IL7R ALPHA SIGNALLING DOMAIN, CHIMERIC ANTIGEN RECEPTOR AND USES THEREOF FIELD OF THE INVENTION The present invention relates to Interleukin-7 receptor-α (IL7Rα) signalling domain, receptors, especially chimeric antigen receptor (CAR), nucleic acids, expression vectors, engineered immune cell such as a T cell, compositions and method of using such CARs and cells expressing them in treating diseases. BACKGROUND The IL-7/IL7R signalling pathway plays critical roles in the development, maintenance, and proliferation of T lymphocytes. The interleukin-7 receptor (IL7R) is expressed in various cell types, including naive and memory T cells, and plays a critical role in the development of immune cells. IL7R is a heterodimer and consists of two subunits, interleukin-7 receptor-α (IL7Ra) and common-γ chain receptor. Upon binding of IL-7, IL-7Ra dimerizes with the common cytokine g chain and triggers kinase activation. The signalling of IL-7/IL-7R is mainly transduced by the Janus kinase JAK-STAT and Phosphoinositide 3-kinase (PI3K)-Ak strain transforming (AKT) pathways in T cells. Chimeric antigen receptor (CAR) is a synthetic cell receptor consisting of an antigen binding domain, a transmembrane domain and an intracellular signalling domain. Adoptive cell transfer therapy with T cells modified to express chimeric antigen receptors (CARs) has shown effectiveness and promising in trials of cancer treatment. However, numerous challenges remain including exhaustion of modified immune cells. Thus, a need exists for improved immune cell therapies with improved properties of modified immune cells. SUMMARY OF THE INVENTION In the first aspect, the present invention provides an IL7Ra signalling domain comprising a mutation at the amino acid position that corresponds to position 300 of the human wild type IL7Ra (i.e. SEQ ID NO:1). In some embodiments, the mutation is selected from a list comprising P300H, P300A, P300W, P300E, P300L and P300Q, preferably the mutation is P300H. In some embodiments, the IL7Ra signalling domain as described herein has a length of 20-30 amino acids, which comprises BOX1 (i.e. SEQ ID NO: 80). In some embodiments, the IL7Ra signalling domain does not have any mutation compared to SEQ ID NO:1. In some preferred embodiments, the IL7Ra signalling domain comprises SEQ ID NO:188 The present invention also provides an IL7Ra signalling domain, preferably according to the IL7Ra signalling domain as described above, which is able to activate STAT3 and STAT5.
Therefore in an embodiment, the present invention provides an IL7Ra signalling domain comprising a mutation at position 36 of SEQ ID NO:5 and/or which is able to activate STAT3 and STAT5. In a preferred embodiment, the IL7Ra signalling domain comprises the mutation selected from a list comprising P36H, P36A, P36W, P36E, P36L, and P36Q when referring to SEQ ID NO:5 and/or is able to activate STAT3 and STAT5. In some embodiments, the IL7Ra signalling domain comprises the following STAT3 binding site: YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF or YRHA (represented as SEQ ID NOs:104-166), preferably wherein this STAT3 binding site is present at amino acid positions that corresponds to positions 456- 459 of the human wild type IL7Ra (SEQ ID NO:1). In an embodiment, the IL7Ra signalling domain as described herein, comprising a STAT3 binding site, is such that the IL7Ra signalling domain comprises two mutations compared to the human wild type IL7Ra counterpart represented by SEQ ID NO:1; these two mutations being at amino acids positions that correspond to positions 457 and 458 of the human wild type IL7Ra (i.e. SEQ ID NO:1). The amino acid positions 457 and 458 of the human wild type IL7Ra correspond to amino acid positions 79 and 80 of a truncated IL7Ra signaling domain such as SEQ ID NO:2, 3, 4 or 5. The amino acid present at positions 457 and 458 of the human wild type IL7Ra are Q (Glutamine) and N (Asparagine) respectively. In a preferred embodiment, the two mutations present in the IL7Ra signaling domain correspond to mutations Q457R and N458H when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). Therefore in an embodiment, the present invention provides an IL7Ra signalling domain comprising mutations at positions 79 and 80 of SEQ ID NO:5. In a preferred embodiment, the IL7Ra signalling domain comprises the mutations Q79R and N80H in SEQ ID NO:5. In an embodiment, the IL7Ra signalling domain as described herein is represented by an amino acid sequence which has at least 80% identity with SEQ ID NO:2, 3 and/or 4, preferably wherein the mutation corresponding to the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in the sequence derived from SEQ ID NO:2 and 4, the mutations corresponding to the Q457R and N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) are still present in the sequence derived from SED ID NO:3 and 4 and the mutations corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:4. In some embodiments, The IL7Ra signalling domain as defined herein is represented by an amino acid sequence which has at least 80% identity with any one of SEQ ID NOs: 178-187, preferably wherein the mutation corresponding to the P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) is still present in the sequence derived from SEQ ID NOs: 178-187, and the mutations corresponding to any one of P300A, P300W, P300E, P300L and P300Q
mutation, and Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in the sequence derived from SEQ ID NOs: 179, 181, 183, 185 and 187. Provided herein is also an IL7Ra signalling domain, preferably as defined above, which is able to activate STAT4. Said IL7Ra signalling domain comprises the following STAT4 binding site: - YLPSNID (SEQ ID NOs:189), preferably wherein this STAT4 binding site is present at amino acid positions that corresponds to positions 460-466 of the human wild type IL7Ra (SEQ ID NO:1), - TX1X2GYL (SEQ ID NOs:190), preferably wherein this STAT4 binding site is present at amino acid positions that corresponds to positions 460-465 of the human wild type IL7Ra (SEQ ID NO:1), or - GYKPQIS (SEQ ID NO: 191), preferably wherein this STAT4 binding site is present at amino acid positions that corresponds to positions 460-466 of the human wild type IL7Ra (SEQ ID NO:1). In some embodiments, the IL7Ra signalling domain which is able to activate STAT4 as described herein, is represented by an amino acid sequence which has at least 80% identity with SEQ ID NOs:192-203, preferably wherein the mutation corresponding to the P300H, P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) and/or the STAT4 binding site is still present in the sequence derived from SEQ ID NOs:192-203, more preferably the mutation P300H and the STAT4 binding site are still present. In a second aspect, the present invention provides a receptor comprising the IL7Ra signaling domain as defined above, said receptor comprises a transmembrane domain. In an embodiment, this receptor comprises an extracellular domain. In an embodiment, this extracellular domain is an antigen binding domain. In an embodiment, this receptor further comprises an additional signalling domain and/or a co- stimulatory domain. In a preferred embodiment, this receptor is a CAR. In an embodiment of this second aspect, the present invention provides a CAR comprising the IL7Ra signalling domain as described in the first aspect, said CAR comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising said IL7Ra signalling domain and a CD3z domain, optionally a co-stimulatory domain. In a further embodiment of this second aspect, the present invention provides a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising an IL7Ra signalling domain and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT3 binding site is present in the intracellular signalling domain of the CAR. Therefore the activation of STAT3 is not directly mediated by the IL7Ra signalling domain and said STAT3 binding site is not present in the IL7Ra signalling domain. In a further embodiment of this aspect, the present invention provides a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising an IL7Ra signalling domain and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT4 binding site is present in the intracellular signalling domain of the CAR. Therefore the activation of STAT4 is not directly mediated by the IL7Ra signalling domain and said STAT4 binding site is not
present in the IL7Ra signalling domain. In some embodiments, the STAT4 binding site is as defined above in the first aspect, and more preferably as defined in SEQ ID NO: 189 (YLPSNID). In an embodiment of the second aspect, the antigen recognized by the antigen binding domain is a tumor associated or tumor specific antigen, preferably wherein the antigen is CD19 or ROR1. In some embodiments, the co-stimulatory domain is 41BB and/or CD28. In a further aspect, the present invention provides a nucleic acid encoding the IL7Ra signalling domain of the first aspect, or the receptor of the second aspect. In a further aspect, the present invention provides an expression vector comprising the nucleic acid as described above. In a further aspect, the present invention provides a cell comprising the nucleic acid as described above or the expression vector as described above, preferably wherein the cell expresses the encoded receptor, or CAR and more preferably wherein the cell is a T cell. In a further aspect, the present invention provides a composition comprising the IL7Ra signalling domain, the receptor, the CAR, the nucleic acid, the expression vector, the cell as described above, preferably wherein the composition is a pharmaceutical composition. In a further aspect, the present invention provides a receptor, a CAR, a nucleic acid, an expression vector, a cell or a composition as described above for use as a medicament, preferably for treating cancer. In an embodiment, the IL7Ra signalling domain is as defined above and the patient is immunocompromised and/or shows signs of T cells exhaustion, preferably wherein the IL7Ra signalling domain comprises the P300H, P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1), more preferably the IL7Ra signalling domain comprises the P300H mutation. All publications, patents, and patent applications mentioned in this application are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Predicted model of IL7R with molecular modeling tool. Intracellular and transmembrane part have their hydrophobicity-colored membranes. Figure 2. Calculated model of IL7R with Amber16 FF. Figure 3. Intracellular domain with selected Box1, Box2, possible membrane-interaction and spatially differentiating sequences. A. View from the Box2 side (“profile”). B. view from the transmembrane domain (“top”).
Figure 4. The secondary structure prediction of the C-terminus of the intracellular domain of IL7Ra. A. The original sequence. B. The suggest truncation’s sequence. Figure 5. CAR T-cells (A) and K562.CD19 (B) gain fold upon repetitive stimulation at 1:1 E:T ratio. Figure 6. CAR T-cells (A) and K562.CD19.CPL (B) gain fold upon repetitive stimulation at 1:1 E:T ration. C. Correlation between AUC of CAR-T cells and AUC of target cell line K562.CD19.CPL+ in rechallenge assay (P (two-tailed) =0.0028), Spearman r=-1). Figure 7. The portion of CCR7+ CAR T-cells in different starting materials from donor 1 and donor 2. Figure 8. CAR-T rechallenge assay at 1:1 E:T ratio, 6 stimulations at 3-day intervals. A. CCR7high CAR-T expansion fold; B. CCR7low CAR-T expansion fold; C. K562.CD19.CPL+- cells gain under the CCR7high CAR-T surveillance; D. K562.CD19.CPL+- cells gain under the CCR7low CAR-T surveillance. Figure 9. High tumor burden cytotoxicity assay of 5th generation CAR-Ts in comparison with 2nd generation anti-CD19 CAR-T (BB.Z) for CCR7high (A) and CCR7low (B) CAR-T. The effector-to-target ratio was 1:5. K562.CD19.CPL cell line that stably expresses multiple immune checkpoint ligands. Figure 10. CAR T-cells (A) and K562.CD19.CPL+ (B) gain fold upon repetitive stimulation at 1:1 E:T ratio. Figure 11. High tumor burden cytotoxicity assay of CD28-based CAR-Ts in comparison with 2nd generation anti-CD19 CAR-Ts (BB.Z and 28.Z+) and IL2RB-containing 5th generation CAR-T. The effector-to-target ratio was 1:1.1:5 and 1:10 and tumor cell lines were Nalm-6 (A) and K562.CD19.CPL cell line that stably expresses multiple immune checkpoint ligands (B). Figure 12. Antigen-induced activation assessment of different CAR designs normalized at CAR surface expression level using NFAT (GFP) Jurkat Reporter Cell. Figure 13. Mean fluorescence intensity (MFI) of phosphorylated STAT3 (A) and STAT5 (B) within the CD8+ CAR T-cells assessed by flow cytometry and STAT3 (C) and STAT5(D) within CD4+ cells. Figure 14. STAT phosphorylation dynamics during CAR T-cell activation - pSTAT3 (A) and pSTAT5 (B). Figure 15. Correlation between AUC of CAR-T cells and AUC of target cell lines for K562.CD19.CPL+ (A) (P (two-tailed)=0.0694, Spearman r=-0.6905) and Nalm6 (B) ((P (two-tailed)=0.0022, Spearman r=- 0.9286) in rechallenge assay. Figure 16. IL7R fragment molecular dynamics simulations. A. Distribution of the average 12Cα-26Cα distances during the MD simulation of the part of signaling domain. B. Color scale of the differences between calculated “energies” of the residues’ interactions for WT and P300H during the MD. C. Mutant similarity to P300H. WT is shown as a red cross. D. Closed conformation of the receptor signaling domain. The Cα–Cα distance between residues L12 and H26 is short (~7.69 Å), reflecting a compact, folded state. This conformation likely corresponds to an inactive or less active state of the domain. E.
Open conformation of the receptor signaling domain. The Cα–Cα distance between residues L12 and A26 is extended (~25.4 Å), indicating a more open and relaxed structure. This state may facilitate enhanced signaling activity. Figure 17. Correlation between AUC of CAR-T cells and AUC of target cell lines for K562.CD19.CPL. Figure 18. Correlation between AUC of CAR-T cells and AUC of target cell lines for Raji (human B lymphocyte cell line) DETAILED DESCRIPTION OF THE INVENTION Engineered cells hold great potential both for research and therapeutic applications. For example, certain engineered immune cells have provided landmark advances in the treatment of some types of cancer for which no effective treatments were previously available. Despite increased efforts to generate new and more advanced engineered cells, a number of challenges remain which limit the success in the field. Examples of these challenges include difficulties in generating sufficient numbers of the desired engineered cells, limited proliferative ability or lifespan of the engineered cells, limited fitness of the engineered cells, limited induction of effector function upon antigen recognition, exhaustion, and limited sensitivity to cell lines with low antigen density on the surface. Accordingly, disclosed herein are IL7Ra signaling domains, receptors comprising this IL7Ra signaling domain, especially CAR, engineered immune cells comprising these receptors, especially comprising these CARs, compositions and methods using such signaling domains, receptors, especially CARs. The invention is based in part on the surprising discovery that using a truncated IL7Ra signalling domain comprising a mutation at the amino acid position corresponding to position 300 of the human wild type IL7Ra (SEQ ID NO:1) (which corresponds to position 36 of the truncated IL7Ra SEQ ID NO:2, 3, 4 or 5), and/or wherein said IL7Ra signalling domain is able to activate STAT3 and STAT5 when used in a receptor, especially in a CAR, and expressed into T cells leads to CAR-T cells with attractive properties: it greatly prevents and overcomes the functional exhaustion of the CAR T-cells, improves the proliferation, survival and expansion of said CAR T-cells, improves the capability to control tumor population cells by said CAR T-cells, improves the antitumor activity and/or cytotoxicity capacity of said CAR T-cells, improves the persistence potential of the CAR-T cells, and improves the safety potential of said CAR-T which can be activated without systemic toxicity. The improved properties of said CAR- T cells can be attributed at least in part to such truncated IL7Ra domain with a single mutation corresponding to amino acid position 300 of the human wild type IL7Ra (corresponds to amino acid position 36 of the truncated IL7Ra having SEQ ID NO:5) according to the present invention. The invention is further based in part on the surprising discovery that using a truncated IL7Ra signalling domain able to activate STAT4 (optionally STAT3 and/or STAT5 and/or optionally comprising a mutation at the amino acid position corresponding to position 300 of the human wild type IL7Ra (SEQ ID NO:1) (which corresponds to position 36 of the truncated IL7Ra SEQ ID NO:2, 3, 4 or 5) when used in a receptor, especially in a CAR, and expressed into T cells leads to CAR-T cells with attractive
properties: it greatly prevents and overcomes the functional exhaustion of the CAR T-cells, improves the proliferation, survival and expansion of said CAR T-cells, improves the capability to control tumor population cells by said CAR T-cells, improves the antitumor activity and/or cytotoxicity capacity of said CAR T-cells, improves the persistence potential of the CAR-T cells, and improves the safety potential of said CAR-T which can be activated without systemic toxicity. Moreover, in one embodiment, said CAR is expected to have an increased sensitivity to cell lines with low antigen density on the surface. The improved properties of said CAR-T cells can be attributed at least in part to such truncated IL7Ra domain with the ability to activate STAT4 (and optionally activate STAT3 and/or STAT5 and/or optionally due to the presence of a single mutation corresponding to amino acid position 300 of the human wild type IL7Ra (corresponds to amino acid position 36 of the truncated IL7Ra having SEQ ID NO:5) according to the present invention. I. General Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles "a", "an", "the" and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional embodiments “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included. A “wild type” protein amino acid sequence can refer to a sequence that is naturally occurring and encoded by a germline genome. A species can have one wild type sequence, or two or more wild type sequences (for example, with one canonical wild type sequence and one or more non-canonical wild type sequences). A wild type protein amino acid sequence can be a mature form of a protein that has been processed to remove N-terminal and/or C-terminal residues, for example, to remove a signal peptide. An amino acid sequence that is “derived from” a wild type sequence or other amino acid sequence disclosed herein can refer to an amino acid sequence that differs by one or more amino acids compared to the reference amino acid sequence, for example, containing one or more amino acid insertions, deletions, or substitutions as disclosed herein. The terms “derivative,” “variant,” “variations” and “fragment,” when used herein with reference to a polypeptide, refers to a polypeptide related to a wild type polypeptide, for example either by amino acid sequence, structure (e.g., secondary and/or tertiary),
activity (e.g., enzymatic activity) and/or function. Derivatives, variants, variations and fragments of a polypeptide can comprise one or more amino acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild type polypeptide. A part or fragment of a polypeptide may correspond to at least 30%, at least 40% of the total length of a polypeptide, such as a polypeptide having an amino acid sequence identified by a specific SEQ ID NO., or having at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the length (in amino acids) of the polypeptide. In an embodiment, a derivative, variant or fragment of a given amino acid sequence defined herein still exhibits at least one of its activities to at least a certain extent. In this context, “to at least a certain extent” means at least 30%, 46%, 50%, 60%, 70%, 80%, 90%, 100% or 120%, 150%, 200%. It will be clear to the skilled person that an activity refers to any of the activities of the given amino acid sequence defined herein. For example, when referring to an IL7Ra signalling domain, activity may refer to STAT3 binding, STAT3 activation, STAT4 binding, STAT4 activation, or STAT5 activation. For example, when referring to a CAR of the invention, activity may refer to any of the activities specifically disclosed in the section entitled Receptor or Methods of treating a disease in a subject. Within the context of the present application, a protein is represented by an amino acid sequence, and correspondingly a nucleic acid molecule or a polynucleotide is represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, it should be understood that for each reference to a specific amino acid sequence using a unique sequence identifier (SEQ ID NO.), the sequence may be replaced by: a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with the reference amino acid sequence. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%. Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred embodiment an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% with the given amino acid sequence, respectively. In an embodiment, an amino acid sequence defined herein as having at least 60% identity or similarity with a given sequence still exhibits at least one of its activities to at least a certain extent. In this context, “to at least a certain extent” means at least 30%, 46%, 50%, 60%, 70%, 80%, 90%, 100% or 120%, 150%,
200%. It will be clear to the skilled person that an activity refers to any of the activities of the given amino acid sequence defined herein. For example, when referring to an IL7Ra signalling domain, activity may refer to STAT3 binding, STAT3 activation, STAT4 binding, STAT4 activation, or STAT5 activation. For example, when referring to a CAR of the invention, activity may refer to any of the activities specifically disclosed in the section entitled Receptor or Methods of treating a disease in a subject. The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences, or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred embodiment, sequence identity is calculated based on the full length (in amino acids or nucleotides) of two given SEQ ID NOs or based on a portion thereof, more preferably based on the full length. A portion of a full-length sequence may be referred to as fragment, and preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of the length (in amino acids or nucleotides) of a reference sequence. "Identity" also refers to the degree of sequence relatedness between two amino acid or between two nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in an embodiment, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10 , end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment ( in an embodiment, MAFFT v7Default value is: BLOSUM62 [bl62], Gap Open: 1.53, Gap extension: 0.123, Order: aligned , Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2 , Perform FFTS: none is used). "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are listed below: - Acidic Residues: Asp (D) and Glu (E) - Basic Residues: Lys (K), Arg (R), and His (H) - Hydrophilic Uncharged Residues: Ser (S), Thr (T), Asn (N), and Gln (Q) - Aliphatic Uncharged Residues: Gly (G), Ala (A), Val (V), Leu (L), and Ile (I)
- Non-polar Uncharged Residues: Cys (C), Met (M), and Pro (P) - Aromatic Residues: Phe (F), Tyr (Y), and Trp (W)
Alcohol group-containing residues S and T Aliphatic residues I, L, V, and M Cycloalkenyl-associated residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Very small residues A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and R For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and, Val to Ile or Leu. The term "heterologous" refers to an entity that is not native to the cell or species of interest.
The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues. As used herein, the terms "target site", "target sequence", or “nucleic acid locus” refer to a nucleic acid sequence that defines a portion of a nucleic acid sequence to be modified or edited and to which a homologous recombination composition is engineered to target. The terms "upstream" and "downstream" refer to locations in a nucleic acid sequence relative to a fixed position. Upstream refers to a position in a sequence that is 5' (i.e., nearer the 5' end of the strand) relative to the fixed position, and downstream refers to the region that is 3' (i.e., nearer the 3' end of the strand) relative to the fixed position. As used herein, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. As used herein, the disclosure of numerical ranges by numerical endpoints includes all numbers encompassed by that range (e.g., “1 to 5” includes but is not limited to 1, 1.25, 1.5, 1.75, 2, 2.3, 2.5, 2.8, 3, 3.1,3.3, 3.8, 3.9, 4, 4.25, 4.5, 4.75 and 5). Unless otherwise indicated, all numbers used herein to express quantities, amounts, dimensions, measurements, and the like should be understood as encompassing the specific quantities, amounts, dimensions, measurements and so on, and also as encompassing such instances modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical descriptions set forth herein may vary while remaining well within the teachings of the present disclosure. At the very least, each numerical value should be construed in view of the number of significant digits and by applying routine rounding techniques. As various changes could be made in the above-described cells and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense. II. IL7Ra In a first aspect, the present invention provides an IL7Ra signalling domain comprising a mutation at the amino acid position that corresponds to position 300 of the human wild type IL7Ra (SEQ ID NO:1), and/or which is able to activate STAT3 and STAT5. Therefore, provided herein is an IL7Ra signalling domain comprising a mutation at the amino acid position that corresponds to position 300 of the human wild type IL7Ra (SEQ ID NO:1). In some embodiments, the mutation presented in the IL7Ra signaling domain corresponds to the mutation selected from a list comprising P300H, P300A, P300W, P300E, P300L and P300Q when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In a preferred embodiment, the mutation presents in the IL7Ra signaling domain corresponds to the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). The P300H mutant is expected to stabilize an “open” conformational state more effectively, potentially facilitating enhanced signaling activity. P300H, P300A, P300W, P300E, P300L and P300Q mutations are also expected to perform similar/same function as P300H, to stabilize an “open” conformational
state more effectively, potentially facilitating enhanced signaling activity, as these mutations at P300 have similar properties as the P300Hmutation: ● P300A – closest overall to H by both metrics ● P300W – small Δ distance and good energetic match ● P300E – almost identical matrix to H, slightly larger distance ● P300L and P300Q– structurally and energetically close The IL7Ra signalling domain of the invention may be denominated “a truncated IL7Ra signalling domain”. Provided herein is also an IL7Ra signalling domain which is able to activate STAT3 and STAT5. Provided herein is also an IL7Ra signalling domain comprising a mutation at the amino acid position that corresponds to position P300 of the human wild type IL7Ra (SEQ ID NO:1) and which is able to activate STAT3 and STAT5. The amino acid position 300 of the human wild type IL7Ra corresponds to amino acid position 36 of a truncated human wild type IL7Ra represented by SEQ ID NO:5. The amino acid at position 300 of the human IL7Ra is Proline. Therefore in an embodiment, the present invention provides an IL7Ra signalling domain comprising a mutation at position 36 of SEQ ID NO:5 and/or is able to activate STAT3 and STAT5. In an embodiment, the mutation present in the IL7Ra signaling domain corresponds to the mutation selected from a list comprising P36H, P36A, P36W, P36E, P36L and P36Q in SEQ ID NO:5 and/or is able to activate STAT3 and STAT5. In a preferred embodiment, the IL7Ra signalling domain comprises the mutation P36H in SEQ ID NO:5 and/or is able to activate STAT3 and STAT5. Cytokines exert a vast array of immunoregulatory actions critical to human biology and disease. Through binding to specific cell surface receptors (or cytokine receptors), they initiate signals that are critical to a diverse spectrum of functions, including induction of immune responses, cell proliferation, differentiation, and survival. Each cytokine binds to a specific receptor on the surface of its target cell. Cytokine receptors possess a conserved extracellular region (cytokine receptor homology domain [CDH]) and several structural modules, including extracellular immunoglobulin or fibronectin type III– like domains, transmembrane domains, and intracellular homology regions. The cytokine receptors can be grouped into six major families based on common structural features: class I cytokine receptors, class II cytokine receptors, TNF receptors, IL-1 receptors, tyrosine kinase receptors, and chemokine receptors. The class I cytokine receptors, also known as the hematopoietin receptors or type I membrane proteins, constitute the largest group among the cytokine receptors. The class I cytokine receptors can be further grouped into homodimeric receptors, characterized by their use of two identical receptor chains, and heterodimeric (or non-homodimeric) receptors. A common structure in non-homodimeric receptors is a cytokine-specific chain (nominally the “alpha” chain) that recognizes cytokine with high affinity, and the resulting dimer will then recruit a “shared” chain in order to initiate signaling. The intracellular domains of the class I cytokine receptors are constitutively associated with tyrosine kinases of the Janus kinase
(JAK) family, and to a more restricted degree the TYK kinase. After the JAK/TYK kinases are activated by ligand-induced receptor oligomerization, they cross phosphorylate each other and the intracellular domains of the receptors. The phosphorylated tyrosine residues in the receptors then serve as the docking sites for a second family of proteins, the signal transducer and activator of transcription (STAT) proteins. Binding of STATs to the intracellular domains of the receptors leads to their tyrosine phosphorylation and subsequent dissociation from the receptors. The phosphorylated STATs form dimers and translocate into the nucleus, where they bind to DNA recognition sequences and act as transcription factors for the expression of cytokine-responsive genes, often leading to proliferation and/or differentiation. Interleukin-7 (IL-7) was discovered in the last century and noted for its growth-promoting effects on progenitors of B cells in vivo. IL-7 is a 25-kDa soluble globular peptide. IL-7 is produced by cells, such as fetal liver cells, stromal cells in the bone marrow (BM), and thymus and other epithelial cells, including keratinocytes and enterocytes. IL-7R is a heterodimeric complex consisting of the α-chain (CD127) and the common cytokine receptor γ chain, shared with the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL- 21, and expressed in a variety of cells. The precursor IL-7Rα protein includes a signal peptide (20 amino acids) and has 459 amino acids in total. The mature protein undergoes several post-translational modifications including glycosylation (6 potential N-glycosylation sites in the extracellular domain) and dissulfide bond formation. The extracellular domain has 219 amino acids (spanning from amino acids 21 to 239), the transmembrane domain has 25 amino acids (spanning from amino acids 240 to 264), and the cytoplasmic tail spans from amino acids 265 - 459 (195 amino acids). In the extracellular domain of IL-7Rα, it displays 4 paired cysteines in 2 fibronectin type III-like domains and, closer to the transmembrane domain, a WSxWS motif. The intracellular domain has a Box 1 motif and tyrosines (e.g. Y401, Y449, Y456) involved in signal transduction (Lin et al., 1995; Venkitaraman and Cowling, 1994). IL-7Ra is expressed in early thymocytes, T cells, pre-B cells, BM macrophages, and other immune cells. The binding of IL-7 to its receptor triggers the activation of JAK1 and JAK3. As a result, JAK1 and JAK3 are phosphorylated. Subsequently, the activated JAK kinases phosphorylate tyrosine residues of several downstream targets, among them the cytoplasmic domain of the IL-7R. Phosphorylation of the IL-7Ra chain is critical for the next stage of signal transduction because it contributes to the recruitment of the STAT proteins. Phosphorylated JAK1 and JAK3 allows the recruitment of STAT5 and its phosphorylation. In human PBMC, the Y449 residue of the IL-7R chain has been implicated in recruitment of STAT5. As a transcription factor, phosphorylated STAT5 dimerizes and translocates into the nucleus, mediating the expression of downstream targeted genes associated with the survival and proliferation of T cells. This results in changes in the expression of B-cell lymphoma 2 (Bcl−2) family members, such as increased expression of the anti−apoptotic molecules Bcl-xl, Mcl-1, and Bcl −2 and decreased expression of the pro−apoptotic molecules Bax, Bim, and Bad. In immune cells, cytokines such as IL-6, IL-11, and IL-22 can induce JAK1- and JAK3-mediated STAT3 activation, while IL-7 induces dominantly JAK1 mediated STAT5 activation. STAT-3 is activated in a very similar manner, upregulating Bcl-xl, c-myc, survivin, cyclin D1, Bcl6 genes. STAT3 has been recently shown to play crucial roles in Tex term cell development in cancer. Mainly, IL-10 and IL-21, activate STAT3, promoting
tumor-specific Tex term cell-associated gene expression, and suppressing Tex Prog cell-related gene expression, resulting in the development and enhanced effector functions of Tex int cells in tumors. The IL-21-STAT3-BATF pathway is necessary for sustained effector function enhancement and survival of Tex term cells of CD8+ T cells during chronic antigen stimulation. STAT-3 genes enrichment has been noted in complete responders CLL patients after CD19 CAR-T cell therapy [Fraietta J.A. et al, 2018]. Jak-1 molecule has been shown to activate all STAT proteins, including Stat3 and Stat 5. Another crucial pathway involved in the IL-7/IL-7R signalling is the PI3K-AKT pathway. When IL-7 binds to the IL7R, p85, a regulatory component of PI3K, is recruited to IL7Ra and then induces the phosphorylation of Y449 in the IL-7Ra cytoplasmic tail, which triggers the activation of PI3K and AKT. Activated AKT in turn activates the transcription factor Forkhead box protein 1 (FOXO1) to further regulate the expression of genes involved in cell cycle regulation, such as p27kip1, and genes involved in the regulation of glucose metabolism, such as glucose transporter-1 (Glut-1) and hexokinase II (HKII). Deficiencies in IL-7 or IL-7R can lead to severely impaired immune cell development. In the ensuing decades, the discovery of relevant signaling pathways was accompanied by recognition that IL-7 plays an indispensable role in the development and maintenance of many other immune cells. The vital regulatory functions of IL-7 throughout the entire immune system have become increasingly evident. In the context of the present invention, the terms “IL-7 Receptor alpha”, “IL-7 Receptor a” “IL-7 Receptor α”, “IL-7R alpha”, “IL-7Ra” and “IL-7Rα” may be used interchangeably. In some embodiments, the IL7Ra signalling domain may be derived from a wild type full length human IL7Ra. The wild type full length human IL7Ra has a sequence as below: SEQ ID NO:1 Full length wild type human IL7Ra: 459 amino acids in total MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNIT NLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVY REGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVR SIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLP DHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTN STLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ In the context of the present invention, the IL7Ra signalling domain as provided herein is a truncated protein. The “truncated IL7Ra protein” has less amino acids than the full length or wild type IL7Ra (e.g. wild type human IL7Ra of SED ID NO:1). Example of a truncated IL7Ra signaling domain: SEQ ID NO:167
KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQL EESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPH VYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ In some embodiments, the IL7Ra signalling domain provided herein has a length of less than 95 amino acids. In some embodiments, the IL7Ra signalling domain has a length of between 10 to 20, or 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, 70 to 80, 80 to 90, or 90 to 95 amino acids. In some embodiments, the IL7Ra signalling domain has a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some preferred embodiments, the IL7Ra signalling domain has a length of 20, 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 or 88 number of amino acids. In an embodiment, the IL7Ra signalling domain has a length of 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, 70 to 80, 80 to 90, or 90 to 95 amino acids, and both BOX1 (i.e. SEQ ID NO:80) and BOX2 (i.e. SEQ ID NO:81) of SEQ ID NO:1 are conserved. In an embodiment, the IL7Ra signalling domain has a length of 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, and BOX1 of SEQ ID NO:1 is conserved. In some embodiments, the IL7Ra signalling domain has a length of 20-30 amino acids, which comprises BOX1 (i.e. SEQ ID NO: 80). In some embodiments, the IL7Ra signalling domain having a length of 20-30 amino acids and comprising BOX1 (i.e. SEQ ID NO: 80), does not have any mutation compared to SEQ ID NO:1. An exemplary truncated IL7Ra signalling domain (SEQ ID NO: 188) having a length of 25AA and comprising BOX1, without any mutation compared to SEQ ID NO:1, is shown as below: SEQ ID NO:188: VWPSLPDHKGGGGSPQQEEAYVTMS In some preferred embodiment, the IL7Ra signalling domain comprises SEQ ID NO:188. In some preferred embodiments, the the IL7Ra signalling domain is represented as SEQ ID NO:188. In the context of the present invention, deletion of a part of wild type human IL7Ra signaling domain enables/facilitates JAK1-JAK1 transphosphorylation by bringing JAK1 molecules into proximity to each other. In the context of the present invention, deletion of a part of wild type human IL7Ra signaling domain enables/facilitates to bring STATs docking site into the proximity of JAK1, thereby facilitating STATs phosphorylation or STATs activation. An exemplary truncated IL7Ra signalling domain is shown as below: SEQ ID NO: 5 Truncated human wild type IL7Ra (81AA) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ
SEQ ID NO:5 is derived from the human wild type full length IL7Ra (i.e. SEQ ID NO:1) by deleting the extracellular, transmembrane and part of the intracellular part. SEQ ID NO:5 does not comprise any mutations compared to SEQ ID NO:1. Alignment of SEQ ID NO:1 and SEQ ID NO:5 using EMBOSS Needle Pairwise Sequence Alignment (PSA) (https://www.ebi.ac.uk/jdispatcher/psa/emboss_needle). Aligned sequence 1 (upper sequence) is SEQ ID NO:1, and aligned sequence 2 (below sequence) is SEQ ID NO:5. 1 MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNG 50 1 -------------------------------------------------- 0 51 SQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFL 100 1 -------------------------------------------------- 0 101 LIGKSNICVKVGEKSLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTF 150 1 -------------------------------------------------- 0 151 NTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAM 200 1 -------------------------------------------------- 0 201 YEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSF 250 1 -------------------------------------------------- 0 251 FSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNP 300 |||||||||||||||||||||||||||||||||||| 1 --------------KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNP 36 301 ESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCP 350 |||||||||||||||||||||||||||||| 37 ESFLDCQIHRVDDIQARDEVEGFLQDTFPQ-------------------- 66 351 SEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHV 400 67 -------------------------------------------------- 66 401 YQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYV 450
451 TMSSFYQNQ 459 ||||||||| 73 TMSSFYQNQ 81 Positions of example mutations or conserved amino acids in SEQ ID NO: 5 (Truncated human wild type IL7Ra (81AA)) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ
*Underlined positions correspond to positions P36, Q79, N80 and Q81 in SEQ ID NO:5 (or P300, Q457, N458 and Q459 when referring to the human wild type IL7Ra SEQ ID NO:1). * Bold letters correspond to positions 68-71 and 78 in SEQ ID NO:5 (or positions 446, 447,448, 449 and 456 when referring to the wild type human IL7Ra (SEQ ID NO:1)) SEQ ID Corresponding amino acid positions in SEQ ID NO:1 and SEQ ID NO:5 1 300 446 447 448 449 456 457 458 459 5 36 68 69 70 71 78 79 80 81 In some embodiments, the IL7Ra signalling domain has a length of 20 to 30, or 30 to 40, or 40 to 50, or 50 to 60, or 60 to 70, 70 to 80, 80 to 90, or 90 to 95 amino acids, and one or more of the amino acids that correspond to positions 401, 446, 447, 448, 449 and/or 456 of the wild type human IL7Ra are conserved in the truncated IL7Ra signalling domain of the present invention. Amino acid positions 446, 447,448, 449 and 456 of the wild type human IL7Ra (SEQ ID NO:1) correspond to amino acid positions 68, 69, 70, 71 and 78 of SEQ ID NO: 5. In some embodiments, these amino acids correspond to Y401, E446, E447, A448, Y449 and Y456 of a wild type human IL7Ra (SEQ ID NO:1). Y401, Y449 and Y456 are docking sites for the binding/recruitment of signalling molecules: Y449 is needed for the recruitment/binding of STAT5. The truncated IL7Ra signalling domain according to the present invention is able to drive, allow, trigger the activation, preferably the recruitment and activation of JAK kinase. In some embodiments, the JAK is JAK1. The activation of JAK, in particular JAK1, is preserved via preservation of a 9-aa motif termed Box1 (VWPSLPDHK (SEQ ID NO:80)) which is homologous within the type I cytokine receptor family and can bind Jak1. Activated JAK1 or tyrosine phosphorylated JAK1 may be assessed using any standard/known technique available to the skilled person, for example by western blotting using antibodies against phosphorylated JAK1 or against phosphorylated tyrosine. The truncated IL7Ra signalling domain according to the present invention is able to drive, allow, trigger the recruitment and activation of the STAT proteins as in the wild type IL7R. In some embodiments, the STAT proteins are STAT5. In an embodiment, STAT5 is activated by activated JAK1. In an embodiment, Y449 of the IL7Ra is phosphorylated and serves as a docking site for STAT5. In an embodiment, STAT5 is activated after having been recruited at this docking site. STAT5 may be considered to be activated when a detectable signal of their activation will be detected using techniques known to the skilled person. STAT activation may be assessed using any standard/known technique available to the skilled person. As an example, STAT5 activation may be assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labeled STAT5 binding site (T4 polynucleotide kinase), or western blotting using antibodies against tyrosine phosphorylated STAT5. In some embodiments, the IL7Ra signalling domain according to the invention is able to drive, allow, trigger the activation, preferably the recruitment and activation of the STAT proteins. In some embodiments, the STAT protein is STAT3. In some embodiments, the IL7Ra signalling domain
according to the invention is able to recruit and activate STAT3, which is not the case of the human wild type IL7Ra. The human wild type IL7Ra comprises a motif (SEQ ID NO:7: YQNQ) at positions 456 to 459 of the human wild type IL7Ra. This motif does not allow the detectable activation of STAT3 by the human wild type IL7Ra. This motif might allow some binding/recruiting of STAT3, but no activation of STAT3 is detectable. However, surprisingly, this motif could be modified by mutation to generate a new motif that allows a detectable activation of STAT3 by the truncated IL7Ra of the invention. Since STAT3 can be activated when this new motif is present in the IL7Ra signaling domain of the invention, it is assumed that this motif is a binding/recruiting site for STAT3. This new motif is represented by SEQ ID NO:6: YX1X2Q, wherein X1 may be any amino acid. In some embodiment, X1 may be Phenylalanine (F) or Leucine (L) or Arginine (R), and X2 may be Lysine (K) or Proline (P) or Histidine (H). In some embodiments, the Q may be mutated into any other amino acid, such as a Proline (P), Tyrosine (Y), Aspartic acid (N), Phenylalanine (F) or Alanine (A). This YX1X2Q motif is present at positions 78-81 of a truncated IL7Ra defined herein as SEQ ID NO:2, 3, 4 or 5. Therefore in an embodiment the STAT3 binding/recruiting site is represented by SEQ ID NO:6: YX1X2Q, wherein: X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally Q is mutated/substituted into P, T, Y, N, F or A. This YX1X2Q motif is present at positions 78-81 of a truncated IL7Ra defined herein as SEQ ID NO:2, 3, 4 or 5. The binding/recruitment of STAT3 to/by the IL7Ra signalling domain of the present invention is therefore made possible via a STAT3 binding site (represented as YX1X2Q (SEQ ID NO:6)) present in said IL7Ra signalling domain. In some embodiments, the STAT3 binding site, present in the IL7Ra signalling domain of the invention, results from mutation(s) compared to the human wild type IL7Ra signaling domain counterpart (= YQNQ (SEQ ID NO:7)). SEQ ID NO: 7 is present at positions 456-459 of the human wild type IL7Ra. In some embodiments, mutations are present at positions 457 and/or 458 of the human wild type IL7Ra (SEQ ID NO:1). In some embodiments, the amino acids are substituted by any other amino acids. Some non- limiting examples of mutations are Q457F, Q457L or Q457R and/or N458K, N458P or N458H. In some embodiments, a further mutation may be present at position 459 when referring to the human wild type IL7Ra. The amino acid at position 459 may be substituted by any other amino acid. Some non-limiting examples of a mutation/substitution at position 459 may be Q459P, Q459Y, Q459A, Q459N and Q459F. In some embodiments, the motif present in the IL7Ra signaling domain of the invention which is able to recruit/bind STAT3 comprises any of the following sequences:
YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA (SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF. In a preferred embodiment, the mutations are such that they result in the STAT3 binding/recruiting site YRHQ (SEQ ID NO:8) being present in the IL7Ra signalling domain, corresponding to positions 456- 459 when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). STAT3 may be considered to be activated when a detectable signal of its activation will be detected using techniques known to the skilled person. STAT activation may be assessed using any standard/known technique available to the skilled person. In the context of the present invention, the recruited/bound STAT3 may be further activated by the phosphorylated and activated JAK1.In the context of this application, it may be understood that STAT3 and/or STAT5 is recruited to the “binding site” present in the IL7Ra signaling domain of the present invention. A “binding site” may be understood as a “recruiting site”. In some embodiments, the IL7Ra signalling domain according to the invention is able to recruit and activate STAT5 and is able to activate STAT3. In the context of the present invention, STAT3 may be activated directly or indirectly by the IL7Ra signalling domain of the present invention. Direct activation may mean that STAT3 is activated/phosphorylated by direct binding to the IL7Ra signalling domain of the present invention, while indirect activation may mean that STAT3 is activated/phosphorylated by another molecule, such as a kinase. In this situation, STAT3 does not interact/bind directly with the IL7Ra.This other molecule may bind to the IL7Ra and indirectly recruit STAT3 in the vicinity of the IL7Ra. Activation of STAT3 may be realized by phosphorylation of the tyrosine residue at Tyr705, serine phosphorylation at position 727 (by molecules from MEK/ERK and NOTCH1 pathways) and acetylation at lysine 685 (by CBP/P300). Tyr705 Phopshorylation may be fulfilled by the conventional pathway through JAK-molecules or non-canonical pathway. The latter (non-canonical pathway) may be realized by Src-kinases (c-Src, Yes, Fyn, Fgr, Yrk, Lyn, Blk, Hck, and Lck) and ABL-kinase. Acetylation at lysine 685 may induce STAT3 homodimerization even in the absence of pY075 and pS727. Acetylation may be measured for example by western blotting using anti-acetyl STAT3 antibodies, or by means of LC- MS/MS analysis of tryptic peptides. Examples of anti-acetyl-STAT3 antibodies include acK685 (or Acetyl-Stat3 (Lys685) Antibody #2523, Cell signaling Technology), acK87 (Polyclonal Rabbit anti Human STAT3 Antibody Acetyl Lys87, WB) LS C413302 (LS Bio), acK707 and acK709 (as described in Yan S. Xu et al. Scientific Reports (2016)). STAT3 may also be subject to reversible S-palmitoylation on cysteine 108. DHHC7 palmitoylates STAT3 and promotes its membrane recruitment and phosphorylation. Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates
phosphorylated STAT3 (p-STAT3) and enables its translocation to the nucleus. STAT3 may also be activated upon the enhanced production of cytokines, for example, IL-2 may increase STAT3 in bystander CAR-T cells. The deletion of site(s) for negative regulator proteins (SOCS2 and CIS) in the IL7Ra signaling domain of the present invention may enable the sustained function of STAT3 signalling pathway. In a preferred embodiment, the activation of STAT3 is assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labelled STAT3 binding site, or western blotting using antibodies against tyrosine phosphorylated STAT3. In a preferred embodiment, the IL7Ra signaling domain according to the invention is able to recruit and activate both STAT3 and STAT5. STAT3 may be recruited via the STAT3 binding site and STAT5 may be recruited via the STAT5 docking/binding site present or preserved in the IL7Ra signalling domain of the present invention. STAT3 and STAT5 may be further activated by phosphorylated/activated JAK1. In the context of the present invention, the STAT3 binding site, may be present in the IL7Ra signalling domain, it may result from mutation(s) introduced in the IL7Ra signalling domain compared to the human wild type IL7Ra signaling domain counterpart. The motif represented as YQNQ (SEQ ID NO:7) is the wild type motif present in the human wild type IL7Ra. In an embodiment, this motif is mutated to create a more effective STAT3 binding site. This more effective binding/recruiting site of STAT3 is represented by YX1X2Q (SEQ ID NO:6). SEQ ID NO:6 and 7 are located at positions 456-459 of the human wild type IL7Ra (i.e. SEQ ID NO:1). These positions 456-459 of the human wild type IL7Ra correspond to positions 78-81 of SEQ ID NO:2, 3, 4 or 5. SEQ ID NO: 6 and SEQ ID NO:7 have been defined earlier herein, and preferred derived sites or motifs have been disclosed herein. In some embodiments, the mutations present in the IL7Ra signaling domain of the invention correspond to the mutations Q457R and/or N458H when referring to the human wild type IL7Ra (SEQ ID NO:1). These mutations Q457R and N458H, result in the STAT3 binding/recruiting site YRHQ (SEQ ID NO:8). In some embodiments, the mutations present in the IL7Ra signaling domain of the invention corresponding to the mutations Q457R and N458H when referring to the human wild type IL7Ra (SEQ ID NO:1) may be additional mutations to the mutation at position P300, wherein the mutation at P300 is selected from a list comprising P300H, P300A, P300W, P300E, P300L and P300Q when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In some preferred embodiments, the IL7Ra signaling domain of the invention comprises the P300H mutation and additional two mutations Q457R and N458H when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). Activated STAT3 and STAT5 further dimerize in the cytoplasm and translocate into the nucleus where they act as transcription factors for genes associated with their target genes comprising a STAT3 or a STAT5 binding site respectively. In an embodiment, such target genes are able to induce or are linked to the survival and proliferation of T cells. In other embodiments, alternative motifs are created to bind/recruit STAT3. Such alternative motifs are represented by SEQ ID NO:6: YX1X2Q, wherein:
X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally Q is mutated/substituted into P, T, Y, N, F or A. This YX1X2Q motif is present at positions 78-81 of a truncated IL7Ra defined herein as SEQ ID NO:2, 3, 4 or 5. In some embodiments, the motif present in the IL7Ra signaling domain of the invention which is able to recruit/bind STAT3 comprises any of the following sequences: YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA (SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF. In a preferred embodiment, the mutations are such that they result in the STAT3 binding/recruiting site YRHQ (SEQ ID NO:8) being present in the IL7Ra signalling domain, corresponding to positions 456- 459 when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). Exemplary IL7Ra signaling domains of the present invention: SEQ ID NO:2 Human truncated IL7Ra signalling domain (81 aa) with P36H mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:3 Human truncated IL7Ra signalling domain (81 aa) with Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ SEQ ID NO:4: Human truncated IL7Ra signalling domain (81 aa) with P36H, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ SEQ ID NO: 5 Human wild type truncated IL7Ra signaling domain (81AA) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ Exemplified mutated positions in wild type or truncated human IL7Ra signaling domain:
Position in human wild type IL7Ra Position in truncated IL7Ra (SEQ ID NO:1) (SEQ ID NO:2-5) P300H P36H in SEQ ID NO:2 or 4 Q457R Q79R in SEQ ID NO:3 or 4 N458H N80H in SEQ ID NO:3 or 4 In some embodiments, the IL7Ra signalling domain according to the present invention, comprises a mutation at the amino acid position that corresponds to position 300 when referring to the human wild type IL7Ra (SEQ ID NO:1), and/or is able to activate STAT3 and STAT5. In some embodiments, the mutation present at P300 when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is selected from a list comprising P300H, P300A, P300W, P300E, P300L and P300Q. In a preferred embodiment, the mutation present in the IL7Ra signaling domain corresponds to the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). Below some embodiments have been exemplified using the mutations of the IL7Ra signalling domain corresponding to the Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) forming the motif YRHQ in the ILR7a signaling domain of the invention. It is to be understood to a skilled person that any other motif as exemplified therein (derived from SEQ ID NO:6 or 7 or literally disclosed herein or designed by a person skilled in the art) could be used to design an IL7Ra signaling domain of the invention starting from the human wild type IL7Ra (i.e. SEQ ID NO:1). Examples of such alternative motifs are represented by SEQ ID NO:6: YX1X2Q, wherein X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally Q is mutated/substituted into P, T, Y, N, F or A. This YX1X2Q motif is present at positions 78-81 of a truncated IL7Ra defined herein as SEQ ID NO:2, 3, 4 or 5. In some embodiments, the motif present in the IL7Ra signaling domain of the invention which is able to recruit/bind STAT3 comprises any of the following sequences: YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA(SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF.
In some embodiments, the mutations are such that they result in the STAT3 binding/recruiting site YRHQ (SEQ ID NO:8) being present in the IL7Ra signalling domain, corresponding to positions 456- 459 when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In some embodiments, the IL7Ra signalling domain as described herein, comprising “at least one”, “at least two”, “at least three”, “at least four”, “at least five” mutations or more compared to the human wild type counterpart IL7Ra represented by SEQ ID NO:1. In some embodiments, the IL7Ra signalling domain as described herein, comprising “at least one” mutation compared to the human wild type counterpart IL7Ra represented by SEQ ID NO:1 said mutation being distinct from Q457R and N458H mutations when referring to the human wild type IL7Ra. In some embodiments, the “at least one” mutation is present at the amino acid position that corresponds to position 300 (or P300) of SEQ ID NO:1. In some embodiments, the “at least one” mutation presented in the IL7Ra signaling domain corresponds to the mutation selected from a list comprising P300H, P300A, P300W, P300E, P300L and P300Q when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In some preferred embodiments, the “at least one” mutation is present at the amino acid position that corresponds to the P300H mutation when referring to the human wild type IL7Ra. Within the context of the present application, one can either refer to the position of an amino acid in the wild type human IL7Ra (SEQ ID NO:1) or the actual position in a given IL7Ra signaling domain of the invention (for example any one of SEQ ID NO: 2-5). For example, the P300H mutation in SEQ ID NO:1 is the P36H mutation in an IL7Ra signaling domain of the present invention represented as any one of SEQ ID NO: 2 or 4. The P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 2 or 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). SEQ ID NO:5 is the truncated human wild type IL7Ra signalling domain that does not have any mutation compared to the full length human wild type IL7Ra (i.e. SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:2. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from SEQ ID NO:2 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting IL7Ra signaling domain variant is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). The IL7Ra signalling domain as defined above may have a length of
less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. Preferably, the P36H in SEQ ID NO:2 (corresponding to the P300H mutation in SEQ ID NO:1) is still present in the sequence derived from SEQ ID NO:2 as described above. The P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 2) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to SEQ ID NO:2 or the sequence derived from SEQ ID NO:2. In some embodiments, the IL7Ra signaling domain may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to SEQ ID NO:2 or a sequence derived from SEQ ID NO:2. Preferably, the P36H in SEQ ID NO:2 (corresponding to the P300H mutation in SEQ ID NO:1) is still present in the sequence derived from SEQ ID NO:2 as described above. The P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 2) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain according to the present invention comprises SEQ ID NO:2. In an embodiment, the IL7Ra signaling domain according to the present invention is SEQ ID NO:2. In some embodiments, the IL7Ra signalling domain as described herein, comprising a STAT3 binding site, preferably wherein the IL7Ra signalling domain comprises two, three, four, five mutations (or at least two or at least three or at least four or at least five) compared to the human wild type IL7Ra counterpart represented by SEQ ID NO:1. In some embodiments, the IL7Ra signalling domain as described herein, comprising a STAT3 binding site, preferably wherein the IL7Ra signalling domain comprises two mutations (or at least two) compared to the human wild type IL7Ra counterpart represented by SEQ ID NO:1. An IL7Ra signaling domain comprising at least one (or at least two) STAT3 binding/recruiting site is encompassed by the present invention. In some embodiments, the IL7Ra signalling domain as described herein, comprising a STAT3 binding site, preferably wherein the IL7Ra signalling domain comprises two mutations (or at least two) compared to the human wild type IL7Ra counterpart represented by SEQ ID NO:1; these two mutations being at amino acids Q79 and N80 of SEQ ID NO:3 or 4 (that correspond to positions Q457 and N458 of SEQ ID NO:1). Within the context of the present application, one can either refer to the position of an amino acid in the wild type human IL7Ra (SEQ ID NO:1) or using the actual position in a given IL7Ra signaling domain (for example SEQ ID NO: 2 or 3 or 4 or 5). For example, the Q457R and N458H mutations in SEQ ID NO:1 correspond to Q79R and N80H mutations in an IL7Ra signaling domain of the present invention represented as any one of SEQ ID NO:3 or 4.
In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:3. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from SEQ ID NO:3 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting IL7Ra signaling domain variant is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to SEQ ID NO:3 or the sequence derived from SEQ ID NO:3. In some embodiments, the IL7Ra signaling domain may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to SEQ ID NO:3 or the sequence derived from SEQ ID NO:3. Preferably, the mutations Q79R and N80H present in SEQ ID NO:3 (corresponding to the Q457R and/or N458H mutations when referring to the human wild type IL7Ra represented by SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:3. In some embodiments, the IL7Ra signaling domain according to the present invention comprises SEQ ID NO:3. In an embodiment, the IL7Ra signaling domain according to the present invention is SEQ ID NO:3. In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:4. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from SEQ ID NO:4 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting IL7Ra signaling
domain variant is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). In this context, the IL7Ra signalling domain encoded by the nucleic acid variants derived from any one of SEQ ID NOs: 60-62 is functional as described herein. In some embodiments, the IL7Ra signaling domain may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to SEQ ID NO:4 or the sequence derived from SEQ ID NO:4. In some embodiments, the IL7Ra signaling domain may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to SEQ ID NO:4 or the sequence derived from SEQ ID NO:4. Preferably, the P36H, Q79R and N80H mutations present in SEQ ID NO:4 (corresponding to P300H, Q457R and/or N458H mutations in the human wild type IL7Ra represented by SEQ ID NO:1)) are still present in the sequence derived from SEQ ID NO:4 as described above. In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). More preferably, one of the P36H and Q79R mutations, P36H and N80H mutations or Q79R and N80H mutations present in SEQ ID NO:4 (corresponding to the P300H and Q457R mutations, P300H and N458H mutations, or Q457R and N458H mutations when referring to the human wild type IL7Ra represented by SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:4 as described above. Most preferably, the P36H, Q79R and N80H mutations present in SEQ ID NO:4 (corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra represented by SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:4 as described above. In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain according to the present invention comprises SEQ ID NO:4. In an embodiment, the IL7Ra signaling domain according to the present invention is SEQ ID NO:4. In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence that is at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs:2-4. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least
88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:2 or 4, and the mutation corresponding to the P300H mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) is still present in said sequence derived from SEQ ID NO:2 or 4. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 2 or 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:3 or 4, and the mutation corresponding to the Q457R or N458H mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) is still present in said sequence derived from SEQ ID NO:3 or 4. In some preferred embodiment, both mutations corresponding to the Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in said sequence derived from SEQ ID NO:3 or 4. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to SEQ ID NO:4, and any one or more of the mutations corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in said sequence derived from SEQ ID NO:4. In some preferred embodiment, the mutations corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in said sequence derived from SEQ ID NO:3 or 4. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 3 or 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In an embodiment, the IL7Ra signalling domain as described herein, wherein this IL7Ra signalling domain is represented by an amino acid sequence which has at least 80% identity with SEQ ID NO:2,
3 and/or 4, preferably wherein the mutation corresponding to the P300H mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) is still present in the sequence derived from SEQ ID NO:2 and 4, the mutations corresponding to the Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in the sequence derived from SED ID NO:3 and 4 and the mutations corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:4. In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO: 4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence represented by SEQ ID NO:2 and has the mutation corresponding to the P300H mutation when referring to the human wild type IL7Ra (SEQ ID NO:1). In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence represented by SEQ ID NO:3 and has the mutations corresponding to the Q457R and N458H mutations when referring to the human wild type. In some embodiments, the IL7Ra signaling domain according to the present invention has an amino acid sequence represented by SEQ ID NO:4 and has the mutations corresponding to the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1). In some embodiments, the P300H mutation (in SEQ ID NO:1) or P36H mutation (in SEQ ID NO:4) may be replaced with any one of P300H, P300A, P300W, P300E, P300L and P300Q mutations (when referring to SEQ ID NO:1). Some other exemplary IL7Ra signaling domains of the present invention: SEQ ID NO:178 Human truncated IL7Ra signalling domain (81 aa) with P36A mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNAESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:179: Human truncated IL7Ra signalling domain (81 aa) with P36A, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNAESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ SEQ ID NO:180 Human truncated IL7Ra signalling domain (81 aa) with P36W mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNWESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:181: Human truncated IL7Ra signalling domain (81 aa) with P36W, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNWESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ
SEQ ID NO:182 Human truncated IL7Ra signalling domain (81 aa) with P36E mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNEESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:183: Human truncated IL7Ra signalling domain (81 aa) with P36E, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNEESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ SEQ ID NO:184 Human truncated IL7Ra signalling domain (81 aa) with P36L mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNLESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:185: Human truncated IL7Ra signalling domain (81 aa) with P36L, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNLESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ SEQ ID NO:186 Human truncated IL7Ra signalling domain (81 aa) with P36Q mutation KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNQESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQ SEQ ID NO:187: Human truncated IL7Ra signalling domain (81 aa) with P36Q, Q79R and N80H mutations. KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNQESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQ In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs: 178-187. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. In some embodiments, the IL7Ra signalling domain is represented by an amino acid sequence which has at least 80% identity with any one of SEQ ID NOs: 178-187, preferably wherein the mutation corresponding to the P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) is still present in the sequence derived from SEQ ID NOs: 178-187, and
the mutations corresponding to any one of P300A, P300W, P300E, P300L and P300Q mutation, and Q457R and N458H mutations when referring to the human wild type IL7Ra (SEQ ID NO:1) are still present in the sequence derived from SEQ ID NOs: 179, 181, 183, 185 and 187. In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from any one of SEQ ID NOs:178-187 by 0, 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, 31, 32, 33, 34, or 35 amino acids. ). In some embodiments, the mutation at P300 when referring to SEQ ID NO:1 (or P300A, P300W, P300E, P300L or P300Q) is still present in the sequence derived from any one of SEQ ID NOs: 178-187. In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from any one of SEQ ID NOs:179, 181, 183, 185 and 187 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting IL7Ra signaling domain variant is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). In some embodiments, the mutation at P300 when referring to SEQ ID NO:1 (or P300A, P300W, P300E, P300L or P300Q) is still present in the sequence derived from any one of SEQ ID NOs: 178-187. In some embodiments, the resulting IL7Ra signaling domain variant is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5) and the mutation at P300 when referring to SEQ ID NO:1 (or P300A, P300W, P300E, P300L or P300Q) is still present in the sequence derived from any one of SEQ ID NOs: 179, 181, 183, 185 and 187. In this context, the IL7Ra signalling domain derived from any one of SEQ ID NOs: 178-187 is functional as described herein. In some embodiments, the IL7Ra signaling domain may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to any one of SEQ ID NOs: 178- 187, or the sequence derived from SEQ ID any one of SEQ ID NOs: 178-187. In some embodiments, the IL7Ra signaling domain may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to any one of SEQ ID NOs: 178-187, or the sequence derived from any one of SEQ ID NOs: 178-187. Preferably, the P36A, P36W, P36E, P36L or P36Q mutation present in any one of SEQ ID NOs: 178-187 (corresponding to P300A, P300W, P300E, P300L, or P300Q mutations in the human wild type IL7Ra represented by SEQ ID NO:1) is still present in the sequence derived from any one of SEQ ID NOs: 178-187. Preferably, the resulting IL7Ra signaling domain variant from any one of SEQ ID NOs: 179, 181, 183, 185 and 187 is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). More preferably, the P36A, P36W, P36E, P36L or P36Q mutation present in any one of SEQ ID NOs: 178- 187 (corresponding to P300A, P300W, P300E, P300L, or P300Q mutations in the human wild type IL7Ra represented by SEQ ID NO:1) is still present in the sequence derived from any one of SEQ ID NOs: 179, 181, 183, 185 and 187, and the resulting IL7Ra signaling domain variant from any one of SEQ ID NOs: 179, 181, 183, 185 and 187 is still able to activate STAT3 and STAT5 as described above (preferably able to recruit and activate STAT3 and STAT5). Preferably, the following mutations:
P36A, Q79R and N80H mutations in SEQ ID NO:179, P36W, Q79R and N80H mutations in SEQ ID NO:181, P36E, Q79R and N80H mutations in SEQ ID NO:183, P36L, Q79R and N80H mutations in SEQ ID NO:185, or P36Q, Q79R and N80H mutations in SEQ ID NO:187, present in SEQ ID NO: 179, 181, 183, 185 or 187 (corresponding to: P300A, Q457R and N458H mutations: P300W, Q457R and N458H mutations: P300E, Q457R and N458H mutations: P300L, Q457R and N458H mutations: or P300L, Q457R and N458H mutations: in the human wild type IL7Ra represented by SEQ ID NO:1) are still present in the sequence derived from SEQ ID NO:179, 181, 183, 185 or 187 as described above. In some embodiments, the IL7Ra signaling domain according to the present invention comprises any one of SEQ ID NOs: 178-187. In an embodiment, the IL7Ra signaling domain according to the present invention is any one of SEQ ID NOs: 178-187. The truncated IL7Ra signalling domain according to the present invention is beneficial when incorporated in any receptor and is particularly advantageous to use in any chimeric antigen receptor signalling cassettes. The advantageous effects are in part attributed to its shorter amino acids length compared to the length of the human wild type full length IL7Ra (e.g. SEQ ID NO:1), its capacity to recruit and activate STAT5, its capacity to activate STAT3, preferably recruit and activate STAT3 (resulted for example from mutations at Q457 and N458 when referring to the human wild type IL7Ra, i.e. SEQ ID NO:1), and/or from the mutation at P300 when referring to the human wild type IL7Ra, i.e. SEQ ID NO:1. Provided herein is also a truncated IL7Ra signalling domain which is able to bind and activate STAT4, preferably to activate STAT4. In some embodiments, the truncated IL7Ra signaling domain is an IL7Ra signaling domain as defined in the first aspect and any of the embodiments described herein. STAT4 is a key signaling molecule, which is essential for signal transduction by IL-12, IL-23, IL27, IL35 and type I IFN signaling in T cells, NK-cells, NKT-cells, gamma-delta T-cells, monocytes and Dendritic cells (doi: 10.7150/ijbs.41852). STAT4 protein contains six domains that have different functions in the JAK-STAT pathway: 1. N-terminal domain: dimerizes inactivated STATs and promotes nuclear translocation; 2. helical coiled coil: provides a carbonized hydrophilic surface and binds to regulatory factors; 3. DNA-binding domain: binds to an enhancer of the GAS family; 4. linker domain: involves in
the DNA binding process; 5. Src homology (SH2) domain: binds specifically to the cytokine receptor after tyrosine phosphorylation 6. C-terminal transactivation domain: activates transcriptional process . Additionally, with or without C-terminal transactivation, there are two spliced STAT4 transcripts, including STAT4α and STAT4β. STAT4α induces more IFN-γ production than that by STAT4β, whereas STAT4β proliferates more vigorously in response to IL12 stimulation (doi: 10.7150/ijbs.41852). In T- cells, NK-cells, NK-T cells and gamma-delta T-cells STAT4 is predominantly activated in response to IL-12 enhancing their activation, proliferation and cytotoxicity (https://doi.org/10.3389/fimmu.2020.575597). Upon binding to IL12R, the JAK2 and TYK2 are linked to IL12Rβ2 and IL12Rβ1, and then STAT4 is phosphorylated on tyrosine 693 (PMID: 8943379). Moreover, STAT4 is phosphorylated on serine 721 during activation of the p38/MKK6 signaling pathway (PMID: 10961885). STAT4 signaling promotes differentiation of naive CD4+ T-cells towards Th1 cells, production of IFN-γ and augmentation of cell-mediated immune responses by T and NK-cells. Moreover, IL-12 triggers downregulation of IFNγR2 with a concomitant decrease in susceptibility to IFNγ-induced apoptosis of tumor-infiltrating CD8+ T cells (doi: 10.4049/jimmunol.1300652). Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation- dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-γ production by APC as well as IFN-γ production by DC during Ag presentation (https://doi.org/10.4049/jimmunol.166.7.4446). IL-12, both in vivo and in vitro, by the means of STAT4 signaling induces a rapid reduction of tumor supportive macrophage activities (IL-10, MCP-1, migration inhibitory factor, and TGFbeta production) and a concomitant increase in proinflammatory and immunogenic activities (TNF-alpha, IL-15, IL-18 production and MHCII upregulation). Similar shifts in functional phenotype are induced by IL-12 in tumor-infiltrating macrophages isolated from the primary tumor mass and in TAMs isolated from lung containing metastases, spleen, and peritoneal cavity. The ability of IL-12 to initiate this functional conversion may contribute to early amplification of the subsequent destructive antitumor immune response (DOI: 10.4049/jimmunol.178.3.1357). Different combinations of STAT4 are activated by a variety of cytokines, including interleukin (IL)12, type I interferon (IFN-I), IL23, IL2, IL27, and IL35, etc. IL12 is produced by B cells and antigen- presenting cells and is secreted as a pro-inflammatory cytokine in the form of a heterodimer. IL12 receptor (IL12R) is composed of two different subunits, including IL12Rβ1 and IL12Rβ2. Upon binding to IL12R, the JAK2 and TYK2 are linked to IL12Rβ2 and IL12Rβ1, and then STAT4 is phosphorylated on tyrosine 693. Moreover, STAT4 is phosphorylated on serine 721 during activation of the p38/MKK6 signaling pathway. The IL12-JAK-STAT4 pathway increases IFNγ production and Th1 cell differentiation. Several other genes that require STAT4 for transcriptional activation have been identified, including activator protein 1 (AP1), IL10, ERM, IFN regulating factor (IRF)-1/4/8, IL18Rα, IL12β2, and Rux. STAT4 binds c-Jun, and then interacts with AP1-relevant promoter. A conserved STAT4- binding element was found in the fourth intron of the IL10 gene. The ETS transcription factor, EMR, is selectively expressed in Th1 cells. ERM can modulate IFNγ gene transcription with STAT4 or some STAT4 inducible factors. IRF1 gene is induced via IL12-dependent transactivation of IRF1 in human natural killer (NK) and T cells. Additionally, it suggests that IL12 may further strengthen innate
immune responses by inducing the expression of IRF4 and IRF8 genes. IL12 induces the binding of STAT4 to the IL12Rβ2 enhancer to form a positive feedback loop of IL12/STAT4 axis during T cell receptor (TCR) stimulation. STAT4 binds directly to the IL18Rα locus and alters its acetylation, reducing metastatic binding and DNA methylation transiently and resulting in high expression of IL18Rα in Th1 cells. The promoter regions of Runx1 and Runx3 are targets of STAT4 to promote the antiviral activity of NK cells. Therefore, the IL12/STAT4 axis is vital for inflammatory cytokines secretion that participates in many diseases and anti-tumor responses. IL12 synergizes with IL18 to enhance both cytotoxicity and IFNγ production. A lot of CAR T-cells constructs incorporating STAT4 signaling through IL12, IL-23, IL-27 have been developed (doi: 10.1038/s41467-023-37646-y, doi:10.1136/jitc-2021- 003633,DOI: 10.1016/j.ymthe.2021.10.011, doi: 10.4161/2162402X.2014.994446). To reprogram the functional capacities specifically of engineered CAR T cells, IL12 may be inserted into the extracellular moiety of a CD28-ζ CAR; both the CAR endodomain and IL12 may be functionally active. This activity may be evidenced by antigen-redirected effector functions and STAT4 phosphorylation, respectively. The IL12-CAR reprogrammed CD8+ T cells toward a so far not recognized natural killer (NK) cell-like signature and a CD94+CD56+CD62Lhigh phenotype closely similar, but not identical, to NK and cytokine induced killer (CIK) cells. In contrast to conventional CAR T cells, IL12-CAR T cells are expected to acquire antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen. Simultaneous signaling through both the CAR endodomain and IL12 are required for inducing maximal NK-like cytotoxicity. Antigen-negative tumors may be attacked by IL12-CAR T cells, but not by conventional CAR T cells. One of the advantages of IL12-CAR is increased CAR-T cell survival, proliferation, and persistence (DOI: https://doi.org/10.1101/2023.01.06.522784), Th1 conversion (https://doi.org/10.1016/j.ymthe.2021.10.011), decreased expression of immune checkpoint receptors (DOI: 10.1038/s41587-019-0398-2), superior killing capacity and in vivo tumour control (https://doi.org/10.1016/j.ymthe.2021.10.011), even in tumor models with low antigen density (DOI: https://doi.org/10.1101/2023.01.06.522784). IL-12 triggers NK-cell transition of CAR T-cells and acquisition of antigen-independent, NK-like cytotoxicity toward cancer cells, mediated, at least in part, by CD94 (https://doi.org/10.1016/j.ymthe.2021.10.011). The inventors of the present invention have surprisingly discovered that using a truncated IL7Ra signalling domain wherein said truncated IL7Ra signalling domain is able to activate STAT4 and STAT5 (preferably bind and activate STAT4 and STAT5) when used in a receptor, especially in a CAR, and expressed into T cells leads to CAR-T cells with attractive properties. In some embodiments, it (i.e. the truncated IL7Ra signalling domain which is able to activate STAT4 and STAT5 when used in a receptor, or a CAR comprising said truncated IL7Ra signalling domain) greatly prevents and overcomes the functional exhaustion of the CAR T-cells, improves the proliferation, survival and expansion of said CAR T-cells, improves the capability to control tumor population cells by said CAR T-cells, improves the
antitumor activity and/or cytotoxicity capacity of said CAR T-cells, improves the persistence potential of the CAR-T cells, improves the safety potential of said CAR-T which can be activated without systemic toxicity, and improves sensitivity of the CAR towards cell lines with low antigen density on the surface. In some embodiments, the truncated IL7Ra signalling domain able to activate STAT4 and STAT5 (preferably bind and activate STAT4 and STAT5) comprises a mutation at the amino acid position corresponding to position 300 of the human wild type IL7Ra (SEQ ID NO:1) (which corresponds to position 36 of the truncated IL7Ra SEQ ID NOs:192 for example). In some embodiments, the improved properties of said CAR-T cells can be attributed at least in part to such truncated IL7Ra domain having a STAT4 binding site according to the present invention. In some embodiments, the improved properties of said CAR-T cells can be attributed at least in part to such truncated IL7Ra domain with a single mutation corresponding to amino acid position 300 of the human wild type IL7Ra (corresponds to amino acid position 36 of the truncated IL7Ra having any one of SEQ ID NO:192-203), according to the present invention. In some embodiments, the IL7Ra signalling domain according to the invention is able to drive, allow, trigger the activation, preferably the recruitment and activation of the STAT proteins. In some embodiments, the STAT protein is STAT4. In some embodiments, the IL7Ra signalling domain according to the invention is able to recruit and activate STAT4, which is not the case of the human wild type IL7Ra. In some embodiments, the STAT proteins are STAT3 and STAT4. In some embodiments, the IL7Ra signalling domain according to the invention is able to recruit and activate both STAT3 and STAT4, which is not the case of the human wild type IL7Ra. In the context of the present invention, the STAT4 binding/recruiting site may be represented by YLPSNID (SEQ ID NOs: 189), TX1X2GYL (wherein X1 and X2 may be any amino acid, each chosen independently from the other, SEQ ID NO: 190) or GYKPQIS (SEQ ID NO: 191). In some embodiments, the STAT4 binding/recruiting site in the IL7Ra signaling domain according to the invention is YLPSNID (SEQ ID NOs: 189) which is present at positions 82-88 when referring to any one of SEQ ID NOs: 192-203. In some preferred embodiments, the Y (tyrosine) is phosphorylated Y or pY. In this context, the motif YLPSNID (SEQ ID NOs: 189) is the motif present in the IL-12 beta 2 subunit of the IL-12 receptor complex, wherein the Y is present at position 800 of the IL-12R beta 2 subunit. Alternatively, the motif used in the IL7Ra signaling domain may be derived therefrom. The Stat4 SH2 domain may be directly recruited to a tyrosine present in a motif, wherein the tyrosine is present as the first residue of said motif. In an embodiment, the motif is YLPSNID (SEQ ID NOs: 189) and the tyrosine recruiting the Stat4 SH2 is the first tyrosine of this motif. In some embodiments, the STAT4 binding/recruiting in the IL7Ra signaling domain according to the invention is TX1X2GYL (SEQ ID NO: 190). In this context, X1 and X2 can be any amino acid, each chosen independently from the other. In some preferred embodiments, X1 is not an H, and X2 is not an D. In some preferred embodiments, the Y (tyrosine) is phosphorylated Y or pY.
In some embodiments, the STAT4 binding/recruiting site in the IL7Ra signaling domain according to the invention is GYKPQIS (SEQ ID NO: 191). In some preferred embodiments, the Y (tyrosine) is phosphorylated. The motif GYKPQIS (SEQ ID NO: 191) is present in the IL-23R, and the Y is the conserved tyrosine residue Y484 of the IL23R. The Stat4 SH2 domain may be directly recruited to a motif comprising a tyrosine in the second place such as the motif present in the IL23R and represented by GYKPQIS (SEQ ID NO: 191), wherein the tyrosine corresponds to Y484 of the IL23R. Example of IL7Ra signaling domain comprising STAT4 binding site: SEQ ID NO: 192: truncated IL7Ra signaling domain + P36H and STAT4 binding site KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 193: truncated IL7Ra signaling domain + P36A and STAT4 binding site KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNAESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 194: truncated IL7Ra signaling domain + P36W and STAT4 binding site KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNWESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 195: truncated IL7Ra signaling domain + P36E and STAT4 binding site KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNEESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 196: truncated IL7Ra signaling domain + P36L and STAT4 binding site KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNLESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 197: truncated IL7Ra signaling domain + P36Q and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNQESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYQNQYLPSNID SEQ ID NO: 198: truncated IL7Ra signaling domain + P36H + STAT3 and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID SEQ ID NO: 199: truncated IL7Ra signaling domain + P36A + STAT3 and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNAESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID SEQ ID NO: 200: truncated IL7Ra signaling domain + P36W + STAT3 and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNWESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID SEQ ID NO: 201: truncated IL7Ra signaling domain + P36E + STAT3 and STAT4 binding sites
KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNEESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID SEQ ID NO: 202: truncated IL7Ra signaling domain + P36L + STAT3 and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNLESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID SEQ ID NO: 203: truncated IL7Ra signaling domain + P36Q + STAT3 and STAT4 binding sites KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNQESFLDCQIHRVDDIQARDEVEGFLQDTFPQQE EAYVTMSSFYRHQYLPSNID *Underlined means P36 mutation. In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs:192-203, preferably to any one of SEQID NO:s 192-197, more preferably to SEQ ID NO: 192. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. Preferably, the mutation at position P36 (P36H, P36A, P36W, P36E, P36L, or P36Q) in any one of SEQ ID NO: 192-203 (corresponding to the P300 mutation in SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NOs:192-203 as described above. In some more preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1, or P36H when referring to any one of SEQ ID NOs:192, and more preferably both the mutation P300H and a STAT4 binding site are still present. In an embodiment, a STAT4 binding site is represented by any of SEQ ID NO:189, 190 or 191 (or derived therefrom). In some embodiments, the IL7Ra signaling domain comprises a sequence that differs from any one of SEQ ID NOs:192-203 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting IL7Ra signaling domain variant is still able to activate STAT4 and STAT5 as described above. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. Preferably, the mutation at position P36 (P36H, P36A, P36W, P36E, P36L, or P36Q) in any one of SEQ ID NO: 192-203 (corresponding to the P300 mutation in SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NOs:192-203 as described above. In some preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1, and more preferably the mutation P300H and a STAT4 binding site are still present. In
an embodiment, a STAT4 binding site is represented by any of SEQ ID NO:189, 190 or 191 (or derived therefrom). In some embodiments, the IL7Ra signaling domain may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to any one of SEQ ID NOs:192-203, or the sequence derived from any one of SEQ ID NOs:192-203. In some embodiments, the IL7Ra signaling domain may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to any one of SEQ ID NOs:192-203 or a sequence derived from any one of SEQ ID NOs:192-203. Preferably, the mutation at position P36 (P36H, P36A, P36W, P36E, P36L, or P36Q) in any one of SEQ ID NO: 192-203 (corresponding to the P300 mutation in SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NOs:192-203 as described above. In some preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1, and more preferably the mutation P300H and a STAT4 binding site are still present. In an embodiment, a STAT4 binding site is represented by any of SEQ ID NO:189, 190 or 191 (or derived therefrom). In some embodiments, the IL7Ra signalling domain is represented by an amino acid sequence which has at least 80% identity with SEQ ID NOs:192-203, preferably wherein the mutation corresponding to the P300H, P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) and/or the STAT4 binding site is still present in the sequence derived from any one of SEQ ID NOs:192-203. In some embodiments, both the STAT3 and STAT4 bindings sites are still present in the sequence derived from any one of SEQ ID NOs:198-203. In some embodiments, both the STAT3 and STAT4 bindings sites, and the P300 mutations are still present in the sequence derived from any one of SEQ ID NOs:198-203. In some preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1. In an embodiment, a STAT4 binding site is represented by any of SEQ ID NO:189, 190 or 191 (or derived therefrom). In some embodiments, the IL7Ra signaling domain according to the present invention comprises any one of SEQ ID NOs: 192-203, preferably any one of SEQ ID Nos: 192-197. In some preferred embodiments, the IL7Ra signaling domain according to the present invention comprises SEQ ID NO:192. In some embodiments, the IL7Ra signaling domain according to the present invention is any one from SEQ ID NOs: 192-203, preferably any one of SEQ ID Nos: 192-197, more preferably the IL7Ra signaling domain is SEQ ID NO:192. The binding/recruitment of STAT4 to/by the IL7Ra signalling domain of the present invention is therefore made possible via a STAT4 binding site (represented as any one of SEQ ID NO:189-191) present in said IL7Ra signalling domain. In some preferred embodiments, the STAT4 binding site is represented SEQ ID NO:189, more preferably the Y is phosphorylated tyrosine (or pY). STAT4 may be considered to be activated when a detectable signal of its activation will be detected using techniques known to the skilled person. STAT activation may be assessed using any standard/known technique available to the skilled person. In the context of the present invention, the recruited/bound STAT4 may be further activated by the phosphorylated and activated JAK2 and/or TYK2. In the context of this application, it may be understood that STAT4 and/or STAT5 is recruited to
the “binding site” present in the IL7Ra signaling domain of the present invention. A “binding site” may be understood as a “recruiting site”. In some embodiments, the IL7Ra signalling domain according to the invention is able to recruit and activate STAT5 and is able to activate STAT4. In the context of the present invention, STAT4 may be activated directly or indirectly by the IL7Ra signalling domain of the present invention. Direct activation may mean that STAT4 is activated/phosphorylated by direct binding to the IL7Ra signalling domain of the present invention, while indirect activation may mean that STAT4 is activated/phosphorylated by another molecule, such as a kinase. In this situation, STAT4 does not interact/bind directly with the IL7Ra. This other molecule may bind to the IL7Ra and indirectly recruit STAT4 in the vicinity of the IL7Ra. Activation of STAT4 may be realized by phosphorylation of the tyrosine residue at Tyr693. phosphorylation may be measured by western blot or flow cytometry Phospho-STAT4 (Y693) antibodies. For example, BD Phosflow™ PE Mouse Anti-Stat4 (pY693) from BD Biosciences may be used. Activation of STAT4 may also be detected using Luciferase reporters’ assay, Electrophoretic Mobility Shift Assay (EMSA), or ChIP-qPCT to confirm genomic binding to target genes. In a preferred embodiment, the IL7Ra signaling domain according to the invention is able to recruit and activate both STAT4 and STAT5. Activated STAT4 and STAT5 further dimerize in the cytoplasm and translocate into the nucleus where they act as transcription factors for genes associated with their target genes comprising a STAT4 or a STAT5 binding site respectively. In an embodiment, such target genes are able to induce or are linked to the survival and proliferation of T cells. III. Receptor In a second aspect of the present invention, there is provided a receptor comprising an IL7Ra signaling domain of the first aspect. This receptor comprises a transmembrane domain. In an embodiment, this receptor comprises an extracellular domain. In an embodiment, this extracellular domain comprises an antigen binding domain. In an embodiment, this receptor further comprises an additional signalling domain and/or a co-stimulatory domain. In a preferred embodiment, this receptor is a CAR. In an embodiment, the receptor is an homodimeric receptor. In another embodiment, the receptor is a heterodimeric receptor. As used herein, the term ''heterodimeric receptor'' includes any receptor which is a macromolecular complex formed by two protein monomers which are different to each other. The term may further be understood to include functional heterodimeric fragments or parts of receptors. As non-limiting examples, the term includes a signal transduction moiety of a B-cell receptor (which is an Ig-α/Ig-β heterodimer (CD79)), B-cell receptor heavy and light chain, a Toll-like receptor 1 and 2 heterodimer, an integrin like ανβ5, a phagocytic receptor Mac-1, an MHC, a CD94, a T-cell receptor (TCR), an alpha
beta (αβ) TCR, a gamma delta (γδ) TCR, and any other receptor or functional fragment or part thereof that may occur as a heterodimer. The receptor may be a synthetic receptor (or engineered receptor). In an embodiment, the synthetic receptor is a cytokine receptor. In some embodiments, said synthetic receptors do not comprise activation and costimulatory domains. In this context, said synthetic receptors, preferably synthetic cytokine receptors, may function to provide cytokine receptor-like signalling. In an embodiment, a synthetic cytokine receptor comprises the IL7Ra signaling domain as defined earlier and a conserved extracellular region (cytokine receptor homology domain [CDH]) and several structural modules, including extracellular immunoglobulin or fibronectin type III–like domains, transmembrane domains, and optionally additional intracellular homology regions. In an embodiment, the engineered cytokine receptor may be a class I cytokine receptor, class II cytokine receptor, TNF receptor, IL-1 receptor, tyrosine kinase receptor, and chemokine receptor. In an embodiment, the engineered cytokine receptor is a class I cytokine receptor. In an embodiment, the engineered class I cytokine receptor is a homodimeric receptor. In another embodiment, the engineered class I receptor is a non-homodimeric receptor. In some embodiments, said synthetic receptors, preferably synthetic cytokine receptors, may be activated by a membrane-bound antigen, and/or a soluble antigen. In this context, synthetic cytokine receptors or SyCyR technology may be used to integrate features of traditional T cell receptors (TCRs) and chimeric antigen receptors (CARs) to provide potent and specific cytotoxic activity. Alternatives to SyCyR Technology may include Chimeric Antigen Receptors (CARs), T Cell Receptor Fusion Constructs (TRuCs), T cell receptor gene editing (TCR-GE), T cell engagers (TCEs) or natural killer (NK) cell-based therapies. In a further embodiment, the present invention provides a CAR comprising the IL7Ra signalling domain as described above (in the first aspect), said CAR further comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising said IL7Ra signalling domain defined above and optionally a co-stimulatory domain. In some embodiments, the CAR further comprises an immunoreceptor Tyrosine-Based Activation Motifs (ITAMs). In some embodiments, said intracellular signalling domain further comprises a CD3z signaling domain. In another embodiment of this aspect, the present invention provides a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain. In an embodiment, the CAR of the invention is able to directly activate STAT3 without needing the IL7Ra signalling domain. In other words, in one embodiment, the CAR of this invention may comprise a STAT3 binding site in its intracellular signalling domain, therefore the activation of STAT3 by the CAR of the invention is not directly mediated by the IL7Ra signalling domain and said STAT3 binding site is not present in the IL7Ra signalling domain.
STAT3 binding/recruiting sites have been earlier defined herein. In short, such motifs are represented by SEQ ID NO:6: YX1X2Q, wherein: X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally Q is mutated/substituted into P, T, Y, N, F or A. In some embodiments, the motif which is able to recruit/bind STAT3 comprises any of the following sequences: YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA (SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF. In a preferred embodiment, the STAT3 binding/recruiting site is YRHQ (SEQ ID NO:8). In some embodiments of this aspect, the present invention provides a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain. In an embodiment, the CAR of the invention is able to directly activate STAT4 without needing the IL7Ra signalling domain. In other words, in one embodiment, the CAR of this invention may comprise a STAT4 binding site in its intracellular signalling domain, therefore the activation of STAT4 by the CAR of the invention is not directly mediated by the IL7Ra signalling domain and said STAT4 binding site is not present in the IL7Ra signalling domain. In some embodiments, the STAT4 binding site is present after CD3z signaling domain. STAT4 binding/recruiting sites have been earlier defined herein. In short, such motifs are represented by YLPSNID (SEQ ID NOs: 189), TX1X2GYL (SEQ ID NO: 190) or GYKPQIS (SEQ ID NO: 191). In some embodiments, the Y in the STAT4 binding site is phosphorylated. In some embodiments, the STAT4 binding site is TX1X2GYL (SEQ ID NO: 190), and X1 and X2 can be any amino acid, each chosen independently from the other. In some preferred embodiments, X1 is not an H, and X2 is not an D. In a preferred embodiment, the STAT4 binding/recruiting site is YLPSNID (SEQ ID NOs: 189), and more preferably the Y in the STAT4 binding site is phosphorylated.
In some embodiments, the STAT4 binding domain in the CAR is able to drive, allow, trigger the activation, preferably the recruitment and activation of the STAT proteins, preferably the STAT protein is STAT4. In some embodiments, the STAT4 binding domain according to the invention is able to recruit and activate STAT4. The binding/recruitment of STAT4 to/by the IL7Ra signalling domain of the present invention is therefore made possible via a STAT4 binding site (preferably represented by any one of SEQ ID NO:189-191 or is derived thereform) present in said CAR, preferably wherein the Y in said STAT4 binding site is phosphorylated Y. The binding/recruitment of STAT4 to/by the IL7Ra signalling domain of the present invention is therefore made possible via a STAT4 binding site (preferably represented by any one of SEQ ID NO:189-191 or derived therefrom) present in said IL7Ra signalling domain. In some preferred embodiments, the STAT4 binding site is represented SEQ ID NO:189, more preferably the Y is phosphorylated tyrosine (or pY). STAT4 may be considered to be activated when a detectable signal of its activation will be detected using techniques known to the skilled person. STAT activation may be assessed using any standard/known technique available to the skilled person. In the context of the present invention, the recruited/bound STAT4 may be further activated by the phosphorylated and activated JAK2 and/or TYK2. In the context of this application, it may be understood that STAT4 and/or STAT5 is recruited to the “binding site” present in the CARs of the present invention. A “binding site” may be understood as a “recruiting site”. In some embodiments, the IL7Ra signalling domain according to the invention is able to recruit and activate STAT5 and is able to activate STAT4. In the context of the present invention, STAT4 may be activated directly or indirectly by the CARs of the present invention. Direct activation may mean that STAT4 is activated/phosphorylated by direct binding to the intracellular domain of the CARs of the present invention, while indirect activation may mean that STAT4 is activated/phosphorylated by another molecule, such as a kinase. In this situation, STAT4 does not interact/bind directly with the intracellular domain of the CARs of the present invention. This other molecule may bind to the IL7Ra and indirectly recruit STAT4 in the vicinity of the intracellular domain of the CARs of the present invention. Activation of STAT4 may be realized by phosphorylation of the tyrosine residue at Tyr693. phosphorylation may be measured by western blot or flow cytometry Phospho-STAT4 (Y693) antibodies. Activation of STAT4 may also be detected using Luciferase reporters’ assay, Electrophoretic Mobility Shift Assay (EMSA), or ChIP-qPCT to confirm genomic binding to target genes. Activated STAT4 and STAT5 further dimerize in the cytoplasm and translocate into the nucleus where they act as transcription factors for genes associated with their target genes comprising a STAT4 or a STAT5 binding site respectively. In an embodiment, such target genes are able to induce or are linked to the survival and proliferation of T cells.
In a preferred embodiment, the CARs according to the invention is able to recruit and activate both STAT4 and STAT5. In some embodiments, the CARs comprising an intracellular domain comprising a STAT4 binding site is expected to enhance CAR sensitivity towards cell lines with low antigen density on the surface of the target cells, to enhance the cytotoxicity activity of the cell comprising said CAR, to enhance/improve the acquired antigen-independent, human leukocyte antigen E (HLA-E) restricted cytotoxic capacities eliminating antigen-negative cancer cells in addition to eliminating cancer cells with CAR cognate antigen, and to improve the resistance of the cell comprising said CAR to immunosuppression by tumour microenviroment and durability of the response. In some embodiment, such CAR constructs are expected to have advantage in other cell like NK-cells, NK-T cells, gamma delta T-cells as well as DC and macrophages. In some embodiments, said improvements can be attributed in part to additional STAT4-mediated phosphorylation. In this context, in some embodiments said CARs comprising an intracellular domain comprising a STAT4 binding site may be used in any immune cells as described herein to generate engineered immune cells. Non-limiting examples of immune cells that may express CARs containing an intracellular domain with a STAT4 binding site, as described herein, include NK cells, NKT cells, γδ T cells, dendritic cells (DCs), and macrophages. As used herein, the term "chimeric antigen receptor" or "CAR" or “chimeric immunoreceptors”, “chimeric T cell receptors”, “engineered T cell receptor”, “recombinant T cell receptor” or “artificial T cell receptors” refers to an artificial exogenous antigen recognition receptor that can induce signaling in an engineered cell that expresses the CAR upon binding of the CAR to an antigen, for example, an antigen associated with a cancer or infectious disease. A CAR generally induces signalling in the engineered cell that expresses the CAR but not in a cell that expresses or presents the antigen bound by the CAR. In some aspects the current invention encompasses the use of any CAR sequence known in the art as long as it is associated with the IL7Ra signalling domain of the first aspect. Non-limiting aspects of chimeric antigen receptors are described in, e.g., Kershaw et al, Nature Reviews Immunol.5(l2):928-940, 2005; Eshhar et al, Proc. Natl. Acad. Sci. U.S.A. 90(2):720-724, 1993; Sadelain et al, Curr. Opin. Immunol. 21(2): 215-223, 2009; WO 2015/142675; WO 2015/150526; and WO 2014/134165. A CAR comprises at least one antigen binding domain, at least one transmembrane domain, and at least one intracellular signalling domain (comprising one or more co-stimulatory domains and a cytotoxicity induction domain). In some cases, a CAR comprises an (extracellular) hinge domain or stalk region. Some embodiments of any of the chimeric antigen receptors described herein can further include a dimerization domain and/or a peptide tag. Several methods can be used to determine the KD values of any of the CARs described herein are known in the art (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.).
The ability of a CAR to bind an antigen or a target (such as BCMA) can be compared with the ability of a negative control CAR to bind the same antigen or target. A CAR that binds the same antigen or target using a suitable assay with 25%, 50%, 100%, 200%, 1000% or higher increased affinity relative to the negative control CAR, is said to “specifically bind to” or “specifically interact with” the target compound. Suitable assays include flow cytometry, Surface Plasmon Resonance (SPR) technology/ BIACORE instrument or Kinetic Exclusion Assay (KinExA®). 1. Antigen-binding domain An "antigen" is a molecule or molecular structure that an antigen receptor or an antigen-binding protein can recognize (for example, bind to). An antigen can be or can comprise, for example, a peptide, a polypeptide, a carbohydrate, a chemical, a moiety, a non-peptide antigen, a phosphoantigen, a tumor- associated antigen, a neoantigen, a tumor microenvironment antigen, a microbial antigen, a viral antigen, a bacterial antigen, an autoantigen, a glycan-based antigen, a peptide-based antigen, a lipid- based antigen, or any combination thereof. In some embodiments, an antigen is capable of inducing an immune response. In some examples, an antigen binds to an antigen receptor or antigen-binding protein, or induces an immune response, when present in a complex e.g., presented by MHC. In some cases, an antigen adopts a certain conformation in order to bind to an antigen receptor or antigen- binding protein, and/or to induce an immune response, e.g., adopts a conformation in response to the presence or absence of one or more metabolites. Antigen can refer to a whole target molecule, a whole complex, or a fragment of a target molecule or complex that binds to an antigen receptor or an antigen- binding protein. Antigen receptors that recognize antigens include exogenous antigen-recognition receptors disclosed herein and other antigen-recognition receptors, such as endogenous T cell receptors. As used herein the terms “antigen binding domain,” “antigen recognition domain”, “extracellular targeting domain” and “target recognition domain” may be used interchangeably. Non-limiting examples of a CAR targeting domain may be derived from a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, or a functional derivative, variant or fragment thereof, including, but not limited to, a Fab, a Fab', a F(ab')2, an Fv, a single-chain Fv (scFv), minibody, a diabody, and a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL), a DARPin, a monobody, a VVH (Variable Heavy domain of Heavy chain), or single domain antibodies, an affibody, a non-antibody domain, a VNAR domain, a (SCFV)2, a BiTE and any combination thereof. A non-antibody CAR targeting domain can be from or derived from a receptor or a receptor ligand, for example, APRIL can be used to target BCMA. A single-chain Fv or scFv fragment includes a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. In other examples, the linker can be a single amino acid. In some examples, the linker can be a chemical bond. See, e.g., Pluckthun, Antibodies from E. coli. In Rosenberg M. & Moore GP. (Eds.), The Pharmacology of Monoclonal Antibodies, Vol.113, pp.269-315, Spinger-Verlag, New York, 1994. ScFv-Fc fragments include an scFv attached to an Fc domain. For example, an Fc domain can be attached, e.g., to the C-terminus of the
scFv. The Fc domain can follow the VL or VH, depending on the orientation of the variable domains in the scFv. The Fc domain can be any Fc domain known in the art. In some examples, the Fc domain is an IgGl, IgG2, IgG3, or IgG4 Fc domain (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc domain). BiTEs are antigen-binding domains that include two VL and two VH in a single polypeptide that together form two scFvs, which can each bind to different epitopes on the same antigen or each bind to different antigens. See, e.g., Baeuerle et al, Curr. Opin. Mol. Ther.11 :22-30, 2009; Wolf et al, Drug Discovery Today 10: 1237-1244, 2005; and Huehls et al, Immunol. Cell Biol.93:290-296, 2015. A VHH domain is a single monomeric variable antibody domain found in camelids, and a VNAR domain is a single monomeric variable antibody domain found in cartilaginous fish. In some aspects the antigen binding domain may selectively bind any tumor antigens well known in the art. Examples include but are not limited to glioma associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, Interleukin-11 receptor alpha (IL-11Ra), Interleukin-13 receptor subunit alpha-2 (IL-13Ra or CD213A2), epidermal growth factor receptor (EGFR), B7H3 (CD276), Kit (CD117), carbonic anhydrase (CA-IX), CS-1 (also referred to as CD2 subset 1), Mucin 1, cell surface associated (MUC1), B cell maturation antigen (BCMA), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) bcr-abl, Receptor tyrosine-protein kinase ERBB2 (HER2/neu), β-human chorionic gonadotropin, alphafetoprotein (AFP), anaplastic lymphoma kinase (ALK), CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, adrenoceptor beta 3 (ADRB3), thyroglobulin, tyrosinase; ephrin type-A receptor 2 (EphA2), Receptor for Advanced Glycation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), synovial sarcoma, X breakpoint 2 (SSX2), A kinase anchor protein 4 (AKAP-4), lymphocyte-specific protein tyrosine kinase (LCK), proacrosin binding protein sp32 (OY-TES1), Paired box protein Pax-5 (PAX5), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), C-type lectin-like molecule-1 (CLL-1 or CLECL1), fucosyl GM1, hexasaccharide portion of globoH glycoceramide (GloboH), MN-CA IX, Epithelial cell adhesion molecule (EPCAM), EVT6-AML, transglutaminase 5 (TGS5), human telomerase reverse transcriptase (hTERT), polysialic acid, placenta-specific 1 (PLAC1), intestinal carboxyl esterase, LewisY antigen, sialyl Lewis adhesion molecule (sLe), lymphocyte antigen 6 complex, locus K 9 (LY6K), heat shock protein 70-2 mutated (mut hsp70-2), M-CSF, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinaserelated protein 2 (TRP-2), Cytochrome P4501B1 (CYP1B1), CCCTC- Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), prostase, prostate-specific antigen (PSA), paired box protein Pax-3 (PAX3), prostatic acid phosphatase (PAP), Cancer/testis antigen 1 (NY-ESO-1), Cancer/testis antigen 2 (LAGE-1a), LMP2, neural cell adhesion molecule (NCAM), tumor protein p53 (p53), p53 mutant, Rat sarcoma (Ras) mutant, glycoprotein 100 (gp100), prostein, OR51E2, pannexin 3 (PANX3), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), , high molecular weight-melanoma-associated antigen (HMWMAA), Hepatitis A virus cellular receptor 1 (HAVCR1), vascular endothelial growth factor receptor 2 (VEGFR2), Platelet-derived growth factor receptor beta (PDGFR-beta), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), survivin, telomerase, sperm protein 17
(SPA17), Stage-specific embryonic antigen-4 (SSEA-4), tyrosinase, TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1), prostate-carcinoma tumor antigen-1 (PCTA-1), melanoma inhibitor of apoptosis (ML-IAP), MAGE, Melanoma-associated antigen 1 (MAGE-A1), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma antigen recognized by T cells 1 (MelanA/MART1), X Antigen Family, Member 1A (XAGE1), elongation factor 2 mutated (ELF2M), ERG (TMPRSS2 ETS fusion gene), N-Acetyl glucosaminyl- transferase V (NA17), neutrophil elastase, sarcoma translocation breakpoints, mammary gland differentiation antigen (NY-BR-1), ephrinB2, CD20, CD22, CD23, CD24, CD30, CD32B, CD33, CD37, CD38, CD44v6, CD70, CD97, CD171, CD179a, CD200, CD229, androgen receptor, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, ganglioside GD2 (GD2), siglec-6, o-acetyl-GD2 ganglioside (OAcGD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), G protein-coupled receptor class C group 5, member D (GPRC5D), G protein-coupled receptor 20 (GPR20), chromosome X open reading frame 61 (CXORF61), folate receptor (FRa), folate receptor beta, Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (Flt3), Tumor-associated glycoprotein 72 (TAG72), Tn antigen (TN Ag or (GalNAcα-Ser/Thr)), angiopoietin-binding cell surface receptor 2 (Tie 2), tumor endothelial marker 1 (TEM1 or CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), uroplakin 2 (UPK2), mesothelin, Protease Serine 21 (Testisin or PRSS21), epidermal growth factor receptor (EGFR), fibroblast activation protein alpha (FAP), Olfactory receptor 51E2 (OR51E2), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); B-cell receptor (BCR), IgM receptor, EGF-like module containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), B cell-activating factor receptor (BAFF-R). In some aspects the antigen binding domain is selective for BCMA, CD19, PSA, HER-2/neu, MUC1, Carcinoembryonic antigen (CEA), CA-125, Epithelial tumor antigen (ETA), Tyrosinase, Melanoma-associated antigen (MAGE). In some preferred embodiment, the antigen binding domain may selectively bind CD19, BCMA and/or CEA. In some embodiments, a chimeric antigen receptor (CAR) described herein can bind to a single antigen (e.g., any of the exemplary antigens described herein or known in the art). In some embodiments, an antigen-binding domain described herein can bind to two or more different antigens (e.g., two or more of any of the exemplary antigens described herein or known in the art). Non-limiting examples of antigens include: BCMA, glypican-3, HER2, A33 antigen, 9-0-acetyl-GD3, CA19-9 marker, BhC CA- 125 marker, carboanhydrase IX (MN/CA IX), calreticulin, CCR5, CCR8, CD2, CD3,CD5, CD16, CD19, CD20, CD22, CD24, CD25, CD27, CD28, CD30, CD33, CD38, CD40L, CD44, CD44V6, CD63, CD70, CRTAM, (PD-l),
LTBR,
0X40, activating forms of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxicity receptors, NTB-A, PEN-5, carcinoma embryonic antigen (CEA; CD66e), desmoglein 4, E-cadherin neoepitope, endosialin, ephrin A2 (EphA2), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), fucosyl GM1, GD2, GD3, GM2, gangboside GM3, Globo H, glycoprotein 100, HER2/neu, HER3, HER4, insulin-like growth factor receptor 1, Lewis-Y, L Ly-6, melanoma-specific chondroitin-sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, Mullerian inhibitory substance (MIS) receptor type II, plasma cell antigen, poly SA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF-a precursor, 2B4 (CD244), 2-integrins, KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KLRGI, LAIR-l, NKG2A, NKR-P IA, Siglec-3, Siglec-7, Siglec-9, TCRa, TCRB, TCR5K, TIM1, LAG3, LAIR1, PD-1H, TIGIT, TIM2, and TIM3. Additional examples of antigens are known in the art. In some preferred embodiments, the chimeric antigen receptor can bind CD19, BCMA and/or CEA. Some exemplary antigen binding domains are provided in table 1. In some embodiments, the antigen binding domain target sequence may comprise a sequence which has at least 60 to 100%, or at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity or identity to any one of SEQ ID NOs: 26-28. In some embodiments, the antigen binding domain target domain comprises a sequence of any one of SEQ ID NOs: 26-28. In some embodiments, the antigen binding domain target domain has a sequence of any one of SEQ ID NOs: 26-28.
Table 1: Antigen binding domains SEQ ID Tumor SEQUENCE NO Antigen 26 CD19 MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYL NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATY FCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLV APSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTS VTVSSAAA(EQKLISEEDLGS) 27 BCMA MALPVTALLLPLALLLHAARPDIVLTQSPPSLAMSLGKRATISCRASESVTILGS HLIHWYQQKPGQPPTLLIQLASNVQTGVPARFSGSGSRTDFTLTIDPVEEDD VAVYYCLQSRTIPRTFGGGTKLEIKGSTSGSGKPGSGEGSTKGQIQLVQSGP ELKKPGETVKISCKASGYTFTDYSINWVKRAPGKGLKWMGWINTETREPAYA YDFRGRFAFSLETSASTAYLQINNLKYEDTATYFCALDYSYAMDYWGQGTSV TVSS 28 CEA MDMRVPAQLLGLLLLWLRGARCDIQLTQSPSSLSASVGDRVTITCKASQDVG TSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPED IATYYCQQYSLYRSFGQGTKVEIKRGGSGSGGSGSGGSGSEVQLVESGGG VVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYA PSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQG TPVTVSSAKPBCMA In some embodiments, the antigen recognized by the antigen-binding domain is human CD19, and the antigen-binding domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity or identity to SEQ ID NO:26. In some embodiments, the antigen-binding domain comprises a sequence of SEQ ID NO:26. In some embodiments, the antigen-binding domain has a sequence of SEQ ID NO:26. In some embodiments, the antigen recognized by the antigen-binding domain is human BCMA, and the antigen-binding domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity or identity to SEQ ID NO:27. In some embodiments, the antigen-binding domain comprises a sequence of SEQ ID NO:27. In some embodiments, the antigen-binding domain has a sequence of SEQ ID NO:27.
In some embodiments, the antigen recognized by the antigen-binding domain is human CEA and the antigen-binding domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity or identity to SEQ ID NO:28. In some embodiments, the antigen-binding domain comprises a sequence of SEQ ID NO:28. In some embodiments, the antigen-binding domain has a sequence of SEQ ID NO:28. Any of the antigen-binding domains described herein can bind to an antigen with a dissociation equilibrium constant (KD) of less than 1 x 107 M, less than 1 x 108 M, less than 1 x 109 M, less than 1 x 1010 M, less than 1 x 1011 M, less than 1 x 1012 M, or less than 1 x 1013 M. In some embodiments, the antigen-binding protein complexes provided herein can bind to a first and/or second antigen with a KD of about 1 x 104 M to about 1 x 106 M, about 1 x 105 M to about 1 x 107 M, about 1 x 106 M to about 1 x 108 M, about 1 x 107 M to about 1 x 109 M, about 1 x 108 M to about 1 x 1010 M, or about 1 x 109 M to about 1 x 1011 M (inclusive). A variety of different methods known in the art can be used to determine the KD value of an antigen-binding domain (e.g., an electrophoretic mobility shift assay, a filter binding assay, surface plasmon resonance, and a biomolecular binding kinetics assay, etc.). 2. Hinge In the context of the present invention, the CAR according to the invention may comprise a hinge domain. The “stalk domain” or “the hinge domain” or “spacer” as used herein comprises the region between the antigen binding domain and the transmembrane domain. The hinge domain provides stability for efficient CAR expression and activity and flexibility to access target antigens and helps minimize steric hindrance. The optimal length and sequence of the spacer varies widely based on the particular CAR and target antigen. In some embodiments, the hinge may be an immunoglobulin-based hinge. In some embodiments, the hinge may not be based on an immunoglobulin hinge. In some embodiments, the hinge sequence may be a sequence from a human protein, a fragment thereof, or a short oligo- or polypeptide linker. In some embodiments, the hinge sequence may be derived from a non-human protein. In some embodiments, hinge domain may be an artificially designed sequence. In some embodiments, the encoded hinge domain may be 10-300 amino acids in length, or about 10 to about 300 amino acids in length. In some embodiments, the length of the hinge domain may range from between 10 to 25, or 25 to 50, or 50 to 75, or 75 to 100, or 100 to 125, or 125 to 150, 150 to 175, 175 to 200, 200 to 225, or 225 to 250, or 250 to 275, or 275-300 and intermediate number of amino acids. As described herein, the hinge may extend less than 20, 15, or 10 nanometers from the surface of the cytotoxic cell. Thus, suitability for a stalk can be influenced by both linear lengths, the number of amino acid residues and flexibility of the hinge.
In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain is a sequence from the same endogenous single-chain polypeptide from which the transmembrane domain is derived. In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain is or includes a hinge region sequence of an antibody such as, without limitation, a human antibody (e.g., IgG1, IgG2, IgG3, or IgG4). Non-limiting examples of hinge or stalk domains include a hinge IgG4, CD8 and CD28 stalk as provided in Table 2. In some embodiments of a CAR comprising an extracellular hinge sequence (e.g., a CD28 hinge sequence), the hinge sequence is coterminal with the transmembrane domain. In some embodiments of a CAR comprising an extracellular hinge sequence (e.g., a CD28 hinge sequence), the extracellular hinge sequence is from the same protein as the transmembrane domain. In some embodiments of a CAR comprising an extracellular hinge sequence (e.g., a CD28 stalk sequence), the extracellular hinge sequence is from a different protein as the transmembrane domain. In some embodiments of the present invention, the hinge domain of the CAR as describe herein may comprise or has a sequence which has at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% similarity or identity to any one of SEQ ID NOs:29-31. In some preferred embodiments, the hinge or the stalk domain comprises a sequence of any one of SEQ ID NOs: 29-31. In some preferred embodiments, the hinge domain is SEQ ID NO:29 or 30. Table 2: Hinge/Stalk sequences SEQ ID Name SEQUENCE NO 29 IgG4 hinge ESKYGPPCPPCP 30 CD28 hinge IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 31 CD8 stalk TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IgG4 hinge- ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV 35 CH2-CH3 VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVL TVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQ KSLSLSLGK 3. Linker Any two neighboring domains of a chimeric antigen receptor can be separated by a linker sequence known in the art.
In some embodiments, one or more amino acids between the extracellular antigen-binding domain and the transmembrane domain is or comprises a linker sequence (e.g., a non-naturally occurring linker sequence, e.g., GS or any of the other linker sequences described herein). In some embodiments, the linker sequence between the antigen-binding domain and the transmembrane domain can be 1 amino acid to 50 amino acids, 2 amino acid to 40 amino acids, 3 amino acid to 30 amino acids, 4 amino acid to 20 amino acids, 5 amino acid to 10 amino acids, in length. In some embodiments, a linker sequence between the antigen-binding domain and the transmembrane domain can be or can include one or more of an IgG1, IgG2, IgG3, or IgG4 CH1, CH2, and CH3 domain. In some embodiments, the linker between the antigen-binding domain and the transmembrane domain can be or can include CH2-CH3 human IgG1 domains. In some embodiments, the linker sequence between the antigen-binding domain and the transmembrane domain can be or include a portion of the human CD8 extracellular sequence that is proximal to the human CD8 transmembrane domain. In some embodiments, the linker sequence between the antigen-binding domain and the transmembrane domain can be or include a human IgG1 hinge sequence. In some embodiments, a linker sequence can be present between the transmembrane domain and a costimulatory domain. In some embodiments, a linker sequence (e.g., any of the linker sequences described herein or known in the art) can be present between the costimulatory domain and the ITAM. In some embodiments, the linker sequence and/or the additional linker sequence comprises a sequence of (SG)n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker sequence and/or the additional linker sequence comprises a sequence of (GS)n, where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the linker sequence and/or the additional linker sequence comprises a sequence of (SGGS)n [SGGS=SEQ ID NO: 32], where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13. In some embodiments, the linker sequence and/or the additional linker sequence comprises a sequence of (SGGGS)n [SGGGS=SEQ ID NO:33], where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the linker sequence and/or the additional linker sequence comprises a sequence of (SGGGGS)n [SGGGGS = SEQ ID NO: 34], where n can be 1, 2, 3, 4, 5, 6, 7, 8, or 9. Table 3: linker sequences SEQ ID NO: Description Sequence Linker RT Linker RSG 37 Flexible linker TSGS 38 Flexible linker GGGGS 39 Flexible linker GGGS Flexible linker GG 41 Flexible linker KESGSVSSEQLAQFRSLD 42 Flexible linker EGKSSGSGSESKST 43 Flexible linker GSAGSAAGSGEF 44 Rigid linker EAAAK
45 Rigid linker EAAAR 46 Rigid linker PAPAP 47 Rigid linker AEAAAKEAAAKA 48 Rigid linker ILTHDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTV QIHDITG Flexible linker GGS 50 Flexible linker SLNGGGGSGGGGSGGGGSGGGGSGGGGSTS 51 Flexible linker SGGSGGGGSGGGSGGGGSLQ 52 Flexible linker SGGGSGGGGSGGGGSGGGGSGGGSLQ 53 Flexible linker GGGGSGGGGSGGGGS AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPE VTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY 54 CH2-CH3 human RWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR IgG1 domains EPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLS PGKKD human CD8 55 sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA transmembrane CDI domain 56 human IgG1 hinge sequence AEPKS PDKTHTCPPCPKDPK 4. Transmembrane domain In the context of the present invention, the encoded CAR may comprise a transmembrane domain that is attached to the extracellular domain. The transmembrane domain sequence may be derived from a natural or recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. Non-limiting examples of transmembrane domains include the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g., CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and fragments thereof. As described herein, exemplary transmembrane domain may include at least the transmembrane region(s) and fragments thereof of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG/Cbp, and CD19. In some embodiments, a chimeric antigen receptor includes a transmembrane domain, or portion thereof, from an endogenous polypeptide, where the endogenous polypeptide is selected from the group of: an alpha chain of a T cell receptor, a b chain of the T cell receptor, a z chain of the T cell receptor, CD28 (also known as Tp44), CD3s, CD35 , CD3y, CD33, CD37 (also known as GP52-40 or TSPAN26), CD64 (also known as FCGR1A), CD80 (also known as B7, B7-1, B7.1, BB1, CD28LG, CD28LG1, and LAB7), CD45 (also known as PTPRC, B220, CD45R, GP180, L-CA, LCA, LY5, T200,
and protein tyrosine phosphatase, receptor type C), CD4, CD5 (also known as LEU1 and Tl), CD8a (also known as Leu2, MAL, and p32), CD9 (also known as BTCC-l, DRAP-27, MIC3, MRP-l, TSPAN- 29, and TSPAN29), CD 16 (also known as FCGR3 andFCG3), CD22 (also known as SIGLEC-2 and SIGLEC2), CD86 (also known as B7-2, B7.2, B70, CD28LG2, and LAB72), CD134 (also known as TNFRSF4, ACT35, RP5-902P8.3, IMD16, 0X40, TXGP1L, and tumor necrosis factor receptor superfamily member 4), CD137 (also known as TNFRSF9, 4-1BB, CDwl37, ILA, and tumor necrosis factor receptor superfamily member 9), CD27 (also known as S152, S152.LPFS2, T14, TNFRSF7, and Tp55), CD152 (also known as CTLA4, ALPS5, CELIAC3, CTLA-4, GRD4, GSE, IDDM12, and cytotoxic T-lymphocyte associated protein 4), PD1 (also known as PDCD1, CD279, PD-l, SLEB2, hPD-l, hPD-l, hSLEl, and Programmed cell death 1), ICOS (also known as AILIM, CD278, and CVID1), CD272 (also known as BTLA and BTLA1), CD30 (also known as TNFRSF8, D1S166E, and Ki-l), GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D), HVEM (also known as TNFRSF14, RP3- 395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2), DAP 10, and CD 154 (also known as CD40LG, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, and CD40 ligand). The letters “CD” is the previous sentence stand for “Cluster of Differentiation.” E.g., CD3 stands for “Cluster of Differentiation 3.” In some embodiments, a chimeric antigen receptor includes a transmembrane domain, or portion thereof, from an endogenous mammalian (e.g., human) polypeptide (e.g., a mammalian or human homolog of any of the polypeptides listed above). A transmembrane domain can include one, two, three, four, five, six, seven, eight, nine, or ten contiguous amino acid sequences that each traverse a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell. As is known in the art, a transmembrane domain can, e.g., include at least one (e.g., two, three, four, five, six, seven, eight, nine, or ten) contiguous amino acid sequence (that traverses a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell) that has a-helical secondary structure in the lipid bilayer. In some embodiments, a transmembrane domain can include two or more contiguous amino acid sequences (that each traverse a lipid bilayer when present in the corresponding endogenous polypeptide when expressed in a mammalian cell) that form a b-barrel secondary structure in the lipid bilayer. Additional examples and features of transmembrane domains are known in the art. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues. In some aspects the transmembrane domain may further include one or more additional amino acid sequences including but not limited to one or more amino acids associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular T cell signaling domain and/or T cell costimulatory domain of the CAR. An exemplary linker sequence includes one or more glycine-serine doublets.
In some embodiments of any of the CARs described herein, the transmembrane domain comprises or has a transmembrane domain of CD28, CD3 epsilon, CD4, CD5, CD6, CD8a, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, or CD154 or the derivatives thereof. In some embodiments, the transmembrane domain may be selected from the transmembrane domains of CD8, CD137 and CD28 as provided in Table 4. In some preferred embodiments, the encoded transmembrane domain may comprise a sequence which has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity or similarity to any one of SEQ ID NOs: 57-59. In some preferred embodiments, the CARs described herein has a transmembrane domain of CD28 as represented by SEQ ID NOs: 58. Table 4: Transmembrane sequences SEQ Name SEQUENCE ID NO 57 CD8 transmembrane IYIWAPLAGTCGVLLLSLVITLYC 58 CD28 transmembrane FWVLVVVGGVLACYSLLVTVAFIIFWV 59 CD137 (4-1BB) IISFFLALTSTALLFLLFFLTLRFSVV transmembrane 5. Intracellular signalling domain The intracellular signalling domain of a CAR can induce or reduce an activity of an engineered cell comprising the CAR. An intracellular signalling domain of a CAR can be or can comprise a truncated portion of a signalling domain of another molecule. In some cases, the intracellular domain of the CAR can be involved in regulating primary activation of a TCR complex in either a stimulatory manner or an inhibitory manner. In some embodiments, the intracellular signalling domain of the CAR is involved in inducing T cell activation and/or a cytotoxic response against cells that express the antigen that is bound by the CAR. In some embodiments, the CAR may include one or more intracellular T cell signaling domains for activation of at least one of the normal T-cell effector functions. Exemplary T cell signaling domains are provided herein, and are known in the art. In some embodiments, an entire intracellular T cell signaling domain can be employed in a CAR. In some embodiments, it may not be necessary to use the entire signaling domain. In some embodiments, the signaling domain may be synthetically designed to comprise multiple and chimeric signaling domains. Examples of intracellular T cell signaling domains for use in the CAR include the cytoplasmic sequences of the T cell receptor (TCR) and co-stimulatory molecules that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability. T cell receptor signaling domains regulate primary activation of the T cell receptor complex either in a stimulatory way, or in an inhibitory way. As described herein, a primary intracellular signaling domain/sequence produces an intracellular signal when an extracellular domain, e.g., an antigen
binding domain, to which it is fused binds an antigen. In some embodiments, it may be derived from a primary stimulatory molecule, e.g., it comprises intracellular sequence of a primary stimulatory molecule. It comprises sufficient primary stimulatory molecule sequence to produce an intracellular signal, e.g., when an antigen binding domain to which it is fused binds an antigen. The CARs of the present invention can include primary cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs that are known as immunoreceptor tyrosine- based activation motifs or ITAMs. In some embodiments, the signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain, a chimeric ITAM or partial ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences that can be included in a disclosed CAR include those from TCR zeta (CD3 zeta), FcRγ(FCER1G), FcεRIβ (MS4A2), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, FcεRI, DAP10, DAP12, CEACAM4 and CEACAM3 proteins. In several aspects, the cytoplasmic signaling molecule in the CAR includes an intracellular T cell signaling domain from CD3 zeta. In some examples of any of the CARs described herein, going in the N-terminal to the C-terminal direction, the intracellular portion of the CAR includes a co-stimulatory domain and an intracellular signaling domain. In some examples of any of the CARs described herein, going in the N-terminal to the C-terminal direction, the intracellular portion of the CAR includes an intracellular signaling domain and a co-stimulatory domain. In some examples of any of the CARs described herein, going in the C-terminal to the N-terminal direction, the intracellular portion of the CAR includes a co-stimulatory domain and an intracellular signaling domain. In some examples of any of the CARs described herein, going in the C-terminal to the N-terminal direction, the intracellular portion of the CAR includes an intracellular signaling domain and a co-stimulatory domain. IL7Ra signalling domain The CAR, in particular the intracellular signalling domain of the CAR, according to the present invention comprises a IL7Ra signaling domain as defined in the first aspect. In an embodiment, the CAR comprises a IL-7Ra signalling domain which does not comprise a STAT3 binding domain. In some embodiments, the IL7Ra signalling domain of the intracellular signalling domain of the CAR according to the invention, is a truncated protein. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5. In some embodiments, said truncated IL7Ra signalling domain is able to recruit and activate STAT3. In some embodiments, said truncated IL7Ra signalling domain is able to recruit and activate STAT4. In some embodiments, said truncated IL7Ra signalling domain of the intracellular signalling domain of the CAR, is able to activate, preferably recruit and activate STAT3. In some embodiments, said truncated IL7Ra signalling domain of the intracellular signalling domain of the CAR, is able to activate, preferably recruit and activate STAT4.
In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5, and is able to activate STAT3. In a preferred embodiment, said truncated IL7Ra signalling domain is able to recruit and activate both STAT3 and STAT5. In some embodiments, said truncated IL7Ra signalling domain is not able to recruit and activate STAT3. In this embodiment, this is the CAR which is able to recruit and activate STAT3. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5, and is able to activate STAT4. In a preferred embodiment, said truncated IL7Ra signalling domain is able to recruit and activate both STAT4 and STAT5. In some embodiments, said truncated IL7Ra signalling domain is not able to recruit and activate STAT4. In this embodiment, this is the CAR which is able to recruit and activate STAT4. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5, and is able to activate STAT3 and STAT4. In a preferred embodiment, said truncated IL7Ra signalling domain is able to recruit and activate all of STAT3, STAT4 and STAT5. In some embodiments, said truncated IL7Ra signalling domain is not able to recruit and activate STAT3 and/or STAT4. In this embodiment, this is the CAR which is able to recruit and activate STAT3 and/or STAT4. In some embodiments, the CAR, in particular the intracellular signalling domain of the CAR, according to the present invention comprises a IL7Ra signaling domain as defined in the first aspect, preferably the CAR comprises a IL-7Ra signalling domain which does not comprise a STAT4 binding domain. In this embodiment, the CAR comprises a STAT4 binding domain in its intracellular signaling domain, but not within the IL7Ra signaling domain. In some embodiments, the STAT4 binding site is present after the IL7Ra signaling domain in said CAR, and/or is present after CD3z domain. In some embodiments, the IL7Ra signalling domain of the intracellular signalling domain of the CAR according to the invention, is a truncated protein. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5. In some embodiments, said truncated IL7Ra signalling domain is able to recruit and activate STAT3 and STAT4. In some embodiments, said truncated IL7Ra signalling domain of the intracellular signalling domain of the CAR, is able to activate, preferably recruit and activate STAT3 and STAT4. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5, and is able to activate STAT3 and STAT4. In a preferred embodiment, said truncated IL7Ra signalling domain is able to recruit and activate STAT3, STAT4 and STAT5. In some embodiments, said truncated IL7Ra signalling domain is not able to recruit and activate STAT3 and STAT4. In this embodiment, this is the CAR which is able to recruit and activate STAT3 and STAT4. In this embodiment, the CAR comprises at least one STAT3 and at least one STAT4 binding site as defined herein in the intracellular signaling domain but not in the IL7Ra signaling domain. In some embodiments, the STAT4 binding site is present after the IL7Ra signaling domain in said CAR, and/or is present after CD3z domain.
In some preferred embodiments, the STAT3 binding site is YRHQ (SEQ ID NO:8). In some preferred embodiments, the STAT4 binding site is YLPSNID (SEQ ID NO:189), and preferably the Y is phosphorylated Y (or pY). Shortest truncated IL7Ra signaling domain In some embodiments, the IL7Ra signalling domain of the intracellular signalling domain of the CAR according to the invention, is a truncated protein, wherein the truncated protein has a length of 20-30 amino acids, which comprises BOX1 (i.e. SEQ ID NO: 80). In some embodiment, the IL7Ra signalling domain does not have any mutation compared to SEQ ID NO:1. In some embodiments, the IL7Ra signalling comprises SEQ ID NO:188. In some embodiments, the IL7Ra signalling domain of the intracellular signalling domain of the CAR according to the invention, is a truncated protein, wherein the truncated protein is represented by SEQ ID NO:188 (VWPSLPDHKGGGGSPQQEEAYVTMS). In this embodiment, said truncated IL7Ra signaling domain is not able to recruit/bind/activate STAT3 and/or STAT4. In this embodiment, this is the CAR which is able to recruit and activate STAT3 and/or STAT4. In this embodiment, said truncated IL7Ra signaling domain does not have any mutation compared to SEQ ID NO:1. The inventors have surprising found that a CAR as defined herein, where the IL7Ra signaling domain is represented by SEQ ID NO:188, when used in a receptor, especially in a CAR, and expressed into T cells leads to CAR-T cells with attractive properties: it is expected to prevent and overcome the functional exhaustion of the CAR T-cells, improve the proliferation, survival and expansion of said CAR T-cells, improve the capability to control tumor population cells by said CAR T-cells, improve the antitumor activity and/or cytotoxicity capacity of said CAR T-cells, improve the persistence potential of the CAR-T cells, and improve the safety potential of said CAR-T which can be activated without systemic toxicity. These improved properties may be attributed to the ability of the short truncated IL- 7Rα intracellular signaling domain to significantly enhance the phosphorylation of STAT proteins, such as STAT5, as well as other STAT proteins that may be recruited/bound to the CAR, including STAT3 and STAT4. An exemplary CAR according this this embodiment may be represented as SEQ ID NO: 227. In some embodiments, said truncated IL7Ra signalling domain is still able to recruit and activate STAT5. In some embodiments, said shortest truncated IL7Ra signalling domain comprises an additional STAT3 and/or STAT4 binding site, and is therefore able to recruit and activate STAT3 and/or STAT4. In a preferred embodiment, said truncated IL7Ra signalling domain is able to recruit and activate STAT3 and/or STAT4, and STAT5. In some preferred embodiments, the STAT3 binding site is YRHQ (SEQ ID NO:8). In some preferred embodiments, the STAT4 binding site is YLPSNID (SEQ ID NO:189), and preferably the Y is phosphorylated Y (or pY).
Co-stimulatory domain In the context of the present invention, the chimeric antigen receptor (CAR) may include one or more (such as two, three, four, or five or more) costimulatory domain(s). In normal lymphocytes, T cell activation is mediated by two classes of intracellular signalling domains. Primary signalling is initiated via MHC-mediated antigen-dependent activation via the T cell receptor (e.g., a TCR/CD3 complex). A secondary or costimulatory signal is provided by a different receptor that includes a costimulatory signalling domain, which acts in an antigen-independent manner. Signals generated through the signalling domain of the TCR alone are insufficient for complete T cell activation; a co-stimulatory signal is also required. Any costimulatory domain, or portion thereof, that serves to provide a costimulatory signal is suitable for use in accordance with the CARs, compositions and methods disclosed herein. As used herein, a costimulatory signalling domain produces an intracellular signal when an extracellular domain, e.g., an antigen binding domain to which it is fused, or coupled by a dimerization switch, binds cognate ligand. In some embodiments, the co-stimulatory domain may be derived from, be a functional fragment of, analog of or modified from a costimulatory molecule. It can comprise the entire intracellular region or a fragment of the intracellular region of a costimulatory molecule which is sufficient for generation of an intracellular signal, e.g., when an antigen binding domain to which it is fused, or coupled by a dimerization switch, binds cognate antigen. The costimulatory domain may include a sequence of amino acids from any isoform of an endogenous mammalian (e.g., human) transmembrane polypeptide having a costimulatory domain including, e.g., an isoform of: CD27 (also known as S152, S152.LPFS2, T14, TNFRSF7, and Tp55), CD28 (also known as Tp44), 4-1BB (also known as TNFRSF9, CD137, CDwl37, ILA, and tumor necrosis factor receptor superfamily member 9), OX40 (also known as TNFRSF4, ACT35, RP5-902P8.3, IMD16, CD134, TXGP1L, and tumor necrosis factor receptor superfamily member 4), CD30 (also known as TNFRSF8, D1S166E, and Ki-l), CD40L (also known as CD40LG, CD154, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, and CD40 ligand), CD40 (also known as Bp50, CDW40, TNFRSF5, p50, CD40 (protein), and CD40 molecule), PD-1 (also known as PDCD1, CD279, PD-l, SLEB2, hPD-1, hPD-1, hSLEl, and Programmed cell death 1), PD-L1 (also known as CD274, B7-H, B7H1, PD-L1, PDCD1L1, PDCD1LG1, PDL1, CD274 molecule, and Programmed cell death 1 ligand 1), ICOS (also known as AILIM, CD278, and CVID1), LFA-1 (also known as Lymphocyte function-associated antigen 1), CD2 (also known as LFA-2, SRBC), CD7 (also known as GP40, LEU-9, TP41, Tp40, and CD7 molecule), CD160 (also known as BY55, NK1, NK28, and CD 160 molecule), LIGHT (also known as TNFSF14, CD258, HVEML, LIGHT, LTg, TR2, TNLG1D, and tumor necrosis factor superfamily member 14), BTLA (also known as CD272 and BTLA1), TIM3 (also known as HAVCR2, HAVcr-2, KIM-3, TIM3, TIMD-3, TIMD3, Tim-3, CD366, and hepatitis A virus cellular receptor 2), CD244 (also known as 2B4, NAIL, NKR2B4, Nmrk, SLAMF4, and CD244 molecule), CD80 (also known as B7, B7-1, BB1, CD28LG, CD28LG1, LAB7, and CD80 molecule), LAG3 (also known as CD223 and lymphocyte activating 3), NKG2C (also known as CD314, D12S2489E, KLR, NKG2-D, NKG2D, and killer cell lectin like receptor Kl), GITR (also known as TNFRSF18, RP5-902P8.2, AITR, CD357, and GITR-D), HVEM (also known
as TNFRSF14, RP3-395M20.6, ATAR, CD270, HVEA, HVEM, LIGHTR, and TR2), TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLRIO, CARD11, CD54 (ICAM), CD83, DAP 10, LAT, LCK, SLP76, TRIM, ZAP70, CD49D, CD29, OX40 (CD134), Cytokine receptor, ITGA6, ITGB2, Integrin, VLA- 6, CD19, CD49f, ITGB7, ICOS (CD278), CD4, ITGAD, ICAM-1, Toll ligand receptor, CD8 alpha, CD11d, TNFR2, LFA-1 (CD11a/, BTLA, CD8 beta ITGAE, TRANCE, CD18, RANKL, CD2, CDS, IL2R beta, CD103, DNAM1 (CD226), CD7 ICAM-1, IL2R gamma, ITGAL, SLAMF4 (CD244, 2B4), LIGHT GITR, IL7R, alpha,CD11a, CD84, BAFFR, ITGA4 ITGAM, CD96 (Tactile), B7-H3, HVEM (LIGHTR), VLA1, CD11b, CEACAM1, KIRDS2, CD49a, ITGAX, CRTAM, Ly9, (CD229), CD160, (BY55), PSGL1, CD100, (SEMA4D), CD69, SLAMF6, (NTB-A, Ly108), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and B7-H3 (also known as CD276, 4Ig-B7-H3, B7H3, B7RP-2, and CD276 molecule) (including, without limitation, a mammalian or human homolog of any of these polypeptides). Accordingly, the chimeric antigen receptor provided herein includes a costimulatory domain, or portion thereof, from an endogenous mammalian (e.g., human) transmembrane polypeptide (e.g., a mammalian or human homolog of any of the polypeptides listed above). In some embodiments, the costimulatory signaling domain, has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% similarity or identity with the corresponding residues of a naturally occurring stimulatory molecule. The intracellular signaling sequences within the cytoplasmic domain may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In some embodiments, a glycine-serine doublet can be used as a suitable linker. In some embodiments a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker. In some embodiments, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In some embodiments, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In some embodiments of any of the CARs described herein, the co-stimulatory domain is or includes the co-stimulatory domain of 4-1BB, CD28, CD2, CD4 or CD8. In some embodiments, the chimeric antigen receptor (CAR) described herein includes a human 4-1BB costimulatory domain (SEQ ID NO:57). In some embodiments, the chimeric antigen receptor (CAR) according to the invention includes a costimulatory domain, or portion thereof, from human CD28 (SEQ ID NO:58). In some embodiments, a costimulatory domain is or includes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similar or identical to SEQ ID NO: 57 or 58, or a fragment thereof.
Table 5: Exemplary Intracellular signaling region sequences. SEQ ID Name SEQUENCE NO CD137 (4-1BB) 57 costimulatory KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL domain CD28 58 costimulatory RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPE 59 CD3z MGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG LYQGLSTATKDTYDALHMQALPPR ITAM domain The CARs according to the present invention may include primary cytoplasmic signaling sequences that act in a stimulatory manner, which may contain signaling motifs that are known as immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are typically repeated (e.g., two or more times) in the cytoplasmic tails of certain cell surface proteins of the immune system, and are typically separated by between six and eight amino acids. In some embodiments, a chimeric antigen receptor includes an ITAM, or portion thereof, from an endogenous mammalian (e.g., human) polypeptide, wherein endogenous mammalian (e.g., human) polypeptide is selected from the group of: Oϋ3z (also referred to as CD3 zeta), CD35 (CD3 delta), CD3s (CD3 epsilon), CD3y (CD3 gamma), DAP12, FCsRly (Fc epsilon receptor I gamma chain), FcRy, FcRft, CD35, CD22, CD79A (antigen receptor complex- associated protein alpha chain), CD79B (antigen receptor complex-associated protein beta chain), and CD66d. As will be appreciated by those of ordinary skill in the art, certain polypeptides have two or more isoforms that differ at least in their primary polypeptide sequence. For example, different isoforms can be generated as a result of alternative splicing. In some embodiments the signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain, a chimeric ITAM or partial ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences that can be included in a disclosed CAR include those from TCR zeta (CD3 zeta), FcRγ(FCER1G), FcεRIβ (MS4A2), CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, FcεRI, DAP10, DAP12, CEACAM4 and CEACAM3 proteins. In some embodiments, the cytoplasmic signaling molecule in the CARs as described herein includes an intracellular T cell signaling domain from CD3 zeta (or CD3z). In some embodiments, the ITAM domain of the chimeric antigen receptor according to the present invention may include a sequence of amino acids having one or more (e.g., two, three, four, or five) amino acid substitutions, deletions, or additions as compared to an ITAM of one or more of an ITAM in an endogenous mammalian (e.g., human) transmembrane protein as described above. In some embodiments, the CAR intracellular signaling domain comprises at least 2 ITAM domains (e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 ITAM domains). In some embodiments, the two or more ITAMs are
identical (e.g., they have the same amino acid sequence). In some embodiments, the two or more ITAMs are not identical. In some embodiments, a chimeric antigen receptor includes an ITAM that is a chimeric ITAM having portions of an ITAM from two or more endogenous mammalian (e.g., human) transmembrane polypeptides as described above, such that the two or more ITAM portions together constitute a functional ITAM. In some embodiments, such a portion of a chimeric ITAM can include one or more amino acid substitutions, deletions, or additions as compared to a corresponding portion of a wild type ITAM. In some embodiments, the CAR comprises a CD3 zeta signaling, a CD28 signaling domain; a CD137 (4-1BB) signaling domain, derivatives or fragments thereof or a combination of two or more thereof. In some embodiments, the cytoplasmic domain includes the signaling domain of CD3-zeta and the signaling domain of CD28. In some embodiments, the cytoplasmic domain includes the signaling domain of CD3 zeta and the signaling domain of CD137. In some embodiments, the cytoplasmic domain includes the signaling domain of CD3-zeta and the signaling domain of CD28 and CD137. The order of the one or more T cell signaling domains on the CAR can be varied as needed by the person of ordinary skill in the art. In some embodiments, the intracellular region of the CAR can include the ITAM containing primary cytoplasmic signaling domain (such as CD3-zeta or CD3z) by itself or combined with any other desired cytoplasmic domain(s) useful in the context of a CAR. In some embodiments, an ITAM comprises a sequence that is at least 80% (e.g., at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, at least 99%, or 100% identical to: the sequence of SEQ ID NO: 59 (or a portion thereof). In some exemplary embodiments, the CAR according to the invention may comprise one or more of the signaling domains listed in Table 5. In some embodiments, the signaling domain comprises a sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity or identity to any one or more from SEQ ID NOs: 57-59. In some embodiments, one cytoplasmic domain is linked to a second cytoplasmic domain. In other aspects, one cytoplasmic domain is linked to two or more other cytoplasmic domains. The cytoplasmic domains can be the same or different. For example, the cytoplasmic domain of a co-stimulatory molecule can be linked to the cytoplasmic domain of one or more of the CD3 chains of the T cell receptor, for example to one or more of the zeta, eta, delta, gamma or epsilon CD3 chains of the T cell receptor. In an embodiment, the invention provides a CAR that comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT3 binding site as described herein is present in the intracellular signalling domain of the CAR, therefore the activation of STAT3 is not directly mediated by the IL7Ra and said STAT3 binding site is not present
in the IL7Ra signalling domain. An IL7Ra signalling domain that may be used in this CAR does not comprise a STAT3 binding site and is not able to activate STAT3. In some embodiments, one or more STAT3 binding sites are present in the IL7Ra signalling domain as earlier described herein. In some alternative embodiments, the CAR, comprises one or more STAT3 binding sites and these sites are not present in the IL7Ra signalling domain. In an embodiment, a STAT3 binding site is present in the CD3z of the CAR. In some embodiments, one or more STAT3 binding sites may be present between the IL7Ra signalling domain and the CD3z domain. The STAT3 binding is still within the intracellular signalling domain of the CAR. In some embodiments, one or more STAT3 binding sites are present after CD3z. STAT3 binding sites have been earlier described herein by reference to a mutated site present in the human wild type IL7Ra. In short, such motifs are represented by SEQ ID NO:6: YX1X2Q, wherein: X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally: Q is mutated/substituted into P, T, Y, N, F or A. In some embodiments, the motif which is able to recruit/bind STAT3 comprises any of the following sequences: YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA(SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF. In a preferred embodiment, the STAT3 binding/recruiting site is YRHQ (SEQ ID NO:8). In an embodiment, the invention provides a CAR that comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT4 binding site as described herein is present in the intracellular signalling domain of the CAR, therefore the activation of STAT4 is not directly mediated by the IL7Ra and said STAT4 binding site is not present in the IL7Ra signalling domain. An IL7Ra signalling domain that may be used in this CAR does not comprise a STAT4 binding site and is not able to activate STAT4. In some embodiments, one or more STAT4 binding sites are present in the IL7Ra signalling domain as earlier described herein. In some alternative embodiments, the CAR, comprises one or more STAT4
binding sites and these sites are not present in the IL7Ra signalling domain. In an embodiment, a STAT4 binding site is present in the CD3z of the CAR. In some embodiments, one or more STAT4 binding sites may be present between the IL7Ra signalling domain and the CD3z domain. The STAT4 binding is still within the intracellular signalling domain of the CAR. In some embodiments, one or more STAT4 binding sites are present after CD3z. STAT4 binding sites have been earlier described herein by reference to a mutated site present in the human wild type IL7Ra. In this context, the STAT4 binding/recruiting site may be represented by YLPSNID (SEQ ID NOs: 189), TX1X2GYL (SEQ ID NO: 190) or GYKPQIS (SEQ ID NO: 191). In some embodiments, the Y (tyrosine) in the STAT4 binding/recruiting site is phosphorylated. In some embodiments, the STAT4 binding/recruiting site is TX1X2GYL (SEQ ID NO: 190), and X1 and X2 may be any amino acid, each chosen independently from the other. In some preferred embodiments, X1 is not an H, and X2 is not an D. In some embodiments, the STAT4 binding site is YLPSNID (SEQ ID NO:189). In an embodiment, the antigen recognized by the antigen binding domain of the CAR according to the present invention is a targeting tumor associated or tumor specific antigen, preferably wherein the antigen is CD19 or ROR1. In an embodiment, the co-stimulatory domain is 4-1BB and/or CD28. In another embodiment, the antigen recognized by the antigen binding domain of the CAR according to the present invention is CD19 and the co-stimulatory domain is CD28. In an embodiment, the CAR further contains CD3z. In a non-limiting example, the C-terminus of a 4-1BB costimulatory domain (e.g. SEQ ID NO:57) is joined to the N-terminal residue of the cytoplasmic domain of CD3 zeta (e.g. SEQ ID NO:59) (i.e., linked head-to-tail), resulting in a CAR with antigen binding domain and transmembrane segments linked to the cytoplasmic domains of 4-1BB and CD3-zeta. In some embodiments, the IL7Ra signaling domain (such as any one of the sequences described in the first aspect, such as SEQ ID NO:2-4 or sequences derived therefrom) of the present invention is present in between the 4-1BB costimulatory domain and the CD3z domain. In some embodiments, the N-terminus of the IL7Ra signaling domain is linked to C- terminus of the co-stimulatory domain and the C-terminus of the IL7Ra signaling domain is linked to the N-terminus of the CD3 zeta. In some embodiments the CAR comprises one or more STAT3 binding site as described herein (preferably SEQ ID NO:8 or any other STAT3 binding site disclosed herein) is present in the intracellular signaling domain of the CAR, preferably in the co-stimulatory domain, the IL- 7Ra signaling domain, the CD3z domain and/or in the linker therein between. In some embodiments, the P300H mutation as described above when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (or the P36H mutation in SEQ ID NO:2 or 4) is still present in said IL7Ra signaling domain. In a non-limiting example, the C-terminus of a CD28 costimulatory domain (e.g. SEQ ID NO:58) is joined to the N-terminal residue of the cytoplasmic domain of CD3 zeta (e.g. SEQ ID NO:59) (i.e., linked head-
to-tail), resulting in a CAR with antigen binding domain and transmembrane segments linked to the cytoplasmic domains of CD28 and CD3-zeta. In some embodiments, the IL7Ra signaling domain (such as any one of the sequences described in the first aspect, such as SEQ ID NO:2-4 or sequences derived therefrom) of the present invention is present in between the CD28 costimulatory domain and the CD3z domain. In some embodiments, the N-terminus of the IL7Ra signaling domain is linked to C-terminus of the co-stimulatory domain and the C-terminus of the IL7Ra signaling domain is linked to the N- terminus of the CD3 zeta. In some embodiments of the CAR according to the present invention, one or more STAT3 binding site as described herein (preferably SEQ ID NO:8 or any other STAT3 binding site disclosed herein) are present in the intracellular signaling domain of the CAR, preferably in the co- stimulatory domain, the IL-7Ra signaling domain, the CD3z domain and/or in the linker therein between. In some embodiments, the STAT3 binding site may be present at the C-terminus of CD3z. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (or the P36H mutation in SEQ ID NO:2 or 4) is still present in said IL7Ra signaling domain. In a non-limiting example, the C-terminus of a 4-1BB costimulatory domain (e.g. SEQ ID NO:57) is joined to the N-terminal residue of the cytoplasmic domain of CD3 zeta (e.g. SEQ ID NO:59) (i.e., linked head-to-tail), resulting in a CAR with antigen binding domain and transmembrane segments linked to the cytoplasmic domains of 4-1BB and CD3-zeta. In some embodiments, the IL7Ra signaling domain (such as any one of the sequences described in the first aspect, such as SEQ ID NO:178-187 or sequences derived therefrom) of the present invention is present in between the 4-1BB costimulatory domain and the CD3z domain. In some embodiments, the N-terminus of the IL7Ra signaling domain is linked to C-terminus of the co-stimulatory domain and the C-terminus of the IL7Ra signaling domain is linked to the N-terminus of the CD3 zeta. In some embodiments the CAR comprises one or more STAT3 binding site as described herein (preferably SEQ ID NO:8 or any other STAT3 binding site disclosed herein) is present in the intracellular signaling domain of the CAR, preferably in the co-stimulatory domain, the IL-7Ra signaling domain, the CD3z domain and/or in the linker therein between. In some embodiments, the P300A, P300W, P300E, P300L, or P300Q mutation as described above when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (or the P36A, P36W, P36E, P36L, P36Q mutation in any one of SEQ ID NO:178-187) is still present in said IL7Ra signaling domain. In some preferred embodiments, the P300A, P300W, P300E, P300L, or P300Q mutation as described above when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (or the P36A, P36W, P36E, P36L, P36Q mutation in any one of SEQ ID NO:178-187) and the Q457R and N458H mutations when referring to SEQ ID NO:1 are still present in said IL7Ra signaling domain derived from any one of SEQ ID Nos: 179, 181, 183, 185 and 187. In a non-limiting example, the C-terminus of a CD28 costimulatory domain (e.g. SEQ ID NO:58) is joined to the N-terminal residue of the cytoplasmic domain of CD3 zeta (e.g. SEQ ID NO:59) (i.e., linked head- to-tail), resulting in a CAR with antigen binding domain and transmembrane segments linked to the cytoplasmic domains of CD28 and CD3-zeta. In some embodiments, the IL7Ra signaling domain (such as any one of the sequences described in the first aspect, such as SEQ ID NO: 192-203 or sequences derived therefrom) of the present invention is present in between the CD28 costimulatory domain and
the CD3z domain. In some embodiments, the N-terminus of the IL7Ra signaling domain is linked to C- terminus of the co-stimulatory domain and the C-terminus of the IL7Ra signaling domain is linked to the N-terminus of the CD3 zeta. In some embodiments of the CAR according to the present invention, one or more STAT4 binding site as described herein (preferably SEQ ID NO:189) are present in the intracellular signaling domain of the CAR, preferably in the co-stimulatory domain, the IL-7Ra signaling domain, the CD3z domain and/or in the linker therein between. In some embodiments, the STAT4 binding site may be present at the C-terminus of CD3z. In some embodiments, the P300A, P300W, P300E, P300L, or P300Q mutation as described above when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (or the P36A, P36W, P36E, P36L, P36Q mutation in any one of SEQ ID NO:192- 203) is still present in said IL7Ra signaling domain. In some embodiments, the STAT4 binding site represented as SEQ ID NO:189 in any one of SEQ ID Nos: 179, 181, 183, 185 and 187 is still present in said IL7Ra signaling domain in said CAR. The non-limiting examples of a CAR as described herein may further comprise a CD19 antigen-binding domain (e.g. SEQ ID NO:26) which is linked to the intracellular signaling domains via a hinge domain and a transmembrane domain. The hinge domain may be an IgG4 hinge (SEQ ID NO: 29) or a CD28 hinge (e.g. SEQ ID NO:30), and the transmembrane domain may be a CD28 transmembrane domain (e.g. SEQ ID NO:58). In some embodiments, the CAR according to the present invention comprises a sequence that is at least 60%, at least 61%, at least 62 %, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs: 14-17, 19- 23, and 95-98, 227-228. In some embodiments, the resulting CAR is able to activate STAT3 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT4 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT5 as described above. Preferably, the corresponding P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NO: 14-17, 19-23, 95-98, and 227-228 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) may be replaced with P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In some embodiments, the CAR according to the present invention comprises a sequence that differs from any one of SEQ ID NOs: 14-17, 19-23, 95-98, and 227-228 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting CAR is able to activate STAT3 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT4 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT5 as described above.
Preferably, the corresponding P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NO: 14-17, 19-23, 95-98, and 227-228 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) may be replaced with P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). In some embodiments, the CAR according to the present invention may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to any one of SEQ ID NOs: 14-17, 19-23, and 95-98 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98. In some embodiments, the CAR according to the present invention may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to any one of SEQ ID NOs: 14-17, 19-23, 95-98, 227-228 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, 95-98, 227-228. Preferably, the corresponding P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) is still present in the sequence derived from any one SEQ ID NO: 14-17, 19-23, 95-98, 227-228 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) can be replaced with P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36A, P36W, P36E, P36L, or P36Q in SEQ ID NO: 178-187) In some embodiments, the CAR according to the present invention may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to any one of SEQ ID NOs: 14-17, 19-23, and 95-98 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98. In some embodiments, the CAR according to the present invention may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to any one of SEQ ID NOs: 14-17, 19-23, and 95-98 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98. Preferably, the corresponding P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) is still present in the sequence derived from any one SEQ ID NO: 14-17, 19-23, and 95-98 as described above. Preferably, the corresponding Q457R and/or N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to Q79R and N80H mutations in SEQ ID NOs:3 or 4) is still present in the sequence derived from SEQ ID NO:3 as described above. More preferably, the Q457R and N458H mutations are both still present in the sequence derived from any one SEQ ID NO: 14-17, 19-23, and 95-98 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) can be replaced with P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36A, P36W, P36E, P36L, or P36Q in SEQ ID NO: 178-187). In some embodiments, the CAR according to the present invention may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to SEQ ID NOs: 14-17, 19-23, and
95-98 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98. In some embodiments, the CAR according to the present invention may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids shorter compared to SEQ ID NOs: 14- 17, 19-23, and 95-98 or the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98. Preferably, the P300H and Q457R and/or N458H mutations (corresponding to P36H, Q79R and N80H mutations in SEQ ID NO:4) when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) are still present in the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98 as described above. More preferably, the P300H, Q457R and N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) are still present in the sequence derived from any one of SEQ ID NOs: 14-17, 19-23, and 95-98 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36H in SEQ ID NO:2 or 4) can be replaced with P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) (corresponding to P36A, P36W, P36E, P36L, or P36Q in SEQ ID NO: 178-187). In some exemplary embodiments, the CAR sequence has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity or similarity as any of the sequences SEQ ID NOs: 14-17, 19-23, 95-98, and 227-228 as provided in Table 6. In some exemplary embodiments, the CAR of the present invention has a sequence of any one of SEQ ID NOs: 14-17, 19-23, 95-98, and 227-228. In some embodiments, the CAR according to the present invention comprises a sequence that is at least 60%, at least 61%, at least 62 %, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NO:228. In some embodiments, the resulting CAR is able to activate STAT4 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT3, STAT4 and STAT5 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in said CAR. In some embodiments, the mutation at P300H when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) may be replaced with P300A, P300W, P300E, P300L, or P300Q.
In some embodiments, the CAR according to the present invention comprises a sequence that differs from any one of SEQ ID NO: 228 by 0, 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, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the resulting CAR is able to activate STAT4 and STAT5 as described above. In some embodiments, the resulting CAR is able to activate STAT3, STAT4 and STAT5 as described above. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in said CAR. In some embodiments, the mutation at P300H when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) may be replaced with P300A, P300W, P300E, P300L, or P300Q. In some embodiments, the CAR according to the present invention may further comprise additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in addition to SEQ ID NOs: 228 or the sequence derived from any one of SEQ ID NOs: 228. In some embodiments, the CAR according to the present invention may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids compared to SEQ ID NOs: 228 or the sequence derived from SEQ ID NOs: 228. In some embodiments, the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) is still present in said CAR. In some embodiments, the mutation at P300H when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) may be replaced with P300A, P300W, P300E, P300L, or P300Q. Table 6: Examples of CAR sequences SEQ ID Name Sequences NO 10 BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (2nd generation anti- GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD19 CAR-T with RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG IgG4 hinge, CD28 RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE transmembrane, RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 41BB co-stimulatory and CD3z domains) 11 IL7.BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra KRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQ incorporated IHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN between CD28TM CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDC and 41BB domains) RESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVA QGQPILTSLGSNQEEAYVTMSSFYQNQAEQKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR 12 BB.IL7.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV CD3z domains) QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTL NPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRSGRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK
GHDGLYQGLSTATKDTYDALHMQALPPR BB.IL7.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV CD3z domains; QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR additional STAT3 SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTL binding motif (YRHQ) NPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRSGRVKFSR at the end of CD3z) SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDAYRHQALPPR BB.IL7tr.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains) QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR BB.IL7tr.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD additional STAT3 KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG binding motif (YRHQ) MKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR at the end of CD3z) BB.IL7tr(mut).Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra) MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR BB.IL7tr(mut).Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR STAT3 binding motif (YRHQ) at the end of CD3z]) 28.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (2nd generation anti- LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD19 CAR-T with RPGPTRKHYQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQ CD28 hinge, QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN CD28TM, CD28 co- PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL stimulatory and CD3z STATKDTYDYRHQQALPPR domains; additional STAT3 binding motif
(YRHQ) at the end of CD3z 28.IL7tr(mut).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains) GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 28.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY additional STAT3 QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK binding motif (YRHQ) NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ at the end of CD3z) GLSTATKDTYDAYRHQALPPR 28.IL7tr(mut)+.Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGYRHQRVKFSRSA P300H mutation in DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG IL7Ra; additional KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH STAT3 binding motif DGLYQGLSTATKDTYDALHMQALPPR (YRHQ) after IL7Ra) 28.IL7tr(mut)++.Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYRHQRSGRVKFSRSADAPAY P300H mutation in QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK IL7Ra; additional NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ STAT3 binding motif GLSTATKDTYDALHMQALPPR (YRHQ) in IL7Ra (mutations: Q457R; N458H).) 28.IL7tr(mut).Z++ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY P300H mutation in QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK IL7Ra; additional NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ STAT3 binding motif GLSTATKDTYDALHMQALPPRYRHQ (YRHQ) after CD3z.) 28.IL7tr(+14).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE CD3z domains.) GFLQDTFPQQLEESEKQRLLGSNQEEAYVTMSSFYQNQRS GRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG
ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 28.IL2d.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (28-ΔIL2RB-z(YXXQ) LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR - RPGPTRKHYQPYAPPRDFAAYRSNCRNTGPWLKKVLKCNT(Ttruncated IL2RB PDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEIS containing 5th PLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRSGRV generation CAR-T KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD [Nature Medicine PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR volume 24, RGKGHDGLYQGLSTATKDTYDAYRHQALPPR pages352–359 (2018)] BB.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (BB.Z GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (2nd generation anti- RV CD19 CAR-T with KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD IgG4 hinge, CD28 PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR transmembrane, RGKGHDGLYQGLSTATKDTYDAYRHQALPPR 41BB co-stimulatory and CD3z domains; additional STAT3 binding motif (YRHQ) at the end of CD3z) 28.BB.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (3rd generation with LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD28, 41BB co- RPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRP stimulatory and CD3z VQTTQEEDGCSCRFPEEEEGGCELRTKKRIKPIVWPSLPDH domains; truncated KKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE IL7Ra incorporated GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY between CD28 and QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK CD3z domains; NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ P300H mutation in GLSTATKDTYDAYRHQALPPR IL7Ra; additional STAT3 binding motif (YRHQ) after CD3z BB.IL7tr(mut)+.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGYRHQRVKFSRSADAPAYQQGQNQLYNELNLGRREE P300H mutation in YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA IL7Ra; additional YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP STAT3 binding motif PR (YRHQ) after IL7Ra) BB.IL7tr(mut)++.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYRH CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR STAT3 binding motif (YRHQ) in IL7Ra (mutations: Q457R; N458H).)
98 BB.IL7tr(mut).Z++ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRYR STAT3 binding motif HQ (YRHQ) after CD3z.) 227 28.IL7tr(JS).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (minimal IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR truncation including RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK the JAK1-STAT5 KTLEHLCVWPSLPDHKGGGGSPQQEEAYVTMSRSGRVKF domain incorporated SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP between CD28 and EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR CD3z domains, with GKGHDGLYQGLSTATKDTYDAYRHQALPPR STAT3 binding motif) 228 28.IL7tr(mut).STAT4. IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV Z+ LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE GFLQDTFPQQEEAYVTMSSFYQNQYLPSNIDRSGRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDAYRHQALPPR 231 28.Z (2nd generation IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVV anti-CD19 CAR-T GGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH with CD28 hinge, YQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQQGQNQLYNELNL CD28TM, CD28 co- GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE stimulatory and CD3z AYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR domains WITHOUT STAT3 binding site. (FDA approved axi- cel, also known as axicabtagene ciloleucel (Yescarta)) *Underlined sequences are IL7Ra (and truncated versions) or truncated IL2RB sequences. *Bold letters are P300H mutation (or P36H mutation in SEQ NO:2 or 4) or STAT3 binding site YXXQ (SEQ ID NO:6 or YRHQ of SEQ ID NO:8). * Bold and Italic letters are STAT4 binding site represented by SEQ ID NO:189 In some embodiments, the CARs comprising the IL7Ra signaling domain according to the present invention may be TCR-based CARs. Some non-limiting examples of TCR-based CARs include STAR (synthetic T cell receptor and antigen receptor), HIT (HLA-independent T cell) receptor and TRuC (T cell receptor fusion construct), as described in Dennis Christoph Harrer et al. (2023). A HIT typically comprises the following domains:
- an extracellular antigen-binding domain including an antibody-derived binding domain (e.g. a single-chain variable fragment (scFv) or a VHH that specifically recognizes a target antigen on the surface of a tumor or infected cell), or ligand-binding domains (such as designed ankyrin repeat proteins or DARPins), - a spacer/linker region (such as portions of the human IgG Fc region or portions of CD8α), - transmembrane domain (such as transmembrane domain derived from natural receptors like CD28, CD3-zeta), and - an intracellular signaling domain(s), including a primary signaling domain (e,g, derived from the CD3-zeta chain of the TCR complex) or a costimulatory domains (e.g. costimulatory molecules such as CD28, 4-1BB (CD137), or OX40 (CD134)) TRuC construct typically comprises the following components: - an antigen-binding domain, such as single-chain variable Fragment (scFv), - TCR Fusion, wherein the scFv is fused to one of the subunits of the TCR complex (e.g., CD3ζ, CD3ε, CD3γ, or CD3δ), - a transmembrane domain, and - an intracellular signaling domain, such as CD3ζ ITAMs. STAR may include the following components: - an extracellular antigen-binding domain including an antibody-derived binding domain (e.g. a single-chain variable fragment (scFv) or a VHH that specifically recognizes a target antigen on the surface of a tumor or infected cell), or Ligand-Binding Domains (such as designed ankyrin repeat proteins or DARPins), - a transmembrane domain, and - an intracellular signaling domain(s), including a primary signaling domain (e,g, derived from the CD3-zeta chain of the TCR complex) or a costimulatory domains (e.g. costimulatory molecules such as CD28, 4-1BB (CD137), or OX40 (CD134)) These TCR-based CARs may present additional advantageous characteristics such as superior antigen sensitivity, enhanced degranulation, recruitment of signaling hubs, less tonic signaling, less exhaustion, and/or enhanced therapeutic efficacy. IV. Polynucleotides The terms ‘’nucleic acid’’, ‘’nucleic acid molecule’’, and ‘’polynucleotide’’ are used interchangeably herein. The terms “nucleic acid encoding . . .”, or “nucleic acid molecule encoding . . . ” should be understood as referring to the sequence of nucleotides which encodes a polypeptide. A polynucleotide described herein may comprise one or more nucleic acids each encoding a polypeptide, all operably linked to (i.e., in a functional relationship with) one or more regulatory
sequences, such as a promoter. Such a polynucleotide may alternatively be referred to herein as a ‘’nucleic acid construct’’ or ‘’construct’’. As used herein, a regulatory sequence refers to any genetic element that is known to the skilled person to drive or otherwise regulate expression of nucleic acids in a cell. Such sequences include without limitation promoters, transcription terminators, enhancers, repressors, silencers, kozak sequences, polyA sequences, and the like. A regulatory sequence can, for example, be inducible, non-inducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. Non-limiting examples of suitable promoters include EF1α, MSCV, EF1 alpha-HTLV-1 hybrid promoter, Moloney murine leukemia virus (MoMuLV or MMLV), Gibbon Ape Leukemia virus (GALV), murine mammary tumor virus (MuMTV or MMTV), Rous sarcoma virus (RSV), MHC class II, clotting Factor IX, insulin promoter, PDX1 promoter, CD11, CD4, CD2, gp47 promoter, PGK, Beta- globin, UbC, MND, and derivatives (i.e. variants) thereof. Examples of these promoters are further described in Poletti and Mavilio (2021), Viruses 13:8;1526, Kuroda et al. (2008), J Gene Med 10(11):1163-1175, Milone et al. (2009), Mol Ther 17:8;1453-1464, and Klein et al. (2008), J Biomed Biotechnol 683505, all of which are incorporated herein by reference in their entireties. A polynucleotide described herein may be multicistronic. ‘’Multicistronic’’ (alternatively referred to herein as ‘’polycistronic’’) can refer to the transcription of the polynucleotide resulting in an mRNA from which at least two distinct polypeptides are translated. This, for example, may be achieved by a polynucleotide comprising at least two nucleic acids encoding distinct polypeptides, preferably operably linked to the same promoter. In some embodiments, at least two, at least three, at least four, at least five, or at least six, preferably at least three or at least four, polypeptides are expressed by a polynucleotide described herein. A polynucleotide described herein may be tricistronic (i.e., three distinct polypeptides may be expressed). A polynucleotide described herein may be tetracistronic (i.e., four distinct polypeptides may be expressed). A multicistronic polynucleotide may comprise additional nucleotide sequences facilitating the co-expression of the encoded polypeptides, such as cis-acting regulatory elements described later herein. A polynucleotide may be incorporated in a vector as described later herein. Accordingly, in a further aspect, the present invention also provides a nucleic acid encoding any one of: - the IL7Ra signalling domain or functional fragments or variants thereof as described above in the first aspect, - the receptor comprising the IL7Ra signalling domain or functional fragments or variants thereof as described above in the second aspect, - the CAR comprising the IL7Ra signalling domain or functional fragments or variants thereof as described herein, - preferably the CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT3 binding site is present in the
intracellular signalling domain of the CAR, therefore the activation of STAT3 is not directly mediated by the IL7Ra and said STAT3 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. In some other preferred embodiments, the nucleic acid encodes the CAR comprising the IL7Ra signalling domain or functional fragments or variants thereof as described herein, wherein the CAR comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT4 binding site is present in the intracellular signalling domain of the CAR, therefore the activation of STAT4 is not directly mediated by the IL7Ra and said STAT4 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. A sequence of a coding DNA may be generated using “Reverse Translate” tool (https://www.bioinformatics.org/sms2/rev_trans.html). Reverse Translate accepts a protein sequence as input and uses a codon usage table to generate a DNA sequence representing the most likely non- degenerate coding sequence. In some embodiments, one or more nucleic acids as described above may be combined in an expression construct and operably linked to the same promoter. In some embodiments, the polynucleotide further comprises one or more cis-acting regulatory sequences. Expression of the IL7Ra signaling domain or the CAR as described therein may be assessed by any standard technique available to the skilled person such as western blotting, flow cytometry, FACS, and the like. Further non-limiting examples are provided in the examples. In some embodiments, the nucleic acid comprises or has a sequence encoding an IL7Ra signalling domain comprising or having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence to SEQ ID NO:2, 3 or 4. In some preferred embodiments, the nucleic acids comprising a nucleotide sequence encoding a polypeptide have at least 60%, 70%, 80%, 90%, 95%, or 100% identity or similarity with SEQ ID NOs:2. Preferably, such nucleic acid is such that the encoded amino acid sequence still comprises the mutation P36H. In some embodiments, the nucleic acid encoding the IL7Ra signalling domain comprises or has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs:60-62. In some embodiments, the nucleic acid encoding the IL7Ra signalling domain comprises or has a sequence selected from any one of SEQ ID NOs: 60-62. The encoded IL7Ra signalling domain by the nucleic acid variant derived from any one of SEQ ID NOs: 60-62 is able to activate STAT3 and STAT5, as described herein. In some embodiments, the nucleic acid comprises or has a sequence encoding the CAR comprising or having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence to any one of SEQ ID NOs: 14-17, 19-23, and 95-98. In some embodiments, the nucleic acid encoding the CAR comprises or has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 63-71, and 100-103. In some embodiments, the nucleic acid encoding the CAR comprises a sequence selected from any one of SEQ ID NOs:63-71, and 100-103. The encoded CAR by the nucleic acid variant derived from any one of SEQ ID NOs: 63- 71, and 100-103 exhibits at least one function of the CAR of the present invention as described herein. In some embodiments, the nucleic acid comprises or has a sequence encoding an IL7Ra signalling domain comprising or having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence to any one of SEQ ID NOs:178-187. In some preferred embodiments, the nucleic acids comprising a nucleotide sequence encoding a polypeptide have at least 60%, 70%, 80%, 90%, 95%, or 100% identity or similarity with any one of SEQ ID NOs: 178-187. Preferably, such nucleic acid is such that the encoded amino acid sequence still comprises the mutation P36A, P36W, P36E, P36L, or P36Q when referring to SEQ ID NOs: 178-187. In some embodiments, the nucleic acid encoding the IL7Ra signalling domain comprises or has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %,
at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 204-213. In some embodiments, the nucleic acid encoding the IL7Ra signalling domain comprises or has a sequence selected from any one of SEQ ID NOs: 204-213. The encoded IL7Ra signalling domain by the nucleic acid variant derived from any one of SEQ ID NOs: 204-213 is able to activate STAT3 and STAT5, as described herein. In some embodiments, the nucleic acid comprises or has a sequence encoding the CAR comprising or having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence to any one of SEQ ID NOs: 229- 230. In some embodiments, the nucleic acid encoding the CAR comprises or has at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 229-230. In some embodiments, the nucleic acid encoding the CAR comprises a sequence selected from any one of SEQ ID NOs:229-230. The encoded CAR by the nucleic acid variant derived from any one of SEQ ID NOs: 229-230 exhibits at least one function of the CAR of the present invention as described herein. V. Vector In another aspect, the present invention provides an expression vector comprising the nucleic acid as described above, or more specifically a nucleic acid encoding any one of: - the IL7Ra signalling domain or functional fragments or variants thereof as described above in the first aspect, - the receptor comprising the IL7Ra signalling domain or functional fragments or variants thereof as described above, - the CAR comprising the IL7Ra signalling domain or functional fragments or variants thereof as described above, - preferably the CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT3 binding site is present in the
intracellular signalling domain of the CAR, therefore the activation of STAT3 is not directly mediated by the IL7Ra and said STAT3 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. In some other preferred embodiments, the vector comprising the nucleic acid encodes the CAR comprising the IL7Ra signalling domain or functional fragments or variants thereof as described herein, wherein the CAR comprises an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain, wherein a STAT4 binding site is present in the intracellular signalling domain of the CAR, therefore the activation of STAT4 is not directly mediated by the IL7Ra and said STAT4 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. A “vector” may be any genetic element, e.g., a plasmid, chromosome, virus, transposon, behaving either as an autonomous unit of polynucleotide replication within a cell. (i.e., capable of replication under its own control) or being rendered capable of replication by insertion into a cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, bacteriophages and cosmids. A “vector” according to the present invention may be a polynucleotide capable of inducing the expression of a recombinant protein (e.g., a chimeric transmembrane protein, a protein, and/or a chimeric antigen receptor) in a mammalian cell. A vector provided herein may be, e.g., in circular or linearized form. Non-limiting examples of vectors include plasmids, SV40 vectors, adenoviral viral vectors, and adeno-associated virus (AAV) vectors. A viral vector can be a DNA or an RNA virus, with either episomal or integrated genomes after delivery to the cell. Non-limiting examples of vectors include lentiviral vectors or retroviral vectors, e.g., gamma-retroviral vectors. See, e.g., Carlens et al, Exp. Hematol.28(10: 1137-1146, 2000; Park et al, Trends Biotechnol. 29(l l):550-557, 2011; and Alonso-Camino et al, Mol. Ther. Nucleic Acids 2:e93, 2013. Non-limiting examples of retroviral vectors include those derived from Moloney murine leukemia virus, myeloproliferative sarcoma virus, murine embryonic stem cell virus, murine stem cell virus, spleen focus forming virus, or adeno-associated virus. Non-limiting examples of retroviral vectors are described in, e.g., U.S. Patent Nos. 5,219,740 and 6,207,453; Miller et al., BioTechniques 7:980-990, 1989; Miller, Human Gene Therapy 1 :5-14, 1990; Scarpa et al, Virology 180:849-852, 1991; Bums et al, Proc. Natl. Acad. Sci.U.S.A.90:8033-8037, 1993; and Boris-Lawrie et al, Cur. Opin. Genet. Develop.3: 102-109, 1993. Exemplary lentiviral vectors are described in, e.g., Wang et al., J. Immunother.35(9):689-70l, 2003; Cooper et al, Blood 101: 1637-1644, 2003; Verhoeyen et al., Methods Mol. Biol.506:97-114, 2009; and Cavalieri et al, Blood l02(2):497-505, 2003. Other non-limiting examples of viral vectors include poxvirus vectors, herpesvirus vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, Epstein- Bar virus vectors, herpes simplex virus vectors, hemagglutinating virus of Japan (HVJ) vectors, and Moloney murine leukemia virus vectors. Further exemplary vectors, in which any of the nucleic acids provided herein may be inserted, are described in, e.g., Ausubel et al, Eds.“Current Protocols in
Molecular Biology” Current Protocols, 1993; and Sambrook et al, Eds.“Molecular Cloning: A Laboratory Manual,” 2nd ed., Cold Spring Harbor Press, 1989. A vector may contain polynucleotide sequences which are necessary to effect ligation or insertion o f the vector into a desired host cell and to affect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors can be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. A vector can comprise a selectable marker gene. In some embodiments, the vector is an “episomal expression vector” or “episome,” which is able to replicate in a host cell and persists as an extrachromosomal segment of DNA within the host cell in the presence of appropriate selective pressure. The vectors as provided herein comprises a nucleic acid sequence comprising or having a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical sequence to any one of SEQ ID NOs:60-71, 204-226, and 229-230. In some embodiments, the vector comprises a nucleic acid sequence selected from SEQ ID NOs:60-71, 204-226, and 229-230. In some embodiments, the vector may include a nucleic acid sequence encoding a chimeric antigen receptor, which binds specifically to a tumor antigen well known in the art. Examples include but are not limited to glioma associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, Interleukin-11 receptor alpha (IL-11Ra), Interleukin-13 receptor subunit alpha-2 (IL-13Ra or CD213A2), epidermal growth factor receptor (EGFR), B7H3 (CD276), Kit (CD117), carbonic anhydrase (CA-IX), CS-1 (also referred to as CD2 subset 1), Mucin 1, cell surface associated (MUC1), B cell maturation antigen (BCMA), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) bcr-abl, Receptor tyrosine-protein kinase ERBB2 (HER2/neu), β-human chorionic gonadotropin, alphafetoprotein (AFP), anaplastic lymphoma kinase (ALK), CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, adrenoceptor beta 3 (ADRB3), thyroglobulin, tyrosinase; ephrin type-A receptor 2 (EphA2), Receptor for Advanced Glycation Endproducts (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), synovial sarcoma, X breakpoint 2 (SSX2), A kinase anchor protein 4 (AKAP-4), lymphocyte-specific protein tyrosine kinase (LCK), proacrosin binding protein sp32 (OY-TES1), Paired box protein Pax-5 (PAX5), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), C-type lectin-like molecule-1 (CLL-1 or CLECL1), fucosyl GM1, hexasaccharide portion of globoH glycoceramide (GloboH), MN-CA IX, Epithelial cell adhesion molecule (EPCAM), EVT6-AML, transglutaminase 5 (TGS5), human telomerase reverse transcriptase (hTERT), polysialic acid, placenta-specific 1 (PLAC1), intestinal carboxyl
esterase, LewisY antigen, sialyl Lewis adhesion molecule (sLe), lymphocyte antigen 6 complex, locus K 9 (LY6K), heat shock protein 70-2 mutated (mut hsp70-2), M-CSF, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), Ras Homolog Family Member C (RhoC), Tyrosinaserelated protein 2 (TRP-2), Cytochrome P4501B1 (CYP1B1), CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), prostase, prostate-specific antigen (PSA), paired box protein Pax-3 (PAX3), prostatic acid phosphatase (PAP), Cancer/testis antigen 1 (NY-ESO-1), Cancer/testis antigen 2 (LAGE-1a), LMP2, neural cell adhesion molecule (NCAM), tumor protein p53 (p53), p53 mutant, Rat sarcoma (Ras) mutant, glycoprotein 100 (gp100), prostein, OR51E2, pannexin 3 (PANX3), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), , high molecular weight-melanoma-associated antigen (HMWMAA), Hepatitis A virus cellular receptor 1 (HAVCR1), vascular endothelial growth factor receptor 2 (VEGFR2), Platelet- derived growth factor receptor beta (PDGFR-beta), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), survivin, telomerase, sperm protein 17 (SPA17), Stage-specific embryonic antigen-4 (SSEA-4), tyrosinase, TCR Gamma Alternate Reading Frame Protein (TARP), Wilms tumor protein (WT1), prostate-carcinoma tumor antigen-1 (PCTA-1), melanoma inhibitor of apoptosis (ML-IAP), MAGE, Melanoma-associated antigen 1 (MAGE-A1), melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma antigen recognized by T cells 1 (MelanA/MART1), X Antigen Family, Member 1A (XAGE1), elongation factor 2 mutated (ELF2M), ERG (TMPRSS2 ETS fusion gene), N-Acetyl glucosaminyl-transferase V (NA17), neutrophil elastase, sarcoma translocation breakpoints, mammary gland differentiation antigen (NY-BR-1), ephrinB2, CD20, CD22, CD23, CD24, CD30, CD32B, CD33, CD37, CD38, CD44v6, CD70, CD97, CD171, CD179a, CD200, CD229, androgen receptor, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, ganglioside GD2 (GD2), siglec-6, o-acetyl-GD2 ganglioside (OAcGD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1- 4)bDGlcp(1-1)Cer), G protein-coupled receptor class C group 5, member D (GPRC5D), G protein- coupled receptor 20 (GPR20), chromosome X open reading frame 61 (CXORF61), folate receptor (FRa), folate receptor beta, Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-Like Tyrosine Kinase 3 (Flt3), Tumor-associated glycoprotein 72 (TAG72), Tn antigen (TN Ag or (GalNAcα-Ser/Thr)), angiopoietin-binding cell surface receptor 2 (Tie 2), tumor endothelial marker 1 (TEM1 or CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), uroplakin 2 (UPK2), mesothelin, Protease Serine 21 (Testisin or PRSS21), epidermal growth factor receptor (EGFR), fibroblast activation protein alpha (FAP), Olfactory receptor 51E2 (OR51E2), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); B-cell receptor (BCR), IgM receptor, EGF-like module containing mucin- like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor- like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1), B cell-activating factor receptor (BAFF-R).
In the context of the present invention, the vector as described herein may be polycistronic (or multicistronic). ‘’Multicistronic’’ or “polycistronic” may refer to the transcription of the polynucleotide resulting in an mRNA from which at least two distinct polypeptides (or coding sequences) are translated, and optionally the at least two distinct polypeptides (or coding sequences) are under the same promoter. In the context of the present invention, provided herein may also be sets of vectors that include a first vector that includes a sequence that encodes any of the IL7Ra signaling domain as described herein, and a second vector that includes a sequence that encodes the chimeric antigen receptor as described herein. In some embodiments, one or both of the first vector and the second vector is a lentiviral or an adenoviral vector. In some embodiments, the second vector further includes a promoter sequence and/or an enhancer sequence that is operably linked to the sequence encoding the chimeric antigen receptor. In some embodiments, the second vector further includes a poly(A) sequence operably linked to the sequence encoding the IL7Ra signaling domain or the chimeric antigen receptor. As used herein, a regulatory sequence refers to any genetic element that is known to the skilled person to drive or otherwise regulate expression of nucleic acids in a cell. Such sequences include without limitation promoters, transcription terminators, enhancers, repressors, silencers, kozak sequences, polyA sequences, and the like. A regulatory sequence can, for example, be inducible, non-inducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. The promoter sequence may be selected from the group consisting of EF1α, MSCV, EF1 alpha-HTLV- 1 hybrid promoter, Moloney murine leukemia virus (MoMuLV or MMLV), Gibbon Ape Leukemia virus (GALV), murine mammary tumor virus (MuMTV or MMTV), Rous sarcoma virus (RSV), MHC class II, clotting Factor IX, insulin promoter, PDX1 promoter, CD11, CD4, CD2, gp47 promoter, PGK, Beta- globin, UbC, and MND, preferably from MSCV, MMLV, EF1α, and MND. In some aspects, the promoter sequence is a derivative sequence (i.e. variant sequence) of a promoter sequence described herein. In some embodiments, the promoter sequence comprises a sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID Nos: 72-75. Examples of promoters are further described in Poletti and Mavilio (2021), Viruses 13:8;1526, Kuroda et al. (2008), J Gene Med 10(11):1163-1175, Milone et al. (2009), Mol Ther 17:8;1453-1464, and Klein et al. (2008), J Biomed Biotechnol 683505. Table 7: Promoter sequences SEQ ID N SEQUENCE NO ame 72 tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaatacata MSCV actgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaaacaggat promoter atctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcccgccctc agcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaaatgaccctgtgccttatttga
actaaccaatcagttcgcttctcgcttctgttcgcgcgtttctgctccccgagctcaataaaagagcccacaacc cctcact 73 aatgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaaaatac ataactgagaatagaaaagttcagatcaaggtcaggaacagatggaacagctgaatatgggccaaacag gatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggaacagctgaatatgggccaa MMLV acaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcggtcc promoter agccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccctgtgcct tatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttatgctccccgagctcaataaaagagccc acaacccctcactcggggcgccagtcctccgattgactgagtcgcccgggtacccgtgtatccaataaaccc tcttgcagttgcatccgacttgtggtctcgctgttccttgggagggtctcctctgagtgattgactacccgtcagcg ggggtctttcatt 74 gctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcg gcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgc ctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggttt gccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgt gccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtggg agagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggcgctgggg ccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaattttt EF1α gatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttc promoter ggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctg cgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcg cgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaa gatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcg ggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgcttcatgtgactccacggagtac cgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtctttaggttggggggaggggtttt atgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctcct tggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttca ggtgtcgtga 75 tttatttagtctccagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctaggatcaaggtta ggaacagagagacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggctcagg MND gccaagaacagttggaacagcagaatatgggccaaacaggatatctgtggtaagcagttcctgccccggct promoter cagggccaagaacagatggtccccagatgcggtcccgccctcagcagtttctagagaaccatcagatgtttc cagggtgccccaaggacctgaaatgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttc gcgcgcttctgctccccgagctcaataaaagagccca Cis-acting regulatory elements as described herein are facilitators of co-expression and include sequences that ensure that the component nucleic acid sequences (for example, the nucleic acid encoding the heterodimeric receptor monomers and the nucleic acid encoding the CAR as described herein) are translated from the single mRNA transcribed from the polynucleotide. A cis-acting regulatory element may be, for example, selected from (but is not limited to) an internal ribosome entry site (IRES) sequence or a sequence encoding a 2A-self cleaving peptide. In some embodiments, the nucleotide sequence inserted between each of the nucleic acids is a sequence encoding a 2A self-cleaving peptide or is an IRES sequence. An IRES sequence functions by allowing the assembly of a new translation initiation complex after the ribosome dissociates from the mRNA following the synthesis of the first polypeptide. Suitable IRES sequences will be known to the skilled person and examples are further available in public databases such as IRESite: The database of experimentally verified IRES structures, described in Mokrejš et al., Nucleic Acids Res. 2006; 34(Database issue): D125–D130, which is incorporated herein by reference in its entirety. Non-limiting examples of IRES’s known in the art include Picornavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, and Hepesvirus IRES.
The nucleotide sequence inserted between each of the nucleic acids may a sequence encoding a 2A self-cleaving peptide. 2A self-cleaving peptides (abbreviated herein as ‘’2A peptides’’) may be advantageous for expression of multicistronic polynucleotides described herein due to their small size and self-cleavage ability, which allows for facilitation of polypeptide co-expression. 2A peptides are typically composed of 16–22 amino acids and originate from viral RNA. 2A peptide-mediated polypeptide cleavage is typically triggered by ribosomal skipping of the peptide bond between the proline (P) and glycine (G) in the C-terminal of a 2A peptide, resulting in the polypeptide located upstream of the 2A peptide to have extra amino acids on its C-terminal end while the peptide located downstream the 2A peptide has an extra proline on its N-terminal end. Examples of nucleic acid sequences encoding 2A peptides may be found in Xu Y., et al (2019), and Pincha M., et al, (2011) (supra). Non-limiting examples of suitable 2A peptides are F2A (2A peptide derived from the foot-and- mouth disease virus), E2A (2A peptide derived from the equine rhinitis virus), P2A (2A peptide derived from the porcine teschovirus-1), or T2A (2A peptide derived from the Thosea asigna virus). In some embodiments, the 2A self-cleaving peptide is a F2A peptide. In some embodiments, the 2A self-cleaving peptide is an E2A peptide. In some embodiments, the 2A self-cleaving peptide is a P2A peptide. In some embodiments, the 2A self-cleaving peptide is a T2A peptide. The skilled person understands that a polynucleotide described herein may also comprise nucleotide sequences encoding different 2A self- cleaving peptides. As a non-limiting example, in a tricistronic construct, a P2A peptide-encoding sequence may be inserted between the nucleic acid encoding the first and the second polypeptide, and a T2A peptide-encoding sequence may be inserted between the nucleic acid encoding the second and third polypeptide. Accordingly, polynucleotides comprising nucleotide sequences encoding multiple different 2A self-cleaving peptides are also provided. An exemplary polynucleotide comprises a P2A peptide-encoding sequence and a T2A peptide-encoding sequence. A further exemplary polynucleotide comprises a nucleotide sequence encoding a 2A self-cleaving peptide having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 77 or 79 or comprises a nucleotide sequence encoding a 2A self-cleaving peptide represented by an amino acid sequence having an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity or similarity with SEQ ID NO: 76 or 78. Table 8: Sequences for cis-acting regulatory elements SEQ ID NO Name SEQUENCE 76 P2A ATNFSLLKQAGDVEENPGP
77 P2A DNA gccaccaatttcagcctgctgaaacaggctggcgacgtggaagagaaccctgggccc 78 T2A EGRGSLLTCGDVEENPGP 79 T2A DNA gaaggccgcggcagcctgctgacctgcggagacgtggaagaaaaccctggcccg In some embodiments, the polynucleotide provided herein may comprise at least two or more cis-acting regulatory elements. In some embodiments, the polynucleotide may comprise at least a first cis-acting regulatory element and a second cis-acting regulatory element such that the first and the second cis-acting regulatory elements are each independently selected from the group consisting of F2A, E2A, P2A, T2A or any combination thereof. In some embodiments, the polynucleotide may comprise at least a first cis-acting regulatory element and a second cis-acting regulatory elements such that the first and second cis-acting regulatory elements are each independently selected from the group consisting of Picornavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, Hepesvirus IRES, and combinations thereof. The vectors provided herein further include a poly(A) sequence, which is operably linked and positioned 3’ to the sequence encoding the chimeric transmembrane protein, the protein, or the chimeric antigen receptor. Non-limiting examples of a poly(A) sequence include those derived from bovine growth hormone (Woychik et al, Proc. Natl. Acad. Sci. U.S.A.81(13): 3944-3948, 1984, and U.S. Patent No. 5,122, 458), mouse^-globin, mouse-a-globin (Orkin et al, EMBO J. 4(2): 453-456, 1985), human collagen, polyoma virus (Batt et al, Mol. Cell Biol.15 (9): 4783 -4790, 1995), the Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy chain gene polyadenylation signal (U.S. Patent Application Publication No.2006/0040354), human growth hormone (hGH) (Szymanski et al. , Mol. Therapy 15(7): 1340-1347, 2007), SV40 poly(A) site, e.g., SV40 late and early poly(A) site (Schek et al, Mol. Cell Biol. 12(12):5386-5393, 1992). In some embodiments, the poly(A) sequence includes a highly conserved upstream element (AATAAA). The AATAAA sequence can, e.g., be substituted with other hexanucleotide sequences with homology to AATAAA which are capable of signaling polyadenylation as described in e.g, WO2006/012414A2. A poly(A) sequence can, e.g., be a synthetic polyadenylation site. See, e.g, Levitt el al, Genes Dev. 3(7): 1019-1025, 1989). Additional examples and aspects of vectors are also known in the art. A polynucleotide vector useful for the methods and compositions described herein can be a good manufacturing practices (GMP) compatible vector. For example, a GMP vector can be purer than a non-GMP vector. In some cases, purity may be measured by bioburden. For example, bioburden can be the presence or absence of aerobes, anaerobes, sporeformers, fungi, or combinations thereof in a vector composition. In some cases, a pure vector can be endotoxin low or endotoxin free. Purity can also be measured by double-stranded primer-walking sequencing. Plasmid identity can be a source of determining purity of a vector. A GMP vector of the invention can be from 10% to 99% more pure than a non-GMP vector. A GMP vector can be from 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% more pure than a non-
GMP vector as measured by the presence of bioburden, endotoxin, sequencing, or combinations thereof. A variety of different methods known in the art can be used to introduce any of the nucleic acids and vectors disclosed herein into a mammalian cell (e.g., any of the mammalian cells described herein, e.g., any of the T cells (e.g., human T cells) described herein). Non-limiting examples of methods that can be used to introduce a nucleic acid or vector into a mammalian cell include lipofection, transfection, electroporation, microinjection, calcium phosphate transfection, dendrimer-based transfection, cationic polymer transfection, cell squeezing, sonoporation, optical transfection, impalection, hydrodynamic delivery, magnetofection, viral transduction (e.g., adenoviral and lentiviral transduction), and nanoparticle transfection. Additional methods of introducing a nucleic acid or vector into a mammalian cell are known in the art. VI. Cell In another aspect, the present invention provides for a cell comprising the nucleic acid as described above or the expression vector as described above, preferably wherein the cell expresses the encoded receptor, the encoded CAR, and more preferably wherein the cell is a T cell. In the context of the invention such CAR-T cells may be named CAR-T cells of the invention or engineered T cells of the invention. A “genetically modified” or “modified ” cell or “modified cell to express” in the context of this aspect refers to a cell in which the nuclear, organellar or extrachromosomal nucleic acid sequences of a cell has been transformed, modified or transduced using recombinant DNA technology to comprise a heterologous nucleic acid molecule, and is used interchangeably with “engineered cell,” “transformed cell,” and “transduced cell.” A genetically modified cell as disclosed herein expresses a protein encoded by a nucleic acid molecule engineered in such manner to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and/or a substitution of at least one nucleotide in a sequence encoding at least one heterologous protein. Engineered cells as disclosed herein comprise at least one polynucleotide and/or vector as described herein, and preferably express the polypeptides encoded by the polynucleotide(s) and/or vector(s). Accordingly, in some embodiments, provided herein is an engineered cell or populations of such cells that express one or more CARs comprising the IL7Ra signaling domain as described herein. In some embodiments, provided herein are mammalian cells that include any of the nucleic acids or vectors described herein. Also provided herein are mammalian cells that include any of the sets of vectors described herein. In some embodiments, the mammalian cell is previously obtained from a subject (e.g., a human subject, e.g., a human subject identified or diagnosed as having a cancer) or is a daughter cell of a mammalian cell that was previously obtained from a subject (e.g., a human subject, e.g., a human subject identified or diagnosed as having a cancer). In some embodiments, the mammalian cell is an immune cell. In some embodiments, the mammalian cell is a human cell.
Non-limiting examples of include a T cell (e.g., a human T cell). Additional examples of mammalian cells include a mast cell, a macrophage, a neutrophil, a dendritic cell, a basophil, an eosinophil, and a natural killer cell. Macrophages and dendritic cells may be referred to as “antigen presenting cells” or “APCs,” which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC interacts with a TCR on the surface of a T cell. An engineered immune cell provided herein may comprise additional edits and or modifications in comparison to naturally occurring counterparts of the same cell. A “T cell” is an immune cell that matures in the thymus and produces T cell receptors (TCRs). T cells can be naïve (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells I (antigen- experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naïve T cells) and effector memory T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naïve T cells or TCM). Effector T cells (TE) refers to an antigen- experienced CD8+ or CD4+ T lymphocytes that has decreased expression of CD62L, CCR7, CD28, and are positive for granzyme and perforin as compared to TCM. Non limiting examples of T cells (e.g., human T cells) may include, e.g., an immature thymocyte, a peripheral blood lymphocyte, a helper T cell, a naive T cell, a T cell precursor, a lymphoid progenitor cell, a memory T cell, a TH17 cell, a TH22 cell, a TH9 cell, a TH2 cell, a TH1 cell, a TH3 cell, gamma-delta T cell, an alpha beta T cell, a regulatory T cell (Treg cell), and a tumor-infiltrating T cell. Additional sources of immune cells include double negative T-cells, natural killer cell, B cell, dendritic cells, NK-T cells,monocyte and hematopoietic and induced pluripotent stem cells, cord blood. Expression of one or more of the peptides or proteins of the present invention in an engineered cell or population thereof can be used as a strategy to overcome limitations that hamper the production and use of engineered cells, for example, low expression, limited cytotoxic effect, limited immune stimulatory effect, limited proliferative ability or lifespan of the engineered cells, limited induction of effector function upon engineered cell recognition of antigen, and engineered cell exhaustion. Accordingly, in some embodiments, provided herein are immune cells comprising at least one polynucleotide and/or vector as described herein, and preferably express the polypeptides encoded by the polynucleotide(s) and/or vector(s). in some preferred embodiments, the immune cells are T-cells, and the expressed polypeptides is a CAR as described above. In some more preferred embodiments, the T-cells are CD8+ or CD4+ T cells. Accordingly, in some embodiments, there provides an engineered CAR-T cells that express a receptor comprising the IL7Ra signalling domain. In an embodiment, said receptor is a CAR as defined earlier.
In an embodiment, an immune cell, preferably a T cell comprises a polynucleotide and/or vector as earlier described herein and preferably expresses a CAR comprising the IL7Ra signalling domain or functional fragments or variants thereof as described above. In another embodiment, an immune cell, preferably a T cell comprises a polynucleotide and/or vector as earlier described herein and preferably expresses a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain. In a preferred embodiment, a STAT3 binding site is present in the intracellular signalling domain of the CAR, therefore the activation of STAT3 is not directly mediated by the IL7Ra and said STAT3 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. In some embodiments, said CAR-T cells express a CAR comprising the IL7Ra signaling domain as earlier described herein, said IL7Ra signaling domain has a sequence at least 60%, at least 61%, at least 62 %, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs: 2-4. Preferably, the P36H mutation is still present in the sequence derived from SEQ ID NO: 2 (this mutation corresponds to the P300H mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1)). Preferably, the Q79R and N80H mutations are still present in the sequences derived from SEQ ID NO:3 or 4 (these mutations correspond to the Q457R and N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1). More preferably, the P36H, Q79R and N80H mutations are still present in the sequence derived from SEQ ID NOs: 4 (these mutations correspond to theP300H and Q457R and N458H mutations when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1) . In some embodiments, the CAR comprises one or more STAT3 binding site in its intracellular signaling domain. In short, such motifs are represented by SEQ ID NO:6: YX1X2Q, wherein: X1 is any amino acid, preferably X1 is F, L or R, X2 is K, P or H and optionally Q is mutated/substituted into P, T, Y, N, F or A. In some embodiments, the motif which is able to recruit/bind STAT3 comprises any of the following sequences:
YFKQ, YFKP, YFKT, YFKY, YFKN, YFKF, YFKA, YFPQ, YFPP, YFPT, YFPY, YFPN, YFPF, YFPA, YFHQ, YFHP, YFHT, YFHY, YFHN, YFHF, YFHA, YLKQ, YLKP, YLKT, YLKY, YLKN, YLKF, YLKA, YLPQ, YLPP, YLPT, YLPY, YLPN, YLPF, YLPA, YLHQ, YLHP, YLHT, YLHY, YLHN, YLHF, YLHA, YRKQ, YRKP, YRKT, YRKY, YRKN, YRKF, YRKA, YRPQ, YRPP, YRPT, YRPY, YRPN, YRPF, YRPA, YRHQ, YRHP, YRHT, YRHY, YRHN, YRHF, YRHA (SEQ ID NOs:104-166). Some non-limiting and preferred examples of STAT3 binding site may be YRHQ, YFKQ, YLQP, YDKP, YVNY, YVTA, YYLN, YDKP, YIYF, YYNF, or YYVF. In a preferred embodiment, the STAT3 binding/recruiting site is YRHQ (SEQ ID NO:8). In some embodiments, said CAR-T cells express a CAR having a sequence that is at least 60%, at least 61%, at least 62 %, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs: 14-17, 19-23, and 95-98. In some embodiments, said CAR-T cells express a CAR comprising the IL7Ra signaling domain as earlier described herein, said IL7Ra signaling domain has a sequence represented as SEQ ID NO: 188 having 25AA. In some other embodiments, an immune cell, preferably a T cell comprises a polynucleotide and/or vector as earlier described herein and preferably expresses a CAR comprising an antigen binding domain, a transmembrane domain, an intracellular signalling domain comprising the IL7Ra signalling domain of the first aspect and a signalling CD3z domain, and optionally a co-stimulatory domain. In a preferred embodiment, a STAT4 binding site is present in the intracellular signalling domain of the CAR, therefore the activation of STAT4 is not directly mediated by the IL7Ra and said STAT3 binding site is not present in the IL7Ra signalling domain or functional fragments or variants thereof as described above. In some embodiments, the IL7Ra signaling domain comprises a sequence that is at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to or similar to any one of SEQ ID NOs:192-203, preferably to any one of SEQID NO:s 192-197, more preferably to SEQ ID NO: 192. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids. The IL7Ra signalling domain as defined above may have a length of less than 95 amino acids, for example a length of 60 to 70, 70 to 80, or 80 to 90 number of amino acids.
Preferably, the mutation at position P36 (P36H, P36A, P36W, P36E, P36L, or P36Q) in any one of SEQ ID NO: 192-203 (corresponding to the P300 mutation in SEQ ID NO:1) is still present in the sequence derived from any one SEQ ID NOs:192-203 as described above. In some more preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1, or P36H when referring to any one of SEQ ID NOs:192, and more preferably both the mutation P300H and the STAT4 binding site are still present. In some embodiments, the IL7Ra signalling domain is represented by an amino acid sequence which has at least 80% identity with SEQ ID NOs:192-203, preferably wherein the mutation corresponding to the P300H, P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (SEQ ID NO:1) and/or the STAT4 binding site is still present in the sequence derived from any one of SEQ ID NOs:192-203. In some embodiments, both the STAT3 and STAT4 bindings sites are still present in the sequence derived from any one of SEQ ID NOs:198-203. In some embodiments, both the STAT3 and STAT4 bindings sites, and the P300 mutations are still present in the sequence derived from any one of SEQ ID NOs:198-203. In some preferred embodiments, the mutation at P300 is P300H when referring to SEQ ID NO:1. The STAT4 binding/recruiting site may be represented by YLPSNID (SEQ ID NOs: 189), TX1X2GYL (SEQ ID NO: 190) or GYKPQIS (SEQ ID NO: 191). In some embodiments, the STAT4 binding/recruiting site in the IL7Ra signaling domain according to the invention is YLPSNID (SEQ ID NOs: 189) which is present at positions 82-88 when referring to any one of SEQ ID NOs: 192-203. In some preferred embodiments, the Y (tyrosine) is phosphorylated Y or pY. In this context, the motif YLPSNID (SEQ ID NOs: 189) is the motif present in the IL-12 beta 2 subunit of the IL-12 receptor complex, wherein the Y is present at position 800 of the IL-12R beta 2 subunit. Alternatively, the motif used in the IL7Ra signaling domain may be derived therefrom. The Stat4 SH2 domain may be directly recruited to a tyrosine present in a motif, wherein the tyrosine is present as the first residue of said motif. In an embodiment, the motif is YLPSNID (SEQ ID NOs: 189) and the tyrosine recruiting the Stat4 SH2 is the first tyrosine of this motif. In some embodiments, the STAT4 binding/recruiting in the IL7Ra signaling domain according to the invention is TX1X2GYL (SEQ ID NO: 190). In this context, X1 and X2 can be any amino acid, each chosen independently from the other. In some preferred embodiments, X1 is not an H, and X2 is not an D. In some preferred embodiments, the Y (tyrosine) is phosphorylated Y or pY. In some embodiments, the STAT4 binding/recruiting site in the IL7Ra signaling domain according to the invention is GYKPQIS (SEQ ID NO: 191). In some preferred embodiments, the Y (tyrosine) is phosphorylated. The motif GYKPQIS (SEQ ID NO: 191) is present in the IL-23R, and the Y is the conserved tyrosine residue Y484 of the IL23R. The Stat4 SH2 domain may be directly recruited to a motif comprising a tyrosine in the second place such as the motif present in the IL23R and represented by GYKPQIS (SEQ ID NO: 191), wherein the tyrosine corresponds to Y484 of the IL23R.
In some embodiments, the IL7Ra signaling domain according to the present invention comprises any one of SEQ ID NOs: 192-203, preferably any one of SEQ ID Nos: 192-197. In some preferred embodiments, the IL7Ra signaling domain according to the present invention comprises SEQ ID NO:192. In some embodiments, the IL7Ra signaling domain according to the present invention is any one from SEQ ID NOs: 192-203, preferably any one of SEQ ID Nos: 192-197, more preferably the IL7Ra signaling domain is SEQ ID NO:192. A target biological outcome or biological parameter and/or function of a CAR expressing immune cell can include a cytotoxic response, e.g., against cancer/tumor cell. A cytotoxic response may be determined directly (e.g., by measuring cell lysis, cell population or survival of target cells). Alternatively, or in addition, a cytotoxic response may be determined by measuring the production of molecules associated with such a response, for example a production of a cytokine such as interferon gamma (IFNγ). Suitable measurement assays, for example luminescence assays to determine cytotoxicity and ELISA to determine IFNγ production are known to the skilled person and further non-limiting examples are provided in the experimental section. In some embodiments, the cytotoxic response can be measured by any of the methods known in the art some of which are provided herein in the examples (e.g., a luciferase assay). As detailed in the Examples below, a luciferase cytotoxicity assay can comprise use of a target cell population (e.g., an immortalized cancer cell line) that is genetically engineered to express a luciferase, which becomes detectable upon cell lysis. Therefore, a cytotoxic response may be easily determined by monitoring a fluorescent (luciferase) signal. Exemplary luciferase expressing target cell populations that may be used in these methods can include RPMI- 8226 LucTOM cells, Daudi cells, MM1S tumor cells and HT-29 LucTOM cells. Through the application, the wording “target biological outcome” may be replaced by “biological parameter and/or biological function” and comprises one or more of cytotoxicity, antitumor activity, and/or tumor cell killing, and/or proliferation, cellular survival, or persistence. A target biological outcome (i.e. a biological parameter and/or biological function) can be or can comprise, for example, cellular proliferation, cellular survival, magnitude of immune effector function, duration of immune effector function, cytotoxic effects on a cell (e.g., a cancer cell), production of inflammatory mediators, an anti- cancer immune response, cellular differentiation, cellular dedifferentiation. In an aspect, the biological parameter and/or function is selected from proliferation, cellular survival, cytotoxicity, antitumor activity, persistence and/or tumor cell killing and/or proliferation. The methods of determining these biological parameters and functions are well known in the art. In some embodiments, the engineered cells, preferably CAR-T cells, expressing CARs having a IL7Ra signaling domain as provided herein (i.e. also named CAR-T cells of the invention) exhibit significantly enhanced CAR T-cell expansion, reduced functional exhaustion of the CAR T-cells, increased cytotoxicity, improved antitumor activity, improved tumor cell killing, improved proliferation, improved cellular survival, and/or improved persistence, as compared to T cells that express a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the invention. In some embodiments, a target biological function of an engineered cell, preferably a CAR-T cell, is
elicited by or directed against cells that express or present an antigen recognized by the antigen- recognition domain of the CAR. For example, in some embodiments, where a target biological function comprises a cytotoxic response against cancer cells, the engineered cells can kill cancer cells based on recognition of an antigen by an antigen-recognition domain. Expression of the CAR and/or the IL7Ra signaling domain may be assessed by any standard technique available to the skilled person, such as western blotting, flow cytometry, FACS, and the like. Further non-limiting examples are provided in the examples. In some embodiments, upon exposure to a cell expressing the antigen, the target biological function of the engineered cell, preferably the CAR-T cell , expressing a CAR having a IL7Ra signaling domain as provided herein (i.e. also named CAR-T cells of the invention) is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold than a corresponding cell (or a control cell) that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. In the context of the present invention, a control cell as described herein may also be a corresponding cell that expresses: - identical CARs with full length IL7Ra - identical CARs without IL7Ra signaling domain - identical CARs with truncated IL7Ra signaling domain but without the P300H/P36H mutation as described herein, - identical CARs with truncated IL7Ra signaling domain but without STAT3 binding sites in the intracellular signaling domain of the CAR, - identical CARs with truncated IL7Ra signaling domain but without STAT4 binding sites in the intracellular signaling domain of the CAR, or- identical CARs except that truncated IL7Ra is replaced with truncated IL2RB. In some embodiments, upon exposure to a cell expressing the antigen, at least one or more of the following cellular activities of the CAR-T cell of the invention is increased compared to the corresponding cellular activity in a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention.: expansion, proliferation, cellular survival, cytotoxicity, tumor control, antitumor activity, persistence and/or tumor cell killing. In this context, “is increased” may mean is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold. This increase may also be more durable than for a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein.
For example, a CAR-T cell of the invention may survive or be persistent or may expand or may proliferate or may exhibit cytotoxic activity or may control tumor cell for a longer period of time than a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. The period of time may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% longer. In some embodiments, upon exposure to a cell expressing the antigen, CAR-T cells of the invention will be able to phosphorylate and preferably activate both STAT3 and STAT5. The phosphorylation and preferably activation of STAT3 is higher in the CAR-T cells of the invention than in a control CAR-T cells that express a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. In some embodiments, the CAR-T cells are CD4+ CAR-T cells. In some embodiments, the CAR-T cells are CD4+ and CD8+ CAR-T cells. This increase may be assessed by comparison with other controls as earlier defined herein. “Higher” may mean that the amount of phosphorylated/activated STAT3 in the CAR-T cells of the present invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more than that in a control cell, as defined herein. STAT3/5 activation may be assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labelled STAT3 or STAT5 binding site, or western blotting using antibodies against tyrosine phosphorylated STAT3/5, or by the means of flow cytometry analysis using antibodies against Y- phosphorylated STAT3/STAT5, or using STAT3/STAT5 singalling reporter cell lines. In some embodiments, upon exposure to a cell expressing the antigen, CAR-T cells of the invention will be able to phosphorylate and preferably activate both STAT4 and STAT5. The phosphorylation and preferably activation of STAT4 is higher in the CAR-T cells of the invention than in a control CAR-T cells that express a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. In some embodiments, the CAR-T cells are CD4+ CAR-T cells. In some embodiments, the CAR-T cells are CD4+ and CD8+ CAR-T cells. This increase may be assessed by comparison with other controls as earlier defined herein. “Higher” may mean that the amount of phosphorylated/activated STAT4 in the CAR-T cells of the present invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more than that in a control cell, as defined herein. STAT4/5 activation may be assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labelled STAT4 or STAT5 binding site, or western blotting using antibodies against tyrosine phosphorylated STAT4/5, or by the means of flow cytometry analysis using antibodies against Y- phosphorylated STAT4/STAT5, or using STAT4/STAT5 signalling reporter cell lines. In some embodiments, upon exposure to a cell expressing the antigen, CAR-T cells of the invention will be able to phosphorylate and preferably activate STAT3, STAT4 and STAT5. The phosphorylation and preferably activation of STAT4 is higher in the CAR-T cells of the invention than in a control CAR-T cells that express a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. In some embodiments, the CAR-T cells are CD4+ CAR-T cells. In some embodiments, the CAR-T cells are CD4+ and CD8+ CAR-T cells. This increase may be assessed by comparison with other controls as earlier defined herein. “Higher” may mean that the
amount of phosphorylated/activated STAT3 and STAT4 in the CAR-T cells of the present invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more than that in a control cell, as defined herein. STAT3, STAT4 and STAT5 activation may be assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labelled STAT3, STAT4 or STAT5 binding site, or western blotting using antibodies against tyrosine phosphorylated STAT3, STAT4, and STAT5, or by the means of flow cytometry analysis using antibodies against Y-phosphorylated STAT3, STAT4 and STAT5, or using STAT3, STAT4 and STAT5 signalling reporter cell lines. The phosphorylation and preferably activation of STAT5 is higher in the CAR-T cells of the invention than in CAR-T cells that express a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. In some embodiments, the CAR-T cells are CD4+ CAR-T cells. In some embodiments, the CAR-T cells are CD4+ and CD8+ CAR-T cells. This increase may be assessed by comparison with other controls as earlier defined herein. “Higher” may mean that the amount of phosphorylated/activated STAT3 or STAT4 in the CAR-T cells of the present invention is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or 300% more than that in a control cell, as defined herein. STAT3/4/5 activation may be assessed by EMSA (Electrophoretic Mobility Shift Assay) using a labelled STAT3, STAT4 or STAT5 binding site, or western blotting using antibodies against tyrosine phosphorylated STAT3/4/5, or by the means of flow cytometry analysis using antibodies against Y-phosphorylated STAT3/STAT4/STAT5, or using STAT3/STAT4/STAT5 signaling reporter cell lines . In the context of the present invention, the STAT3, STAT4and/or STAT5 phosphorylation and preferably activation in the engineered cells, preferably CAR-T cells, are triggered in an antigen-dependent manner, or upon antigen engagement by the antigen-binding domain of the CAR of said engineered CAR-T cells. The CAR of the present invention comprising the IL7Ra signaling domain as described herein preserves the endogenous function of a wild type IL7Ra signaling domain to activate STAT5 promotes the proliferation and survival of the engineered cells comprising said CAR. Said property of the CARs may confer the one or more improved/enhanced target biological function as described above. In some embodiments, upon exposure to a cell expressing the antigen, at least one or more of the following cellular activities of the CAR-T cell of the invention is increased compared to the corresponding cellular activity in a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention.: expansion, proliferation, cellular survival, cytotoxicity, tumor control, antitumor activity, persistence and/or tumor cell killing. This improvement may be assessed by comparison with other controls as earlier defined herein. In this context, “is increased” may mean is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000- fold. This increase may also be more durable than for a CAR-T cell that expresses a CAR identical to
the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein For example, a CAR-T cell of the invention may survive or be persistent or may expand or may proliferate or may exhibit cytotoxic activity or may control tumor cell for a longer period of time than a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein. The period of time may be at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% longer. Said property of the CARs according to the present invention may be attributed to their STAT3 activation property present in the intracellular signaling domain of the CARs, preferably its STAT3 recruitment and activation binding site present in the intracellular signaling domain of the CARs. The activation of STAT3 is either directly triggered via the IL7Ra signalling domain present in the CAR or in the CAR itself and not in the IL7Ra.This property has another advantage that the elevation levels of pSTAT3/pSTAT5 resemble the one induced by native cytokines stimulation but only upon CAR-T activation. As a result, systemic toxicity is not expected. Additional property of the CARs of the present invention may be attributed to their STAT4 activation property present in the intracellular signaling domain of the CARs, preferably its STAT4 recruitment and activation binding site present in the intracellular signaling domain of the CARs. The activation of STAT4 is either directly triggered via the IL7Ra signalling domain present in the CAR or in the CAR itself and not in the IL7Ra. This property has another advantage that the elevation levels of pSTAT4/pSTAT5, optionally also pSTAT3, resemble the one induced by native cytokines stimulation but only upon CAR- T activation. As a result, systemic toxicity is not expected. An additional property of the CARs of the present invention may also be attributed to the IL7Ra signaling domain and to the mutation present at position P300, when referring to the wild type human IL7Ra (i.e. SEQ ID NO1), optionally the mutation is selected from a list comprising P300H, P300A, P300W, P300E, P300L, and P300Q, preferably P300H mutation in the intracellular signaling domain of the CARs. CAR comprising a IL7Ra signalling domain comprising this mutation exhibit advantageous properties for treating immunocompromised cancer patients and/or for treating cancer patients showing signs of T cell exhaustion. In some embodiments, upon exposure to a cell expressing the antigen, the capability of the CAR-T cells of the present invention to eliminate target tumor/cancer cells is increased of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50- fold, at least 100-fold, or at least 1000-fold compared to corresponding capabilities of a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein. In some embodiments, upon exposure to a cell expressing the antigen, the capability of the CAR-T cells of the present invention to eliminate target tumor/cancer cells under a condition of high tumor/cancer
burden is increased of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10- fold, at least 20-fold, at least 50-fold, at least 100-fold, or at least 1000-fold compared to corresponding capabilities of a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein High tumor burden is a significant adverse prognostic factor that negatively impacts progression-free survival and overall outcomes in patients with various lymphomas. As an example, high tumor burden in lymphoma may be assessed using PET imaging based on the high tumor-to-background ratio of hypermetabolic lymphomas. As another example, a high tumor burden situation may be defined when an effector-to-target (E:T) ratio (e.g.: the ratio between effector CAR-T cells and target tumor cells) is at least 1:5, more specifically 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In some embodiments, upon exposure to a cell expressing the antigen, the capability of the CAR-T cells of the present invention to eliminate immunosuppressive target tumor/cancer cells is increased at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50- fold, at least 100-fold, or at least 1000-fold compared to the capability of a CAR-T cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein. In this context, the immunosuppressive target tumor/cancer cells may have multiple immune checkpoint receptor ligands (e.g. PD-L1, PD-L2, CD86, or GAL9). Said immunosuppressive target tumor/cancer cells may be present in heavily pretreated patients. In some embodiments, the current disclosure also encompasses a plurality, i.e., a population of engineered T-cells expressing the same or different polynucleotides as provided in the present invention. For example, the present invention also encompasses mixtures of CAR-T cells co-expressing different CARs with or without the IL7Ra signaling domain and/or the variants thereof. This increase may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the population of cells provided herein individually or together exhibit enhanced target biological function in comparison to a population of cells expressing the respective CARs alone without the IL7Ra signaling domain and/or the variants thereof as described herein. In some embodiments, these cell populations may comprise cells having one or more additional modifications that improve biological function. In some embodiments, the present aspect also encompasses a population of cells at least one cell of which comprises a polynucleotide disclosed here and preferably express a corresponding encoded polypeptide. In some embodiments, the population may only comprise engineered cell or plurality of engineered cells as provided herein. In some embodiments, the population of cells may further comprise additional cells not comprising the polynucleotide provided herein. For example, in certain aspects, the cell population comprises γδ T cells, αβ T cells and NK cells and at least a portion of the T cells (γδ T cells and/or αβ T cells) comprise one or more polynucleotides provided herein. In some embodiments,
a cell population as described herein comprises engineered T cells and other engineered or non- engineered immune system cells. In some exemplary embodiments, the cell population comprises at least 5% to 10%, or 10% to 20%, or 20% to 30%, or 30% to 40%, or 40% to 50%, or 50% to 60%, or 60% to 70%, or 70% to 80%, or 80% to 90%, or 90% to 100% of the engineered cells provided herein. VII. Composition Also provided herein are compositions that comprise any of the nucleic acids, vectors, polypeptides, proteins, receptors, CARs, sets of nucleic acids, sets of vectors, or mammalian cells described herein. In another aspect of the invention, provided herein is a composition that includes any of the nucleic acids or sets of nucleic acids described herein, or any of the vectors or sets of vectors provided herein, polypeptides encoded by the polynucleotides and/or vectors; cells (or populations of cells) comprising the polynucleotides and/or vectors; or cells expressing the polypeptides, either individually or in any combination(s). In some embodiments, a composition can be any of the mammalian cells, preferably T cells, described herein (e.g., any of the mammalian cells described herein previously obtained from a subject, e.g., a subject identified or diagnosed as having a cancer) comprising a nucleic acid encoding any of the chimeric transmembrane proteins and/or any of the chimeric antigen receptors described herein. In a composition including any of the CAR-T cells described herein, the composition may further include a cell culture medium. The compositions may also include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range. The compositions may also include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate. In some embodiments, the composition is a pharmaceutical composition which further include a pharmaceutically acceptable solvent, carrier or buffer (e.g., phosphate-buffered saline). The compositions can also include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers, excipients, diluents, fillers, salts, buffers, detergents (e.g., a nonionic detergent, such as Tween-20 or Tween- 80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and/or other materials suitable for inclusion in a pharmaceutical composition, and which are vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected to not affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer’s solutions, dextrose solution, and Hank’s solution. In addition, the pharmaceutical composition or formulation may
also include other carriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers and the like. Examples of suitable aqueous and non-aqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethyl cellulose colloidal solutions, tragacanth gum and injectable organic esters, such as ethyl oleate, and/or various buffers. Other carriers are well-known in the pharmaceutical arts. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the present invention is contemplated. The compositions may also include large, slowly metabolized macromolecules, such as proteins, polysaccharides like chitosan, polylactic acids, polyglycolic acids and copolymers (e.g., latex functionalized sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Suitability for carriers and other components of pharmaceutical compositions is determined based on the lack of significant negative impact on the desired biological properties of the active compound or CAR-T cells of the present invention (e.g., less than a substantial impact (e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.) on target cancer cell killing). The pharmaceutical compositions of the present invention may also comprise pharmaceutically acceptable antioxidants for instance (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. The pharmaceutical compositions of the present invention may also comprise isotonicity agents, such as sugars, polyalcohols, such as mannitol, sorbitol, glycerol or sodium chloride in the compositions. The pharmaceutical compositions of the present invention may also contain one or more adjuvants appropriate for the chosen route of administration such as preservatives, wetting agents, emulsifying agents, dispersing agents, preservatives or buffers, which may enhance the shelf life or effectiveness of the pharmaceutical composition. The antibodies of the present invention may be prepared with carriers that will protect the antibodies against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with a wax, or other materials well-known in the art. Methods for the preparation of such
formulations are generally known to those skilled in the art. See, e.g., SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In some instances, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. A summary of pharmaceutical compositions described herein is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). The pharmaceutical compositions described herein can be administered by any suitable administration route, including but not limited to, parenteral (e.g., intravenous, intratumoral, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, oral, or rectal administration routes. In some instances, the pharmaceutical composition is formulated for parenteral (e.g., intravenous, intratumoral, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial) administration. A composition that comprises any of the CAR-T cells as described herein may be preferably formulated for intravenous or intraarterial administration. In some preferred embodiments, the composition is administered by via intravenous route. For parenteral administration, agents of the present invention are typically formulated as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water, oil, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin. Peanut oil, soybean oil, and mineral oil are all examples of useful materials. In general, glycols, such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for injectable solutions. Agents of the invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time. Administration can also be by surgical deposition of a bolus or pellet of cells or positioning of a medical device. The pharmaceutical compositions described herein may be formulated into any suitable dosage form, including but not limited to, aqueous dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for administration to a subject to be treated. In some embodiments, the pharmaceutical compositions are formulated into solutions (for example, for IV administration). In some cases, the
pharmaceutical composition is formulated as an infusion. In some cases, the pharmaceutical composition is formulated as an injection. Pharmaceutical compositions for injection must typically be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to achieve high drug concentration. The carrier may be an aqueous or non-aqueous solvent or dispersion medium containing for instance water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin. Sterile injectable solutions may be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients e.g. as enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, examples of methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. A therapeutically effective amount of a composition of the disclosure can be administered to a subject. A “therapeutically effective amount” can refer to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the inventive nucleic acid sequences to elicit a desired response in the individual. In the context of this aspect, cells (e.g. the CAR-T cells according to the present invention comprised in the composition) administered to a subject in need thereof can be autologous to the subject. Cells administered to a subject in need thereof can be allogeneic to the subject, for example, fully HLA- matched, HLA matched at 1, 2, 3, 4, 5, 6, 7, or 8 HLA alleles, or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 HLA alleles. Cells administered to a subject in need thereof can be haploidentical to the subject. Cells administered to a subject in need thereof can be from a donor that is related to the subject. Cells administered to a subject in need thereof can be from a donor that is not related to the subject. In some embodiments, pharmaceutical compositions include a vector for introduction of a polynucleotide into cells in vitro, ex vivo, or in vivo. The polynucleotide compositions can result in the generation of a polypeptide that said polypeptide encodes for (e.g. a CAR comprising IL7Ra signaling domain) in the cells (e.g. T cells) within at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6
hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, 60 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days of administration of the composition to the cells. The CAR-T cells composition, when administered to the subject in need thereof, may result in the persistent generation of the CAR-T cells of the present invention in the subject for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days. Also provided are kits that include one or more of any of the compositions and pharmaceutical compositions described herein. In some embodiments, a kit can further include instructions for performing any of the methods described herein. VIII. Methods of treating a disease in a subject In a further aspect, the present invention provides a polynucleotide, a vector comprising said polynucleotide, a cell comprising, preferably expressing proteins encoded by the polynucleotide or a population of cells comprising said cell for the manufacture of a medicament or pharmaceutical composition for treating a disease or condition. In other words, there is provided a receptor, a CAR, a nucleic acid, an expression vector or a cell as described above for use as a medicament for treating a disease or a condition. In some embodiments, said receptor, CAR, nucleic acid, expression vector or cell as described above is for use for treating cancer. In some embodiments, said receptor, CAR, nucleic acid, expression vector or cell according to the present invention is administered to a subject with cancer. In some embodiments, the current disclosure also comprises methods of increasing expression or function of a CAR. In some embodiments the current disclosure further encompasses methods of enhancing the biological function of a cell comprising the compositions provided herein. In some embodiments, the present invention further encompasses methods of enhancing the target biological function of a cell population comprising the compositions provided herein. In some embodiments the present invention further encompasses methods of using the compositions provided herein for use in treatment of disease or conditions. As used herein, the term "subject" may include a mammal or a human e.g., humans, other primates, pigs, rodents, such as mice and rats, rabbits, guinea pigs, hamsters, horses, cows, cats, dogs, sheep, chickens and goats. Human and veterinary applications are anticipated by the present disclosure. Both pediatric and adult subjects are included. For example, in any of the methods described herein, the subject can be at least 6 months old (e.g., 6 months or older, 12 months or older, 18 months or older,
2 years or older, 4 years or older, 6 years or older, 10 years or older, 13 years or older, 16 years or older, 18 years or older, 21 years or older, 25 years or older, 30 years or older, 35 years or older, 40 years or older, 45 years or older, 50 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, 85 years or older, 90 years or older, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16 ,18, 20, 21, 24, 25, 27, 28, 30, 33, 35, 37, 39, 40, 42, 44, 45, 48, 50, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, or more years old). In some embodiments, the subject is suffering from a cancer. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the receptors, preferably CARs of the disclosure include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included. In one embodiment, the cancer to be treated in a solid tumor, e.g., a solid tumor described herein. In some embodiments, the present invention provides for a method of increasing one or more target biological outcome of an engineered immune cells (e.g. a CAR-T cells comprising a IL7Ra signaling domain of the present invention). The one or more target biological outcome includes but not limited to the cytotoxicity, antitumor activity, tumor cell killing capability, proliferation, expansion, survival, and persistence of the engineered immune cells. In this context, said method comprising introducing the polynucleotide or vectors provided herein to an immune cell, preferably a T cell. In some embodiments, the present invention also encompasses methods of making engineered cells (e.g. CAR-T cells comprising a IL7Ra signaling domain of the present invention) disclosed herein, using the polynucleotides and vectors provided herein. Cells can be obtained from any suitable source for the generation of engineered cells. Cells can be primary cells. Cells can be recombinant cells. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Cells can be derived from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. Cells can also be obtained from a cell therapy bank. Cells can also be obtained from whole food, apheresis, or a tumor sample of a subject. A cell can be a tumor infiltrating lymphocytes (TIL). In some cases, an apheresis can be a leukapheresis. A desirable cell population can also be selected prior to modification. A selection can include at least one of: magnetic separation, flow cytometric selection, antibiotic selection. The one or more cells can be any blood cells, such as peripheral blood mononuclear cell (PBMC), lymphocytes, monocytes or macrophages. The one or more cells can be any immune cells such as a lymphocyte, a T cell, immature thymocyte, a peripheral blood lymphocyte, a helper T cell, a naive T cell, a pluripotent TH cell precursor, a lymphoid progenitor cell, a memory T cell, a TH17 cell, a TH22 cell, a TH9 cell, a TH2 cell, a TH1
cell, a TH3 cell, a regulatory T cell (Treg cell), a tumor-infiltrating T cell, and double negative T-cells, an alpha-beta T cell, a gamma-delta T cell, a Jurkat cell, CD4+ T cell, CD8+ T cell, a T effector cell, a lymphocyte, a B cell, an NK cell, an NKT cell, a myeloid cell, a monocyte, a macrophage, or a neutrophil. In the context of the present invention, the cells can be cultured, expanded and activated using methods known in the art. For instance, conditions appropriate for T cell culture can include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640, TexMACS (Miltenyi) or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum. In some cases, serum- free or cytokine-free medium is used. Cells may be maintained under conditions necessary to support growth; for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). Methods of making engineered cells may comprise stimulation, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) sometimes in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule can be used. In some cases, a population of T cells can be CD3-CD28 co- stimulated, for example, contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions that can stimulate proliferation of the T cells. T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005. As an example, T cells may be activated using the T Cell TransAct Kit (Miltenyi Biotec). Methods of making engineered cells can comprise the use of a vector described herein to introduce a polynucleotide described herein. A variety of enzymes can catalyze insertion of foreign DNA into a host genome. Non-limiting examples of gene editing tools and techniques include CRISPR, TALEN, zinc finger nuclease (ZFN), meganuclease, Mega-TAL, and transposon-based systems. A CRISPR system can be utilized to facilitate insertion of a polynucleotide sequence encoding a membrane protein or a component thereof into a cell genome. For example, a CRISPR system can introduce a double stranded break at a target site in a genome. There are at least five types of CRISPR systems which all incorporate RNAs and CRISPR-associated proteins (Cas). Types I, III, and IV assemble a multi-Cas protein complex that is capable of cleaving nucleic acids that are complementary to the crRNA. Types I and III both require pre-crRNA processing prior to assembling the processed crRNA into the multi-Cas protein complex. Types II and V CRISPR systems comprise a single Cas protein complexed with at least one guiding RNA. A transposon-based system maybe be utilized for insertion of a polynucleotide or a component thereof into a genome.
Methods to introduce gene editing components into a cell include, but are not limited to, electroporation, sonoporation, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viral vectors. Viral vector delivery systems can include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. Examples of viral vectors include, but are not limited to, retroviral vectors, lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirus vectors and adeno-associated virus (AAV) vectors, helper-dependent adenovirus vectors, hybrid adenovirus vectors, Epstein-Bar virus vectors, herpes simplex virus vectors, hemagglutinating virus of Japan (HVJ) vectors, and Moloney murine leukemia virus vectors. In some embodiments, the engineered cells of the present invention used in the method as described herein exhibit enhanced expression and functionality of CAR when said CAR is according to the invention as described above. Methods of assessing the level of expression of CAR are well known in the art. Non-limiting examples of suitable assays are western blotting, FACS, florescence imaging, or ELISA. In some aspects an engineered cell may exhibit at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least-100 fold, or at least-1000 fold enhanced expression of CAR compared to a cell expressing the CAR alone without said IL7Ra signalling domain of the present invention. This increase may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the engineered cells of the present invention used in the method as described herein exhibit one or more enhanced target biological outcome in comparison to a cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain. The one or more target biological outcome includes but is not limited to: cytotoxicity, antitumor activity, tumor cell killing capability, proliferation, expansion, survival, and persistence of the engineered immune cells. Methods of assessing target biological outcome differ based on the aspects being considered and are well known in the art. For instance, if the desired biological functionality is enhanced in vitro cytotoxicity, methods include in vitro rechallenge assay, NFAT-reporter system 51Cr-release assay, bioluminescent assays, cell-based flow cytometry assay, cytokine release assay, tumor killing assays etc. In some embodiments, an engineered cell may exhibit at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 enhanced cytotoxicity compared to a cell that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain. This increase may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the engineered cells of the present invention used in the method as described herein exhibit least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold enhanced/improved cell survival (e.g., as compared to the cell survival in a control subject or a control population of subjects having the same cancer and administered engineered cells that expresses a CAR identical to the CAR of the invention
except that it does not comprise the IL7Ra signaling domain). This increase may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the engineered cells of the present invention used in the method as described herein exhibit least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold reduced cell exhaustion (e.g., as compared to the cell exhaustion in a control subject or a control population of subjects having the same cancer and administered a different engineered cells that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain. This increase may be assessed by comparison with other controls as earlier defined herein. Cell exhaustion may can be evaluated by the expression level of transcription factors (TCF-1, T-bet, EOMES, BATF, NFAT, PRDM1, NR4A, TOX, Foxo1, Zeb2, Id3), or inhibitory receptors (PD1, LAG-3, CD244 (2B4), CD160, TIM-3, CTLA-4, SLAMF6), or reduced or complete loss of of IL-2 ,TNF, IFN-γ production, or altered metabolic activity (i.e. reduced glucose uptake, switch to fatty acid oxidation and loss of mitochondrial mass and function), measured using techniques known to a skilled person. In some embodiments, the engineered cells of the present invention used in the method as described herein exhibit least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2-fold, at least 3-fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 50 fold, at least 100 fold, or at least 1000 fold enhanced or improved sensitivity (e.g., as compared to the cell sensitivity towards target cells with low antigen density on the surface in a control subject or a control population of subjects having the same cancer and administered a different engineered cells that expresses a CAR identical to the CAR of the invention except that it does not comprise the IL7Ra signaling domain or the STAT4 binding site in said CAR). “Low antigen density” may mean that the antigen density on the target cells is least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than a control target cell expressing the same antigen. This increase may be assessed by comparison with other controls as earlier defined herein. Cell sensitivity may can be evaluated by techniques known to a skilled person, for example, cytotoxicity assays using flow cytometry, cytokine release assays, assessing T cell activation markers, proliferation assays, luciferase-based killing assays. Increased proliferation can be determined by measuring the incorporation of either tritiated thymidine or orotic acid to measure DNA synthesis following ligand binding to the CAR-expressing cells disclosed herein. The incorporation of bromodeoxyuridine into newly synthesized DNA can be measured by immunological staining and the detection of dyes, or by ELISA (Enzyme-linked immunosorbent assay) (Doyle et al., Cell and Tissue Culture: Laboratory Procedures, Wiley, Chichester, England, (1994)). The mitotic index of cells can be determined by staining and microscopy, by the fraction labeled mitoses method or by fluorescence activated cell sorting (FACS) analysis. The increase in cell size which accompanies progress through the cell cycle can be measured by centrifugal elutriation (Faha et al., J. Virol.67:2456- 2465, 1993). Increases in the number of cells may also be measured by counting the cells, with or without the addition of vital dyes. In addition, signal transduction can be measured by the detection of
phosphotyrosine, the in vitro activity of tyrosine kinases from activated cells, c-myc induction, or calcium mobilization. Cell survival can be measured by flow cytometry using antibodies against cell surface antigens. One way of assessing T cell activation is the production of cytokines. In some embodiments, CD28 co- stimulation increases cytokine production by increasing transcription of cytokine genes and stabilizing cytokine mRNAs. In other embodiments, CD8+ T cells expressing the CARs disclosed herein have a greater capacity for cytokine production. Specific, non-limiting examples of cytokines include IL-2, IL-4, and γ-IFN. CAR T-cell activation may also be assessed using a NFAT-reporter system, which is a rapid and easily standardized method for evaluating the functional activity of different receptor designs. Animal models can be used to assess in vivo activity of the T-cells. Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models. In some embodiments, any assay including assessment of tumor size, shrinkage, tumor marker assays, metastasis assays can be used. In some embodiments, the engineered T-cells (e.g. the CAR-T cells as provided herein) are at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, at least 100% more effective in inducing a positive biological response to tumors than T-cells that do not express the CAR of the present invention. Method for treatment and use Also provided herein are methods of treating a disease in a subject, said method comprising administering a therapeutically effective amount of any one of CAR, a nucleic acid, an expression vector or a cell as described above. The subject may be a mammalian subject (e.g., a human, a mouse, a rabbit, a rat, a horse, a dog, a monkey, or an ape). In some embodiment, the subject is a human subject. In some embodiment the human subject has a cancer. Accordingly, in a further aspect, the present invention provides a receptor, a CAR, a nucleic acid, an expression vector or a cell as described above for use as a medicament, preferably for treating cancer. In some embodiments, the cell is a T cell (e.g., a CD8+ T cell, a CD4+ T cell, a memory T cell, a Treg cell, and a natural killer T cell). In some examples, the T cell is a T cell previously obtained from a subject (e.g., a subject that has been identified or diagnosed as having a cancer, e.g., any of the cancers described herein). Some embodiments of these methods further include obtaining the T cell from the subject.In some embodiments, the disease is cancer. Non-limiting examples of cancer that can be treated using any of the methods provided herein include: hepatocellular carcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma, lymphoma, anal cancer, appendix cancer, teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, bronchial tumor, carcinoid tumor, cardiac tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloproliferative neoplasm, colon cancer, colorectal cancer, craniopharyngioma, bile duct cancer, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, eye cancer, fallopian tube
cancer, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, germ cell tumor, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, hypophamgeal cancer, pancreatic cancer, kidney cancer, laryngeal cancer, chronic myelogenous leukemia, lip and oral cavity cancer, lung cancer, melanoma, Merkel cell carcinoma, mesothelioma, mouth cancer, oral cancer, osteosarcoma, ovarian cancer, penile cancer, pharyngeal cancer, prostate cancer, rectal cancer, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, gastric cancer, testicular cancer, throat cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer. Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hod’kin's disease, non-Hod’kin's lymphoma (indolent and high grade forms), multiple myeloma, Waldens’rom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia. Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, E’ing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, W’lms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases). In some embodiments, the subject has cancer and has been pretreated. In this context, such subjects may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower number of T-cells due to the pre-treatment (such as toxic chemotherapy) than a healthy subject or a subject having the same/similar disease but who has not been pretreated. The CAR-T cells of the invention may therefore be used as a second or further line of treatment of a cancer patient. The first line of treatment may be chemotherapy.
In some embodiments, the cancer/tumor cells from the subject to be treated may express multiple immune checkpoint ligands (such as PD-L1, PD-L2, CD86 and GAL9). In this context, the effectiveness of a CAR-T, which does not comprise a CAR of the present invention, mediated cytotoxicity may be hampered by the multiple immune checkpoint ligands in said subjects. In some embodiments, the subject is immunocompromised, and/or shows signs of T-cell exhaustion, preferably wherein the IL7Ra signalling domain comprises the P300H, P300A, P300W, P300E, P300L, or P300Q mutation when referring to the human wild type IL7Ra (i.e. SEQ ID NO:1), more preferably the IL7Ra signalling domain comprises the P300H mutation. The number of T-cells may be measured using known techniques to the skilled person. An immunocompromised subject or a subject with T-cell exhaustion may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% lower number of T-cells than a healthy subject or a subject having the same/similar cancer but is not immunocompromised or does not show T-cell exhaustion. In some embodiments of any of these methods, the methods result in a decrease in the number of cancer/tumor cells in a subject. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g., about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) reduction in the cancer/tumor cells in a subject (e.g., as compared to the cancer/tumor cells in the subject prior to treatment). In some embodiments of any of these methods, the methods result in a decrease in the tumor burden (e.g., a decrease in tumor mass and/or volume of a solid tumor) in a subject. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g., about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) reduction in the tumor burden in a subject (e.g., as compared to the tumor burden in the subject prior to treatment). In some embodiments, the methods result in a decrease in the rate of progression of a cancer in the subject. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g., about 99%, about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) reduction in the rate of progression of a cancer in a subject (e.g., as compared to the rate of progression of a cancer in the subject prior to treatment or in a control subject or a control population of subjects having the same cancer and administered no treatment or a different treatment).
In some embodiments of any of these methods, the methods result in an increase of the time of survival of a subject having cancer. For example, any of the methods described herein can result in an about 1% to about 800% (e.g. about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, about 20%, about 10%, or about 5% (inclusive); about 5% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, about 20%, or about 10% (inclusive); about 10% to about 800%, about 750%, about 700%, about 650%, about 600%, about 550%, about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, about 100%, about 80%, about 60%, about 40%, or about 20%) increase in the time of survival of a subject (e.g., as compared to the time of survival for a control subject or a population of control subjects having the same cancer and receiving no treatment or a different treatment). Also provided herein are methods of inducing cell death in a cancer cell of a subject in need thereof that include administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. Also provided herein are methods of decreasing the risk of developing metastasis in a subject having a cancer that include administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g. about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) decrease in the risk of developing metastasis in the subject (e.g., as compared to the risk of developing metastasis in a control subject or a control population of subjects having the same cancer and administered no treatment or a different treatment). Also provided herein are methods of increasing the cell survival of an engineered cell, preferably a CAR-T cell, in a subject having a cancer that includes administering to the subject a therapeutically effective amount of any of the engineered cells described herein. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g. about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) increase of survival of the engineered cell in the subject (e.g., as compared to the cell survival in a control subject or a control population of subjects having the same cancer and administered with similar engineered CAR-T cells which does not express the IL7Ra signaling domain of the present invention). This increase may be assessed by comparison with other controls as earlier defined herein.
Also provided herein are methods of decreasing the exhaustion of an engineered cell, preferably a CAR-T cell, in a subject having a cancer that include administering to the subject a therapeutically effective amount of any of the engineered cells described herein. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g. about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) decrease of exhaustion of the engineered cell in the subject (e.g., as compared to the cell exhaustion in a control subject or a control population of subjects having the same cancer and administered with similar engineered CAR-T cells which does not express the IL7Ra signaling domain of the present invention). This increase may be assessed by comparison with other controls as earlier defined herein. Also provided herein are methods of decreasing the risk of systemic toxicity in a subject having a cancer that include administering to the subject a therapeutically effective amount of any of the mammalian cells described herein. For example, any of the methods described herein can result in at least about 1% to about 99% (e.g. about 98%, about 96%, about 94%, about 92%, about 90%, about 88%, about 86%, about 84%, about 82%, about 80%, about 78%, about 76%, about 74%, about 72%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%) decrease in the risk of developing a systemic toxicity in the subject (e.g., as compared to the risk of developing a systemic toxicity in a control subject or a control population of subjects having the same cancer and administered no treatment or a different treatment). In some aspect, the current disclosure encompasses use of the methods presented herein to combat any cancer for which an effective CAR is known. In some aspect, the current disclosure encompasses use of the methods presented herein to combat any cancer for which a novel effective CAR is developed. For example, the disclosure provides methods of treating cancer by providing to the subject in need thereof immune cells (e.g., T cells) that are engineered to co-express CD19 CAR, e.g., with an anti- CD19 binding domain known or described herein, wherein the cancer cells express CD19. In one embodiment, the cancer to be treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL (Mantle cell lymphoma, or MM (multiple myeloma). In some embodiments, the engineered cells as disclosed herein which expresses the CAR according to the present invention as described herein, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage used for administering a cell, or composition of cells expressing the CAR without said IL7Ra signaling domain. This change of dose may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the dosage may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% lower that the recommended dosage for cells expressing CAR without said IL7Ra signaling domain for the same patient.
In some embodiments, the engineered cells as disclosed herein which expresses the CAR of the invention as described herein, can be administered in a frequency that is higher, lower or the same than the frequency used for administering a cell, or composition of cells expressing the CAR without said IL7Ra signaling domain. This frequency of administration may be assessed by comparison with other controls as earlier defined herein. In some embodiments, the frequency may be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% lower that the recommended frequency for cells expressing CAR without said IL7Ra signaling domain for the same patient. In further aspects, a CAR-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, checkpoint inhibitors such as pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo) and atezolizumab (Tecentriq), peptide vaccine, or any combination thereof. Cells administered to a subject in need thereof can be autologous to the subject. Cells administered to a subject in need thereof can be allogeneic to the subject, for example, fully HLA-matched, HLA matched at 1, 2, 3, 4, 5, 6, 7, or 8 HLA alleles, or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 HLA alleles. Cells administered to a subject in need thereof can be haploidentical to the subject. Cells administered to a subject in need thereof can be from a donor that is related to the subject. Cells administered to a subject in need thereof can be from a donor that is not related to the subject. In certain aspects, cryopreserved cells (e.g., engineered cells) are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure. In an aspect, a composition comprising an engineered cell can include a dosage form of a cell, e.g., a unit dosage form. EXAMPLE 1 The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. I. MATERIALS AND METHODS CAR T cell production Peripheral blood mononuclear cells were obtained from healthy donors by density gradient centrifugation using Lymphocyte Separation Medium, Density 1077 g/ml (Capricorn Scientific). Total
CD4+ and CD8+ cells subsets were separately isolated using the CD4+ the CD8+ T Cell Isolation Kits (Miltenyi Biotec). The T cells were activated using the T Cell TransAct Kit (Miltenyi Biotec). The complete culture media for T cells contained 90% RPMI-1640 (Capricorn Scientific), 10% heat-inactivated FBS (Gibco), 10 ng/ml IL-15, and 10 ng/ml IL-7 (Miltenyi Biotec). Cell culture media was replenished every 48–72 hours. T cells were transduced (MOI 5) 48 hours post-activation using lentiviral vectors coding for the corresponding CAR construct and a truncated version of the human EGFR protein as a reporter gene. After 12 days, CAR T-cells were analyzed for transduction efficiency using an anti-human EGFR antibody (R&D Systems, Cetuximab biosimilar) and collected for subsequent use. Cell lines NALM6 (American Type Culture Collection; CRL-3273) and K562 (American Type Culture Collection; CCL-243) were subcultured according to ATCC recommendations. К562.CD19 is a derivate of the K562 cell line and was generated by stable transduction with lentivirus (LV) vector coding for CD19 protein expression cassette. К562.CD19.CPL+ cell line is a derivate of К562.CD19 generated by a stable transduction with LV’s coding for PDL1, PDL2, CD86 and GAL9 proteins expression cassettes. Flow cytometry analysis The following antibodies were used for surface antigen expression analysis by flow cytometry (Beckman Coulter, CytoFlex): CD4 FITC (Miltenyi Biotec, clone REA623), CD8 FITC (Miltenyi Biotec, clone REA734), anti-human EGFR AlexaFluor488 (R&D Systems, Cetuximab biosimilar), PDL1 PE (Miltenyi Biotec, clone REA1197), Galectin-9 PE (Miltenyi Biotec, clone REA435), PDL2 PE (Miltenyi Biotec, clone REA985), CD86 PE/Cy7 (Elabscience, clone BU63). DAPI (Invitrogen) was used as a viability dye. The phosphorylation status of STAT3 and STAT5 proteins in CAR T-cells was evaluated by intracellular flow cytometry analysis. In given case only CD8+ CAR T-cells were analyzed to reduce the influence of auto- and paracrine stimulation by cytokines secreted by the CD4+ CAR-T population upon its activation. Shortly, CD8+ CAR T-cells were rested in a cytokine-free growth medium for 48h and subsequently co-cultivated with K562 or K562.CD19 at an effector-to-target ratio of 1 to 1. After 2h of co-cultivation, the cells were fixed with IC Fixation Buffer (Invitrogen) and permeabilized by ice-cold methanol. The following antibodies were used: anti-pSTAT3 PE (Tyr705) (BD Biosciences, clone 4/P- Stat3), Alexa Fluor 647-anti-pSTAT5 Alexa Fluor 647 (Tyr694) (BD Biosciences, clone 47/Stat5). In vitro cytotoxicity assay Before initiation of the cytotoxicity assay CAR T-cells were rested in cytokine-free growth medium for 48h. After that 2 x 104 CAR T-cells were co-cultivated with K562.CD19.CPL+ or NALM6 cells in cytokine-free growth medium at an effector to target ratios 1:1, 1:5, 1:10. At 144h post-initiation of co- cultivation, CAR T-cells and target cells expansion and viability were evaluated by flow cytometry. In vitro rechallenge assay
Before initiation of the rechallenge assay CAR T-cells were rested in cytokine-free growth medium for 48h. After that, 2 x 104 CAR T-cells were cocultivated with 2 x 104 K562.CD19 or K562.CD19.CPL+ cells in cytokine-free growth medium. CAR T-cells and target cells expansion and viability were evaluated by flow cytometry every 72h. A rechallenge of CAR T-cells was performed every 72h by adding 2 x 104 corresponding viable target cells to the co-culture. The rationale behind the truncation strategy The comprehensive three-dimensional conformation of IL7R remains elusive. Presently, only the extracellular segment has been experimentally resolved, with documented structures represented by PDB entries 7OPB (Cao et al., 2022) and 3UP1 (McElroy et al., 2012). Conversely, the transmembrane domain and the intracellular portion lack experimentally derived structures, and their spatial configurations are currently reliant on predictions generated by AlphaFold methodology (referenced at https://www.uniprot.org/uniprotkb/P16871/entry; see Figure 1). The model observation reveals that the intracellular domain of the receptor predominantly exhibits a coiled or disordered structural arrangement. However, a notable discrepancy arises in this model, wherein a substantial portion of the intracellular motif exhibits coordinates proximal to the extracellular domain—a circumstance incompatible with the presence of the cellular membrane. In response to this discrepancy, a corrective measure was undertaken through a minimization process applied to the intracellular domain of the system, yielding a refined and more physiologically plausible predicted structure (see Figure 2). In accordance with the findings presented by (Ferrao & Lupardus, 2017) and (Winer et al., 2022), the Box1 and Box2 regions within IL7R correspond to the VWPSLPDHK (SEQ ID NO:80) and DCQIHRVDDIQARD (SEQ ID NO:81) sequences, respectively. Box1 exhibits a markedly hydrophobic nature, whereas Box2 is characterized by a positive charge and less well-defined coordinates. Notably, these two boxes are spatially delineated by a short linker and an extended non-structured sequence at the C-terminal region. This sequence encompasses a membrane-anchored loop, suggesting its potential involvement as a regulatory element that affixes the intracellular segment of the uneffected receptor to the cellular membrane. According to (Kasai et al., 2018) the optimal functionality of the intracellular segment necessitates the presence of specific structural elements, namely Box1, Box2, and two tyrosine residues (Y449 and Y456) designated as phosphorylation sites. In order to obtain a maximally compact and fully operational intracellular component, the decision was made to retain these critical structural features, along with a spatially differentiating loop denoted as EVEGFLQDTFPQQ (SEQ ID NO:82), situated between Box1 and Box2. This loop is primarily located right after Box 2 sequence on the C-terminus but spatially separates 2 boxes one from another and seems to be an important functional part (see Figure 3). To ensure the preservation of the phosphorylation environment, particularly concerning Y449 as the initial phosphorylation site for STAT docking, it was imperative to maintain the secondary structure
composition of the residue in its native state. Employing the PSIPRED algorithm, a judicious selection was made of the minimal sequence preceding Y449 (E446, E447, A448), thereby conserving the secondary structure composition at this critical site, as illustrated in Figure 4. The final truncated IL7R sequence is presented in bold, while the deleted IL7R region is underlined: >IL7Rtr (SEQ ID NO:5) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQL EESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPH VYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ Thus, truncated IL7R preserves all the necessary for JAK docking and STAT phosphorylation functional elements of IL7R, but has a 60% shorter amino acid sequence which is beneficial for the usage in chimeric antigen receptor signaling cassettes. We also hypothesized that the mutual positioning of Box1 and Box2 is crucial for efficient JAK1 binding and subsequent STAT phosphorylation, so we tried to alter these patterns. According to amino acids’ Ramachandran plots, proline has the most severe restrictions in phi\psi angles due to its pyrrolidine ring, so we mutated proline 300 between Box1 and Box2 to test our hypothesis to histidine (P300H mutation). Native IL7Ra molecule is associated with JAK1 and triggers mainly STAT5 response upon signaling (Wang Y. et al, 2019). Several studies have shown a superiority of its incorporation into CAR-T cells in comparison to second-generation CAR-T cells (Shum T. et al, 2017). But, data of transcriptomic profiling from clinical studies revealed that CAR T cells from complete-responding patients after CD19 CAR-T cells were enriched in STAT3 gene signatures (Fraietta J.A. et al., 2018). Later novel preclinical studies have been able to show the superiority of STAT3 over STAT5-based CAR-T cells in terms of killing capacity, proliferation, stem cell memory CAR T cells maintenance, persistence, and CAR-T cells exhaustion prevention (Wang Y. et al, 2019), (Saxby C.P. et al, 2023). JAK1 has been shown to phosphorylate different STAT-binding sites for subsequent phosphorylation of different STATs (STAT1, STAT3, and STAT5) (Liu K.D. et al., 1997). Taking into account, that STAT-binding sites from one receptor can be replaced with binding sites for different STATs from other receptors and thereby activate nonphysiological STATs (Morris R. et al., 2018), we have introduced the STAT3 binding motif into the backbone of our CAR. CAR-T sequences and schematic designs As a reference for comparison for our 41BB-containing IL7R-containing variants, we chose 2nd gen anti-CD19 CAR-T (FMC63 scFv, IgG4 hinge, CD28 transmembrane domain, 41BB co-stimulatory domain and CD3z signaling) - here and thereafter referred as BB.Z. For CD28-based IL7R CAR we used FMC63-CD28hinge-CD28-CD3z CAR as a reference - 28.Z. We constructed and validated several IL7R-based 5th-generation CAR cassettes (Table 1). Table 1: CAR sequences
SEQ ID Name Sequences NO 9 Leader and antibody MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTIS sequences (the same CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS for all designs) GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKL EITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSV TCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYN SALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYG GSYAMDYWGQGTSVTVSSAAA(EQKLISEEDLGS) 10 BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (2nd generation anti- GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD19 CAR-T with RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG IgG4 hinge, CD28 RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE transmembrane, RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 41BB co-stimulatory and CD3z domains) 11 IL7.BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra KRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQ incorporated IHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPN between CD28TM CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDC and 41BB domains) RESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVA QGQPILTSLGSNQEEAYVTMSSFYQNQAEQKRGRKKLLYIF KQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSAD APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR 12 BB.IL7.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV CD3z domains) QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTL NPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRSGRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALHMQALPPR 13 BB.IL7.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (full-length IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV CD3z domains; QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSR additional STAT3 SLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTL binding motif (YRHQ) NPVAQGQPILTSLGSNQEEAYVTMSSFYQNQRSGRVKFSR at the end of CD3z) SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDAYRHQALPPR 14 BB.IL7tr.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains) QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
BB.IL7tr.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD additional STAT3 KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG binding motif (YRHQ) MKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR at the end of CD3z) BB.IL7tr(mut).Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra) MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR BB.IL7tr(mut).Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDAYRHQALPPR STAT3 binding motif (YRHQ) at the end of CD3z) 28.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (2nd generation anti- LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD19 CAR-T with RPGPTRKHYQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQ CD28 hinge, QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN CD28TM, CD28 co- PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL stimulatory and CD3z STATKDTYDAYRHQALPPR domains; additional STAT3 binding motif (YRHQ) at the end of CD3z)) 28.IL7tr(mut).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains) GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GLSTATKDTYDALHMQALPPR 28.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY additional STAT3 QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK binding motif (YRHQ) NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ at the end of CD3z) GLSTATKDTYDAYRHQALPPR 28.IL7tr(mut)+.Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK
between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGYRHQRVKFSRSA P300H mutation in DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG IL7Ra; additional KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH STAT3 binding motif DGLYQGLSTATKDTYDALHMQALPPR (YRHQ) after IL7Ra) 28.IL7tr(mut)++.Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYRHQRSGRVKFSRSADAPAY P300H mutation in QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK IL7Ra; additional NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ STAT3 binding motif GLSTATKDTYDALHMQALPPR (YRHQ) in IL7Ra (mutations: Q457R; N458H).) 28.IL7tr(mut).Z++ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY P300H mutation in QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK IL7Ra; additional NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ STAT3 binding motif GLSTATKDTYDALHMQALPPRYRHQ (YRHQ) after CD3z.) 28.IL7tr(+14).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE CD3z domains.) GFLQDTFPQQLEESEKQRLLGSNQEEAYVTMSSFYQNQRS GRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 28.IL2d.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (28-ΔIL2RB-z(YXXQ) LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR - truncated IL2RB RPGPTRKHYQPYAPPRDFAAYRSNCRNTGPWLKKVLKCNT containing 5th PDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEIS generation CAR-T PLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRSGRV [Nature Medicine KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD volume 24, PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR pages352–359 RGKGHDGLYQGLSTATKDTYDAYRHQALPPR (2018)]) BB.Z+ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (BB.Z GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (2nd generation anti- RV CD19 CAR-T with KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD IgG4 hinge, CD28 PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR transmembrane, RGKGHDGLYQGLSTATKDTYDAYRHQALPPR 41BB co-stimulatory and CD3z domains; additional STAT3 binding motif (YRHQ) at the end of CD3z)
95 28.BB.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (3rd generation with LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD28, 41BB co- RPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRP stimulatory and CD3z VQTTQEEDGCSCRFPEEEEGGCELRTKKRIKPIVWPSLPDH domains; truncated KKTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE IL7Ra incorporated GFLQDTFPQQEEAYVTMSSFYQNQRSGRVKFSRSADAPAY between CD28 and QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK CD3z domains; NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ P300H mutation in GLSTATKDTYDAYRHQALPPR IL7Ra; additional STAT3 binding motif (YRHQ) after CD3z 96 BB.IL7tr(mut)+.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGYRHQRVKFSRSADAPAYQQGQNQLYNELNLGRREE P300H mutation in YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA IL7Ra; additional YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP STAT3 binding motif PR (YRHQ) after IL7Ra) 97 BB.IL7tr(mut)++.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYRH CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR STAT3 binding motif (YRHQ) in IL7Ra (mutations: Q457R; N458H).) 98 BB.IL7tr(mut).Z++ ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVTRK (Truncated IL7Ra RGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE incorporated LRTKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNHESF between 41BB and LDCQIHRVDDIQARDEVEGFLQDTFPQQEEAYVTMSSFYQN CD3z domains; QRSGRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD P300H mutation in KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG IL7Ra; additional MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRYR STAT3 binding motif HQ (YRHQ) after CD3z.) *Underlined sequences are IL7Ra (and truncated versions) or truncated IL2RB sequences. *Bold letters are P300H mutation (or P36H mutation in SEQ ID NO:2 or 4) or STAT3 binding site YXXQ (SEQ ID NO:6) (YRHQ = SEQ ID NO:8). II. RESULTS 1. Design of the truncated IL7Ra As an initial design, we introduced the IL7Ra signaling domain (amino acids: 265-459, UniProt: P16871) into 2nd gen. CAR under the membrane prior to 4-1BB and CD3z domains with or without additional
STAT3 binding motifs at the C-terminal end of CD3z and performed in vitro rechallenge assay, using K562.CD19 as a tumor model (Figure 5). IL7Rα-based CAR T-cells were not able to control CD19-positive cells and didn't expand at all. We hypothesized that IL7Rα did not provide STAT signaling upon antigen ligation, but abrogated CAR NFAT/NFκB signaling for CD3ζ and 4-1BB. IL7Rα truncation and repositioning next to 4-1BB and CD3ζ could solve the issues, so we designed and assembled a number of alternative variants with IL7Rα located between 4-1BB and CD3ζ, truncated from 194 amino acids in length to 81 amino acids with or without P300H mutation between Box1 and Box2 JAK-binding domains. In order to emphasize the differences between different CAR cassettes, we "armored" the K562.CD19 tumor model cell line with multiple immune checkpoint receptor ligands (PD-L1, PD-L2, CD86, and GAL9). The resulting re-challenge assay results are presented in Figure 6. According to the data, 2nd generation CAR T-cells were not able to expand and control the tumor population, facing multiple immune checkpoint ligands. Contrary to them, IL7Rα(mut) had a 4-fold peak expansion during the 3rd re-challenge assay and effectively controlled the immunosuppressive tumor cell line. It was previously shown that CCR7 (an early differentiation status marker) expression on the surface of CAR T-cell products is a favorable marker for the success of adoptive T-cell immunotherapy (https://doi.org/10.1186/s40425-017-0216-7). However, for many patients, it is not possible to collect a significant portion of CCR7high+ T-cells for CAR-T manufacturing. We intentionally generated 5th generation CAR T-cells from two donors (donor 1 - CCR7high and donor 2 - CCR7low) (Figure 7) to elucidate whether IL7Rα signaling in our design could further enhance CAR T-cell potency. Six rounds of repetitive stimulation of CAR T-cells with K562.CD19.CPL cells revealed excellent expansion potential (up to 50 times) of BB.IL7tr(mut).Z+ and BB.IL7tr(mut).Z CAR T-cells. They significantly outperformed all other designs for CCR7high starting material (Figure 8A) and for CCR7low starting material (Figure 8B) in terms of expansion potential and tumor control (Figure 8C for CCR7high and Figure 8D for CCR7low). High tumor burden is a significant adverse prognostic factor that negatively impacts progression-free survival and overall outcomes in patients with various lymphomas. To simulate and study the effects of high tumor burden conditions in a controlled laboratory setting, we performed an in vitro cytotoxicity assay spanning 6 days. This experiment aimed to evaluate the ability of different CAR T-cell designs to eliminate target cells under conditions mimicking a high tumor burden scenario. Specifically, we used an effector-to-target (E:T) ratio of 1:5, which represents a high tumor burden situation where the number of target cells (tumor cells) outnumbers the effector cells (CAR T-cells) by a factor of 5. This challenging ratio tests the potency and efficacy of the CAR T-cell designs in an unfavorable setting with a relatively low number of effector cells compared to the tumor cell population.
The cytotoxicity assay was performed separately for two different starting T-cell populations: CCR7high T-cell material (Figure 9A) and CCR7low T-cell material (Figure 9B). CCR7 is a marker of early T-cell differentiation status, and its expression level can impact the functionality and persistence of CAR T- cells. By evaluating the performance of the CAR designs using both CCR7high and CCR7low starting populations, we aimed to investigate the potential influence of the T-cell differentiation status on the ability to control high tumor burden situations. This experimental setup allowed us to compare the cytotoxic potency and tumor control capabilities of different CAR T-cell designs under simulated high tumor burden conditions, providing valuable insights into their potential efficacy in challenging clinical scenarios characterized by a high cancer cell load. It was revealed that in CCR7high CAR-T cells all CAR variants that contain IL7 with the P300H mutation were able to expand and eradicate a high amount of the tumor cells, contrary to CCR7low CAR T-cells. In that case (figure 9B) only BB.IL7tr(mut).Z (SEQ ID NO: 16) and BB.IL7tr(mut).Z+ (SEQ ID NO: 17) were expanded during co-cultivation and controlled tumor population emphasizing the importance of IL7Ra truncation for CAR-T performance. The inclusion of the IL7tr(mut) domain into the cytoplasmic moiety of the CAR improves the antitumor activity and expansion potential of T-cells, which is particularly critical for heavily pretreated patients. It is well-known that in such patients, T-cells are present in low numbers and are significantly exhausted due to toxic chemotherapy (Mark Leick et al. 2019), which poses a significant portion of patients for failure to obtain a desirable amount of CAR-T cells. Our CAR design incorporates the IL7tr domain to provide pro-survival signals and is able to maintain the T-cell population, preventing terminal differentiation and activation-induced cell death. This mechanism helps to control tumors at lower E:T, which is not seen in 2-generation CAR-T cells. This means that we can use a lower number of T-cells even with a bad immunophenotype and subpopulation composition (adverse risk factor) in the starting material to obtain an efficient CAR T-cell product (Cytotherapy.2022 Sep;24(9):869-87). To further enhance the potency of IL7tr(mut) domain incorporation into the CAR cassette, we assembled a CD28 co-stimulatory domain-containing CAR. Well-known limitations of CD28-based CAR-T cells include AICD and early exhaustion, resulting in a median persistence of only one month according to clinical trials. B-ALL, CLL, and mantle cell lymphoma are B-cell neoplasms where CAR-T cell persistence plays a significant role. Fixing these issues with our truncated IL7R could significantly enhance the clinical efficacy of CD28-based CAR-T cell therapy. We also included a state-of-art 5th generation CAR with JAK–STAT signaling domain 28-ΔIL2RB- z(YXXQ) ((SEQ ID NO: 25)) in the re-challenge and 6-day tumor challenge experiments (Nature Medicine volume 24, pages 352–359 (2018)) as 5th generation CAR-T reference. As the antigen-bearing cell line harbors multiple immune checkpoint ligands, preventing effective CAR- T-mediated cytotoxicity, most of the CAR designs were not able to control K562.CD19.CPL+ growth during 6 rechallenge cycles, except 28.IL7(tr).mut.Z (SEQ ID NO:19) and 28.IL7(tr).mut.Z+ (SEQ ID NO: 20) variants that completely outperform 28.IL2d.Z+ competitor not to mention 2nd generations
BB.Z and 28.Z. CAR-T expansion fold was the highest for CD28-based 5th generation CAR T variants with 28.IL7(tr).mut.Z and 28.IL7(tr).mut.Z+ leading the chart. This data allows us to suggest the higher expansion and persistence potential for these CAR designs even for the environment rich for immune checkpoint ligands. 2. Assessment of the properties and activities of the CAR-T cells To evaluate CAR-T cytotoxic capacity in high tumor burden conditions we performed a 6-day long co- cultivation assay at three effector to target (E:T) ratios (CAR+ T cells to tumor cells), ranging from 1:1 up to 1 to 10 (Figure 11) against Nalm-6 (Figure 11A) and K562.CD19.CPL+ immunosuppressive cell lines (Figure 11B). Not surprisingly all CAR T designs were able to eradicate Nalm-6 at 1:1 E:T ratio, but only 28.IL7tr(mut).Z+ controlled Nalm-6 population at 1:10 E:T ratio preventing any Nalm-6 growth. Immunosuppressive K562.CD19.CPL+ cells’ growth was also totally controlled only by 28.IL7tr(mut).Z+ CAR T product for up to 1:5 E:T ratio. Initial assessment of CAR T-cell activation using the NFAT-reporter system is a rapid and easily standardized method for evaluating the functional activity of different receptor designs. The data obtained using this approach allow us to semi-quantitatively assess the intensity of T-cell activation and make preliminary conclusions about the sensitivity of the studied CAR designs to different densities of antigen molecules on the surface of target cells. By using the Jurkat reporter cell line with the NFAT-controlled GFP expression, we were able to compare the activation strength of different CAR designs on CD19-positive cell lines with the different antigen expression levels (from 9813 MESF for CII to 151560 MESF for K562.CD19). It was revealed that CD28-based CAR possesses a higher magnitude of activation that 4-1BB-based CARs which was expected and is in good agreement with the literature data. Activation of CAR T-cells was equivalent to K562.CD19 with a high density of antigen and CII, with only 9813 CD19 molecules on its surface. Surprisingly, IL7tr incorporation between CD3z and cell's inner membrane and CD3z and the resulting distancing didn’t diminish CD3z activation level, unlike additional STAT3 domain incorporation at the end of CD3z domain for IL7-based 5th generation CAR-T. However additional STAT3 domain at CD3z of 28.IL2Rb.Z+ didn’t alter its activation level in comparison with the 28.Z CAR. To achieve an optimal magnitude of activation and preserve expansion and persistence potential of IL7tr(mut)-based CAR, we designed alternative versions with additional STAT3 binding domain at different positions (28.IL7tr(mut)+.z (SEQ ID NO:21), 28.IL7tr(mut)++.z(SEQ ID NO:22), 28.IL7tr(mut).z++(SEQ ID NO:23)). Here we compared the dynamics of STATs activation-induced phosphorylation in our lead designs (28.IL7tr(mut).Z and 28.IL7tr(mut).Z+) with a highly similar design (28.IL7tr(+14).Z (SEQ ID NO:24)) and 28.IL2d.Z+. As you can see, our peak values of pSTAT3 and pSTAT5 are higher for our designs. Surprisingly, 28.IL7tr(mut).Z without any additional STAT-binding domains showed a similar level of
STAT3 phosphorylation as 28.IL2d.Z+, that contains extra STAT3-binding motif at CD3z. 28.IL7tr(+14).Z from Patent EP4190807 exhibited a lowest level of pSTAT3\pSTAT5. We also compared variants with the different positions of an extra STAT3-binding motif and with 3rd generation 28.41BB-based CAR-T harboring our IL7tr(mut) domain in re-challenge assay (SEQ ID: 21, 22, 23). Before initiation of the rechallenge assay CAR T-cells were rested in cytokine-free growth medium for 48h. After that, at the first round of rechallenge 0.5 x 104 CAR T-cells were cocultivated with 2.5 x 104 targets cells in a cytokine-free growth medium for 144h. At next rounds rechallenge was performed by 0.5 x 104 K562.CD19.CPL+ or 1.5 x 104 Nalm6 cells correspondingly. CAR T-cells expansion and target cells persistence were evaluated by flow cytometry before every next round of rechallenge and at the final point. Calculation of AUC (area under the curve) was performed using the trapezoidal rule (Figure 15). It was revealed that there is indeed a correlation between CAR-T expansion potential and its ability to control tumor cell population during multiple rounds of CAR-T incubation with target cell lines. IL7tr(mut) based variants: 28.BB.ILtr(mut).Z+ (SEQ ID NO:95), 28.IL7tr(mut).Z(SEQ ID NO:19), 28.IL7tr(mut).Z+(SEQ ID NO:20) and 28.IL7tr(mut)++.Z(SEQ ID NO:22) were able to control Nalm6 and K562.CD19.CPL+ more effectively than 28.IL7tr(+14).Z(SEQ ID NO:24) and 28.IL2d.Z+(SEQ ID NO:25) and expanded more significantly. According to pSTAT3\pSTAT5 measurement data - BB.IL7tr(mut).Z+ (SEQ ID NO:17) CAR showed a significant upregulation of pSTAT3 and pSTAT5 in comparison with 2nd generation BB.Z CAR-T both in CD4+ and CD8+ T-cells (figure 13). It confirmed our initial hypothesis that such a design could promote STAT3\STAT5 phosphorylation in an antigen-dependent manner thus resulting in a full T-cell activation (signals 1-3) upon antigen engagement. The elevation levels of pSTAT3/pSTAT5 resemble native cytokines stimulation, but only upon CAR-T activation without systemic toxicity. This should confer expansion and persistence advantages of IL7tr(mut)-containing CARs. Moreover, pSTAT5 signaling could exclude the need for lymphodepletion and alleviate signs of CRS and neurotoxicity, facilitating in vivo CAR delivery [doi: 10.1016/j.ymthe.2023.07.015. Epub 2023 Jul 21.]. EXAMPLE 2 Initial experimental data indicated that the P300H mutant of the receptor’s signaling domain exhibits superior functional activity compared to the wild-type (WT, denoted P). To explore the structural basis of this behavior, we turned to molecular dynamics (MD) simulations as a tool to probe conformational and interactional differences between these variants. Our hypothesis was that the functional activity of the receptor is directly related to the conformational states accessible to its intracellular signaling domain. Specifically, we postulated that this domain exists in a dynamic equilibrium between two extreme structural states: a compact, “closed” conformation and an extended, “open” conformation, with a number of intermediate forms. As a surrogate marker for this
conformational variability, we used the distance between the Cα atoms of residues L286 and position 300 (corresponding to residues 12 and 26 in our molecular dynamics model). This segment of the domain has been observed to undergo folding and unfolding transitions during simulations, and the 12– 26 Cα–Cα distance thus serves as a relevant dynamic scoring parameter. First MD simulations were conducted on the WT and P300H mutant (His at position 26 in MD model) versions of the 34-amino acid part of the signaling domain. These simulations revealed that the H mutant favors a significantly more open and conformationally stable structure compared to WT (Fig. 16A). From the statistical analysis of the simulations, we can state following findings: ● The average Cα–Cα distance between residues 12 and 26 was ~18.9 Å for P300H and ~16.3 Å for WT. ● P300H maintained this larger separation with ~1.9 Å standard deviation, whereas WT showed higher fluctuation with ~3.3 Å deviation. ● P300H spent over 96% of the simulation time in a conformation with a distance above 15 Å, while WT only achieved this in ~65% of frames. ● WT occasionally adopted highly compact states with distances as low as ~7.7 Å, unlike P300H, which never fell below ~11.2 Å. These observations support the idea that the P300H mutant stabilizes an “open” conformational state more effectively, potentially facilitating enhanced signaling activity. In attempt to understand the possible mechanism of the observed finding we analyzed the interaction profile of each residue in modeled domain. Thus, basing on the distance, mutual position and residues type we verified if there is an interaction, specified its type and collected all frames in which these interactions appeared. After, we converted these interactions events into a numerical form as a multiplication of the average interaction energy (kcal/mol) on the percent of time this interaction existed (eq 1). As a result, we’ve obtained a symmetrical table of “energies” between residues that can be compared to each other (Fig.16B). Such approach allowed to use this comparison as a fingerprint in the search of a possible similar mutants. Thus, thirteen additional single-point mutants (replacing Proline at position 300 (26 in MD model) with other amino acids) were simulated using the same MD approach (Fig.16C). Each mutant was evaluated using two metrics: 1. RMSD of its interaction matrix compared to H (lower is better). 2. 12–26 Cα Δ distance distribution alongside the MD vs P300H mutant distribution.
The WT showed intermediate similarity (RMSD ~1.01, Δ distance ~2.66 Å), indicating that several mutants more closely mimic H's favorable conformation and interaction landscape. Based on a composite ranking of both RMSD and Δ distance, we identified possible candidates with similar to P300H properties: ● P300A – closest overall to H by both metrics ● P300W – small Δ distance and good energetic match ● P300E – almost identical matrix to H, slightly larger distance ● P300L and P300Q– structurally and energetically close Additional information:
where ^^,^,^ is the fraction of frames in which residues i and j are involved in interaction type k, the average energy (kcal/mol) associated with that interaction type. The energies used in this analysis represent estimated average stabilizing forces (in kcal/mol) for common non-covalent interactions in biomolecular systems, as summarized in the table 2 below: Table 2 Interaction Type Average Energy (kcal/mol) Hydrophobic 1.5 HBAcceptor 3.5 HBDonor 3.5 XBAcceptor 1.0 XBDonor 1.0 Cationic 5.0 Anionic 5.0 CationPi 4.0 PiCation 4.0 FaceToFace 1.5 EdgeToFace 1.0 PiStacking 2.0 MetalDonor 10.0 MetalAcceptor 10.0 VdWContact 0.5
These values reflect general trends in molecular interaction strengths. For instance, hydrophobic contacts (~1.5 kcal/mol) represent entropic stabilization due to exclusion of water, whereas hydrogen bonds and ionic interactions range from 3–5 kcal/mol and are more directional. Pi-stacking and cation– pi interactions contribute moderate affinity, and metal coordination, often observed in enzymatic or structural motifs, can exceed 10 kcal/mol. Although these energy values are not meant to be precise physical measurements, the calculated composite interaction energies provide a useful heuristic for comparing residue pair interaction strengths across simulations and conditions. The analyzed signaling domain consists primarily of two α-helices that preserve their secondary structure across all simulation frames. The mutable residue 26 (300), which was systematically substituted in the mutants, is positioned near the end of one of these helices. To symmetrically monitor conformational changes involving both helices, we selected L12 (L286)—a stable residue flanking the second helix—as the reference point. Measuring the Cα–Cα distance between these two residues (L12–pos26) provides a sensitive indicator of domain compaction or extension throughout the dynamics. In the closed state (Fig.16D), the signaling domain adopts a compact fold where the N-terminal and C-terminal segments come into close proximity—this is visually confirmed by the short distance between residues L12 (L286) and 26 (300). In contrast, the open state (Fig.16E) is characterized by a pronounced spatial separation between these termini, forming an extended loop conformation. This structural divergence likely reflects distinct functional modes of the receptor. EXAMPLE 3 I. MATERIALS AND METHODS CAR T Cell Production Peripheral blood mononuclear cells (PBMCs) were isolated from a healthy donor using density gradient centrifugation with Lymphocyte Separation Medium, Density 1077 g/mL (Capricorn Scientific). CD4⁺ and CD8⁺ T cell subsets were separately isolated using the CD4⁺ and CD8⁺ T Cell Isolation Kits (Miltenyi Biotec). T cells were activated using the T Cell TransAct Kit (Miltenyi Biotec). The complete culture medium for T cells consisted of 90% RPMI-1640 (Capricorn Scientific), 10% heat-inactivated fetal bovine serum (FBS; Capricorn Scientific), supplemented with 10 ng/mL IL-15 and 10 ng/mL IL-7 (Miltenyi Biotec). Culture medium was replenished every 48–72 hours. Forty-eight hours post-activation, T cells were transduced at a multiplicity of infection (MOI) of 5 using lentiviral vectors encoding the chimeric antigen receptor (CAR) construct along with a truncated form of human EGFR as a reporter gene. Twelve days after transduction, CAR T cells were analyzed for transduction efficiency using an anti-human EGFR antibody (R&D Systems, Cetuximab biosimilar) and harvested for downstream applications.
CARs used in Example 3 (Figure 16) SEQ ID Name Sequences NO 10 BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (2nd generation anti- GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD19 CAR-T with RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG IgG4 hinge, CD28 RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE transmembrane, RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 41BB co-stimulatory and CD3z domains) 18 28.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (2nd generation anti- LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD19 CAR-T with RPGPTRKHYQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQ CD28 hinge, QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN CD28TM, CD28 co- PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL stimulatory and CD3z STATKDTYDAYRHQALPPR domains; additional STAT3 binding motif (YRHQ) at the end of CD3z)) 20 28.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYRHQRSGRVKFSRSADAPAY additional STAT3 QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK binding motif (YRHQ) NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ at the end of CD3z) GLSTATKDTYDAYRHQALPPR 24 28.IL7tr(+14).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE CD3z domains.) GFLQDTFPQQLEESEKQRLLGSNQEEAYVTMSSFYRHQRS GRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 25 28.IL2d.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (28-ΔIL2RB-z(YXXQ) LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR - truncated IL2RB RPGPTRKHYQPYAPPRDFAAYRSNCRNTGPWLKKVLKCNT containing 5th PDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEIS generation CAR-T PLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRSGRV [Nature Medicine KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD volume 24, PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR pages352–359 RGKGHDGLYQGLSTATKDTYDAYRHQALPPR (2018)])
227 28.IL7tr(JS).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (minimal IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR truncation including RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK the JAK1-STAT5 KTLEHLCVWPSLPDHKGGGGSPQQEEAYVTMSRSGRVKF domain incorporated SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP between CD28 and EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRR CD3z domains, with GKGHDGLYQGLSTATKDTYDAYRHQALPPR STAT3 binding motif) *Underlined sequences are IL7Ra (and truncated versions) or truncated IL2RB sequences. *Bold letters are P300H mutation (or P36H mutation in SEQ ID NO:2 or 4) or STAT3 binding site YXXQ (SEQ ID NO:6) (YRHQ = SEQ ID NO:8). Table 3: CARs used in Figure 17. SEQ ID Name Sequences NO 10 BB.Z ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWVKR (2nd generation anti- GRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD19 CAR-T with RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG IgG4 hinge, CD28 RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE transmembrane, RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR 41BB co-stimulatory and CD3z domains) 18 28.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (2nd generation anti- LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR CD19 CAR-T with RPGPTRKHYQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQ CD28 hinge, QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN CD28TM, CD28 co- PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL stimulatory and CD3z STATKDTYDAYRHQALPPR domains; additional STAT3 binding motif (YRHQ) at the end of CD3z)) 20 28.IL7tr(mut).Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE CD3z domains; GFLQDTFPQQEEAYVTMSSFYRHQRSGRVKFSRSADAPAY additional STAT3 QQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK binding motif (YRHQ) NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ at the end of CD3z) GLSTATKDTYDAYRHQALPPR 24 28.IL7tr(+14).Z IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (Truncated IL7Ra LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR incorporated RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK between CD28 and KTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE CD3z domains.) GFLQDTFPQQLEESEKQRLLGSNQEEAYVTMSSFYRHQRS GRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRR GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
25 28.IL2d.Z+ IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV (28-ΔIL2RB-z(YXXQ) LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR - truncated IL2RB RPGPTRKHYQPYAPPRDFAAYRSNCRNTGPWLKKVLKCNT containing 5th PDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEIS generation CAR-T PLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRSGRV [Nature Medicine KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRD volume 24, PEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR pages352–359 RGKGHDGLYQGLSTATKDTYDAYRHQALPPR (2018)]) 228 28.IL7tr(mut).STAT4. IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV Z+ LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR RPGPTRKHYQPYAPPRDFAAYRSRTKKRIKPIVWPSLPDHK KTLEHLCKKPRKNLNVSFNHESFLDCQIHRVDDIQARDEVE GFLQDTFPQQEEAYVTMSSFYQNQYLPSNIDRSGRVKFSR SADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEM GGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDAYRHQALPPR 231 28.Z (2nd generation IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV anti-CD19 CAR-T LVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPR with CD28 hinge, RPGPTRKHYQPYAPPRDFAAYRSRSGRVKFSRSADAPAYQ CD28TM, CD28 co- QGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN stimulatory and CD3z PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL domains WITHOUT STATKDTYDALHMQALPPR STAT3 binding site. (FDA approved axi- cel, also known as axicabtagene ciloleucel (Yescarta)) *Underlined sequences are IL7Ra (and truncated versions) or truncated IL2RB sequences. *Bold letters are P300H mutation (or P36H mutation in SEQ ID NO:2 or 4) or STAT3 binding site YXXQ (SEQ ID NO:6) (YRHQ = SEQ ID NO:8). * Bold and Italic letters are STAT4 binding site SEQ ID NO:189 Cell Lines К562.CD19 is a derivate of the K562 (ATCC no. CCL-243) cell line and was generated by stable transduction with lentivirus (LV) vector coding for CD19 protein expression cassette. К562.CD19.CPL+ cell line is a derivate of К562.CD19 generated by a stable transduction with LV’s coding for PDL1, PDL2, CD86 and GAL9 proteins expression cassettes. Raji – Burkitt’s lymphoma (ATCC no. CCL-86). Flow Cytometry Analysis Surface marker expression was evaluated by flow cytometry using the CytoFLEX instrument (Beckman Coulter). The following antibodies were used: CD4-FITC (Miltenyi Biotec, clone REA623), CD8-FITC (Miltenyi Biotec, clone REA734), Anti-human EGFR-AlexaFluor488 (R&D Systems, Cetuximab biosimilar). DAPI (Invitrogen) was used as a viability dye.
In Vitro Cytotoxicity Assay CAR T cells were rested in cytokine-free growth medium for 48 hours prior to the assay. A total of 2 × 10⁴ CAR T cells were co-cultured with target cells (K562.CD19.CPL and Raji) at effector-to-target (E:T) ratios of 3:1, 1:1, and 1:3 in cytokine-free medium (RPMI-1640 supplemented with 10% FBS). After 24 hours of co-culture, the number and viability of target cells were evaluated by flow cytometry. In Vitro Rechallenge Assay CAR T cells were rested in cytokine-free medium for 48 hours before the assay. A total of 1 × 10⁴ CAR T cells were co-cultured with 1 × 10⁴ target cells (K562.CD19.CPL, Raji) in cytokine-free growth medium. The expansion and viability of CAR T cells and target cells were assessed by flow cytometry every 72 hours. Rechallenge was performed every 72 hours by adding 1 × 10⁴ viable target cells to the co-culture. II. RESULTS We have performed rechallenge assays involving CAR constructs comprising the shortest IL7Ra which only comprises JAK1 and STAT-binding site with the GS linker between them (28.IL7tr(JS).Z+). We have surprisingly found that this CAR outperformed 2nd generation and 5th generation competitors (except for 28.IL7tr(mut).Z+), in terms of killing activity as well as proliferation in the model of K562 cancer cell expressing multiple checkpoint ligands (K562.CD19.CPL). This variant does not contain the P36H mutation, as it lacks that amino acid entirely. Regarding CAR constructs comprising truncated IL7Ra with both the P300H and STAT4 binding sites (the construct 28.IL7tr(mut).STAT4.Z+), we have been able to demonstrate an advantage in proliferation in rechallenge assays with the Raji cancer cell line, compared to CAR constructs comprising truncated IL7Ra with P300H (P36H). It seems that STAT4 binding site enhances proliferation potential in comparison with 28.IL7tr(mut).Z+ (without STAT4 site). It significantly outperformed all 2nd generation and 5th generation competitors. Interestingly, the difference became evident not in the short-term CTA, but in the chronic re-stimulation assay, which better resembles in vivo conditions.