Field of the invention

The present invention relates to the field of cell immunotherapy and more particularly to a new generation of chimeric antigen receptors (CAR), allowing the control of immune cells endowed with such CARs through the interaction with small molecules. More particularly, the present invention relates to chimeric antigen receptors which comprise in at least one ectodomain a molecular switch controlling the antigen binding function of the receptor. The present invention thus provides more controlled and potentially safer engineered CAR endowed immune cells, such as T-lymphocytes.

Backfiround of the invention

Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen- specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.

Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010).

However, although numerous clinical studies have demonstrated the potential of adoptive transfer of CAR T cells for cancer therapy, they have also raised the risks associated with the cytokine-release syndrome (CRS) and the "on-target off-tumor" effect. To date, few strategies have been developed to pharmacologically control CAR engineered T-cells and mainly rely on suicide mechanisms. Such suicide strategies leading to a complete eradication of the engineered T-cells will result in the premature end of the treatment.

Consequently, there is a great need of implementing non-lethal control of engineered CAR T-cells to improve the CAR T-cell technology and its safety.

Summary of the invention

Here, the inventors have developed a system where controlled variations in the conformation of the extracellular portion of a CAR containing the antigen-binding domain could be obtained upon addition of small molecules. This integrated system switches the interaction between the antigen and the antigen binding domain between on/off states. Particularly, by being able to control the confirmation of the extracellular portion of a CAR, downstream functions of an immune cell, such as cytotoxicity of T cell, endowed with such chimeric antigen receptor can be directly modulated. This system provides for novel more controlled and potentially safer engineered CAR endowed immune cells.

The present invention thus provides in a first aspect a chimeric antigen receptor (CAR) characterized in that it comprises: a) at least one ectodomain which comprises: i) an extracellular antigen binding domain; and ii) a switch domain comprising at least a first multimerizing ligand-binding domain and a second multimerizing ligand-binding domain which are capable of binding to a predetermined multivalent ligand to form a multimer comprising said two binding domains and the multivalent ligand to which they are capable of binding; b) at least one transmembrane domain; and c) at least one endodomain comprising a signal transducing domain and optionally a co-stimulatory domain; wherein the switch domain is located between the extracellular antigen binding domain and the transmembrane domain.

The present invention provides in further aspect polynucleotides and vectors comprising one or more nucleic acid sequences encoding a chimeric antigen receptor of the present invention.

The present invention provides in a further aspect an immune cell, and more particularly an isolated immune cell, which comprises (e.g., expresses on its surface) at least one chimeric antigen receptor of the present invention. The present invention provides in a further aspect methods for engineering an immune cell of the present invention.

The present invention provides in a further aspect the use of immune cells of the present invention in therapy, such as for use in the treatment of cancer.

The architecture of the CAR of the inventions provides for a reliable functionality since the first multimerizing ligand-binding domain and second multimerizing ligand-binding domain constituting the molecular switch are present on the same polypeptide molecule. This architecture makes the CAR system of the invention independent from factors which may otherwise have an influence on the proper expression and positioning of the ligand-binding domains on the cell surface if both are present on different polypeptide molecules, something which is difficult to control.

Brief description of the figures

Figure 1. (A) Schematic illustration of a single chain chimeric antigen receptor according to the present invention. (B) Schematic illustration of the CAR ectodomains used in the examples. Both lower structures incorporate FRB and FKBP domains according to the present invention.

Figure 2. Schematic illustration of a multi-chain chimeric antigen receptor according to the present invention Figure 3. (A) Percentages of live cells positive for surface detection of mcCAR in function of presence of vehicle (DMSO) or rapamycin. The detection of the Fab'2 region of the scFv is shown. (B) The fold increase in the median fluorescence intensity (MFI) upon addition of the small molecule rapamycin as depicted in the whole live cell population.

Figure 4. Fold increase in the median fluorescence intensity (MFI) upon addition of the small molecule AP21967 used in association with the T2098L mutant FKBP/FRB* construct as depicted in the whole live cell population.

Figure 5. (A) The effect of the AP21967 rapalog on the cytolytic capacites of the of the CAR T cells toward model antigen presenting cell was assessed in a flow-based cytotoxicity assay. The CD19pos and CD19neg target cell viability was measured after coculture with engineered CAR T-cells in presence or absence of AP21967. Effector/target ratios was set to 20:1. NT represents non-transfected T-cells. (B) Diagram showing the percentage of Daudi CD19 positive cell lysis when using increasing concentration of rapalog AP21967 as per the present invention to induce CAR activity.

Figure 6. Competition experiment between AP21967 (10 nM) and tacrolimus (0 to 500 nM) as described in Example 3.

Figure 7. Fold increase in the median fluorescence intensity (MFI) upon addition of the small molecule AP21967 used in association with the T2098L mutant FKBP/FRB* construct as depicted in the whole live cell population.

Figure 8. (A) Determination of the AP21967 EC50 with CD19 targeting engineered CAR. T-cells transfected with three doses (D, Di/ ₂ and Di/ ₄ ) of mRNA coding for the engineered CAR were treated with increasing amount of AP21967 rapamycin synthetic analog. The Fab'2 region of the scFv is detected. (B) Determination of the AP21967 EC50 with CD123 targeting engineered mcCAR. T-cells transfected with three doses (D, Di/ ₂ and Di/ ₄ ) of mRNA coding for the engineered mcCAR were treated with increasing amount of AP21967 rapalog. The scFv is detected using a recombinant CD123 fused to an Fc fragment. Figure 9. Percentages of live cells positive for surface detection of FKBP/FRB*-mcCAR in function of presence of increasing dose of AP21967.

Figure 10. Treatment schedule of MOLM-13-Luc treated with T cells CAR CD123+ in conjunction with repeated doses of rapalog AP21967 as described in Example 8. Figure 11. Survival curve of MOLM-13-Luc mice following treatment as illustrated in

Figure 10 and Example 8.

Figure 12. Graph body weight of MOLM-13-Luc mice following treatment as illustrated in Figure 10 and Example 8.

Detailed description of the invention

Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et a I, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I -IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Chimeric antigen receptors of the invention The present invention thus provides in a first aspect a chimeric antigen receptor

(CAR) characterized in that it comprises: a) at least one ectodomain which comprises: i) an extracellular antigen binding domain; and ii) a switch domain comprising at least a first multimerizing ligand- binding domain and a second multimerizing ligand-binding domain which are capable of binding to a predetermined multivalent ligand to form a multimer comprising said two binding domains and the multivalent ligand to which they are capable of binding; b) at least one transmembrane domain; and c) at least one endodomain comprising a signal transducing domain and optionally a co-stimulatory domain; wherein the switch domain is located between the extracellular antigen binding domain and the transmembrane domain.

Upon simultaneous binding of the multivalent ligand to the first and second multimerizing ligand-binding domains, a multimeric, such as dimeric, complex is formed which leads to a conformational change within the ectodomain of the chimeric antigen receptor, which in turn improves the surface exposition of the antigen binding domain.

A "multimerizing ligand" or "multimerizer", is a multivalent ligand which is capable of simultaneously binding to at least two binding partners, such as the first and second multimerizing ligand-binding domains, causing upon binding a multimerization, such as dimerization, of the binding partners. More particularly, a "multivalent ligand" is a molecule, preferably a small molecule, which possesses at least two binding sites allowing the simultaneous binding of at least two binding partners, such as the first and second multimerizing ligand-binding domains. The terms "multimerizing ligand", "multimerizer" and "multivalent ligand" can be used herein interchangeably, and include the terms "dimerizing ligand", "dimerizer" and "bivalent ligand", respectively. A "dimerizing ligand", "dimerizer" or "bivalent ligand", is a molecule, preferably a small molecule, which possesses two binding sites allowing the binding of two ligand binding partners, such as the first and second multimerizing ligand-binding domains, thus causing the dimerization thereof.

A "small molecule", as used herein, is a low molecular weight (<2000 daltons) organic compound. Non-limiting examples of small molecules which find application in the present invention include the macrolide rapamycin and its analogs, also known as "rapalogs", such as AP21967, Deforolimus (AP23573), everolimus (RADOOl), and temsirolimus (CCI-779). Other non-limiting examples of small molecules which find application in the present invention include tacrolimus (FK506), FK506 derivatives, such as FK1012, FK506 analogs, such as AP1903. Yet other non-limiting examples of small molecules which find application in the present invention include coumermycin, gibberellin, HaXs, AP1510, AP20187 and AP21967.

A "multimerizing ligand" may also be a peptide or nucleic acid (natural or synthetic). According to certain embodiments, the first multimerizing ligand-binding domain and the second multimerizing ligand binding domain are derived from a chemical induced dimerization (CID) system.

CID systems are based on the mechanism by which two polypeptides bind only in the presence of a certain small molecule or other dimerizing agent. A variety of CID systems is known and may be employed in accordance with the present invention. Non-limiting examples of suitable CID systems are provided in Table 1 below.

First multimerizing ligand- Second multimerizing

Dimerizer(s) binding domain ligand-binding domain

Rapamycin, rapalogs

FKBP12 FRB

(AP21967, AP23573, RAD001, CCI-779)

FKBP12 FKBP12 FK1012, AP1510

FKBP12 (F36V) FKBP12(F36V) AP1903, AP20187

FKBP12 FRB(T2098L) Rapamycin, AP21967

FKBP12 CalcineurinA (CnA) FK506

FKBP12 Cyclophilin (CyP) FKCsA

GyrB GyrB Coumermycin

GAI GID1A Gibberellin

GAI GID1B Gibberellin GAI GID1C Gibberellin

Snap-tag Halo-tag HaXs

Snap-tag CLIP-tag sc

DHFR DHFR BisMTX

glucocorticoid receptor

DHFR Dex-Mtx, Dex-TMP ligand-binding domain

PYL1 ABI1

S-(+)-abscisic acid (ABA)

(PYLcs, amino acids 33 to (ABIcs, amino acids 126 to

209) 423)

AP21967 is a rapamycin analog with heterodimerizing activity. Dimerizer AP21967 heterodimerizes FKBP12 with a variant of FRB which contains a single amino acid substitution (T2098L). AP1903 is a tacrolimus analog with homodimerizing activity. Dimerizer AP1903 homodimerizes a variant of FKBP12 which contains a single amino acid substitution (F36V).

According to certain embodiments, Tacrolimus analogs, like AP1903 or other macrolide immunosuppressants, can be used to further modulate CAR mediated activation of the immune cells as per the present invention. They can act as small molecule competitors offering additional control of the engineered CAR T-cells. As an illustration of the possibility to tune the amount of CAR locked in an on-state at the cell surface, tacrolimus (FK506) was used by the inventors as a small molecule known to bind to the FKBP12 without enabling to form a complex with the FRB (see Example 4 and Figure 6). AP21967 (or rapamycin) and tacrolimus have identical FKBP12 binding core and compete for the same binding site within the FKBP moiety (Wilson, K.P. et al. 1995. Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin. Acta Crystallogr D Biol Crystallogr 51, 511- 521). As shown in Figure 9, T-cells transfected with the engineered CAR incubated with a fixed amount of AP21967 and increasing amount of tacrolimus (0 to 10 mM, preferably 0 to 500 nM) show decreased surface detection of the CAR. Accordingly, the method according to the present invention aiming to switch-on CAR induced activation of an immune cell, can comprise an additional step where said induction is modulated by contacting said immune cells in-vivo or in-vitro with a tacrolimus analog or macrolide immunosuppressant. This may be done to tune, reduce or suppress the activation of the immune cells by the CAR object of the present invention. The dimerizer HaXS is described in, e.g., Erhart, D. et al. (2013), "Chemical development of intracellular protein heterodimerizers", Chemistry & Biology 20: 549-557.

The dimerizer sc is described in, e.g., Gaultier, A. et al. (2009), "Selective cross-linking of interacting proteins using self-labeling tags", J Am Chem Soc. 131(49):17954-62.

The dimerizer BisMTX is described in, e.g., Kopytek, S. J. et al. (2000), "Chemically induced dimerization of dihydrofolate reductase by a homobifunctional dimer of methotrexate", Chemistry & Biology 7:313-321.

The dimerizer Dex-Mtx is described in, e.g., Lin, H.N., et al. (2000), "Dexamethasone- methotrexate: An efficient chemical inducer of protein dimerization in vivo", Journal of the American Chemical Society, 122(17):4247-4248. The dimerizer Dex-TMP is described in, e.g., Gallagher, S.S. et al. (2007) "An orthogonal dexamethasone-trimethoprim yeast three-hybrid system", Anal Biochem. 363(l):160-2.

The first multimerizing ligand-binding domain and second multimerizing ligand- binding domain can be the same or different. Thus, according to certain embodiments, the first multimerizing ligand-binding domain and second multimerizing ligand-binding domain are the same. According to other certain embodiments, the first multimerizing ligand- binding domain and second multimerizing ligand-binding domain are different.

According to certain embodiments, the first multimerizing ligand-binding domain and second multimerizing ligand-binding domain are selected from the pairs of multimerizing ligand-binding domains consisting of: FK506 binding protein (FKBP12):FKBP-rapamycin binding domain of mTOR (FRB), FKBP12:FKBP12, FKBP (F36V): FKBP (F36V), FKBP12:FRB (T2098L), FKBP12:Calcineurin A (CnA), FKBP12:Cyclophilin (CyP), GyrB:GyrB, GA GIDIA, GAI:GID1B, GAI:GID1C, Snap-tag:Halo-tag, Snap-tag:CLIP-tag, DHFR:DHFR, glucocorticoid receptor ligand-binding domain:DHFR and PYL1:ABI1. According to certain embodiments, the first multimerizing ligand-binding domain is

FKBP12 (SEO ID NO: 1) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEO ID NO: 1).

According to particular embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEO ID NO: 1). According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEO ID NO: 1).

According to certain embodiments, the second multimerizing ligand-binding domain is also FKBP12 (SEO ID NO: 1) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEO ID NO: 1).

According to particular embodiments, the second multimerizing ligand-binding domain is FKBP12 (SEO ID NO: 1).

According to other particular embodiments, the second multimerizing ligand-binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEO ID NO: 1).

According to certain embodiments, the second multimerizing ligand-binding domain is FRB (SEO ID NO: 2) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEO ID NO: 2). According to particular embodiments, the second multimerizing ligand-binding domain is FRB (SEQ ID NO: 2).

According to other particular embodiments, the second multimerizing ligand-binding domain is a variant of FRB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEQ. ID NO: 2).

According to other particular embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is FRB (SEQ ID NO: 2) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEQ ID NO: 2).

According to more particular embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is FRB (SEQ ID NO: 2) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEQ ID NO: 2).

According to other more particular embodiments, the first multimerizing ligand- binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is FRB (SEQ ID NO: 2).

According to other more particular embodiments, the first multimerizing ligand- binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is a variant of FRB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEQ ID NO: 2).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is FRB (SEQ ID NO: 2) .

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is a variant of FRB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FRB (SEQ. ID NO: 2).

A variant of FKBP12 may differ from FKBP12 (SEQ ID NO: 1) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from FKBP12 (SEQ ID NO: 1) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s).

Suitably, a variant of FKBP12 is capable of binding to rapamycin or a rapalog, such as AP21967, AP23573, RAD001 or CCI-779. More specifically, such variant comprises a rapamycin or rapalog binding sequence. A non-limiting example of a FKBP12 variant is one which contains a single amino acid substitution (F36V) as shown in SEQ ID NO: 3. Such variant of FKBP12 may for example be used as first and second multimerizing ligand-binding domains, forming a homodimer upon binding of the dimerizer AP1903.

Hence, according to certain embodiments, the first multimerizing ligand-binding domain is FKBP12(F36V) having the amino acid sequence as set forth in SEQ ID NO: 3 and the second multimerizing ligand-binding domain is FKBP12(F36V) having the amino acid sequence as set forth in SEQ ID NO: 3.

A variant of FRB may differ from FRB (SEQ ID NO: 2) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from FRB (SEQ ID NO: 2) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s).

Suitably, a variant of FRB is capable of binding to rapamycin or a rapalog, such as AP21967, AP23573, RAD001 or CCI-779. More specifically, such variant comprises a rapamycin or rapalog binding sequence. Non-limiting examples of FRB variants include ones which contain one or more amino acid substitution at the amino acid positions selected from L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108. According to certain embodiments, a variant of FRB comprises an amino acid substitution at T2098, where T2098 is replaced by leucine (T2098L), e.g., SEQ ID NO: 4, or phenylalanine (T2098F). According to certain other embodiments, a variant of FRB comprises an amino acid substitution at E2032, where E2032 is replaced by phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I) or leucine (E2032L). According to certain other embodiments, a variant of FRB comprises an amino acid substitution at E2032 and at T2098, where E2032 is replaced by any amino acid, and where T2098 is replaced by any amino acid. According to certain other embodiments, a variant of FRB comprises an E2032I and a T2098L substitution. According to certain other embodiments, a variant of FRB comprises an E2032L and a T2098L substitution.

A variant of FRB having the T2098L substitution may for example be used as second multimerizing ligand-binding domain, forming a heterodimer with FKBP12 or variant thereof upon binding of the dimerizer AP21967. Hence, according to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is a variant of FRB comprising an amino acid substitution at T2098, where T2098 is replaced by leucine (SEQ. ID NO: 4).

According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Calcineurin A (CnA) (SEQ ID NO: 5) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CnA (SEQ ID NO: 5). According to certain embodiments, the first multimerizing ligand-binding domain is

FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Calcineurin A (CnA) (SEQ ID NO: 5) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CnA (SEQ ID NO: 5).

According to certain embodiments, the first multimerizing ligand-binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Calcineurin A (CnA) (SEQ ID NO: 5) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CnA (SEQ ID NO: 5). According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Calcineurin A (CnA) (SEQ ID NO: 5).

According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is a variant of Calcineurin A (CnA) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CnA (SEQ ID NO: 5).

A variant of Calcineurin A (CnA) may differ from CnA (SEQ ID NO: 5) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from CnA (SEQ ID NO: 5) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of CnA is capable of binding to FK506. More specifically, such variant comprises a FK506 binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Cyclophilin (CyP) (SEQ ID NO: 6) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CyP (SEQ ID NO: 6). According to certain embodiments, the first multimerizing ligand-binding domain is

FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Cyclophilin (CyP) (SEQ ID NO: 6) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CyP (SEQ ID NO: 6).

According to certain embodiments, the first multimerizing ligand-binding domain is a variant of FKBP12 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is Cyclophilin (CyP) (SEQ ID NO: 6) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CyP (SEQ ID NO: 6). According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ. ID NO: 1), and the second multimerizing ligand-binding domain is Cyclophilin (CyP) (SEQ ID NO: 6).

According to certain embodiments, the first multimerizing ligand-binding domain is FKBP12 (SEQ ID NO: 1), and the second multimerizing ligand-binding domain is a variant of Cyclophilin (CyP) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CyP (SEQ ID NO: 6).

A variant of Cyclophilin (CyP) may differ from CyP (SEQ ID NO: 6) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from CyP (SEQ ID NO: 6) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of CyP is capable of binding to FKCsA. More specifically, such variant comprises a FKCsA binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain and second multimerizing ligand-binding domain are selected from GyrB (SEQ ID NO: 7), a coumermycin binding fragment thereof, or variants thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7) or the coumermycin binding fragment thereof.

According to certain embodiments, the first multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is selected from GyrB (SEQ ID NO: 7), a coumermycin binding fragment thereof, or variants thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7) or the coumermycin binding fragment thereof.

According to certain embodiments, the first multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB, and the second multimerizing ligand-binding domain is selected from GyrB (SEQ ID NO: 7), a coumermycin binding fragment thereof, or variants thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7) or the coumermycin binding fragment thereof. According to certain embodiments, the first multimerizing ligand-binding domain is GyrB (SEQ. ID NO: 7) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7).

According to particular embodiments, the first multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of GyrB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7).

According to more particular embodiments, the first multimerizing ligand-binding domain is GyrB, and the second multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7).

According to other particular embodiments, the first multimerizing ligand-binding domain is GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is a variant of GyrB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of GyrB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7), and the second multimerizing ligand-binding domain is a variant of GyrB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7).

According to certain embodiments, the first multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the coumermycin binding fragment of GyrB, and the second multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GyrB (SEQ ID NO: 7) or the coumermycin binding fragment thereof.

According to particular embodiments, the first multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB, and the second multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the coumermycin binding fragment of GyrB.

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of a coumermycin binding fragment of GyrB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the coumermycin binding fragment of GyrB. According to other particular embodiments, the first multimerizing ligand-binding domain is a coumermycin binding fragment of GyrB, and the second multimerizing ligand- binding domain is a coumermycin binding fragment of GyrB.

A variant of GyrB may differ from GyrB (SEQ. ID NO: 7) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from GyrB (SEQ ID NO: 7) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of GyrB is capable of binding to coumermycin. More specifically, such variant comprises a coumermycin binding sequence.

A variant of a coumermycin binding fragment of GyrB may differ from a coumermycin binding fragment of GyrB in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from a coumermycin binding fragment of GyrB in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, such variant is capable of binding to coumermycin. More specifically, such variant comprises a coumermycin binding sequence. A coumermycin binding fragment the may be used according to the present invention may be the 24KDa amino terminal subdomain of GyrB.

According to certain embodiments, the first multimerizing ligand-binding domain is GAI (SEQ. ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIA (SEQ ID NO: 9) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIA (SEQ ID NO: 9).

According to particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIA (SEQ ID NO: 9) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIA (SEQ ID NO: 9).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIA (SEQ ID NO: 9) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIA (SEQ ID NO: 9).

According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 6), and the second multimerizing ligand-binding domain is GIDIA (SEQ ID NO: 9).

According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIA having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIA (SEQ ID NO: 9). According to more particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ. ID NO: 8), and the second multimerizing ligand-binding domain is GIDIA (SEQ ID NO: 9).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIA having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIA (SEQ ID NO: 9). A variant of GAI may differ from GAI (SEQ ID NO: 8) in the substitution of one or more

(such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from GAI (SEQ ID NO: 8) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of GAI is capable of binding to gibberellin. More specifically, such variant comprises a gibberellin binding sequence.

A variant of GIDIA may differ from GIDIA (SEQ ID NO: 9) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from GIDIA (SEQ ID NO: 9) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of GIDIA is capable of binding to gibberellin. More specifically, such variant comprises a gibberellin binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIB (SEQ ID NO: 10) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIB (SEQ ID NO: 10).

According to particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIB (SEQ ID NO: 10) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIB (SEQ ID NO: 10).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIB (SEQ ID NO: 10) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIB (SEQ ID NO: 10).

According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIB (SEQ ID NO: 10).

According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIB (SEQ ID NO: 10).

According to more particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIB (SEQ ID NO: 10).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIB having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIB (SEQ ID NO: 10).

A variant of GIDIB may differ from GIDIB (SEQ ID NO: 10) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from GIDIB (SEQ ID NO: 10) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of GID1B is capable of binding to gibberellin. More specifically, such variant comprises a gibberellin binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is GAI (SEQ. ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIC (SEQ ID NO: 11) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIC (SEQ ID NO: 11). According to particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIC (SEQ ID NO: 11) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIC (SEQ ID NO: 11).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIC (SEQ ID NO: 11) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIC (SEQ ID NO: 11). According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is GIDIC (SEQ ID NO: 11).

According to other particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ ID NO: 8) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIC having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIC (SEQ ID NO: 11). According to more particular embodiments, the first multimerizing ligand-binding domain is GAI (SEQ. ID NO: 8), and the second multimerizing ligand-binding domain is GIDIC (SEQ ID NO: 11).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of GAI having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GAI (SEQ ID NO: 8), and the second multimerizing ligand-binding domain is a variant of GIDIC having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with GIDIC (SEQ ID NO: 11). A variant of GIDIC may differ from GIDIC (SEQ ID NO: 11) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from GIDIC (SEQ ID NO: 11) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of GIDIC is capable of binding to gibberellin. More specifically, such variant comprises a gibberellin binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is Halo-tag (SEQ ID NO: 13) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Halo-tag (SEQ ID NO: 13).

According to particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is Halo-tag (SEQ ID NO: 13) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Halo-tag (SEQ ID NO: 13).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of Snap-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is Halo-tag (SEQ ID NO: 13) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Halo-tag (SEQ. ID NO: 13).

According to other particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is Halo-tag (SEQ ID NO: 13).

According to other particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is a variant of Halo-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Halo-tag (SEQ ID NO: 13).

According to more particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is Halo-tag (SEQ ID NO: 13).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of Snap-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is a variant of Halo-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Halo-tag (SEQ ID NO: 13).

A variant of Snap-tag may differ from Snap-tag (SEQ ID NO: 12) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from Snap-tag (SEQ ID NO: 12) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of Snap-tag is capable of binding to HaXs. More specifically, such variant comprises a HaXs binding sequence.

A variant of Halo-tag may differ from Halo-tag (SEQ ID NO: 13) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from Halo-tag (SEQ ID NO: 13) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of Halo-tag is capable of binding to HaXs. More specifically, such variant comprises a HaXs binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ. ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is CLIP-tag (SEQ ID NO: 14) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CLIP-tag (SEQ ID NO: 14). According to particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is CLIP-tag (SEQ ID NO: 14) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CLIP-tag (SEQ ID NO: 14).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of Snap-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is CLIP-tag (SEQ ID NO: 14) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CLIP-tag (SEQ ID NO: 14). According to other particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is CLIP-tag (SEQ ID NO: 14).

According to other particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ ID NO: 12) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is a variant of CLIP-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CLIP-tag (SEQ ID NO: 14). According to more particular embodiments, the first multimerizing ligand-binding domain is Snap-tag (SEQ. ID NO: 12), and the second multimerizing ligand-binding domain is CLIP-tag (SEQ ID NO: 14).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of Snap-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with Snap-tag (SEQ ID NO: 12), and the second multimerizing ligand-binding domain is a variant of CLIP-tag having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CLIP-tag (SEQ ID NO: 14). A variant of CLIP-tag may differ from CLIP-tag (SEQ ID NO: 14) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from CLIP-tag (SEQ ID NO: 14) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of CLIP-tag is capable of binding to SC. More specifically, such variant comprises a SC binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to particular embodiments, the first multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to more particular embodiments, the first multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15), and the second multimerizing ligand-binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

A variant of DHFR may differ from DHFR (SEQ ID NO: 15) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from DHFR (SEQ ID NO: 15) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of DHFR is capable of binding to BisMTX. More specifically, such variant comprises a BisMTX binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with said glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is DHFR (SEQ. ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to particular embodiments, the first multimerizing ligand-binding domain is a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of a glucocorticoid receptor ligand-binding domain having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with said glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15).

According to other particular embodiments, the first multimerizing ligand-binding domain is a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with said glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

According to more particular embodiments, the first multimerizing ligand-binding domain is a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16), and the second multimerizing ligand-binding domain is DHFR (SEQ ID NO: 15). According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of a glucocorticoid receptor ligand-binding domain having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with a glucocorticoid receptor ligand-binding domain (SEQ. ID NO: 16), and the second multimerizing ligand-binding domain is a variant of DHFR having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with DHFR (SEQ ID NO: 15).

A variant of a glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) may differ from said glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from DHFR (SEQ ID NO: 15) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of said glucocorticoid receptor ligand-binding domain (SEQ ID NO: 16) is capable of binding to Dex-Mtx or Dex-TMP. More specifically, such variant comprises a Dex- Mtx or Dex-TMP binding sequence.

According to certain embodiments, the first multimerizing ligand-binding domain is PYLl (SEQ ID NO: 17) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is ABIl (SEQ ID NO: 18) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ABIl (SEQ ID NO: 18).

According to particular embodiments, the first multimerizing ligand-binding domain is PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is ABIl (SEQ ID NO: 18) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ABIl (SEQ ID NO: 18).

According to other particular embodiments, the first multimerizing ligand-binding domain is a variant of PYLl having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is ABIl (SEQ ID NO: 18) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ABIl (SEQ ID NO: 18).

According to other particular embodiments, the first multimerizing ligand-binding domain is PYLl (SEQ ID NO: 17) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is ABIl (SEQ ID NO: 18).

According to other particular embodiments, the first multimerizing ligand-binding domain is PYLl (SEQ ID NO: 17) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is a variant of ABIl having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ABIl (SEQ ID NO: 18).

According to more particular embodiments, the first multimerizing ligand-binding domain is PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is ABIl (SEQ ID NO: 18).

According to other more particular embodiments, the first multimerizing ligand- binding domain is a variant of PYLl having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with PYLl (SEQ ID NO: 17), and the second multimerizing ligand-binding domain is a variant of ABIl having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with ABIl (SEQ ID NO: 18).

A variant of PYLl may differ from PYLl (SEQ ID NO: 17) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from PYLl (SEQ ID NO: 17) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of PYLl is capable of binding to S-(+)-abscisic acid (ABA). More specifically, such variant comprises a S-(+)- abscisic acid (ABA) binding sequence.

A variant of ABIl may differ from ABIl (SEQ ID NO: 18) in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from ABIl (SEQ ID NO: 18) in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitably, a variant of ABI1 is capable of binding to S-(+)-abscisic acid (ABA). More specifically, such variant comprises a S-(+)- abscisic acid (ABA) binding sequence.

The first and second multimerizing ligand-binding domains may either be directly fused to each other or may be separated by a peptide linker.

Thus, according to certain embodiments, are separated by a peptide linker. The peptide linker may be composed of up to 50 amino acids, such as up to 25 amino acids. According to certain embodiments, the peptide linker is composed of 5 to 25 amino acids. Non-limiting examples of peptide linkers that may be employed according to the invention include a four-EAAAR- linker (SEQ ID NO: 19), a -GS-4x-EAAAR- linker (SEQ ID NO: 20) and variants thereof. Thus, according to particular embodiments, the peptide linker is a four- EAAAR- linker (SEQ ID NO: 19) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the four-EAAAR- linker (SEQ ID NO: 19). According to other particular embodiments, the peptide linker is a— GS-4X-EAAAR- linker (SEQ ID NO: 20) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the -GS-4x- EAAAR- linker (SEQ ID NO: 20).

According to certain other embodiments, the first and second multimerizing ligand- binding domains are in direct fusion (e.g., the C-terminus of the first multimerizing ligand- binding domain is in direct fusion with the N-terminus of the second multimerizing ligand- binding domain). With "direct fusion" it is meant that there is no peptide linker between the first and second multimerizing ligand-binding domains, that is the two domains are linked by a carbon-nitrogen bond.

The first and second multimerizing ligand-binding domains may be arranged in any possible order, that is the first multimerizing ligand-binding domain may be located N- terminal to the second multimerizing ligand-binding domain, or the first multimerizing ligand-binding domain may be located C-terminal to the second multimerizing ligand-binding domain. Thus, according to certain embodiments, the first multimerizing ligand-binding domain is located N-terminal to the second multimerizing ligand-binding domain. According to other certain embodiments, the first multimerizing ligand-binding domain is located C- terminal to the second multimerizing ligand-binding domain.

A chimeric antigen receptor according to the present invention may further comprise a hinge within the at least one ectodomain. The hinge is suitably located between the switch domain and the transmembrane domain.

The term "hinge" or "hinge region" used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the switch domain. In particular, a hinge is used to provide more flexibility and accessibility for the extracellular ligand-binding domain. A hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. A hinge may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region. Alternatively the hinge may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. Non-limiting examples of hinges which may be used in accordance to the invention include a part of human CD8 alpha chain, FcyRllla receptor or IgGl.

According to certain embodiments, the hinge is selected from the group consisting of CD8a hinge, IgGl hinge and FcyRllla hinge.

According to particular embodiments, the hinge is a CD8a hinge (SEQ ID NO: 21) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the CD8a hinge (SEQ. ID NO: 21). According to more particular embodiments, the hinge is a CD8a hinge (SEQ ID NO: 21). According to other more particular embodiment, the hinge is a variant of a CD8a hinge having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the CD8a hinge (SEQ ID NO: 21). A variant of a CD8a hinge may differ from said CD8a hinge in the substitution of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said CD8a hinge in the addition or deletion of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s).

According to other particular embodiments, the hinge is a IgGl hinge (SEQ ID NO: 22) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the IgGl hinge (SEQ ID NO : 22). According to more particular embodiments, the hinge is a IgGl hinge (SEQ. ID NO : 22). According to other more particular embodiment, the hinge is a variant of a IgGl hinge having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the IgGl hinge (SEQ ID NO : 22). A variant of a IgGl hinge may differ from said IgGl hinge in the substitution of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said IgGl hinge in the addition or deletion of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s).

According to other particular embodiments, the hinge is a FCYRI I ICX hinge (SEQ ID NO : 23) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the FcyRllla hinge (SEQ ID NO : 23). According to more particular embodiments, the hinge is a FcyRllla hinge (SEQ ID NO : 23). According to other more particular embodiment, the hinge is a variant of a FcyRllla hinge having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the FcyRllla hinge (SEQ ID NO : 23). A variant of a FcyRllla hinge may differ from said FcyRllla hinge in the substitution of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said FcyRllla hinge in the addition or deletion of one or more (such 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s).

The "extracellular antigen binding domain" comprised by the ectodomain of the chimeric antigen receptor may be any oligo- or polypeptide that is capable of binding a ligand, more specifically an antigen. Preferably, the domain will be capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus examples of cell surface markers that may act as ligands include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells. In particular, the extracellular ligand-binding domain can comprise an antigen binding fragment derived from an antibody against an antigen of the target.

Thus, according to certain embodiments, the extracellular antigen binding domain comprises an antibody or antigen binding fragment thereof. The antigen binding fragment may be, for example, a scFv or a Fab. According to particular embodiments, the extracellular antigen binding domain is a scFv, preferably one derived from a monoclonal antibody against an antigen of a target. More specifically, the extracellular ligand-binding domain may comprise a single chain antibody fragment (scFv) comprising the light (VL) and the heavy (VH) variable fragment of a target antigen specific monoclonal antibody, optionally joined by a peptide linker composed of, e.g., 5 to 25 amino acids (such as a GGGGSGGGGSGGGGS-linker as shown in SEQ. ID NO: 24).

According to other particular embodiments, the extracellular antigen binding domain is a Fab, preferably one derived from a monoclonal antibody against an antigen of a target. As non-limiting examples, the antigen of the target can be any cluster of differentiation molecules (e.g. CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138), a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvlll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma- associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RUl, RU2 (AS), intestinal carboxyl esterase, , M-CSF, prostase, prostase specific antigen (PSA), PAP, NY- ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-l, IGF-II, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (fap); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7-1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, , endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), or a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an HBV-specific antigen, an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, a fungi-specific antigen or a bacterium-specific antigen as well as any derivate or variant of these surface markers. Antigens are not necessarily surface marker antigens but can be also endogenous small antigens presented by HLA class I at the surface of the cells.

According to certain embodiments, the extracellular antigen binding domain may be directed against CD19. Such extracellular antigen binding domain may be a scFV derived from a CD19 monoclonal antibody, such as 4G7 (Peipp, Saul et al., 2004). According to particular embodiments, said scFV comprises the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin gamma 1 heavy chain (SEQ ID NO: 25) and the variable fragments of the CD19 monoclonal antibody 4G7 immunoglobulin kappa light chain (SEQ. ID NO: 26 or SEQ ID NO: 27), optionally linked by a peptide linker. According to certain embodiments, the extracellular antigen binding domain is directed against CD123. Such extracellular antigen binding domain may be a scFV derived from a CD123 monoclonal antibody.

According to other certain embodiments, the extracellular antigen binding domain is directed against ROR1. Such extracellular antigen binding domain may be a scFV derived from a ROR1 monoclonal antibody.

According to other certain embodiments, the extracellular antigen binding domain is directed against BCMA. Such extracellular antigen binding domain may be a scFV derived from a BCMA monoclonal antibody.

According to other certain embodiments, the extracellular antigen binding domain may be directed against CD20. Such extracellular antigen binding domain may be a scFV derived from a CD20 monoclonal antibody.

According to other certain embodiments, the extracellular antigen binding domain may be directed against CD33. Such extracellular antigen binding domain may be a scFV derived from a CD33 monoclonal antibody. A chimeric antigen receptor according to the invention comprises at least one ectodomain comprising a signal transducing domain and optionally a co-stimulatory domain

The signal transducing domain or intracellular signaling domain of the CAR of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response. In other words, the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines. In the present application, the term "signal transducing domain" refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.

Preferred examples of signal transducing domain for use in single or multi-chain CAR can be the cytoplasmic sequences of the Fc receptor or T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivate or variant of these sequences and any synthetic sequence that as the same functional capability. Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co- stimulatory signal. Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs. ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases. Non-limiting xxamples of ITAM which can be employed in accordance with the invention can include those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b and CD66d.

According to certain embodiments, the signaling domain comprises the CD3zeta signaling domain, or the intracytoplasmic domain of the FcsRI beta or gamma chains.

According to particular embodiments, the signaling domain comprises a CD3 zeta signaling domain. According to more particular embodiments, the signaling domain comprises the CD3 zeta signaling domain as set forth in SEQ. ID NO: 28. According to other more particular embodiments, the signaling domain comprises a variant of the CD3 zeta signaling domain as set forth in SEQ ID NO: 28 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with CD3 zeta signaling domain as set forth in SEQ ID NO: 28. A variant of a CD3 zeta signaling domain may differ from said CD3 zeta signaling domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said CD3 zeta signa ling domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a varia nt is one which has the same or similar function and activity as seen for the CD3 zeta signaling domain (SEQ. I D NO : 28).

According to other particular embodiments, the signaling domain comprises the intracytoplasmic domain of the FcsRI beta or gamma chains or a variant thereof.

According to more particular embodiments, the signaling domain comprises the intracytoplasmic domain of the FcsRI beta chain (SEQ I D NO : 29). According to other more particular embodiments, the signaling domain comprises a variant of the intracytoplasmic domain of the FcsRI beta chain (SEQ I D NO : 29) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with intracytoplasmic domain of the FcsRI beta chain (SEQ I D N O : 29). A variant of the intracytoplasmic domain of the FCERI beta chain may differ from said intracytoplasmic domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said intracytoplasmic domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function and activity as seen for the intracytoplasmic domain of the FCERI beta chain (SEQ I D NO : 29). According to other more particular embodiments, the signaling domain comprises the intracytoplasmic domain of the FcsRI gamma chain (SEQ I D NO : 30). According to other more particular embodiments, the signaling domain comprises a variant of the intracytoplasmic domain of the FcsRI gamma chain (SEQ I D NO : 30) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with intracytoplasmic domain of the FcsRI gamma chain (SEQ I D NO : 30). A variant of the intracytoplasmic domain of the FcsRI gamma chain may differ from said intracytoplasmic domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said intracytoplasmic domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function and activity as seen for the intracytoplasmic domain of the FcsRI gamma chain (SEQ I D NO : 30). According to certain embodiments, the CAR of the present invention comprises in at least one endodomain a co-stimulatory domain.

The co-stimulatory domain may be any cytoplasmic domain of a costimulatory molecule. A co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response.

"Co-stimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like. A co- stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD- L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-1 BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. A "co-stimulatory molecule" refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.

A "co-stimulatory signal" as used herein refers to a signal, which in combination with primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules. The co-stimulatory domain may, for example, be the cytoplasmic domain from a costimulatory molecule selected from CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, CD8, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and any combination thereof. Thus, according to certain embodiments, the co-stimulatory domain is a co- stimulatory domain from 4-1BB. According to particular embodiments, the co-stimulatory domain is a co-stimulatory domain from 4-1BB as set forth in SEQ ID NO: 31. According to other particular embodiments, the co-stimulatory domain is variant of the co-stimulatory domain from 4-1BB as set forth in SEQ ID NO: 31 having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with co-stimulatory domain from 4-1BB as set forth in SEQ ID NO: 31. A variant of the co-stimulatory domain from 4-1BB may differ from said co-stimulatory domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said co-stimulatory domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function and activity as seen for the co-stimulatory domain from 4- 1BB (SEQ ID N0: 31).

A chimeric antigen receptor according to the invention comprises at least one transmembrane domain. The distinguishing features of appropriate transmembrane domains comprise the ability to be expressed at the surface of a cell, preferably in the present invention an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and to interact together for directing cellular response of immune cell against a predefined target cell. The at least one transmembrane domain can be derived either from a natural or from a synthetic source. The at least one transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the at least one transmembrane domain can be a subunit of the T cell receptor such as α, β, γ or δ, polypeptide constituting CD3 complex, IL2 receptor p55 (a chain), p75 (β chain) or γ chain, subunit chain of Fc receptors, in particular Fey receptor III or CD proteins. Alternatively, the at least one transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. The at least one transmembrane domain may, for example, be derived from the CD8 alpha chain. Thus, according to certain embodiments, the at least one transmembrane domain is a CD8 alpha transmembrane domain. According to particular embodiments, the at least one transmembrane domain is a CD8 alpha transmembrane domain (SEQ ID NO: 32). According to other particular embodiments, the at least one transmembrane domain is a variant of a CD8 alpha transmembrane domain (SEQ. ID NO: 32) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the human CD8 alpha transmembrane domain (SEQ ID NO: 32). A variant of the CD8 alpha transmembrane domain may differ from said transmembrane domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said transmembrane domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function as seen for the CD8 alpha transmembrane domain (SEQ ID NO: 32). Alternatively, the at least one transmembrane domain may be derived from 4-lBB.

Thus, according to certain embodiments, the at least one transmembrane domain is a 4-lBB transmembrane domain (SEQ ID NO: 33). According to particular embodiments, the at least one transmembrane domain is a 4-lBB transmembrane domain (SEQ ID NO: 33). According to other particular embodiments, the at least one transmembrane domain is a variant of a 4- IBB transmembrane domain (SEQ ID NO: 33) having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with the 4-lBB transmembrane domain (SEQ ID NO: 33). A variant of the 4-lBB transmembrane domain may differ from said transmembrane domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said transmembrane domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function as seen for the 4-lBB transmembrane domain (SEQ ID NO: 33).

Alternatively, the at least one transmembrane domain may be derived from the Fes receptor chains or variant thereof. Particularly, the transmembrane domain may be selected from the transmembrane domains of the FcsRI α, β and γ chains, fragments or variants thereof. Thus, according to certain embodiments, the at least one transmembrane domain is the transmembrane domain from the alpha chain of high-affinity IgE receptor (FcsRI) (SEQ. ID NO: 34). According to certain other embodiments, the at least one transmembrane domain is a variant of the transmembrane domains of the FcsRI a chain having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FCERI a (SEQ ID NO: 34). A variant of the transmembrane domain of the FcsRI a chain may differ from said transmembrane domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said transmembrane domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function as seen for the transmembrane domains of the FcsRI a chain (SEQ ID NO: 34).

In case that the chimeric antigen receptor is a multi-chain CAR, which is composed of at least two different polypeptide chains, each of which contains at least one transmembrane domain, the transmembrane domains may, for example, be selected from the transmembrane domains of the FcsRI α, β and γ chains, fragments or variants thereof.

Thus, the at least one transmembrane domain comprised by a first polypeptide chain comprising at least one ectodomain in accordance with the invention may be the transmembrane domain from the alpha chain of high-affinity IgE receptor (FcsRI) (SEQ ID NO: 34) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FcsRI a (SEQ ID NO: 34). The at least one transmembrane domain comprised by a second polypeptide chain comprising at least one endodomain in accordance with the invention may be the transmembrane domain from the gamma or beta chain of FcsRI (SEQ ID NO: 35 and 36, respectively) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FcsRI γ or β (SEQ ID NO: 35 and 36, respectively). The at least one transmembrane domain comprised by a third polypeptide chain comprising at least one endodomain in accordance with the invention may be the transmembrane domain from the gamma or beta chain of FcsRI (SEQ ID NO: 35 and 36, respectively) or a variant thereof having at least 80, such as at least 85, at least 90, at least 95, at least 98 or at least 99%, sequence identity with FcsRI γ or β (SEQ ID NO: 35 and 36, respectively). A variant of the transmembrane domain of the FcsRI γ or β chain may differ from said transmembrane domain in the substitution of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Alternatively, or in addition, such variant may differ from said transmembrane domain in the addition or deletion of one or more (such as 1 to 10, 1 to 5, or 1 to 3) amino acid residue(s). Suitable, a variant is one which has the same or similar function as seen for the transmembrane domains of the FcsRI γ or β chain (SEQ. ID NO: 35 or 36, respectively).

A chimeric antigen receptor according to the present invention may be a single chain CAR. A single chain CAR is a chimeric antigen receptor wherein all domains of which said CAR is composed are located on one polypeptide chain. A non-limiting illustration of a single chain CAR according to the present invention is shown in Figure 1A.

Alternatively, a chimeric antigen receptor according to the present invention may be a multi-chain CAR. A non-limiting illustration of a multi-chain CAR according to the present invention is shown in Figure 2. According to this architecture, at least on ectodomain and the at least one endodomain are born on different polypeptide chains. The different polypeptide chains are anchored into the membrane in a close proximity allowing interactions with each other. In such architectures, the signaling and co-stimulatory domains can be in juxtamembrane positions (i.e. adjacent to the cell membrane on the internal side of it), which is deemed to allow improved function of co-stimulatory domains. The multi-subunit architecture also offers more flexibility and possibilities of designing CARs with more control on T-cell activation. For instance, it is possible to include several extracellular antigen recognition domains having different specificity to obtain a multi-specific CAR architecture. It is also possible to control the relative ratio between the different subunits into the multichain CAR. This type of architecture has been recently described by the applicant in PCT/US2013/058005.

Accordingly, a multi-chain CAR according to the invention may be one which comprises:

A) a first polypeptide chain comprising a) at least one ectodomain which comprises: i) an extracellular antigen binding domain; and ii) a switch domain comprising at least a first multimerizing ligand-binding domain and a second multimerizing ligand-binding domain which are capable of binding to a predetermined multivalent ligand to form a multimer comprising said two binding domains and the multivalent ligand to which they are capable of binding; and aa) at least one transmembrane domain; and

B) a second polypeptide chain comprising b) at least one endodomain comprising a signal transducing domain and optionally a co-stimulatory domain; and bb) at least one transmembrane domain.

According to certain embodiments, a multi-chain CAR of the invention may further comprise:

C) a third polypeptide chain comprising c) at least one endodomain comprising a co-stimulatory domain; and cc) at least one transmembrane domain.

The assembly of the different chains as part of a single multi-chain CAR is made possible, for instance, by using the different alpha, beta and gamma chains of the high affinity receptor for IgE (FcsRI) (Metzger, Alcaraz et al. 1986). Such multi-chain CARs can be derived from FcsRI, by replacing the high affinity IgE binding domain of FcsRI alpha chain by an ectodomain as detailed herein, whereas the N and/or C-termini tails of FcsRI beta and/or gamma chains are fused to an ectodomain as detailed herein comprising a signal transducing domain and co-stimulatory domain, respectively. The extracellular ligand binding domain has the role of redirecting T-cell specificity towards cell targets, while the signal transducing domains activate the immune cell response. The fact that the different polypeptide chains derived from the alpha, beta and gamma polypeptides from FcsRI are transmembrane polypeptides sitting in juxtamembrane position, provides a more flexible architecture to CARs, improving specificity towards the antigen target and reducing background activation of immune cells.

Thus, according to particular embodiments, the first polypeptide chain (A) comprising the ectodomain comprises the transmembrane domain from the alpha chain of high-affinity IgE receptor (FcsRI), whereas the second polypeptide chain (B) comprising the endodomain which comprises the signal transducing domain comprises the transmembrane domain from the gamma or beta chain of FcsRI, such as the transmembrane domain from the gamma chain of FcsRI. If present, the third polypeptide chain (C) comprising the endodomain which comprises the co-stimulatory domain comprises the transmembrane domain from the gamma or beta chain of FcsRI, such as the transmembrane domain from the beta chain of FceRI.

The conformation of the at least one ectodomain of the chimeric antigen receptor is preferably such that in absence of the corresponding multimerizing ligand the extracellular binding domain is not capable of binding to the targeted antigen. The binding of the multimerizing ligand to the switch domain then results in a conformational change which exposes the extracellular binding domain in a manner that allows its binding to the targeted antigen (this mechanism may be referred to as switch on). The appropriate conformation can be determined on the basis of the cytolytic activity (cytotoxicity) of an immune cell expressing said CAR. With "cytolytic activity" it is meant the percentage of cell lysis of target cells conferred by an immune cell expressing said CAR.

A method for determining the cytotoxicity is described below:

With adherent target cells: 2 x 10 ⁴ specific target antigen (STA)-positive or STA- negative cells are seeded in 0.1ml per well in a 96 well plate. The day after the plating, the STA-positive and the STA-negative cells are labeled with CellTrace CFSE and co-cultured with 4 x 10 ⁵ T cells for 4 hours. The cells are then harvested, stained with a fixable viability dye (eBioscience) and analyzed using the MACSQuant flow cytometer (Miltenyi).

The percentage of specific lysis is calculated using the following formula:

% viable target cells upon coculture with CAR modified T cells

% viable control cells upon coculture with CAR modified T cells

% cell lysis = 100%

% viable target cells upon coculture with non modified T cells

% viable control cells upon coculture with non modified T cells With suspension target cells: STA-positive and STA-negative cells are respectively labeled with CellTrace CFSE and CellTrace Violet. About 2 x 10 ⁴ RORl-positive cells are co- cultured with 2 x 10 ⁴ STA-negative cells with 4 x 10 ⁵ T cells in 0.1ml per well in a 96-well plate. After a 4 hour incubation, the cells are harvested and stained with a fixable viability dye (eBioscience) and analyzed using the MACSQuant flow cytometer (Miltenyi).

The percentage of specific lysis is calculated using the previous formula.

"Specific target antigen (STA)-positive cells" means cells which express the target antigen for which the chimeric antigen receptor shows specificity, whereas "STA-negative cells" means cells which do not express the specific target antigen. By way of a non-limiting example, if the CAR is directed against CD19, the specific target antigen is thus CD19. Accordingly, CD19-positive and CD19-negative cells are to be used to determine the cytolytic activity. Hence, the above-described cytotoxicity assay will have to be adapted to the respective target cells depending on the antigen-specificity of the chimeric antigen receptor expressed by the immune cell.

Similar methods for assaying the cytolytic activity are also described in, e.g., Valton et al. (2015) or Poirot et al. (2015). According to certain embodiments, a chimeric antigen receptor according to the present invention confers a modulated cytolytic activity to an immune cell expressing same in the presence of a corresponding multimerizing ligand compared to the cytolytic activity of said immune cell in the absence of the multimerizing ligand.

According to particular embodiments, a chimeric antigen receptor of the present invention is one which confers an increased cytolytic activity to an immune cell expressing same in the presence of a corresponding multimerizing ligand compared to the cytolytic activity of said immune cell in the absence of the multimerizing ligand. By "increased cytolytic activity" it is meant that the % cell lysis of target cells conferred by the immune cell expressing said CAR increases by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, in the presence of the multimerizing ligand compared to the % cell lysis of target cells conferred by the immune cell in the absence of the multimerizing ligand.

According to other particular embodiments, a chimeric antigen receptor of the present invention is one which confers a decreased cytolytic activity to an immune cell expressing same in the presence of a corresponding multimerizing ligand compared to the cytolytic activity of said immune cell in the absence of the multimerizing ligand. By "decreased cytolytic activity" it is meant that the % cell lysis of target cells conferred by the immune cell expressing said CAR decreases by at least 10%, such as at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%, in the presence of the multimerizing ligand compared to the % cell lysis of target cells conferred by the immune cell in the absence of the multimerizing ligand.

By "corresponding multimerizing ligand" is meant a multimerizing ligand which is bound by both the first multimerizing ligand-binding domain and the second multimerizing ligand-binding domain, and thus promotes multimerization (e.g., dimerization) between the first and second multimerizing ligand-binding domains. By way of a non-limiting example, if the first multimerizing ligand-binding domain is KBP12 and the second multimerizing ligand- binding domain is FRB, then the "corresponding ligand-binding domain" may be rapamycin.

Polynucleotides, vectors: The present invention also relates to polynucleotides and vectors that comprise one or more nucleotide sequences encoding a chimeric antigen receptor according to the invention. The present invention provides polynucleotides, including DNA and RNA molecules, which comprise one or more nucleotide sequences encoding a chimeric antigen receptor. In case the chimeric antigen receptor is a multi-chain CAR, at least one polynucleotide is provided which comprises two or more nucleotide sequence encoding the polypeptide chains composing the multi-chain CAR according to the invention. According to certain embodiments, a composition is provided comprising a first polynucleotide comprising a nucleotide sequence encoding a first polypeptide chain and a second polynucleotide comprising a nucleotide sequence encoding a second polypeptide chain. Optionally, the composition comprises a third polynucleotide comprising a nucleotide sequence encoding a third polypeptide chain.

The polynucleotide(s) may be comprised by an expression cassette or expression vector (e.g. a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell). According to certain embodiments, the different nucleotide sequences can be included in one polynucleotide or vector which comprises a nucleotide sequence encoding ribosomal skip sequence such as a sequence encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picornaviruses, causes a ribosomal "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see Donnelly et al., J. of General Virology 82: 1013-1025 (2001); Donnelly et al., J. of Gen. Virology 78: 13-21 (1997); Doronina et al., Mol. And. Cell. Biology 28(13): 4227-4239 (2008); Atkins et al., RNA 13: 803-810 (2007)). By "codon" is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA. As non-limiting example, in the present invention, 2A peptides have been used to express into the cell the different polypeptides of the multi-chain CAR.

To direct, transmembrane polypeptide such as FcsR into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in polynucleotide sequence or vector sequence. The secretory signal sequence may be that of FcsR, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). In a preferred embodiment the signal peptide comprises the residues 1 to 25 of the FcsRI alpha chain. Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. Preferably, the nucleotide sequences of the present invention are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species by codons that are generally frequent in highly expressed genes of such species, such codons encoding the amino acids as the codons that are being exchanged.

Methods for engineering an immune cell

The present invention further relates to methods of preparing immune cells for immunotherapy comprising introducing into said immune cells a CAR according to the present invention and expanding said cells. In particular, a method for engineering an immune cell is provided, said method comprises:

(i) Providing an immune cell, such as such as T cell; and

(ii) Expressing on the surface of said immune cell at least one chimeric antigen receptor according to the present invention.

According to certain embodiments, the method comprises:

(a) Providing an immune cell;

(b) Introducing into said cell at least one polynucleotide or vector according to the present invention; and

(c) Expressing a chimeric antigen receptor of the invention in said cell.

In a preferred embodiment, said polynucleotides are included in lentiviral vectors in view of being stably expressed in the cells.

In order to enhance, for example, an antitumor effect, it is contemplated to further express on the surface of the immune cell at least one co-stimulatory receptor. Thus, the method for engineering an immune cell may comprises (iii) expressing on the surface of the immune cell at least one co-stimulatory receptor.

According to certain embodiments, the method further comprises: (d) Introducing into said cell at least one polynucleotide comprising a nucleotide sequence encoding a co-stimulatory receptor; and

(e) Expressing said at least one co-stimulatory receptor.

A "co-stimulatory receptor", as used herein, is meant to be a member of a family of receptors that modulate the activation of T-lymphocytes by the T cell receptor (TCR). The receptors are responsive to one or more B7 antigens found on antigen presenting cells, and, depending upon the specific ligand-receptor combination, modulate a variety of T-cell functions such as the rate of clonal expansion, cell survival and cytokine production. Non- limiting examples of suitable co-stimulatory receptors to be expressed by an immune cell according to the invention include NKG2D (UniProtKB: P26718) and DAP10 (UniProtKB: Q.9UBK5).

According to certain embodiments, the immune cell expresses on its surface at least NKG2D.

According to certain other embodiments, the immune cell expresses on its surface at least DAP10.

The expression of the at least one co-stimulatory receptor may be transient or constitutively. Thus, according to certain embodiments, the at least one co-stimulatory receptor is transiently expressed by the immune cell. According to other certain embodiments, the at least one co-stimulatory receptor is constitutively expressed by the immune cell.

Delivery methods

The different methods described above involve introducing CAR into a cell. As non- limiting example, said CAR can be introduced as transgenes encoded by one plasmidic vector. Said plasmid vector can also contain a selection marker which provides for identification and/or selection of cells which received said vector.

Polypeptides may be synthesized in situ in the cell as a result of the introduction of polynucleotides encoding said polypeptides into the cell. Alternatively, said polypeptides could be produced outside the cell and then introduced thereto. Methods for introducing a polynucleotide construct into cells are known in the art and including as non-limiting examples stable transformation methods wherein the polynucleotide construct is integrated into the genome of the cell, transient transformation methods wherein the polynucleotide construct is not integrated into the genome of the cell and virus mediated methods. Said polynucleotides may be introduced into a cell by for example, recombinant viral vectors (e.g. retroviruses, adenoviruses), liposome and the like. For example, transient transformation methods include for example microinjection, electroporation or particle bombardment. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells.

Engineered immune cells

The present invention also relates to immune cells, e.g., isolated immune cells, or cell lines susceptible to be obtainable by said method to engineer cells.

In particular, an immune cell, e.g. isolated immune cell, according to the present invention comprises at least one CAR of the present invention. According to certain embodiments, said immune cell, e.g. isolated immune cell, comprises a population of CARs each one comprising different extracellular ligand binding domains. In particular, said immune cell, e.g. isolated immune cell, comprises one or more exogenous polynucleotide sequences encoding polypeptide(s) composing at least one CAR. Genetically modified immune cells of the present invention are activated and proliferate independently of antigen binding mechanisms.

An "immune cell", as referred to herein, means a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response. Said immune cell according to the present invention can be derived from a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human cells are CD34+ cells. Said immune cell can also be a dendritic cell, killer dendritic cell, a mast cell, a NK-cell, a B-cell or a T-cell selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T- lymphocytes or helper T-lymphocytes. According to particular embodiments, said immune cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T- lymphocytes. According to more particular embodiments, said immune cell can be derived from CD4+ T-lymphocytes. According to certain embodiments, the immune cell is a human immune cell, such as a human T-lymphocyte.

Since rapamycin directly inhibits immune cells, such as T cells, through interaction with the cytosolic protein FK-binding protein 12 (FKBP12) followed by inhibition of mTOR (mammalian target of rapamycin) by the FKBP12/rapamycin complex, it may thus be desirable to inhibit the formation of the endogenous FKBP12/rapamycin/mTOR complex.

The inhibition of the formation of the endogenous FKBP12/rapamycin/mTOR complex may be achieved, e.g., by introducing one or more amino acid substitution, including an amino acid substitution at position 2035, within the mTOR protein sequence (NCBI Reference Sequence: NP_004949.1; SEO ID NO: 37). Techniques for introducing an amino acid substation within the amino acid sequence of a protein are well known to a skilled person, and include as a non-limiting example site directed mutagenesis.

An immune cell of the present invention may thus be further modified to comprise within the endogenous mTOR protein at least an amino acid substitution at position 2035, wherein serine is replaced by another amino acid. Thus, according to certain embodiments, the immune cell comprises within the amino acid sequence of the endogenous mTOR protein one or more amino acid substitutions, including an amino acid substitution at position 2035 wherein serine is replaced by another amino acid, such as lie.

The inhibition of the formation of the endogenous FKBP12/rapamycin/mTOR complex may also be achieved, e.g., by inactivating the endogenous FKBP12 gene. By "inactivating" or "inactivation of" a gene it is intended that the gene of interest (e.g., the FKBP12 gene) is not expressed in a functional protein form. Techniques for inactivating a gene are well-known to those of skill in the art, and include as non-limiting example the use of specific rare-cutting endonucleases targeting this gene, such as TAL-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease. Further non-limiting examples include Cas9/Crispr or argonaute (Ago) based systems, such as disclosed in WO2014/191128 and Swarts et al (2014), respectively.

An immune cell of the present invention may thus be further modified to inactivate the endogenous FKBP12 gene. Thus, according to certain embodiments, the immune cell comprises an inactivated FKBP12 gene. Such immune cell thus does not express the FKPB12 protein.

To keep immune cells in a proliferation state, avoiding a precocious re-administration of new engineered immune cells, it may be appropriate to use virus-specific T cells (VSTs). Without being cytotoxic in their native form, VSTs are stimulated by endogenous viral antigen by engagement of their native receptors, and then are allowed to proliferate. Expansion and persistence would occur irrespectively of the presence of the CAR target antigen. When engineered according to the present invention, i.e. bearing the ectodomain switch system, the VSTs may benefit from their properties of proliferation without the presence of the CAR target antigen, while non-VSTs T cells would not proliferate and finally die. Donor-derived virus-specific T cells engineered to express a CD19 specific chimeric antigen receptor and the generation thereof has been described in Cruz et al. (2013).

Thus, according to certain embodiments, the immune cell is a virus-specific T cell (VST), preferably isolated from a donor.

Prior to expansion and genetic modification of the immune cells of the invention, a source of cells can be obtained from a subject through a variety of non-limiting methods. 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. In certain embodiments of the present invention, any number of T cell lines available and known to those skilled in the art, may be used. In another embodiment, said cell can be derived from a healthy donor, from a patient diagnosed with cancer or from a patient diagnosed with an infection. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics. In the scope of the present invention is also encompassed a cell line obtained from a transformed T- cell according to the method previously described. Modified cells resistant to an immunosuppressive treatment and susceptible to be obtained by the previous method are encompassed in the scope of the present invention.

An immune cell according to the present invention may further be modified to be allogenic. Thus, according to certain embodiments, the immune cell further comprise at least one inactivated gene selected from the group consisting of CD52, GR, TCR alpha, TCR beta, HLA gene, immune check point genes such as PDl and CTLA-4, or can express a pTalpha transgene. More particularly, the immune cell may comprise at least one inactivated gene selected TCR alpha or TCR beta genes. Such inactivation renders the TCR not functional in the cells. This strategy is particularly useful to avoid Graft versus Host Disease (GvHD). Methods for inactivating genes are known in the art, and include the use of rare-cutting endonucleases which able to selectively inactivate by DNA cleavage, preferably by double- strand break, the gene(s) of interest. Said genes may thus be inactivated by transforming the immune cell with a polynucleotide comprising a nucleotide sequence encoding a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break a gene selected from the group consisting of CD52, GR, TCR alpha, TCR beta, HLA gene, immune check point genes such as PDl and CTLA-4. Said rare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease or a TALE-nuclease. According to particular embodiments, said rare-cutting endonuclease is a TALE-nuclease. Preferred methods and relevant TALE-nucleases have been described in WO2013176915. According to other particular embodiments, said rare-cutting endonuclease is RNA-guided endonuclease such as Cas9 or DNA-guided endonuclease, such as Argonaute based techniques as described in WO2014189628.

An immune cell according to the present invention may further be modified to be resistant to chemotherapy drugs. Thus, according to certain embodiments, the immune cell further comprises at least one inactivated gene responsible for the cell's sensitivity to the drug (drug sensitizing gene(s)), such as the dcK and/or HPRT genes. Methods for inactivating genes are known in the art, and include the use of rare-cutting endonucleases which able to selectively inactivate by DNA cleavage, preferably by double-strand break, the gene(s) of interest. Said gene(s) may thus be inactivated by transforming the immune cell with a polynucleotide comprising a nucleotide sequence encoding a rare-cutting endonuclease able to selectively inactivate by DNA cleavage, preferably by double-strand break at least one gene responsible for the cell's sensitivity to the drug (drug sensitizing gene(s). Said rare- cutting endonuclease can be a meganuclease, a Zinc finger nuclease or a TALE-nuclease. According to particular embodiments, said rare-cutting endonuclease is a TALE-nuclease. Preferred methods and relevant TALE-nucleases have been described in WO2013176915. According to other particular embodiments, said rare-cutting endonuclease is RNA-guided endonuclease such as Cas9 or DNA-guided endonuclease, such as Argonaute based techniques as described in WO2014189628.

Alternatively, the resistance to drugs can be conferred to an immune cell, such as a T cell, by expressing a drug resistance gene. Variant alleles of several genes such as dihydrofolate reductase (DHFR), inosine monophosphate dehydrogenase 2 (IMPDH2), calcineurin or methylguanine transferase (MGMT) have been identified to confer drug resistance to an immune cell according to the invention.

Activation and expansion of T cells

Whether prior to or after genetic modification of the immune cells, even if the genetically modified immune cells of the present invention are activated and proliferate independently of antigen binding mechanisms, the immune cells, particularly T-cells of the present invention can be further activated and expanded generally using methods as described, for example, in U.S. Patents 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. T cells can be expanded in vitro or in vivo.

Generally, the immune cells of the invention are expanded by contact with an agent that stimulates a CD3 TCR complex and a co-stimulatory molecule on the surface of the T cells to create an activation signal for the T-cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13- acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T-cell.

As non-limiting examples, T cell populations may be stimulated in vitro 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) 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 is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPM I Media 1640 or, X-vivo 5, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (I L-2), insulin, I FN-g , 1L-4, 1L-7, GM-CSF, -10, - 2, 1L-15, TGFp, and TNF- or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanoi. Media ca n include RPM I 1640, A1M-V, DMEM, M EM, a-M EM, F-12, X-Vivo 1, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% C02). T cells that have been exposed to varied stimulation times may exhibit different characteristics

According to certain embodiments, said cells can be expa nded by co-culturing with tissue or cells. Said cells can also be expanded in vivo, for example in the subject's blood after administrating said cell into the subject.

Therapeutic applications

I mmune cells obtainable in accordance with the present invention are intended to be used as a medicament, and in particular for treating cancer in a patient (e.g. a human patient) in need thereof. Accordingly, the present invention provides immune cells for use as a medicament. Particularly, the present invention provides immune cells for use in the treatment of a cancer. Also provided are compositions, particularly pharmaceutical compositions, which comprise at least one immune cell of the present invention. In certain embodiments, a composition may comprise a population of immune cells of the present invention. The treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment. By autologous, it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor. By allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.

The invention is particularly suited for allogenic immunotherapy, insofar as it enables the transformation of immune cells, such as T-cells, typically obtained from donors, into non-alloreactive cells. This may be done under standard protocols and reproduced as many times as needed. The resultant modified immune cells may be pooled and administrated to one or several patients, being made available as an "off the shelf" therapeutic product.

The treatments are primarily to treat patients diagnosed with cancer. Particular cancers to be treated according to the invention are those which have solid tumors, but may also concern liquid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included. According to certain embodiments, the immune cell(s) or composition is for use in the treatment of a cancer, and more particularly for use in the treatment of a solid or liquid tumor. According to particular embodiments, the immune cell(s) or composition is for use in the treatment of a solid tumor. According to other particular embodiments, the immune cell(s) or composition is for use in the treatment of a liquid tumor. According to particular embodiments, the immune cell(s) or composition is for use in the treatment of a cancer selected from the group consisting of lung cancer, small lung cancer, breast cancer, uterine cancer, prostate cancer, kidney cancer, colon cancer, liver cancer, pancreatic cancer, and skin cancer. According to more particular embodiments, the immune cell(s) or composition is for use in the treatment of lung cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of small lung cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of breast cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of uterine cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of prostate cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of kidney cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of colon cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of liver cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of pancreatic cancer. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of skin cancer.

According to other particular embodiments, the immune cell(s) or composition is for use in the treatment of a sarcoma.

According to other particular embodiments, the immune cell(s) or composition is for use in the treatment of a carcinoma. According to more particular embodiments, the immune cell or composition is for use in the treatment of renal, lung or colon carcinoma.

According to other particular embodiments, the immune cell(s) or composition is for use in the treatment of leukemia, such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), and chronic myelomonocystic leukemia (CMML). According to more particular embodiments, the immune cell(s) or composition is for use in the treatment of acute lymphoblastic leukemia (ALL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of acute myeloid leukemia (AML). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of chronic lymphocytic leukemia (CLL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of chronic myelogenous leukemia (CML). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of chronic myelomonocystic leukemia (CMML).

According to other particular embodiments, the immune cell(s) or composition is for use in the treatment of lymphoma, such as B-cell lymphoma. According to more particular embodiments, the immune cell(s) or composition is for use in the treatment of primary CNS lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Hodgkin's lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Non- Hodgkin's lymphoma. According to more particular embodiments, the immune cell(s) or composition is for use in the treatment of diffuse large B cell lymphoma (DLBCL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Follicular lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of marginal zone lymphoma (MZL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Mucosa-Associated Lymphatic Tissue lymphoma (MALT). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of small cell lymphocytic lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of mantle cell lymphoma (MCL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Burkitt lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of primary mediastinal (thymic) large B-cell lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Waldenstrom macroglobulinemia. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of nodal marginal zone B cell lymphoma (NMZL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of splenic marginal zone lymphoma (SMZL). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of intravascular large B-cell lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Primary effusion lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of lymphomatoid granulomatosis. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of T cell/histiocyte-rich large B-cell lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of primary diffuse large B-cell lymphoma of the CNS (Central Nervous System). According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of primary cutaneous diffuse large B-cell lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of EBV positive diffuse large B-cell lymphoma of the elderly. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of diffuse large B-cell lymphoma associated with inflammation. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of ALK-positive large B-cell lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of plasmablastic lymphoma. According to other more particular embodiments, the immune cell(s) or composition is for use in the treatment of Large B-cell lymphoma arising in HHV8- associated multicentric Castleman disease.

According to certain embodiments, the immune cell(s) or composition is for use in the treatment of a viral infection, such as an HIV infection or HBV infection. According to certain embodiment, the immune cell of originates from a patient, e.g. a human patient, to be treated. According to certain other embodiment, the immune cell originates from at least one donor.

The treatment can take place in combination with one or more therapies selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

According to certain embodiments, immune cells of the invention can undergo robust in vivo immune cell expansion upon administration to a patient, and can persist in the body fluids for an extended amount of time, preferably for a week, more preferably for 2 weeks, even more preferably for at least one month. Although the immune cells according to the invention are expected to persist during these periods, their life span into the patient's body are intended not to exceed a year, preferably 6 months, more preferably 2 months, and even more preferably one month.

The administration of the immune cells or composition according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The immune cells or composition described herein may be administered to a patient subcutaneously, intradermal^, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intra lymphatic injection, or intraperitoneally.

According to certain embodiments, the immune cells or composition are/is administered by intravenous injection.

According to other certain embodiments, the immune cell(s) or composition is administrated parenterally.

According to certain other embodiments, the immune cell(s) or composition is administered intratumorally. Said administration can be done by injection directly into a tumor or adjacent thereto.

The administration of the cells or population of cells can consist of the administration of 104-109 cells per kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight including all integer values of cell numbers within those ranges. The cells or population of cells can be administrated in one or more doses. In another embodiment, said effective amount of cells are administrated as a single dose. In another embodiment, said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. According to certain embodiments, immune cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or nataliziimab treatment for MS patients or efaliztimab treatment for psoriasis patients or other treatments for PML patients. In further embodiments, the T cells of the invention may be used in combination with 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, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (Liu et al., Cell 66:807-815, 1 1; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Citrr. Opin. mm n. 5:763-773, 93). In a further embodiment, the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH, In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded genetically engineered immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

Also encompassed within this aspect of the invention are methods for treating a patient in need thereof, comprising a) providing at least one immune cell of the present invention, preferably a population of said immune cell; and b) administering said immune cell or population to said patient. Also encompassed within this aspect of the invention are methods for preparing a medicament using at least one immune cell of the present invention, and preferably a population of said immune cell. Accordingly, the present invention provides the use of at least one immune cell of the present invention, and preferably a population of said immune cell, in the manufacture of a medicament. Preferably, such medicament is for use in the treatment of a disease as specified above. It is particularly envisaged that the immune cell of the present invention is used (or is for use) in combination the multivalent ligand capable of binding to the first and second multimerizing ligand-binding domains. In this respect, the present invention contemplates administering an effective amount of the multivalent ligand of the first and second multimerizing ligand-binding domains to said patient. The multivalent ligand, such as rapamycin, may be administered to said patient, for example, at a dose of about 0.01 to 10 mg/kg body weight. According to certain embodiments, the multivalent ligand is administered at a dose of about 0.01 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.01 to 4 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.01 to 3 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.01 to 2 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.01 to 1 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.05 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.05 to 4 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.05 to 3 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.05 to 2 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.05 to 1 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.1 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.1 to 4 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.1 to 3 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.1 to 2 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.1 to 1 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.5 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.5 to 4 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.5 to 3 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.5 to 2 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 0.5 to 1 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 1 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose of about 2 to 5 mg/kg body weight. According to certain other embodiments, the multivalent ligand is administered at a dose or of about 2,5 to 5 mg/kg body weight.

The administration of the multivalent invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The multivalent ligand may be administered to a patient subcutaneously, intradermal^, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intra lymphatic injection, or intraperitoneally.

According to certain embodiments, the multivalent ligand is administered by intravenous injection.

According to certain other embodiments, the multivalent ligand is administered intratumorally, optionally together an immune cell or a population of an immune cell according to the present invention. Such approach prevents or limits the activation (on- switching) of the CAR containing immune cell outside the tumor (e.g., a solid tumor) to be treated.

Other definitions

- "ectodomain" refers to a part of a chimeric antigen receptor of the present invention which extends into the extracellular space (the space outside a cell). - "endodomain" refers to a part of a chimeric antigen receptor of the present invention which extends into the cytoplasm of a cell.

- Amino acid residues in a polypeptide sequence are designated herein according to the one-letter code, in which, for example, Q. means Gin or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.

- "Substitution" or "substituted" refers to modification of a polypeptide by replacing one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a polypeptide sequence is an amino acid substitution.

- "Conservative substitution" refers to a substitution of an amino acid residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; an amino acid having an aromatic side chain is substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain is substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

- "Non-conservative substitution" refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties. Non- conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid. - "Deletion" or "deleted" refers to modification of a polypeptide by removal of one or more amino acids in the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the polypeptide while retaining polypeptide function. Deletions can be directed to the internal portions and/or terminal portions of the polypeptide, in various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

- "Insertion" or "inserted" refers to modification of the polypeptide by addition of one or more amino acids to the reference polypeptide. Insertions can comprise addition of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the reference polypeptide.

- Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.

- "As used herein, "nucleic acid" or "polynucleotides" refers to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single stranded or double stranded.

- By " delivery vector" or " delivery vectors" is intended any delivery vector which can be used in the present invention to put into cell contact ( i.e "contacting") or deliver inside cells or subcellular compartments (i.e "introducing") agents/chemicals and molecules (proteins or nucleic acids) needed in the present invention. It includes, but is not limited to liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other appropriate transfer vectors. These delivery vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, or penetrating peptides. In these later cases, delivery vectors are molecule carriers.

- The terms "vector" or "vectors" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A "vector" in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV- BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

- By "lentiviral vector" is meant HIV-Based lentiviral vectors that are very promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells. By "integrative lentiviral vectors (or LV)", is meant such vectors as non limiting example, that are able to integrate the genome of a target cell. At the opposite by "non integrative lentiviral vectors (or NILV)" is meant efficient gene delivery vectors that do not integrate the genome of a target cell through the action of the virus integrase.

- Delivery vectors and vectors can be associated or combined with any cellula r permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques.

- By "cell" or "cells" is intended any eukaryotic living cells, primary cells and cell lines derived from these organisms for in vitro cultures.

- By "primary cell" or "primary cells" are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. As non-limiting examples cell lines can be selected from the group consisting of CHO- Kl cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells. All these cell lines can be modified by the method of the present invention to provide cell line models to produce, express, quantify, detect, study a gene or a protein of interest; these models can also be used to screen biologically active molecules of interest in research and production and various fields such as chemical, biofuels, therapeutics and agronomy as non-limiting examples. - By "stem cell" is meant a cell that has the capacity to self-renew and the ability to generate differentiated cells. More explicitly, a stem cell is a cell which can generate daughter cells identical to their mother cell (self-renewal) and can produce progeny with more restricted potential (differentiated cells).

- By "NK cells" is meant natural killer cells. NK cells are defined as large granula r lymphocytes and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes.

- by "mutation" is intended the substitution, deletion, insertion of up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty five, thirty, fourty, fifty, or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence. The mutation can affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

- by "variant(s)", it is intended a polypeptide variant obtained by mutation or replacement of at least one residue in the amino acid sequence of the parent molecule. - By "gene" is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region. The gene may further comprise a terminator, enhancers and/or silencers. - As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome. The term "locus" can refer to the specific physical location of a rare-cutting endonuclease target sequence on a chromosome. Such a locus can comprise a target sequence that is recognized and/or cleaved by a rare-cutting endonuclease according to the invention. It is understood that the locus of interest of the present invention can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e. in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples. - By "fusion protein" is intended the result of a well-known process in the art consisting in the joining of two or more genes which originally encode for separate proteins or part of them, the translation of said "fusion gene" resulting in a single polypeptide with functional properties derived from each of the original proteins.

-"identity", "percentage of sequence identity," "% sequence identity" and "percent identity" are used herein to refer to comparisons between an amino acid sequence and a reference amino acid sequence. The "% sequence identify", as used herein, is calculated from the two amino acid sequences as follows: The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default BLOSUM62 matrix with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (for each additional null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference amino acid sequence. For example, polypeptides having at least 80%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated.

- "Reference sequence" or "reference amino acid sequence" refers to a defined sequence to which another sequence is compared.

- The term "subject" or "patient" as used herein includes all members of the animal kingdom including non-human primates and humans. - The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description. Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and sub ranges within a numerical limit or range are specifically included as if explicitly written out.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

Examples

Example 1: Development of a small molecule (rapamycin) switch-on mcCAR19 and mcCAR123 - mRNA delivery - surface detection

Constructs and mRNA preparation

All constructs originated from the pCLS24707 (SEQ ID NO: 38) which encode the a- chain (SEQ. ID NO: 39), β-chain (SEQ ID NO: 40) and γ-chain (SEQ ID NO: 41) of the multichain CAR (mcCAR). The sequences coding for the FRB domain (SEQ ID NO: 2) and FKBP domain (SEQ ID NO: 1) were synthetized de novo (GeneCust). The scFV, hinge-transmembrane- intracytoplasmic alpha chain domain, the FRB and the FKBP were further amplified by PCR to generate golden gate assembly compatible fragments (SEQ ID NO: 42 to 45). In addition an FRB-FKBP and an FKBP-FRB fragment were generated (SEQ ID NO: 46 to 47) using a four- EAAAR- linker (SEQ ID NO: 19) or a -GS-4x-EAAAR- linker (SEQ ID NO: 20) and standard molecular biology procedures. Fragments were then assembled using round of restriction and ligation leading to pCLS26563, pCLS26564, pCLS26881, and pCLS27123 (SEQ ID NO: 48 to 51). The respective amino acid sequences encoded by these constructs are shown in SEQ ID NOs: 52 to 55. All individual chains were amplified by PCR using oligo pairs a-chain-F/ a-chain-R, β- chain-F/ β-chain-R and y-chain-F/ γ-chain-R (SEQ. ID NO: 56 to 61) prior to mRNA synthesis. mRNA encoding the different a-chains, β-chain, γ-chain were in vitro transcribed from the PCR product and polyadenylated using the mMessage mMachine T7 Ultra kit (Life technologies) following the manufacturer's instructions. RNAs were purified with RNeasy columns (Qjagen), eluted in cytoporation medium T and quantified by measuring absorbance at 260 nm using a Nanodrop ND-1000 spectrophotometer. Quality of the RNA was verified on a denaturing formaldehyde/MOPS agarose gel.

Transfection T lymphocytes were transfected by electrotransfer of messenger RNA using an

AgilePulse MAX system (Harvard Apparatus) 3 to 6 days after activation. Following removal of activation beads, cells were pelleted, resuspended in cytoporation medium T at >28xl06cells/ml. 5xl06cells were mixed with 6.9 μg total RNA (2.5 μg a chain, 1.9 μg β chain and 2.5 μg y chain) or with 8.4 μg total RNA (4 μg modified a chain, 1.9 μg β chain and 2.5 μg y chain) into a 0.4 cm cuvette. The electroporation consisted of two 0.1 ms pulses at 1200 V followed by four 0.2ms pulses at 130V. Following electroporation, cells were diluted into 2mL culture medium and incubated at 37°C/ 5% C0 ₂ . 2 hours after mRNA electrotransfer, vehicle (DMSO) or Rapamycin (100 nM) was added for 19 hours.

Flow cytometry First labelling for the detection of the a-chain was performed with anti-Fab'2-Biotin

(goat anti-mouse IgG, Fab'2 fragment specific, 115-066-072, Jackson Immunoresearch) in PBS FBS2%, EDTA 2mM, azide 0.1% for 20 min at 4°C followed by a two washing steps with PBS FBS2% EDTA 2mM azide 0.1%. Second labelling was performed with Streptavidin-APC in PBS FBS2% EDTA 2mM azide 0.1% for 20 min at 4°C followed by a washing step in PBS FBS2% EDTA 2mM azide 0.1% and a washing step in PBS. Cell viability was monitored using the efluor450 (ebioscience 65-0863-14) in PBS for 20min 4°C, followed by a washing step with PBS FBS2% EDTA 2mM azide 0.1% and fixed in 2% PFA. Flow cytometry was performed using the MACSQ.UANT (Miltenyi Biotec) and data analysis was performed with the FlowJo software. The data obtained clearly indicated an improved surface exposition in presence of rapamycin when the FRB-FKBP or the FKBP-FRB domains were incorporated in the a-chain (Figure 3A and B).

Example 2: Development of a small molecule (AP21967) switch-on mcCARCD19 - mRNA delivery - surface detection

Constructs, mRNA preparation and flow cytometry

To design an integrated system to switch the scFv/antigen interaction between on/off states, either the FRB, the FKBP12, or fusion of the FRB and FKBP12 were inserted between the CD8a hinge and the scFv domains (Figure IB). As a starting experiment, primary T cell with mRNAs encoding each chain of the multichain CAR (mcCAR) were transfected. Upon addition of rapamycin, changes in the detection of the extracellular hinge domain were monitored by tracking the Fab'2 domain of CD19-targeting scFv (100 nM, 20 h). In the absence of the small molecule (rapamycin), it was found that a high level of surface detection could only be achieved for the wild type mcCAR and the FKBP-mcCAR with above 90% of positive cells with an overall high MFI as shown in Figure 3A. The presence of both FKBP and FRB in the stalk region virtually abolished surface detection of the CD19 ScFV, independently of their reciprocal position (below 40 % of positive cells, with up to 40 fold decrease in MFI when compared to the mcCAR). Interestingly, while the addition of rapamycin barely effected the mcCAR, FRB-mcCAR and FKB-mcCAR constructs, when considering the percentage of positive cells or the MFI (Figure 3B), it strongly improved (up to 15 fold when considering the MFI and 3 fold when considering the percentage of positive cell) the surface detection of the FKBP/FRB-mcCAR and FRB/FKBP-mcCAR constructs, turning the system from an off to an on state. This variation of detection upon addition of rapamycin may results from different factors, including stabilization of the CAR chain that is containing the switch on component. However it has to be noted that the small molecule was always required to efficiently turn-on the detection of the FKBP/FRB-CAR of the CAR at the surface of the T-cell.

Synthetic non-immunosupressing AP21967 rapamycin synthetic analog was also tested, which binds to the FKBP12 but does not promote the binding to the FRB domain of imTOR. Accordingly, T2098L mutation was introduced in the FRB domain (referred as FRB*) to allow the FKBP/AP21967/FRB* complex to be formed.

The T2098L mutation in the FRB domain was introduced in the FKBP-FRB domain using commercially available kits (Agilent) and standard molecular biology procedures leading to the FKBP-FRB* domain (SEQ ID NO: 62). Assembly of the alpha chain containing the scFv, the FKBP-FRB* domain, hinge-transmembrane-intracytoplasmic domain leaded to pCLS27039 (SEQ. ID NO: 63). The amino acid sequence encoded by this construct is shown in SEQ ID NO: 64. FRB* refers to a variant of FRB having the T2098L mutation (SEQ ID NO: 4). mRNA preparation, transfection and flow cytometry measurement of surface presentation was performed as described in example 1 using AP21967 instead of Rapamycin.

The data obtained clearly indicated an improved surface exposition in presence of AP21697 when the FKBP-FRB* domains was incorporated in the a-chain (Figure 4).

To evaluate the AP21967 usable dose range for the switch-on system a dose response assay was performed (Figure 8A). The results obtained indicated a maximum signal induction at 100 nM and an EC50 value of approximately ΙΟηΜ (8.2-10.1 nM) that was independent from the amount of transfected engineered CAR. To validate the portability of the switch-on approach, a CAR targeting CD123 was also engineered. As demonstrated by a similar EC50 value of 10 nM (7.3-8.7 nM, Figure 8B), it was found that the nature of the scFv did not influence the switch-on properties. Remarkably, the EC50s are in range with rapamycin concentrations reported in peripheral blood or tumor tissue of patients, suggesting that the switch-on system may be sensitive to clinically relevant concentration.

Example 3: Development of a small molecule (AP21967) switch-on mcCAR - mRNA delivery - Induced cytotoxicity The cytolytic activity of engineered T-cells endowed with the FKBP-FRB* mcCAR CD19 from example 2, was assessed using a flow cytometry-based cytotoxicity assay. In this assay target cells presenting the CAR target antigen (Daudi CD19 positive) are labelled with CellTrace™ CFSE or and control cells with CellTrace™ violet. The mixed target cell populations (1:1 ratio) was co-incubate at 37°C with various ratio of engineered effector CAR T cells (Effector/Target ratio of 20:1) in a final volume of X-Vivo-15 media lOOuL, for a fixed time periods (5h) in presence of vehicle (Ethanol) or AP21967 (100 nM).

The whole cell population was recovered, washed in PBS and labeled with eFluor780 viability marker before being fixed by 2% PFA. Fixed cells were analyzed by flow cytometry to determine their viability. Flow cytometry and data analysis were performed as described in example 1.

The data obtained clearly indicated an improved switched-on cytolytic activity in presence of AP21697 (Figure 5A).

A dose response was also performed (0, 1, 5, 10, 33, 100 nM) of the AP21967 and measured the resulting cytolytic capacities of the engineered CAR T-cells. It resulted that the level of target cell killing correlated, as expected, with variation of the AP21967 (Figure 5B). It was calculated an EC50 of approximately ΙΟηΜ (12.7 nM), in range of the one determined using the surface detection. The level of ta rgeted cell killing also correlated with the level of CAR detection (Figure 9). Altogether, the results presented here provide the proof of principle of engineering the hinge domain of a CAR molecule to create an integrated switch-on system for logic gating strategies.

Example 4: development of a small molecule (AP21967) switch-on mcCAR - mRNA delivery - other small molecule competition tunin mRNA preparation, transfection and flow cytometry measurement of surface presentation was performed as described in example 2, incubating transfected T-cells simultaneously with 10 nM of AP21967 and increasing amounts Tacrolimus.

Conditions: AP21967: 10 nM, Tacrolimus: 0 nM, 10 nM, 30 nM, 100 nM or 500 nM . T- cells were incubated for 20 hours at 37°C/ 5% C02.

The data obtained clearly indicated the possibility to tune the surface presentation of the engineered CAR, due to the small molecule AP21967, with a second small molecule (Tacrolimus) when the FKBP-FRB* domains was incorporated in the a-chain (Figure 6). Example 5: Development of a small molecule (AP21967) switch-on scCAR - mRNA delivery - surface detection

The CAR extracellular domains (alpha chain) presented in example 2 were used as template to prepare plasmid DNA encoding single chain CARs (scCARs). The CD8 alpha transmembrane domain (SEQ ID NO: 32), the intracytoplasmic signalling region of the ζ-chain of the CD3-T cell receptor (SEQ. ID NO: 28) and the signalling domains from co-stimulatory 4- 1BB (CD137) (SEQ ID NO:31) were used to complete the CARs. scCARs were assemble by Golden Gate cloning using round of restriction and ligation, according to standard molecular biology procedures, leading to pCLS27572 (FKBP-FRB*) (SEQ ID NO:65), pCLS27603 (FKBP) (SEQ ID NO:66), pCLS27604 (FRB*) (SEQ ID NO:67). The respective amino acid sequences encoded by these constructs are shown in SEQ ID NOs: 68 to 70. mRNA preparation (using oligo pair scCAR-F (SEQ ID NO: 71) and scCAR-R (SEQ ID NO: 72) that are located in the CAR and on the plasmid respectively) and transfection and flow cytometry measurement of surface presentation was performed as described in example 1 using AP21967 instead of Rapamycin. Primary labelling for the detection of the scCARs was performed with Fc-tagged recombinant CD123 (Lake Pharma) in PBS FBS2%, EDTA 2mM, azide 0.1% for 20 min at 4°C followed by a two washing steps with PBS FBS2% EDTA 2mM azide 0.1%. Secondary labelling was performed with PE labeled Goat Anti-Mouse IgG (subclasses l+2a+2b+3) Fey Fragment Specific (Jackson Immunoresearch) in PBS FBS2% EDTA 2mM azide 0.1% for 20 min at 4°C followed by a washing step in PBS FBS2% EDTA 2mM azide 0.1% and a washing step in PBS. Following the extracellular labelling, the cell viability was monitored using the efluor450 or efluor780 (ebioscience) in PBS for 20min 4°C, followed by a washing step with PBS FBS2% EDTA 2mM azide 0.1% and fixed in PFA 2%.

The data obtained clearly indicated an improved surface exposition in presence of AP21697 when the FKBP-FRB* domains was incorporated in the a-chain (Figure 7).

Example 6: Development of a small molecule (Rapamycin) switch-on mcCAR in combination with mTOR genome editiri Rapamycin directly inhibit T cells through interaction with the cytosolic protein FK- binding protein 12 (FKBP12) followed by inhibition of mTOR by the FKBP12/rapamycin complex.

Designer nucleases that create a single or a double strand break that target the sequence surrounding the triplet (or the triplet itself) coding for the amino acid (Serine) 2035 of Serine/threonine-protein kinase mTOR are designed and constructed/produced. To perform the gene correction/mutation of position 2035, a donor DNA containing the desired mutated base(s) surrounded by two homology arms of the endogenous sequence was designed. Additional silent mutations are added to prevent cleavage of the donor DNA, or the corrected/mutated genomic DNA by the designer nuclease.

T cells are transfected or transduced with genetic material coding for the designer nuclease and the donor DNA. T cells that contained the desired mutation at the endogenous locus are then selected or isolated. Improved expansion properties of the engineered T cell in presence of rapamycin are recorded and compared to the non-engineered T cell. The switch on CARs presented in example 1 are then implemented in the newly engineered T cells and improved surface presentation of the CARs and cytolytic properties in presence of rapamycin is recorded.

Example 7: Development of a small molecule (Rapamycin) switch-on mcCAR in combination with FKBP12 fienome editin (knock-out)

Rapamycin directly inhibit T cells through interaction with the cytosolic protein FK- binding protein 12 (FKBP12) followed by inhibition of mTOR by the FKBP12/rapamycin complex.

Designer nucleases that create a double strand break that target the sequence of the FKBP12 gene are designed and constructed/produced. T cells are transfected or transduced with genetic material coding for the designer nuclease. T cells that contained the desired knock-out at the endogenous locus are then selected or isolated. Improved expansion properties of the engineered T cell in presence of rapamycin are recorded and compared to the non-engineered T cell. The switch on CARs presented in example 1 are then implemented in the newly engineered T cells and improved surface presentation of the CARs and cytolytic properties in presence of rapamycin is recorded.

Example 8: Antitumor activity study of human modified inductible CD123 CAR+ T cells in nofi mice intravenously injected with molml3-luc tumor cells

The aim of the study was to demonstrate the anti-tumor activity in vivo of human T- cells genetically modified by Cellectis to express an inducible Chimeric Antigen Receptors (CAR) directed against the human CD123 antigen. This inducible system works with a non- immunosupressing rapamycin synthetic analog (AP21967, developed by ARIAD, # 635055).

The anti-tumor activity of human T-cells expressing a switch-on mcCAR-CD123 as described in Example 1 , was assessed in NOG mice intravenously injected with MOLM13-Luc tumour cells. Repeated injections of rapamycin synthetic analog AP21967 was performed as shown in Figure 10 (3 mg/kg/inj) into the peritoneal cavity of mice (Intraperitoneal^, IP). To establish the MOLM13-Luc cell line, MOLM13 cells (DSMZ ACC 554) have been transduced with a lentivirus encoding the GFP and the firefly luciferase (amsbio LVP438- PBS). The GFP-positive cells have been selected with Neomycin (ref 10131-027, Gibco, Life Technologies, Saint-Aubin, France). MOLM13-Luc cells are grown in suspension at 37°C in a humidified atmosphere (5% C02, 95% air) into culture medium RPMI 1640 containing 2.05 mM L-glutamine (ref: BE12-702F, Lonza) supplemented with 15% fetal bovine serum (ref: 3302, Lonza, Verviers, Belgium), lOOU/mL Penicillin and 100μg/mL Streptomycin (ref: DE17- 602E, Lonza). The cells are counted in a hemocytometer and their viability is assessed by 0.25% trypan blue exclusion assay and are passed twice weekly (0.8 millions/mL) in fresh culture medium. The day of injection to mice, frozen human T-cells transformed with the switch-on mcCAR-CD123 CAR are assessed to be within a range of 1-2.106 live cells per mL.

Eighteen (18) healthy female NOG (NOD.Cg-Prkdcscidll2rgtmlSug/JicTac) mice, 7-8 weeks old, were obtained from Taconic (Ry, Danemark) and bred according to NRC Guide for the Care and Use of Laboratory Animals The intravenous injection of MOLM13-Luc cells (0.25x106 cells/mouse) were performed on D-7.

The cell injection and treatment were scheduled as follows:

Group 1 (No transduced T cells/Vehicle) will receive a single IV injection of x No transduced T-cells (200 μΙ_ in RPMI 1640) on DO, followed by 2 daily IP injections of vehicle for 10 consecutive days (2Q.lDxlO),

Group 2 (No transduced T cells/AP21967) will receive a single IV injection of x No transduced T-cells (200 μΙ_ in RPMI 1640) on DO, followed by 2 daily IP injections of AP21967 at 3 mg/kg/inj for 10 consecutive days (2Q.lDxlO), Group 3 (CAR T cells/Vehicle) will receive a single IV injection of x modified CAR T-cells including X CAR-positive T-cells (200 μΙ_ in RPMI 1640) on DO, followed by 2 daily IP injections of vehicle for 10 consecutive days (2Q.lDxlO),

Group 4 (CAR T cells/AP21967) will receive a single IV injection of x modified CAR T- cells including X CAR-positive T-cells (200 μΙ_ in RPMI 1640) on DO, followed by 2 daily IP injections of AP21967 for 10 consecutive days (2QlDxlO).

T cell injection took place at DO, D2, D4, D7, D10, D15 and D21, while the mice were injected twice a day with rapalog.

Mice were monitored daily with respect to their body weight measurements, clinical and mortality records, and treatment were recorded on Vivo Manager ^® database (Biosystemes, Dijon, France). Survival curves and Graph body weight are respectively reported in Figure 11 and Figure 12, from where it is apparent that the mcCAR-CD123 CAR induced by AP21967 increased the survival of the mice.

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