer, BD Biosciences, #554655). Cells P39375-EP were washed once more with FACS-Buffer , before being analyzed using a BD LSRFortessa™ Cell Analyzer.

[0423] 1.17 Phospho-STAT staining

[0424] In preparation of the assay, 200,000 cells were washed with advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) without added cytokines and starved for 4-12 hours in the same medium in a 96 well plate. The cells were stimulated with serial dilutions of cytokines or small molecule ligands (IL2, IL7 pathways: 120min; IL12, RECOMBINANT RECEPTOR pathways: 40 min) and incubated for the indicated time intervals at 37 °C in a 5 % CO2 atmosphere incubator. Immediately after, cells were fixed and permeabilized (BD Phosflow™ Fix Buffer I, BD Biosciences, #557870) for 15 minutes at 37 °C. Following centrifugation at 300 x g for 2 minutes, the cells were resuspended in Phosflow Perm Buffer III (BD Biosciences, 558050) and incubated at 4 °C for 30 minutes or at -20 °C overnight. The cells were washed two more times in DPBS before being stained with pre-diluted antibody solution using a murine anti-pSTAT4 antibody conjugated with AF647 (BD, #558137) and murine anti-pSTAT3 antibody conjugated with PE (BD, #562072) or murine anti-pSTAT5 antibody conjugated with AF647 (BD, #562076) for 60 minutes at 4 °C. After washing the cells once more in FACS-buffer (PBS containing 2 % FBS, 10 % 0.5 M EDTA, pH 8 and 0.5 g*L1NaN3)), the acquisition was done on a BD LSRFortessa™ Cell Analyzer.

[0425] 1.18 Phospho-Nf-KB staining

[0426] Phosphorylation of Nuclear factor kappa-light-chain-enhancer of activated B cells (Nf-KB) was used to assess D18R signaling. In preparation of the assay, 200.000 cells were washed with advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) without adding cytokines and starved for 4-12 hours in the same medium in a 96 well plate. The cells were stimulated with serial dilutions of IL18 (R&D Systems, #9124-IL-050 / CF) or DOTAM ligands and incubated for 60 minutes at 37 °C in a 5 % CO2 atmosphere incubator. Afterwards, cells were fixed and permeabilized (BD Phosflow™ Fix Buffer I, BD Biosciences, #557870) for 15 minutes at 37 °C. Following centrifugation at 300 x g for 2 minutes, the cells were resuspended in Phosflow Perm Buffer III (BD Biosciences, 558050) and incubated at 4 °C for 30 minutes. The cells were washed two more times in DPBS before being stained with pre-diluted antibody solution using a murine anti-NF-KB p65 (pS529) antibody (BD,# 558422 for 60 minutes at 4 °C. After washing the cells once more in FACS-buffer (PBS containing 2 % FBS, 10 % 0.5 M EDTA, pH 8 and 0.5 g*L1NaN3)), the acquisition was done on a BD LSRFortessa™ Cell Analyzer.

[0427] 1.19 Homogeneous Time Resolved Fluorescence assay

[0428] Homogeneous Time Resolved Fluorescence (HTRF) assay was used as an alternative to ELISA for assessing cytokine secretion. The Human IFN gamma kit (Perkin Elmer CisBio, #62HIIFNGPET) was used according to the manufacturer's protocol to determine secreted Interferon-gamma (I FNy) concentration in P39375-EP cell culture supernatants. In brief, 100.000 cells were washed with advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) + 50 IU m1IL2 (Miltenyi, #130-097-748) + 25 ng*mL1IL7 (Miltenyi, #130-095-364) + 50 ng*mL1IL15 (Miltenyi, #130-095- 766). The cells were stimulated with serial dilutions of IL12 (R&D Systems #10018-IL-050), or IL18 (R&D Systems, #9124-IL-050 / CF) or DOTAM and incubated for 24 hours at 37 °C in a 5 % CO2 atmosphere incubator. IFNy EU Cryptate - and IFNy XL antibodies were diluted 20-fold in 20x in detection buffer #3. Next, supernatants were combined 4:1 with a pre-mixed 1 :1 dilution of donor and acceptor antibodies and incubated for 4- to 12 hours at room temperature. A standard calibration curve was acquired using recombinant IFNy (part of CisBio IFNy HTRF kit, #62HIIFNGPET) using serial 1 :3 dilutions from 4000 pg*mL1to 0 pg*mL1obtained in the same assay. FRET-Fluorescence emission was determined at 665 nm (acceptor) and 620 nm (donor) in a Tecan Spark M10 plate reader. The data was normalized to the FRET-donor signal, background subtracted and fitted to the IFNy standard curve.

[0429] 1.20 Incucyte killing assay

[0430] Target cells (MKN-45 or HPAF-II) were engineered with a nuclear-localized mKate2 (NLR), using commercially available lentivirus (Incucyte® Nuclight Red Lentivirus (puro), #4625, Sartorius). High expressing clones were sorted by fluorescence-assisted cell sorting (FACS) using a BD FACSAria II flow cytometer. Single clones were expanded and if possible, stable expression of RFP was verified for 8 weeks. Engineered target cells (MKN-45-NLR or HPAF-II-NLR) were seeded in RPMI 1640 (Fisher Scientific, # 11875093) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) in a 96-well plate with edge reservoir (Fisher Scientific, # 167425) and incubated for at least 2 hours. Engineered cells were washed two times in DPBS, resuspended in RPMI 1640 (Fisher Scientific, # 11875093) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061). Cell populations were normalized to equal percentage of CAR-positive cells before being added to the target cells. Engineered T cells were used at different effector to target cells ratios (E:T) with fixed 10 pM concentration of the targeting IgG for adaptorbased CARs. Target cell killing was monitored for 7 days in an Incucyte® Live-Cell Analysis System (Sartorius). Data was normalized to the initial cancer cell count or integrated red fluorescence.

[0431] 1 .21 Proliferation assay

[0432] Engineered T cells were labeled with CellTrace™ Violet Cell Proliferation Kit (Invitrogen, #C34557) following the manufacturer protocol. The cells were then aliquoted at 200’000 cells per well in a 96 flat bottom plate. The test compounds were added in a serial dilution and the cells were incubated for 5-6 days at 37 °C in a 5 % CO2 incubator. To analyze proliferation, the cells were resuspended and washed twice with PBS before being fixed in BD Cytofix ™ Fix Buffer (BD, #554655). The cells were then washed twice and resuspended in a FACS buffer before being analyzed with a BD LSRFortessa™ Cell Analyzer.

[0433] 1.22 Multiplex cytokine-release assay P39375-EP

[0434] Cytokine release from engineered T cells was assessed by flow cytometry-based multiplex immunoassay (BioLegend, #741 187) . In preparation of the assay, 100’000 cells were washed with advanced RPMI 1640 (Fisher Scientific, #12633012) + 10 % FBS (Sigma-Aldrich) + 1x GlutaMAX™ (Fisher Scientific, #35050061) without added cytokines and starved for 4-12 hours in the same medium in a 96 well plate. After starvation, the cells were stimulated with dilutions of DOTAM or IL12 as a control and incubated for 16 hours at 37 °C in a 5 % CO2 atmosphere incubator. After incubation, 100 pL supernatants were collected and optionally stored at -80 °C. The cytokine array measurement was performed using a commercial kit (Legendplex(™) Human CD8 / NK panel kit) according to the suppliers manual. In brief, 12.5 pL of supernatants or standard solution was combined with 12.5 pL assay buffer, and 12.5 pL assay beads and incubated at room temperature for 2 hours while shaking at 450 rpm in an orbital shaker. Next the beads were washed 1x with 200 pL wash buffer and further complexed with 12.5 pL detection antibodies for 1 hour at RT, while shaking at 450 rpm. After addition of 12.5 pL streptavidin-PE-conjugate and incubation for another 30 min, the beads were washed 2x in 200 pL wash buffer and finally acquired on a BD Symphony™ A5 Cell Analyzer. Fitting to standard curve and data analysis was performed using Biolegends' accompanying online tool (https: / / leqendplex.qoqnit.com / ) and in GraphPad Prism.

[0435] Example 2: Analysis of signal transduction in reporter cells engineered with D2R and D7R

[0436] HEK-Blue™ IL2 cells (InvivoGen, #hkb-il2) are genetically engineered to express all components of a functional, human IL2 signaling pathway, i.e., IL2Ra, IL2Rp, IL2Ry, JAK1 , JAK3 and STAT5 as well as a STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene (Fig. 4A). The reporter cells were modified by transient transfection using Lipofectamine LTX (#15338500, Invitrogen) to introduce different D2R and D7R variants with different extracellular receptor designs (Fig. 2).

[0437] D2Rv1 contains the membrane-proximal extracellular domain 2 (D2) of the WT IL2Rpy receptor chains to which the antibody variable fragments were fused via a short linker (Fig. 3A). These D2 domains were omitted in D2Rv2, i.e., the extracellular domains of the D2Rv2 consist only of antibody VH and VL domains which were fused to the 3 extracellular amino acids of the transmembrane-domains of IL2Rp and IL2Ry, respectively. Likewise, D7Rv1 and D7Rv2 also differ by the presence (D7Rv1) or absence (D7Rv2) of the membrane-proximal extracellular D2 domains (Fig. 3B).

[0438] To assess whether DOTAM receptors would be able to induce STAT5 signaling, transiently transfected HEK-Blue™ IL2 cells were stimulated with serial dilutions of DOTAM or IL2 (Proleukin, Novartis), which served as a positive control. Dose-dependent STAT5 signaling upon DOTAM stimulation was observed for both D2R variants (Fig. 4B), without any noticeable difference between D2Rv1 and D2Rv2. (Fig. 2). The results demonstrate the functionality of D2Rs and that D2Rs tolerate different designs with regard to the extracellular domains.

[0439] For D7R-modified HEK-Blue™ IL2 cells, exhibited lower but still statistically significant STAT5 signaling (D7Rv1 P-value=0.0021 ; D7Rv2 P-value<0.0001 , t-test), which is in line with the lower STAT5 signaling of WT IL7R compared to IL2R. Again, no difference between D7Rv1 and D7Rv2 was observed suggesting that different members of the common gamma chain receptor family exhibit similar design constraints in P39375-EP the context of chimeric cytokine receptors. Moreover, DOTAM signaling was specific to D2R- and D7R- transfected cells, since no STAT5 signaling was observed in non-transfected HEK-Blue™ IL2 cells.

[0440] Surface expression of the engineered receptor was shown through labeling via DOTAM-FITC or Fc(PGLALA)-DOTAM-Alexa-647 and detection by flow cytometry (Fig. 4D). Expression of the engineered receptor was also assessed indirectly by the expression of the fluorescent reporter RFP which is placed downstream of the DOTAM receptor transgene and the proteins are separated by a self-cleaving 2A peptide (Fig. 3A).

[0441] Example 3: Analysis of STAT5 phosphorylation in human primary T cell engineered with D2Rs

[0442] Analysis of phosphorylation of signal transduction and activators of transcription factors (STATs, pSTATs for phosphorylated STATs) induced by DOTAM / D2R signaling was done by intracellular pSTAT5 staining and flow cytometry. Expression of D2R variants in primary cells was assessed indirectly by RFP expression, and stepwise D2R staining with DOTAM-biotin and Streptavidin-AF647 conjugate at 37 °C. (Fig. 5A)

[0443] T cells transduced with D2Rs were stimulated with serial dilutions of DOTAM for 120 min, followed by immediate fixation (BD, #557870), permeabilization (BD, #558050) and intracellular staining using a murine anti-pSTAT5 antibody conjugated with AF647 (BD, #562076). Cells were gates on lymphocytes, single cells (FSC-A vs FSC-H), single cells (SSC-A vs SSC-H) and, where applicable, RFP positive cells.

[0444] D2Rv3-modified T cells (Fig. 3A, D2Rv3 is identical to D2Rv2 except for the signal peptides) demonstrate a dose-dependent increase in STAT5 phosphorylation when stimulated with serial dilutions of DOTAM, although not to the extent of IL2 (Proleukin, Novartis) (Fig. 5B,C,D,E). The different engineered versions of D2R, i.e. , D2Rv3-8 (Fig. 3A,C), result in varying levels of STAT5 phosphorylation, all of which are still lower than the levels induced by natural IL-2 stimulation (Fig. 7B,C,D,E).

[0445] Example 4: Analysis of DOTAM-induced cellular proliferation in human primary D2R T cells

[0446] D2R-engineered T cells were labeled with CellTrace™ Violet dye to analyze DOTAM-induced proliferation. Cells were incubated for 6 days with a serial dilution of DOTAM or IL2 (Proleukin, Novartis) as a control. The cells were subsequently analyzed by flow cytometry to assess their proliferation profile. Proliferation was measured by a decrease in CellTrace™ Violet dye which is diluted out of the cells as a result of cell division.

[0447] To assess if transduced cells can be specifically expanded by DOTAM, the cells were seeded at different cell densities with respect to the percentage of RFP+ cells. More specifically, RPF+ cells were mixed with mock transduced cells to achieve 5 %, 20 %, 50 % and ~80 % RFP+ cell populations (Fig. 6A). Thereafter, the cells were incubated with different concentrations of DOTAM for 6 days at 37 °C 5 % CO2. The results indicate that D2Rv3-modified RFP+ T cells not only proliferate but can be specifically expanded, e.g., from 5 % to ~ 50 % RFP+ cells within 6 days in a given cell population (Fig. 6A). When incubated with IL2 (Proleukin, Novartis), the percentages of RFP+ cells and corresponding MFI do not change (Fig. 6A). When incubated with DOTAM, D2R+ RFP+ T cells proliferate, as assessed by CellTrace™ Violet staining and P39375-EP subsequent flow cytometry analysis. Higher molar concentrations of DOTAM are needed to induce a similar level of proliferation in RFP+ cells compared to IL2 (Proleukin, Novartis) (Fig. 6B,C). However, DOTAM enables specific proliferation of RFP+ cells (Fig. 6A), highlighting the orthogonality of the effect of DOTAM on D2R engineered cells.

[0448] In another experiment, different D2R ICD deletion variants, i.e., D2Rv4-8 (Fig. 3C) were compared. Expression was assessed only indirectly via RFP. All tested D2R-modified T cells were comparable with regard to RFP expression (Fig. 7A). Subsequent functional analysis revealed a varying degree of T cell proliferation (Fig. 8A,C) induced by the different D2R variants. IL2 (Proleukin, Novartis) was again used as a control (Fig. 8B,D) and none of the ICD-engineered D2R variants D2Rv4-8 outperformed the parental D2Rv3 variant. The proliferation data with D2Rv3-8 is largely in line with the STAT5 phosphorylation data (Fig. 7B,C).

[0449] Example 5: Expression and DOTAM-induced internalization of D12R in human primary T cells

[0450] D12R T cells were produced and cultured as described above. Expression of all tested D12R variants after transduction of human healthy donor primary T cells was indirectly assessed by RFP expression. D12R receptor constructs (Fig. 9A-C) exhibit transduction rates >70 % with respect to RFP (Fig. 10A,B). Cell surface expression of D12R was demonstrated by 2 different staining methods. Direct staining with DOTAM-FITC at 37 °C (Fig. 10A) implied that only a small fraction of RFP+ cells were D12R+, or the receptor expression was low. We hypothesized that D12Rs either exhibit low cell surface expression or our DOTAM-FITC staining method is suboptimal. To confirm that the DOTAM-FITC staining is specific for D12Rs, we pre-blocked D12Rs first with DOTAM and then applied the DOTAM-FITC staining. Indeed, preblocking D12Rs with DOTAM abolished the detection with DOTAM-FITC, indicating that the DOTAM-FITC staining was specific. However, indirect, stepwise D12R staining with DOTAM-biotin and Streptavidin- AF647 conjugate at 37 °C, later confirmed that over 90 % of RFP+ cells are also D12R+ (Fig. 10B). This finding stands in stark contrast to the data obtained with D2Rs described in Examples 2 and 3, where only low levels of D2Rs could be detected on the cell surface using any of the two staining methods. Notably, both, D2Rs as well as D12Rs followed the same design principles and only differ with regard to the cytokine receptor chains. Hence, we concluded that there are large differences in cell surface expression levels of D12R and D2R in engineered primary T cells. This finding is largely in line with published reports in which cytokine receptor expression on T cells were compared (7).

[0451] Internalization of D12R in engineered T cells upon stimulation with DOTAM-biotin was assessed indirectly by measuring the reduction of D12R staining by flow cytometry at 37 °C compared to 4 °C over time, using the DOTAM-biotin / Streptavidin-AF647 conjugated staining described above. No internalization of cell surface receptors is assumed to occur at 4 °C. 200'000 cells per condition were stained at 4 °C with DOTAM-biotin for each condition (4 °C and 37 °C). Afterwards, DOTAM-biotin-labeled cells were either incubated at 37 °C or 4 °C. Samples were taken at different time points and kept on ice. All samples were then stained with Streptavidin-AF647 conjugate at 4 °C and analyzed by flow cytometry. At the onset of the experiment, D12R cell surface staining was strong for both conditions (Fig. 10C). However, a ~ 10 % loss of D12Rs cell surface staining was observed within 30 minutes (Fig. 10C) for the D12R cells incubated at 37 °C, indicating a rapid initial internalization phase. Afterwards, the levels of D12Rs on the cell surface P39375-EP was relatively stable, implying that no further internalization occurs after the initial rapid internalization phase (Fig. 10C).

[0452] Individual chains of the D12R (D12Rv1A and D12Rv1 B, Fig. 9B) can be detected using a purified protein of the counterpart variable domain of the anti-DOTAM antibody and DOTAM-biotin, which can then be conjugated with Streptavidin-AF647 (Panels 4 and 5). This staining is specific to the individual chain (Fig. 10D), however, it is only possible in T cells which are separately transduced with constructs encoding only one D12R chain, i.e., either D12Rv1A or D12Rv2A. Otherwise, formation of DOTAM / D12R complexes interfere with the staining of individual chains. The staining may indicate that D12Rv1A exhibits higher cell surface expression levels than D12Rv2A in this specific setting.

[0453] Example 6: Analysis of STAT4 phosphorylation in human primary T cell engineered with D12Rs

[0454] Analysis of STAT4 phosphorylation induced by D12R signaling was done by flow cytometry. T cells transduced with D12R (Fig. 9A,B) were stimulated with serial dilutions of DOTAM for 40 min, followed by immediate fixation, permeabilization (see Example 2 and methods for details) and intracellular staining using a murine anti-pSTAT4 antibody conjugated with AF647 (BD, #558137). Cells were gates on lymphocytes, single cells (FSC-A vs FSC-H), single cells (SSC-A vs SSC-H) and, where applicable, RFP+ cells.

[0455] D12R-modified cells showed dose-dependent phosphorylation of STAT4 when stimulated with DOTAM (Fig. 11 A,B), although to a lower extent than IL12 which signals via endogenous IL12Rs (Fig. 11 A). A kinetic analysis of STAT4 phosphorylation was performed by incubating D12R T cells with DOTAM or IL12 for 1 h before washing the cells with PBS and subsequent pSTAT4 analysis at different time points. The cells were stained for pSTAT4 at different time points, i.e., at 60, 120, 240, 360 min or 24 h after the start of the experiment and analyzed by flow cytometry (Fig. 11 C). The analysis revealed that phosphorylation of STAT4 is highest within the first 2 hours of incubation and then decreases rapidly, akin to the stimulation with IL12.

[0456] Example 7: Functional screening of D12R-mediated IL12 signaling in engineered primary T cells

[0457] Functional analysis of D12Rs was done by flow cytometric analysis of secreted cytokines and cell surface markers which are known to be upregulated upon IL12 signaling. These include 1118Ra (8) and IL12Rp2 via a STAT4 mediated positive feed-forward loop (10), and secretion of IFNy. T cells transduced with a D12Rv1 and a RFP reporter protein (Fig. 12A) were stimulated with serial dilutions of DOTAM for 24 h. Following incubation, the cells were stained (Panel 1) and fixed for flow cytometric analysis. Cells were gated on lymphocytes, singlets (FSC-A vs FSC-H) and living cells expressing CD3 and, where applicable, RFP positive cells.

[0458] D12Rs are transduced and expressed in primary T cells (Fig. 12B). The cells show dose-dependent upregulation of IL18Ra upon stimulation with DOTAM (Fig. 12C). There is a statistically insignificant baseline effect when no DOTAM is present (paired t test: P-value=0.1447). Similarly, the IL12Rp2 is upregulated by stimulation of the D12Rv1 with DOTAM (Fig. 12D), consistent with a previously reported P39375-EP feed-forward mechanism, triggered by STAT4 signaling (10). In this example, D12R T cells show no baseline activation of IL12Rp2, similar to the wild type cells. In wildtype cells stimulated with IL12, the feedforward mechanism cannot be visualized in this manner because IL12 interferes with the anti-IL12Rp2 antibody (Biolegend, #394205).

[0459] The D12R-engineered T cells show dose-dependent secretion of IFNy when stimulated with DOTAM and (Fig. 12E). Notably, cells engineered with a D12R show a statistically insignificant baseline IFNy release without stimulation (paired one-way Anova: 95 % Cl, P-value = 0.2084). The EC50 obtained with DOTAM / D12R is generally higher than the ones obtained with IL12 signaling through WT IL12R, although the maximum signal obtained with DOTAM was reaching the level as with IL12.

[0460] Example 8: Validation of DOTAM-induced IL12 signaling in human primary T cells co-expressing CARs and D12Rs

[0461] Analysis of engineered T cells co-expressing an anti-human lgG1 Fc P329G adaptor CAR (P329G-CAR) and D12R but no downstream RFP reporter (Fig. 13A), was done by flow cytometric analysis of secreted cytokines and cell surface markers which are known to be upregulated upon IL12 signaling, as explained in Example 7. Following stimulation and incubation, the cells were stained (Panel 2) and fixed for flow cytometric analysis. Cells were gated on lymphocytes, singlets (FSC-A vs FSC-H) and living cells expressing CD3 and CAR.

[0462] Notably, transduction efficiency and CAR expression by P329G-CAR-D12R T cells was lower compared to cells expressing only the CAR (Fig. 13B). P329G-CAR-D12R T cells showed dose-dependent upregulation of IL18Ra upon stimulation with DOTAM (Fig. 13C). Similarly, IL12Rp2 was upregulated upon stimulation of the D12R with DOTAM (Fig. 13D). In this example, D12R T cells show no baseline activation of IL12Rp2.

[0463] CAR-D12R engineered immune cells also show dose-dependent secretion of IFNy when stimulated with DOTAM (Fig. 13E). Notably, cells engineered with a D12R show no baseline IFNy release without stimulation. Unlike with D12R T cells, maximum induction of IFNy release could not be achieved in P329G- CAR-D12R T cells, which may be attributable to the lower transduction efficiency (Fig. 13B).

[0464] Example 9: Cytotoxicity assays using CAR+D12R T cells

[0465] T cell mediated killing of target cells was assessed in the context of the P329G adaptor-CAR (Fig. 9C). Prior to the preparation of the killing assay, transduction efficiency of the immune cells was assessed by flow cytometry, as described in previous examples (Fig. 14A, 15A). Target cells, MKN-45-NLR or HPAF-II- NLR, were seeded in a 96-well plate with edge reservoir and incubated for at least 2 hours to adhere to the plate. Engineered cells were washed two times in PBS and normalized to the amount of CAR-positive cells by addition of wild-type cells before being added to the target cells. Engineered T cells were seeded at varying effector to target ratio (E:T) with a constant dilution of targeting IgG for adaptor-based CAR-T cells. Target cell killing was monitored for 5-7 days in an Incucyte® live-cell analysis system. Data was normalized to the initial cell count. Under suboptimal killing conditions, i.e., at low E:T ratios, the addition of 50 nM of P39375-EP

[0466] DOTAM to the P329G-CAR-D12R / tumor cell line co-culture leads to a 20-40 % increase in target cell killing, similar to the addition of IL12 (Fig. 14B,C; Fig. 15B,C).

[0467] Example 10: Expression and functional screening of D18Rs in human primary T cells

[0468] Primary T cells engineered with different D18Rs (Fig. 16, 17A) were produced and cultured according to methods mentioned in previous examples and methods. D18Rv1 contains the membrane-proximal extracellular domain 3 (D3) of the WT IL18 receptor alpha (IL18Ra) and IL18 receptor accessory protein (IL18RAP) to which the antibody variable domains were fused via a short linker. D18Rv2 also contains the D3 domains of the WT 1118Ra and IL18RAP but a slightly different sequence (compare protein and DNA sequences of D18Rv1_RFP, D18Rv2_RFP). These D3 domains were omitted in D18Rv3 compared to D18Rv1 and D18Rv2, i.e., the extracellular domains of the D18Rv3 consist only of antibody VH and VL domains which were fused via a short linker to the 3 extracellular amino acids of the transmembranedomains of IL18Ra and IL18RAP, respectively. Analysis of D18R T cells was done by flow cytometric analysis of RFP reporter expression (Fig. 18A) and HTRF-based cytokine quantification (Fig. 18B). Expression of D18R versions differs between 25-40 %. While both D18Rv1- and D18Rv2 T cells stimulated with DOTAM lead to release of IFNy, D18Rv3 T cells do not respond to DOTAM (Fig. 18B). IL18 (R&D Systems, #9124-IL-050 / CF) is used as a control. This exemplifies that some DOTAM receptor formats benefit from the extracellular domain to activate a signaling cascade downstream of the orthogonal D18R. D18Rv1 and D18Rv2-mediated IFNy response is additionally shown to be cooperative with IL12 stimulation (Fig. 18B) which is in line with published data (11).

[0469] Example 11 : Analysis of D18R expression, DOTAM -mediated D18R signaling and IFNy secretion in human primary T cells

[0470] T cells engineered to co-express CARs and D18Rs were analyzed by flow cytometry for CAR expression. CAR, and CAR-D18R constructs exhibited similar transduction efficiencies, 97.5 % and 87 %, respectively. However, the expression of the CAR in CAR-D18R cells, as determined by the MFI of the CAR+ population, was lower (Fig. 19A). Functional analysis of CAR-D18R cells was done by staining of phosphorylated Nf- KB, and HTRF-based IFNy quantification. Only CAR-D18R T cells but not the control CAR T cells, show dose-dependent phosphorylation Nf-KB upon stimulation with DOTAM (Fig 19B). DOTAM / D18R- and IL18 / IL18R-mediated phosphorylation of Nf-KB resulted in similar EC50 (Fig. 19B). Likewise, IFNy secretion mediated by D18R was highly comparable to the WT IL18R system in CAR-D18R T cells (Fig. 19C).

[0471] Example 12: Expression and functional screening of D23R in human primary T cells

[0472] Primary T cells engineered with IL23-derived D23Rs (Fig. 20, 21A) were produced and cultured according to methods mentioned in previous examples and methods. D23R T cells were produced and cultured according to methods mentioned in previous examples and methods. Cell surface expression D23Rs was assessed indirectly by GFP / RFP reporter expression. D23R chains were encoded on 2 different plasmids (Fig. 20B) and co-transduced in T cells. The double-positive population was greater than 60 % (Fig. 21 B). P39375-EP

[0473] The functionality of D23Rs was assessed by intracellular staining of phosphorylated STAT3, STAT4, STAT5 and flow cytometry analysis. While the cell population only positive for either the chimeric IL12Rp1 chain (D23RA) or the chimeric IL23R chain (D23RB) did not bind DOTAM, the double positive population did (Fig. 21 B). The expression of D23RB itself but not D23RA led to high baseline levels of phosphorylated STAT3 (Fig. 21 C). Only the double positive population shows dose-dependent phosphorylation of STAT3 when stimulated with DOTAM (Fig. 21 C). Similarly but not as pronounced, expression of D23RB shows a baseline STAT4 phosphorylation and dose-dependent additional phosphorylation of STAT4 when stimulated with DOTAM (Fig. 21 D). Conversely, D23Rs shows a minimal baseline phosphorylation and strong dose-dependent additional phosphorylation of STAT5 when stimulated with DOTAM (Fig.21 E).

[0474] Example 13: Analysis of DOTAM-mediated increased Granzyme B release from D23R-engineered human primary T cells

[0475] Analysis of granzyme B-release from D23R-modified T cells was done by flow cytometric analysis using a commercially available multiplex cytokine assay (Legendplex assay, #741187, Biolegend). T cells engineered with a D23R, but not WT T cells, respond to stimulation with DOTAM by increased granzyme B release (Fig. 22).

[0476] The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sktodowska-Curie grant agreement No 955575.

[0477] Example 14: Characterization of a further DOTAM binder comprising an improved variable heavy domain

[0478] Functional analysis of other variable domain frameworks for DOTAM-IL12R constructs was done using methods previously outlined. In brief, primary T cells were transduced using conventional lentiviral particles encoding D12R variants followed by an RFP reporter gene. Transduction efficiency was assessed by measuring the frequency of RFP positive T cells using flow cytometry. IL12 signaling via DOTAM / D12R variants was assessed by flow cytometry and measurement of cytokine secretion from supernatants, based on previously outlined markers (IL18Ra, IFNg). Cells were gated on lymphocytes, singlets (FSC-A vs FSC- H) and living cells expressing CD3 and the RFP reporter. Both DOTAM-IL12R variants showed dosedependent upregulation of IL18Ra and IFNg secretion upon stimulation with DOTAM. For one variant (V2) the ECmax of IL18Ra upregulation was reduced compared to V1 and IL12 control (Fig. 23).

[0479] Example 15: Functional analysis of the D2R was conducted using various variable domain framework (VH) constructs.

[0480] HEK-Blue™ IL2 cells, transfected with D2R constructs as previously described, were stimulated with varying concentrations of DOTAM. DOTAM-IL2R constructs B2 (D1AK2285), B3 (D1AK2283), B4 (D1AK2074), and B5 (D1AK2072) exhibited constitutive activity even without DOTAM and showed a small P39375-EP dose-dependent response to DOTAM. In contrast, D2R B1 (D1AJ2672) displayed minimal constitutive activity and a dose-response to DOTAM. HEK-Blue™ IL2 cells unmodified were used as a negative control. Depicted are technical average values from triplicates, error bars indicate standard deviation (Fig. 24).

[0481] IV. NUMBERED CLAUSES

[0482] 1 . A recombinant receptor polypeptide comprising:

[0483] (i) an extracellular portion comprising an antigen binding domain capable of binding to a small molecule;

[0484] (ii) an intracellular portion comprising the intracellular domains of a cytokine receptor; and

[0485] (iii) a transmembrane domain that joins the extracellular portion to the intracellular portion.

[0486] 2. The recombinant receptor polypeptide of clause 1 , wherein the antigen binding domain of the extracellular portion comprises a light chain variable region (VL) or a heavy chain variable region (VH) of an antibody capable of specific binding to said small molecule.

[0487] 3. The recombinant receptor polypeptide of clause 1 or 2, wherein the small molecule is DOTAM or a DOTAM derivative.

[0488] 4. The recombinant receptor polypeptide of any of clauses 1-3, wherein DOTAM or DOTAM derivative chelates a two-fold positively charged metal ion, in particular wherein the charged metal ion is Ca2+.

[0489] 5. The recombinant receptor polypeptide of any of clauses 1-4, wherein the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108.

[0490] 6. The recombinant receptor polypeptide of any of clauses 1-4, wherein the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 165, and a HCDR 3 of SEQ ID NO: 108.

[0491] 7. The recombinant receptor polypeptide of any of clauses 1-6, wherein the VL comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 11 1.

[0492] 8. The recombinant receptor polypeptide of any of clauses 1-5, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2.

[0493] 9. The recombinant receptor polypeptide of any of clauses 1-7, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence of selected from the group consisting of SEQ ID NOs: 2, 157, 160, 161 , 162, and 163.

[0494] 10. The recombinant receptor polypeptide of any of clauses 1-9, wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8. P39375-EP

[0495] 11. The recombinant receptor polypeptide of any of clauses 1-10, wherein the cytokine receptor is IL2R, IL7R, IL12R, IL18R or IL23R.

[0496] 12. The recombinant receptor polypeptide of any of clauses 1-11 , wherein the transmembrane domain comprises the transmembrane domain of the cytokine receptor.

[0497] 13. The recombinant receptor polypeptide of any of clauses 1-12, further comprising a signal sequence.

[0498] 14. The recombinant receptor polypeptide of clause 13, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 26.

[0499] 15. The recombinant receptor polypeptide of any of clauses 1-14, further comprising one or more linkers.

[0500] 16. The recombinant receptor polypeptide of clause 15, wherein the linker is a GS linker, a 2A linker, an a- helical linker, a glycine-alanine polymer linker, an alanine-serine polymer linker, or an lgG4-Fc linker.

[0501] 17. The recombinant receptor polypeptide of any of clauses 1-16, further comprising a tag polypeptide, e.g. a Flag tag.

[0502] 18. The recombinant receptor polypeptide of any of clauses 1-17, further comprising a marker, e.g. a fluorescent polypeptide, e.g. GFP, eGFP, or RFP.

[0503] 19. The recombinant receptor polypeptide of any of clauses 1-18, comprising from the N-terminus to the C- terminus:

[0504] Optional signal peptide - optional tag - optional linker - antigen binding domain - optional linker - transmembrane domain - intracellular portion comprising the intracellular domain(s) of a wild-type cytokine receptor chain - optional linker - optional marker.

[0505] 20. The recombinant receptor polypeptide of any of clauses 1-19, wherein the cytokine receptor is IL2R.

[0506] 21. The recombinant receptor polypeptide of clause 20, wherein the extracellular domain comprises an amino acid sequence selected from SEQ ID NO: 27 and SEQ ID NO: 31 , or an amino acid sequence having at least 80% sequence identity thereto.

[0507] 22. The recombinant receptor polypeptide of clause 20 or 21 , wherein the extracellular domain comprises an amino acid sequence of SEQ ID NO: 28 or SEQ ID NO: 32, located between the transmembrane domain and the antigen binding domain.

[0508] 23. The recombinant receptor polypeptide of any of clauses 20-22, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 29 and SEQ ID NO: 33, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of confining the recombinant receptor polypeptide to the membrane.

[0509] 24. The recombinant receptor polypeptide of any of clauses 20-23, wherein the intracellular domain comprises an amino acid sequence selected from SEQ ID Nos: 30, 34, 35, 36, 37, 38 and 39, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling.

[0510] 25. The recombinant receptor polypeptide of any of clauses 20-24, wherein the recombinant receptor polypeptide comprises an amino acid sequence selected from SEQ ID Nos: 112-120, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling. P39375-EP

[0511] 26. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of clauses 20-25 and a second recombinant receptor polypeptides according to any one of clauses 20-25, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0512] 27. The recombinant receptor of clause 26, capable of initiating a signal transduction pathway analogous to IL2R, upon binding the small molecule to the antigen binding domains of the extracellular portion.

[0513] 28. The recombinant receptor of clause 26 or 27, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

[0514] 29. The recombinant receptor of any of clauses 26-28, comprising i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence selected from SEQ ID Nos: 30, 36, 37, 38 and 39, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence selected from SEQ ID Nos: 34 and 35, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling.

[0515] 30. The recombinant receptor of clause 29, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0516] 31 . The recombinant receptor of clause 29, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0517] 32. The recombinant receptor of clause 29, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0518] 33. The recombinant receptor of clause 29, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH P39375-EP comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0519] 34. The recombinant receptor of any one of clauses 30-33, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0520] 35. The recombinant receptor of any one of clauses 30-33, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, 160, 161 , 162, and 163; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0521] 36. The recombinant receptor of any of clauses 26-35, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 112, 114, 1 16, 1 17, 118, 119, and 120, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling.

[0522] 37. The recombinant receptor of any of clauses 26-35, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, the amino acid sequence of SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling.

[0523] 38. The recombinant receptor polypeptide of any of clauses 1-19, wherein the cytokine receptor is IL7R.

[0524] 39. The recombinant receptor polypeptide of clause 38, wherein the extracellular domain comprises an amino acid sequence selected from SEQ ID NO: 31 and SEQ ID NO: 40, or an amino acid sequence having at least 80% sequence identity thereto.

[0525] 40. The recombinant receptor polypeptide of clause 38 or 39, wherein the extracellular domain comprises an amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 41 , located between the transmembrane domain and the antigen binding domain

[0526] 41. The recombinant receptor polypeptide of any of clauses 38-40, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 33 and SEQ ID NO: 42, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of confining the recombinant receptor polypeptide to the membrane. P39375-EP

[0527] 42. The recombinant receptor polypeptide of any of clauses 38-41 , wherein the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 34 and SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity thereto.

[0528] 43. The recombinant receptor polypeptide of any of clauses 38-42, wherein the recombinant receptor polypeptide comprises an amino acid sequence selected from SEQ ID Nos: 113, 115, 128 and 129, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling.

[0529] 44. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of claims 38-43 and a second recombinant polypeptide according to any one of claims 38-43, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0530] 45. The recombinant receptor of clause 44, capable of initiating a signal transduction pathway analogous to the wild-type cytokine receptor, upon binding the small molecule to the antigen binding domains of the extracellular portion.

[0531] 46. The recombinant receptor of clause 44 or 45, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

[0532] 47. The recombinant receptor of any of clauses 44-46, comprising i) a first recombinant receptor polypeptide comprising an intracellular domain comprising the amino acid sequence of SEQ ID Nos: 34, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling.

[0533] 48. The recombinant receptor of clause 47, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0534] 49. The recombinant receptor of clause 47, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0535] 50. The recombinant receptor of clause 47, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH P39375-EP comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0536] 51. The recombinant receptor of clause 47, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0537] 52. The recombinant receptor of any one of clauses 48-51 , wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0538] 53. The recombinant receptor of any one of clauses 48-51 , wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, 160, 161 , 162, and 163; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0539] 54. The recombinant receptor of any of clauses 44-53, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 128 and SEQ ID NO: 129, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling.

[0540] 55. The recombinant receptor polypeptide of any of clauses 1-19, wherein the cytokine receptor is IL18R.

[0541] 56. The recombinant receptor polypeptide of clause 55, wherein the extracellular domain comprises an amino acid sequence selected from SEQ ID Nos: 44, 47 and 57, or an amino acid sequence having at least 80% sequence identity thereto.

[0542] 57. The recombinant receptor polypeptide of clause 55 or 56, wherein the extracellular domain comprises an amino acid sequence of SEQ ID NO: 121 or SEQ ID NO: 122, located between the transmembrane domain and the antigen binding domain.

[0543] 58. The recombinant receptor polypeptide of any of clauses 55-57, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 45 and SEQ ID NO: 48, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of confining the recombinant receptor polypeptide to the membrane. P39375-EP

[0544] 59. The recombinant receptor polypeptide of any of clauses 55-58, wherein the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 46 and SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL18 signaling.

[0545] 60. The recombinant receptor polypeptide of any of clauses 55-59, wherein the recombinant receptor polypeptide comprises an amino acid sequence selected from SEQ ID NO: 123, 124, 125, 126 and 127, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of I L18 signaling.

[0546] 61. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of claims 55-60 and a second recombinant polypeptide according to any one of claims 55-60, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0547] 62. The recombinant receptor of clause 61 , capable of initiating a signal transduction pathway analogous to the wild-type cytokine receptor, upon binding the small molecule to the antigen binding domains of the extracellular portion.

[0548] 63. The recombinant receptor of clause 61 or 62, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

[0549] 64. The recombinant receptor of any of clauses 61-63, comprising i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 46, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL18 signaling; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 49, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL18 signaling.

[0550] 65. The recombinant receptor of clause 64, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0551] 66. The recombinant receptor of clause 64, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0552] 67. The recombinant receptor of clause 64, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH P39375-EP comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0553] 68. The recombinant receptor of clause 64, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0554] 69. The recombinant receptor of any one of clauses 65-68, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0555] 70. The recombinant receptor of any one of clauses 65-68, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, 160, 161 , 162, and 163; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0556] 71 . The recombinant receptor of any of clauses 61-70, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 123, 125, and 126, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 124 and SEQ ID NO: 127, or an amino acid sequence having at least 80% sequence identity thereto.

[0557] 72. The recombinant receptor polypeptide of any of clauses 1-19, wherein the cytokine receptor is IL12R.

[0558] 73. The recombinant receptor polypeptide of clause 72, wherein the extracellular domain comprises an amino acid sequence selected from SEQ ID NO: 14 and SEQ ID NO: 15, or an amino acid sequence having at least 80% sequence identity thereto.

[0559] 74. The recombinant receptor polypeptide of clause 72 or 73, wherein the extracellular domain comprises an amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 9, located between the transmembrane domain and the antigen binding domain.

[0560] 75. The recombinant receptor polypeptide of any of clauses 72-74, wherein the transmembrane domain comprises an amino acid sequence selected from SEQ ID NO: 5 and SEQ ID NO: 10, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of confining the recombinant receptor polypeptide to the membrane. P39375-EP

[0561] 76. The recombinant receptor polypeptide of any of clauses 72-75, wherein the intracellular domain comprises an amino acid sequence selected from SEQ ID NO: 6 and SEQ ID NO: 11 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling.

[0562] 77. The recombinant receptor polypeptide of any of clauses 72-76, wherein the recombinant receptor polypeptide comprises an amino acid sequence selected from SEQ ID NO: 130 and SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling.

[0563] 78. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of claims 72-77 and a second recombinant polypeptide according to any one of claims 72-77, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0564] 79. The recombinant receptor of clause 78, capable of initiating a signal transduction pathway analogous to the wild-type cytokine receptor, upon binding the small molecule to the antigen binding domains of the extracellular portion.

[0565] 80. The recombinant receptor of clause 78 or 79, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

[0566] 81 . The recombinant receptor of any of clauses 78-80, comprising i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 1 1 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling.

[0567] 82. The recombinant receptor of clause 81 , wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0568] 83. The recombinant receptor of clause 81 , wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0569] 84. The recombinant receptor of clause 81 , wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH P39375-EP comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0570] 85. The recombinant receptor of clause 81 , wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0571] 86. The recombinant receptor of any one of clauses 82-85, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0572] 87. The recombinant receptor of any one of clauses 82-85, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, 160, 161 , 162, and 163; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0573] 88. The recombinant receptor of any of clauses 78-87, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto.

[0574] 89. The recombinant receptor of any of clauses 78-87, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 158, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto.

[0575] 90. The recombinant receptor polypeptide of any of clauses 1-19, wherein the cytokine receptor is IL23R.

[0576] 91. The recombinant receptor polypeptide of clause 90, wherein the extracellular domain comprises an amino acid sequence of SEQ ID NO: 50, located between the transmembrane domain and the antigen binding domain.

[0577] 92. The recombinant receptor polypeptide of clause 90 or 91 , wherein the transmembrane domain comprises an amino acid sequence of SEQ ID NO: 51 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of confining the recombinant receptor polypeptide to the membrane. P39375-EP

[0578] 93. The recombinant receptor polypeptide of any of clauses 90-92, wherein the intracellular domain comprises an amino acid sequence selected from SEQ ID NO:6 and SEQ ID NO: 52, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL23 signaling.

[0579] 94. The recombinant receptor polypeptide of any of clauses 90-93, wherein the recombinant receptor polypeptide comprises an amino acid sequence selected from SEQ ID NO: 130 and SEQ ID NO: 132, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL23 signaling.

[0580] 95. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of claims 90-94 and a second recombinant polypeptide according to any one of claims 90-94, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

[0581] 96. The recombinant receptor of clause 95, capable of initiating a signal transduction pathway analogous to the wild-type cytokine receptor, upon binding the small molecule to the antigen binding domains of the extracellular portion.

[0582] 97. The recombinant receptor of clause 95 or 96, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

[0583] 98. The recombinant receptor of any of clauses 95-97, comprising i) a first recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid comprising SEQ ID NO: 6, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL23 signaling; and ii) a second recombinant receptor polypeptide comprising an intracellular domain comprising an amino acid sequence comprising SEQ ID NO: 52, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL23 signaling.

[0584] 99. The recombinant receptor of clause 98, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0585] 100. The recombinant receptor of clause 98, wherein i) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

[0586] 101. The recombinant receptor of clause 98, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH P39375-EP comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0587] 102. The recombinant receptor of clause 98, wherein i) the second recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VH comprising a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 164, and a HCDR 3 of SEQ ID NO: 108; and wherein ii) the first recombinant receptor polypeptide comprises an extracellular domain comprising an antigen binding domain, said domain comprising a VL comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 1 11.

[0588] 103. The recombinant receptor of any one of clauses 99-102, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0589] 104. The recombinant receptor of any one of clauses 99-102, wherein i) the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, 160, 161 , 162, and 163; and / or ii) the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0590] 105. The recombinant receptor of any of clauses 95-104, wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 132, or an amino acid sequence having at least 80% sequence identity thereto.

[0591] 106. A nucleic acid, or a plurality of nucleic acids, encoding a recombinant receptor polypeptide or a recombinant receptor according to any one of clauses 1-105, and optionally further comprising a nucleic acid sequence encoding a chimeric antigen receptor.

[0592] 107. An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to clause 106.

[0593] 108. A cell comprising a chimeric receptor polypeptide according to any of clauses 1-105, a nucleic acid or a plurality of nucleic acids according to clause 106, or an expression vector or a plurality of expression vectors according to clause 107.

[0594] 109. The cell of clause 108, wherein the cell is a eukaryotic cell.

[0595] 110. The cell of clause 109, wherein the eukaryotic cell is an animal cell.

[0596] 111. The cell of clause 1 10, wherein the animal cell is a mammalian cell. P39375-EP

[0597] 112. The cell of clause 1 11 , wherein the mammalian cell is a human cell.

[0598] 113. The cell of clause 11 1 or 112, wherein the cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell.

[0599] 114. The cell of clause 1 13, wherein the cell is an immune cell.

[0600] 115. The immune cell of clause 1 14, wherein the immune cell is a tumor infiltrating lymphocyte (TIL), B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a Natural Killer T (NKT) cell, or other T cell.

[0601] 116. The cell of clause 1 14 or 115, further comprising a chimeric antigen receptor (CAR).

[0602] 117. The cell of any of clauses 114-116, further comprising an engineered TCR.

[0603] 118. The cell of any of clauses 108-117, wherein the cell is a transduced T cell capable of expressing the recombinant receptor polypeptide according to any one of clauses 1-105.

[0604] 119. The cell of any of clauses 108-118, wherein the cell expresses an antigen binding receptor comprising an anchoring transmembrane domain and an extracellular domain, wherein the extracellular domain comprises an antigen binding moiety comprising

[0605] (i) a heavy chain variable domain (VH) comprising a heavy chain complementary determining region (HCDR) 1 of SEQ ID NO: 133, a HCDR 2 of SEQ ID NO: 134 or SEQ ID NO: 135, and a HCDR 3 of SEQ ID NO: 136, and

[0606] (ii) a light chain variable domain (VL) comprising a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 137, a LCDR 2 of SEQ ID NO: 138 and a LCDR 3 of SEQ ID NO: 139.

[0607] 120. The cell of clause 119, wherein the VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 140, SEQ ID NO: 141 , SEQ ID NO: 142 and SEQ ID NO: 143.

[0608] 121. The cell of clause 119 or 120, wherein the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 144 or SEQ ID NO: 145.

[0609] 122. The cell of any of clauses 119-121 , wherein the anchoring transmembrane domain is a transmembrane domain selected from the group consisting of the CD8, the CD4, the CD3z, the FCGR3A, the NKG2D, the CD27, the CD28, the CD137, the 0X40, the ICOS, the DAP10 or the DAP12 transmembrane domain or a fragment thereof, in particular wherein the anchoring transmembrane domain is the CD8 transmembrane domain or a fragment thereof and that is capable of confining the antigen binding receptorto the membrane.

[0610] 123. The cell of any of clauses 119-122, wherein the antigen binding receptor further comprises at least one stimulatory signaling domain and / or at least one co-stimulatory signaling domain.

[0611] 124. The cell of any of clauses 119-123, wherein in the antigen binding receptor at least one stimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD3z, of FCGR3A and of NKG2D, or fragments thereof that retains stimulatory signaling activity, in particular P39375-EP wherein the at least one stimulatory signaling domain is the CD3z intracellular domain or a fragment thereof that retains CD3z stimulatory signaling activity.

[0612] 125. The cell of any of clauses 119-124, wherein in the antigen binding receptor the at least one costimulatory signaling domain is individually selected from the group consisting of the intracellular domain of CD27, of CD28, of CD137, of 0X40, of ICOS, of DAP10 and of DAP12, or fragments thereof that retain costimulatory signaling activity.

[0613] 126. The cell of any of clauses 119-125, wherein the antigen binding receptor comprises at least one CD28 costimulatory domain or a fragment thereof that retains CD28 co-stimulatory activity, and / or at least one CD137 costimulatory domain or a fragment thereof that retains CD137 co-stimulatory activity.

[0614] 127. The cell of any of clauses 119-126, wherein the antigen binding receptor comprises a stimulatory signaling domain comprising the intracellular domain of CD3z, or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises a co-stimulatory signaling domain comprising the intracellular domain of CD28, or a fragment thereof that retains CD28 co-stimulatory signaling activity.

[0615] 128. The cell of any of clauses 119-127, wherein the antigen binding receptor comprises one stimulatory signaling domain comprising the intracellular domain of CD3z, or a fragment thereof that retains CD3z stimulatory signaling activity, and wherein the antigen binding receptor comprises one co-stimulatory signaling domain comprising the intracellular domain of CD137, or a fragment thereof that retains CD137 co-stimulatory signaling activity.

[0616] 129. The cell of any of clauses 119-128, wherein the antigen binding moiety is connected at the C-terminus to the N-terminus of the anchoring transmembrane domain, optionally through a peptide linker.

[0617] 130. The cell of any of clauses 119-129, wherein in the antigen binding receptor the light chain variable domain (VL) of the antigen binding moiety is connected at the C-terminus to the N-terminus of the anchoring transmembrane domain, optionally through a peptide linker, and / or wherein the heavy chain variable domain (VH) is connected at the C-terminus to the N-terminus of the light chain variable domain (VL), optionally through a peptide linker.

[0618] 131. A pharmaceutical composition comprising the cell of any one of clauses 108-130, and optionally a pharmaceutically acceptable excipient.

[0619] 132. A recombinant receptor or a recombinant receptor polypeptide according to clauses 1-105, or a nucleic acid according to clause 106, or an expression vector according to clause 107, or a cell according to any of clauses 108-130, or a pharmaceutical composition according to clause 131 , for use in a method of medical treatment of a disease or prophylaxis of a disease.

[0620] 133. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, expression vector, cell, or pharmaceutical composition for use according to clause 132, wherein the method is a cell therapy, e.g. an adoptive cell therapy.

[0621] 134. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, expression vector, cell, or pharmaceutical composition for use according to clause 132 or 133, wherein the chimeric receptor P39375-EP polypeptide, nucleic acid, expression vector, cell, or pharmaceutical composition is administered intravenously, intratumorally, or subcutaneously.

[0622] 135. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, expression vector, cell, or pharmaceutical composition for use according to any of clauses 132-134, wherein the disease is a cancer,

[0623] 5 an autoimmune disease, or an infection.

[0624] 136. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, expression vector, cell, or pharmaceutical composition for use according to clause 135, wherein the cancer is a solid tumor.

[0625] 137. A method for modulating the activity of an immune cell, said method comprising: administering, to an immune cell, the nucleic acid or plurality of nucleic acids of clauses 87, or the expression vector or plurality

[0626] 10 of expression vectors of clause 107.

[0627] 138. The method of clause 137, wherein the immune cell is a tumor infiltrating lymphocyte (TIL), B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell (Treg), a helper T cell (Th), a cytotoxic T cell (Tctl), an effector T cell, a memory T cell, a Natural Killer T (NKT) cell, or other T cell.

[0628] 15 139. A method of treating a disease in a subject, comprising administering to the subject the cell according to claim 108-130.

[0629] 140. A method for depleting or killing a cell, comprising contacting the cell with a cell according to any one of claims 108-130.

[0630] 141. The recombinant receptor according to any one of claims 1-105, the nucleic acid according to claim

[0631] 20 106, the expression vector according to claim 107, or the cell according to any one of claims 108-130 for the manufacture of a medicament for the treatment of a disease, in particular wherein the disease is a cancer, an autoimmune disease, or an infection.

[0632] 25 IV. EXEMPLARY SEQUENCES

[0633] Table 1 : CDR definition according to Kabat P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP P39375-EP

Claims

P39375-EPClaims1 . A recombinant receptor polypeptide comprising:(i) an extracellular portion comprising an antigen binding domain capable of binding to a small molecule;(ii) an intracellular portion comprising the intracellular domains of a cytokine receptor; and(iii) a transmembrane domain that joins the extracellular portion to the intracellular portion.

2. The recombinant receptor polypeptide of claim 1 , wherein the antigen binding domain of the extracellular portion comprises a light chain variable region (VL) or a heavy chain variable region (VH) of an antibody capable of specific binding to said small molecule.

3. The recombinant receptor polypeptide of claim 1 or 2, wherein the small molecule is DOTAM or a DOTAM derivative.

4. The recombinant receptor polypeptide of any of claims 1 -3, wherein(a) the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 107, and a HCDR 3 of SEQ ID NO: 108, and / or wherein the VL comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 ; or(b) the VH comprises a heavy chain complementarity determining region (HCDR) 1 of SEQ ID NO: 106, a HCDR 2 of SEQ ID NO: 165, and a HCDR 3 of SEQ ID NO: 108, and / or wherein the VL comprises a light chain complementarity determining region (LCDR) 1 of SEQ ID NO: 109, a LCDR 2 of SEQ ID NO: 110, and a LCDR 3 of SEQ ID NO: 111 .

5. The recombinant receptor polypeptide of any of claims 1 -4, wherein the VH comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 157, SEQ ID NO: 160, SEQ ID NO: 161 , SEQ ID NO: 162, and SEQ ID NO: 163, and wherein the VL comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8.

6. The recombinant receptor polypeptide of any of claims 1 -5, wherein the cytokine receptor is IL2R, IL7R, IL12R, IL18R or IL23R.

7. A recombinant receptor comprising a first recombinant receptor polypeptides according to any one of claims 1-6 and a second recombinant polypeptide according to any one of claims 1 -6, wherein the receptor is capable of specific binding to the small molecule, in particular wherein the small molecule is DOTAM.

8. The recombinant receptor of claim 7, wherein the first recombinant receptor polypeptide comprises the VL, and the second recombinant receptor polypeptide comprises the VH, or wherein the first recombinant receptor polypeptide comprises the VH, and the second recombinant receptor polypeptide comprises the VL.

9. The recombinant receptor of claim 8, whereinP39375-EP a) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 112, 1 14, 1 16, 1 17, 118, 119, and 120, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling; or b) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 113 and SEQ ID NO: 115, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 128 and SEQ ID NO: 129, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL7 signaling; or c) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID Nos: 123, 125, and 126, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 124 and SEQ ID NO: 127, or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL18 signaling; or d)(i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling and that is capable of IL12 signaling; or e) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 130, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 132, or an amino acid sequence having at least 80% sequence identity thereto thereto and that is capable of IL23 signaling; or f) i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence selected from SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, and SEQ ID NO: 168, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, the amino acid sequence of SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL2 signaling. g) wherein i) the first recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 158, or an amino acid sequence having at least 80% sequence identity thereto; and ii) the second recombinant receptor polypeptide comprises, e.g. consists of, an amino acid sequence comprising SEQ ID NO: 131 , or an amino acid sequence having at least 80% sequence identity thereto and that is capable of IL12 signaling and that is capable of IL12 signaling.

10. A nucleic acid, or a plurality of nucleic acids, encoding a recombinant receptor polypeptide or a recombinant receptor according to any one of claims 1-9, and optionally further comprising a nucleic acid sequence encoding a chimeric antigen receptor.P39375-EP11 . An expression vector, or a plurality of expression vectors, comprising a nucleic acid or a plurality of nucleic acids according to claim 10.

12. A cell comprising a chimeric receptor polypeptide according to any of claims 1 -9, a nucleic acid or a plurality of nucleic acids according to claim 10, or an expression vector or a plurality of expression vectors according to claim 11 .

13. A pharmaceutical composition comprising the cell of claim 12, and optionally a pharmaceutically acceptable excipient.

14. A recombinant receptor or a recombinant receptor polypeptide according to any one of claims 1-9, or a nucleic acid according to claim 10, or an expression vector according to claim 11 , or a cell according to claim 12, or a pharmaceutical composition according to claim 13, for use in a method of medical treatment of a disease or prophylaxis of a disease.

15. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, vector, cell, or pharmaceutical composition for use according to claim 14, wherein the method is a cell therapy, e.g. an adoptive cell therapy.

16. The recombinant receptor polypeptide, recombinant receptor, nucleic acid, vector, cell, or pharmaceutical composition for use according to claim 14 or 15, wherein the disease is a cancer, an autoimmune disease, or an infection.

17. A method of treating a disease in a subject, comprising administering to the subject the cell according to claim 12.

18. A method for depleting or killing a cell, comprising contacting the cell with a cell according to claim 12.

19. The recombinant receptor according to any one of claims 1-9, the nucleic acid according to claim 10, the expression vector according to claim 11 , or the cell according to claim 12 for the manufacture of a medicament for the treatment of a disease, in particular wherein the disease is a cancer, an autoimmune disease, or an infection.

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