Field of the invention

The present invention generally relates to a new generation of Chimeric Antigen Receptor (CAR) specific for tumor cells expressing IL-3 Receptor alpha (also named cluster of differentiation 123 (CD123)). In particular, the present invention also relates to engineered immune cells expressing the anti-CD123 CAR of the invention, and said engineered immune cells for their use in the treatment of pathologies related to CD123+ cancer cells, with more specificity for cancer cells, with less side effects such as GVHD, cytokine releasing syndrome (CRS) than previous engineered immune cells, in particular in patients treated with anti-cancer drugs affecting the survival of immune cells. Background of the invention

Induction treatments for lymphoproliferative diseases such as leukemia and i n particular for acute myeloid leukemia (AM L) have remained largely unchanged for nearly 50 years. Such standard induction chemotherapy ca n induce complete remissions, but patients eventually relapse and succumb to the disease, calling for the development of novel therapeutics for AM L, in particular for relapsed refractory AM L.

Similar observations account for aggressive lymphoproliferative diseases such as Blastic plasmacytoid dendritic cell neoplasm (BPDCN). These diseases remain of poor prognosis. I mmunophenotyping of these cancerous cells have revealed that the interleukin 3 receptor alpha chain (I L-3Ra; CD123 - NCBI reference: NP_001254642) is a potential immunotherapeutic target since it is over-expressed on these tumor cells as compared to normal cells. Additionally, two phases I trials for CD123-specific therapeutics have been completed with both drugs displaying good safety profiles (ClinicalTrials.gov I D: NCT00401739 - anti-CD123 monoclonal antibody- and NCT00397579 - a diphtheria toxin/interleukin 3 fusion protein)). The selectivity of CD123 targeting drugs for cancer cells over non-cancerous cells remains to be improved. Alternative, more potent and specific therapies targeting CD123 are required to reach a sustainable and long lasting anti-leukemic activity, with no relapse and less side effects.

A possibly more potent alternative therapy for the treatment of Leukemia could be the use of immune cells expressing chimeric antigen receptors (CARs) that selectively direct immune cells' specificity towards cell surface tumor associated antigens (TAAs) in an M HC- independent manner (Jena, Dotti et al. 2010) and destroy them. Moreover, because engineered immune leaving cells may exert a continuous action and survey on cancer cells, (at least until it is cleared by the host immune system in the case of "allogeneic" cells), it provides an important means of fighting replicating cancer cells.

CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single or multiple fusion molecule(s). I n general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody variable fragment (scFv), comprising the light a nd heavy va riable fragments of a monoclonal antibody (VL and VH respectively) joined by a flexible linker or ca n be based on receptor domains. Recent advances in the development of CAR demonstrated the feasibility of adding sequences specifically recognized by a monoclonal antibody into the extracellular domain of CARSs to control the number of engineered cells in vivo using the monoclonal antibody specific for said sequence \N 02016120216 Al. I n mouse models of human ca ncer, CARs can redirected the CAR-expressing cells against antigens expressed at the surface of tumor cells with an efficiency dependent on the nature and length of each domain (Condomine M .et al., 2015 Plos One 10(6):e0130518). So far autologous transfer of CAR- expressing specific T cells, alone, has been shown to be successful in treating specific forms of cancer despite side effects such as cytokine storm, non specific destruction of healthy cell populations, or unwanted specific immune reactions (Park, Rosenberg et al. 2011).

To make these treatments available for a broad population of patients, so ca lled "allogenic" T cells expressing a CAR have been prepared for their use in human suffering cancer. I n that case, immune cells isolated from a healthy donor are engineered (mainly by inactivating the expression of M HC and/or TCR gene) to modify the cell surface expression of molecules involved in the rejection of engineered cells by the host and/or those involved in the attack of healthy host cells/tissues by engineered cells. Thus, CAR that not only replace the TCR but also direct the T cell against a tumor expressing antigen. I mmunotherapy using CAR-expressing so called "allogeneic" T cells (also ca lled universal or "off the shelf" T cells) was recently implemented and the two first patients treated with such cells are still in remission about two years after treatment.

The extent to which such engineered cells still proliferate and survive in hosts remains largely unknown, especially in patients already treated with chemotherapy agents that affects immune cell functioning and/or survival. Moreover, there are aspects of such therapy that may be improved, such as efficiency and/or specificity against cancer cells over healthy cells, persistence and capacity to destroy cancer cells nested in tissues, in solid tumors, to overcome relapsed cancer cells, means of control of the cytokine release syndrome. Thus, there is still a need for developing efficient, more specific and safer treatments for these pathologies, in particular for their aggressive or refractory/relapsed forms.

Contrary to what was believed previously, that is to say that using a neutralizing antibody for preparing a CAR and cells expressing it, would be more efficient than using a non blocking antibody, the inventors' tested the hypothesis that a CAR made from a discriminating non blocking antibody linked to appropriate hinge TM and intracellular domains capable together of activating a CTL activity, would be more efficient than a CAR made using a neutralizing Ab.

The inventors identified two anti-CD123 antibodies, namely 6H6 and 9F5, that unexpectedly discriminate CD123 overexpressing cancer cells from CD123 healthy cells and were more efficient than previous neutralizing anti-CD123 Ab, when derived as a CAR expressed at the cell surface of an immune cell. When engineered as a CAR in "allogeneic immune T cells", this new generation of anti- CD123 CAR, were more specific and efficient in the treatment of AML and of BPDCN with less side effects as compared to previous treatments.

Summary of the invention

The present invention provides a CD123 specific chimeric antigen receptor (CD123 CAR) comprising:

• i) an extracellular binding domain comprising : a scfv that specifically binds to CD123 comprising CDRs or a VH and a VL from a non blocking anti-CD123 antibody, a hinge, · ii) a transmembrane domain, preferably from CD8 alpha and

• iii) an intracytoplasmic domain, preferably comprising a CD3zeta domain and a 4-1BB costimulatory domain,

• iv) a monoclonal antibody (mAb)-specific epitope.

The present invention also provides a CD123 CAR as above further comprising a transduction enhancer. In a preferred embodiment said scfv specifically binds to CD123 expressed or over expressed on cancerous cells,

In a preferred embodiment said scfv is comprising a VH and a VL from 6H6 antibody (6H6) separated by a linker (L), optionally said scfv is humanized. In a preferred embodiment said scfv is comprising a VH and a VL from 9F5 antibody

(9F5), separated by a linker (L), optionally said scfv is humanized,

The present invention provides a CD123 CAR as above wherein L comprises a sequence (GGGGS)n with n = 1 -10, more preferably a linker L of sequence (GGGGS) ₃ .

The present invention provides a CD123 CAR as above wherein said extracellula r domain comprises at least two monoclonal antibody (mAb)-specific epitopes, preferably four monoclonal antibody (mAb)-specific epitopes, more preferably four monoclonal antibody (mAb)-specific epitopes inserted into the linker L of the scfv and/or into the hinge.

The present invention provides a CD123 CAR as above, wherein said molecular antibody (mAb)-specific epitope, is a mAb-specific epitope specifically recognized by an monoclonal antibody selected from ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, Q.BEND-10 and ustekinumab, preferably from rituximab (R) and/or from Q.BEN10 (Q.).

The present invention provides a CD123 CAR as above, wherein the mAb-specific epitope is selected from CPYSNPSLC, NSELLSLINDMPITNDQKKLMSNN, CQFDLSTRRLKC, CQYNLSSRALKC, CVWQRWQKSYVC, CVWQRWQKSYVC, SFVLNWYRMSPSNQTDKLAAFPEDR, SGTYLCGAISLAPKAQJKE, ELPTQGTFSNVSTNVSPAKPTTTA, ELPTQGTFSNVSTNVSPAKPTTTA, GQN DTSQTSSPS.

The present invention provides a CD123 CAR as above, comprising 2 mAb-specific epitopes having an amino acid sequence of CPYSNPSLC or 3 mAb-specific epitopes comprising an amino acid sequence of CPYSNPSLC and one mAb-specific epitope comprising an amino acid sequence of ELPTQGTFSNVSTNVSPAKPTTTA. The present invention provides a CD123 CAR as above, comprising: an extracellular binding domain comprising : a scfv that specifically binds to CD123 over expressed on cancer CD123+ cells comprising CDRs from 6H6 or from 9F5, a hinge,

• ii) a transmembrane domain from CD8 alpha,

• iii) an intracytoplasmic domain comprising a CD3zeta domain and a 4- 1BB costimulatory domain,

• iv) at least two monoclonal antibody (mAb)-specific epitopes. The present invention provides a CD123 CAR as above, comprising: a hinge from CD8 alpha, a hinge from IgGl a hinge from Fcgamma III, as in PCT/EP2015/057331.

I n particular aspects, the present invention provides a CD123 CAR comprising no sequence having identity with the human CD28 NP_006130.1 or comprising no sequence having identity with a fragment thereof.

The present invention provides a CD123 CAR as above, wherein said intracellular domain comprises an intracellular death domain as in PA201770037 or USP62/436749 or a domain conferring resistance to hypoxia as in WO2015092024 .

The present invention provides a polynucleotide encoding a CD123 specific chimeric antigen receptor (CD123 CAR) according to any one of the above.

The present invention provides a polynucleotide encoding a CD123 specific chimeric antigen receptor (CD123 CAR) comprising successively, a sequence coding a self-cleaving peptide, preferably a self-cleaving 2A peptide or an I RES and an open reading frame encoding a CD123 CAR of the invention.

The present invention provides a polynucleotide comprising a TALEN -binding sequence, a 2A peptide in frame with an endogenous gene open reading frame. The present invention provides an expression vector comprising a polynucleotide according to the above.

The present invention provides an expression vector further comprising an exogenous promotor controlling the expression of the CD123 CAR. The present invention provides a viral particle comprising a polynucleotide according to any one the above or an expression vector according to any one of the above.

Under certain aspects, the present invention provides a viral particle as above comprising a viral protein from AAV6 and an Inverted terminal repeat (ITR) from AAV2 and a polypeptide or an expression vector according to the present invention. An engineered immune cell expressing at the cell surface a CD123 CAR according to any one of the above is another object of the present invention.

Thus, the present invention provides an engineered immune cell as above endowed with a polynucleotide according to the above, allowing a CD123 CAR according to any one of the above to be expressed at the cell surface. The present invention provides an engineered immune cell according to the above wherein said polynucleotide is under the control of an endogenous promotor.

The present invention provides an engineered immune cell according to the above comprising at least one edited gene, preferably an inactivated T Cell Receptor (TCR) gene, more preferably a TCR knocked out (KO) gene, even more preferably an alpha TCR knocked out (KO) gene comprising a knock in sequence coding a CD123 CAR of the invention.

The present invention provides an engineered immune cell as above comprising a CD52 KO gene.

The present invention provides an engineered immune cell as above comprising a human deoxycytidine kinase (dCK) KO gene. The present invention provides an engineered immune cell according to any one of the above wherein the expression of at least one MHC protein, is suppressed, preferably by inactivation, more preferably by inactivation of the gene encoding the beta 2 microglobulin protein or the Class II Major Histocompatibility Complex Transactivator (CIITA), using an endonuclease targeting said gene. The present invention provides an alpha beta TCR inactivated - MHC deficient engineered immune cells expressing a Chimeric Antigen Receptors (CAR) specific for CD123+ cancer cells in which the extracellular ligand binding domain comprises a scFV derived from a non blocking CD123 monoclonal antibody, preferably from 6H6 or 9F5, conferring specific immunity (cytolytic activity) against CD123 overexpressing cells.

The present invention provides an engineered immune cell as any one of the above comprising a human PD1 KO gene.

The present invention provides a pharmaceutical composition comprising a pharmaceutical excipient and the engineered immune cell according to any one the above. The present invention provides an engineered immune cell as any one of the above or the pharmaceutical composition as above for use in the treatment of a patient in a need thereof.

The present invention provides an engineered immune cell according for use in the treatment of acute myelogenous leukemia (AML) of BPDCN, preferably refractory/relapsed AML or BPDCN, or for use before, or during a bone marrow transplant.

The present invention provides an engineered immune cell according to the above for use in the treatment of CD123 expressing acute myelogenous leukemia (AML), preferably refractory/relapsed CD123 expressing AML, CD123 expressing BPDCN, or for use before or during a bone marrow transplant. The present invention provides an engineered immune cell according to any one of the above for use in the treatment of CD123 overexpressing acute myelogenous leukemia (AML), preferably refractory/relapsed CD123 over expressing AML, CD123 overexpressing BPDCN, or for use before or during a bone marrow transplant.

The present invention provides an engineered immune cell according to any one of the above for use in the treatment of CD123 expressing refractory anemia with excess blasts (RAEB), acute myeloid leukemia (AML), acute lymphoid leukemia (ALL).

The present invention provides an engineered immune cell as above or the pharmaceutical composition as above for use in the treatment of AML patients with cytogenetic abnormalities and/or TP53 mutations. The present invention provides an engineered immune cell as above or the pharmaceutical composition as above for use in the treatment of AML patients in remission.

The present invention provides an anti-CD123 CAR with advantageous properties when expressed in T cells. The present invention is drawn to new anti-CD123 chimeric antigen receptors (anti-

CD123 CAR), comprising a VH and a VL from a non blocking anti-CD123 antibody, preferably from 6H6 or from 9F5 monoclonal antibody, optionally humanized that discriminates CD123 - expressing cancer cells from CD123 expressing -healthy cells.

The present invention relates to an anti-CD123 CAR which extracellular binding domain comprises a (scFv) prepared using a VH and a VL from anti CD123 antibody that discriminates CD123+ - positive cancer cells from CD123+-healthy cells, optionally humanized, a hinge, an intracellular domain and a costimulatory domain, a suicide domain, optionally inserted into the hinge and/or into the scfv.

The invention also relates to a humanized anti-CD123 CAR comprising at least one extracellular ligand binding domain comprising a scfv from humanized 6H6 antibody or from humanized 9F5 antibody, a hinge, an intracellular domain and a costimulatory domain, a suicide domain inserted into the hinge, and into the linker of said scfv from humanized 6H6 antibody or from humanized 9F5 antibody.

In particular embodiments, the suicide domain comprises a mAb-specific epitope recognized by a specific monoclonal antibody (mAb-specific epitope) allowing cell sorting and/or cell depletion as described in PCT/EP2016/051467.

Single-chain or multi-chain anti-CD123 CAR comprising a scfv from 6H6 or from 9H5 Ab were prepared based on and as disclosed in PCT/EP2015/063656 and PCTUS2013/058005. The scFv of the invention, which is formed of the VH and VL polypeptides from non blocking antibodies discriminating CD123+ cancer cells from CD123+ healthy cells, such as Ab 6H6 or 9F5, may itself have different structures depending on the position of inserted mAb specific epitope(s) and on the nature of linkers and hinge. The structure of the CAR allowing cell sorting and cell depletion, comprises at least one epitope recognized by a monoclonal antibody (named mAb-driven sorting/depletion system or mAb-specific epitope recognized by a specific monoclonal antibody or mimotope) and is inserted into the extracellular binding domain of the anti-CD123 CAR within the Linker between the VH and the VL of the scFv and/or in the hinge.

The mAb-specific epitopes, allowing cell sorting and cell depletion, may further be combined to a linker Li.

Examples of epitope recognized by a monoclonal antibody (or mAb-specific epitope) may be one of the following epitopes: CPYSNPSLC, NSELLSLINDMPITNDQKKLMSNN, CQFDLSTRRLKC, CQYNLSSRALKC, CVWQRWQKSYVC, CVWQRWQKSYVC,

SFVLNWYRMSPSNQTDKLAAFPEDR, SGTYLCGAISLAPKAQJKE, ELPTQGTFSNVSTNVSPAKPTTTA, ELPTQGTFSNVSTNVSPAKPTTTA, GQN DTSQTSSPS, preferably ELPTQGTFSNVSTNVSPAKPTTTA and/or CPYSNPSLC, more preferably three CPYSNPSLC and one ELPTQGTFSNVSTNVSPAKPTTTA.

Such epitope has the specificity to be recognized by a specific antibody (preferably a monoclonal antibody (mAb), optionally humanized.

The present invention encompasses a combination treatment comprising a cell expressing a CAR of the invention and an antibody allowing said T cells to be destroyed in vivo upon successive administration.

Examples of monoclonal antibody (mAb) in combination treatment are, optionally humanized, that recognizes the mAb-specific epitope are ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab, preferably rituximab (R) and/or from QBEN10 (Q).

The present invention also relates to a method for sorting and/or depleting the engineered immune cells endowed with the anti-CD123 CARs of the invention. Several epitope-mAb couples can be used to generate such system; in particular, those already approved for medical use, e.g. CD20/rituximab, Q.BEN10, as a non-limiting example.

The CD123 specific CAR of the invention which scfv is from or derived from, ie prepared using CDRs or using a VH and a VL from 6H6 antibody or from 9F5 antibody is designated CD123 specific CAR or anti-CD123 CAR, or 123 CAR, or "CAR of the invention" indiscriminately.

The present invention generally provides an engineered immune cell endowed with the anti-CD123 CAR of the invention. Thus, in specific embodiments, an engineered immune cell endowed with the anti-

CD123 CAR comprising a scfv derived from humanized 6H6 a ntibody or from huma nized 9F5 antibody is provided.

6H6 and 9F5 are mouse anti-human CD123 antibody available through for example (BioLegend) for 6H6, and (BDbiosciences) for 9F5 and as such may be humanized as previously described to make a scfv (see US9573988 B2), the new anti-CD123 antibody comprises at leat the 6 CD of 6H6 or of 9F5 antibodies or the VH and VL optionally humanized of 6H6 or 9F5.

I nterleukin 3 receptor alpha chain (CD123) has been identified as being frequently over-expressed on Leukemia tumor cells, especially in the case of acute myeloid leukemia (AM L), compared to normal cells of the same lineage. The inventors have generated an engineered immune cell expressing a CD123 CAR from 6H6 or from 9F5 that discriminates CD123-positive (CD123+) cancer cells from CD123+- healthy cells (UCART123) and selectively destroy cancer cells.

I n particular embodiments, the UCART123 of the invention expresses at the cell surface a CD123 CAR comprising a scFV from 6H6 or 9F5 antibody, a hinge, a transmembrane domain from CD8a, and intracellular domains conferring engineered cells the capacity to proliferate in vivo, to reach CD123+ cancer cells specifically, to alter the survival of CD123+ cancer cells, and much less that of healthy CD123+ cells (healthy CD123+ cells can ultimately recover from the treatment), to persist in host, to survey potential emergence of CD123+ cance r cells. An immune cell engineered to express a CD123 specific CAR of the invention, that discriminated CD123+ cancer cells from CD123+healthy cell may comprise additional marker or suicide domain allowing their specific and targeted destruction as in PA2017 700 37 or in WO2016120216 (using a monoclonal Ab). In the present invention, a specific and selective alpha beta TCR KO CD123 CAR expressing cell was prepared using a nonblocking antibody specific for the IL-3 receptor alpha subunit expressed on CD123+cancer cells, and used for the treatment of patients suffering AML, B-cell lymphoproliferative disorder (BC-LPD) or BPDCN. Preferably said cell is a T cells. In preferred embodiments, said cells is endowed with: a CD123 CAR at the cell surface, said CD123 CAR was prepared using 6H6 or 9F5, or with humanized sequences derived from this 6H6 or 9F5 antibody, a gene encoding an anti-CD123 CAR of the invention inserted into the TRAC gene, using in particular viral vectors more particularly AAV6/2 vectors or AAV6/2 particles comprising an anti-CD123 CAR of the invention and a TALEN specific for the TRAC gene.

Concomitantly, these cells expressing an anti-CD123 CAR of the invention allow the destruction and clearance of CD123+precancerous cells stopping the progression and emergence of refractory/ relapsed cancer. Under this aspect, the CD123 CAR of the present invention is used for the prevention of relapse CD123+ cancer.

Under one important aspect, due to their capacity to proliferate in vivo and reach tissues or niches, the cells of the invention eradicate even aggressive lymphoproliferative disorder.

According to a method for preparing a cell of the invention, cells, preferably T-cells from a donor, who may be a patient, are transformed with a polynucleotide encoding a CAR of the invention, following a non-specific activation in vitro (e.g. with anti CD3/CD28 coated beads and recombinant IL2).

Using a TALEN, the polynucleotides encoding a CAR of the invention may be inserted in frame into transcriptionally active genes as described in 62/410,187, into genes of interest or into the TCRA gene or downstream an IRES allowing its transcription when inserted into transcriptionally active genes.

In one embodiment, T-cells were engineered to create less-alloreactive T-cells, by disruption of a component of the T cell receptor TCR (αβ - T-Cell receptors) to reduce Graft versus host reaction. This is achieved for example, by inserting the polynucleotides encoding a CAR of the invention in frame into transcriptionally active TRAC gene (TRAC means constant part of the alpha subunit of the T cell receptor gene).

In other embodiments, T-cells were further engineered by editing (deleting and/or overexpressing) specific combination(s) of genes identified in PA2017 70603, to create tolerogene T cells resistant to anti-cancer drugs, for example to be used in combination with classical anti-cancer drugs, such as purine analogs (PNA) and/or cyclophosphamides.

The resulting engineered T-cells displayed reactivity against CD123 positive cancer cells not against CD123+ healthy cells in patients treated with PNA, showing that the cells of the present invention contribute to antigen dependent activation and proliferation, even in the presence of PNA, making them useful for immunotherapy.

The resulting engineered T-cells of the invention displayed reactivity in-vivo against CD123 positive cancer cells and significantly reduce the number of cancer cells (tumoral mass) in vivo.

In a particular embodiment, engineered T-cells of the invention can be used for a therapeutic treatment, making them useful for immunotherapy as a first treatment (induction), as a consolidation treatment, as a treatment in combination with classical anticancer chemotherapy, as a bridging treatment before transplant of bone marrow cells.

The polypeptides and polynucleotide sequences encoding the CARs of the present invention are detailed in the present specification. The engineered immune cells of the present invention are particularly useful for therapeutic applications such as for treating CD123+ cancers specifically.

The engineered immune cells of the present invention may be useful for therapeutic applications such as for treating cancers from CD123+ precursors that transformed intp cancer cells during hematopoiesis. The engineered immune cell of the invention comprising for the least an anti-CD123 CAR of the invention may be used as a treatment of a patient which immune cells fully match the engineered immune cells (autologous transfer). The engineered immune cells of the invention may further comprise at least one edited gene, preferably an inactivated TCR alpha and/or beta gene(s), for allogeneic transfer ie transfer to any individuals in need thereof.

The engineered immune cells of the invention may further comprise at least one edited gene, preferably an inactivated TCR alpha and/or beta gene(s), and an inactivated beta 2microglobulin gene.

Engineered immune cells of the invention optionally comprise a T cell receptor alpha inactivated gene, preferably a T cell receptor alpha knocked out gene (UCART123), more preferably a T cell receptor alpha knocked out gene comprising a CAR encoding sequence knocked in said TCR alpha gene.

According to one aspect, the present invention relates to UCART123 comprising at least one additional edited gene, preferably a human deoxycytidine kinase inactivated gene (dCK), a CD52 inactivated gene, or a sur expressed P450 cytochrome gene or a combination thereof.

The present invention provides engineered immune cells for the treatment of a disease or a condition associated with overexpression or expression of IL-3 Receptor alpha, preferably of acute myeloid leukemia (AML) or Blastic plasmacytoid dendritic cell neoplasm (BPDCN) or their relapse/refractory forms.

In one embodiment, the above engineered immune cells of the invention are provided for their used as a treatment of AML, or of a complication of AML is provided, preferably a CD123+ AML, or a CD123+ relapse/refractory forms of AML.

In one embodiment, the above engineered immune cells of the invention are provided for their use as a treatment of Blastic plasmacytoid dendritic cell neoplasm (BPDCN) or for a complication of BPDCN, preferably a CD123+ BPDCN, or a CD123+ relapse/refractory forms of BPDCN.

As compared to previous non or less specific treatments, the engineered immune cells endowed with the CD123 CARs according to the invention show high efficiency for treating several forms of AML as compared to previous CD123 CAR, and induce less damage of non cancerous cells, thus, less side effects than previous treatments in patients.

As compared to previous nonspecific treatments, the engineered immune cells endowed with the CD123 CARs of the invention prevents cancer cells homing and therefore prevent cancer cells relapse due to the nesting of cancer cells in the tissues that engineered immune cells cannot easily access.

Finally, the invention encompasses the engineered immune cells of the invention for use in a therapeutic method where number, activity and survival of the engineered immune cells endowed with anti-CD123 CARs is modulated by redosing (re-injecting) and/or depleting the cells by using an antibody that directs the external ligand binding domain of said CARs. This allows controlling the activity of infused cells.

Brief description of the figures

Figure 1 Representation of a CAR of the invention (123 CAR) comprising a peptide signal that is cleaved upon expression at the cell surface, a scfv from a non blocking, a hinge from FcRIII, IgGl or CD8alpha, a transmembrane domain from CD8alpha, and two intracellular domains : a costimulatory domain from 4-1BB and an intracellular domain from CD3zeta, inclusing at least one (2 or 3 or 4) monoclonal antibody specific epitope (star) in the hinge and /or scfv L region. L: linker between VH and VL, preferably a L of sequence (GGGGS)n with n=l to 4, preferably n=3. Stars may be linked to the CAR structure by linker sequence corresponding to GGGS or SGGGGS or GSGGGGS, TM : transmembrane domain.

Figure 2 A CD123 CAR immune T cell of the invention comprising a VH from 6H6 or 9F5 optionally humanized, a linker L, a VL from 6H6 or 9F5, optionally humanized, a suicide domain (e.g. 3 sequences CPYSNPSLCS, and a one sequence ELPTQGTFSNVSTNVSPAKPTTTA) or two CPYSNPSLCS, is disclosed. Binding on target cells triggers a signal which is transduced to the T cells activating the cytolytic activity of the T cells. In turn, the T cells lyses the target cell.

Table 1: Examples of individual sequence of the

various domains included into a single or

amultichain CAR of the invention, Functional domains Amino acid sequence

CD8a signal peptide MALPVTALLLPLALLLHAARP

Alternative signal peptide M ETDTLLLWVLLLWVPGSTG

CD8a hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

IgGl hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLM IARTPEVTCVVVD

VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK

CD8a transmembrane IYIWAPLAGTCGVLLLSLVITLYC

domain

41BB transmembrane 1 ISFFLALTSTALLFLLFFLTLRFSVV

domain

41BB intracellular domain KRGRKKLLYI FKQPFM RPVQTTQEEDGCSCRFPEEEEGGCEL

CD3ζ intracellular domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG

GKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR

Linker GGGGSGGGGSGGGGS

Examples of mAb-specific epitopes (and their corresponding mAbs) that can be used in the extracellular domain of the CAR of the invention

Rituximab

Mimotope CPYSNPSLC

Palivizumab

Epitope NSELLSLINDMPITNDQKKLMSNN

Cetuximab

Mimotope 1 CQFDLSTRRLKC

Mimotope 2 CQYNLSSRALKC

Mimotope 3 CVWQRWQKSYVC

Mimotope 4 CMWDRFSRWYKC

Nivolumab

Epitope 1 SFVLNWYRMSPSNQTDKLAAFPEDR

Epitope 2 SGTYLCGAISLAPKAQIKE

QBEND-10

Epitope ELPTQGTFSNVSTNVSPAKPTTTA

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, pharmacology, immunology, biochemistry, genetics, and molecular biology.

All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

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 al, 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).

In general, the present invention provides a CAR prepared using a specific parts of a non blocking antibody that combined to adequate structural elements (hinge, TM, intracellular domains) allow a cell expressing said CAR to be activated and lyses a target cells.

In particular embodiments, the CD123 CAR of the present invention comprises CDRs from 6H6 (as available in BioLegend), or CDRs from 9F5 (available in BDbiosciences) anti human CD123 antibody, or a VH and a VL from 6H6 or a VL from 9F5 anti human CD123 antibody and triggers degranulation, IFN gamma secretion and/or CTL activity of T cells expressing at the cell surface said preferred CD123 CAR of the invention, upon binding with human CD123 as expressed by AML cancer cells.

The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific chimeric antigen receptor ("CD123 CAR" or "CAR") of the invention, the gene encoding said CD123 CAR is inserted into the TRC or dCK gene. In general, an engineered immune cell of the invention comprises a TALEN- inactivated TCR gene (constant region of the TRC alpha subunit, TRAC gene) comprising a polypeptide encoding at least a CD123 CAR of the invention downstream a 2A peptide or an IRES sequence, express at the cell surface a CD123 CAR of the invention and undetectable level of alpha beta TCR. A CD123 CAR of the invention comprises an extra cellular ligand binding-domain comprising a VH and a VL from a non blocking monoclonal anti-CD123 antibody or humanized VH and humanized VL sequence thereof, a hinge from CD8 alpha, from IgGl or from FcRIII, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB. Any non blocking antibody that allows discriminating pathological cells from healthy cells may be part of the present invention and constitute part of the CAR of the invention when combined with appropriate domains, allowing a T cells expressing it to be activated and destroy a target cells upon binding to the target.

Unexpectedly, the invention provides a CAR prepared using a non-blocking antibody that triggers a CTL activity upon binding to its target, in particular to CD123. The present invention also discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific chimeric antigen receptor ("CD123 CAR" or "CAR") of the invention.

In particular embodiments, an engineered immune cell comprising an extra cellular ligand binding-domain comprising a VH and a VL from a monoclonal anti-CD123 antibody 9F5 or humanized VH and humanized VL sequence thereof, a hinge from CD8 alpha, from IgGl or from FcRIII, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB, is disclosed.

The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific chimeric antigen receptor ("CD123 CAR" or "CAR") of the invention comprising an extra cellular ligand binding-domain comprising a VH and a VL from a monoclonal anti-CD123 antibody 6H6 or humanized VH and humanized VL sequence thereof, a hinge from CD8 alpha, from IgGl or from FcRIII, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

In another embodiment, the present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific chimeric antigen receptor ("CD123 CAR" or "CAR") comprising an extra cellular ligand binding-domain comprising a CDRs from a monoclonal anti-CD123 antibody 6H6, a hinge from CD8 alpha from IgGl or from FcRIII, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

In another embodiment, the present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific chimeric antigen receptor ("CD123 CAR" or "CAR") comprising an extra cellular ligand binding-domain comprising a CDRs from a monoclonal anti-CD123 antibody 9F5, a hinge from CD8 alpha, from IgGl or from FcRIII, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

Preferably the present invention discloses an engineered immune cell (TCR KO and/or dCK KO) expressing a CD123 specific CAR of the invention, more preferably an engineered immune cell (TCR KO) expressing a CD123 specific CAR, and even more preferably an engineered immune cell (TCR KO and dck KO) expressing a CD123 specific CAR. Advantageously the present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific CAR and a suicide domain such as R R8, of BEN10.

The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific CAR having one of the polypeptide structure comprising a hinge from CD8alpha, or IgGl or FcyRllla, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB.

The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific CAR comprising no sequence from CD28. The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific CAR having one of the polypeptide structure comprising a hinge from CD8alpha, or IgGl or FcyRllla, a transmembrane domain from CD8 alpha, a cytoplasmic domain including a CD3 zeta signaling domain and a co-stimulatory domain from 4-1BB, and no sequence from CD28. Another most preferred embodiment discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CAR comprising at least one epitope recognized by a specific monoclonal antibody.

Another most preferred embodiment discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CAR comprising at least one epitope recognized by rituximab.

Another most preferred embodiment discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CAR comprising at least one epitope recognized by OBEN 10.

Another most preferred embodiment discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CAR comprising at least one epitope recognized by rituximab and OBEN 10.

The present invention discloses an engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific 123 CAR which extracellular binding domain is modified in such a way to allow both cell sorting and cell depletion using at least one specific monoclonal antibody. This structure named "mAb-driven sorting/depletion system" or "epitope specific for a monoclonal antibody" or "mimotope" is a selected epitope inserted within the extracellular domain of the anti-CD123 CAR of the invention, in particular into the anti- CD123 scFv; and/or between the TM and the hinge; this epitope having a specificity to be recognized by a specific antibody (preferably mAb). Given the fact that mainly the external ligand binding domain of the CAR is modified to include the epitope, different CAR architectures can be envisioned: single-chain or multi-chain. The chimeric scFv of the invention, which is formed of the VH and VL polypeptides and of the specific epitope(s) may itself have different structures depending on the position of insertion of the epitope and the use of linkers. The present invention also relates to the resulting method for sorting and/or depleting the engineered immune cells endowed with the modified CARs.

In some embodiments, the extracellular binding domain comprises the following sequence (including mimotopes) (Nterm is located on the left hand side):

Vi-Li-V ₂ -(L)x-Epitopel-(L) _x ;

Vi-Li-V ₂ -(L)x- Epitope 1 -(L) _x -Epitope2-(L) _x ;

Vi-Li-V ₂ -(L)x- Epitope 1 -(L) _x -Epitope2-(L)x-Epitope3-(L) _x ;

(L)x- Epitope 1 -(L)x-Vi-Li-V ₂ ; (L)x- Epitope 1 -(L) _x -Epitope2-(L) _x -Vi-Li-V ₂ ;

Epitope 1 -(L)x-Epitope2-(L)x-Epitope3-(L) _x -Vi-Li-V ₂ ;

(L)x- Epitope 1 -(L)x-Vi-Li-V ₂ -(L)x-Epitope2-(L) _x ; (L)x- Epitope 1 -(L)x-Vi-Li-V ₂ -(L)x-Epitope2-(L)x-Epitope3-(L) _x ; (L)x- Epitope 1 -(L) _x -Vi-Li-V ₂ -(L)x-Epitope2-(L)x-Epitope3-(L)x-Epitope4-(L) _x ; (L)x- Epitope 1 -(L)x-Epitope2-(L)x-Vi-Li-V ₂ -(L)x-Epitope3-(L) _x ; (L)x-Epitopel-(L)x-Epitope2-(L)x-Vi-Li-V ₂ -(L)x-Epitope3-(L)x-Epitope4-(L) _x ; Vi-(L)x- Epitope 1 -(L) _x -V ₂ ;

Vi-(L)x- Epitope 1 - (L) _x -V ₂ -(L)x-Epitope2-(L)x;

Vi-(L)x- Epitope 1 -(L) _x -V2-(L)x-Epitope2-(L)x-Epitope3-(L) _x ;

Vi-(L)x-Epitopel-(L)x-V ₂ -(L)x-Epitope2-(L)x-Epitope3-(L)x-Epitope4-(L)x; (L)x- Epitope 1 -(L) _x -Vi-(L)x-Epitope2-(L)x-V ₂ ;

(L)x- Epitope 1 - (L)x- V i - (L) _x - Epitope2 - (L) _x - V ₂ - (L) _x - Epitope3 - (L) _x ;

Vi -Li -V ₂ - -L-Epitopel ;

Vi -Li -V ₂ - -L-Epitopel-L;

Vi -Li -V ₂ - -L- Epitope 1 -L-Epitope2;

Vi -Li -V ₂ - -L- Epitope 1 -L-Epitope2-L;

Vi -Li -V ₂ - -L- Epitope 1 -L-Epitope2-L-Epitope3;

Vi -Li -V ₂ - -L- Epitope 1 -L-Epitope2-L-Epitope3-L;

Vi -Li -V ₂ - -Epitope 1 ;

Vi -Li -V ₂ - -Epitope 1-L;

Vi -Li -V ₂ - -Epitope 1 -L-Epitope2;

Vi -Li -V ₂ - -Epitope 1 -L-Epitope2-L;

Vi -Li -V ₂ - -Epitope 1 -L-Epitope2-L-Epitope3;

Vi -Li -V ₂ - -Epitope 1 -L-Epitope2-L-Epitope3-L;

Epitope I-V1-L1-V2;

Epitope I-L-V1-L1-V2;

L-Epitopel-Vi-Li-V ₂ ;

L-Epitope 1 -L-Vi-Li-V ₂ ;

Epitope 1 -L-Epitope2-Vi-Li-V ₂ ;

Epitope 1 -L-Epitope2-L-Vi-Li-V ₂ ; L-Epitopel-L-Epitope2-Vi-L ₁ -V ₂ ; L-Epitope 1 -L-Epitope2-L-Vi-Li-V ₂ ; Epitope 1 -L-Epitope2-L-Epitope3-Vi-Li-V2; Epitope 1 -L-Epitope2-L-Epitope3-L-Vi-Li-V2; L-Epitope 1 -L-Epitope2-L-Epitope3-Vi-Li-V2; L-Epitopel-L-Epitope2-L-Epitope3-L-V ₁ -Li-V ₂ ;

Vi-L-Epitopel-L-V ₂ ;

L-Epitope 1 -L-Vi-L-Epitope2-L-V ₂ ;

Vi-L- Epitope 1 -L-V ₂ -L-Epitope2-L;

Vi-L-Epitopel-L-V ₂ -L-Epitope2-L-Epitope3;

Vi-L- Epitope 1 -L-V ₂ -L-Epitope2-Epitope3;

Vi-L- Epitope 1 -L-V ₂ -L-Epitope2-L-Epitope3-Epitope4;

L-Epitope 1 -L-Vi-L-Epitope2-L-V ₂ -L-Epitope3-L;

Epitope 1 -L-Vi-L-Epitope2-L-V ₂ -L-Epitope3-L;

L-Epitopel-L-V ₁ -L-Epitope2-L-V ₂ -L-Epitope3;

L-Epitope 1 -L-Vi-Li-V ₂ -L-Epitope2-L; L-Epitope 1 -L-Vi-Li-V ₂ -L-Epitope2-L-Epitope3; L-Epitope 1 -L-Vi-Li-V2-L-Epitope2-Epitope3, or, Epitopel-L-V ₁ -Li-V ₂ -L-Epitope2-L-Epitope3-Epitope4.

wherein,

Vi and V2 are VH and VL of an ScFv (i.e , Vi is VL and V ₂ is V _H or Vi is V _H and V2 is VL); preferably from 6H6 or 9F5, more preferably from 6H6 or 9F5 and humanized, or comprising CDR from 6H6 or from 9F5

Li is any linker suitable to link the VH chain to the VL chain in an ScFv; L is a linker, preferably comprising glycine and serine residues, and each occurrence of L in the extracellular binding domain can be identical or different to other occurrence of L in the same extracellular binding domain, and, x is 0 or 1 and each occurrence of x is independently from the others; and, epitope 1, epitope 2 and epitope 3 are mAb-specific epitopes and can be identical or different.

In some embodiments, the extracellular binding domain comprises the following sequence (Nterm is located on the left hand side): V _H -Li-V _L -L-Epitopel-L-Epitope2-L;

L-Epitopel-L-V _H -L-Epitope2-L-V _L -L-Epitope3-L;

V _L -L ¹ -V _H -L-Epitopel-L-Epitope2-L; or,

L-Epitopel-L-V _L -L-Epitope2-L-V _H -L-Epitope3-L wherein L, LI, epitope 1, epitope 2 and epitope 3 are as defined above.

Li is a linker comprising Glycine and/or Serine. In some embodiment, Li is a linker comprising the amino acid sequence (Gly-Gly-Gly-Ser) _n or (Gly-Gly-Gly-Gly-Ser) _n , where n is 1, 2, 3, 4 or 5. In some embodiments Li is (Gly ₄ Ser) ₄ or (Gly ₄ Ser) ₃ .

L is a flexible linker, preferably comprising Glycine and/or Serine. In some embodiments, L has an amino acid sequence selected from SGG, GGS, SGGS, SSGGS, GGGG, SGGGG, GGGGS, SGGGGS, GGGGGS, SGGGGGS, SGGGGG, GSGGGGS, GGGGGGGS, SGGGGGGG, SGGGGGGGS, or SGGGGSGGGGS preferably SGG, SGGS, SSGGS, GGGG, SGGGGS, SGGGGGS, SGGGGG, GSGGGGS or SGGGGSGGGGS. In some embodiment, when the extracellular binding domain comprises several occurrences of L, all the Ls are identical. In some embodiments, when the extracellular binding domain comprises several occurrences of L, the Ls are not all identical. In some embodiments, L is SGGGGS. In some embodiments, the extracellular binding domain comprises several occurrences of L and all the Ls are SGGGGS. In some embodiments, Epitope 1, Epitope 2 and Epitope 3 are identical or different.

In a preferred embodiments, Epitope 1, Epitope 2 are identical or different and are selected from mAb-specific epitopes specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, Q.BEND-10, alemtuzumab or ustekinumab, preferably those already approved for medical use, such as rituximab as a non-limiting example.

The invention encompasses therapeutic methods wherein the number, activation and/or survival of the engineered immune cells endowed with a CD123 CAR of the present invention is controlled by using an antibody (at least one) that directly binds to an epitope specific for said antibody located within in the CD123 CARs of the invention.

The present invention encompasses embodiments disclosing an engineered immune cell (TCR KO and/or dck KO) expressing one of the CD123 specific CAR discloses herein.

Engineered immune cell

An engineered immune cell is meant to be a cell from a healthy donor or from a patient, that expressed temporarily at least one rare cutting endonuclease, preferably a TALEN ^® , more preferably a TALEN ^® binding to the following sequences ttgtcccacagATATC and/or CCGTGTACCAGCTGAGA, and comprises a genomic TCR gene comprising a mutation, a deletion or an insertion, preferably an insertion into the TRAC gene (encoding the constant region of the alpha TCR subunit) said insertion comprises a polynucleotide, said

polynucleotide comprises i) at least an IRES or an open reading frame encoding a self- cleaving peptide and ii) at least a sequence encoding a CAR, preferably a CD123 CAR, more preferably a CD123 CAR from 6H6 or 9F5 non blocking antibody, of the invention.

The present invention encompasses embodiments disclosing vectors comprising a polynucleotide or inserted sequence comprising i) at least an IRES or an open reading frame encoding a self-cleaving peptide and ii) at least a sequence encoding a CAR, preferably a CD123 CAR of the invention.

The present invention encompasses embodiments disclosing a method for preparing of engineered immune cell (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses herein.

In particular, the present invention encompasses embodiments disclosing vectors allowing integration /insertion and expression of a sequence, preferably a sequence coding CD123 specific CAR of the invention into specific genomic loci, as disclosed in PA201670240.

For instance, the inserted exogenous coding sequence(s) can have the effect of reducing or preventing the expression, by the engineered immune cell of at least one protein selected from PD1 (Uniprot Q151 16), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG 3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), BTLA (Uniprot Q7Z6A9), CD160 (Uniprot 095971 ), TIGIT (Uniprot Q495A1 ), CD96 (Uniprot P40200), CRTAM (Uniprot 095727), LAIR1 (Uniprot Q6GTX8), SIGLEC7 (Uniprot Q9Y286), SIGLEC9 (Uniprot Q9Y336), CD244 (Uniprot Q9BZW8), TNFRSF10B (Uniprot 014763), TNFRSF10A (Uniprot 000220), CASP8 (Uniprot Q14790), CASP10 (Uniprot Q92851 ), CASP3 (Uniprot P42574), CASP6 (Uniprot P55212), CASP7 (Uniprot P55210), FADD (Uniprot Q13158), FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Uniprot P84022), SMAD4 (Uniprot Q13485), SMAD10 (Uniprot B7ZSB5), SKI (Uniprot P12755), SKIL (Uniprot P12757), TGIF1 (Uniprot Q15583), IL10RA (Uniprot Q13651 ), IL10RB (Uniprot Q08334), HMOX2 (Uniprot P30519), IL6R (Uniprot P08887), IL6ST (Uniprot P40189), EIF2AK4 (Uniprot Q9P2K8), CSK (Uniprot P41240), PAG1 (Uniprot Q9NWQ8), SIT1 (Uniprot Q9Y3P8), FOXP3 (Uniprot Q9BZS1 ), PRDM1 (Uniprot Q60636), BATF (Uniprot Q16520), GUCY1A2 (Uniprot P33402), GUCY1A3 (Uniprot Q02108), GUCY1 B2 (Uniprot Q8BXH3) and GUCY1 B3 (Uniprot Q02153). The gene editing introduced in the genes encoding the above proteins is preferably combined with an inactivation of TCR in CAR T cells. Preference is given to inactivation of PD1 , CISH and/or CTLA4, in combination with the expression of non-endogenous immunosuppressive polypeptide, such as a PD1 - ligand 1 or 2 and/or CTLA-4 Ig. List of genes involved into immune cells inhibitory pathways

Inhibiting suppressive cyto ines/metabolites

According to another aspect of the invention, the inserted exogenous coding sequence has the effect of reducing or preventing the expression of genes encoding or positively regulating suppressive cytokines or metabolites or receptors thereof, in particular TGFbeta (Uniprot:P01 137), TGFbR (Uniprot:P37173), IL10 (Uniprot:P22301 ), IL10R (Uniprot: Q13651 and/or Q08334), A2aR (Uniprot: P29274), GCN2 (Uniprot: P15442) and PRDM1 (Uniprot: 075626).

Preference is given to engineered immune cells in which a sequence encoding IL-2, IL-12 or IL-15 replaces the sequence of at least one of the above endogenous genes.

Inducing resistance to chemotherapy drugs

According to another aspect of the present method, the inserted exogenous coding sequence has the effect of reducing or preventing the expression of a gene responsible for the sensitivity of the immune cells to compounds used in standard of care treatments for cancer or infection, such as drugs purine nucleotide analogs (PNA) or 6-Mercaptopurine (6MP) and 6 thio-guanine (6TG) commonly used in chemotherapy. Reducing or inactivating the genes involved into the mode of action of such compounds (referred to as "drug sensitizing genes") improves the resistance of the immune cells to same.

Examples of drug sensitizing gene are those encoding DCK (Uniprot P27707) with respect to the activity of PNA, such a clorofarabine et fludarabine, FLAG treatment (see below), HPRT (Uniprot P00492) with respect to the activity of purine antimetabolites such as 6MP and 6TG, and GGH (Uniprot Q92820) with respect to the activity of antifolate drugs, in particular methotrexate.

This enables the cells to be used after or in combination with conventional anti-cancer chemotherapies.

Resistance to immune-suppressive treatments

According to another aspect of the present invention, the inserted exogenous coding sequence has the effect of reducing or preventing the expression of receptors or proteins, which are drug targets, making said cells resistant to immune-depletion drug treatments. Such target can be glucocorticoids receptors or antigens, to make the engineered immune cells resistant to glucocorticoids or immune depletion treatments using antibodies such as Alemtuzumab, which is used to deplete CD52 positive immune cells in many cancer treatments.

Also, the method of the invention can comprise gene targeted insertion in endogenous gene(s) encoding or regulating the expression of CD52 (Uniprot P31358) and/or GR (Glucocorticoids receptor also referred to as NR3C1 - Uniprot P04150).

Improving CAR positive immune cells activity and survival

According to another aspect of the present invention, the inserted exogenous coding sequence can have the effect of reducing or preventing the expression of a surface antigen, such as BCMA, CS1 and CD38, wherein such antigen is one targeted by a CAR expressed by said immune cells.

This embodiment can solve the problem of CAR targeting antigens that are present at the surface of infected or malignant cells, but also to some extent expressed by the immune cell itself.

According to a preferred embodiment the exogenous sequence encoding the CAR or one of its constituents is integrated into the gene encoding the antigen targeted by said CAR to avoid self-destruction of the immune cells.

Engineered immune cells and populations of immune cells The present invention is also drawn to the variety of engineered immune cells obtainable according to one of the method described previously under isolated form or as part of populations of cells.

According to a preferred aspect of the invention the engineered cells are primary immune cells, such as NK cells or T-cells, which are generally part of populations of cells that may involve different types of cells. In general, population deriving from patients or donors isolated by leukapheresis from PBMC (peripheral blood mononuclear cells).

The present invention encompasses immune cells comprising any combinations of the different exogenous coding sequences and gene inactivation, which have been respectively and independently described above. Among these combinations are particularly preferred those combining the expression of a CAR under the transcriptional control of an endogenous promoter that is steadily active during immune cell activation and preferably independently from said activation, and the expression of an exogenous sequence encoding a cytokine, such as IL-2, IL-12 or IL-15, under the transcriptional control of a promoter that is up- regulated during the immune cell activation.

By endogenous promotor is meant any one of the promoter controlling one of the following genes: symbol description

II21 interleukin 21

11 interleukin 3

Ccl4 isopentenyl-diphosphate delta isomerase 2

II21 granzyme C

Gp49a chemokine (C-C motif) receptor 8

CxcllO interleukin 2

Nr4a3 interleukin 1 receptor, type 1

tumor necrosis factor (ligand) superfamily,

Lilrb4 member 4

Cd200 neuronal calcium sensor 1 Cdknla CDK5 and Abl enzyme substrate 1 transmembrane and tetratricopeptide repeat

Gzmc containing 2

LON peptidase N-terminal domain and ring

Nr4a2 finger 1

Cish glycoprotein 49 A

Nr4al polo-like kinase 2

Tnf lipase, endothelial

Ccr8 cyclin-dependent kinase inhibitor 1A (P21)

Ladl grainyhead-like 1 (Drosophila)

Slamfl cellular retinoic acid binding protein II

Crabp2 adenylate kinase 4

Furin microtubule-associated protein IB

acyl-CoA synthetase long-chain family

Gadd45g member 6

Bcl2ll zinc finger E-box binding homeobox 2

Ncsl CD200 antigen

Ciart carboxypeptidase D

Ahr thioredoxin reductase 3

Spryl myosin IE

Tnfsf4 RNA binding protein with multiple splicing 2 mitogen-activated protein kinase kinase 3,

MyolO opposite strand

Dusp5 PERP, TP53 apoptosis effector

Myc myosin X

Psrcl immediate early response 3

St6galnac4 folliculin interacting protein 2

leukocyte immunoglobulin-like receptor,

Nfkbid subfamily B, member 4

circadian associated repressor of

Bst2 transcription

Txnrd3 RAR-related orphan receptor gamma

Plk2 proline/serine-rich coiled-coil 1

Gfil cysteine rich protein 2

Piml cAMP responsive element modulator

Pvtl chemokine (C-C motif) ligand 4

nuclear receptor subfamily 4, group A,

Nfkbib member 2

Gnl2 transglutaminase 2, C polypeptide

synapse defective 1, Rho GTPase, homolog 2

Cd69 (C, elegans)

Dgat2 sprouty homolog 1 (Drosophila)

Atf3 activating transcription factor 3

pogo transposable element with KRAB

Tnfrsf21 domain

tumor necrosis factor receptor superfamily,

Lonrfl member 21

Cablesl cytokine inducible SH2-containing protein Cpd lymphotoxin A

Qtrtdl FBJ osteosarcoma oncogene

signaling lymphocytic activation molecule

Polr3d family member 1

Kcnq5 syndecan 3

Fos mitochondrial ribosomal protein L47

Slcl9a2 ladinin

Hifla E2F transcription factor 5

Ill5ra ISG15 ubiquitin-like modifier

Nfkbl aryl-hydrocarbon receptor

Phlda3 diacylglycerol O-acyltransferase 2

Mtrr FBJ osteosarcoma oncogene B

pleckstrin homology-like domain, family A,

Pogk member 3

potassium voltage-gated channel, subfamily

Map2k3os Q, member 5

tumor necrosis factor receptor superfamily,

Egr2 member 10b

Isgl5 Mirl7 host gene 1 (non-protein coding) glucose-fructose oxidoreductase domain

Perp containing 1

lpo4 plexin Al

MphosphlO heat shock factor 2

Plk3 carbohydrate sulfotransferase 11

growth arrest and DNA-damage-inducible 45

Ifitm3 gamma

solute carrier family 5 (sodium-dependent

Polrlb vitamin transporter), member 6

Uspl8 interferon induced transmembrane protein 3

Toplmt DENN/MADD domain containing 5A

Dkcl plasminogen activator, urokinase receptor solute carrier family 19 (thiamine

Polrlc transporter), member 2

Cdk6 ubiquitin domain containing 2

nuclear receptor subfamily 4, group A,

Ier3 member 3

Lta zinc finger protein 52

Ptprs SH3 domain containing ring finger 1

Fnip2 dihydrouridine synthase 2

cyclin-dependent kinase 5, regulatory subunit

Asnal 1 (p35)

processing of precursor 7, ribonuclease P

Mybbpla family, (S, cerevisiae)

lllrl growth factor independent 1

Dennd5a interleukin 15 receptor, alpha chain

E2f5 BCL2-like 1

protein tyrosine phosphatase, receptor type, cll S Fosl2 plasmacytoma variant translocation 1

Atad3a fos-like antigen 2

Bax BCL2-associated X protein

solute carrier family 4, sodium bicarbonate

Phf6 cotransporter, member 7

tumor necrosis factor receptor superfamily,

Zfp52 member 4

Crtam chemokine (C-X-C motif) ligand 10

Nopl4 polo-like kinase 3

CD3E antigen, epsilon polypeptide associated el protein

tumor necrosis factor (ligand) superfamily,

Gramdlb member 11

polymerase (RNA) III (DNA directed)

Ifi27l2a polypeptide D

TnfrsflOb early growth response 2

DnaJ (Hsp40) homolog, subfamily C, member

Rpl7ll 2

Eifla DNA topoisomerase 1, mitochondrial

Nfkb2 tripartite motif-containing 30D

DnaJ (Hsp40) homolog, subfamily C, member

Heatrl 21

SAM domain, SH3 domain and nuclear

Utp20 localization signals, 1

solute carrier family 5 (inositol transporters),

Chstll member 3

Ddx21 mitochondrial ribosomal protein L15

Hsf2 dual specificity phosphatase 5

Bccip apoptosis enhancing nuclease

Tagap ets variant 6

DIM1 dimethyladenosine transferase 1-like

Sdc3 (S, cerevisiae)

Sytl3 2'-5' oligoadenylate synthetase-like 1

UTP18, small subunit (SSU) processome

Gtpbp4 component, homolog (yeast)

Crip2 BRCA2 and CDKN1A interacting protein

Sh3rfl synaptotagmin-like 3

5-methyltetrahydrofolate-homocysteine

Nsfllc methyltransferase reductase

URB2 ribosome biogenesis 2 homolog (S,

Gtf2fl cerevisiae)

ubiquitin-conjugating enzyme E2C binding

Slc4a7 protein

Etv6 lysine (K)-specific demethylase 2B

queuine tRNA-ribosyltransferase domain

Trim30d containing 1

Ddx27 ubiquitin specific peptidase 31

eukaryotic translation initiation factor 2-

Pwp2 alpha kinase 2 Chchd2 ATPase family, AAA domain containing 3A adhesion molecule, interacts with CXAD

Myole antigen 1

Eif5b SUMO/sentrin specific peptidase 3

ESF1, nucleolar pre-rRNA processing protein,

Stat5a homolog (S, cerevisiae)

deoxynucleotidyltransferase, terminal,

Cops6 interacting protein 2

D19Bwgl357e TGFB-induced factor homeobox 1

Aatf eukaryotic translation initiation factor 1A

Aen interferon-stimulated protein

Amical pleiomorphic adenoma gene-like 2

PWP2 periodic tryptophan protein homolog

Wdr43 (yeast)

furin (paired basic amino acid cleaving

Cct4 enzyme)

Nifk tumor necrosis factor

Tgm2 apoptosis antagonizing transcription factor

Eroll interferon, alpha-inducible protein 27 like 2A

ST6 (alpha-N-acetyl-neuraminyl-2,3-beta- galactosyl-l,3)-N-acetylgalactosaminide

Gfodl alpha-2,6-sialyltransferase 4

Ak4 methyltransferase like 1

Sdadl notchless homolog 1 (Drosophila)

Dimtl mitochondrial ribosomal protein L3

Esfl UBX domain protein 2A

guanine nucleotide binding protein-like 2

Cd3eap (nucleolar)

Samsnl programmed cell death 11

Tnfrsf4 cyclin-dependent kinase 8

Mettll eukaryotic translation initiation factor 5B

Cd274 RNA terminal phosphate cyclase-like 1

Ubtd2 NSFL1 (p97) cofactor (p47)

nuclear factor of kappa light polypeptide lcos gene enhancer in B cells inhibitor, delta

M-phase phosphoprotein 10 (U3 small

Kdm2b nucleolar ribonucleoprotein)

Larp4 GRAM domain containing IB

Eif3d EROl-like (S, cerevisiae)

nuclear receptor subfamily 4, group A,

Tnfaip3 member 1

Maplb surfeit gene 2

N(alpha)-acetyltransferase 25, NatB auxiliary

Cdv3 subunit

Plac8 yrdC domain containing (E,coli)

La ribonucleoprotein domain family, member

Mrpl3 4

Surf2 SDA1 domain containing 1

Ubxn2a importin 4 Utpl8 inducible T cell co-stimulator solute carrier family 7 (cationic amino acid

Isg20 transporter, y+ system), member 1

arsA arsenite transporter, ATP-binding,

Dnajc2 homolog 1 (bacterial)

Jak2 polymerase (RNA) 1 polypeptide C

Slc7al spermatogenesis associated 5

Syde2 ubiquitin specific peptidase 18

Slc5a6 placenta-specific 8

Dnttip2 general transcription factor IIF, polypeptide 1 nuclear factor of kappa light polypeptide

Idi2 gene enhancer in B cells inhibitor, beta

Dus2 PHD finger protein 6

RRN3 RNA polymerase 1 transcription factor

Pitrml homolog (yeast)

Plxnal cytotoxic and regulatory T cell molecule

COP9 (constitutive photomorphogenic)

Cdk5rl homolog, subunit 6 (Arabidopsis thaliana) asparagine-linked glycosylation 3 (alpha-1,3-

Ube2cbp mannosyltransferase)

Tnfsfll tryptophanyl-tRNA synthetase

Pop7 hypoxia up-regulated 1

Psme3 family with sequence similarity 60, member A

Mirl7hg bone marrow stromal cell antigen 2

nuclear factor of kappa light polypeptide

Tsrl gene enhancer in B cells 2, p49/pl00

UTP20, small subunit (SSU) processome bpms2 component, homolog (yeast)

Mrpl47 CD274 antigen

Rab8b proviral integration site 1

signal transducer and activator of

Plagl2 transcription 5A

Grhll CD69 antigen

Zeb2 pitrilysin metallepetidase 1

sept-02 cyclin-dependent kinase 6

Slc5a3 DEAD (Asp-Glu-Ala-Asp) box polypeptide 27

Naa25 polymerase (RNA) 1 polypeptide B

tumor necrosis factor, alpha-induced protein

Plaur 3

Metapl nodal modulator 1

Alg3 NOP14 nucleolar protein

Mrpll5 ribosomal protein L7-like 1

Oasll methionyl aminopeptidase 1

Rorc hypoxia inducible factor 1, alpha subunit

Nomol Janus kinase 2

nuclear factor of kappa light polypeptide

Tgifl gene enhancer in B cells 1, pl05

Lipg reticuloendotheliosis oncogene rn3 septin 2

nucleolar protein interacting with the FHA

Dnajc21 domain of MKI67

elongation factor Tu GTP binding domain

Yrdc containing 2

Acsl6 myelocytomatosis oncogene

Spata5 dyskeratosis congenita 1, dyskerin

carnitine deficiency-associated gene

Urb2 expressed in ventricle 3

Nlel GTP binding protein 4

Wars HEAT repeat containing 1

proteaseome (prosome, macropain) activator

Crem subunit 3 (PA28 gamma, Ki)

La ribonucleoprotein domain family, member

Larpl 1

DNA segment, Chr 19, Brigham & Women's

Eif2ak2 Genetics 1357 expressed

eukaryotic translation initiation factor 3,

Hyoul subunit D

Senp3 TSR1 20S rRNA accumulation

Tmtc2 MYB binding protein (P160) la

T cell activation Rho GTPase activating

Fosb protein

Pdcdll RAB8B, member RAS oncogene family

Usp31 DEAD (Asp-Glu-Ala-Asp) box polypeptide 21

Cdk8 chaperonin containing Tcpl, subunit 4 (delta)

coiled-coil-helix-coiled-coil-helix domain

Eftud2 containing 2

Fam60a WD repeat domain 43

Another preferred combination is the insertion of an exogenous sequence encoding a CAR or one of its constituents under the transcription control of the hypoxia-inducible factor 1 gene promoter (Uniprot: Q16665).

PHARMACEUTICAL COMPOSITION

The present invention encompasses a pharmaceutical composition comprising an engineered immune cell (TCR KO and/or dck KO) expressing one of any one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle.

The present invention encompasses a pharmaceutical composition comprising an engineered immune cell (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle for use as a medicament.

The present invention encompasses an embodiment disclosing a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle for use as a medicament.

The present invention discloses a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising at least two epitopes specific for a monoclonal Ab, as any one disclosed in patent application PA 2015 70044 which is incorporated herein by reference in its entirety, a pharmaceutically acceptable vehicle for use as a medicament in combination with said monoclonal Ab.

In preferred embodiments, a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising at least two epitopes specific for rituximab and said a pharmaceutical composition comprises rituximab.

In preferred embodiments, a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising three epitopes specific for rituximab and an epitope specific for OBEN and said a pharmaceutical composition comprises rituximab and BEN10.

The present invention discloses a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and any one of the engineered immune cell (TCR KO and/or dck KO) expressing a CD123 specific CAR as described above and another drug, preferably a purine analogue and more preferably a FLAG treatment.

Examples of purine analogues according to the invention may be pentostatin, fludarabine 2-deoxyadenosine, cladribine, clofarabine, Nelarabine, preferably pentostatin, fludarabine monophosphate, and 2-chlorodeoxyadenosine (2-CDA). Examples of FLAG treatments that may be associated with the CD123 T cells of the invention are as follows: Standard FLAG without additions, FLAG-IDA, Mito-FLAG, FLAMSA.

An Example of FLAG treatment according to the invention may be:

Standard FLAG without additions

FLAG-IDA

Mito-FLAG

FLAMSA

THERAPEUTIC APPLICATION

The following combination treatments are disclosed herein: An engineered immune cell as any one described above, a composition comprising said engineered immune cell as disclosed above, for use in therapy to prevent or treat a condition.

The present invention discloses an engineered immune cell of the invention, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the patient is a human.

The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the condition is a pre-malignant or malignant cancer condition characterized by CD123- expressing cells.

The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the condition is a condition which is characterized by an overabundance of CD123-expressing cells. The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the malignant cancer condition is a haematological cancer condition.

The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the haematological cancer condition is leukemia or malignant lymphoproliferative disorders.

The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein said leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

The present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein the leukemia is acute myelogenous leukemia (AML), preferably refractory /relapsed AML. In one embodiment, the present invention discloses an engineered immune cell, a composition comprising said engineered immune cell as disclosed above, for use in therapy as above, wherein said hematologic cancer is a malignant lymphoproliferative disorder.

The present invention discloses an engineered immune cell for use in therapy as above, wherein said malignant lymphoproliferative disorder is lymphoma.

The present invention discloses an engineered immune cell for use in therapy as above, wherein said lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). From 10 ^"2 to 10 ⁸ CD123 CAR expressed in TCR KO and dck KO T cells of the invention/kg, in combination with a FLAG treatment without addition, for use in the treatment of AM L, preferably refractory relapsed AML.

From 10 ^"2 to 10 ⁸ CD123 CAR expressed in TCR KO and dck KO T cells of the invention/kg, in combination with a FLAG treatment without addition, for use in the treatment of BPDCN. From 10 ^"2 to 10 ⁸ CD123 CAR expressed in TCR KO and dck KO T cells of the invention/kg, in combination with a FLAG treatment without addition.

From 10 ^"2 to 10 ⁸ CD123 CAR expressed in TCR KO and dck KO T cells of the invention/kg, in combination with fludarabine (from 20 mg/kg to 50 mg/kg), for use in the treatment of AML, preferably refractory relapsed AML. From 10 ^"2 to 10 ⁸ CD123 CAR expressed in TCR KO and dck KO T cells of the invention/kg, in combination with fludarabine (from 20 mg/kg to 50 mg/kg), for use in the treatment of BPDCN.

The present invention discloses a method of impairing a hematologic cancer cell comprising contacting said hematologic cancer cell with an engineered cell according to the invention in an amount effective to cause impairment of said cancer cell (from 10 ^"2 to 10 ⁸ cells/kg).

In particular embodiments, an amount effective to cause impairment of said cancer cell may be less than 10 ^"2 cells/kg, more than 10 ^"2 cells/kg, more than 10 ¹ cells/kg, more than 10 ° cells/kg, more than 10 ¹ cells/kg, more than 10 ² cells/kg, more than 10 ³ cells/kg, more than 10 ⁴ cells/kg, more than 10 ⁵ cells/kg, more than 10 ⁶ cells/kg, more than 10 ⁷ cells/kg, more than 10 ⁸ cells/kg. The present invention discloses a method of engineering an immune cell comprising:

• Providing an immune cell from a donor,

• Knocking out the TCR gene,

• Expressing at the surface of said cell at the CD123 specific chimeric antigen receptor according to the invention as any one of the above.

A donor may the patient suffering a cancer himself (for autologous adoptive transfer) or another individual (for adoptive transfer of allogenic T cells).

The present invention discloses a method of engineering an immune cell as above comprising:

Providing an immune cell from a donor,

Knocking out the TCR gene, and the dck gene and inserting the CD123 CAR in the TCR (trac) gene

Expressing at the surface of said cell the CD123 specific chimeric antigen receptor according to any one of the above by introducing into said cell at least one polynucleotide encoding said CD123 specific chimeric antigen receptor.

In particular embodiment, said method comprises expressing at the cell surface a suicide domain.

In preferred embodiments, said method comprises expressing at the cell surface a suicide domain, preferably a suicide domain recognized by one of the following antibodies ibritumomab, tiuxetan, muromonab-CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and ustekinumab.

In another embodiment said method further comprises a step of binding said engineered immune cell of the invention to a specific monoclonal antigen as those disclosed herein selected from ibritumomab, tiuxetan, muromonab- CD3, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, alemtuzumab, bevacizumab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND- 10 and ustekinumab.

This is reducing the number of active cells in vivo.

The present invention discloses a method of treating a subject in need thereof comprising:

• Providing an immune cell expressing at the surface a CD123 specific Chimeric Antigen Receptor according to any one of the above or a composition comprising the same,

• Administrating said immune cells to said patient.

In another embodiment, said composition is associated to a FLAG treatment, a FLAG treatment without addition.

In a preferred embodiment, said composition further comprises a purine analogue, fludarabine.

In one embodiment said subject in need thereof suffers AML, preferably refractory relapsed AML, BPDNL, or must have bone marrow transplantation.

The present invention discloses a method of treating a subject in need thereof as above, wherein an immune cell is provided from a donor.

The present invention discloses a method of treating a subject in need thereof as above, wherein said immune cell is provided from the patient himself.

By the term " antibody" as used herein, is meant an antibody or antibody fragment which is generated using, for example a recombinant DNA technology, such as, for example, an antibody or antibody fragment expressed by a bacteriophage, a yeast expression system or a mammalian cell expression system. The term should also be construed to mean an antibody or antibody fragment which has been generated by the synthesis of a DNA molecule encoding the antibody or antibody fragment and which DNA molecule expresses an antibody or antibody fragment protein, or an amino acid sequence specifying the antibody or antibody fragment, wherein the DNA or amino acid sequence has been obtained using recombinant or synthetic DNA or amino acid sequence technology which is available and well known in the art.

As used herein, the term "conservative sequence modifications" or "humanization" or "humanized antibody" or "humanized antibody fragment", "humanized VH or humanized VL" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the CAR and/or that do not significantly affect the activity of the CAR containing the modified amino acid sequence and reduce or abolish a human anti- mouse antibody (HAMA) response. In a preferred embodiment, amino acid modifications significantly improve the binding characteristics of the CAR and/or significantly improve the activity of cells expressing the CAR containing the modified amino acid sequence and reduce or abolish a human anti- mouse antibody (HAMA) or anti-CAR response.

Such conservative modifications include amino acid substitutions, additions and deletions in said antibody fragment in said CAR and/or any of the other parts of said CAR molecule. Modifications can be introduced into an antibody, into an antibody fragment or in any of the other parts of the CAR molecule of the invention by standard techniques known in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis or by employing optimized germline sequences. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested for the ability to bind CD 123 using the functiona l assays described herein.

In one embodiment said scfv comprises at least one, preferably two epitopes binding to a monoclonal antibody. In one embodiment said scfv comprises at least 3 epitopes binding to a monoclonal antibody.

In one embodiment said scfv comprises at least 4 epitopes binding to a monoclonal antibody.

The signal transducing domain or intracellular signaling domain of a CAR according to the present 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. Thus, 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 a CAR can be the cytoplasmic sequences of the 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 has 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. Examples of ITAM used in the invention can include as non limiting examples those derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the signaling transducing domain of the CAR can comprise the CD3zeta signaling domain, a ITAM from the CD3zeta signaling domain.

In particular embodiment the signal transduction domain of the CAR of the present invention comprises a co-stimulatory signal molecule, or a co-stimulatory domain. 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, 4-1BB, 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. Preferred costimulatory molecules include 4-1BB.

Any of the above may be a transduction enhancer. A transduction enhancer means an intracellular domain that facilitates the CAR transduction (as compare to the same CAR without transduction enhancer. In one embodiment, the signal transduction domain of the CAR of the present invention in particular the co-stimulatory molecules do not include and CD28 (NP_006130.1).

In one preferred embodiment, the CAR of the present invention does not include a sequence of human CD28 (NP_006130.1) and/or from any other CD28.

In another preferred embodiment, the signal transduction domain of the CAR of the present invention comprises a part of co-stimulatory signal molecule selected from the group consisting of fragments of human 4-1BB (GenBank: AAA53133.).

A CAR according to the present invention is expressed on the surface membrane of the cell. Thus, such CAR further comprises a 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 transmembrane domain can be derived either from a natural or from a synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. As non-limiting examples, the transmembrane polypeptide can be a subunit of the T-cell receptor such as α, β, or δ, polypeptide constituting CD3 complex, IL2 receptor p55 (a chain), p75 (β chain) or chain, subunit chain of Fc receptors, in particular Fc receptor III or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.

In a preferred embodiment said transmembrane domain (TM) is derived from the human CD8 alpha chain (e.g. NP_001139345.1), IgGl, lgG4, FcyRllla.

In a more preferred embodiment said TM domain comprises a transmembrane domain (TM) from human CD8a.

In one embodiment the CD123 CAR of the invention does not comprise a TM domain from 4-1BB, preferably of sequence IISFFLALTSTALLFLLFFLTLRFSW.

A CAR according to the present invention comprises a hinge region between said extracellular ligand-binding domain and said transmembrane domain. The term "hinge region" used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, hinge region is used to provide more or less 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. Hinge region 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 from all or part of an antibody constant region.

In the present case,

A CAR according to the present invention comprises a hinge from 10 to 25 amino acids, or less. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In embodiments said hinge domain comprises a part of human CD8 alpha chain, FcRllla receptor or IgGl respectively.

In a more preferred embodiment, said hinge domain comprises a part of human FcRllla receptor.

The present invention also relates to an isolated immune cell which comprises a population of CARs each one comprising different extracellular ligand binding domains.

According to the invention, the immune cells expressing the anti-CD123 CAR of the invention trigger an anti-cancer immune response, no or reduce GVHD and proliferate even in the presence of purine analogue of FLAG treatment.

In a preferred embodiment, the immune cells expressing the CAR of the invention endowed with the anti-CD123 CAR of the invention does trigger an immune response which does not comprise a human anti-mouse antibody (HAMA) response.

According to the invention, an efficient amount of the engineered immune cell of the invention can be administered to a patient in need thereof at least once, alone or in combination with another treatment.

Polynucleotides, vectors

The present invention also relates to polynucleotides, vectors encoding the above described CAR according to the invention. The polynucleotide may consist in 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 adeno associated vector, a lentivirus for transfection of a mammalian host cell.

VECTORS

Preferably the present invention encompasses vectors allowing integration /insertion of a sequence, preferably a sequence coding CD123 specific CAR of the invention into the TRAC gene or into any one of the genes cited here.

The present invention encompasses vectors allowing the preparation of engineered immune cell (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above.

Self cleaving peptide

In a particular embodiment, the different nucleic acid sequences can be included in one polynucleotide or vector which comprises a nucleic acid sequence encoding ribosomal skip sequence such as a sequence encoding a sele-cleaving peptide, such as 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 and Elliott 2001; Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). 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. Signal sequences

To direct transmembrane polypeptide 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 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).

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 nucleic acid 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.

In a more preferred embodiment the claimed invention is directed to a vector allowing a stable integration of the CAR of the invention into the genome, preferably into the TRAC gene.

Cells

Cell according to the present invention refers to a cell of hematopoietic origin functionally involved in the initiation and/or execution of innate and/or adaptative immune response. Cell according to the present invention is preferably a T-cell obtained from a donor. Said T cell according to the present invention can be derived from a stem cell. The stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, totipotent stem cells or hematopoietic stem cells. In a preferred embodiment, cells are human cells, in particular human stem cells. In a more preferred embodiment, cells are human T cells, in particular human engineered T cells.

Representative human stem cells are CD34+ cells. Said isolated 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. In another embodiment, said cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. In a preferred embodiment, said cell can be derived from the group consisting of engineered CD4+ T- lymphocytes and engineered CD8+ T-lymphocytes.

Prior to expansion and genetic modification of the 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 is preferably derived from a healthy donor. In another embodiment, said cell is part of a mixed population of cells which present different phenotypic characteristics.

Preferably, isolation and preparation of stem cells does not require the destruction of at least one human embryo. The immune cells can originate from the patient, in view of operating autologous treatments, or from one or several donors in view of producing allogeneic cells, which can be used in allogeneic treatments.

General Methods of engineering immune cells endowed with CARs:

The present invention encompasses the method of preparing immune cells for immunotherapy comprising introducing ex-vivo into said immune cells the polynucleotides or vectors encoding the CD123 CAR previously described in WO2014/130635, and in WO2013176916 incorporated herein by reference.

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

In a more preferred embodiment, said polynucleotide is included in a lentiviral vector in view of being stably expressed in the immune cells.

In an even more preferred embodiment, said polynucleotides are included in an adeno associated vector in view of being stably integrated into the TRAC gene or any one of the genes disclosed in USP62/410,187 and then, expressed in the cells. According to further embodiments, said method further comprises the step of genetically modifying said cell to make them more suitable for allogeneic transplantation and to reduce GVHD response.

Modifying T-cell by inactivating at least one gene encoding a T-cell receptor (TCR) component.

According to a main aspect, the CD123 CAR immune cell made less allogeneic, by inactivating a T-cell receptor (TCR) alpha gene as described in WO 2013/176915, and combined with the inactivation of a gene encoding or regulating HLA or β2ιη protein expression. Accordingly, the risk of graft versus host syndrome and graft rejection is significantly reduced.

Accordingly, when the immune cells are T-cells, the present invention also provides methods to engineer T-cells that are less allogeneic.

Methods of making cells less allogenic can comprise the step of inactivating at least one gene encoding a T-Cell Receptor (TCR) component, in particular TCRalpha and the two TCRbeta genes.

Methods disclosed in WO2013/176915 to prepare CAR expressing immune cell suitable for allogeneic transplantation, by inactivating one or more component of T-cell receptor (TCR), are all incorporated herein by reference.

The present invention encompasses an anti-CD123 CAR expressing immune cell wherein the gene expressing the alpha component of T-cell receptor (TCR) has been inactivated by inserting a polynucleotide encoding an anti-CD123 CAR of the invention. Thus, the present invention provides an anti-CD123 CAR expressing T cell wherein the CAR is derived from 6H6, or 9F5 anti-CD123 antibody and wherein the T-cell receptor (TCR) is inactivated in all cells expressing the CAR. By inactivating a gene it is intended that the gene of interest is not expressed in a functional protein form. In particular embodiments, the genetic modification of the method relies on the expression, in provided cells to engineer, of one rare-cutting endonuclease such that said rare-cutting endonuclease specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused by the rare- cutting endonuclease are commonly repaired through the distinct mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Mechanisms involve rejoining of what remains of the two DNA ends through direct re-ligation (Critchlow and Jackson 1998) or via the so-called microhomology-mediated end joining (Betts, Brenchley et al. 2003; Ma, Kim et al. 2003). Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions and can be used for the creation of specific gene knockouts. Said modification may be a substitution, deletion, or addition of at least one nucleotide. Cells in which a cleavage-induced mutagenesis event, i.e. a mutagenesis event consecutive to an NHEJ event, has occurred can be identified and/or selected by well-known method in the art. In a particular embodiment, the step of inactivating at least a gene encoding a component of the T-cell receptor (TCR) into the cells of each individual sample comprises introducing into the cell a rare-cutting endonuclease able to disrupt at least one gene encoding a component of the T-cell receptor (TCR). In a more particular embodiment, said cells of each individual sample are transformed with nucleic acid encoding a rare-cutting endonuclease capable of disrupting at least one gene encoding a component of the T-cell receptor (TCR), and said rare-cutting endonuclease is expressed temporarily into said cells.

Said rare-cutting endonuclease can be a meganuclease, a Zinc finger nuclease, CRISPR/Cas9 nuclease, Argonaute nuclease, a TALE-nuclease or a MBBBD-nuclease. In a preferred embodiment, said rare-cutting endonuclease is a TALE-nuclease. By TALE-nuclease is intended a fusion protein consisting of a DNA-binding domain derived from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain to cleave a nucleic acid target sequence (Boch, Schoize et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Cermak, Doyle et al. 2011; Geissler, Schoize et al. 2011; Huang, Xiao et al. 2011; Li, Huang et al. 2011; Mahfouz, Li et al. 2011; Miller, Tan et al. 2011; Morbitzer, Romer et al. 2011; Mussolino, Morbitzer et al. 2011; Sander, Cade et al. 2011; Tesson, Usal et al. 2011; Weber, Gruetzner et al. 2011; Zhang, Cong et al. 2011; Deng, Yan et al. 2012; Li, Piatek et al. 2012; Mahfouz, Li et al. 2012; Mak, Bradley et al. 2012). In the present invention new TALE- nucleases have been designed for precisely targeting relevant genes for adoptive immunotherapy strategies. Preferred TALE-nucleases recognizing and cleaving the target sequence are described in PCT/EP2014/075317. In particular, additional catalytic domain can be further introduced into the cell with said rare-cutting endonuclease to increase mutagenesis in order to enhance their capacity to inactivate targeted genes. More particularly, said additional catalytic domain is a DNA end processing enzyme. Non limiting examples of DNA end- processing enzymes include 5-3' exonucleases, 3-5' exonucleases, 5-3' alkaline exonucleases, 5' flap endonucleases, helicases, phosphatase, hydrolases and template-independent DNA polymerases. Non limiting examples of such catalytic domain comprise of a protein domain or catalytically active derivate of the protein domain selected from the group consisting of hExol (EX01_HUMAN), Yeast Exol (EX01_YEAST), E.coli Exol, Human TREX2, Mouse TREX1, Human TREX1, Bovine TREX1, Rat TREX1, TdT (terminal deoxynucleotidyl transferase) Human DNA2, Yeast DNA2 (DNA2_YEAST). In a preferred embodiment, said additional catalytic domain has a 3'-5'-exonuclease activity, and in a more preferred embodiment, said additional catalytic domain is TREX, more preferably TREX2 catalytic domain (WO2012/058458). In another preferred embodiment, said catalytic domain is encoded by a single chain TREX2 polypeptide. Said additional catalytic domain may be fused to a nuclease fusion protein or chimeric protein according to the invention optionally by a peptide linker.

Endonucleolytic breaks are known to stimulate the rate of homologous recombination. Thus, in another embodiment, the genetic modification step of the method further comprises a step of introduction into cells of an exogeneous nucleic acid comprising at least a sequence homologous to a portion of the target nucleic acid sequence, such that homologous recombination occurs between the target nucleic acid sequence and the exogeneous nucleic acid. In particular embodiments, said exogenous nucleic acid comprises first and second portions which are homologous to region 5' and 3' of the target nucleic acid sequence, respectively. Said exogenous nucleic acid in these embodiments also comprises a third portion positioned between the first and the second portion which comprises no homology with the regions 5' and 3' of the target nucleic acid sequence. Following cleavage of the target nucleic acid sequence, a homologous recombination event is stimulated between the target nucleic acid sequence and the exogenous nucleic acid. Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used within said donor matrix. In a particular embodiment, the homologous sequence can be from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared nucleic acid homologies are located in regions flanking upstream and downstream the site of the break and the nucleic acid sequence to be introduced should be located between the two arms. As used herein, a cell which is "resistant or tolerant" to an agent means a cell which has been genetically modified so that the cell proliferates and is active in the presence of an amount of an agent that inhibits or prevents proliferation of a cell without the genetic modification.

In a preferred embodiment, a drug sensitizing gene which can be inactivated to confer drug resistance to the T-cell is the human deoxycytidine kinase (dCK) gene. This enzyme is required for the phosphorylation of the deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA). Purine nucleotide analogs (PNAs) are metabolized by dCK into mono-, di- and tri-phosphate PNA. Their triphosphate forms and particularly clofarabine triphosphate compete with ATP for DNA synthesis, acts as proapoptotic agent and are potent inhibitors of ribonucleotide reductase (RNR) which is involved in trinucleotide production.

Preferably, the inactivation of dCK in T cells is mediated by TALE nuclease. To achieve this goal, several pairs of dCK TALE-nuclease have been designed, assembled at the polynucleotide level and validated by sequencing. Examples of TALE-nuclease pairs which can be used according to the invention are depicted in PCT/EP2014/075317.

This dCK inactivation in T cells confers resistance to purine nucleoside analogs (PNAs) such as clofarabine and fludarabine.

In more preferred embodiments, the dCK inactivation in T cells is combined with an inactivation of TRAC genes and insertion into the TRAC of the CD123 encoding vector of the invention rendering these double knock out (KO) T cells both resistant to drug such as clofarabine and less allogeneic than the same cell with an intact TCR. This features are particularly useful for a therapeutic goal, allowing "off-the-shelf" allogeneic cells for immunotherapy in conjunction with chemotherapy to treat patients with cancer preferably refractory relapsed AML, or BPDNL . One example of TALE-nuclease dCK/TRAC pairs which gave success in the invention is described in PCT/EP2014/075317, in particular, the target sequences in the 2 loci (dCK and TRAC).

According to another aspect, the CD123 CAR expressing T-cell of the invention can be further genetically engineered to improve its resistance to immunosuppressive drugs or chemotherapy treatments, which are used as standard care for treating cancer.

Several cytotoxic agents (anti-cancer drugs) such as anti-metabolites, alkylating agents, anthracyclines, DNA methyltransferase inhibitors, platinum compounds and spindle poisons have been developed to kill cancer cells. However, the introduction of these agents with novel therapies, such as immunotherapies, is problematic as these drugs affect the functioning/survival of immune T cells. For example, chemotherapy agents can be detrimental to the establishment of robust anti-tumor immunocompetent cells due to the agents' non-specific toxicity profiles. Small molecule-based therapies targeting cell proliferation pathways may also hamper the establishment of anti-tumor immunity. If chemotherapy regimens that are transiently effective can be combined with novel immunocompetent cell therapies then significant improvement in anti-neoplastic therapy might be achieved (for review (Dasgupta, McCarty et al. 2011). These drugs are considered as standard care drugs against cancer.

To improve cancer therapy and selective engraftment of allogeneic TCR KO, CD123 CAR expressing T-cells, drug resistance is conferred to said allogeneic T cells to protect them from the toxic side effects of chemotherapy agent. The drug resistance of T-cells also permits their enrichment in or ex vivo, as T-cells which express the drug resistance gene will survive and multiply relative to drug sensitive cells.

Methods for engineering T-cells resistant to chemotherapeutic agents are disclosed in PCT/EP2014/075317 which is fully incorporated by reference herein.

A method of engineering allogeneic CD123 CAR expressing T-cells suitable for combination therapy with purine analogues comprising :

• Providing an anti-CD123 CAR expressing T cell with an inactivated TCR • Modifying said anti-CD123 CAR expressing T-cell to confer drug resistance to said anti-CD123 CAR expressing T-cell; preferably to confer resistance to purine analogues

• Expanding said engineered anti-CD123 CAR expressing T-cell in the presence of said drug said drug is a purine analogue selected from pentostatin, fludarabine 2-deoxyadenosine, cladribine, clofarabine, Nelarabine, preferably pentostatin, fludarabine monophosphate, and 2-chlorodeoxyadenosine (2- CDA). Gene expression conferring drug resistance to anti-CD123 CAR-expressinq immune cells of the invention

In a particular embodiment, said drug resistance can be conferred to the T-cell of the invention by the expression of at least one drug resistance gene. Said drug resistance gene refers to a nucleic acid sequence that encodes "resistance" to an agent, such as a chemotherapeutic agent (e.g. methotrexate). In other words, the expression of the drug resistance gene in a cell permits proliferation of the cells in the presence of the agent to a greater extent than the proliferation of a corresponding cell without the drug resistance gene or survival in the presence of said drug. The expression of the drug resistance gene in a cell permits proliferation of the cells in the presence of the agent and does not affect its activity. A drug resistance gene of the invention can encode resistance to anti-metabolite, methotrexate, vinblastine, cisplatin, alkylating agents, anthracyclines, cytotoxic antibiotics, anti-immunophilins, their analogs or derivatives.

In one embodiment, a drug resistance gene confers resistance to a drug (or an agent), in particular an anti-cancer drug selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP 16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, cytarabine, anthracyclines, 6- thioguanine, hydroxyurea, prednisone, and combination thereof.

Combination treatment Any one of the following drug may be combined to the engineered cells of the invention, to improve the treatment against a CD123+ expressing cells involved directly or indirectly into a pathology.

The terms "therapeutic agent", "chemotherapeutic agent", or "drug" or "anti-cancer drug" as used herein also refers to a medicament, preferably a compound or a derivative thereof. A drug preferably reduces the proliferative status of the cell and/or kills the cell. Examples of chemotherapeutic agents or "anti-cancer drug" include, but are not limited to, alkylating agents (e.g., Busulfan, Carboplatine, Chlorambucil, Cisplatine, Cyclophosphamide, Ifosfamide, Melphalan, Mechlorethamine, Oxaliplatine, Uramustine, · Temozolomide, Fotemustine), metabolic antagonists (e.g., purine nucleoside antimetabolite such as clofarabine, fludarabine or 2'-deoxyadenosine, methotrexate (MTX), 5-fluorouracil or derivatives thereof, Azathioprine, Capecitabine, Cytarabine, · Floxuridine, · Fluorouracile, · Gemcitabine, · Methotrexate, Pemetrexed), antitumor antibiotics (e.g., mitomycin, Adriamycin, Bleomycine ,· Daunorubicine, · Doxorubicine, · Epirubicine, · Hydroxyurea, · Idarubicine, · Mitomycin C, · Mitoxantrone), plant-derived antitumor agents (e.g., vincristine, vindesine, Taxol, Vinblastine, · (Vinorelbine), · Docetaxel, · Paclitaxel), topoisomerase inhibitor (Irinotecan, · Topotecan, · Etoposide).

In a preferred embodiment, a therapeutic agent, a chemotherapy drug as used herein refers to a compound or a derivative thereof that may be used to treat cancer, in particular to treat a hematopoietic cancer cell and more particularly AML, and even more particular refractory relapsed AML thereby reducing the proliferative status of the cancer cell and/or killing the cancer cell.

Other Examples of chemotherapeutic agents include, but are not limited to Aracytine, Cytosine Arabinoside, Amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof.

In other embodiments of the present invention, cells of the invention are administered to a patient in conjunction with a drug (or an agent) selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone, and combination thereof.

Such agents may further include, but are not limited to, the anti-cancer agents TRI M ETHOTRIXATE™ (TMTX), TEMOZOLOMIDE™, RALTRITREXED™, S-(4-Nitrobenzyl)-6- thioinosine (NBMPR),6-benzyguanidine (6-BG), bis-chloronitrosourea (BCNU) and CAMPTOTHECIN™, or a therapeutic derivative of any thereof.

In particular embodiments an anti-CD123 CAR expressing T cell with a TCR and a dck KO gene, is administered to a patient, in combination with at least one therapeutic agent selected from Aracytine, Cytosine Arabinoside, Amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid and combination thereof.

In the present invention a combination treatment comprises infusion of a mAb for sorting and/or depletion purpose(s). In a preferred embodiment, said mAb is ixutimab.

The maximum dose of Rituximab to be administered preferably by intravenous route is 2,250 mg/m2. It is no administered as an intravenous push or bolus.

Multiple drug resistance of anti-CD123 CAR-expressinq immune cells

In another particular embodiment, the inventors sought to develop an "off-the shelf" immunotherapy strategy, using allogeneic T-cells, in particular allogenic anti-CD123 CAR expressing T-cell resistant to multiple drugs to mediate selection of engineered T-cells when the patient is treated with different drugs. The therapeutic efficiency can be significantly enhanced by genetically engineering multiple drug resistance allogeneic T-cells. Such a strategy can be particularly effective in treating tumors that respond to drug combinations that exhibit synergistic effects. Moreover multiple resistant engineered T-cells can expand and be selected using minimal dose of drug agents.

Thus, the method according to the present invention can comprise modifying T-cell of the invention to confer multiple drug resistance to said T-cell of the invention. Said multiple drug resistance can be conferred by either expressing more than one drug resistance gene or by inactivating more than one drug sensitizing gene. In another particular embodiment, the multiple drug resistance can be conferred to said T-cell by expressing at least one drug resistance gene and inactivating at least one drug sensitizing gene. In particular, the multiple drug resistance can be conferred to said T-cell by expressing at least one drug resistance gene such as mutant form of DHFR, mutant form of IMPDH2, mutant form of calcineurin, mutant form of MGMT, the ble gene, and the mcrA gene and inactivating at least one drug sensitizing gene such as HPRT gene. In a preferred embodiment, multiple drug resistance can be conferred by inactivating HPRT gene and expressing a mutant form of DHFR; or by inactivating HPRT gene and expressing a mutant form of IMPDH2; or by inactivating HPRT gene and expressing a mutant form of calcineurin; by inactivating HPRT gene and expressing a mutant form of MGMT; by inactivating HPRT gene and expressing the ble gene; by inactivating HPRT gene and expressing the mcrA gene.

In one embodiment, the present invention provides allogenic anti-CD123 CAR expressing T-cell expressing more than one drug resistance gene or wherein more than one drug sensitizing gene is inactivated.

Isolated cell

The present invention relates to an isolated cell expressing a CD123 CAR which binds to CD123. Thus, the invention relates to an anti-CD123 CAR expressing isolated cell. In a particular embodiment, said anti-CD123 CAR expressing isolated cell is resistant to at least one drug and comprises at least one disrupted gene encoding a T-cell receptor component.

In a preferred embodiment, the present invention relates to an isolated T cell expressing a CAR which binds to CD123 and is resistant to at least one purine analogue and comprises a disrupted gene encoding a T-cell receptor alpha component.

In another particular embodiment, said anti-CD123 CAR expressing isolated T cell comprises at least one disrupted drug sensitizing gene such as dCK or HPRT gene.

IMMUNE CHECK POINTS

The present invention provides allogeneic TCR KO with an insertion of a polypeptide comprising a CAR of the invention, expressing an anti-CD123 CAR of the invention, wherein at least one gene expressing one or more gene selected from the genes encoding a TCR beta subunit, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCDl, LAG 3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIRl, SIGLEC7, SIGLEC9, CD244, TNFRSFIOB, TNFRSFIOA, CASP8, CASPIO, CASP3, CASP6, CASP7, FADD, FAS, TGFBRM, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HM0X2, IL6R, IL6ST, CSK, PAG1, SIT1, F0XP3, PRDM1 (orblimpl), BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, is inactivated as referred to in WO2014/184741.

According to further aspect of the invention, the immune cells can be further manipulated to make them more active or limit exhaustion, by inactivating genes encoding proteins that act as "immune checkpoints" that act as regulators of T-cells activation, such as the following gene selected from CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCDl, LAG 3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIRl, SIGLEC7, SIGLEC9, CD244, TNFRSFIOB, TNFRSFIOA, CASP8, CASPIO, CASP3, CASP6, CASP7, FADD, FAS, TGFBRM, TGFBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, CSK, PAG1, SIT1, FOXP3, PRDM1 (orblimpl), BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, preferably, said gene is PDCDl or CTLA-4.

The present invention encompasses the isolated anti-CD123 CAR-immune cells or cell lines obtainable by the method of the invention, more particularly isolated cells comprising any of the proteins, polypeptides, allelic variants, altered or deleted genes or vectors described herein. This object is provided for the treatment of a cancer, in particular AML, refractory

AML, relapse AML, BPDCN.

In another aspect, the present invention concerns the method for treating or preventing cancer in the patient by administrating at least once an engineered immune cell obtainable by the above methods. One preferably two alleles of above genes are modified in engineered T cells of the invention; the locus is verified for each preparation of TALEN used to KO or KO by Kl said gene/allele. In preferred embodiments said method of further engineer the immune cells involves introducing into said T cells polynucleotides, in particular mRNAs, encoding specific rare- cutting endonuclease to selectively inactivate the genes mentioned above by DNA cleavage. In a more preferred embodiment said rare-cutting endonucleases are TALE-nucleases or Cas9 endonuclease. TAL-nucleases have so far proven higher specificity over the other types of rare-cutting endonucleases, making them the endonucleases of choice for producing of the engineered immune cells on a large scale. The initial method is that disclosed in Hendel et al., 2015. Trends Biotechnol. Feb; 33(2): 132-140; Pattanavak V. 2014. Methods Enzvmol. 546: 47-78, further adapted to TALEN.

Delivery methods

The different methods described above involve expressing a protein of interest such as drug resistance gene, rare-cutting endonuclease, Chimeric Antigen Receptor (CAR), in particular an anti-CD123 CAR into an isolated cell. As non-limiting example, said protein of interest can be expressed in the cell by its introduction as a transgene preferably encoded by at least one plasmid vector. Polypeptides may be expressed 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 include 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, cell fusion. Said polynucleotides may be included in vectors, more particularly plasmids or virus, in view of being expressed in cells. Said plasmid vector can comprise a selection marker which provides for identification and/or selection of cells which received said vector.

Different transgenes can be included in one vector. Said vector can comprise a nucleic acid 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.

In a more preferred embodiment of the invention, polynucleotides encoding polypeptides according to the present invention can be mRNA which is introduced directly into the cells, for example by electroporation. The inventors determined the optimal condition for mRNA electroporation in T-cell. The inventor used the cytoPulse technology which allows, by the use of pulsed electric fields, to transiently permeabilize living cells for delivery of material into the cells. The technology, based on the use of PulseAgile (BTX Havard Apparatus, 84 October Hill Road, Holliston, MA 01746, USA) electroporation waveforms grants the precise control of pulse duration, intensity as well as the interval between pulses (U.S. patent 6,010,613 and International PCT application WO2004083379). All these parameters can be modified in order to reach the best conditions for high transfection efficiency with minimal mortality. Basically, the first high electric field pulses allow pore formation, while subsequent lower electric field pulses allow moving the polynucleotide into the cell. The different methods described above involve introducing a CD123 CAR of the invention into a cell. As non-limiting example, said CAR can be introduced as transgenes encoded by one plasmid 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.

Activation and expansion of T cells

Whether prior to or after genetic modification of the T 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 T 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 RPMI 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 (IL-2), insulin, IFN-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 can include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, 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

In another particular embodiment, said cells can be expanded 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.

Pharmaceutical Compositions

The present invention encompasses a pharmaceutical composition comprising an engineered immune cell (TCR KO and/or dck KO) expressing one of any one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle. The pharmaceutical composition of the present disclosure may further comprise a pharmaceutically acceptable carrier or vehicle. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, etc. Compositions comprising such carriers can be formulated by well-known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose.

The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. An example of a dosage for administration might be in the range of 0.24 μg to 48 mg of cells according to the invention, preferably 0.24 μg to 24 mg, more preferably 0.24 μg to 2.4 mg, even more preferably 0.24 μg to 1.2 mg and most preferably 0.24 μg to 240 mg units per kilogram of body weight per day. Particularly preferred dosages are recited herein below. Progress can be monitored by periodic assessment.

The present invention encompasses a pharmaceutical composition comprising an engineered immune cell (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle for use as a medicament.

The present invention encompasses an embodiment disclosing a pharmaceutical composition comprising between from 10 ^~2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a pharmaceutically acceptable vehicle for use as a medicament.

The present invention discloses a pharmaceutical composition comprising between from 10 ^~2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising at least two epitopes specific for a monoclonal Ab, as any one disclosed in patent application PA 2015 70044 which is incorporated herein by reference in its entirety, a pharmaceutically acceptable vehicle for use as a medicament in combination with said monoclonal Ab.

I n preferred embodiments, a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising at least two epitopes specific for rituximab and said a pharmaceutical composition comprises rituximab.

I n preferred embodiments, a pharmaceutical composition comprising between from 10 ^"2 to from 10 ¹⁰ /kg of engineered immune cells (TCR KO and/or dck KO) expressing individually any (each) one of the CD123 specific CAR discloses above and a suicide domain comprising three epitopes specific for rituximab and an epitope specific for OBEN and said a pharmaceutical composition comprises rituximab and BEN10.

The present invention discloses a pharmaceutical composition comprising a pharmaceutically acceptable vehicle and any one of the engineered immune cell (TCR KO Kl CAR and/or dck KO) expressing a CD123 specific CAR as described above and another drug, preferably a purine analogue and more preferably a FLAG treatment.

Examples of purine analogues according to the invention may be pentostatin, fludarabine 2-deoxyadenosine, cladribine, clofarabine, Nelarabine, preferably pentostatin, fludarabine monophosphate, and 2-chlorodeoxyadenosine (2-CDA). Exam ples of FLAG treatments that may be associated with the CD123 T cells of the invention are as follows: Standard FLAG without additions, FLAG-I DA, Mito-FLAG, FLAMSA.

An Example of FLAG treatment according to the invention may be:

Standard FLAG without additions

Drug Dose Mode Days

30 mg/m ² a IV infusion over 30 min, every 12 hours in 2

(FL)udarabine Days 1-5

day divided doses

IV infusion over 4 hours, every 12 hours in 2

(A)ra-C 2000 mg/m ² Days 1-5

divided doses, starting 4 hours after the end

FLAG-IDA

Mito-FLAG recovery

FLAMSA

Provided herein are pharmaceutical compositions comprising the genetically engineered immune cells of the invention, e.g., genetically engineered TCR KO- dCK KO, CD123 CAR T cells Kl expressing at the cell surface, a CD123 CAR from 6H6 or from 9F5 anti-CD123 antibody.

I n another embodiment, pharmaceutical compositions comprising the genetically engineered immune T cells of the invention and at least a PNA are provided.

I n another embodiment, pharmaceutical compositions comprising the genetically engineered immune T cells of the invention, rituximab and at least a PNA are provided.

I n a preferred embodiment, the present invention provides a kit of parts, comprising any one of the following part: a pharmaceutical composition comprising the genetically engineered immune T cells of the invention, a monoclonal antibody, a drug. In a more preferred embodiment, the present invention provides a kit of parts, a part comprising any one of the following: a pharmaceutical composition comprising the genetically engineered immune CD123 T cells of the invention, a monoclonal antibody rituximab, a purine analogue or a FLAG treatment. The treatment with the engineered immune cells according to the invention may be in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.

Preferably, the treatment with the engineered immune cells according to the invention may be administered in combination (e.g., before, simultaneously or following) with one or more therapies against cancer selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine ,Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, combination of arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof. According to a preferred embodiment of the invention, said treatment can be administrated into patients undergoing an immunosuppressive treatment. Indeed, the present invention preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In this aspect, the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.

Administration

The administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection. In accordance with this disclosure, the term "pharmaceutical composition" relates to a composition that can be administered to an individual. In a preferred embodiment, the pharmaceutical composition comprises a composition for parenteral, transdermal, intraluminal, intra-arterial, intrathecal or intravenous administration (iv) or for direct injection into a cancer. It is in particular envisaged that said pharmaceutical composition is administered to the individual via infusion or injection iv. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous (iv), subcutaneous, intraperitoneal, intramuscular, topical or intradermal administration, preferably iv or intra tumor for the engineered cells of the present invention. The CD123 CAR+ cell pharmaceutical compositions of the disclosure may be administered locally or systemically. Administration will generally be parenteral, e.g., intravenous; In a preferred embodiment, the pharmaceutical composition is administered subcutaneously and in an even more preferred embodiment intravenously. Preparations for parenteral administration include sterile aqueous solutions, suspensions, and emulsions comprising a non aqueous solvent. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present disclosure might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition of the disclosure might comprise, in addition to the CD123+ engineered cells (as described in this disclosure), further biologically active agents, depending on the intended use of the pharmaceutical composition.

Any of the compositions described herein may be comprised in a kit of parts. In a non-limiting example, one or more cells according to the invention for use in cell therapy and/or the reagents to generate one or more cells for use in cell therapy that harbors recombinant expression vectors may be comprised in a kit. The kit components are provided in suitable container means.

Some components of the kits may be packaged either in aqueous media or in lyophilized form and under frozen packages. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly useful. In some cases, the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.

In a more preferred embodiment one and/or more liquid solutions is cryoresistant, even more preferably is cryoresistant and protect the engineered immune cells of the invention during freezing and thawing procedures.

However, the components of the kit or part of it may be provided as dried powder(s) or as a tablet. When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

In particular embodiments, cells that are to be used for cell therapy are provided in a kit, and in some cases the cells are essentially the sole component of the kit. The kit may comprise reagents and materials to make the desired cell. In specific embodiments, the reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and/or DNA that encodes a CAR molecule as described herein and/or regulatory elements therefor.

In particular embodiments, there are one or more apparatuses in the kit suitable for extracting one or more samples from an individual. The apparatus may be a syringe, scalpel, and so forth.

In some cases, the kit, in addition to cell therapy embodiments, also includes another cancer therapy, such as chemotherapy, hormone therapy, and/or immunotherapy, for example. The kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual. Preferably, said other cancer therapy is a purine analogue, a FLAG treatment.

Therapeutic uses of engineered T-cells Comprising a CD123 CAR of the invention

In various embodiments CAR constructs, nucleic acid sequences, vectors, host cells, as contemplated herein and/or pharmaceutical compositions comprising the same are used for the prevention, treatment or amelioration of a disease, such as a cancerous disease. In particular embodiments, the pharmaceutical composition of the present disclosure may be particularly useful in preventing, ameliorating and/or treating cancer, including cancer having liquid tumors, for example.

In particular embodiments, provided herein are a method of treating an individual for cancer, comprising the step of providing a therapeutically effective amount of a plurality of any of engineered cells of the disclosure to the individual.

In certain aspects, the cancer is a solid tumor, and the tumor may be of any size, but in specific embodiments, the solid tumors are about 2 mm or greater in diameter. In certain aspects, the method further comprises the step of providing a therapeutically effective amount of an additional cancer therapy to the individual.

As used herein "treatment" or "treating," includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. "Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

As used herein, "prevent," and similar words such as "prevented," "preventing" etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition. In particular embodiments, the present invention contemplates, in part, cells, CAR constructs, nucleic acid molecules and vectors that can administered either alone or in any combination using standard vectors and/or gene delivery systems, and in at least some aspects, together with a pharmaceutically acceptable carrier or excipient.

In specific embodiments, viral vectors may be used that are specific for certain cells or tissues and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art. The compositions prepared according to the disclosure can be used for the prevention or treatment or delaying the above identified diseases.

Furthermore, the disclosure relates to a method for the prevention, treatment or amelioration of a tumorous disease comprising the step of administering to a subject or individual in the need thereof an effective amount of immune cells, e.g., T cells or cytotoxic T lymphocytes, harboring a CD123 CAR; a nucleic acid sequence encoding the same; a vector comprising a nucleotide sequence encoding said CD123 CAR, as described herein and/or produced by a process as described herein.

The administration of the composition(s) of the disclosure is useful for all stages and types of lymphoproliferative disorder, including for minimal residual disease, early cancer, advanced cancer, and/or relapsed and/or refractory cancer.

The disclosure further encompasses co-administration protocols with other compounds, e.g. PNA, specific antibody, targeted toxins or other compounds, which act via immune cells. The clinical regimen for co-administration of the inventive compound(s) may encompass co-administration at the same time, before or after the administration of the other component. Particular combination therapies include chemotherapy, radiation, surgery, hormone therapy, or other types of immunotherapy. Particular doses for therapy may be determined using routine methods in the art.

However, in specific embodiments, the engineered CD123+ T cells of the invention are delivered to an individual in need thereof once, although in some cases it is multiple times, including 2, 3, 4, 5, 6, or more times. When multiple doses are given, the span of time between doses may be of any suitable time, but in specific embodiments, it is days or weeks, between the doses. It may be months in chronically lymphodepleted patients. Doses and origins of the T cell donor are selected to avoid any undesirable side effects. Preferably cells were engineered to avoid any undesirable effect. The time between doses may vary in a single regimen and may depend on the recipient (patient in need thereof). In particular embodiments, the time between doses is 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days or weeks. In specific cases, it is between 4-8 or 6-8 weeks, for example.

In specific embodiments, one regimen includes one of the following dose regimen of CD123 T cell of the invention :

1/m ² ,

1 1 x 10/m ² , 1 1 x 10 ² /m ² ,

1 1 x 10 ³ /m ² ,

1 1 x 10 ⁴ /m ² ,

1 1 x 10 ⁵ /m ² ,

1 1 x 10 ⁶ /m ² , 2 3 x 10 ⁶ /m ² ,

3 1 x 10 ⁷ /m ² 4 3 x 1 0 ⁷ /m ² ,

5 1 x 1 0 ⁸ /m ² , or from 10 ^"2 to from 10 ¹⁰ cells /kg. In an alternative embodiment, the T cells are provided to the individual in the following dose regimen:

Dose Level CD123 CTL Dose l l x l 0 ⁶ /m ² , 2 1 x 1 0 ⁷ /m ² , 3 1 x 1 0 ⁸ /m ² ,

In an alternative embodiment, the T cells are provided to the individual in the following dose regimen: from :

6xl0 ² /kg to from 5xl0 ⁸ cells /kg, An efficient amount of the CD123 CAR engineered immune cell of the invention corresponds to a dose that is adapted to the status of each treated individual.

Therapeutic applications

In embodiments, isolated cell(s) obtained by the different methods or cell derived (after one to 50 in vitro passages) from said isolated cell of the invention can be used as a medicament. In another embodiment, said medicament can be used for treating cancer, particularly for the treatment of CD123 expressing cell lymphomas or leukemia.

In another embodiment, said isolated cell(s) according to the invention or cell line derived from said isolated cell(s) can be used in the manufacture of a medicament for treatment of a cancer in a patient in need thereof. In a particular embodiment, an anti-CD123 CAR expressing T cell is provided as a medicament for the treatment of AML, of an AML subtype, of an AML-related complication, of an AML-related condition.

In another embodiment, said medicament can be used for treating a CD123- expressing cell-mediated pathological condition or a condition characterized by the direct or indirect activity of a CD123-expressing cell. In other words, the invention is related to an anti-CD123 CAR expressing T cell for its use as a medicament to treat a condition linked to the detrimental activity of CD123-expressing cells, in particular to treat a condition selected from AML, AML subtype, AML-related complication, and AML-related conditions; I n another aspect, the present invention relies on methods for treating patients in need thereof, said method comprising at least one of the following steps:

• providing an immune-cell obtainable by any one of the methods previously described; · Administrating said transformed immune cells to said patient,

On one embodiment, said T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time.

I n a preferred embodiment said T cell is destroyed by administration of a drug or a n antibody that selectively destroys said T cells. I n a more preferred embodiment genetically engineered TCR KO dCK KO CD123 CAR T

Kl, cells of the invention having a CD123 CAR from 6H6 or from 9F5 anti-CD123 antibody at the cell surface are used as a medicament/ a treatment and then cleared up from the patient using an antibody recognizing the suicide domain RQ.R8, or an antibody recognizing a CD20 epitope, using preferably rituximab and/or using an antibody Q.BEN 10 that recognizes a CD34 epitope.

Said 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 a re originating from a donor, preferably a healthy donor and preferably a compatible (HLA matching donor.

Compatible donor means a donor which immune (histocompatible) cells if engrafted into a patient will induce low or no adverse immune reaction as compared to non histo compatible HLA mismatch cells.

Conditions that may be treated using the CD123 expressing CAR cells of the present invention are found in hematologic cancers, such as leukemia or malignant lymphoproliferative disorders. Lymphoproliferative disorder ca n be lymphoma, in particular multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). Cancers that may be treated using the cells of the invention or a pharmaceutical composition comprising said cell may comprise nonsolid tumors (such as hematological tumors, including but not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, BPDNL, AML, refractory relapse AML, or before bone marrow transplantation.

Types of cancers to be treated with the CD123 cell expressing CARs of the invention include, but are not limited leukemia or lymphoid malignancies. Adult tumors/cancers and pediatric tumors/cancers are also included.

Any other CD123-mediating or CD123-involving malignant lymphoproliferative disorders disclosed herein may be improved with the anti-CD123 CAR-expressing cells of the present invention.

In a preferred embodiment, the cancer that may be treated using the anti-CD123 CAR -expressing cells of the present invention is leukemia (AML), a disease associated to leukemia or a complication thereof. Leukemias that can be treated using the anti-CD123 CAR -expressing cells of the present invention can be acute myelogenous leukemia (AML), chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

AML or AML subtypes that may be treated using the anti-CD123 CAR-expressing cells of the present invention may be in particular, acute myeloblastic leukemia, minimally differentiated acute myeloblastic leukemia, acute myeloblastic leukemia without maturation, acute myeloblastic leukemia with granulocytic maturation, promyelocytic or acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, myelomonocytic together with bone marrow eosinophilia, acute monoblastic leukemia (M5a) or acute monocytic leukemia (M5b), acute erythroid leukemias, including erythroleukemia (M6a) and very rare pure erythroid leukemia (M6b), acute megakaryoblastic leukemia, acute basophilic leukemia, acute panmyelosis with myelofibrosis, whether involving CD123-positive cells.

Subtypes of AML that may be treated using the anti-CD123 CAR-expressing cells of the present invention also include, hairy cell leukemia, Philadelphia chromosome-positive acute lymphoblastic leukemia. AML may be classified as AML with specific genetic abnormalities. Classification is based on the ability of karyotype to predict response to induction therapy, relapse risk, survival.

Accordingly, AML that may be treated using the anti-CD123 CAR-expressing cells of the present invention, may be AM L with a translocation between chromosomes 8 and 21, AML with a translocation or inversion in chromosome 16, AML with a translocation between chromosomes 9 and 11, APL (M3) with a translocation between chromosomes 15 and 17, AML with a translocation between chromosomes 6 and 9, AML with a translocation or inversion in chromosome 3, AML (megakaryoblastic) with a translocation between chromosomes 1 and 22.

The present invention also provides an anti-CD123 CAR expressing T cell for the treatment of patients with specific cytogenetic subsets of AML, such as patients with t(15;17)(q22;q21) identified using all-trans retinoic acid (ATRA)16-19 and for the treatment of patients with t(8;21)(q22;q22) or inv(16)(pl3q22)/t(16;16)(pl3;q22) identified using repetitive doses of high-dose cytarabine.

Preferably, the present invention provides an anti-CD123 CAR expressing T cell for the treatment of patients with aberrations, such as— 5/del(5q),—7, abnormalities of 3q, or a complex karyotype, who have been shown to have inferior complete remission rates and survival, in combination with a FLAG. GROUP OF PATIENTS

The present invention provides an anti-CD123 CAR expressing T cell for the treatment of patients with "high risk AML".

Children older than age 2 with AML are considered as higher risk AML.

As high risk AML Children those with a WBC count more than 100,000 cells per cubic millimeter at diagnosis, undifferentiated AML (M0) and acute megakaryoblastic leukemia (M7) are usually harder to treat. The object of the present invention is particularly suited for the treatment of these children. Similarly Children whose leukemia cells are missing a copy of chromosome 7 (known as monosomy 7) have a poorer prognosis and may be treated with the pharmaceutical composition of the present invention.

Children who first have myelodysplastic syndrome ("smoldering leukemia") or whose leukemia is the result of treatment for another cancer may be treated with the pharmaceutical composition of the present invention.

The present engineered cells are particula rly well adapted for these poor prognosis, difficult to treat children suffering AML or refractory AM L.

I n a preferred embodiment, the invention provides an anti-CD123 CAR expressing T cell of as a treatment for AM L in patients over 60 years or in pediatric patients.

I n still another preferred embodiment, the present invention is used as a treatment in AM L patients with low, poor or unfavorable status that is to say with a predicted survival of less than 5 years survival rate. I n this group, patients suffering AM L with the following cytogenetic characteristics : -5; 5q; -7; 7q-;llq23; non t(9;ll); inv(3); t(3;3); t(6;9); t(9;22) is associated with poor-risk status (Byrd J.C. et al., December 15, 2002; Blood : 100 (13) and is especially contemplated to be treated according to the present invention or with an object of the present invention.

I n one embodiment, the anti-CD123 CAR expressing T cell of present invention may be used as induction therapy, as post remission therapy of AM L or as a consolidation therapy in patient with AM L.

I n another preferred embodiment, TCR KO, dck KO, anti-CD123 CAR expressing T cell of the present invention is used for preventing cancer cells development occurring in particular after anti-cancer treatment, during bone marrow depletion or before bone marrow transplantation, after bone marrow destruction. AM L COM PLICATIONS

I n one particular embodiment the invention provides a medicament that improves the health condition of a patient, in particular a patient undergoing a complication related to AM L. A complication or disease related to AML may include a preceding myelodysplasia phase, secondary leukemia, in particular secondary AML, high white blood cell count, and absence of Auer rods. Among others, leukostasis and involvement of the central nervous system (CNS), Hyperleukocytosis, residual disease, are also considered as a complication or disease related to AML.

AML ASSOCIATED DISEASES

In one embodiment, the present invention also provides a TCR KO, anti-CD123 CAR expressing T cell for the treatment of a pathological condition related to AML.

The present invention provides a therapy (a TCR KO, anti-CD123 CAR expressing T cell for AML related myeloid neoplasms, for acute myeloid leukemia and myelodysplastic syndrome, a treatment of relapsed or refractory acute myeloid leukemia, a treatment of relapsed or refractory acute promyelocytic leukemia in adults, a treatment for acute promyeloid leukaemia, a treatment of acute myeloid leukemia in adults over 60 years.

According to another aspect, the present invention provides a composition for the treatment of AML associated diseases, in particular hematologic malignancy related to AML.

Hematologic malignancy related to AML conditions include myelodysplasia syndromes (MDS, formerly known as "preleukemia") which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML In another embodiment, the invention provides a medicament that improves the health state of a patient suffering multiple myeloma.

Other pathological conditions or genetic syndromes associated with the risk of AM L can be improved with the adequate use of the present invention, said genetic syndromes include Down syndrome, trisomy, Fanconi anemia, Bloom syndrome, Ataxia-telangiectasia, Diamond-Blackfan anemia, Schwachman-Diamond syndrome, Li-Fraumeni syndrome, Neurofibromatosis type 1, Severe congenital neutropenia (also called Kostmann syndrome)

Other CD123-mediated pathological conditions According to another aspect, the present invention provides a composition for the treatment of CD123+cell-mediated diseases. These CD123+cell mediated diseases include inflammation, autoimmune diseases.

In particular, the present invention can be used for the treatment of CD123+cell mediated diseases such as inflammation of the gastrointestinal mucosae and more particularly, inflammatory bowel diseases, nasal allergy, inflammation of the skin such as juvenile dermatomyositis, hematodermia.

The present invention can be used as a medicament for the treatment of CD123+cell mediated diseases such as autoimmune diseases in particular Kikushi disease. Preferably, the present invention provides a treatment for a recurrent infection including infection due to viruses such as Epstein-Barr virus, herpes simplex virus, in particular oncogenic viruses, HHV-8, HHV-6, HTLV or HIV, parasitic infection such as infection due to Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, or Plasmodium malariae.

In particular, the present invention provides a treatment for Epstein-Barr virus lymphadenitis, herpes simplex virus lymphadenitis.

In another aspect, the present invention provides a composition for the treatment of systemic lupus erythematosus lymphadenitis , tuberculosis, cystic fibrosis, hepatitis, biliary atresia, in particular virus-induced hepatitis or biliary atresia in children, autoimmune hepatitis; primary biliary cirrhosis. Any of these CD123-mediated diseases may be improved using the object of the present invention.

Composition comprisinfi an enfiineered T cells accordin to the invention for use as a medicament and method

The present invention also provides a composition comprising a genetically engineered immune TCR KO - CD123 CAR T Kl cells or a genetically engineered immune TCR KO - dCK KO CD123 CAR T Kl cells for its use or a method for treating a cancer such as BPDNL or as a treatment before bone marrow transplant.

The present invention also provides a composition as above for its use or a method for inhibiting the proliferation or reducing a CD123-expressing cell population or activity in a patient. An exemplary method includes contacting a population of cells comprising a CD123- expressing cell with a CD 123 CART cell of the invention that binds to the CD123-expressing cell.

In a more specific aspect, the present invention provides a composition for its use or a method for inhibiting the proliferation or reducing the population of cancer cells expressing CD 123 in a patient, the methods comprising contacting the CD123-expressing cancer cell population with a CD 123 CART cell of the invention that binds to the CD123- expressing cell, binding of a CD 123 CART cell of the invention to the CD123-expressing cancer cell resulting in the destruction of the CD123-expressing cancer cells.

In certain aspects, the CD 123 CART cell of the invention reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% (to undetectable level) in a subject with or animal model for a CD123+-mediated disease such as myeloid leukemia or another cancer associated with CD123-expressing cells, relative to a negative control. The present invention also provides a composition for its use or a method for preventing, treating and/or managing a disorder or condition associated with CD123- expressing cells (e.g., associated with a hematologic cancer), the methods comprising administering to a subject in need a CD 123 CART cell of the invention that binds to the CD123-expressing cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with CD123-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies, IBD, and asthma) and cancers (such as hematological cancers, in particular AML or AML complications).

The present invention also provides a composition for its use or a method for preventing, treating and/or managing a disease associated with CD123-expressing cells, the method comprising administering to a subject in need a CD 123 CART cell of the invention that binds to the CD123-expressing cell. In one aspect, the subject is a human. Non-limiting examples of diseases associated with CD123-expressing cells include Acute Myeloid Leukemia (AML), myelodysplasia, B-cell Acute Lymphoid Leukemia, T-cell Acute Lymphoid Leukemia, hairy cell leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia, Hodgkin lymphoma. The present invention provides a composition for its use or a method for treating or preventing relapse of cancer associated with CD123-expressing cells, the method comprising administering to a subject in need thereof a CD 123 CART cell of the invention that binds to the CD 123- expressing cell. In another aspect, the methods comprise administering to the subject in need thereof an effective amount of a CD 123 CART cell of the invention that binds to the CD123-expressing cell in combination with an effective amount of another therapy.

In one aspect, CD 123 is considered to be a "cancer stem cell" marker in AM L. Therefore, a CD 123 CART cell of the invention can prevent relapse of AML, or even treat AML that is mostly CD 123-negative but with a "stem" population of CD 123+ cells (a CD123- expressing cells).

In one aspect, the invention provides compositions and methods for treating subjects that have undergone treatment for a disease or disorder associated with elevated expression levels of CD 19, and exhibits a disease or disorder associated with elevated levels of CD123. In one aspect, B-cell acute lymphoid leukemia (ALL) is an example of disease requiring a serial treatment using CART cells. For example, treatment with anti-CD 19 CAR T cells can sometimes result in CD19-negative relapse, which can be treated with anti-CD123 CAR T cells of the invention. Alternatively, the present invention includes dual targeting of B- ALL using CART cells comprising an anti-CD 19 CAR and an anti-CD 123 CAR. The administration of the cells or population of cells can consist of the administration of from 10 ⁴ -10 ⁹ 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.

In another embodiment, said effective amount of cells or composition comprising those cells are administrated parenterally. Said administration can be an intravenous administration. Said administration can be directly done by injection within a tumor.

In certain embodiments of the present invention, 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 antivira l therapy, cidofovir and interleukin-2, Cytarabine (also known as ARA-C) or natalizimab 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) (Henderson, Naya et al. 1991; Liu, Albers et al. 1992; Bierer, Hollander et al. 1993). 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 immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery. In certain embodiments of the present invention, anti-CD123 CAR expressing cells are administered to a patient in conjunction (e.g., before, simultaneously or following) with a drug selected from Aracytine, Cytosine Arabinoside, amsacrine, Daunorubicine, Idarubicine, Novantrone, Mitoxantrone, Vepeside, Etoposide (VP16), arsenic trioxyde, transretinoic acid, mechlorethamine, procarbazine, chlorambucil, and combination thereof. In these embodiments anti-CD123 CAR expressing cells may be resistant to the particular drug or combination of drugs that is (are) administered in conjunction with anti-CD123 CAR expressing cells.

The "resistant" CD123 CAR expressing cells disclosed in such embodiments are not destroyed when administered to a patient treated with said drug and still can recognize and lyse a CD123+ cancer cells in such drug-treated patients.

In other embodiments of the present invention, anti-CD123 CAR expressing cells are administered to a patient in conjunction with a drug selected from cytarabine, anthracyclines, 6-thioguanine, hydroxyurea, prednisone, and combination thereof. Other definitions

- Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.- 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.

- Amino acid substitution means the replacement of one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a peptide sequence is an amino acid substitution.

- Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: an 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 chimeric antigen receptor (CAR) is intended molecules that combine a binding domain against a component present on the target cell, for example an antibody-based specificity for a desired antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-target cellular immune activity. Generally, CAR consists of an extracellular single chain antibody (scFv Fc) fused to the intracellular signaling domain of the T cell antigen receptor complex zeta chain (scFv Fc^) and have the ability, when expressed in T cells, to redirect antigen recognition based on the monoclonal antibody's specificity. One example of CAR used in the present invention is a CAR directing against CD123 antigen and can comprise as non limiting example the amino acid sequences disclosed in PCT/EP 2016/051471.

- The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Endonucleases do not cleave the DNA or RNA molecule irrespective of its sequence, but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as "target sequences" or "target sites". Endonucleases can be classified as rare-cutting endonucleases when having typically a polynucleotide recognition site greater than 12 base pairs (bp) in length, more preferably of 14-55 bp. Rare-cutting endonucleases significantly increase HR by inducing DNA double- strand breaks (DSBs) at a defined locus (Perrin, Buckle et al. 1993; Rouet, Smih et al. 1994; Choulika, Perrin et al. 1995; Pingoud and Silva 2007). Rare-cutting endonucleases can for example be a homing endonuclease (Paques and Duchateau 2007), a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus and Carroll 2005), a Cas9 endonuclease from CRISPR system (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) or a chemical endonuclease (Eisenschmidt, Lanio et al. 2005; Arimondo, Thomas et al. 2006). In chemical endonucleases, a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences (Kalish and Glazer 2005). Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention.

- By a "TALE-nuclease" (TALEN) is intended a fusion protein consisting of a nucleic acid-binding domain typically derived from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain to cleave a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, like for instance l-Tevl, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease like for instance l-Crel and l-Onul or functional variant thereof. In a more preferred embodiment, said nuclease is a monomeric TALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of l-Tevl described in WO2012138927. Transcription Activator like Effector (TALE) are proteins from the bacterial species Xanthomonas comprise a plurality of repeated sequences, each repeat comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from new modular proteins recently discovered by the applicant in a different bacterial species. The new modular proteins have the advantage of displaying more sequence variability than TAL repeats. Preferably, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, Nl for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In another embodiment, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. TALE-nuclease have been already described and used to stimulate gene targeting and gene modifications (Boch, Scholze et al. 2009; Moscou and Bogdanove 2009; Christian, Cermak et al. 2010; Li, Huang et al. 2011). Engineered TAL-nucleases are commercially available under the trade name TALEN™ (Cellectis, 8 rue de la Croix Jarry, 75013 Paris, France).

The rare-cutting endonuclease according to the present invention can also be a Cas9 endonuclease. Recently, a new genome engineering tool has been developed based on the RNA-guided Cas9 nuclease (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012; Cong, Ran et al. 2013; Mali, Yang et al. 2013) from the type II prokaryotic CRISPR (Clustered Regularly Interspaced Short palindromic Repeats) adaptive immune system (see for review (Sorek, Lawrence et al. 2013)). The CRISPR Associated (Cas) system was first discovered in bacteria and functions as a defense against foreign DNA, either viral or plasmid. CRISPR- mediated genome engineering first proceeds by the selection of target sequence often flanked by a short sequence motif, referred as the proto-spacer adjacent motif (PAM). Following target sequence selection, a specific crRNA, complementary to this target sequence is engineered. Trans-activating crRNA (tracrRNA) required in the CRISPR type II systems paired to the crRNA and bound to the provided Cas9 protein. Cas9 acts as a molecular anchor facilitating the base pairing of tracRNA with cRNA (Deltcheva, Chylinski et al. 2011). In this ternary complex, the dual tracrRNAxrRNA structure acts as guide RNA that directs the endonuclease Cas9 to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence for homology between the target sequence and the crRNA. In addition to the target sequence-crRNA complementarity, DNA targeting requires the presence of a short motif adjacent to the protospacer (protospacer adjacent motif - PAM ). Following pairing between the dual-RNA and the target sequence, Cas9 subsequently introduces a blunt double strand brea k 3 bases upstream of the PAM motif (Garneau, Dupuis et al. 2010).

Rare-cutting endonuclease ca n be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (Stoddard 2005). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the invention may for example correspond to a LAGLI DADG endonuclease, to a HN H endonuclease, or to a GIY-YIG endonuclease. Preferred homing endonuclease according to the present invention can be an \-Crel variant.

- 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, peptides developed by Diatos. I n these cases, delivery vectors are molecule carriers. By "delivery vector" or "delivery vectors" is also intended delivery methods to perform transfection. - 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 vira l vector, a plasmid, a RNA vector or a linea r 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 "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 repeat variant, a variant, a DNA binding variant, a TALE-nuclease variant, a polypeptide variant obtained by mutation or replacement of at least one residue in the amino acid sequence of the parent molecule.

- by "functional variant" is intended a catalytically active mutant of a protein or a protein domain; such mutant may have the same activity compared to its parent protein or protein domain or additional properties, or higher or lower activity.

-"identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 70%, 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.

- "similarity" describes the relationship between the amino acid sequences of two or more polypeptides. BLASTP may also be used to identify an amino acid sequence having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence similarity to a reference amino acid sequence using a similarity matrix such as BLOSUM45, BLOSUM62 or BLOSUM80. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score. BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means. A polynucleotide encoding such a functional variant would be produced by reverse translating its amino acid sequence using the genetic code.

- "signal-transducing domain" or "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 igand (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-IBB, 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. 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 term "extracellular ligand-binding domain" as used herein is defined as an oligo- or polypeptide that is capable of binding a ligand. 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. "Universal" means that cells of the invention are "off the shelve" engineered cells wherein at least the TCR was inactivated, preferably by deletion of the TRAC gene using the TALEN ^® technology. Inactivated means preferentially that a genomic sequence is deleted, inserted or mutated, more preferentially deleted or inserted. Consequently, cells may be administered to a patient and induce no or very reduced Graft versus host disease (GVHD) (intensity grade 0 to 2) as compared to a GVHD measured in a immunohistoincompatible individual after "allogeneic" transplantation or transfer of immune cells with no alteration of the genomic DNA encoding a sub unit of the TCR.

Preferably "Universal" means that cells of the invention are "off the shelve" engineered cells wherein the TCR was inactivated, preferably by deletion of the TRAC gene and insertion of an open reading frame using the TALEN ^® technology. In a more preferred embodiment, the insertion comprises a CD123 CAR of the invention. In an even more preferred embodiment, the insertion comprises a CD123 CAR of the invention and said cells comprise a genetic modification of the beta 2 M gene inactivation the expression of M HC Class I molecules and a genetic modification modifying the expression of MHC class II molecules.

The term "subject" or "patient" as used herein includes all members of the animal kingdom including non-human primates and humans.

The term "relapsed" refers to a situation where a subject or a mammal, who has had a remission of cancer after therapy has a return of cancer cells. The term "refractory or resistant" refers to a circumstance where a subject or a mammal, even after intensive treatment, has residual cancer cells in his body.

The term "drug resistance" refers to the condition when a disease does not respond to the treatment of a drug or drugs. Drug resistance can be either intrinsic (or primary resistance), which means the disease has never been responsive to the drug or drugs, or it can be acquired, which means the disease ceases responding to a drug or drugs that the disease had previously responded to (secondary resistance). In certain embodiments, drug resistance is intrinsic. In certain embodiments, the drug resistance is acquired.

The term "hematologic malignancy" or "hematologic cancer" refers to a cancer of the body's blood- bone marrow and/or lymphatic tissue. Examples of hematological malignancies include, for instance, myelodysplasia, leukemia, lymphomas, such as cutaneous Lymphomas, non-Hodgkin's lymphoma, Hodgkin's disease (also called Hodgkin's lymphoma), and myeloma, such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndromes (MDSs), myeloproliferative disorders (MPD), and multiple myeloma (MM).

The term "leukemia" refers to malignant neoplasms of the blood-forming tissues, including, but not limited to, chronic lymphocytic leukemia or chronic lymphoid leukemia, chronic myelocytic leukemia, or chronic myelogenous leukemia, acute lymphoblastic leukemia, acute myeloid leukemia or acute myelogenous leukemia (AML) and acute myeloblasts leukemia.

In general, a primary cell is a cell isolated from a blood sample or a biopsy and then optionally further cultured in vitro. A cell line is a cellular culture of a transformed ie cancerous cell, preferably a homogenous cellular culture of a transformed ie cancerous cell (wherein a marker is represented by a Gaussian curve).

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 subranges within a numerical limit or range are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

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.

GENERAL METHOD In general, the CD123 CAR T cells of the invention were prepared using cells purified from Buffy coat or leukapheresis samples from different donors. The process and products satisfies the requirement of the Good Manufacturing Practices (FDA 21 CFR and EU GMP). The clinical assay was conducted under Good Clinical Practices (UK GCP or USA GCP)

Blood 2014; 124(21):4689.

CAR constructions:

CDRs from7G3, Klon43, 6H6, 9F5, were inserted into humanized anti-CD123 scfv previously described in WO2016120220 and fused to various CAR architectures comprising different hinge lengths, and intracellular domains to produce a CD123 CAR that will trigger a CTL response upon binding to CD123 expressed in Cancer cells (AML samples vs Control (CD123+ normal cell sample). Viral Vectors, including Recombinant AAV6 donor vectors were prepared to insert the

CD123 CARs of the invention into the endogenous genomic TCR, more specifically onto the part of the gene encoding the TCR alpha subunit. The location for insertion was chosen to ultimately prevent the expression of the alpha-beta TCR at the cell surface.

• Primary T-cell cultures

T cells were purified from Buffy coat samples provided by EFS (Etablissement Frangais du Sang, Paris, France) using Ficoll gradient density medium. The PBMC layer was recovered and T cells were purified using a commercially available T-cell enrichment kit. Purified T cells were activated in X-Vivo™-15 medium (Lonza) supplemented with 20ng/mL Human IL-2, 5% Human, and Dynabeads Human T activator CD3/CD28 at a bead ell ratio 1:1 (Life Technologies).

• CAR mRNA transfection

Transfections were done at Day 4 or Day 11 after T-cell purification and activation. 5 millions of cells were transfected with 15μg of mRNA encoding the different CAR constructs. CAR mRNAs were produced using T7 mRNA polymerase transfections done using Cytopulse technology, by applying two 0.1 mS pulses at 3000V/cm followed by four 0.2 mS pulses at 325V/cm in 0.4cm gap cuvettes in a final volume of 200μΙ of "Cytoporation buffer T" (BTX Harvard Apparatus). Cells were immediately diluted in X-Vivo™-15 media and incubated at 37°C with 5% C0 ₂ . IL-2 was added 2h after electroporation at 20ng/mL.

Production of CD123 U CART cells by INSERTING in frame a CD123 CAR into the TRAC locus with TALEN

To disrupt the TRAC locus and place a CD123-specific CAR under its transcriptiona l control (TRAC-CAR) we used a TRAC TALEN targeting the first exon of TRAC locus and an adeno-associated virus (AAV) vector repair matrix encoding a self-cleaving T2A peptide followed by the CAR cDNA as previously described using Crispr /Cas 9 system (MacLeod et al., Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells, Molecular Therapy (2017), http://dx.doi.Org/10.1016/j.ymthe.2017.02.00, Eyquem J, Mansilla-Soto J, Giavridis T, van der Stegen SJ, Hamieh M, Cunanan KM, Odak A, Gonen M, Sadelain M, Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature. 2017 Mar 2;543(7643):113-117. doi: 10.1038/nature21405. Epub 2017 Feb 22).

PBMCs were thawed and activated using Transact human T activator CD3/CD28 beads. 3 days after their activation, T cells were passed to be transfected 4 hours later at the earliest.

T cells were then transfected by electrotransfer of 1 μg of mRNA encoding TRAC TALEN per million cells using an AgilePulse system (Harvard Apparatus) into a 0.4 cm cuvette. Following electroporation, cells were immediately diluted in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% CTS™ Immune Cell SR at the concentration of 4x106 cells/mL and incubated in 12 well-plates (500 μΙ per well) at 37°C in the presence of 5% C02.

Each recombinant AAV6 donor vector was added to the culture 1.5h after electroporation at the multiplicity of infection of 104 to 3x104 vg/cell. Subsequently, cells were cultured overnight at 30°C in X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% CTS™ Immune Cell SR and cultured back in the standard conditions starting from the day after (37°C, 1x106 cells/mL, X-Vivo-15 media supplemented by 20 ng/ml IL-2 and 5% CTS™ Immune Cell SR). Cells were then expanded in the standard conditions and passed every 2 to 3 days. 4 days after transfection/transduction TRAC knock-out and CAR expression were assessed by flow cytometry.

TCR and CAR expressions were assessed by flow cytometry on viable T cells using CD4, CD8, TCRa-β mAb, CD123 recombinant protein (full length) in combination with a marker of cell viability. The frequency of positive cells is indicated in each panel. D4, d7 and dll correspond to the day post-transduction.

Alternatively, transduction of T-cells was performed using AAV6 particles comprising a gene encoding a self-cleaving peptide to be inserted in frame with the TRAC gene or an IRES and a gene expressing a CD123 CAR of the invention.

• Degranulation assay (CD107a mobilization)

T-cells were incubated in 96-well plates (40,000 cells/well), together with an equal amount of cells expressing various levels of the CD123 protein. Co-cultures were maintained in a final volume of ΙΟΟμΙ of X-Vivo™-15 medium (Lonza) for 6 hours at 37°C with 5% C0 ₂ . CD107a staining was done during cell stimulation, by the addition of a fluorescent anti- CD107a antibody at the beginning of the co-culture, together with ^g/ml of anti-CD49d, ^g/ml of anti-CD28, and lx Monensin solution. After the 6h incubation period, cells were stained with a fixable viability dye and fluorochrome-conjugated anti-CD8 and analyzed by flow cytometry. The degranulation activity was determined as the % of CD8+/CD107a+ cells, and by determining the mean fluorescence intensity signal (MFI) for CD107a staining among CD8+ cells.

• IFN gamma release assay

T-cells were incubated in 96-well plates (40,000 cells/well), together with cell lines expressing various levels of the CD123 protein. Co-cultures were maintained in a final volume of ΙΟΟμΙ of X-Vivo™-15 medium (Lonza) for 24 hours at 37°C with 5% C0 ₂ . After this incubation period the plates were centrifuged at 1500 rpm for 5 minutes and the

supernatants were recovered in a new plate. IFN gamma detection in the cell culture supernatants was done by ELISA assay. The IFN gamma release assays were carried by starting the cell co-cultures 24h after mRNA transfection.

• Cytotoxicity assay T-cells were incubated in 96-well plates (100,000 cells/well), together with 10,000 target cells (expressing CD123) and 10,000 control (CD123+ or CD123- normal ) cells in the same well, or a mixture of normal and cancer cells. Target and control cells were labelled with different fluorescent intracellular dyes (CFSE or Cell Trace Violet) before co-culturing them with CAR+ T-cells. The co-cultures were incubated for 4 hours at 37°C with 5% C0 ₂ . After th incubation period, cells were counted, labelled with a fixable viability dye and analyzed by flow cytometry. Viability of each cellular population (target cells or CD123neg control cells) was determined and the % of specific cell lysis was calculated. · Anti-tumor mouse model

Immuno-deficient NOG mice were intravenously (iv) injected with CD123 + AML cells or CD123 + normal cell AML xenograft mouse model. Optionally, mice received an anti-cancer treatment that is PNA or FLAG. Mice were then iv injected (either 2 or 7 days after injection of the tumor cell line) with different doses of CAR+ T-cells to be tested and compared (different CD123 CAR scfv, and the CD123 of the present invention) or with T-cells that were not transduced with the CAR lentiviral vector. The tumoral mass was evaluated at D7, 14, 21, 28 and 40 after T-cell injection in order to follow tumoral progression/ CART Cell efficiency.

Results

Cytotoxicity was evaluated by multi-parameter flow cytometry at 24 hours after co-cultures of AML primary samples and UCART123 or control cells were established. Different co- culture ratios (E:T) were evaluated. At later time points, the results show on average, more than 90% of cell death in leukemia cells that were co-cultured with UCART123 (6H6 or 9F5) at all E:T ratios (Error! Reference source not found.). In contrast, TCRa KO T-cells control cells induce significantly less cell death when co-cultured with AML cells.

The activity was prolonged with the 6H6 or 9F5 CAR expressing cells and the amount of normal cell increased in all mice tested as compared to previous anti-CD123 construction.

CLINICAL STUDY

CD123 CAR T cells

CD123 CAR + immune cells were prepared using mAbs against CD123 [7G3 (BDbiosciences), Klon43, (as a control), 6H6 (BioLegend), 9F5 (BDbiosciences), with 3 different hinge CD8alpha, IgGl and FcgammaR. Humanized version of these CARs were prepared and tested in parallel.

Expression was analyzed in a primary cell background ie TCR alpha KO B2M KO T CAR Kl cells.

The percentage of cells expressing a CD123 CAR and activity was determined using CD123+ AML and normal MNC samples, along with appropriate controls.

A Phase I dose escalation trial was designed to evaluate the safety and the biologic efficacy of these allogeneic TCR b2M KO /CD123 CAR Kl specific cytotoxic T -lymphocytes (CTL) (CD123 CAR T cell of the invention "6H6" or "9F5") targeting the CD123 molecule (CD123CAR) in patients diagnosed with AML, relapsed/refractory Acute myeloid Leukemia (AML), blastic plasmacytoid dendritic cell neoplasm.

Each patient received at least one dose of donor derived, genetically modified CTL and was monitored for toxicity and detection of transduced CTL as well as disease specific markers.

Any of the following may vary individually upon medical indication.

Study Interventional

Type:

Study Endpoint Classification: Safety/Efficacy Study

Design: Intervention Model: Single Group Assignment

Masking: Open Label

Primary Purpose: Treatment

Official A Phase 1 Dose Escalation Trial Using In Vitro Expanded Allogeneic Cytotoxic

T-Lymphocytes (CTLs) Genetically Targeted to CD123-expressing cancer cells Or Relapsed Acute myeloid Leukemia, Blastic plasmacytoid dendritic cell neoplasm.

Key words Acute myeloid Leukemia, Blastic plasmacytoid dendritic cell neoplasm, dosing

Primary Outcome Measures:

Evaluate the safety/persistence of escalating doses (and redosing) of allogeneic specific CTL modified to express artificial chimeric receptors targeting CD123 molecule given for persistence or relapse of AML, for Blastic plasmacytoid dendritic cell neoplasm.

Secondary Outcome Measures:

· To assess the effects (activity specificity) of the adoptively transferred CD123 specific

T-cells on the progression of AML.

• To quantitate the number of CD123 chimeric antigen receptor (CD123 CAR) positive T-cells in the blood at defined intervals post infusion in order to determine their survival and proliferation in the host; To quantitate the number of CD123 chimeric antigen receptor (CD123 CAR) positive T-cells in the blood at defined intervals post infusion of Rituximab (375 mg/m2).

Arms Assi ned Interventions

Experimental: Biological/Genetically Biological: Biological/Genetically Modified T Modified T cells cells

Patients with persistent minimal residual Following completion of the chemotherapy, disease (+MRD) or relapsed AML will receive genetically modified T cells will be given a conditioning chemotherapy regimen intravenously at one of 3 dose levels (10 ² , 10 ⁴ followed by intravenous infusion of and 10 ⁶ ). After the infusion patients will be allogeneic specific cytotoxic T-cells (CTLs) monitored clinically and with serial blood and genetically modified ex vivo to express the marrow evaluations to assess toxicity,

CD123-specific chimeric artificial T-cell therapeutic effects, and the in-vivo survival of receptor. the genetically modified T-cells. Eligibility

Criteria

Inclusion Criteria:

• History of CD123+ leukemia with evidence of bone marrow relapse or persistent.

• Persistent minimal residual disease must be demonstrated by morphology, FISH, flow cytometry or RT-PCR with at least 2 sequential testings separated by at least 1 week.

• No age restriction for patients · KPS or Lansky score > or = to 40

• Renal function (measured prior to conditioning chemotherapy)

• Hepatic function (measured prior to conditioning chemotherapy):

• AST≤ 5 x the institutional ULN Elevation secondary to leukemic involvement is not an exclusion criterion. Leukemic involvement will be determined by the presence of progressive relapse defined by escalating bone marrow or peripheral blood leukemia blasts within the previous month and the absence of initiation of know hepatotoxic medication (e.g. azoles).

• Total bilirubin≤ 2.5 x the institutional ULN

• Adequate cardiac function (e.g. LVEF > 40%) as assessed by ECHO or MUGA or other similar cardia imaging performed within 1 month of enrollment.

• Pulmonary function (measured prior to conditioning chemotherapy):

• Oxygen saturation > 90% on room air Donor Eligibility: • The donor, including a third party donor, must consent to a leukapheresis or whole blood donation(s) obtained at one or more phlebotomies which, in aggregate, will total approximately 250 ml for adults and no more than 5ml/kg per draw from pediatric donors. · Related donors <18 years of age requiring placement of a leukapheresis catheter will donate peripheral blood collected by phlebotomy (including a unit of blood if weight permits) and shall not undergo catheter placement for leukapheresis as this is considered above minimal risk to the donor.

• There is no upper age limit for a donor. However, the minimum age for a related donor is 7 years as this is the youngest age a person can be considered capable of giving assent to participate in a research study.

• Donor's high resolution HLA typing must be available for review.

• CBC within one week of donation. Results of tests must be within a range that would not preclude donating blood or undergoing leukapheresis. · Serologic testing for transmissible diseases will be performed as per institutional guidelines adopted from extant NMDP and FACT guidelines. Donors should be considered eligible to donate leukapheresis or blood based on these guidelines (i.e. blood donation guidelines)

Exclusion Criteria: · Patients with active HIV, hepatitis B or hepatitis C infection.

• Patients with any concurrent active malignancies as defined by malignancies

requiring any therapy other than expectant observation.

• Females who are pregnant.

• Patients will be excluded if they have isolated extra-medullary relapse of ALL. · Patients with active (grade 2-4) acute graft versus host disease (GVHD), chronic

GVHD or an overt autoimmune disease (e.g. hemolytic anemia) requiring

glucocorticosteroid treatment (>0.5 mg/kg/day prednisone or its equivalent) as treatment • Active central nervous system (CNS) leukemia, as defined by unequivocal morphologic evidence of lymphoblasts in the cerebrospinal fluid (CSF) or

symptomatic CNS leukemia (i.e. cranial nerve palsies or other significant neurologic dysfunction) within 28 days of treatment. Prophylactic intrathecal medication is not a reason for exclusion.

• Adult patients (>18 years old) with the following cardiac conditions will be excluded:

• New York Heart Association (NYHA) stage II I or IV congestive heart failure

• Myocardial infarction≤ 6months prior to enrollment

• History of clinically significant ventricular arrhythmia or unexplained syncope, not believed to be vasovagal in nature or due to dehydration.

• History of severe non-ischemic cardiomyopathy with EF≤20%

Administration of Rituximab (Rituxan (R).

The total dose of Rituximab administered by intravenous route during 4 day after an initial rituximab dose of 375 mg/m2 was 2,250 mg/m2.

Premedication was performed before each infusion with acetaminophen and a n antihistamine.

I n another embodiment, the first I nfusion was initiated at a rate of 50 mg/hr. I n the absence of infusion toxicity, infusion rate was 50 mg/hr incremented every 30 minutes, to a maximum of 400 mg/hr.

For the subsequent Infusions, it was performed at a rate of 100 mg/hr. In the absence of infusion toxicity, increased to a maximum of 400 mg/hr to reach 2,250 mg/m2.

The level of cells was below detection at 24 h after the last infusion. I n these patients a second lower injection of CD123 + cells eventually performed.

N umber and activity of the cells is established and sustained or controlled by the use of safety switch (rituximab/QBEN 10).

Fludarabine, cyclophosphamide may be first administered for 3-4 days and then combined to a set or a kit of n pharmaceutical unit doses according to the present invention and used to treat patients with relapsed or refractory cancer As another example: Treatment consisted in Fludarabine (F) 25mg/m(2)/day on days 2-4, Cyclophosphamide © 250 mg/m(2)/day on days 2-4, Mitoxantrone (M) 6 mg/m(2) on day 2, and itiximab 375 mg/m(2) on day 1. For cycles 2-6, FCM started day 1 together with R 500 mg/m(2). Pegfilgrastim (recombinant human G-CSF) is administered with each cycle. Cycles are repeated every 4-6 weeks and each is followed by an administration of a unit dose of engineered cells.

Cells (6.25 x 106 cells/kg were allogenic TCR-negative (less than 3% TCR+) CD123 CAR positive and CD52 or dcK deficient administered 3- 4 times).

The preliminary results are in favor of an improvement with the CD123 CAR+ cells of the invention, as compared to Klon 43 CAR+ allogeneic cells with less side effects, especially for the treatment of over expressing CD123 + cancer cells.

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