RU2775453C2 - Constructed universal immune cells with anti-cd22 chimeric antigen receptor - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions is described, including a constructed cell with a “knockout” human T-cell receptor (TCR-KO), expressing on its surface a chimeric antigen receptor (hereinafter – CAR) specific relatively to differentiation cluster 22 (hereinafter – CD22) (UCART 22), and a protective switch for the prevention or treatment of a patient suffering from CD22+-mediated cancer or CD22+-mediated inflammatory disease, a cell population, and a pharmaceutical composition. In one of implementations, UCART 22 cell expresses anti-CD22 CAR containing an extracellular domain, a transmembrane domain of CD8alpha, and an intracellular signal domain, wherein the specified anti-CD22 CAR has a polypeptide sequence at least 80% identical to SEQ ID NO: 15.

EFFECT: invention expands the arsenal of remedies for the prevention or treatment of a patient suffering from CD22+-mediated cancer or CD22+-mediated inflammatory disease.

14 cl, 9 dwg, 24 tbl, 1 ex

Description

The field of technology to which the invention belongs

The present invention relates generally to the field of immunotherapy, and more particularly to universal CD22-specific chimeric antigen receptor T cells (UCART 22), which are human engineered primary immune cells that contain at least one edited gene, preferably the gene , encoding the TCR subunit, or the CD52 gene, and chimeric antigen receptors (CAR) specific for cluster of differentiation 22 (CD22), (CAR CD22), and methods for constructing these cells. The invention also relates to UCART 22 for use in patients who may or may not be primary cell donors ("allogeneic" or "autologous" CD22 CAR-bearing engineered primary human immune cells) as a treatment for relapsed refractory hematologic cancers . Cells expressing CD22, proposed in the invention, are primarily effective and safe for immunotherapy, in particular against aggressive or recurrent cancer.

Prerequisites for the creation of the invention

In 2000, 45,000 deaths from hematological cancers (non-Hodgkin's lymphoma, leukemia) were expected in the United States (Greenlee et al., CA Cancer J. Clin., 50, 2000, pp. 7-33). The number of deaths reported in 2014 appeared to be similar despite advances in treatments such as chemotherapy, and the prognosis for these cancers remains broadly unchanged (E. K. Mai, U. Bertsch, J Dtirig, C. Kunz, M. Haenel, I. W. Blau, M. Munder, A. Jauch, B. Schurich, T. Hielscher, M. Merz, B. Huegle-Doerr, A. Seckinger, D. Hose, J. Hillengass, M. S. Raab, K. Neben, H.-W. Lindemann, M. Zeis, C. Gerecke, I. G. H. Schmidt-Wolf, K. Weisel, C. Scheid, H. Salwender, and H. Goldschmidt Phase III trial of bortezomib , cyclophosphamide and dexamethasone (VCD) versus bortezomib, doxorubicin and dexamethasone (PAd) in newly diagnosed myeloma Leukemia, 19 March 2015, doi: 10.1038/leu.2015.80.

Unique among the newly explored treatments for these hematologic cancers is the genetic modification of cells with cytolytic capacity, such as T cells, through chimeric antigen receptor (CAR) gene transfer (Jena, Dotti et al., 2010).

CARs are synthetic receptors consisting of a targeting (“targeting”) fragment associated with one or more intracellular signaling domains in one molecule or in several transmembrane domains forming a multimer. In a particular CAR, the binding fragment consists of an antigen-binding domain from a single chain antibody (scFv) that contains the variable fragments of a monoclonal antibody connected by a linker. Binding fragments based on receptor or ligand domains have also been successfully used to obtain CARs.

Signaling domains from costimulatory T cell receptor (TCR) molecules, as well as specific transmembrane and hinge domains, have been added to generate second and third generation CARs that have been successfully tested in humans as therapeutic agents. In these studies, T cells from a patient suffering from hematological (“liquid”) cancer were redirected against malignant cells expressing, for example, CD19 or CD22 (June et al., 2011, Haso et al., 2013) and re-injected into this same patient (Haso W., Lee D.W., Shah N.N., Stetler-Stevenson M., Yuan CM., Pastan I.H., Dimitrov D.S., Morgan R.A, FitzGerald D.J., Barrett D.M., Wayne A.S., Mackall C.L., Orentas R.J. Anti-CD22- chimeric antigen receptors targeting B-cell precursor of acute lymphoblastic leukemia Blood 121(7) February 14, 2013, pp. 1165-1174 doi: 10.1182/blood-2012-06-438002 Epub December 14, 2112

Techniques that allow re-injection of pre-engineered immune cells from one individual into the same individual who typically has cancer are not well adapted to aggressive forms of cancer that can progress rapidly. In addition, this method may be problematic or unpredictable in patients with altered immune systems.

To address this problem, immunotherapy has now been implemented using CAR-expressing so-called "allogeneic" T cells (referred to as generic or "off-the-shelf" T cells), and in the first two patients who treated with these cells, still remains in remission for two years after treatment.

However, this therapy still has aspects to be improved, such as efficacy in the presence of drugs against T cells, efficacy against immune elusive cancer cells, persistence, controls, etc. Thus, it is likely that cancer cells, due to the downregulation of surface antigen expression recognized by CAR, can elude treatment and survive despite the persistence of redirected immunity in treated patients. In addition, one of the major and sometimes fatal side effects found in patients treated with either autologous or allogeneic T cells is cytokine release syndrome.

Thus, there is still a need to develop effective and safe treatment options for these pathologies, especially aggressive or refractory/recurrent forms of hematological cancers.

Brief summary of the invention

When creating the invention, a new effective treatment was developed ("UCART 22"), which includes the use of an engineered primary human T cell with at least one deletion in the TRAC gene, endowed with a chimeric antigen receptor that targets CD22, and a safety marker that allows you to control the number of these cells in vivo and, consequently, the activity of these cells.

An effective new treatment ("UCART 22") involving the use of an engineered primary human T cell with at least a deletion in the TRAC gene provides for at least one additional deletion in the CD52, dCK, beta-2-microglobulin genes.

These novel UCART 22s are most effective for adoptive transfer to a patient suffering from a CD22-mediated pathology, whether or not said patient is a primary immune cell donor, and whether or not said patient is already or not yet undergoing immune-influencing therapy.

The UCART 22 cells of the invention are resistant to at least one chemical drug or antibody drug used to treat CD22-mediated pathology, such as Campath and/or purine nucleotide analogs (PNAs). The UCART 22 cells of the invention can survive and be active in the presence of said drug at a dose that kills more than 80% of the cells.

The engineered, isolated UCART 22 primary immune cells have been found to have distinct and unexpected clinical benefits, including low cytokine release, no or very little graft-versus-host response, and marked activity against refractory recurrent hematologic cancer cells.

In the present description, other advantages will be indicated.

The present invention proposes:

1. Engineered human T-cell receptor (TCR-KO) knockout cell endowed with a CD22-specific chimeric antigen receptor (CAR) (UCART 22), an on-off switch, preferably expressed on cell surface, where said anti-CD22 CAR (CD22 CAR) contains:

I) at least one extracellular domain containing:

- hinge domain from CD8alpha,

- an antigen-binding domain specific for CD22, optionally a leader sequence,

II) a transmembrane domain from SE8alpha and

iii) an intracellular signaling domain of said CD22 CAR having a polypeptide sequence at least 80% identical to SEQ ID NO: 15,

said safety switch contains:

- RQR8 (suicide gene) temporally linked to CD22 CAR by a 2A peptide, or

at least two rituximab Mab specific epitopes, preferably located between the VH and the hinge domain, or 3 rituximab Mab specific epitopes, or 3 rituximab Mab specific epitopes and QBEND-10 Mab specific epitopes associated with CD22 CAR.

2. UCART 22 according to embodiment 1, in which a polynucleotide sequence at least 80% identical to SEQ ID NO: 11 is inserted into the genome, and additionally containing an inactivated TRAC gene with an insertion, deletion or mutation in SEQ ID NO 18, With

an undetectable level of T cell receptor (TCR) on the cell surface as measured by flow cytometry; and an undetectable level of off-target events as measured by guide sequence technology.

UCART 22 according to claim 2, wherein said anti-CD22 CAR is inserted preferably into the TRAC gene, preferably having SEQ ID NO: 18.

3. UCART 22 according to one of the embodiments of the invention 1-2, containing another inactivated gene selected from the dCK gene, the B2M gene, the CD52 gene, preferably the CD52 gene.

4. UCART 22 according to one of the embodiments of the invention 1-3, in which at least one additional gene is inactivated, where the specified gene is selected from a gene encoding an aryl hydrocarbon receptor (aromatic hydrocarbon receptor) (AHR), a transforming growth factor receptor (TGF receptor beta), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, their combination.

5. UCART 22 according to one of the embodiments of the invention 1-4, in which the gene encoding beta-2-microglobulin (B2M) is inactivated.

6. UCART 22 according to one of the embodiments of the invention 1-5, in which the gene encoding the aryl hydrocarbon receptor (AHR) is inactivated.

7. UCART 22 according to one of embodiments 1-6, wherein the gene encoding transforming growth factor receptor beta (TGF receptor beta) is inactivated.

8. UCART 22 according to one of the embodiments of the invention 1-7, in which the gene encoding the interleukin 10 receptor (IL-10 R) is inactivated.

9. UCART 22 according to one of embodiments 1-8, wherein the gene encoding programmed cell death protein 1 (PD1) is inactivated.

5. UCART 22 according to one of the embodiments of the invention 1-4, containing additional scfv, specific for one of the following tumor-associated surface antigens, which are selected from CD19, CD20, CD30, major histocompatibility complex (MCHC) molecule, immunoglobulin (Ig) , CD3, CD5, CD34, CD79, preferably CD79b, CD 138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3 or PAX5, preferably CD19.

6. UCART 22 according to one of embodiments 1-5, wherein said CD22 CAR further comprises an antigen-binding domain specific for CD19, or said UCART 22 further comprises a CD19 CAR, preferably a CD19 CAR, at least 80% identical to SEQ ID NO: 25 or SEQ ID NO: 26.

7. UCART 22 according to one of embodiments 1-5, wherein the anti-CD22 CAR is a single chain CAR or a multi chain CAR.

8. UCART 22 according to one of embodiments 5 or 6, wherein the anti-CD19 CAR is a single chain CAR or a multi chain CAR.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD19.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD20.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD30.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for the major histocompatibility complex (MHC) molecule.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for immunoglobulin (Ig).

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD3.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD5.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD34.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD79, preferably CD79b.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD138.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD80.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for BCMA (CD269).

UCART 22 according to one of the above, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for TNFRSF 17.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for FLT-3.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD19.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD79 or CD79b.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD20.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD30.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for the major histocompatibility complex (MCHC) molecule.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for immunoglobulin (Ig).

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD3.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD5.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD34.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD138.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD80.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for BCMA (CD269).

UCART 22 according to one of the above, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for TNFRSF 17.

UCART 22 according to one of the above paragraphs, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for FLT-3.

9. A population of UCART cells containing UCART 22 according to one of the embodiments of the invention 1-8.

10. A population of UCART cells according to embodiment 9 containing UCART 19, preferably UCART 19, which expresses on the cell surface an anti-CD19 CAR containing SEQ ID NO: 25 or SEQ ID NO: 26.

11. A kit containing UCART 22 and UCART 19 for sequential (at least once) or simultaneous, or sequential (at least once) and then simultaneous administration to a patient who needs it.

12. The kit of embodiment 11 wherein UCART 19 is applied first at least one, two, three, four or more times, and then UCART 22 is applied alone or with UCART 19.

13. The set of embodiments 11 or 12 wherein UCART 19 is applied first at least one, two, three, four or more times, and then UCART 19 is applied alone or in conjunction with UCART 22.

14. The kit of embodiments 11-13 further comprising a lymphodepletion agent that is used prior to UCART.

15. The kit of embodiments 11-14 wherein lymphodepletion is achieved with fludarabine and cyclophosphamide, preferably fludarabine at a dose of 25 mg/m ² iv x 5 doses on days -6 to -2 and cyclophosphamide at a dose of 60 mg/m 2 kg iv as 1 dose per day -5.

16. The kit of embodiments 11-15 comprising at least one other UCART cell directed against a cancer antigen selected from CD79a, CD79b, CD20, CD30, CD52, CD40, CD80, CD86, CD74, VEGF.

17. Pharmaceutical composition containing UCART 22 according to one of the embodiments of the invention 1-8 or a population of cells containing the specified UCART 22 according to the embodiment of the invention 9 or 10 and a pharmaceutically acceptable excipient.

18. Pharmaceutical composition according to embodiment 17 further comprising bryostatin, preferably bryostatin-1.

19. The pharmaceutical composition of Embodiment 17 or 18 or the kit of Embodiment 11-16 for use as a medicament for the prevention or treatment of a patient suffering from a CD22 ⁺ mediated cancer or a CD22 ⁺ mediated inflammatory disease.

20. The pharmaceutical composition of Embodiment 17-18 or the kit of one of Embodiments 11-16 for use as a medicament for the prevention or treatment of a patient suffering from a CD19 ⁺ mediated cancer or a CD19 ⁺ mediated inflammatory disease.

21. The pharmaceutical composition of Embodiment 17-18 or the kit of one of Embodiments 11-16 for use as a medicament for the prevention or treatment of a patient suffering from a CD19CD22-mediated cancer or a CD19 ⁺ CD22 ⁺ -mediated inflammatory disease.

22. Pharmaceutical composition according to embodiment 17-18 or kit according to one of embodiments 11-16 for use according to one of embodiments 19 or 21, where treatment of the patient includes the step of administering the pharmaceutical composition at least twice (redosing, repeated dosing) to avoid the development of relapsed/refractory cancer.

23. Pharmaceutical composition according to embodiment 17-18 or kit according to one embodiment 11-16 for use according to one embodiment 19 or 21 for the treatment of CD22-mediated hematologic cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma , leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably CD22-expressing hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, more preferably recurrent refractory CD22-expressing hematologic o cancer, even more preferably an aggressive form of said CD22-associated hematologic cancer.

24. Pharmaceutical composition according to embodiment 17-18 or kit according to one embodiment 11-16 for use according to one embodiment 19 or 21 for the treatment of CD19-mediated hematologic cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma , leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably CD19-expressing hematologic cancer, hematologic cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, more preferably recurrent refractory expressive th CD19 hematologic cancer, even more preferably an aggressive form of said CD19-associated hematologic cancer.

25. The pharmaceutical composition of Embodiment 17-18 or the kit of Embodiment 11-16 for use according to Embodiment 19-22 for the treatment of relapsed or refractory B cell-expressing ALL, preferably in pediatrics.

26. Pharmaceutical composition according to an embodiment of the invention 17-18 or kit according to one of the embodiments of the invention 11-16 for use according to one of the embodiments of the invention 19-25, where the treatment of the patient includes the introduction of at least one monoclonal antibody (MAb), preferably QBEND-10 and/or rituximab, to a patient at a dose that provides contact of said UCART 22 with at least one specific mAb.

Variants CD22 CAR

The invention provides an anti-CD22 CAR containing a hinge domain selected from FcRIIIα, CD8alpha, IgG1, IgG4 and PD1, preferably from CD8alpha or IgG4.

Described above is an anti-CD22 CAR in which a CD22-specific scfv contains VH and VL linked to each other by an L1 linker, preferably a linker that contains 1-3 "GGGGS" motifs, more preferably one "GGGGS" -motive.

In one of the above variants in the anti-CD22 CAR, said scfv specific for CD22 is linked to the transmembrane domain via a hinge selected from a hinge from FcRIIIα, CD8alpha, IgG1, preferably from CD8alpha.

In one of the above variants, the anti-CD22 CAR contains an intracellular domain, where the specified intracellular domain contains the CD3zeta signaling domain and the 4-1BB signaling domain.

In one of the above embodiments, the anti-CD22 CAR is an anti-CD22 CAR which contains at least one, preferably two, three or four monoclonal antibody (MAb) specific epitopes, preferably two epitopes, inserted into the linker L of the scfv fragment , specific for CD22, and/or in the hinge.

In one of the above embodiments, the anti-CD22 CAR Mab-specific epitope is a polypeptide selected from:

The invention provides a polynucleotide encoding an anti-CD22 CAR as defined in one of the above embodiments, or a polynucleotide at least 80% identical to a polynucleotide encoding an anti-CD22 CAR, a vector containing a polynucleotide corresponding to one of the polynucleotides described above.

As will be described below, other genes have been edited in said UCART 22, primarily the dCK gene, the B2M gene, the CD52 gene.

Proposed UCART

The above UCART 22 is provided, in which at least one additional gene is edited or engineered, said gene is selected from a gene encoding aryl hydrocarbon receptor (AHR), transforming growth factor receptor (TGF receptor beta), interleukin 10 receptor (IL-10 R ), protein 1 programmed cell death, their combination.

Provided is UCART 22 as defined above, wherein at least one additional gene contains a mutation, deletion, or insertion that inactivates activity and/or expression of said gene selected from a gene encoding an aryl hydrocarbon receptor (AHR), a transforming growth factor receptor (TGF receptor beta ), interleukin 10 receptor (IL-10 R), programmed cell death protein 1, their combination.

Provided is UCART 22 as defined above, wherein at least one additional gene contains a mutation, deletion, or insertion that inactivates activity and/or expression of said gene selected from a gene encoding an aryl hydrocarbon receptor (AHR), a transforming growth factor receptor (TGF receptor beta ), interleukin 10 receptor (IL-10 R), a combination thereof.

Provided is UCART 22 as defined above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates activity and/or expression of said gene selected from a gene encoding a transforming growth factor receptor (TGF receptor beta), interleukin 10 receptor ( IL-10 R), programmed cell death protein 1, their combination.

Provided is UCART 22 as defined above, wherein at least one additional gene contains a mutation, deletion or insertion that inactivates activity and/or expression of said gene selected from a gene encoding an aryl hydrocarbon receptor (AHR), an interleukin 10 receptor (IL-10 R ), programmed cell death protein 1, their combination.

Provided is UCART 22 as defined above, wherein at least one additional gene contains a mutation, deletion, or insertion that inactivates activity and/or expression of said gene selected from a gene encoding an aryl hydrocarbon receptor (AHR), a transforming growth factor receptor (TGF receptor beta ), programmed cell death protein 1, their combination.

The genes into which said CAR can be inserted are described in Table 9 below.

Bold indicates preferred genes inactivated in the UCART of the invention.

The above UCART 22 contains an anti-CD22 CAR (UCART 22) as provided in one of the above variants, and a genome-integrated polynucleotide encoding said anti-CD22 CAR (UCART 22) in the CD25 gene.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR or a multi chain CAR.

In one of the above embodiments, UCART 22 is provided wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for one of the following tumor associated surface antigens selected from CD19, CD20, CD30, a major histocompatibility complex molecule (MCGS), immunoglobulin (Ig), CD3, CD5, CD34, CD79, preferably CD79b, CD138, B7-1 (CD80), BCMA (CD269, TNFRSF 17), FLT-3 or PAX5, preferably CD19.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD19.

In one of the above embodiments, UCART 22 is provided which expresses an anti-CD22 CAR and an anti-CD19 CAR, preferably SEQ ID NO: 94 or SEQ ID NO: 95.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD20.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD30.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for a major histocompatibility complex (MCHC) molecule.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional immunoglobulin (Ig) specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD3.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional CD5-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD34.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD79, preferably CD79b.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD138.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for CD80.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for BCMA (CD269).

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for TNFRSF 17.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a single chain CAR containing an additional scfv specific for FLT-3.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD19.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD79 or CD79b.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multichain (mc) CAR containing an additional scfv specific for CD19.

In one of the above embodiments, UCART 22 is provided which expresses anti-CD22 mcCAR and anti-CD19CAR, preferably SEQ ID NO: 94 or SEQ ID NO: 95.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional CD20-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional CD30-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multichain CAR containing an additional scfv specific for a major histocompatibility complex (MCHC) molecule.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multichain CAR containing an additional immunoglobulin (Ig) specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional CD3-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional CD5-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD34.

In one of the above embodiments, UCART 22 is provided, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for CD138.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional CD80-specific scfv.

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for BCMA (CD269).

In one of the above embodiments, UCART 22 is provided, in which the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for TNFRSF 17,

In one of the above embodiments, UCART 22 is provided, wherein the anti-CD22 CAR is a multi-chain CAR containing an additional scfv specific for FLT-3.

Proposed cell population containing UCART 22, presented in one of the above variants.

What is provided is a cell population containing UCART 22 as presented in one of the above variants, and in which cells expressing said anti-CD22 CAR also express anti-CD19 CAR, preferably said anti-CD19 CAR contains on the cell surface the sequence of SEQ ID NO: 25 or SEQ ID NO: 26.

CD22 CAR T cell (UCART 22), optionally combined with a protein kinase C inhibitor such as bryostatin 1 as a pharmaceutical composition of the invention, is particularly useful in the treatment of CLL, ALL, multiple myeloma (MM), blastoid plasmacytoid dendritic cell neoplasia (BPDCN), especially refractory/recurrent ALL, refractory/recurrent CLL and/or aggressive forms of these diseases, more preferably refractory or recurrent B-ALL.

CD22 CAR T cell (UCART 22) derived from the m971 antibody, optionally combined with a protein kinase C inhibitor such as bryostatin 1 as a pharmaceutical composition of the invention, is particularly useful in the treatment of CLL, ALL, multiple myeloma (MM) , blastoid plasmacytoid dendritic cell neoplasia (BPDCN), especially refractory/recurrent ALL, refractory/recurrent CLL and/or aggressive forms of these diseases, more preferably refractory or recurrent B-ALL.

The engineered immune cells of the present invention are not only characterized by a high level of activity in vivo against malignant cells, low release of cytokines, but also by the fact that their number and activity can be controlled, which ensures safety and efficacy in immunotherapy.

The present invention provides an engineered human receptor knockout (TCR-KO) T cell endowed with a chimeric antigen receptor specific for CD22 (CD22 CAR) (UCART 22), preferably expressed on the cell surface, wherein said CD22 CAR contains:

I) at least one extracellular domain containing:

- a hinge domain from CD8alpha,

- an antigen-binding domain specific for CD22, optionally a leader sequence,

II) a transmembrane domain from CD8alpha and

III) intracellular signaling domain.

Said UCART 22 contains at least one additional edited gene, preferably a deletion in the CD52 gene.

The present invention also provides a CD22 UCART as described above, wherein the antigen binding domain comprises a CD22 specific scfv derived from m971 (m971) antibody, preferably said scfv comprises a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, more preferably SEQ ID NO: 15.

One embodiment of the present invention is also an engineered human receptor knockout (TCR-KO) T cell endowed with a chimeric antigen receptor specific for CD22 (CD22 CAR) (UCART 22), preferably expressed on the cell surface. ,

where said CD22 CAR contains:

I) at least one extracellular domain containing:

- a hinge domain from CD8alpha,

- an antigen-binding domain specific for CD22, optionally a leader sequence,

II) a transmembrane domain from CD8alpha and

III) intracellular signaling domain,

said UCART 22 contains at least one additional edited gene, preferably a deletion in the CD52 gene, and wherein the antigen binding domain contains scfv specific for the distal portion of CD22, said CAR has SEQ ID NO: 20.

The above UCART CD22 contains the following sequences:

QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTY

YRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLE

DAFDIWGQGTMVTVSS and

DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ

SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK or sequences 80-99% homologous to each of the indicated sequences.

The present invention also provides the above UCART 22 containing a polypeptide having SEQ ID NO: 15 and a security switch that contains:

- RQR8 temporally linked to CD22 CAR by a 2A peptide, or at least two rituximab Mab-specific epitopes,

preferably three rituximab Mab-specific epitopes, and more preferably three rituximab Mab-specific epitopes and QBEND-10 Mab-specific epitopes associated with CD22 CAR [allowing sorting and/or depletion of immune cells endowed with said CD22 CAR].

The present invention also provides the above UCART 22 containing at least one additional edited gene, preferably a deletion in the CD52 gene or in the dCK gene, or a deletion in the beta-2-microglobulin gene or in the CTIIA gene, even more preferably a deletion in the CD52 gene and/or an insertion in the HIF-1alpha gene conferring resistance to hypoxia.

The present invention also provides the aforementioned UCART 22 containing a deletion in the CD52 gene, used in combination with alemtuzumab.

The present invention also provides the above UCART 22, in which said CD22 CAR is expressed under hypoxic conditions (lower O ₂ concentration is less than 5%, preferably less than 1% O ₂ ).

The present invention also provides the above UCART 22 containing a polynucleotide that encodes a polypeptide containing CD22 CAR and a security switch. In a preferred embodiment, the present invention provides the above UCART 22, which contains a polynucleotide having SEQ ID NO: 22 and SEQ ID NO: 18 in the same gene, preferably in the TRAC gene.

The present invention also provides the above UCART 22 containing a polynucleotide that encodes a polypeptide containing CD22 CAR and a security switch. In a preferred embodiment, the present invention provides the above UCART 22, which contains a polynucleotide having SEQ ID NO: 20 and SEQ ID NO: 18 in the same gene, preferably in the TRAC gene.

The present invention provides a population of cells containing the above UCART 22.

The present invention provides a pharmaceutical composition that contains either UCART 22 as defined above or a cell population containing any of the above UCART 22 and a pharmaceutically acceptable excipient.

In specific embodiments, the invention provides a pharmaceutical composition that contains the UCART 22 of the invention, or a cell population containing the specified UCART 22 in combination with UCART CD 19, and a pharmaceutically acceptable excipient. UCART 19 and UCART 22 can be used at the same time, simultaneously or sequentially, starting with UCART 19 or UCART 22, depending on the level of expression of CD19 and CD22 in patients' cancer cells.

In specific embodiments, the invention provides a pharmaceutical composition that contains UCART 22/19 or UCART 19/22 (bispecific single chain CAR or multichain CAR or cells that express both CD 19 CAR and CD22 CAR) and a pharmaceutically acceptable excipient. The present invention provides the above pharmaceutical composition further comprising a compound from the bryostatin family, preferably bryostatin 1.

The present invention provides a therapeutically effective amount of the above UCART 22 or a pharmaceutical composition containing the above UCART 22 for use as a medicament in the treatment of a patient.

The present invention provides a therapeutically effective amount of the above UCART 22 or the pharmaceutical composition of the above for use as a medicament in the treatment of a patient, wherein the treatment of the patient comprises administering at least two times (repeated dosing) of said therapeutically effective amount of UCART CD22 or of said pharmaceutical composition to avoid relapse.

In a specific embodiment, the present invention provides UCART CD22 as set forth in one of the above items, or a pharmaceutical composition as set forth in one of the above items, for use as a medicament in the treatment of a patient, wherein the treatment of the patient comprises administering the above UCART 22 or the above pharmaceutical composition at least twice to avoid relapse.

The present invention provides a therapeutically effective amount of the above UCART 22 or the pharmaceutical composition of the above for use as a medicament in the treatment of a patient as defined above for a CD22-associated pathology, preferably a CD22 B-cell malignant pathology (e.g., CD22-associated hematologic cancer).

In a specific embodiment, the present invention provides:

a therapeutically effective amount of the above UCART 22 or a pharmaceutical composition as defined above for use as a medicament for the treatment of hematological cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably CD22-expressing hematologic cancer, more preferably recurrent refractory CD22-expressing hematologic cancer, even more preferably an aggressive form of said CD22-associated hematologic cancer.

Preferably, UCART 22 is used to treat patients with relapsed refractory B-cell ALL.

The present invention provides a therapeutically effective amount of the above UCART 22 or the pharmaceutical composition of the above for use as a medicament for the treatment of a patient, wherein said patient is suffering from a cancer selected from alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (eg, medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleura cancer, nose cancer, nasal cancer cavity, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (eg, squamous cell carcinoma head and neck), hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer (eg, non-small cell lung carcinoma), malignant mesothelioma, mastocytoma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, cancer skin, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer.

The present invention proposes:

1. Constructed human receptor knockout (TCR-KO) T cell endowed with a chimeric antigen receptor (CAR) specific for CD22 (CD22 CAR) (UCART 22), preferably expressed on the cell surface,

where said CD22 CAR contains:

I) at least one extracellular domain containing:

- hinge domain from CD8alpha,

- an antigen-binding domain specific for CD22, optionally a leader sequence,

II) a transmembrane domain from CD8alpha and

III) intracellular signaling domain,

said UCART 22 contains at least one additional edited gene, preferably an inactivated CD52 gene, an inactivated dCK gene, an inactivated beta-2-microglobulin gene, or an inserted HIF-1alpha gene.

2. UCART CD22 according to claim 1, in which the antigen-binding domain contains a scfv specific for CD22, where the specified scfv is derived from the m971 (m971) antibody, preferably the specified scfv contains a sequence selected from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, preferably the sequence from SEQ ID NO: 15.

3. UCART 22 according to one of the paragraphs. 1-2, containing a polypeptide having SEQ ID NO: 15 and a security switch, where said security switch contains:

- RQR8 temporally linked to CD22 CAR by peptide 2A, or

at least two rituximab Mab-specific epitopes, preferably three rituximab Mab-specific epitopes, and more preferably three rituximab Mab-specific epitopes and QBEND-10 Mab-specific epitopes associated with CD22 CAR [allowing sorting and/or depletion of immune cells endowed with the specified CD22 CAR].

4. UCART 22 according to one of paragraphs. 1-3 containing an inactivated CD52 gene, even more preferably an inactivated CD52 gene and an inserted HIF-1 alpha gene, which confer resistance to alemtuzumab and hypoxia.

5. UCART 22 according to one of paragraphs. 1-4, where the specified anti-CD22 CAR is expressed on the cell surface at a low concentration of O ₂ (<5% 02).

6. UCART 22 according to one of paragraphs. 1-5, containing a polynucleotide that encodes a polypeptide containing an anti-CD22 CAR according to p.p. 1-5.

UCART 22 according to one of the above items, containing an anti-CD22 CAR on the cell surface, a polynucleotide encoding the specified anti-CD22 CAR, built into the TRAC gene, and an exogenous polynucleotide sequence that encodes IL-12, built into the CD25 genomic sequence, genomic the B2M sequence or into the PD1 genomic sequence.

UCART 22 according to one of the above items, containing an anti-CD22 CAR on the cell surface, a polynucleotide encoding the specified anti-CD22 CAR, built into the TRAC gene, and an exogenous polynucleotide sequence that encodes IL-12, built into the CD25 genomic sequence or into the genomic sequence of PD1, and additionally containing a genomic "off" (KO) gene selected from the gene of the IL-10 receptor, TGFbeta receptor, TIM-3, LAG-3 (see above table 9).

7. UCART 22 according to one of paragraphs. 1-6, additionally containing a polynucleotide having SEQ ID NO: 22, preferably containing the sequences of SEQ ID NO: 18 and 22 in the same TRAC gene.

8. A population of cells containing the specified UCART 22 according to one of paragraphs. 1-7.

9. Pharmaceutical composition containing UCART 22 according to one of paragraphs. 1-7 or a population of cells containing the specified UCART 22 according to claim 8, and a pharmaceutically acceptable excipient.

10. Pharmaceutical composition according to claim 9, additionally containing bryostatin, preferably bryostatin-1.

11. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to claim 9 or claim 10 for use as a medicine for the treatment of a patient.

12. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to claim 9 or claim 10 for use as a medicament for treating a patient according to claim 11, wherein the treatment of the patient comprises administering at least two times (repeated dosing) in said therapeutically effective amount of UCART CD22 or said pharmaceutical composition to avoid relapse.

13. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to claim 9 or claim 10 for use as a medicament for treating a patient according to claim 11 or claim 12, for treating a CD22-related pathology, preferably CD22-related B-cell malignancy.

14. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to p. 9 or p. 10 for use as a drug for the treatment of a patient according to one of p. 11-13 for use in the treatment of hematologic cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably CD22-expressing hematologic cancer, more preferably recurrent refractory CD22-expressing hematologic cancer, even more preferably an aggressive form of said CD22-associated hematologic cancer.

15. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to p. 9 or p. 10 for use as a drug for the treatment of a patient according to one of p. 11-13 for use in the treatment of relapsed and/or refractory CD22 positive B-ALL.

16. A therapeutically effective amount of UCART 22 according to one of paragraphs. 1-7 or a pharmaceutical composition according to p. 9 or p. 10 for use as a drug for the treatment of a patient according to one of p. 11-13, wherein said patient is suffering from a cancer, preferably mediated by CD22 ⁺ cells, selected from alveolar rhabdomyosarcoma, bladder cancer (eg, bladder carcinoma), bone cancer, brain cancer (eg, medulloblastoma), breast cancer, cancer anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleura cancer, nose cancer, nasal cavity cancer, middle ear cancer, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (eg, head and neck squamous cell carcinoma), hypopharynx cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (eg, non-small cell lung carcinoma), malignant mesothelioma, mastocytoma, melanoma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer gland cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureter cancer , preferably liver cancer and lung cancer.

Brief description of the drawings

The drawings show:

in fig. 1 is a schematic representation of an engineered immune cell according to the invention.

The engineered immune cell shown in FIG. 1 may be a T cell endowed with a polynucleotide that encodes a CAR of the invention. Said T cell is also engineered to provide better and safer graft engraftment in the patient. X or Y denotes an edited gene that can be changed by mutation, deleted by deletion and/or have an insertion. For example, a gene expressing a component of the T cell receptor (TCR), such as TCRalpha or TCRbeta, may be deleted by deletion or may contain an insertion, Y may indicate a gene involved in rendering T cells sensitive to immunosuppressive drugs such as dCK ( for resistance to purine nucleotide analogues) or CD52 (for Campas) or HPRT (for 6-thioguanine);

in fig. 2 is a depiction of single and multi-chain CD22 CAR.

In FIG. 2 illustrates examples of CD22 CARs of the invention which comprise a CD22-specific scfv, optionally contain a security switch, hinge and transmembrane domain from CD8alpha, intracellular domains from 4-1BB and CD3zeta, optionally a hypoxia-resistant domain;

in fig. 3 are examples of CD22 CAR designs according to the invention that include a safety switch.

At least rituximab Mab-specific (R) epitopes (black boxes), preferably 3 rituximab Mab-specific epitopes and more preferably 3 rituximab Mab-specific epitopes and QBEND-10 Mab-specific (Q) epitopes (gray box) are embedded in CD22 CAR.

R can be embedded in scfv, between VH and VL domain (or VL and VH domain) and/or in a hinge;

in fig. 4 is an example of a polypeptide encoded by a polynucleotide.

In FIG. 4 shows one example of a polypeptide that can be cleaved so that the guard switch (RQR8) and CD22 CAR can be expressed on the cell surface.

R is a CD20 mimotope (binding to rituximab), Q is a CD34 epitope (binding to QBEND-10);

in fig. 5 - data on the degranulating activity of UCART 22.

The degranulating activity of UCART 22 (scfv-V1 against the proximal domain of CD22) was compared with the degranulating activity of non-transduced (NT) T cells or T cells transduced by a CAR targeting the distal region of CD22 (scfv-V2) in the presence of CD22-positive cells NALM-16 versus CD22-negative SUP-T1 cells;

in fig. 6 - data on the cytotoxic activity of UCART 22.

The cytotoxic activity of UCART 22 (scfv-V1 against CD22 proximal domain) was compared with the cytotoxic activity of non-transduced (NT) T cells or T cells transduced with a CAR targeting the distal CD22 (scfv-V2) in the presence of CD22-positive cells NALM-16 versus CD22-negative SUP-T1 cells;

in fig. 7 - data on the production of interferon gamma by UCART 22 cells;

in fig. 8 - data on the survival of mice in the presence of control cells, UCART 22 (scfv-V1 against the proximal domain of CD22) or CART 22 (no deletion in TRAC);

in fig. 9 is a general strategy for inserting a gene into the TRAC gene using TALEN.

Detailed description of the invention

Unless specifically stated otherwise, all technical and scientific terms used in the present description have the meaning that is generally accepted by experts in the field of gene therapy, biochemistry, genetics and molecular biology.

For the practice or testing of the present invention, you can apply all methods and materials similar or equivalent to those specified in the present description, while this application describes the acceptable methods and materials. All publications, patent applications, patents and other references mentioned in the present description are hereby incorporated by reference in their entirety. In case of discrepancies, the present description, including definitions, shall prevail. In addition, materials, methods and examples are presented for illustration only and are not intended to limit the scope of the invention, unless otherwise indicated.

In the practice of the present invention, unless otherwise indicated, conventional methods of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology known to the person skilled in the art should be used. Such methods are described in detail in the literature, see, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, Wiley and son Inc, Library of Congress, USA, 2000); Molecular Cloning: A Laboratory Manual, 3rd ed. (Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001); Oligonucleotide Synthesis, ed. M.J. Gait, 1984; Mullis and others, US No. 4683195; Nucleic Acid Hybridization (ed. B. D. Harries and S. J. Higgins, 1984); Transcription And Translation (ed. B. D. Hames and S. J. Higgins, 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; series: Methods In ENZYMOLOGY (ed. by J. Abelson and M. Simon, Academic Press, Inc., New York), primarily vol. 154 and vol. 155 (ed. by Wu et al.) and vol. 185 , "Gene Expression Technology)) (ed. D. Goeddel); Gene Transfer Vectors For Mammalian Cells (Ed. J.H. Miller and M.P. Calos, 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (ed. Mayer and Walker, Academic Press, London, 1987); Handbook Of Experimental Immunology, volumes I-IV (ed. D.M. Weir and C.C. Blackwell, 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986)

CD22-specific chimeric antigen receptors

The present invention relates to newly created anti-CD22 chimeric antigen receptors (CAR or CD22 CAR or anti-CD22 CAR), which are chimeric antigen receptors with the ability to specifically bind to CD22, especially the proximal domain of CD22.

The CD22-specific chimeric antigen receptors of the invention comprise an extracellular domain that contains an extracellular ligand-binding domain and a hinge, optionally a suicide domain, a transmembrane domain, and an intracellular domain that contains a signal transduction domain.

The CD22 CAR of the present invention, which is expressed on the cell surface, contains an extracellular domain that contains an extracellular ligand-binding domain.

The term "extracellular ligand-binding domain" in the context of the present description means an oligo - or polypeptide that has the ability to bind to at least one CD22 epitope. Preferably, the extracellular ligand-binding domain should be capable of interacting at least in part with a cell surface molecule that interacts with CD22 and with another cell surface antigen or other membrane bound antigen, or interacting directly with CD22 or interacting with human CD22, more specifically interact directly with the proximal region of human CD22 (amino acid 243 to amino acid 687).

In one embodiment, the CD22 CAR of the present invention contains an extracellular domain that contains an extracellular ligand binding domain that is capable of interacting with the proximal region of CD22 (amino acid 243 to amino acid 687) and with the distal portion of CD22 (aa 20 to aa 425).

According to the present invention, the full-length extracellular domain of CD22 extends from amino acid (aa) 20 to aa 687, the proximal membrane domain of CD22 extends from aa 243 to aa 687, and the distal membrane domain of CD22 extends from aa 20 to aa 425.

In one embodiment, an extracellular ligand-binding domain can be selected to recognize a particular form (glycosized) of CD22 that functions as a cell surface marker on target cells associated with a particular disease state.

In a preferred embodiment of the invention, said extracellular ligand-binding domain comprises at least one single chain antibody fragment (scFv) comprising a variable fragment of the light (V _L ) and heavy (V _H ) chains of a target antigen-specific anti-CD22 monoclonal antibody linked by a linker, primarily a flexible linker. Said V _L and V _H are preferably derived from the m971 antibody shown in Table 2 below. They are preferably linked by a flexible linker which contains, for example, the sequence of SEQ ID NO: 10.

For the purposes of the present invention, specific regions of a full-length human anti-CD22 antibody, i. m971 antibody (m971), which was previously identified using phage display methodology and characterized (Xiao X., No M., Zhu Z., Pastan I., Dimitrov D.S. Identification and characterization of fully human anti-CD22 monoclonal antibodies. mAbs, 1(3), 2009, pp. 297-303), were combined with specific sequences to obtain new CD22 CARs of the invention (see also WO 2014065961, which is incorporated herein by reference).

A preferred embodiment of the invention are CARs that comprise an antigen binding domain comprising, consisting of, or essentially consisting of, a single chain variable fragment (scFv) of the m971 antigen binding domain with the following fragments shown in Table 1.

In a preferred embodiment, the CD22 CAR of the invention comprises the sequence of SEQ ID NO: 15.

In a preferred embodiment, the CD22 CAR of the invention comprises the sequence of SEQ ID NO: 23.

The CD22 CAR sequences of the invention contain a peptide signal, a TM domain from CD8alpha, and a linker between the VH and VL domains from m971.

SCFV

According to the present invention, scfv is a fusion protein of an immunoglobulin heavy (VH domain) and a light chain (VL domain) variable region or a CD22-specific immunoglobulin region, which are linked by a short linker peptide of 10-25 amino acids, preferably having SEQ ID NO: 10.

In a preferred embodiment, said CARs preferably comprise an extracellular ligand-binding domain that contains a polypeptide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12 .

In a preferred embodiment, said CARs preferably comprise an extracellular ligand-binding domain that contains a polypeptide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 13 .

In a preferred embodiment, said CARs preferably comprise an extracellular ligand-binding domain that contains a polypeptide sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 12 , and a polypeptide sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 13.

Hinge

The extracellular domain may also contain a hinge region between said extracellular ligand-binding domain and said transmembrane domain. In the context of the present description, the term "hinge region", as a rule, refers to any oligo - or polypeptide, the function of which is to link the transmembrane domain to the extracellular ligand-binding domain. In particular, the hinge region is used to provide greater flexibility and accessibility of the extracellular ligand binding domain. The hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 10 to 50 amino acids. The hinge region can be derived from complete naturally occurring molecules, or parts thereof, such as the complete extracellular region of CD8 or CD4, or part thereof, or from the entire constant region of an antibody, or part thereof. Alternatively, the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. In a preferred embodiment of the invention, the specified hinge domain contains part of the alpha chain of human CD8, FcyRIIIα receptor or IgG1, respectively, which are designated in the present description as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or more in a preferred embodiment of the invention are hinge polypeptides whose sequences are preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% identical to the sequence of SEQ ID NO: 4, even more preferably 100% identical to the sequence of SEQ ID NO: 4.

An IgG4 or PD1 hinge is part of the present invention and is described in WO 2016/120216 and can be used in the CD22 CAR construct of the invention.

The CD22 CAR of the present invention is anchored in the cell membrane. Thus, CD22 CAR additionally contains a transmembrane domain. The distinguishing features of the respective trans membrane domains are the ability to be expressed on the cell surface, according to the present invention preferably on the surface of an immune cell, especially lymphocytes or natural killer (NK) cells, and the ability to interact with each other, directing the cellular response of the immune cell to a predetermined cell -target. The transmembrane domain can be derived from either a naturally occurring or synthetic source. The transmembrane domain can be derived from any membrane-bound or transmembrane protein. Examples of transmembrane polypeptides include, but are not limited to, a T cell receptor subunit such as the α-, β-, γ-, or δ-subunit, CD3 complexing polypeptide, p55 (α-chain), p75 ( β chain) or IL2 receptor γ chain, Fc receptor subunit chain, in particular Fcγ receptor III, or CD proteins. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine.

In a preferred embodiment of the invention, said transmembrane domain is derived from the alpha chain of human CD8 (eg, NP 001139345.1).

The CD22 CAR of the invention typically further comprises a transmembrane domain (TM), more specifically of CD8α, which is identical to polypeptides having SEQ ID NO: 6 or SEQ ID NO: 7. Preferably, the CAR of the invention contains a TM at least 70%, preferably at least 80%, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 identical % or 100% of the polypeptide sequence of SEQ ID NO: 6.

The CD22 CAR of the invention typically additionally contains a transmembrane domain (TM) from CD8α that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 identical. % or 100% polypeptides having SEQ ID NO: 6. Preferably, the CAR of the invention contains a TM that is at least 70% identical, preferably at least 80%, more preferably at least 90%, 91% identical , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the polypeptide sequence of SEQ ID NO: 6.

The CD22 CAR of the present invention contains an intracellular domain that contains a signal transduction domain or an intracellular signaling domain.

The signal transduction domain or intracellular signaling domain of the CD22 CAR of the present invention is responsible for intracellular signaling after binding of the extracellular ligand-binding domain to the target, resulting in immune cell activation and immune response (cytolytic activity against the target cell). In other words, the signal transduction domain is responsible for activating at least one of the normal effector functions of an immune cell in which CAR is expressed. For example, the effector function of a T cell may be a cytolytic activity or a helper activity, including the secretion of cytokines. Thus, the term "signal transduction domain" refers to the region of the protein that transduces the signal of an effector signaling function and induces the cell to perform a specialized function.

Preferred examples of a signal transduction domain in the CD22 CAR of the invention may be the cytoplasmic sequences of the T cell receptor and co-receptors that act together to initiate signal transduction upon contact with the antigen receptor, any derivative or variant of these sequences, and any synthetic a sequence that has the same functionality. The signal transduction domain contains two distinct classes of cytoplasmic signal sequences, those that initiate antigen-dependent primary activation and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. The primary cytoplasmic signaling sequence may contain signal motifs known as immune receptor activation motifs based on tyrosine, ITAM. ITAMs are well known signaling motifs present in the intracytoplasmic tail of various receptors that serve as binding sites for the syk/zap70 class of tyrosine kinases. Examples of ITAMs useful according to the invention include, but are not limited to, ITAMs derived from TCRzeta, FcRgamma, FcRbeta, FcRepsilon, CB3 gamma, CB3delta, CB3epsilon, CD5, CD22, CD79a, CD79b and CD66d. In a preferred embodiment, the CAR signal transduction domain may comprise a CD3zeta signaling domain that has an amino acid sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% , 99% or 100% of an amino acid sequence selected from the group consisting of SEQ ID NO: 9. Optionally, said CD3zeta signal domain contains a polypeptide sequence that is at least 90%, 91%, 92% 93% 94%, 95%, 96%, 97%, 98%, 99% or 100% SEQ ID NO: 9.

In a more preferred embodiment, the CD22 CAR signal transduction domain consists of the CD3zeta signaling domain of SEQ ID NO: 9 and omits the presence of any sequence from the human CD28 signaling domain. In a specific embodiment, the CAR signal transduction domain of the present invention comprises a co-stimulatory signaling molecule. A costimulatory molecule is a cell surface molecule, other than the antigen receptor or its ligands, that is required for an effective immune response. The term "costimulatory ligand" refers to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby creating a signal that, in addition to the primary signal generated, for example, when the TCR/CD3 complex binds to an MHC molecule , loaded with a peptide, mediates a T cell response, including (but not limited to) activation of proliferation, differentiation, and the like. The costimulatory ligand may 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 receptor beta, 3/TR6, ILT3, ILT4, agonist or antibody that/which binds to Toll-ligand receptor, and a ligand that specifically binds to B7-H3. The term "costimulatory ligand" includes, inter alia, an antibody that specifically binds to a costimulatory molecule present on a T cell, such as (but not limited to) CD27, 4-1BB, OX40, CD30, CD40, PD- 1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, CD83 specific binding ligand.

The term "costimulatory molecule" refers to a cognate binding partner present on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the cell, such as (but not limited to) proliferation. Costimulatory molecules include, but are not limited to, the MHC class I molecule, BTLA, and the Toll receptor ligand. Examples of costimulatory molecules are CD27, CD8, 4-1BB (CD 137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and CD83 specific binding ligand, and the like.

In a preferred embodiment, the CAR signal transduction domain of the present invention comprises a portion of a co-stimulatory signaling molecule consisting of a 4-1BB fragment (GenBank: AAA53133). In particular, the CAR signal transduction domain of the present invention contains an amino acid sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% of an amino acid sequence selected from the group consisting of SEQ ID NO: 8.

In a more preferred embodiment, the CAR signal transduction domain of the present invention does not contain a sequence from CD28 (NP 006130.1).

In an even more preferred embodiment of the invention, all embodiments of the present invention lack a sequence from CD28 (NP 006130.1).

In an even more preferred embodiment, the CD22 CAR signal transduction domain of the present invention contains part of the costimulatory signaling molecule 4-1BB (GenBank: AAA53133) and does not contain a sequence from CD28 (NP 006130.1).

The present invention provides a CD22 specific chimeric antigen receptor (CD22 CAR) comprising:

extracellular domain, which contains:

- a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said binding domain specific for human CD22, which is a single chain variable fragment (scFv),

- hinge, preferably in CD8alpha,

- transmembrane domain, preferably from CD8alpha,

- intracellular domain containing:

a co-stimulatory signaling molecule from human 4-1BB; and an intracellular signaling domain that contains the human CD3zeta signaling domain.

In a preferred embodiment of the invention, the CD22 CAR of the invention does not have a sequence from CD28.

In a preferred embodiment, the CD22 CAR of the invention does not contain any sequence from CD28 and contains the leader sequence, TM domain and hinge from CD8a, preferably does not contain any sequence from CD28 and contains the leader sequence of SEQ ID NO: 1, TM- a domain having SEQ ID NO: 6 and a hinge having SEQ ID NO: 4 from CD8α.

In one embodiment, the CD22 CAR of the invention contains a leader sequence from human CD8α (SEQ ID NO: 1) or a leader sequence at least 95% identical to SEQ ID NO: 1.

In another embodiment, the CD22 CAR of the invention contains a leader sequence of SEQ ID NO: 2 or a leader sequence that is at least 95% identical to SEQ ID NO: 2.

In one embodiment, the present invention provides a CD22 specific chimeric antigen receptor (CD22 CAR) comprising:

- a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said binding domain specific for human CD22, which is a single chain variable fragment (scFv), even more preferably containing VH and VL from m971 ,

- hinge made of human CD8alpha (from CD8α),

- transmembrane domain from human CD8alpha (α),

- a co-stimulatory signaling molecule from human 4-1BB,

- intracellular signaling domain containing the signaling domain of the human CD3zeta.

In one embodiment, the present invention provides a CD22 specific chimeric antigen receptor (CD22 CAR) comprising:

- a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said binding domain specific for human CD22, which is a single chain variable fragment (scFv), even more preferably containing VH and VL from m971 ,

- human FcRIIIα hinge,

- transmembrane domain from human CD8alpha (α),

- a co-stimulatory signaling molecule from human 4-1BB,

- intracellular signaling domain containing the signaling domain of the human CD3zeta.

The present invention also provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising:

- a binding domain specific for CD22, preferably a binding domain specific for human CD22, more preferably said binding domain specific for human CD22, which is a single chain variable fragment (scFv), even more preferably containing VH and VL from m971 ,

- human IgG1 hinge,

- transmembrane domain from human CD8alpha (α),

- a co-stimulatory signaling molecule from human 4-1BB,

- intracellular signaling domain containing the signaling domain of the human CD3zeta.

These last three embodiments of the invention include a CD22 CAR with a signal peptide having SEQ ID NO: 1 or SEQ ID NO: 2, preferably SEQ ID NO: 1.

More preferably, said CD22 CAR does not have any sequence from CD28.

The Scfv of the invention is derived from an antibody specific for CD22, it contains a VH domain separated from the VL domain by a linker, said VH and/or VL domain(s) are involved in binding to CD22.

In a preferred embodiment, the UCART 22 of the invention comprises scfv from the m971 antibody with a single GS4 linker between VH and VL and a 2R between VL and the CD8alpha hinge.

According to the present invention, scfv is a fusion protein of the variable regions of the heavy (VH domain) (SEQ ID NO: 12) and light (VL domain) (SEQ ID NO: 13) chains of an immunoglobulin specific for CD22, m971, which are connected by a linker a peptide, preferably a linker, having SEQ ID NO: 10.

In one embodiment, said scfv of the invention further comprises a leader sequence (or signal peptide), preferably said leader sequence is linked to a VH domain.

An embodiment of the invention in which said leader sequence is linked to the VL domain is part of the present invention.

In one of the embodiments of the invention the VH domain is connected to the hinge, in another embodiment of the invention the VL domain is connected to the specified hinge.

The present invention provides a scfv associated with a hinge of various lengths, preferably a CD8α, IgG1 or FcRIIIα hinge (see FIG. 2), more preferably CD8α.

Preferably, the present invention provides a CD22 CAR comprising:

- a signal peptide, for example, a signal peptide having SEQ ID NO: 2 or from CD8alpha having SEQ ID NO: 1,

- (scFv) containing a VH domain separated from the VL domain by a linker, where the specified VH and VL and the linker are involved in binding to CD22,

- hinge from human CD8 alpha chain or hinge from human IgG1, or hinge from FcRIIIα, preferably from CD8alpha,

- transmembrane domain (TM) from CD8alpha(α),

- a co-stimulatory signaling molecule from human 4-1BB,

- intracellular signaling domain containing the CD3zeta signaling domain.

One of the CD22 CARs according to the invention consists of:

- a leader sequence (for example, a CD8α leader sequence or a CD8α signal peptide),

- anti-CD22 scfv which contains VH, linker and VL or VL, linker and VH, where said VH and VL are derived from m971,

• CD8α hinge,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- an epitope recognized by QBEND-10 and an epitope recognized by rituximab,

- an anti-CD22 scfv that contains a VH, a linker and a VL, where said VH and VL are derived from m971, two consecutive epitopes recognized by rituximab,

• CD8α hinge,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- an epitope recognized by QBEND-10 and an epitope recognized by rituximab,

- an anti-CD22 scfv that contains a VH, a linker and a VL, where said VH and VL are derived from m971, two consecutive epitopes recognized by rituximab,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD22 scfv which contains VH, linker and VL, where said VH and VL are derived from m971,

two consecutive epitopes recognized by rituximab,

• CD8α hinge,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD22 scfv which contains VH, linker and VL, where said VH and VL are derived from m971,

two consecutive epitopes recognized by rituximab,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD22 scfv, which contains VH, linker and VL, where said VH and VL are derived from m971, anti-CD 19 scfv,

two consecutive epitopes recognized by rituximab,

• CD8α hinge,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD22 scfv, which contains VH, linker and VL, where said VH and VL are derived from m971, anti-CD 19 scfv,

two consecutive epitopes recognized by rituximab,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD19 scfv and anti-CD22 scfv, which contains VH, linker and VL, where said VH and VL are derived from m971,

two consecutive epitopes recognized by rituximab,

• CD8α hinge,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

One of the CD22 CARs proposed in the invention contains sequentially located:

- signal peptide CD8α, which can be removed after expression on the cell surface,

- anti-CD19 scfv and anti-CD22 scfv, which contains VH, linker and VL, where said VH and VL are derived from m971,

two consecutive epitopes recognized by rituximab,

• TM CD8α,

• costimulatory signaling molecule from 4-1BB,

• intracellular signaling domain CD3zeta.

Linker-SCFV

The linker of the invention may be, for example, a pentapeptide multimer (GGGGS) _n or (G ₄ S) _n or (Gly ₄ Ser) _n , where n is between 1 and 4, preferably n is 3, 18- the dimensional linker GGSSRSSSSGGGGSGGGG (Andris-Widhopf et al., 2011) and the 20-mer linker (648)4 (Schaefer et al., 2010). The linker of the invention may include sequences with added functionality, such as an epitope tag (Sblattero and Bradbury, Nature Biotechnology 18, 2000, pp. 75-80), at least SEQ ID NO: 20, preferably two, separated by a linker , sequences that enhance the properties of the scFv of the present invention, often in the context of specific antibody sequences.

Among other linkers suitable according to the present invention, mention should be made of the 15-mer peptide linker (RGGRGRGRRSRGGGS) (Zhihong Shen, Heping Yan, Ying Zhang, Raymond L. Mernaugh and Xiangqun Zeng Anal Chem. 80(6), 2008, cc. 1910- 1917).

In a preferred embodiment of the invention, the linker that links the VH sequence to the VL in m971 (or the VL sequence to the VH) is a linker of the formula (G ₄ S) _n in which n is from 1 to 3; preferably n is 3 in the sequence (G ₄ S) ₃ (SEQ ID NO: 10).

One of the embodiments of the invention of the present invention is:

CD22 CAR containing

- human CD8α leader sequence (CD8α leader or CD8α signal peptide) SEQ ID NO: 1,

- anti-CD22 scfv, which contains a VH having SEQ ID NO: 12, a linker having SEQ ID NO: 10, and a VL having SEQ ID NO: 13,

human CD8α hinge having SEQ ID NO: 4,

• TM human CD8α having SEQ ID NO: 6,

• a co-stimulatory signaling molecule from 4-1BB having SEQ ID NO: 8,

• intracellular signaling domain CD3zeta having SEQ ID NO: 9.

One of the embodiments of the invention of the present invention is also:

CD22 CAR containing

- human CD8α leader sequence (CD8α leader or CD8α signal peptide) SEQ ID NO: 1,

- anti-CD22 scfv, which contains a VH having SEQ ID NO: 12, a linker having SEQ ID NO: 10, and a VL having SEQ ID NO: 13,

human FcγRIIIα hinge having SEQ ID NO: 3,

• TM human CD8α having SEQ ID NO: 6,

• a co-stimulatory signaling molecule from 4-1BB having SEQ ID NO: 8,

• intracellular signaling domain CD3zeta having SEQ ID NO: 9.

One of the embodiments of the invention of the present invention is:

CD22 CAR containing

- human CD8α leader sequence (CD8α leader or CD8α signal peptide) SEQ ID NO: 1,

- anti-CD22 scfv, which contains a VL having SEQ ID NO: 13, a linker having SEQ ID NO: 10, and a VH having SEQ ID NO: 12,

human FcyRIIIα hinge having SEQ ID NO: 3,

• TM human CD8α having SEQ ID NO: 6,

• a co-stimulatory signaling molecule from 4-1BB having SEQ ID NO: 8,

• intracellular signaling domain CD3zeta having SEQ ID NO: 9.

One of the embodiments of the invention of the present invention is:

CD22 CAR containing

- human CD8α leader sequence (CD8α leader or CD8α signal peptide) SEQ ID NO: 1,

- anti-CD22 scfv, which contains a VL having SEQ ID NO: 13, a linker having SEQ ID NO: 10, and a VH having SEQ ID NO: 12,

human CD8alpha hinge having SEQ ID NO: 4,

• TM human CD8α having SEQ ID NO: 6,

• a co-stimulatory signaling molecule from 4-1BB having SEQ ID NO: 8,

• intracellular signaling domain CD3zeta having SEQ ID NO: 9.

In one embodiment, the present invention provides a CD22-specific chimeric antigen receptor (CD22 CAR) comprising:

- a signal peptide that has an amino acid sequence that is at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% identical to the polypeptide sequence of SEQ ID NO: 1 or 2; preferably the signal peptide has an amino acid sequence that is at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% identical to the polypeptide sequence of SEQ ID NO: 1,

- VH-domain, separated from the VL-domain by a linker, where these VH and VL are involved in binding to CD22; the specified linker has a sequence identical to at least 90%, 95%, 97%, 99% or 100% of the polypeptide sequence of SEQ ID NO: 10;

said VH domain has a sequence that is at least 90%, 95%, 97%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 12,

said VL domain has a sequence that is at least 90%, 95%, 97%, 99%, or 100% identical to the polypeptide sequence of SEQ ID NO: 13,

- a hinge derived from the alpha chain of human CD8 which has an amino acid sequence that is at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% identical to the polypeptide sequence of SEQ ID NO: four,

- a transmembrane domain derived from CD8alpha(α) that has an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical or 100% of the polypeptide sequence of SEQ ID NO: 6,

- a co-stimulatory signaling molecule derived from human 4-1BB (or intracellular domain 4-1BB) having an amino acid sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95 %, 97%, 99% or 100% of an amino acid sequence selected from the group consisting of SEQ ID NO: 8,

- an intracellular signaling domain that contains a CD3zeta signaling domain having an amino acid sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100 % amino acid sequence selected from the group consisting of SEQ ID NO: 9.

In a preferred embodiment of the invention, the CD22-specific chimeric antigen receptor (CD22 CAR) of the present invention does not contain any sequence from human CD28, primarily from the intracellular signaling domain of human CD28. In a more preferred embodiment of the invention, the CD22-specific chimeric antigen receptor (CD22 CAR) of the present invention does not contain any sequence from human CD28, primarily from the intracellular signaling domain of human CD28, and further comprises a signal peptide from CD8α, preferably fused to VH domain of scfv specific for CD22.

In one embodiment, the present invention provides a CD22 CAR having SEQ ID NO: 15.

In one embodiment, the present invention provides a CD22 CAR having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% polypeptide sequence of SEQ ID NO: 15.

In one embodiment, the present invention provides a CD22 CAR having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % polynucleotide sequence SEQ ID NO: 22.

The CD22 CAR of the invention contains the following sequences:

v1-m971 (FcγRIIIα-CD8αTM-4-1BB.IC-CD3ζ.IC)

V3-m971 (CD8α-CD8αTM-4-1BB.IC-CD3ζ.IC)

In a preferred embodiment, the CD22 CAR of the invention contains the following sequence:

The following are the sequences of the CD22 CAR with the peptide signal from SEQ ID NO: 2, the TM domain from CD8α, and a linker between the VH and VL domain:

M971 V1

M971 V3

One of the embodiments of the present invention are the following sequences:

В указанном CD22 CAR отсутствует сигнальный пептид.

This CD22 CAR lacks a signal peptide.

In one of the embodiments of the invention UCART 22 proposed in the present invention contains the sequence of SEQ ID NO: 20

In one embodiment, the UCART 22 of the present invention contains the sequence of SEQ ID NO: 22.

In a preferred embodiment of the invention, the UCART 22 of the present invention contains the sequence SEQ ID NO: 22 inserted into the human TRAC gene (for example, the human TRAC gene on chromosome 14 - NC_000014.9) and expresses anti-CD22 on the cell surface CAR specific for the proximal region of CD22.

In a more preferred embodiment of the invention, UCART 22 of the present invention contains the sequence of SEQ ID NO: 20 inserted into the human TRAC gene (for example, the human TRAC gene on chromosome 14 - NC_000014.9) and expresses anti- CD22 CAR specific for the distal region of CD22.

In one aspect of the invention, the anti-CD22 binding domain of the CD22 CAR of the invention is an anti-CD22 binding domain specific for the distal region of CD22.

HA22-CAR

The present invention provides a CD22 CAR having the following sequence:

Preferably UCART 22, proposed in the present invention, contains the sequence:

The anti-CD22 binding domain specific for the distal region of CD22 can be expressed alone or in combination with the anti-CD22 binding domain specific for the proximal region of CD22 in UCART 22 of the invention.

In one aspect of the invention, the anti-CD22 binding domain of the CD22 CAR of the invention is an optimized anti-CD22 binding domain.

As used herein, an "optimized" antibody (or scfv) refers to forms of antibodies (or scfv) that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding antibody subsequences) that contain minimal immunoglobulin-derived sequences. Preferably, the antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable regions (CDRs) are replaced with residues from the CDRs to achieve the desired specificity, affinity, and desired potential.

Several amino acid substitutions can be made in the donor CDR which can significantly affect or alter the binding characteristics of the CD22 CAR of the invention. Thus, one of the embodiments of the invention presented in the present description is a CD22 CAR, the binding of which to a CD22 expressing cell (and cytolytic activity) is retained, but the affinity is modified to reduce the intensity of the response (release of cytokines).

Amino acid modifications, as a rule, are conservative modifications, including amino acid substitutions, additions and deletions in the specified antibody fragment in the specified CAR and/or other regions of the specified CAR molecule. Modifications can be introduced into an antibody, into an antibody fragment, or into any of the other regions of the CAR molecule of the invention using standard techniques known in the art such as site-directed mutagenesis, PCR-mediated mutagenesis, or by using optimized germline sequences.

Typically, an optimized CAR should contain substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDRs correspond to the original human immunoglobulin.

Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue that has a similar side chain. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention can be substituted for other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays provided herein.

In a preferred embodiment, the present invention provides a CD22 CAR having conservative sequence modifications (or amino acid sequence change) compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15.

In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 2 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15.

In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 3 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15.

In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 4 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15.

In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 5 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15, optionally containing at least one sequence of SEQ ID NO: 20.

In a more preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 5 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15, and wherein at least one CDR in SEQ ID NO: 15 is conserved.

In a more preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with 1-15 amino acid changes compared to the amino acid sequence of the polypeptide of SEQ ID NO: 15, and at least one CDR in SEQ ID NO: 15 is conserved.

In a preferred embodiment, the CD22 CAR sequence of the invention is modified by changing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids from wt CD22 CAR to reduce HAMA (human response). anti-mouse antibody) without affecting the binding capacity of said CAR to its target (CD22).

In a preferred embodiment, the present invention provides a CD22 CAR having an amino acid sequence with at least 1 amino acid change compared to the amino acid sequence of wt (wt is the m971 sequence), said at least 1 amino acid change does not affect or improves binding and/or activity said CD22 CAR in primary T cells.

The knitting characteristics can be modified using an adapted technique first described by Mitchell Ho, Satoshi Nagata and Ira Pastan. Isolation of anti-CD22 Fv with high affinity by Fv display on human cells, PNAS 103 (25), 2006, cc. 9637-9642; provisional publication June 8, 2006, doi: 10.1073/pnas.0603653103, which is incorporated herein by reference.

These optimized scfvs also carry at least one of the mutations equivalent to the Pro-91-Thr-92 (PT), Gly-91-Ala-92 and Val-91-Phe-92 mutations.

In one embodiment, the present invention provides an anti-CD22 CAR that contains:

- extracellular domain containing a signal peptide,

a ligand-binding domain, optionally optimized, which comprises a VH domain and a VL domain from an anti-CD22 monoclonal antibody having one of the following mutations: Pro-91-Thr-92 (PT), Gly-91-Ala-92, Val-91-Phe -92 or its equivalent,

a hinge containing a CD8alpha (α) hinge,

- transmembrane domain CD8alpha and

- cytoplasmic domain containing the CD3zeta signaling domain and the co-stimulatory domain from 4-1BB.

In a preferred embodiment, the present invention provides the above CD22 CAR, wherein said anti-CD22 monoclonal antibody is derived from an m971 antibody with "hot spots" in the CDR. Preferably it has one of the following mutations: Pro-91-Thr-92 (PT), Gly-91-Ala-92, Val-91-Phe-92.

In a more preferred embodiment, the present invention provides an isolated engineered (TCR- and dCK-KO) immune T cell containing an anti-CD22 CAR that contains

• extracellular domain containing

a signal peptide, a ligand-binding domain that comprises a VH domain and a VL domain from an anti-CD22 monoclonal antibody having at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92 mutation; mutation Val-91-Phe-92,

a hinge containing a CD8alpha (α) hinge,

- transmembrane domain CD8alpha and

- cytoplasmic domain containing the CD3zeta signaling domain and the co-stimulatory domain from 4-1BB.

In a more preferred embodiment, the present invention provides an isolated engineered (TCR- and CD52-KO) immune T cell containing an anti-CD22 CAR that contains

• extracellular domain containing

a signal peptide, a ligand-binding domain that comprises a VH domain and a VL domain from an anti-CD22 monoclonal antibody having at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92 mutation; mutation Val-91-Phe-92,

a hinge containing a CD8alpha (α) hinge,

- transmembrane domain CD8alpha and

- cytoplasmic domain containing the CD3zeta signaling domain and the co-stimulatory domain from 4-1BB.

In a more preferred embodiment, the present invention provides an isolated engineered (TCR-, CD52- and dCK-KO) immune T cell containing an anti-CD22 CAR that contains

• extracellular domain containing

a signal peptide, a ligand-binding domain that comprises a VH domain and a VL domain from an anti-CD22 monoclonal antibody having at least one of the following mutations: Pro-91-Thr-92, Gly-91-Ala-92 mutation; mutation Val-91-Phe-92,

a hinge containing a CD8alpha (α) hinge,

- transmembrane domain CD8alpha and

- cytoplasmic domain containing the CD3zeta signaling domain and the co-stimulatory domain from 4-1BB.

In one aspect of the invention, CAR 22 may be co-expressed on the cell surface with at least one, preferably two, more preferably three monoclonal antibody (MAb) specific epitopes, said Mab specific epitopes may be fused to the trans membrane domain of CD8. In one embodiment, said MAb-specific epitope is an epitope recognized by rituximab and/or QBEND-10 and the peptide co-expressed with CD22 CAR is RQR8.

In another embodiment of the invention, at least one, preferably two, more preferably three, monoclonal antibody (MAb) specific epitopes can be inserted into the linker L of the CD22-specific scfv fragment (binding VH to VL) and/or hinge CD22 CAR.

The monoclonal antibody (MAb)-specific epitope may be one of the following MAb-specific epitopes that are specifically recognized by a monoclonal antibody selected from ibritumomab, tiuxetan, muromonab-CD3, tositumomab, absiximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, алемтузумаба, бевацизумаба, цертолизумаба пэгола, даклизумаба, экулизумаба, эфализумаба, гемтузумаба, натализумаба, омализумаба, паливизумаба, ранибизумаба, тоцилизумаба, трастузумаба, ведолизумаба, адалимумаба, белимумаба, канакинумаба, деносумаба, голимумаба, ипилимумаба, офатумумаба, панитумумаба, QBEND-10 и устекинумаба , preferably from rituximab (R) and/or from QBEND-10 (Q).

The epitope-specific Mab can be used for in vitro cell sorting and/or for in vivo cell depletion of CD22-expressing immune cells.

In specific embodiments, the extracellular binding domain of the CD22 CAR of the invention may comprise one of the following sequences:

V1-L1-V2-(L)x-epitope 1-(L)x-;

V1-L1-V2-(L)x-epitope 1-(L)x-epitope 2-(L)x-;

V1-L1-V2-(L)x-epitope 1-(L)x-epitope 2-(L)x-epitope 3-(L)x-;

(L)x-epitope 1-(L)x-V1-L1-V2;

(L)x-epitope 1-(L)x-epitope 2-(L)x-V1-L1-V2;

epitope 1-(L)x-epitope 2-(L)x-epitope 3-(L)x-V1-L1-V2;

(L)x-epitope 1-(L)x-V1-L1-V2-(L)x-epitope 2-(L)x;

(L)x-epitope 1-(L)x-V1-L1-V2-(L)x-epitope 2-(L)x-epitope 3-(L)x-;

(L)x-epitope 1-(L)x-V1-L1-V2-(L)x-epitope 2-(L)x-epitope 3-(L)x-epitope 4-(L)x-;

(L)x-epitope 1-(L)x-epitope 2-(L)x-V1-L1-V2-(L)x-epitope 3-(L)x-;

(L)x-epitope 1-(L)x-epitope 2-(L)x-V1-L1-V2-(L)x-epitope 3-(L)x-epitope 4-(L)x-;

V1-(L)x-epitope 1-(L)x-V2;

V1-(L)x-epitope 1-(L)x-V2-(L)x-epitope 2-(L)x;

V1-(L)x-epitope 1-(L)x-V2-(L)x-epitope 2-(L)x-epitope 3-(L)x;

V1-(L)x-epitope 1-(L)x-V2-(L)x-epitope 2-(L)x-epitope 3-(L)x-epitope 4-(L)x;

(L)x-epitope 1-(L)x-V1-(L)x-epitope 2-(L)x-V2; or

(L)x-epitope 1-(L)x-V1-(L)x-epitope 2-(L)x-V2-(L)x-epitope 3-(L)x;

where

V1 is VL, preferably SEQ ID NO: 12 and V2 is VH, preferably SEQ ID NO: 13, or V1 is VH and V2 is VL;

L1 denotes a linker that can be used to link the VH chain to the VL chain; preferably having SEQ ID NO: 10;

L is a linker containing glycine and serine residues, and each L occurring in an extracellular binding domain may be identical or different from another L occurring in the same extracellular binding domain, and

x is 0 or 1 and each of the occurring x is chosen independently of each other; and

epitope 1, epitope 2 and epitope 3 are MAT-specific epitopes and may be identical or different.

In one of the embodiments of the invention, the extracellular binding domain contains the following sequence V1-L1-V2-L-epitope1; V1-L1-V2-L-epitope 1-L; V1-L1-V2-L-epitope1-L-epitope2; V1-L1-V2-L-epitope 1-L-epitope 2-L; V1-L1-V2-L-epitope1-L-epitope2-L-epitope3; V1-L1-V2-L-epitope 1-L-epitope 2-L-epitope 3-L; V1-L1-V2 epitope1; V1-L1-V2 epitope 1-L; V1-L1-V2-epitope1-L-epitope2; V1-L1-V2-epitope 1-L-epitope 2-L; V1-L1-V2-epitope1-L-epitope2-L-epitope3; V1-L1-V2-epitope 1-L-epitope 2-L-epitope 3-L; epitope 1-V1-L1-V2; epitope 1-L-V1-L1-V2; L-epitope 1-V1-L1-V2; L-epitope 1-L-V1-L1-V2; epitope1-L-epitope2-V1-L1-V2; epitope 1-L-epitope 2-L-V1-L1-V2; L-epitope 1-L-epitope 2-V1-L1-V2; L-epitope 1-L-epitope 2-L-V1-L1-V2; epitope 1-L-epitope2-L-epitope3-V1-L1-V2; epitope 1-L-epitope 2-L-epitope 3-L-V1-L1-V2; L-epitope 1-L-epitope 2-L-epitope 3-V1-L1-V2; L-epitope 1-L-epitope 2-L-epitope 3-L-V1-L1-V2; V1-L-epitope1-L-V2; L-epitope 1-L-V1-L-epitope 2-L-V2; V1-L-epitope 1-L-V2-L-epitope 2-L; V1-L-epitope1-L-V2-L-epitope2-L-epitope3; V1-L-epitope1-L-V2-L-epitope2-epitope3; V1-L-epitope1-L-V2-L-epitope2-L-epitope3-epitope4; L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L; epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3-L; L-epitope 1-L-V1-L-epitope 2-L-V2-L-epitope 3; L-epitope 1-L-V1-L1-V2-L-epitope 2-L; L-epitope 1-L-V1-L1-V2-L-epitope 2-L-epitope 3; L-epitope 1-L-V1-L1-V2-L-epitope 2-epitope 3 or epitope 1-L-V1-L1-V2-L-epitope 2-L-epitope 3-epitope 4, where

V1 is VL and V2 is VH or V1 is VH and V2 is VL; both VH and VL are optimized VH and VL from m971.

L1 is any linker that can be used to link a VH chain to a VL chain;

L is a linker containing glycine and serine residues, and each L occurring in an extracellular binding domain may be identical or different from another L occurring in the same extracellular binding domain, and

epitope 1, epitope 2 and epitope 3 are MAT-specific epitopes and may be identical or different.

L1 is a linker containing glycine and/or series, preferably L1 is a linker containing 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, or a linker containing the amino acid sequence (Gly ₄ Ser) ₄ or (Gly ₄ Ser) ₃ .

Likewise, L is a linker containing glycine and/or series, preferably L is a linker which has an amino acid sequence selected from

предпочтительно L обозначает

preferably L is

In a preferred embodiment, epitope 1, epitope 2, epitope 3, and epitope 4 are independently selected from MAb-specific epitopes that are specifically recognized by ibritumomab, tiuxetan, muromonab-CD3, tositumomab, absiximab, basiliximab, brentuximab, vedotin, cetuximab, infliximab, rituximab, алемтузумабом, бевацизумабом, цертолизумаба пэголом, даклизумабом, экулизумабом, эфализумабом, гемтузумабом, натализумабом, омализумабом, паливизумабом, ранибизумабом, тоцилизумабом, трастузумабом, ведолизумабом, адалимумабом, белимумабом, канакинумабом, деносумабом, голимумабом, ипилимумабом, офатумумабом, панитумумабом, QBEND-10 и устекинумабом , preferably epitope 1, epitope 2, epitope 3 and epitope 4 are recognized by rituximab or QBEND-10.

Epitope 1, epitope 2, epitope 3 and epitope 4 are MAb-specific epitopes that are specifically recognized by:

Thus, a MAb-specific epitope may comprise one of the polypeptides selected from:

In a preferred embodiment, the MAb-specific epitope is a MAb-specific epitope that has the amino acid sequence ELPTQGTFSNVSTNVSPAKPTTTA and/or CPYSNPSLC (SEQ ID NO: 19).

In a more preferred embodiment, the CD22 CAR of the invention contains 3 MAb-specific epitopes that have the amino acid sequence CPYSNPSLC (R) and one that has the amino acid sequence ELPTQGTFSNVSTNVSPAKPTTTA (Q).

In an even more preferred embodiment of the invention, the CD22 CAR of the invention contains two MAb-specific epitopes having the amino acid sequence CPYSNPSLC (R) located in the NT portion of the hinge, immediately after the VL.

In one aspect of the invention, at least one sequence by which rituximab binds to (R) and/or a sequence by which QBEND-10 binds to (Q) can be inserted into a GGGGSGGGGSGGGGS linker and/or into a hinge such that previously described in WO 2016/120216.

In a specific embodiment, the CD22 CAR of the present invention is a single chain CAR (scCAR).

In specific embodiments, the single chain anti-CD22 CAR of the invention comprises an scfv from m971 and at least one other binding domain, preferably specific for the distal region of CD22, alternatively specific for another B cell antigen, primarily if it is expressed by malignant B cells such as CD34, CD10, CD79a, CD20, IgD, CD5, CD23, CD19, STAT5, CD3, CD30, BCMA. In a specific embodiment, the CD22 CAR of the present invention is a multi-chain CAR (mcCAR). Multi-chain CD22 CARs are part of the present invention and can be obtained according to the method detailed in WO 2014/039523, which is incorporated herein by reference. In a particular embodiment, the VH domain and the VL domain of an immunoglobulin or CD22-specific immunoglobulin region may be on two different and separated (non-covalently linked) chains of a multi-chain CAR.

In the multi-stranded version of CD22, the CAR of the invention contains two, preferably three, transmembrane domains (which are not covalently linked to each other), with at least one transmembrane domain containing the inventive CD22-specific scfv.

In a particular embodiment, the VH domain and the VL domain of an immunoglobulin specific for CD22, preferably from m971, may be present on the same strand of a multi-chain CAR.

An example of the scCD22 CAR and mcCD22 CAR of the invention is shown in FIG. 2.

In a specific embodiment, the VH domain and VL domain of an immunoglobulin specific for CD22, preferably from m971, may be on the same strand of a multichain CAR, and the VH domain and VL domain of another immunoglobulin specific for CD22 may be on the same chain. another mcCAR circuit.

Down-regulation or mutation of target antigens is common in cancer cells, leading to the creation of antigen-free variants that have the ability to elude immunological surveillance. Thus, in order to neutralize the escape of tumors from immunological surveillance and to give the immune cell a higher specificity for the target, the CB22-specific CAR of the invention may contain another extracellular ligand-binding domain to simultaneously bind to other elements in the target, thereby increasing activation and immune cell function. In one embodiment, the extracellular ligand binding domains can be placed in tandem on the same transmembrane polypeptide and optionally separated by a linker.

In another embodiment of the invention, these different extracellular ligand-binding domains can be placed on different transmembrane polypeptides that form a multi-chain CAR. Another embodiment of the present invention is a population of CARs that contain various extracellular ligand-binding domains, one of which is specific for CD22.

In one embodiment, the extracellular binding domain specific for CD22 and the second extracellular binding domain are contained in the same sCAR.

In another embodiment of the invention, the extracellular binding domain specific for CD22 and the second extracellular binding domain are contained in the same mcCAR and belong to the same or to two different and non-covalently linked transmembrane domains of said mcCAR.

Any extracellular binding domain that specifically binds to an antigen associated (co-expressed - even transiently) with CD22 on abnormal cells such as CD34, CD10, CD79a, CD20, IgD, CD5, CD23, CD19 can be used as the second extracellular binding domain. , STAT5, CD3, CD30, BCMA, PAX5, CD19, CD20, CD30, glycosphingolipids, major histocompatibility complex (MCHC) molecule, Ig, CD3, CD34, CD79, preferably CD79a, CD 138, B7-1 (CD80), B7- 2 (CD86), major histocompatibility complex (MCHC) molecule, BCMA (CD269, TNFRSF 17) or FLT-3, Pax5.

The invention also relates to CD22 CAR related nucleic acids, recombinant CD22 CAR expression vectors, engineered TCR-KO cells, preferably T cells that contain at least another edited gene, which are endowed with CD22 CAR, populations of said TCR-KO cells. endowed with CD22 CAR, and pharmaceutical compositions containing CD22 CAR, protein, expression vector, engineered TCR-KO cells, CD52-KO expressing said CD22 CAR of the invention.

Objects of the invention are: CD22 CAR related nucleic acids, recombinant expression vectors, engineered TCR-KO cells containing at least another edited gene selected from a gene conferring resistance to hypoxia, a gene conferring resistance to alemtuzumab, a protease inhibitor, such as bortezomib, a gene conferring resistance to PNA (dCK) and endowed with CD22 CARs and the corresponding nucleic acid, populations of engineered TCR-KO cells that contain at least another edited gene listed below that are endowed with said CD22 CAR, and pharmaceutical compositions containing these objects of the invention, as a drug.

Polynucleotides. vectors:

The present invention relates to polynucleotides, vectors that encode the CD22 CAR described above, proposed in the invention.

The polynucleotide may be contained 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, for transfection of an insect host cell, or a plasmid or viral vector, such as a lentivirus or an adeno-associated virus intended for introduction into a mammalian host cell, preferably a human cell.

In a specific embodiment of the invention, different nucleotide sequences can be included in a single polynucleotide or vector that contains a nucleotide sequence encoding a ribosomal skip sequence, such as a sequence encoding a 2A peptide. Peptides 2A, which have been identified in the aphthovirus subgroup of the picornavirus group, result in a ribosomal "skip" from one codon to the next without forming 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).

Thus, the present invention provides the vector shown in FIG. 4, which encodes RQR8 and anti-CD22CAR linked to peptide 2A.

A “codon” is understood to mean three nucleotides per mRNA (or on the sense strand of a DNA molecule) that are translated by the ribosome into one amino acid residue. Thus, it is possible to synthesize two polypeptides from one contiguous open reading frame in mRNA if the polypeptides are separated by the 2A oligopeptide sequence present in the reading frame. Such ribosomal skip mechanisms are well known in the art and are known to be used in multiple vectors to express multiple proteins encoded by a single messenger RNA.

The vector for expressing the CD22 CAR of the invention in a cell is another object of the present invention. In a preferred embodiment of the invention, said vector provides transient expression of the CD22 CAR of the invention. In a more preferred embodiment of the invention, said vector provides constitutive and stable expression of the CD22 CAR of the invention by inserting a sequence that encodes said CD22 CAR into the cell's genome.

Expression of the CD22 CAR of the invention and/or survival of a cell expressing the CD22 CAR of the invention can be controlled using a gene under the control of an inducible promoter as described by R. Kuhn, F. Schwenk, M. Aguet, K Rajewsky. Inducible gene targeting in mice. Science, vol. 269, no. 5229, September 8, 1995, pp. 1427-1429 DOI: 10.1126/science.7660125, and in the references cited in the cited publication.

In one embodiment, a CD22 CAR is provided wherein the extracellular domain comprises at least two CD20 mimotopes having SEQ ID NO: 19 (CPYSNPSLC) located between the scfv m971 domains and the human CD8alpha hinge. Patent document WO 2016/120216A1 describes a method for obtaining these constructs and is incorporated herein by reference.

In one embodiment, the present invention provides a vector containing a sequence that encodes a CD22 CAR selected from SEQ ID NO: 22.

To direct a transmembrane polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, pre-pro sequence or pre-sequence) is placed in the polynucleotide sequence or in the vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleotide 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 typically placed in the 5' direction relative to the nucleotide sequence encoding the polypeptide of interest, although some secretory signal sequences can be placed elsewhere in the nucleotide sequence of interest (see, for example, Welch et al., US no. 5037743; Holland et al., US No. 5143830). In a preferred embodiment, the signal peptide comprises the amino acid sequences of SEQ ID NOs: 1 and 2.

In a more preferred embodiment, the CAR signal peptide of the invention comprises the amino acid sequence of SEQ ID NO: 1 from human C08alpha.

It should be apparent to those skilled in the art that, given the degeneracy of the genetic code, there may be significant variations in the sequence of these polynucleotide molecules. Preferably, the nucleotide sequences of the present invention have optimized codons for expression in mammalian cells, preferably for expression in human cells. Codon optimization refers to the replacement, in a sequence of interest, of codons that occur relatively infrequently in highly expressed genes in a specified species with codons that tend to occur frequently in highly expressed genes in a specified species, such codons code for the same amino acids. , which are the codons to be replaced.

Methods for constructing immune cells endowed with CD22 CAR:

The present invention provides a method for producing immune cells for immunotherapy, comprising ex vivo introduction into said immune cells of a polynucleotide or vector encoding one of the previously described CD22 CARs of the invention, preferably SEQ ID NO: 15.

In a preferred embodiment of the invention, these polynucleotides are included in the vector, given that it is stably expressed in immune cells.

In other embodiments of the invention, said method further comprises genetically modifying said cell to be suitable for adoptive transfer and/or for use in combination with a drug that affects the survival of said immune cell, primarily for transplantation (also referred to as allotransplantation or homotransplantation), alone or in combination with a drug to which the immune cell becomes resistant.

In the latter case, engineered cells can first be isolated from a donor and used for re-injection into the same donor in combination with a drug to which it is becoming resistant.

To edit a gene, which in the context of the present description means modification of a gene or inactivation of a gene, for example, mutation of a gene, deletion of a gene, insertion of a sequence into a gene, modification of the methylation of a specified gene (including the gene promoter), etc., apply the methods described in the document RA201670503, included in the present description by reference, and illustrated below in the examples.

Methods described in 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.2017.02.005, which is incorporated herein by reference, may also represent possible alternatives to the method used in the present invention to obtain TCR-KO CD22 CAR or cells endowed with CD22 CAR that are resistant to hypoxia as a result of overexpression of HIF -1a.

The method of the present invention relies on cellular homology-directed repair (HDR) mechanisms to "knock" the CD22 CAR into the TRAC gene (coding for the alpha subunit of the TCR), resulting in a more efficient product.

The HDR mechanism with an exogenous DNA sequence has been previously described for T cells using short oligonucleotides paired with CRISPR/Cas9. Other studies have demonstrated that adeno-associated virus (AAV) vectors can be used as a template in combination with site-specific nuclease Crispr/Cas9 or MegaTAL to achieve gene insertion via HDR. The present invention provides a novel method for combining adeno-associated virus (AAV) and TALEN vectors for insertion of a CAR into the TCR gene.

When creating UCART 22, the AAU6 vector can be used after genetic editing using TALEN specific for a gene, such as the TRAC gene (the gene encoding the alpha subunit, or TCR), or any gene described in PCT/EP2017/076798.

Since TALEN is DNA sequence specific and allows integration of the sequence into a gene, preferably into the TRAC gene, the present invention also provides engineered immune cells that contain a CAR coding sequence, preferably CD22 CAR as described above, located strictly in the region of the TRAC gene, determined by the applied TALEN. The sequence in the TRAC gene of this engineered immune cell is unique as a result of the specificity of TALEN.

The present invention provides an engineered immune cell containing the following sequence:

(YYY) _n -ZZZ-(XXX) _m ,

where n has a value from 1 to at least 10

and m has a value from 1 to 100, preferably m>100, and denotes the number of base pairs of the sequence to be integrated,

where ZZZ encodes a self-cleaving peptide, such as a 2A peptide, in frame with a TRAC coding sequence,

Y is A or T or G or C and flanks or contains a TALEN-targeted TRAC gene sequence that contains at least ttgtcccacagATATC, preferably ttgtcccacagATATCCAG, and (XXX) _n is A or T or G or C and part of the exogenous a sequence to be inserted into the TRAC gene, preferably a sequence that encodes a CAR, more preferably a sequence that encodes a CD22 CAR.

In one embodiment, the TRAC gene is deleted by deletion and the inserted gene is expressed under the control of the TRAC promoter.

Additional or alternative sequences such as IRES (internal ribosome entry site); can be "wedge" between the target TALEN and XXX.

In the present invention, the target of TALEN is SEQ ID NO: 21 and CAR CD22 has SEQ ID NO: 22.

In one embodiment, the sequence cleaved by said TALEN is AGAACCCTGACCCTG.

The AGAACCCTGACCCTG sequence may be at least partially conserved (see FIG. 9) in the engineered cell of the invention, depending on the insert sequence.

The present invention provides an engineered immune cell containing the TRAC gene comprising SEQ ID NO: 22.

The present invention provides an engineered immune cell which contains a sequence encoding a CD19 specific CAR or a sequence encoding a CD22 specific CAR inserted into the TRAC gene, preferably anywhere in the locus: AGAACCCTGACCCTG.

Thus, according to specific embodiments of the invention, the engineered immune cell of the invention contains two different CAR coding sequences and expresses these two different CARs. On the cell surface, the TCR level is not detectable.

Thus, according to specific embodiments of the invention, the engineered immune cell of the invention contains two different CAR coding sequences and expresses these two different CARs. On the cell surface, TCR levels and MHC class I molecules are not detectable.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD19 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD34 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD79a is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD79b is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD10 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for IgD is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD5 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD23 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD30 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for BCMA is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for FLT3 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD138 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD80 is inserted.

The engineered immune cell of the invention contains in the genome of the TRAC gene a sequence encoding CD22, preferably SEQ ID NO: 22, and another genomic sequence into which another exogenous gene encoding CAR specific for CD86 is inserted.

Another genomic sequence into which another exogenous gene encoding a second CAR is inserted may be any of the genes described in PCT/EP2017/076798 or WO 2017/069958A2.

Another exogenous gene encoding CAR may be inserted into the TRAC gene in frame with a self-cleaving peptide and with a CD22 CAR sequence, preferably SEQ ID NO:, or into another gene encoding a protein such as that provided in PCT/EP2017/076798 or selected from PD1 (Uniprot Q15116), CTLA4 (Uniprot P16410), PPP2CA (Uniprot P67775), PPP2CB (Uniprot P62714), PTPN6 (Uniprot P29350), PTPN22 (Uniprot Q9Y2R2), LAG3 (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) , FAS (Uniprot P25445), TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), SMAD2 (Uniprot Q15796), SMAD3 (Unipro t 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), GUCY1 A3 (Uniprot Q02108), GUCY1B2 (Uniprot Q8BXH3) and GUCY1B3 (Uniprot Q02153).

Preferably edited (KO) genes in UCART 22 are TNFRSF10B (Uniprot 014763), TNFRSF10A (Uniprot 000220), IL10RA (Uniprot Q13651), IL10RB (Uniprot Q08334, TGFBRII (Uniprot P37173), TGFRBRI (Uniprot Q15582), Q15 (Uniprot ), CTLA4 (Uniprot PI6410), LAG3 (Uniprot P18627), HAVCR2 (Uniprot Q8TDQ0), TIGIT (Uniprot Q495A1).

Preferably, the gene into which the second CAR is inserted is a genomic gene active during T-cell activation, selected from one of those listed in Table A:

or from the safe harbor loci described in PCT/EP2017/076798 or Table AA.

Another gene into which the second CAR can be inserted may be a gene encoding one of the proteins shown in Table B, selected from the following genes...

UCART 22 can be generated using an AAV vector, preferably an AAV6 vector, and even more preferably an AAV6/2 vector for insertion of the CD22 CAR or other gene described herein. AAV vector(s) can be used after genetic editing of a gene, such as the TRAC gene (the gene encoding the alpha subunit, or TCR), or any gene described in PCT/EP 2017/076798, using a specific endonuclease.

Adoptive cell transfer is the transfer of cells into a patient's body. The cells may originate from the patient's own body and then modified before being transferred back (sygenetic transfer), or they may be obtained from another individual. Cells are most commonly obtained from the immune system to carry improved immune functionality and characteristics when the cells are returned to the patient's body. Transfer of autologous cells or cells from a patient minimizes graft-versus-host disease (GVHD) or tissue or organ rejection. Similarly, the transfer of TCR-deficient T cells expressing the CD22 CAR minimizes GVHD. The transfer of TCR-deficient, MHC-deficient T cells expressing the CD22 CAR minimizes GVHD and HVGD.

In one of the embodiments of the invention, the step of genetic modification (construction, engineering) of said immune cell is performed prior to the step of introducing polynucleotides or vectors encoding one of the CD22 CARs of the invention into said cells. According to a first aspect of the invention, an immune cell can be made less allogeneic, for example, by inactivating at least one gene expressing one or more components of the T cell receptor (TCR) specified in WO 2013/176915, which can be combined with inactivating a gene that encodes or regulates the expression of an HLA such as the β2m gene as described in WO 2008/102199 or WO 2015/136001 or WO 2016/201047, all of which are incorporated herein by reference. Thus, the risk of graft-versus-host syndrome and the risk of transplant rejection are significantly reduced.

Thus, according to another aspect of the invention, the immune cells of the invention can be further genetically engineered to increase the resistance of the engineered immune cells to an immunosuppressive drug or chemotherapy treatment that is used as a standard of care in the treatment of CD22-positive malignant cells according to the approach described in WO 2015/75195, which is incorporated herein by reference.

Campas (alemtuzumab) resistance

According to one preferred embodiment of the invention, genes that can be inactivated to confer drug resistance on T cells are the glucocorticoid receptor (GR) and CD52 genes. The genes are inactivated to render the cells resistant to said treatments and to give them a competitive advantage over the patient's own T cells, which are not endowed with specific CD22 CARs.

Inactivation of the CD52 and TRAC genes in the engineered immune cell of the invention is carried out using TALE nuclease or the CRISPR CAS9 system.

According to one more preferred embodiment of the invention, the gene that can be inactivated to confer drug resistance in T cells is CD52 in TCR-KO in immune T cells that are endowed with the CD22 CAR.

According to one preferred embodiment of the invention, the gene that can be inactivated to confer drug resistance on T cells is the glucocorticoid receptor (GR) gene.

Expression of the CD3 gene can also be down-regulated or down-regulated to confer resistance to teplizumab, which is another immunosuppressive drug. The expression of HPRT can also be suppressed or reduced, according to the invention, to confer resistance to 6-thioguanine, a cytostatic agent widely used in chemotherapy, primarily for the treatment of acute lymphoblastic leukemia.

Resistance to purine nucleotide analogs by deletion of the human deoxycytidine kinase (dCK) gene

According to one preferred embodiment of the invention, the gene that can be inactivated to confer drug resistance on T cells is the deoxycytidine kinase (dCK) gene. This enzyme is required for the phosphorylation of the deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG), and deoxyadenosine (dA). Purine nucleotide analogs (PNA) are metabolized by dCK to form PNA mono-, di-, and triphosphate. These triphosphate forms, and above all clofarabine triphosphate, compete with ATP in DNA synthesis, act as a proapoptotic agent, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in the production of trinucleotides.

Inactivation of the dCK gene in the engineered immune cell of the invention is mediated by TALE nuclease or the CRISPR CAS9 system. To achieve this goal, several pairs of TALE-nucleases for dCK were developed, assembled at the polynucleotide level, and their correctness was confirmed by sequencing. Examples of pairs of TALE-nucleases that can be used according to the invention are described in PCT/EP 2014/075317.

This inactivation of dCK in engineered immune cells of the invention confers resistance to purine nucleoside analogs (PNAs) such as clofarabine and fludarabine.

In another preferred embodiment of the invention, inactivation of dCK in engineered immune cells of the invention is combined with inactivation of TRAC genes to produce said double knockout (KO) cells (TCR- or TRAC-KO and dCK-KO) that are resistant to a drug such as clofarabine and are less allogenic.

In another preferred embodiment of the invention, inactivation of CD52 in engineered immune cells of the invention is combined with inactivation of the TRAC gene to produce said double knockout (KO) cells (TCR- or TRAC-KO and CD52-KO) that are resistant to a drug such as campas (alemtuzumab) and are less allogenic.

These dual properties are most valuable for therapeutic purposes, allowing the use of "off-the-shelf" allogeneic cells (UCART 22) for immunotherapy in combination with chemotherapy to treat cancer patients who need it. Said double KO inactivation of dCK/TRAC or CD52/TRAC can be performed simultaneously or sequentially. One example of a pair of TALE-nuclease dCK/TRAC, which provides success according to the invention, is described in PCT/EP 2014/075317, while, in particular, the target sequences are in 2 loci (dCK and TRAC). PCT/EP 2014/075317 is incorporated herein by reference in its entirety.

The present invention provides primary T cells expressing the CD22 CAR having SEQ ID NO: 15, in which the CD52 and TRAC genes are inactivated by deletion for their use in the treatment of CLL, ALL, preferably their aggressive relapsing refractory forms, optionally in patients with lymphoid depletion, more preferably recurrent refractory forms of B-ALL.

According to another aspect of the invention, immune cells can be subjected to additional manipulations to make them more active or to limit their depletion by inactivating genes that encode proteins that act as "immune checkpoints" that are regulators of T cell activation, such as PDCD1 or CTLA -four. Examples of genes that can be down-regulated or down-regulated are shown in Table 9.

The present invention also provides primary T cells expressing the CD22 CAR having SEQ ID NO: 15 in which the CD52, TRAC and dCK genes have been deleted by deletion.

Another embodiment of the present invention are also primary T cells that express CD22 CARs that have been rendered resistant to hypoxia.

The present invention also provides primary T cells expressing the CD22 CAR having SEQ ID NO: 15, in which the CD52, TRAC and dCK genes are deleted and HIF-1a sequences are inserted to render the cells resistant to hypoxia.

Engineered cells resistant to hypoxia

In specific embodiments, the expression and cytolytic activity of the CD22 CAR T cell of the invention is maintained, or the expression of CD22 CAR T cells is induced and activity is maintained under conditions of low oxygen content (hypoxia), (normal oxygen level of 20% O ₂ vs. with a level of 1-5% O ₂ ), and the specified cell still targets and destroys tumor cells when it enters the tissue.

Examples of hypoxia-induced CAR in T cells are described (in WO 2013/123061 or Juillerat A. et al. An oxygen sensitive self-decision making engineered CAR T-cell, Sci. Rep. 7, 39833; doi: 10.1038/srep39833, 2017, both documents incorporated herein by reference): a synthetic OxiTF-specific promoter has been designed to control the expression of the CD22 CAR. OxiTF was designed to activate a synthetic genetic element encoding the CD22 CAR. Upon contact with the tumor (creating hypoxia), engineered T cells can "detect" a decrease in oxygen levels (compared to mean O ₂ levels in the blood) and trigger CD22 CAR expression. Cell surface exposed CD22 CAR can recognize tumor antigen under hypoxic conditions, which ultimately triggers T cell activation and proliferation via the activation and costimulatory domains present in said CD22 CAR. As a result, cells expressing the tumor antigen are lysed by UCART 22 of the invention.

According to the present invention, immune cells can also be engineered to remain effective under conditions of low O ₂ levels (low oxygen is 1-5%) by overexpressing at least one, preferably all, of the following factors: Oct3, Oct4, Sox2, Klf4 and c -Mus, or by editing the HIF-1a factor.

In the present invention, an engineered cell expressing oxygen-sensitive single-stranded CD22 CAR and an engineered cell expressing hypoxia-resistant CD22 CAR were created and successfully tested.

Because CD22 is expressed primarily in CD22-expressing B-cell malignancies, which are "fluid tumors" and therefore is not expected to produce hypoxia, unlike solid tumors, CD22 CAR-expressing engineered immune cells are not expected to be resistant to hypoxia, will be more effective than engineered immune cells expressing CD22 CAR, which are not resistant to hypoxia, against B-ALL from the patient's body. In fact, the UCART 22 of the invention, when reaching clustered nested cancer cells or homing into tissues, can lyse these cells.

Other genes that can be edited in UCART 22, proposed in the present invention, is presented below in table 5:

The present invention provides isolated engineered immune T cells expressing the CD22 CAR having SEQ ID NO: 15, in which the dCK and/or CD52 and TRAC genes have been edited, i. inactivated by deletion, intended for use in the treatment of CLL, ALL, preferably their aggressive recurrent refractory forms, in patients with lymphoid depletion.

The present invention provides isolated engineered immune T cells expressing the CD22 CAR having SEQ ID NO: 15 in which the b2m and TRAC genes have been edited, i. inactivated by deletion, intended for use in the treatment of CLL, ALL, preferably their aggressive recurrent refractory forms, in patients with lymphoid depletion.

The present invention provides isolated engineered immune T cells expressing the CD22 CAR having SEQ ID NO: 15, in which the TRAC and IL-10R genes have been edited, i. inactivated by deletion, intended for use in the treatment of CLL, ALL, preferably their aggressive recurrent refractory forms, in patients with lymphoid depletion.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (resulting in impaired expression of TCR on the cell surface), deficient in the B2M gene (as a result, expression of MHC class I molecules on the cell surface is impaired). ), and in addition to this with the KO gene of the TGFbeta receptor.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (resulting in impaired expression of TCR on the cell surface), deficient in the B2M gene (as a result, expression of MHC class I molecules on the cell surface is impaired). ), and in addition to this with the KO gene of the IL-10 receptor.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (resulting in impaired expression of TCR on the cell surface), deficient in the B2M gene (as a result, expression of MHC class I molecules on the cell surface is impaired). ), and in addition to this with the AHR KO gene.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (resulting in impaired expression of TCR on the cell surface), deficient in the B2M gene (as a result, expression of MHC class I molecules on the cell surface is impaired). ), and in addition to this with the PD1 KO gene.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (resulting in impaired expression of TCR on the cell surface), deficient in the B2M gene (as a result, expression of MHC class I molecules on the cell surface is impaired). ), and in addition to this with the LAG-3 KO gene.

The present invention provides engineered immune T cells expressing CD22 CAR (UCART 22), deficient in the TCR alpha gene (as a result, expression of TCR on the cell surface is impaired), deficient in the B2M gene (as a result, expression of class I GTC molecules on the cell surface is impaired). ), and in addition to this with the TIM-3 KO gene.

The present invention provides engineered immune T cells expressing the CD22 CAR having SEQ ID NO: 15, in which the CD52 and TRAC genes are inactivated by deletion, for use in the treatment of relapsed refractory forms of B-ALL in patients with lymphoid depletion.

In a preferred embodiment of the invention, the method of creating immune cells includes the introduction into these T-cells of polynucleotides, primarily mRNA, encoding a specific, rarely cleaving endonuclease, which has the ability to selectively inactivate the above genes by DNA cleavage.

In a more preferred embodiment of the invention, said rarely cleaving endonucleases are TALE nucleases or Cas9 endonuclease. To date, TAL nucleases have proven to have higher specificity and cleavage efficiency than other types of low-cleavage endonucleases, so they are chosen as endonucleases for producing engineered immune cells in large-scale, constant-cycle production.

Shipping Methods

The various methods described above involve introducing the CD22 CAR of the invention into a cell. As non-limiting examples, said CD22 CAR can be introduced as a transgene encoded by a single plasmid vector of the invention. Said plasmid vector encoding the CD22 CAR of the invention may also contain a selection marker for identification and/or selection of cells into which said vector is delivered.

The method for introducing the CD22 CAR of the invention and subsequently expressing it in an isolated immune cell is known, for example, described in WO 2013/126720 or WO 2015/121454, which are incorporated herein by reference in their entirety.

Polypeptides corresponding to the CD22 CAR of the invention can be synthesized in situ in a cell by introducing polynucleotides encoding said polypeptides into the cell. Alternatively, these polypeptides can be obtained outside the cell and then introduced into it. Techniques for introducing a polynucleotide construct into cells are known in the art and examples include, but are not limited to, stable transformation methods in which the polynucleotide construct is integrated into the genome of the cell, transient transformation methods in which the polynucleotide construct is not integrated into the genome of the cell, and methods based on the use of viruses. These polynucleotides can be introduced into a cell, for example, using recombinant viral vectors (eg, retroviruses such as lentiviruses, adenoviruses, adeno-associated virus), liposomes, and the like. For example, transient transformation methods include microinjection, electroporation, or particle bombardment. These polynucleotides can be included in vectors, more specifically plasmids or viruses, for expression in cells.

In a preferred embodiment of the invention, said CD22 CAR-encoding polynucleotide is inserted into an AAV6-based vector and introduced into a specific gene. Methods for obtaining a vector that provides for the introduction of the CD22 CAR of the invention and subsequent expression in an isolated T cell are known and described, for example, in WO 2013/126720, which is incorporated herein by reference.

Engineered immune cells (UCART)

An engineered immune cell endowed with the CD22 CAR (UCART 22) is another aspect of the present invention.

Preferably said immune cell is an isolated immune cell, more preferably an isolated immune T cell, more preferably an isolated primary immune T cell.

UCART 22 is proposed as a drug, so UCART 22 is proposed as a drug in a therapeutically effective amount.

"Primary immune cell" according to the invention means a cell derived from a tissue, such as a blood sample, or from peripheral blood mononuclear cells (PBMC), and which can be cultured using several passages, freezing at the end before use, said primary immune cell has a limited ability to divide (Raulf-Heimsoth M. T cell - primary culture from peripheral blood. Methods Mol Med. 138, 2008, cc. 17-30. doi: 10.1007/978-1-59745-366-0) no comparison with the transformed or a cancer cell.

The immune cell according to the invention is preferably an immune T or NK cell. Thus, the engineered immune cell of the invention, isolated from a blood sample, is a primary cell and is derived from an immune T cell selected from inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, or helper T lymphocytes, natural killer cells from the group of T cells, preferably from cytotoxic T lymphocytes, further designed.

Engineering involves modifying primary immune cells to contain CD22 CAR and at least one edited gene, preferably said cells are modified to contain CD22 CAR and neither express TCR nor die in the presence of purine nucleotide analogues.

In other words, the engineered immune cells are CD22 CAR-expressing TCR-KO isolated immune T cells containing at least one other edited gene.

In a specific embodiment, the design involves modifying primary immune cells to contain CD22 CAR, preferably said cells are modified to contain CD22 CAR and not die in the presence of purine nucleotide analogs (1-5 µmol/l) or in the presence of alemtuzumab (50 μg/ml) (Valton et al., Molecular Therapy vol. 23, no. 9, September 2015, pp. 1507-1518).

Preferably, said T cell is endowed with a CD22 CAR having SEQ ID NO: 15.

More preferably, said T cell is endowed with a CD22 CAR having SEQ ID NO: 15 and contains at least one sequence of SEQ ID NO: 22.

More preferably, said T cell is endowed with a CD22 CAR having SEQ ID NO: 15 and contains at least one sequence of SEQ ID NO: 22 and at least a portion of the sequence.

More preferably, said T cell is endowed with a CD22 CAR having SEQ ID NO: 18, which includes at least one sequence of SEQ ID NO: 20.

The present invention provides a primary immune T cell expressing the CD22 CAR of the invention and having CTL and/or degranulation activity against a CD22 expressing cell.

The present invention also provides a primary CD22-expressing T cell The CAR of the invention for lysis of a CD22-expressing cell, especially a CD22-expressing cancer cell.

Preferably, cells targeted by a T cell that is endowed with a CD22 CAR having SEQ ID NO: 16 of the invention are effective in treating relapsed/refractory/aggressive ALL or CLL, preferably.

The present invention also relates to isolated cells or cell lines, which can be obtained by this method of constructing cells. In particular, said isolated cell contains at least one inventive CD22 CAR as defined above. In another embodiment of the invention, said isolated cell contains a population of CARs, each of which contains different extracellular ligand-binding domains. In particular, said isolated cell contains an exogenous polynucleotide sequence encoding CAR. The genetically modified immune cells of the present invention are active and can proliferate independently of antigen binding mechanisms.

Also within the scope of the present invention is an isolated immune cell, preferably an isolated immune T cell (T cell), more preferably an engineered isolated immune T cell obtained by one of the previously described methods. The specified immune cell is a cell derived from a hematopoietic cell, functionally involved in the initiation and/or implementation of the innate or adaptive immune response. Said immune cell according to the present invention can be obtained from a stem cell. The stem cells can be mature stem cells, non-human embryonic stem cells, more preferably non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human cells are CD34 ⁺ cells. Said isolated cell may also be a dendritic cell, a dendritic killer cell, a mast cell, an 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 of the invention, the specified cell may be derived from the group consisting of CD4 ⁺ -T-lymphocytes and CD8 ⁺ -T-lymphocytes.

Prior to propagation and genetic modification of the cells of the invention, a source of cells can be obtained from the body of an individual by various methods, without limiting the scope of the invention. Cells can be obtained from numerous sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected area, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments of the present invention, any number of T cell lines available and known to those skilled in the art can be used. In another embodiment of the invention, said cell is preferably obtained from a healthy donor, a patient with a diagnosed cancer, or a patient with a diagnosed infection. In another embodiment of the invention, the specified cell is part of a mixed population of cells with different phenotypic characteristics. Also within the scope of the present invention is a cell line derived from a T cell constructed using the described method. Under the scope of the present invention fall modified cells that are resistant to immunosuppressive treatment, and which can be obtained using the method described above.

As a preferred embodiment of the invention, the present invention provides T cells or a population of T cells endowed with the CD22 CAR of the invention described above, which do not express a functional TCR and which are reactive against CD22-positive cells, intended for adoptive transfer to the patient's body.

As a preferred embodiment of the invention, the present invention provides T cells or a population of T cells endowed with the CD22 CAR of the invention as described above, which do not express a functional TCR and are rendered resistant to chemotherapy, especially purine nucleotide analogs (PNA) .

According to a preferred embodiment of the invention, immune cells endowed with CD22 CAR are engineered to become resistant to chemotherapeutic drugs, especially purine nucleotide analogs (PNAs), allowing them to be used in cancer treatment in combination with adoptive immunotherapy and chemotherapy.

Purine nucleotide analogs are included in chemotherapeutic compositions for many cancer treatments. They are used as the standard of care for leukemia or lymphoma. The most widely used PNAs are clofarabine, fludarabine and cytarabine alone or in combination. PNAs are metabolized by enzymes with deoxycytidine kinase (dCK) activity [EC 2.7.1.74] into PNA mono-, di- and triphosphate. Their triphosphate forms, primarily clofarabine triphosphate, compete with ATP for DNA synthesis, act as a pro-apoptotic agent, and are strong inhibitors of ribonucleotide reductase (RNR), which is involved in the formation of trinucleotides.

Thus, the present invention includes a method for ex vivo production of immune cells, preferably primary T cells, that do not express TCR, are resistant to a purine analogue drug, and that can target CD22-positive cancer cells.

The method of obtaining UCART 22 proposed in the invention may also be the method described in WO 2013/176915 or WO 2014/191128, which are fully incorporated into the present description by reference.

The method for obtaining UCART 22 includes the following steps:

(a) obtaining an immune cell from a donor, preferably an isolated T cell or an isolated population of T cells,

(b) introducing into said immune cell (preferably by transfection or transduction) a nucleotide sequence encoding a rarely cleaving endonuclease specifically targeting

- a gene that expresses an enzyme having deoxycytidine kinase activity (dcK - EC 2.7.1.74), especially the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and/or

- a gene that encodes one of the alpha and/or beta TCR subunits, preferably alpha, and/or

- the gene that codes for human CD52,

(c) expression of said endonuclease in said immune cells to produce targeted inactivation of said gene(s);

(d) propagating the engineered immune cells obtained in step c), optionally in the presence of a purine analogue drug;

(e) introducing into said immune cell a CD22 CAR according to the invention, preferably having SEQ ID NO: 15.

The method for obtaining UCART 22 includes the following steps:

(a) obtaining an immune cell from a donor, preferably an isolated T cell or an isolated population of T cells,

(b) introducing into said immune cell (preferably by transfection or transduction) a nucleotide sequence encoding a rarely cleaving endonuclease specifically targeting

- a gene that expresses an enzyme having deoxycytidine kinase activity (dcK - EC 2.7.1.74), especially the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and/or

- a gene that encodes one of the alpha and/or beta TCR subunits, preferably alpha, and/or

- the gene that codes for human beta-2 microglobulin (B2M),

(c) expression of said endonuclease in said immune cells to produce targeted inactivation of said gene(s);

(d) propagating the engineered immune cells obtained in step c), optionally in the presence of a purine analogue drug;

(e) introducing into said immune cell a CAR of the invention, preferably CD22 CAR alone or in combination with CD19 CAR, even more preferably CD22 CAR which has SEQ ID NO: 15, alone or in combination with CD19 CAR.

In a preferred embodiment, the present invention includes a method for ex vivo production of immune cells, preferably primary T cells, that do not express TCR, are resistant to a purine analogue drug, and that can target CD22 positive cancer cells.

This method includes the following steps:

• (a) obtaining an immune cell from a donor, preferably an isolated T cell (or an isolated population of T cells),

• (b) introducing into said immune cell a CD22 CAR according to the invention, preferably having a SEQ ID selected from SEQ ID NO: 15,

• (c) propagation of the engineered immune cells obtained in step b),

(d) introducing into said immune cell (by transfection or transduction) a nucleotide sequence that encodes a rarely cleaving endonuclease specifically targeting

a gene that expresses an enzyme having deoxycytidine kinase activity (dcK EC 2.7.1.74), especially the human deoxycytidine kinase gene (NCBI Gene ID: 1633), and/or a gene that encodes one of the alpha and/or beta TCR subunits,

(e) expression of said endonuclease in said immune cells to produce targeted inactivation of said gene(s);

(e) propagating the engineered immune cells obtained in step e), optionally in the presence of a purine analogue drug.

A method for ex vivo production of immune cells, preferably primary T cells, that do not express TCR, are resistant to a purine analogue drug, and that can target CD22-positive cancer cells, optionally includes another step of introducing into said immune cell (by transfection or transduction) a nucleotide sequence that encodes a rarely cleaving endonuclease specifically targeting one of the genes listed in Table 9, preferably PD-1, CD279 and more preferably PDCD1 (PD-1, CD279) or CTLA4 (CD152).

The present invention has made progress in generating CD22 CAR-expressing primary T cells resistant to purine nucleotide analogs (dCK-KO), more preferably clofarabine and/or fludarabine, by mediating the inactivation (deletion) of dcK gene expression in these cells, in particular using nucleases, primarily TAL nucleases.

Transfection of T cells with mRNA that encodes a specific TAL nuclease directed against dCK genes, preferably by electroporation as described in WO 2013/176915, induced marked drug resistance while maintaining T cell cytotoxic activity against CD22-bearing cells .

This application also describes TCR-KO, CD22 CAR (preferably SEQ ID NO: 15) primary T cells in which the expression of deoxycytidine kinase is suppressed or inactivated (dCK-KO), intended for the treatment of leukemia or lymphoma, preferably in their aggressive resistant relapsing form, more preferably B-ALL.

According to a preferred embodiment of the invention, immune cells endowed with the CD22 CAR are engineered to be resistant to chemotherapeutic drugs, in particular alemtuzumab (Campas), allowing them to be used in cancer treatment in combination with adoptive immunotherapy and chemotherapy.

Alemtuzumab is used in many cancer treatments. It is used as a standard of care for leukemia or lymphoma, primarily for the treatment of chronic lymphocytic leukemia (CLL), cutaneous T-cell lymphoma (CTCL) and T-cell lymphoma. It is known under the brand names Campath (campas), MabCampath and Campath-1H. It is used in some conditioning regimens in bone marrow transplantation, kidney transplantation, and islet cell transplantation.

It is a monoclonal antibody that binds to CD52, a protein that is present on the surface of mature lymphocytes but not on the stem cells that form lymphocytes. After treatment with alemtuzumab, these CD52-bearing lymphocytes become targets for destruction.

Alemtuzumab is also used as second-line therapy for CLL. It is approved for use by the US Food and Drug Administration in CLL patients who have been treated with alkylating agents and who have not responded to fludarabine therapy.

Thus, the present invention includes a method for ex vivo production of UCART 22, not expressing, as indicated above, TCR, which are resistant to alemtuzumab.

The method for obtaining UCART 22 CD52-KO includes the following steps:

(a) obtaining an immune cell from a donor, preferably an isolated T cell or an isolated population of T cells,

(b) introducing into said immune cell (preferably by transfection or transduction) a nucleotide sequence encoding a rarely cleaving endonuclease specifically targeting

- a gene that encodes one of the alpha and/or beta TCR subunits, preferably alpha, and/or

- the gene that codes for CD52,

(c) expression of said endonuclease in said immune cells to produce targeted inactivation of said gene(s);

(d) propagating the engineered immune cells obtained in step c), optionally in the presence of alemtuzumab;

(e) introducing into said immune cell a CD22 CAR according to the invention, preferably having SEQ ID NO: 15.

In a preferred embodiment of the invention, the present invention includes a method for ex vivo production of immune cells, preferably primary T-cells that do not express TCR, that are resistant to alemtuzumab and that can target CD22-positive cancer cells. This method includes the following steps:

• (a) obtaining an immune cell from a donor, preferably an isolated T cell (or an isolated population of T cells),

• (b) introducing into said immune cell a CD22 CAR according to the invention, preferably having SEQ ID NO: 15,

• (c) propagation of the engineered immune cells obtained in step b),

(d) introducing into said immune cell (by transfection or transduction) a nucleotide sequence that encodes a rarely cleaving endonuclease specifically targeting

a gene that expresses CD52 and a gene that codes for one of the alpha or beta TCR subunits,

(e) expression of said endonuclease in said immune cells to produce targeted inactivation of said gene(s);

(e) propagating the engineered immune cells obtained in step e), optionally in the presence of a purine analogue drug.

In one of the embodiments of the invention, the method of obtaining UCART 22 includes the following steps:

(a) obtaining an immune cell from a donor, preferably an isolated T cell or an isolated population of T cells,

(b) introduction into said immune cell (preferably by transfection or transduction)

a nucleotide sequence encoding a rarely cleaving endonuclease specifically targeting a gene that encodes one of the alpha and/or beta TCR subunits, preferably alpha, and

- a nucleic acid encoding a sequence to be inserted, preferably encoding HIF-1alpha,

(c) expressing said endonuclease in said immune cells to produce a targeted insertion of said sequence to be inserted,

(d) propagating the engineered immune cells obtained in step c), optionally in the presence of a low concentration of O ₂ (5% O ₂ , preferably 1% O ₂ );

(e) introducing into said immune cell a CD22 CAR according to the invention, preferably having SEQ ID NO: 15.

In a preferred embodiment of the invention, the present invention includes a method for ex vivo production of immune cells, preferably primary T-cells that do not express TCR, that are resistant to hypoxia and that can target CD22-positive malignant cells. This method includes the following steps:

• (a) obtaining an immune cell from a donor, preferably an isolated T cell (or an isolated population of T cells),

• (b) introducing into said immune cell a CD22 CAR according to the invention, preferably having SEQ ID NO: 15,

• (c) propagation of the engineered immune cells obtained in step b),

(d) introduction into said immune cell (by transfection or transduction)

- a nucleotide sequence that encodes a rarely cleaving endonuclease specifically targeting a gene that encodes one of the alpha and/or beta TCR subunits, preferably alpha, and

a nucleic acid encoding a sequence to be inserted that encodes HIF-1 alpha,

(e) expression of said endonuclease in said immune cells to produce a targeted insertion of said gene(s);

(e) propagating the engineered immune cells obtained in step e), optionally in the presence of a low concentration of O ₂ (5% O ₂ , preferably 1% O ₂ ).

A method for ex vivo production of immune cells, preferably primary T cells, that do not express TCR, are resistant to either a purine analog drug, alemtuzumab, or hypoxia, and that can target CD22-positive cancer cells, optionally comprising another step of introducing into said immune cell (by transfection or transduction) a nucleotide sequence that encodes a rarely cleaving endonuclease specifically targeting one of the genes listed in Table 9, preferably PD-1, CD279 and more preferably PDCD1 (PD-1, CD279) and CTLA4 (CD 152).

The present invention has made progress in generating CD22 CAR-expressing primary T cells resistant to purine nucleotide analogs (dCK-KO), more preferably clofarabine and/or fludarabine, by mediating the inactivation (deletion) of dcK gene expression in these cells, in particular using nucleases, primarily TAL nucleases.

Transfection of T cells with mRNA that encodes a specific TAL nuclease directed against dCK genes, preferably by electroporation as described in WO 2013/176915, induced marked drug resistance while maintaining T cell cytotoxic activity against CD22-bearing cells .

The same method was used for the deletion of human CD52 using specific TALEN, as described in the present invention.

The present application also provides TCR-KO endowed with CD22 CAR (preferably SEQ ID NO: 15) primary T cells in which deoxycytidine kinase expression is down-regulated or inactivated (dCK-KO) for the treatment of leukemia or lymphoma, preferably in their aggressive resistant relapsing form, more preferably B-ALL.

Such cells are "universal" T cells (or UCARTs).

One embodiment of the invention presented herein is CD22 CAR-expressing (preferably SEQ ID NO: 15) primary T cells in which deoxycytidine kinase expression is down-regulated or inactivated (dCK-KO) for the treatment of leukemia or lymphoma, preferably in their aggressive stable relapsing form; more preferably recurrent B-ALL after autologous transfer.

One embodiment of the invention presented herein is CD22 CAR expressing (preferably SEQ ID NO: 15) primary T cells in which CD52 expression is suppressed or inactivated (CD52-KO) for the treatment of leukemia or lymphoma, preferably in their aggressive stable relapsing form; more preferably recurrent B-ALL after autologous transfer.

One embodiment of the invention presented herein is CD22 CAR expressing (preferably SEQ ID NO: 15) primary T cells in which HIF-1alpha expression is upregulated by insertion of a coding sequence into a TRAC sequence without "turning off" the TCR, intended for the treatment of leukemia or lymphoma, preferably in their aggressive resistant relapsing form; more preferably recurrent B-ALL after autologous transfer.

One embodiment of the invention presented herein is CD22 CAR-expressing (preferably SEQ ID NO: 15) primary T cells in which deoxycytidine kinase and CD52 expression is down-regulated or inactivated (dCK- and CD52-KO) for the treatment of leukemia. or lymphomas, preferably in their aggressive persistent relapsing form; more preferably recurrent B-ALL after autologous transfer.

One embodiment of the invention presented herein is CD22 CAR expressing (preferably SEQ ID NO: 15) primary T cells in which deoxycytidine kinase and CD52 expression is suppressed or inactivated (dCK- and CD52-KO) and HIF-1alpha expression. elevated by insertion of a coding sequence into a TRAC sequence without "turning off" TCRs intended for the treatment of leukemia or lymphoma, preferably in their aggressive, persistent relapsing form; more preferably recurrent B-ALL after autologous transfer.

A method for ex vivo production of immune cells, preferably primary T cells, that do not express TCR are resistant to a purine analog drug and/or alemtuzumab and/or to hypoxia that can target CD22-positive malignant cells, optionally includes another step of introducing into said immune cell (by transfection or transduction) a nucleotide sequence that encodes a low-cleavage endonuclease specifically targeting one of the genes listed in Table 9, preferably PD-1, CD279 and more preferably PDCD1 (PD-1, CD279) or CTLA4 (CD152).

The present invention has made progress in generating CD22 CAR-expressing primary T cells resistant to purine nucleotide analogs (dCK-KO), more preferably clofarabine and/or fludarabine, by mediating the inactivation (deletion) of dcK gene expression in these cells, in particular using nucleases, primarily TAL nucleases.

The present invention has made progress in generating CD22 CAR-expressing hypoxia-resistant primary T cells by targeted insertion of the HIF-1 alpha gene into said cells, in particular using nucleases, especially TAL nucleases.

Transfection of T cells with mRNA that encodes a specific TAL nuclease directed against dCK genes, preferably by electroporation as described in WO 2013/176915, induced marked drug resistance while maintaining T cell cytotoxic activity against CD22-bearing cells .

The present application also provides TCR-KOs endowed with CD22 CAR (preferably SEQ ID NO: 15) hypoxia resistant primary T cells for the treatment of leukemia or lymphoma, preferably in their aggressive resistant relapsing form, more preferably B-ALL.

Activation and reproduction of T cells

Both before and after the implementation of genetic modification of T cells, even if the activation and proliferation of genetically modified immune cells proposed in the present invention does not depend on antigen binding mechanisms, immune cells, especially T cells, proposed in the present invention, can be subjected to additional activation and propagated using the methods described in General, for example, in US No. 6352694; 6534055; 6905680; 6692964; 5858358; 6887466; 6905681; 7144575; 7067318; 7172869; 7232566; 7175843; 5883223; 6905874; 6797514; 6,867,041 and US Patent Application Publication No. 2006/0121005. T cells can be propagated in vitro or in vivo.

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

For example, but not limited to, T cell populations can be stimulated in vitro 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 ( for example, bryostatin) in combination with a calcium ionophore. To costimulate a helper molecule on the surface of T cells, a ligand is used that binds to the helper molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions that stimulate T cell proliferation. Conditions suitable for culturing T cells include appropriate media (e.g., minimal maintenance medium or RPMI 1640 or X-vivo 5 medium (Lonza)), which may contain factors necessary to ensure 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 cell growth supplement known to the person skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. The media can be RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 1 and X-Vivo 20, Optimizer supplemented with amino acids, sodium pyruvate and vitamins and may be serum-free or may contain as supplements in a certain amount of serum (or plasma) or a certain set of hormones, and/or cytokine(s) in an amount sufficient for the growth and reproduction of T cells.

Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures, not in cell cultures that are intended to be administered by infusion to an individual. The target cells are maintained under conditions necessary to support growth, eg at an appropriate temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ). Cells that have been stimulated for different periods of time may have different characteristics.

In another specific embodiment of the invention, these cells can be propagated by co-culturing with tissue or cells. Said cells can also be propagated in vivo, for example, in the blood of an individual after administration of said cell to the individual.

pharmaceutical composition

Another aspect of the present invention is a pharmaceutical composition comprising an engineered (TCR- and dCK-KO) or (TRAC- and CD52-KO) immune T cell that expresses the CD22 CAR of the invention (preferably SEQ ID NO: 15), and pharmaceutically acceptable excipient.

Another embodiment of the present invention is a composition that contains UCART 22 of the present invention (as defined in any of the above embodiments of the invention), in combination with a compound from the bryostatin family, preferably bryostatin-1.

Bryostatins are a group of macrolide lactones from the bryozoans Bugula neritina. The structure of bryostatin 1 was established in the 1980s. To date, 20 different bryostatins have been isolated; as well as some analogues of bryostatin, designated as "bryologs". Briostatins are potent protein kinase C modulators (Wender, Paul A., Jeremy L. Baryza, Chad E. Bennett, F. Christopher Bi, Stacey E. Brenner, Michael O. Clarke, Joshua C. Horan, Cindy Kan, Emmanuel Lacote, Blaise Lippa, Peter G. Nell and d Tim M. Turner, The Practical Synthesis of a Novel and Highly Potent Analogue of Bryostatin, Journal of the American Chemical Society 124 (46), 2002, pp. 13648-13649 DOI: 10.1021/ja027509+) .

Examples of bryostatins suitable for combination with UCART 22 of the invention and methods for preparing these compounds are described in WO 2001/040214A1 or EP 2737904A2, WO 1997/034598, which are incorporated herein by reference.

An example of the dose of bryostatin-1 that can be used in combination with UCART 22, proposed in the present invention, previously described in Varterasian M.L. 1, Mohammad R.M., Shurafa M.S., Hulburd K., Pemberton P.A., Rodriguez D.H., Spadoni V., Eilender D.S., Murgo A., Wall N., Dan M., Al-Katib A.M. Phase II trial of bryostatin 1 in patients with relapsed low-grade non-Hodgkin's lymphoma and chronic lymphocytic leukemia. Clin Cancer Res., 6(3), March 2006, pp. 825-828.

Constructed (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22], or a pharmaceutical composition containing said UCART 22 , as a medicine.

Another aspect of the invention is an engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22] for use in the treatment cancer or reduce inflammation.

Another aspect of the invention is an engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22] for use in the treatment of ALL , CLL, recurrent refractory aggressive forms of CLL or ALL.

What is proposed is an engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22] for the treatment of CD19-expressing recurrent cancer, preferably expressing CD19 B-ALL.

Another embodiment of the invention is a hypoxia resistant engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22], or a pharmaceutical composition containing said UCART 22 as a drug.

Another embodiment of the following object of the present invention is a hypoxia-resistant engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22 ], intended for use in the treatment of cancer or the reduction of inflammation.

Another embodiment of the following object of the present invention is a hypoxia-resistant engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22 ] intended for use in the treatment of ALL, CLL, relapsed refractory aggressive forms of CLL or ALL.

Another embodiment of the invention is a hypoxia-resistant engineered (TRAC- and dCK-KO or TRAC- and CD52-KO) immune T cell expressing the CD22 CAR of the invention (preferably SEQ ID NO: 15) [UCART 22] intended for for use in the treatment of CD19-expressing recurrent cancer, preferably CD19-expressing recurrent B-ALL.

According to another embodiment of the invention, an isolated cell obtained by various methods proposed in the present invention, or a cell line obtained from said isolated cell, can be used as a drug. According to another embodiment of the invention, said medicament can be used for the treatment of cancer, especially for the treatment of B-cell lymphomas and leukemia in a patient in need thereof. According to another embodiment of the invention, said isolated cell of the invention, or a cell line derived from said isolated cell, can be used to prepare a medicament for the treatment of cancer in a patient in need thereof.

The present invention includes autologous engineered cell transfer. In this case, cells are isolated from a single human donor, engineered, and then transferred to the original donor in need.

In this specific embodiment, the cells can be engineered, for example, to be resistant to a drug such as alemtuzumab (Campas) and/or pna (purine nucleotide analog) and optionally to be resistant to hypoxia.

Therapeutic Applications

The term "cancer" refers to a disease characterized by the uncontrolled growth of one or more types of cells.

Examples of cancers are provided herein and include, but are not limited to, liquid tumors or hematologic cancers.

The hematological cancer of the present invention can be selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, preferably CD22-expressing hematologic cancer, more preferably recurrent refractory CD22-expressing hematologic cancer, even more preferably an aggressive form of said CD22-associated hematologic cancer.

In a preferred embodiment, the relapsed or refractory CD22-expressing hematologic cancer is relapsed and/or refractory CD22-expressing or CD22 B-ALL positive.

Thus, the use of a therapeutically effective amount of UCART 22 according to one of the above embodiments of the invention or a therapeutically effective amount of a pharmaceutical composition as defined above is provided as a medicament for the treatment of a patient suffering from relapsed and/or refractory CD22 expressing or positive for CD22 B-ALL.

According to another embodiment of the invention, the use of a therapeutically effective amount of UCART 22 as provided in one of the above embodiments of the invention, or a therapeutically effective amount of a pharmaceutical composition as defined above, is provided as a medicament for the treatment of a patient suffering from CD22-positive hematologic cancer, selected from leukemia and lymphoma, hairy cell leukemia, any acute lymphocytic cancer, acute lymphocytic leukemia (ALL), acute myeloid leukemia, chronic lymphocytic leukemia, B-cell chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma and Burkitt's lymphoma, multiple myeloma.

According to another embodiment of the invention, the use of a therapeutically effective amount of UCART 22 according to one of the above embodiments of the invention, or a therapeutically effective amount of the pharmaceutical composition indicated above, is provided as a drug for the treatment of a patient suffering from CD22-positive cancer, selected from alveolar rhabdomyosarcoma, bladder cancer (eg, bladder carcinoma), bone cancer, brain cancer (eg, medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer, pleura cancer, nasal cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, carcinoid tumors of the gastrointestinal tract, head and neck cancer (eg, head and neck squamous cell carcinoma), hypopharynx cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer (eg, non-small cell lung carcinoma), malignant mesothelioma, mastocytoma, melanoma, nasopharyngeal cancer, ovarian cancer, cancer pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, cancer ureter.

Other examples of CD22-mediated cancers are provided herein and include, but are not limited to, liver cancer, lung cancer (eg, non-small cell lung carcinoma), ovarian cancer, pancreatic cancer, and urothelial cancer.

A specific embodiment of the object of the present invention is the treatment of aggressive forms of these cancers, which grow at least 2 times faster compared to the usual average growth of these cancers in the population.

One of the embodiments of the objects of the present invention is the treatment of leukemia during the treatment of the acceleration phase.

A specific embodiment of the object of the present invention is the treatment of refractory/recurrent diffuse large B-cell non-Hodgkin's lymphoma - metastases of breast cancer in the lung - a triple cancer consisting of chronic lymphocytic leukemia in combination with carcinoma of the bladder and prostate.

Preferably, the cancer is a hematological malignancy (e.g., leukemia or lymphoma, including but not limited to Hodgkin's lymphoma, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, acute lymphocytic cancer, acute myeloid leukemia, B-cell chronic lymphocytic leukemia, hairy cell leukemia , acute lymphocytic leukemia (ALL), and Burkitt's lymphoma).

More preferably the cancer is characterized by the expression of CD22, more preferably the cancer is characterized by the expression of CD22 by the cancer cells, even more preferably by the overexpression of CD22 by the cancer cells.

In one of the embodiments of the invention, these cancer cells are recurrent refractory CD19-negative cancer cells.

In one embodiment, said cancer cells are recurrent refractory CD22-expressing cancer cells.

In a preferred embodiment of the invention, said cancer cells are recurrent refractory CD19-negative, CD22-positive ALL B cells.

ALL B cells contain:

• early B precursors (early pre-B) ALL cells (also called pro-B ALL),

• ordinary cells ALL,

• pre-IN ALL,

• Mature B cells of ALL, also called Burkitt's leukemia or non-Hodgkin's lymphoma in children.

The term “disease associated with CD22 expression” as used herein includes, but is not limited to, a disease associated with CD22 expression, or a condition associated with the activity of cells that express CD22, including tumor cells of various cancers, such as, for example, as expressing CD22 B-ALL.

Cell destruction by lysis is one of the mechanisms by which the CD22 CAR T cells of the invention act against CD22 expressing cells to shrink or eliminate tumors, facilitate infiltration of host immune cells into the tumor site, and/or increase/prolong antitumor responses.

Another object of the present invention are methods for treating patients in need thereof, where said method comprises at least one of the following steps:

• receiving UCART 22, proposed in the invention,

• administering said transformed immune cells to said patient.

In one of the embodiments of the invention, these UCART 22-cells, proposed in the invention, can be subjected to enhanced T-cell propagation in vivo and can be stored for a longer time in the host.

Another object of the present invention are methods for treating patients in need thereof, where said method comprises at least one of the following steps:

• conditioning a patient with cancer,

• receiving UCART 22, proposed in the invention,

• administering said transformed immune cells to said patient.

Conditioning includes lymphodepletion or any suitable conditioning known to the person skilled in the art, preferably also the attending physician, who knows how to determine the necessary medical care for the specified patient.

In a preferred embodiment of the invention, said method further comprises the step of bone marrow transplantation.

In one of the embodiments of the invention, these UCART 22-cells, proposed in the invention, can be subjected to enhanced T-cell propagation in vivo and can be stored for a longer time in the host.

Said treatment may be relieving, curative or prophylactic. It can either be either part of autologous immunotherapy or part of allogeneic immunotherapy. By autologous treatment is meant that the cells, cell line or population of cells that are used to treat patients are obtained from said patient or from a human leukocyte antigen (HLA) compatible donor. By allogeneic treatment is meant that the cells or population of cells that are used to treat patients are not obtained from said patient, but are obtained from a donor.

In one embodiment, the survival of said T cells of the invention in the host is controlled by treatment with an anti-CD20 antibody (rituximab) and/or QBEND-10.

Individual (subject)

The compositions and methods of the present invention can be used to treat an individual who is found to have abnormal cells or tissues expressing CD22, or who is expected to have abnormal cells or tissues expressing CD22. For example, patients who may benefit from the treatment of the invention include patients with B-ALL or CLL, refractory B-ALL, recurrent B-ALL.

In a preferred embodiment of the invention, the patients are children suffering from B-ALL, recurrent B-ALL, refractory B-ALL (pediatric indication).

Treatment with the engineered immune cells of the invention may be combined with one or more types of cancer therapy selected from the group consisting of antibody-based therapy, chemotherapy, cytokine therapy, dendritic cell therapy, gene therapy, hormone therapy. , light-laser therapy and radiation therapy.

Thus, the present invention provides a pharmaceutical composition containing a therapeutically effective amount of UCART 22 for the treatment of children suffering from B-ALL, recurrent B-ALL, refractory B-ALL.

The present invention provides a pharmaceutical composition containing UCART 22 and a pharmaceutically acceptable excipient for the treatment of children suffering from B-ALL, relapsed B-ALL, refractory B-ALL.

In a preferred embodiment, the pharmaceutical composition comprises UCART 22 of the invention and a compound from the bryostatin family, preferably bryostatin-1, a pharmaceutically acceptable excipient for the treatment of children with B-ALL, relapsed B-ALL, refractory B-ALL.

According to a preferred embodiment of the invention, said treatment can be used for patients undergoing immunosuppressive treatment (conditioned treatment), more preferably lymphodepletion. Thus, the present invention preferably relates to cells or cell populations that have been rendered resistant to at least one immunosuppressive agent by inactivating a gene encoding a receptor for said immunosuppressive agent. In this aspect, immunosuppressive treatment or lymphodepletion should promote the selection and expansion of the T cells of the invention in the patient's body and the destruction of CD22-expressing cancer cells.

Administration of the cells or cell populations of the present invention may be by any suitable method, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions provided herein may be administered to a patient subcutaneously, intradermally, 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.

The administration of the cells or cell population may comprise the administration of 10 ⁴ -10 ⁹ cells/kg of body weight, preferably 10 ⁵ to 10 ⁶ cells/kg of body weight, including all integer numbers of cells in the indicated ranges. The cells or cell population can be administered as one or more doses, preferably several subsequent doses (repetitive dosing) to avoid escaping (from immunological surveillance) (recurrent cells). In another embodiment, said effective amount of cells is administered as a single dose or doses. In another embodiment of the invention, said effective amount of cells is administered as more than one dose over a period of time.

The schedule of administration is within the competence of the attending physician and depends on the clinical condition of the patient.

The cells or population of cells can be obtained from any source such as a blood bank or donor. Since individual needs vary, it is within the skill of the art to determine the optimal ranges of effective amounts of the cell type in question for a particular disease or conditions. An effective amount is an amount that provides a therapeutic or prophylactic benefit. The dose administered should depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment, and the nature of the effect desired.

The UCART 22 of the present invention is designed to be effective, but not to activate and limit the cytokine storm. In the case of hypersensitive patients, the present invention may be combined with appropriate medical treatment to prevent or block the cytokine storm, such as the use of anti-IL-6 drugs.

In another embodiment of the invention, the cells or a composition containing these cells is administered parenterally in the indicated effective amount. Said administration may be intravenous administration. Said administration can be carried out directly by injection into the tumor.

In some embodiments, the cells are administered to a patient in conjunction with (e.g., before, concurrently, or after) any number of appropriate treatment options, including (but not limited to) treatment with antiviral therapies such as cidofovir and interleukin-2. , cytarabine (also known as ARA-C), or treatment with natalizumab in patients with MS (multiple sclerosis), or treatment with efalizumab in patients with psoriasis, or other treatments in patients with PML (progressive multifocal leukoencephalopathy). In other embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodestructive agents such as campas, alemtuzumab, anti-CD3 antibodies, or with other antibody-based therapies, cytoxin, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs either inhibit calcium-dependent phosphatase, calcineurin (cyclosporine and FK506), or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin) (Henderson, Naya et al., 1991; Liu, Albers et al., 1992; Bierer, Hollander et al. 1993).

In another embodiment of the invention, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, simultaneously with, or after) bone marrow transplantation, T-cell ablative therapy using either chemotherapeutic agents such as fludarabine, or external beam radiation therapy. (XRT), cyclophosphamide, or antibodies such as OKT3 or Campas. In another embodiment, the cell compositions of the present invention are administered following B cell ablative therapy using agents that interact with CD20, such as Rituxan or QBEND-10, for example. For example, in one of the embodiments of the invention, individuals can be subjected to standard treatment with high dose chemotherapeutic agents, followed by transplantation of peripheral blood cells. In some embodiments, after transplantation, individuals are infused with the expanded immune cells of the present invention. In yet another embodiment of the invention, the expanded cells are administered before or after surgery.

In another embodiment, after transplantation, an agent that interacts with CD20, such as rituxan or ritiximab, is administered to the individual, preferably in combination with an agent that interacts with CD22 and CD20.

Other definitions

- Amino acid residues in a polypeptide sequence are referred to herein with a one-letter code, whereby, for example, Q is Gln or a glutamine residue, R is Arg or an arginine residue, and D is Asp or an aspartic acid residue.

- Amino acid substitution refers to the substitution of one amino acid residue for another, for example, substitution of an arginine residue for a glutamine residue in a peptide sequence is an amino acid substitution.

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

- In the context of the present description, the terms "nucleic acid" or "polynucleotides" refer to nucleotides and/or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments created using polymerase chain reaction (PCR), and fragments created by ligation, cleavage, endonuclease treatment, and exonuclease treatment. Nucleic acid molecules may be composed of monomers that are naturally occurring nucleotides (such as DNA or RNA) or analogs of naturally occurring nucleotides (eg, enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides may have changes in sugar moieties and/or moieties containing pyrimidine or purine bases. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or the sugars may be functionalized to ethers or esters. In addition, the entire sugar moiety can be replaced by sterically and electronically similar structures such as aza-sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety are alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers can be crosslinked by phosphodiester bonds or analogues of such bonds. Nucleic acids can be single stranded or double stranded.

- By chimeric antigen receptor (CAR) is meant molecules in which a binding domain directed against a component present on the target cell is combined, for example, the inherent specificity of an antibody for the desired antigen (for example, tumor antigen), with an activating T-cell receptor intracellular domain, resulting in the formation of a chimeric protein with specific cellular immune activity against the target. Typically, CAR consists of an extracellular single chain antibody (scFvFc) fused to the intracellular T cell antigen receptor complex zeta chain signal domain (scFvFc:ζ) and when expressed in T cells, it has the ability to redirect antigen recognition due to the specificity of the monoclonal antibodies. One example of a CAR useful in the present invention is a CAR directed against the CD22 antigen and may, for example, comprise (but not be limited to) the amino acid sequences: SEQ ID NO: 15-18, preferably SEQ ID NO: 16 or 18, more preferably SEQ ID NO: 16.

- The term "endonuclease" refers to a wild-type enzyme or a variant thereof that has the ability to catalyze the hydrolysis (cleavage) of bonds between nucleic acids in a DNA or RNA molecule, preferably in a DNA molecule. Endonucleases do not cleave a DNA or RNA molecule regardless of its sequence, but they recognize and cleave a DNA or RNA molecule at specific polynucleotide sequences, hereinafter referred to as "target sequences" or "target sites". Endonucleases can be classified as rare cleaving endonucleases, for which, as a rule, the polynucleotide recognition site has a length of more than 12 base pairs (bp), more preferably 14-55 base pairs. Rarely cleaving endonucleases significantly increase homologous recombination (HR) by inducing DNA double-strand breaks (DSB) at a specific locus (Perrin, Buckle et al., 1993; Rouet, Smih et al., 1994; Choulika, Perrin et al., 1995; Pingoud and Silva, 2007). Rarely cleaving endonucleases can be, for example, homing endonuclease (Paques and Duchateau, 2007), a chimeric zinc finger nuclease (ZFN) formed by fusing engineered zinc finger domains with the catalytic domain of a restriction enzyme such as FokI (Porteus and Carroll, 2005), Cas9 endonuclease from the CRISPR system (Gasiunas, Barrangou et al., 2012; Jinek, Chylinski et al., 2012; Cong, Ran et al., 2013; Mali, Yang et al., 2013), or chemical endonuclease (Eisenschmidt , Lanio et al., 2005; Arimondo, Thomas et al., 2006). In chemical endonucleases, a chemical or peptide cleavage moiety is conjugated to either a nucleic acid polymer or other DNA that recognizes a specific target sequence, thereby directing the cleavage activity to a specific sequence. Chemical endonucleases also include synthetic nucleases such as conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), which are known to bind to specific DNA sequences (Kalish and Glazer, 2005). Such chemical endonucleases fall under the term "endonuclease" in the context of the present invention.

- By "TALE-nuclease" (TALEN) is meant a fusion protein that consists of a nucleic acid-binding domain, typically derived from a transcription activator-like effector (TALE), and one nuclease catalytic domain for cleaving a target nucleic acid sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, such as I-TevI, ColE7, NucA and Fok-I. In a specific embodiment, the TALE domain may be fused to a meganuclease, such as I-CreI and I-OnuI, or a functional variant thereof. In a more preferred embodiment of the invention, 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 engineered TAL repeat fusions to the I-TevI catalytic domain described in WO 2012/138927. Transcription activator-like effectors (TALE) are proteins from Xanthomonas bacterial species containing multiple repeat sequences, where each repeat contains di residues (double residues) at positions 12 and 13 (RVD) that are specific for each nucleotide base of the nucleic acid sequence. target acids. Binding domains having similar modular base-per-base (MBBBD) nucleic acid binding properties can also be obtained from novel modular proteins recently discovered by applicants in various bacterial species. The advantage of the new modular proteins is that they are characterized by greater sequence variability than TAL repeats. Preferably, the RVDs associated with recognition of various nucleotides are HD for C recognition, NG for T recognition, NI for A recognition, NN for G or A recognition, NS for A, C, G or T recognition, HG for T, IG recognition. for T recognition, NK for G recognition, HA for C recognition, ND for C recognition, HI for C recognition, HN for G recognition, NA for G recognition, SN for G or A recognition and YG for T recognition, TL for A recognition , VT for A or G recognition, and SW for A recognition. just to increase their specificity. TALE nuclease has been previously described and is used to promote gene targeting and gene modification (Boch, Schoize et al., 2009; Moscou and Bogdanove, 2009; Christian, Cermak et al., 2010; Li, Huang et al., 2011). The engineered TAL nucleases are marketed under the trademark TALEN (Cellectis, 8 rue de la Croix Jarry, 75013, Paris, France).

The rare cleaving endonuclease of the present invention may also be Cas9 endonuclease, zinc finger endonuclease, MegaTAL endonuclease. In recent years, a new tool for genetic engineering based on the RNA-guided nuclease Cas9 has been developed (Gasiunas, Barrangou et al., 2012; Jinek, Chylinski et al., 2012; Cong, Ran et al., 2013; Mali, Yang et al. ., 2013) from the adaptive immune system CRISPR (Clustered R egularly Interspaced Short p alindromic Repeats ) of type II prokaryotes (see review by Sorek, Lawrence et al., 2013). The CRISPR-associated (Cas) system was first discovered in bacteria and its function is to protect against foreign viral or plasmid DNA. In CRISPR-mediated genetic engineering, a target sequence is first selected, often flanked by a short motif sequence, referred to as a protospacer adjacent motif (PAM). After selecting a target sequence, a specific crRNA (CRISPR-RNA) is constructed that is complementary to said target sequence. Type II CRISPR systems require a trans activating crRNA (tracrRNA) paired with the crRNA and associated with an introduced Cas9 protein. Cas9 functions as a molecular anchor facilitating tracRNA base pairing with sRNA (Deltcheva, Chylinski et al., 2011). In this ternary complex, the tracrRNA:crRNA double structure functions as a guide RNA that directs the Cas9 endonuclease to the cognate target sequence. Target recognition by the Cas9-tracrRNA:crRNA complex is initiated by scanning the target sequence to detect homology between the target sequence and the crRNA. In addition to the complementarity of the target sequence and crRNA, DNA targeting requires the presence of a short motif; adjacent to the protospacer (motif adjacent to the protospacer - RAM). After pairing of the double RNA and the target sequence, Cas9 introduces a blunt double strand break at a distance of 3 base pairs in the opposite direction relative to the PAM motif (Garneau, Dupuis et al., 2010).

The rare cleaving endonuclease may be a homing endonuclease, also known as a meganuclease. Such homing endonucleases are well known in the art (Stoddard, 2005). Homing endonucleases recognize the target DNA sequence and create a single or double strand break. Homing endonucleases are highly specific, recognizing target DNA sites 12 to 45 base pairs (bp) long, typically 14 to 40 base pairs long. The homing endonuclease according to the invention may correspond to, for example, LAGLIDADG endonuclease, HNH endonuclease or GIY-YIG endonuclease. Preferably, the homing endonuclease of the present invention may be an I-CreI variant.

- By "delivery vector" or "delivery vectors" is meant any delivery vector that can be used according to the present invention for bringing into contact with a cell (i.e. for "contacting") or delivery into a cell or subcellular compartments ( i.e. for "introduction") agents/chemicals and molecules (proteins or nucleic acids) required for the implementation of the present invention. These include, but are not limited to, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasonic contrast agents), nanoparticles, emulsions, or others. suitable vectors for transfer. Such delivery vectors allow the delivery of molecules, chemicals, macromolecules (genes, proteins) or other vectors such as plasmids, peptides developed by Diatos. In these cases, the delivery vectors are carrier molecules. By "delivery vector" or "delivery vectors" is meant also delivery methods intended for transfection.

- The terms "vector" or "vectors" refer to a nucleic acid molecule that has the ability to transport another nucleic acid to which it is linked. "Vectors" as used in the present invention include, but are not limited to, a viral vector, plasmid, RNA vector, or linear or circular DNA or RNA molecule, which may be composed of chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acids. Preferred vectors are vectors that are capable of autonomously replicating (episomal vector) and/or expressing the nucleic acids to which they are linked (expression vectors). Those skilled in the art are aware of a large number of suitable vectors that are commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (eg adeno-associated virus, primarily aav6), coronavirus, negative strand RNA viruses such as orthomyxovirus (eg influenza virus), rhabdovirus (eg rabies virus and vesicular stomatitis virus) , paramyxovirus (eg, measles virus and Sendai virus), positive-strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses, including adenovirus, herpesvirus (eg, herpes simplex virus types 1 and 2, Epstein- Barr, cytomegalovirus) and smallpox virus (for example, vaccinia virus, avian pox virus and canarypox virus). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovirus, hepadnavirus, and hepatitis virus. Examples of retroviruses are: avian leukemia-sarcoma virus, mammalian C-type, B-type, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin J. M., Retroviridae: The viruses and their replication, in: Fundamental Virology, 3rd ed., B. N. Fields et al., Lippmcott-Raven Publishers, Philadelphia, 1996).

- By "lentiviral vector" is meant HIV-based lentiviral vectors which are very promising in terms of gene delivery due to their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce a wide range of different cell types with high efficiency. Lentiviral vectors are typically generated by transient transfection of producer cells with three (packaging plasmid, envelope plasmid, and transfer plasmid) or more plasmids. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. After entry, the viral RNA undergoes reverse transcription mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which serves as a substrate for viral integration into the DNA of infected cells. The term "integrating lentiviral vectors (or LV)" means, for example (but not limited to), those vectors that are capable of integrating into the genome of a target cell. In contrast, the term "non-integrating lentiviral vectors (or NILV)" refers to efficient gene delivery vectors that are not integrated into the target cell genome by viral integrase.

- Delivery vectors and vectors can be combined or combined with any cell permeabilization techniques such as sonoporation or electroporation, or variations of these techniques.

- Under the cell or cells means any eukaryotic living cells, primary cells and cell lines derived from these organisms to obtain cultures in vitro.

- By "primary cell" or "primary cells" is meant cells taken directly from living tissue (i.e. biopsy material) and prepared for in vitro growth, in a population of which only a small number of doublings have occurred, and therefore they are more representative in terms of major functional components and characteristics of the tissues from which they originate, compared to continuously maintained or artificially immortalized oncogenic cell lines.

Examples of such cell lines may include (but are not limited to) cell lines selected from the group consisting of CHO-K1 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 of these cell lines can be modified using the method proposed in the present invention, creating models of cell lines for obtaining, expressing, quantifying, detecting, studying a gene or protein of interest; these models can also be used to screen biologically active molecules of interest for research and industrial purposes in various fields such as (but not limited to) chemistry, biofuels, therapeutics, and agronomy.

- By "mutation" is meant the substitution, deletion or insertion of one/one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty five, thirty, forty, fifty or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. A mutation may 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)" is meant a repeat variant, variant, DNA-binding variant, TALE nuclease variant, polypeptide variant resulting from a mutation or substitution of at least one residue in the amino acid sequence of the parent molecule.

By "functional variant" is meant a catalytically active mutant of a protein or protein domain; such a mutant may have the same activity as the parent protein or protein domain, or additional properties, or higher or lower activity.

- The term "identity" refers to the sequence identity of two nucleic acid molecules or polypeptides. Identity can be assessed by comparing positions in each of the sequences, which can be aligned for comparison purposes. If a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. The degree of similarity or identity between two nucleotide or amino acid sequences is a function of the number of identical or matching nucleotides at the positions of the nucleotide sequences. To calculate the identity of two sequences, various algorithms and/or programs for comparing the primary structure of sequences can be used, including FASTA or BLAST, which are available as a component of the GCG sequence analysis software package (University of Wisconsin, Madison, Wisconsin), and can be used, for example, using default parameters. For example, one may consider polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with the specific polypeptides provided herein, and preferably having substantially the same functions, as well as polynucleotides encoding such polypeptides. Unless specifically noted otherwise, the similarity scores indicated in the description were obtained using BLOSUM62. When using BLASTP, percent similarity is based on the BLASTP-calculated positive score, and percent sequence identity is based on the BLASTP-calculated identity score. BLASTP-calculated "identities" are the number and proportion of identical residues relative to the total number of residues in high-scoring sequence pairs; and BLASTP-calculated "positive scores" are the number and proportion of residues for which the primary sequence structure comparison scores are positive and are similar to each other. The present application proposes and falls within the scope of the invention amino acid sequences having the indicated degrees of identity or similarity, or any intermediate degree of identity or similarity, with the amino acid sequences presented herein. Polynucleotide sequences of similar polypeptides are derived using the genetic code and can be obtained using conventional methods, in particular by reverse translation of its amino acid sequence using the genetic code.

- The terms "signal transduction domain" or "costimulatory ligand" refer to a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell, thereby providing a signal that, in addition to the primary signal provided, for example, by binding of the TCR complex /CD3 with an MHC molecule loaded with a peptide mediates a T cell response, including (but not limited to) activation of proliferation, differentiation, and the like. The costimulatory ligand can be, 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 receptor beta, 3/TR6, ILT3, ILT4, an agonist or antibody that/which binds to a Toll receptor ligand, and a ligand that specifically binds to B7-H3). CD28 is excluded from this list. A costimulatory ligand also includes, among other things, an antibody that specifically binds to a costimulatory 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 to CD83.

The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the cell, such as (but not limited to) proliferation. Costimulatory molecules include, but are not limited to, the MHC class I molecule, BTLA, and the Toll receptor ligand.

In the context of the present description, the term "costimulatory signal" refers to a signal that, in combination with a primary signal, such as a signal due to TCR/CD3 linkage, leads to T cell proliferation and/or up or down regulation of critical molecules.

- In the context of the present description, the term "extracellular ligand-binding domain" means an oligo - or polypeptide that has the ability to bind to a ligand. Preferably, the domain should be capable of interacting with a cell surface molecule. For example, an extracellular ligand-binding domain can be selected to recognize a ligand that functions as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that can function as ligands are those associated with viral, bacterial and parasitic infections, autoimmune disease, and cancer cells.

In the context of the present description, the term "individual" or "patient" includes all members of the animal kingdom, including primates other than humans and humans. In one embodiment, the patients are patients with aggressive or refractory or recurrent ALL, or aggressive refractory recurrent CLL.

A mammal is any warm-blooded mammalian vertebrate, preferably a human.

"Suicide domain or switches" or "protective on and off switches" refers to a domain, typically a cell surface domain, recognized by a molecule, protein, chemical, antibody for immunoselection of cell expression and ultimately control of cell function and survival.

In the above description of the invention, the approach and method of making and using it is set out so that any person skilled in the art is able to make and use it, this possibility refers primarily to the essence of the invention set forth in the attached claims, which is an integral part of the original descriptions.

Where numerical limits or a numerical range are indicated herein, they include their limits. In addition, all values and sub-ranges within the specified limits or specified numerical range are considered to be included, as if they were specifically indicated.

The foregoing description is provided to enable a person skilled in the art to make and use the invention, and it is set forth in the context of a particular application and the conditions required therefor. Various modifications to the preferred embodiments of the invention will be apparent to those skilled in the art, and the general principles set forth therein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, the present invention should not be considered limited to the present embodiments of the invention, on the contrary, it corresponds to the broadest scope consistent with the principles and features specified in the present description.

General Methods

CAR screening and selection

Primary T cell cultures

du Sang, Париж, Франция), используя среду с градиентом плотности фиколла. Выделяют слой РВМС. Т-клетки активируют в среде Х-Vivo™-15 (фирма Lonza), дополненной человеческим IL-2 в концентрации 20 нг/мл, человеческой сывороткой в концентрации 5%, и используя гранулы Dynabeads® с активатором человеческих Т-клеток CD3/CD28 при соотношении гранулы: клетки 1:1 (фирма Life Technologies).T cells are purified from buffy coat samples obtained from EFS (Etablissement

du Sang, Paris, France) using ficoll density gradient media. Allocate a layer of RVMS. T cells are activated in X-Vivo™-15 medium (Lonza) supplemented with human IL-2 at 20 ng/ml, human serum at 5%, and using Dynabeads® beads with human CD3/CD28 T cell activator at a granule:cell ratio of 1:1 (Life Technologies).

CAR mRNA transfection

Transfections of CAR mRNA encoding each of the CAR constructs are performed on day 4 or day 11 after T cell purification and activation. Cells are immediately diluted in X-Vivo™-15 medium (Lonza) and incubated at 37°C in an atmosphere with 5% CO ₂ . IL-2 is added 2 hours after electroporation at a concentration of 20 ng/ml.

T cell transduction

Vectors encoding the CD22 CAR are introduced into T cells according to the previously described method.

Detection of CARs on the surface of T cells is performed using a recombinant protein that consists of the extracellular domain protein of the human CD22 protein (whole protein, CD22 distal or CD22 proximal) fused to a mouse IgG1 Fc fragment. The binding of this protein to the CAR molecule was determined with a PE-conjugated secondary antibody (Jackson Immunoresearch) targeting the mouse Fc fragment of the protein and analyzed by flow cytometry.

Inactivation of specific gene(s) in primary T cells

Inactivation of the specific gene(s) in primary T cells can be performed before, but preferably after, the introduction of CD22 CAR into the cells using endonucleases such as TAL endonuclease, optionally Crispr Cas 9 endonucleases, suitably engineered. At least one gene is inactivated, one, two or three genes can be inactivated in one step or in several successive steps. In a preferred embodiment, two genes are inactivated, preferably the TCRalpha gene and the gene whose deletion confers resistance to a drug selected from purine nucleotide analogs, alemtuzumab, platinum drugs (cisplatin or carboplatin), anti-topoisomerase I antibody (irinotecan), anti-topoisomerase antibody II (etoposide), methotrexate (folic acid analogs), preferably purine nucleotide analogs, alemtuzumab.

In general, a heterodimeric nuclease, in particular a TALE nuclease, is designed and prepared to target two long sequences (called hemi-targets) separated by a spacer in a target gene.

Each TALE nuclease construct can be cloned into an appropriate mammalian cell expression vector. An mRNA encoding a TALE nuclease that cleaves a targeted genomic sequence can be synthesized from a plasmid carrying the coding sequence upstream of a promoter. Purified T cells are used, previously activated with anti-CD3/CD28 coated beads, and transfected with each of the two mRNAs encoding both halves of TALE nucleases. Cells can be reactivated with soluble anti-CD28 antibody to assess cell proliferation at various time points, and the CD25 activation marker can be determined to assess the activation status of cells.

• Degranulation assay (CD107a mobilization)

Cells are incubated in 96-well plates along with an equal number of cells expressing various levels of targeting protein (CD22). The co-cultures are maintained for 6 hours at 37°C in an atmosphere with 5% CO ₂ . CD107a staining is performed during cell stimulation by adding a fluorescent anti-CD107a antibody at the start of co-culture, along with anti-CD49d, anti-CD28, and 1x monensin as a control. After an incubation period of 6 hours, cells are stained with fixative vital stain and fluorochrome-conjugated anti-CD8 antibody and analyzed by flow cytometry. Degranulating activity is determined as % CD8 ⁺ /CD107a ⁺ cells and based on the determination of the average fluorescence signal intensity (MFI) by staining CD107a in a population of CD8 ⁺ cells. Degranulation assays are performed 24 hours after mRNA transfection.

• IFN gamma release assay

24 hours after mRNA transfection, CD22 CAR-expressing T cells are incubated with cell lines that express various levels of targeting protein for 24 hours at 37°C. Supernatants are isolated and IFN gamma is determined in cell culture supernatants by ELISA analysis.

• Cytotoxicity analysis

Cells are incubated with target cells (expressing various levels of CD22) or with cells (representing a negative control). Target and control cells are labeled with fluorescent intracellular dyes (CFSE or Cell Trace Violet) prior to co-culture with CAR ⁺ -T cells. Co-cultures are incubated for 4 hours at 37°C. After the indicated incubation period, the cells are labeled with fixative vital stain and analyzed by flow cytometry. Determine the viability of each cell population (target cells or cells used as a negative control) and calculate the % specific (specific) cell lysis. Cytotoxicity assays are performed 48 hours after mRNA transfection.

• Mouse model for evaluating antitumor activity Immunocompromised mice are implanted with tumor cells (CD22 B-ALL from patients) or cells expressing targeting protein-luciferase complex in the lateral region. The cells are then implanted into the brains of mice. Serial transplantation in subsequent generations of mice continues to maintain cell lines transplanted in vivo Optionally, mice are subjected to anti-cancer treatment before/or simultaneously with injection of CAR+ T cells (alemtuzumab and/or flu). Mice are then injected iv (either 2 or 7 days after tumor cell line injection) at varying doses of CAR+ T cells to be tested, or T cells that do not express CD22 CAR. Bioluminescent signals are determined on the day of T cell injection (D0), at D7, 14, 21, 28 and 40 after T cell injection, to monitor the development of tumors in various animals.

Phase I Dose Escalation Study to Evaluate the Safety, Propagation, and Persistence of Allogeneic CD22 CART (UCART 22) in Patients with Relapsed or Refractory or MRD+ CD22+ B-Cell Acute Lymphoblastic Leukemia (B-ALL)

Background and rationale

With current multi-drug chemotherapeutic regimens, prolonged survival has been found in >80% of children with acute lymphoblastic leukemia (ALL) and approximately 40% of adults with ALL (1). The effectiveness of additional intensification of chemotherapy has not been proven (2). In the past few years, significant advances have been made in understanding the biology of ALL, which has led to the possibility of using "targeted therapy" (3, 4).

Relapsed/refractory ALL remains a problematic disease. Post-relapse therapies can lead to a secondary CR (complete response) (CR2) in 30-40% of patients with a 5-year OS (overall survival) of only 10%. In reporting the largest study to date of adult patients with relapsed ALL, Fielding and colleagues analyzed the end points (outcomes) for adult patients with relapsed ALL treated according to the MRC study UKALLXII/ECOG E2993 (5). Of the 1508 patients studied, 1372 (91%) achieved CR1, of which 609 (44% of CR1 responders) had a median relapse of 11 months. 5-year OS was achieved in only 7% of patients with disease recurrence. The median OS for patients with disease relapse was 5.5 months. Tavernier et al described the endpoints for 421 ALL patients in whom relapse treatment was first studied in the French LALA-94 study (6). CR2 was achieved in 44% of patients, with a median DFS (relapse-free survival) of 5.2 months and a median OS of 6.3 months. Oriol et al reported outcomes for 263 ALL patients in whom relapse treatment was first studied in 4 consecutive PETNEMA trials (7), CR2 was achieved in 45% of patients, a level similar to that obtained in the French LALA trials. Median OS after relapse was 4.5 months, 5-year OS was achieved in 10%.

CD22 expression is found in >90% of ALL patients and is a valuable therapeutic target. Cellular therapies, such as chimeric antigen receptor (CAR) expressing T cells, are increasingly being used to treat patients with hematologic malignancies (8-16). In patients with relapsing acute lymphoblastic leukemia (ALL), a very high complete response rate (80-90%) has been described with autologous CD19-CART cells (12). Similarly, a response rate of 40-50% has been found in patients with relapsing chronic lymphocytic leukemia (CLL) treated with autologous CD19 CART therapies (9).

In the present study, allogeneic CD22-targeted CART cells were investigated in patients with relapsed and/or refractory CD22 B-ALL.

Tasks

Primary Tasks

Evaluation of the safety and tolerability of allogeneic CD22 CART and determination of the maximum tolerated dose (MTD).

Secondary Tasks

Determination of the effectiveness of allogeneic CD22 CART. Determination of the frequency of occurrence of GVHD.

Research tasks

Determination of reproducibility, phenotype, transport and persistence of infused CART cells.

Inclusion Criteria

1. Relapsed or refractory CD22-positive ALL (for phase extension: patients with MRD+ disease may be included).

2. Patients aged ≥2 years.

3. Assessment of the general condition of the patient using the ECOG ≤2 scale.

4. Normal organ function, including bilirubin ≤2 mg/dl, ALT/AST ≤3×ULN, and creatinine ≤2 mg/dl.

5. Left ventricular ejection fraction (LVEF)≥40%.

Exclusion Criteria

1. The patient is pregnant or breastfeeding.

2. Patients with uncontrolled active infections.

3. Isolated extramedullary recurrence (i.e. testicles, CNS).

4. Active CNS leukemia is known.

Note: Patients with a history of CNS disease who have been treated successfully may be included provided they are in CNS remission for >4 weeks prior to enrollment.

5. Active hepatitis B or active hepatitis C.

6. HIV infection.

7. Active GVHD reaction requiring systemic steroid therapy. Steroid therapy for physiological replacement is acceptable.

8. Receiving DLI (Allogeneic Related Infusion) for 4 weeks of CD22 CART infusion.

9. Allo-SCT (Allogeneic Hematopoietic Stem Cell Transplantation) for 60 days of CD22 CART infusion.

Experience Description

The experience belongs to phase I of the study. Carried out 2 phases of this study. Escalated doses and expanded the number of patients.

Patients received allogeneic CD22 CART after lymphodepletion chemotherapy.

Dose Escalation: Four dose levels were studied according to a standard 3×3 schedule. Overall, 9-18 patients were included.

Once the level of R2PD was identified, the experiment began with the expansion of the number of patients.

A total of 20 patients were then included in the trial (10 R/R ALL; 10 MRD+ post-SCT).

Total sample size: 29-38 patients.

The second trial included allogeneic CART cells targeting CD22 and allogeneic CART cells targeting CD22 and CD19 (2 forms) in patients with CD19+ CD22+ B-ALL.

Tasks

Primary Tasks

Evaluation of the safety and tolerability of allogeneic CD19+ CD22 CART and determination of the maximum tolerated dose (MTD).

Secondary Tasks

Determination of the effectiveness of allogeneic CD22 CART. Determination of the frequency of occurrence of GVHD.

Research tasks

Determination of reproducibility, phenotype, transport and persistence of infused CART cells.

Inclusion Criteria

1. Relapsed or refractory CD22-positive ALL (for phase extension: patients with MRD+ disease may be included).

2. Patients aged ≥2 years.

3. Assessment of the general condition of the patient using the ECOG ≤2 scale.

4. Normal organ function, including bilirubin ≤2 mg/dl, ALT/AST <3×ULN, and creatinine ≤2 mg/dl.

5. Left ventricular ejection fraction (LVEF)≥40%.

Exclusion Criteria

1. The patient is pregnant or breastfeeding.

2. Patients with uncontrolled active infections.

3. Isolated extramedullary recurrence (i.e. testicles, CNS).

4. Active CNS leukemia is known.

Note: Patients with a history of CNS disease who have been treated successfully may be included provided they are in CNS remission for >4 weeks prior to enrollment.

5. Active hepatitis B or active hepatitis C.

6. HIV infection.

7. Active GVHD reaction requiring systemic steroid therapy. Steroid therapy for physiological replacement is acceptable.

8. Receiving DLI within 4 weeks of CD22 CART infusion.

9. Allo-SCT for 60 days of CD22 CART infusion.

Experience Description

The experience belongs to phase I of the study. Carried out 2 phases of this study. Escalated doses and expanded the number of patients.

Patients received allogeneic CD22 CART after lymphodepletion chemotherapy.

Dose Escalation: Four dose levels were studied according to a standard 3×3 schedule. Overall, 9-18 patients were included.

Once the level of R2PD was identified, the experiment began with the expansion of the number of patients.

A total of 20 patients were then included in the trial (10 R/R ALL; 10 MRD+ post-SCT).

Total sample size: 29-38 patients.

Links

1. Inaba H., Greaves M., Mullighan C. G., Acute lymphoblastic leukaemia. Lancet. 381(9881), 2013, pp. 1943-1955.

2. Faderl S., Thomas D.A., O'Brien S., Ravandi F., Garcia-Manero G., Borthakur G. et al., Augmented hyper-CVAD based on dose-intensified vincristine, dexamethasone, and asparaginase in adult acute lymphoblastic leukemia salvage therapy. Clin Lymphoma Myeloma Leuk. 11(1), 2011, pp. 54-59.

3. Mulligan C.G., Genome sequencing of lymphoid malignancies. Blood. 122(24), 2013, pp. 3899-3907.

4. Mullighan C.G., Genomic characterization of childhood acute lymphoblastic leukemia. Semin Hematol. 50(4), 2013, pp. 314-324.

5. Fielding A.K., Richards S.M., Chopra R., Lazarus H.M., Litzow M.R., Buck G., et al., Outcome of 609 adults after relapse of acute lymphoblastic leukemia (ALL); an MRC UKALL12/ECOG 2993 study. Blood. 109(3), 2007, pp. 944-950.

6. Tavernier E., Boiron J.M., Huguet F., Bradstock K., Vey N., Kovacsovics T. et al., Outcome of treatment after first relapse in adults with acute lymphoblastic leukemia initially treated by the LALA-94 trial. Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K. 21(9), 2007, pp. 1907-1914.

7. Oriol A., Vives S., Hernandez-Rivas J.M., Tormo M., Heras I., Rivas C. et al., Outcome after relapse of acute lymphoblastic leukemia in adult patients included in four consecutive risk-adapted trials by the PETHEMA Study Group. Haematologica. 95(4), 2010, pp. 589-596.

8. Kochenderfer J.N., Wilson W.H., Janik J.E., Dudley M.E., Stetler-Stevenson M., Feldman S.A. et al., Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19. Blood. 116(20), 2010, pp. 4099-4102.

9. Porter D.L., Levine B.L., Kalos M., Bagg A., June C.H., Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N Engl J Med. 365(8), 2011, pp. 725-733.

10. Brentjens R.J., Davila M.L., Riviere I., Park J., Wang X., Cowell L.G. et al., CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med. 5(177), 2013, 177ra38.

11. Grupp S.A., Kalos M., Barrett D., Aplenc R., Porter D.L., Rheingold S.R. et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med. 368(16), 2013, pp. 509-518.

12. Maude S.L., Teachey D.T., Porter D.L., Grupp S.A., CD19-targeted chimeric antigen receptor T-cell therapy for acute lymphoblastic leukemia. Blood. 125(26), 2015, pp. 4017-4023.

13. Park J.H., Geyer M.B., Brentjens R.J., CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date. Blood. 127(26), 2016, pp. 3312-3320.

14. Lee D.W., Kochenderfer J.N., Stetler-Stevenson M., Cui Y.K., Delbrook C, Feldman S.A. et al., T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. Lancet. 385(9967), 2015, pp. 517-528.

15. Kochenderfer J.N., Dudley M.E., Kassim S.H., Somerville R.P., Carpenter R.O., Stetler-Stevenson M. et al., Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor. J Clin Oncol. 33(6), 2015, pp. 540-549.

16. Kebriaei P., Singh H., Huls M.H., Figliola M. J., Bassett R., Olivares S. et al., Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells. J Clin Invest. 126(9), 2017, pp. 3363-3376.

Examples

Example 1: Proliferation of cells with inactivated TCRalpha expressing CD22-CAR

A heterodimeric TALE nuclease was constructed and generated targeting two 17 bp sequences (referred to as half-targets) separated by a 15 bp spacer in the T cell receptor alpha chain (TRAC) constant region gene. Each of the half-targets was recognized by repeats of half of the TALE-nucleases presented in Table 6.

Each TALE nuclease construct was subcloned using restriction enzyme digestion in a mammalian cell expression vector under the control of the T7 promoter. An mRNA encoding a TALE nuclease that cleaves the TRAC genomic sequence was synthesized using a plasmid carrying a coding sequence located upstream of the T7 promoter.

Purified T cells, preactivated for 72 h with anti-CD3/CD28 antibody coated beads, were transfected with each of the 2 mRNAs encoding both halves of TRAC_T01 TALE nucleases. 48 hours after transfection, different groups of T cells from the same donor, respectively, were transduced with a vector encoding one of the previously described CD22 CARs of the invention. 2 days after transduction, CD3-NEG cells were purified using anti-CD3 antibody-coated magnetic beads and 5 days post-transduction, cells were reactivated with soluble anti-CD28 antibody (5 μg/ml).

Monitoring of cell proliferation was carried out for a period of time up to 30 days after reactivation, counting the number of cells 2 times a week. The proliferation of cells with inactivated TCR alpha expressing CD22 CAR was comparable to the proliferation of non-activated cells and significantly increased when they were reactivated with anti-CD28 antibody.

Multiple transfection of TALEN mRNA

mRNAs encoding TRAC TALEN (left and right) and mRNAs encoding a TALEN pair specific for the B2M gene were transfected into activated T cells and then transduced with exogenous polynucleotides encoding CAR specific for CD22 or CAR specific for CD19 , or both of them.

Cells with double KO inactivation of TRAC and B2M gene or CD56 expressed an undetectable level of TCR and MHCCI molecules or TCR and CD56. After purification, the cells were transduced again.

Example 2: CD22 CAR-T

• Creation of engineered CD22-targeting CAR T cells for the treatment of refractory recurrent or aggressive ALL or CLL

• CD22 CAR: (Fig. 2)

• m971 construct and m971 variants with reduced CD22 affinity and high selectivity

CD22 CARs were generated and generated using various scfvs. m971 scfv was obtained from antibody 971 (Haso W. ¹ , Lee DW, Shah NN, Stetler-Stevenson M., Yuan C. M., Pastan IH, Dimitrov DS, Morgan RA, FitzGerald DJ, Barrett DM, Wayne AS, Mackall CL , Orentas RJ, Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. December 14, 2012)

CAR architecture was created (FIG. 2) using the 4-1BB co-stimulatory domain, CD3ξ activation domain, transmembrane domain and CD8α hinge, CD8α hinge (SEQ ID NO: 15). The construct contained an FcγRIIIα hinge according to SEQ ID NO: 14.

The constructs were inserted into a vector for stable expression and the generated CARs were screened.

CD22 CARs were m971-V3 CAR (SEQ ID NO: 22) and SEQ ID NO: 24.

Sequences can also be optimized for CD22 binding and treatment of ALL and CLL, preferably their refractory relapsing and aggressive forms.

• CAR expression

CD22 CARs were introduced into primary KO-TCR T cells 5 days after activation with anti-CD3/CD28 antibody-coated beads and IL-2. CAR expression was assessed by flow cytometry. All CARs were expressed on the cell surface. Activity was determined against CD22+ transformed cell lines and against cells from patients with refractory or recurrent CD22+ B-ALL.

To assess the functionality of anti-CD22 CAR B cells expressing CD22 (ALL lines REH, SEM, NALM6-GL, KOPN8, Daudi, Raji and K562 were used, see Haso et al., Blood: 121 (7), 2013 , pp. 1165-1174 details of the experiment). Received cells from patients with refractory or recurrent CD22+ B-ALL.

As expected, all cells expressing CD22 stained positively and were targeted by CD22 CAR from m971 of the invention, as well as CD22 CAR (scfv2 from HA22).

• Degranulation analysis

To validate the CD22 CAR constructs, a degranulation assay in target cells was performed using T cells expressing the CD22 CAR of the invention. The degranulating ability of CART was assessed by flow cytometry. Received data on the expression of CD107a on the plasma membrane of T cells after incubation for 5 h with target cells. The results showed that degranulation was more pronounced with CARm971-(scfv-1) than with CD22 CAR T cells against the distal region of CD22 (SEQ ID NO: 20) scfv-2 (FIG. 5).

• Cytotoxicity analysis

Cytotoxicity assays were performed using the same target cells and T cells expressing the CD22 CARs of the invention. For CD22 CAR, a strong specific lysis of CD22 cells was found with UCART 22 of the invention compared to cells expressing scfv against the distal region of CD22 (scfv2) (FIG. 6).

Interferon gamma analysis

UCART 22 interferon gamma production (scfv-V1 against CD22 proximal domain) was compared with that of non-transduced (NT) cells or T cells transduced with a CAR that targets the distal region of CD22 (scfv-V2) in the presence of CD22-positive NALM-16 cells compared to CD22-negative SUP-T1 cells (FIG. 7).

Mice Survival

Mice survival was increased in the presence of UCART 22 (scfv-V1 against CD22 proximal domain) or CART22 (no TRAC inactivation) compared to control cells (FIG. 8).

Resistance to hypoxia and/or drugs

The engineered UCART 22 cells of the invention were not significantly affected (survival and CTL activity) by alemtuzumab (50 μg/ml) or PNA (flu) compared to unengineered cells, which died within 48 hours after the addition of the drug to the cell wall. culture or after cultivation under hypoxic conditions (less than 5%, preferably less than 1% O ₂ ).

Experiments performed under low O ₂ conditions (<5% or <1%) showed similar results and confirmed that UCART 22 overexpressing HIF-1a had the ability to survive while expressing the CD22 CAR, and were active under hypoxic conditions.

Similar results (survival, CTL activity) were obtained in mice treated with campas (50 μg/ml), confirming drug resistance of UCART 22. The ability of the UCART 22 cells of the invention to reach tissue-nesting cancer cells or to reach cluster-forming cancer cells in vivo is contemplated, since the number of cancer cells that "recovered" from or "avoided" UCART 22 treatment was significantly lower (approx. by 15%), compared with mice treated with UCART 22, not resistant to O ₂ . It follows that the local hypoxia created by liquid tumors may prevent immune cells from fighting them.

Experiments were carried out to decide whether UCART 22 cells could be eliminated if and when required (FIGS. 3 and 4). In mice, rituximab induced a significant decrease in UCART, as evidenced by increased tumor cell fluorescence in mice treated with rituximab and UCART compared with cells alone.

For the first time, these clinical trial results demonstrate that UCAR T cells had a significant beneficial effect on relapsed and refractory ALL in vivo and in vitro, with no or very weak (grade 1) GVHD response and little or no detectable cytokine storm. .

This treatment may be less "toxic" compared to autologous CD22 CART treatment and can be controlled in patients using rituximab and/or QBEND-10.

Cells have been retained in the human body for a long enough time to be active (more than a month) and they can be depleted using QBEND-10.

Examples of derived CD22 CAR polypeptide sequences Framed sequences correspond to the preferred VH and VL sequences. VH and VL can be swapped (hotspot modification) to improve CAR performance as described above.

• v1-m972 (FcγRIIIα-CD8αTM-4-1BB.IC-CD3ζ.IC) (control construct not part of the invention)

• V3-m972 (CD8α-CD8αTM-4-1BB.IC-CD3ζ.IC) (control construct not part of the invention)

• v1-m971 (FcγRIIIα-CD8αTM-4-1BB.IC-CD3ζ.IC)

• v3-m971 (CD8α-CD8αTM-4-1BB.IC-CD3ζ.IC)

results

The CAR construct selected to generate UCART 22 contains an anti-human CD22 m971 scFv (described in Haso et al., 2013), a CD8α hinge (optional) and a transmembrane domain, and a cytoplasmic tail consisting of a 4-1BB costimulatory domain and a signaling domain CD3ζ (Fig. 3).

Another CAR construct contains concatenated m971 human CD22 scFv sequences, two rituximab binding domains (CD20), a CD8α hinge and a transmembrane domain, and a cytoplasmic tail consisting of a 4-1BB costimulatory domain and a CD3ζ signaling domain (Fig. 3).

An alternative CAR construct contains a cascading rituximab binding domain (CD20), a Q-BEN10 binding domain (CD34), m971 human CD22 scFv, two rituximab binding domains (CD20), a CD8α hinge and a transmembrane domain, and a cytoplasmic tail. , consisting of the 4-1BB co-stimulatory domain and the CD3ζ signaling domain (Fig. 3).

For UCART 22, a third-generation recombinant self-inactivating (SIN) lentiviral vector (rLV) was used to generate T cells expressing the anti-CD22 CAR and RQR8-associated depletion mechanism under the control of the human EF1α promoter (FIG. 4). Recombinant lentiviral vectors derived from the HIV virus are genetically stable and there are currently no data on pathological properties attributed to the vectors (Chang and Sadelain, 2007; Wang et al., 2009). In addition, the conditions for transduction of T cells with rLV are known and suitable for maintaining the functional properties of T cells.

For UCART 22, the AAV6 vector can be used after genetic editing of a gene such as the TRAC gene (the gene encoding the alpha subunit or TCR) or any gene described in PCT/EP2017/076798 (FIG. 9).

Variants of the anti-CD22 CAR constructs of the invention containing an additional CD19 scfv (upstream or downstream of CD22 scfv) were also prepared, as well as constructs containing multi-chain CD22 CAR, multi-chain CD22, CD19 CAR (FIG. 2) and inserted into adeno-associated virus 6 (AAV6) lentiviral vector, which allowed the insertion of the CD22 CAR constructs or the CD19 CAR construct described below into the TRAC gene, or into the CD25 gene, or into the beta-2-microglobulin gene (the CAR sequence was flanked at 5'- and 3' ends of the sequences to allow homologous recombination with the TRAC gene, CD25 gene or beta-2-microglobulin gene after specific cleavage of said gene using a specific TAL protein-Fok-1 (TALEN).

Created UCART 22, which additionally contained the TGFbeta receptor KO gene, IL-10 receptor KO gene, AHR KO gene, PD1 KO gene, LAG-3 KO gene, TIM-3 KO gene, or a combination thereof.

Each of the exemplary UCART 22's was created containing additional TGFbeta receptor KO gene or IL-10 receptor KO gene or aryl hydrocarbon receptor (AHR) KO gene or PD1 KO gene or LAG-3 KO gene, or the TIM-3 KO gene.

To evaluate the effect on CD19+ depletion of CD22+ cancer cells, an anti-CD19 construct previously described in: "Waseem Qasim et al., Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells" was also prepared. Science Translational Medicine, January 25, 2017) and additionally containing a suicide gene other than that present in CD22 CAR (described in WO 2016/120216 A1). In the present study, the cytolytic activity was measured:

1) combinations of UCART 19 + UCART 22 (two single-stranded CARs) versus individual cells alone (5×10 ⁶ cells in all cases),

2) TCR-negative cells expressing a bispecific anti-CD22-anti-CD19 CAR (bispecific single chain CAR) compared to individual cells alone (5×10 ⁶ cells in all cases),

3) multi-chain CAR containing both scfv CD22 and CD19 or scfv CD19 CD22 versus single cells alone (5×10 ⁶ cells in all cases).

Expression of RQR8, R2CD22CAR or OR3CD22CAR to confer sensitivity to rituximab/rituximab and QBEND-10

A lentiviral vector cassette that triggers CD22 CAR expression was created to co-express RQR8 (via peptide linker 2A). RQR8 is a depletion receptor ligand that can be activated in the event of uncontrolled adverse events associated with UCART 22 administration. RQR8 is a 136 amino acid artificial cell surface protein that combines antibody-binding epitopes from human CD34 recognized by the QBEND-10 antibody, and human CD20 (Philip et al., 2014). CD20 epitopes present in the construct are recognized by rituximab, allowing deletion of RQR8 expressing cells by complement-mediated cell cytolysis (CDC) and antibody-mediated cell cytotoxicity (ADCC) in the presence of rituximab and/or QBEND-10.

In other experiments, constructs corresponding to anti-CD22 CARs were generated that contained 2 rituximab-recognized epitopes (R) (R2 CD22 CAR: two rituximab-recognized epitopes were inserted immediately after the scfv and immediately before the hinge or instead of the hinge to shorten the extracellular domain compared to with full hinge construct), or "QR3" QR3 CD22 CAR (Q: epitope recognized by QBEND-10, Q-R - at the N-terminus before the VH-linker-VL RR hinge TM-4-1BB-CD3zeta), and tested in against cells expressing different levels of CD22.

Knockout "turning off" the TRAC gene to limit the alloreactivity of UCART 22

A possible limitation of allogeneic adoptive immunotherapy approaches is that recognition of MHC antigens is mismatched between donor and recipient due to the TCRαβ complex of donor cells, which can lead to activation/proliferation of donor T cells and the development of graft-versus-host disease (GvHD) . TCRαβ consists of α- and β-subunits, while TCRα is encoded by one gene, and TCRβ by two homologous genes.

UCART 22 cells were genetically modified to specifically disrupt the T cell receptor α-constant (TRAC) alpha gene. Inactivation of the TRAC gene interfered with cell surface expression of the TCRαβ complex, eliminating TCR-mediated histocompatibility antigen recognition that could lead to GvHD. At the end of the UCART 22 production process, the remaining TCRαβ+ was depleted, ensuring a residual level of ≤3.0% TCRαβ+ cells in the final product, according to the release conditions (criteria).

CD52 gene knockout to confer resistance to alemtuzumab

UCART 22 was designed to be a mixture of CD52 ^- and CD52 ⁺ cells, which allowed the use of alemtuzumab in the mode of lymphodepletion before the introduction of UCART 22. Alemtuzumab is a monoclonal antibody that binds to human CD52. Thus, the CD52 ^- UCART 22 fraction is resistant to alemtuzumab.

TALEN® technology was used to disrupt both the TRAC and CD52 genes. During the preparation of UCART 22, TRAC- and CD52-TALEN® were introduced into cells as messenger ribonucleic acid (mRNA) using an electroporation system. This transient expression allows TALEN® to perform an effective targeted gene knockout prior to cell cleavage. This approach prevents potential risks associated with long-term expression of nucleases in cells injected into the body of patients.

B2M gene knockout to limit UCART 22 alloreactivity

TALEN® technology was used to destroy both TRAC and B2M genes. During the preparation of UCART 22, TRAC- and B2M-TALEN® were introduced into cells as messenger ribonucleic acid (mRNA) using an electroporation system. This transient expression allows TALEN® to perform an effective targeted gene knockout prior to cell cleavage.

Examples

Sources of T cells used in preclinical studies (pharmacology and toxicology)

Description of cell products used in preclinical studies is summarized in Table B.

conclusions

Activation of UCART 22 is triggered by contact between the anti-CD22 chimeric antigen receptor (CD22 CAR) and the CD22 antigen, resulting in destruction of CD22+ B-ALL cells by T-cell mediated cytotoxicity and possibly the production of pro-inflammatory cytokines.

The correctness of the concept has been validated in ongoing trials to develop other UCART products (UCART 19 and UCART 123), demonstrating that UCART cells (CAR T cells deficient in TCRαβ) have the same activity as CAR T cells that do not subjected to gene editing (TCRαβ-positive CAR T cells), in vitro and in vivo in NSG mouse tumor xenograft models (Poirot et al., 2015). This was confirmed for TCRαβ-deficient CD22 CAR T cells and TCRαβ-deficient CAR T cells, MHC I deficiency compared to TCRαβ-positive CD22 CAR T cells.

Data from in vitro pharmacological studies conducted during the UCART 22 preclinical trial are summarized in the table. Experiments have shown:

- Antitumor activity of UCART 22 via CD22 antigen dependent cytotoxicity and cytokine secretion assayed. Activity assays were performed using B-ALL cell lines and primary B-ALL samples.

- Efficient TALEN®-mediated inactivation of the TRAC and CD52 genes.

- Efficient TALEN®-mediated inactivation of the TRAC and beta-2-microglobulin genes.

- Resistance of CD52 ^{- -} T cells to alemtuzumab.

- Efficient elimination of RQR8+ cells or R2 CD22 CAR+ cells with rituximab.

In vivo pharmacological studies (results summarized in Table E) demonstrated:

- Antitumor activity of UCART 22 in vivo against immunocompromised mouse tumor grafts derived using the B-ALL cell line. The in vivo activity of UCART 22 obtained in accordance with GMP requirements was confirmed against the B-ALL cell line.

- Increased in vivo antitumor activity of UCART 22/19 against immunocompromised mouse tumor grafts derived from the B-ALL cell line. The in vivo activity of UCART 22/19 and 19/22, obtained in accordance with the requirements of GMP, was confirmed against the B-ALL cell line.

- The ability of rituximab to eliminate RQR8+ cells in a model created using immunocompetent mice.

To demonstrate the activity of UCART 22 cells, CAR-driven cytotoxicity and cytokine secretion assays were performed against tumor cell lines and primary B-ALL cells.

Evaluation of UCART 22 Activity Using Cytotoxicity Assays Cytotoxic Activity Against B-ALL Cell Lines The potential cytotoxicity of UCART 22 was evaluated against several tumor cell lines derived from B-ALL-affected pediatric and adult patients (Table E). The results demonstrated that all lines MHH-CALL-4, MUTZ-5, SEMK2, PALL-2, LAX2, B-ALL-1, NALM-6 and RS4; 11 are CD22+ cell lines expressing different levels of CD22. Two acute myeloid leukemia (AML) cell lines (OCI-AML2 and MOLM13) lacking CD22 expression were used as negative controls.

The specific cytotoxicity of UCART 22 against CD22+ cells was assessed by measuring the viability of CD22+ target cells after co-culture with UCART 22 cells. -ALL target cells with UCART 22. The results demonstrated the cytotoxic activity of UCART 22 cells against a panel of CD22+ B-ALL tumor cell lines. Even when used at the lowest concentrations, UCART 22 has been shown to have specific cell-lysing activity against all CD22-expressing cell lines.

Cytotoxic activity against primary samples of B-ALL

The cytotoxic activity of UCART 22 was confirmed against several primary B-ALL samples. Characteristics of the primary samples are shown in Table G. Only patient-derived samples containing more than 50% blasts were used in the co-incubation experiments, which included 14 out of 19 samples.

The expression level of CD22 and CD19 was also assessed in all primary B-ALL samples except for Pt1. In 11 samples, the level of CD22 expression on the surface exceeded the level in CD22 ^- -controls and in 4 samples obtained from patients suffering from B-ALL, the level of CD22 expression on the surface was found in the range characteristic of B-ALL cell lines (>1000 CD22 molecules / cell). Samples with high levels of CD22 expression (>60%) also showed CD19 expression, with the exception of two samples of recurrent cancer.

Using the same approach as used for cell lines, CTL activity was confirmed for 8 B-ALL patient samples (more than 10% specific cell lysis).

Analysis of cytokine secretion in the presence of B-ALL cell lines and primary B-ALL samples

The release of interferon-gamma (IFNγ) and other cytokines by UCART 22 cells co-incubated with several cell lines or samples from B-ALL patients was assessed using the BioLegend Legend PLEX 13 cytokine assay. The concentration of IFNγ was found to be the highest of all detected cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22 and factor tumor necrosis alpha (TNF-α)) secreted into the medium after 25 hours of co-incubation of B-ALL cells with either UCART 22 cells or control T cells (NTD DKO).

A high level of IFNγ secretion was detected when UCART 22 was co-incubated with both B-ALL cell lines and B-ALL primary samples.

Secondary in vitro studies

TALEN®-induced inactivation of TRAC, B2M and CD52 genes The possibility of using UCART 22 for allogeneic transplantation depends on the ability to prevent cell surface expression of TCRαβ, MHC I molecules, to eliminate TCR-mediated recognition of histocompatibility antigens, which can lead to GvHD and attack on host CD8+ cells. To effectively eliminate TCRαβ and MHCCI molecules from the cell surface, TALEN® mediated gene editing was used to inactivate the TRAC gene and the beta-2-microglobulin (B2M) gene. In addition, another TALEN® was used during the production of UCART 22 to inactivate the CD52 gene, allowing the use of alemtuzumab in a conditioning regimen that includes lymphodepletion. In specific embodiments, TALEN® has been used during the production of UCART 22 to inactivate the beta-2-microglobulin (B2M) gene, to alter the expression of major histocompatibility complex class I (MCHC class I) molecules, and to prevent host T-cell rejection.

Molecular analysis of the destruction of the genes TRAC, CD52, B2M

To demonstrate targeted modifications of the TRAC and CD52 genes at the molecular level, genomic deoxyribonucleic acid (DNA) was isolated from electroporated T cells using GMP-compliant TRAC- and CD52-TALEN® mRNA or TRAC- and B2M TALEN mRNA. ® and purified to obtain TCRαβ cells MHCCI regions around the TRAC-, B2M-, and CD52-TALEN® cleavage sites were amplified using polymerase chain reaction (PCR) and analyzed by next generation sequencing (NGS, Illumina). In control samples, the same analysis was performed using cells removed prior to electroporation of TALEN® mRNA (day 6). This analysis demonstrated that while no modifications were found in the TRAC and CD52 loci in the control cells at day 6, high frequency modifications were found in the TALEN® treated T cells (Table 6).

Table 3. Percent non-homologous end joining (NHEJ) after TALEN® cleavage at the TRAC and CD52, and B2M loci

Percentages were determined by NGS for TALEN® treated T cells from 3 different batches. Control cells were sampled prior to electroporation of TALEN® mRNA from all batches.

Additional sequence analysis demonstrated that the TALEN®-induced modifications were localized to a small region surrounding the target site, with 93-97% of these modifications being deletions. These deletions tend to be small, with an average of 97% being less than 150 bp and 81% being less than 50 bp.

Functional analysis demonstrating disruption of the TRAC gene in UCART 22

To demonstrate functional inactivation of TCRαβ, the ability of UCART 22 cells to express activation markers such as CD25 and CD69 after phytohemagglutinin (PHA) mediated TCR stimulation was analyzed. UCART 22 was generated using all GMP derived starting products, T cells were isolated either before (TRAC) or after (UCART 22) depletion of TCRαβ+ cells. Cells were reactivated with 0.5 μg/ml PHA for 24 hours. Expression of activation markers (CD25 and CD69) was measured by flow cytometry. While control TRAC cells (65.2% TCRαβ-) were upregulated for CD25 and CD69, no upregulation was found in UCART 22 cells (98.9% TCRαβ-), confirming the absence of the TCRαβ receptor in UCART. 22.

Alemtuzumab resistance in CD52-UCART 22 cells

The possibility of using alemtuzumab, an anti-CD52 monoclonal antibody, in the lymphodepletion regimen depends on the effective elimination of the CD52 glycoprotein from the cell surface of UCART 22 cells to prevent their elimination by alemtuzumab. Thus constructed UCART 22 using CD52-TALEN® was a mixture of CD52+ and CD52- cells. The flow cytometric analysis of generated batches of UCART 22 at the end of the production process showed that, on average, 72% of T cells (CD45+/CD4+ or CD8+ cells) were CD52 ^- cells (range 62.3-76.5%, N= 6).

To demonstrate that CD52.beta. ^- cells are resistant to alemtuzumab, a complement dependent cytotoxicity (CDC) assay was performed. Human T cells with TALEN®-mediated CD52 gene inactivation were treated with rat anti-CD52 at 50 μg/mL, from which the therapeutic antibody alemtuzumab is derived, or with rat immunoglobulin G (IgG) as a control, with or without rabbit complement, for 2 h at 37°C and then analyzed by flow cytometry for viability and CD52 expression.

The results demonstrated that cells without CD52 gene inactivation were specifically eliminated by complement-dependent cytotoxicity in the presence of alemtuzumab.

Resistance to host CD8+ T cells

Class I MHC-deficient UCART 22 cells were resistant to CD8+ T cell-mediated destruction. Interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, enhancing CD8+ T cell-mediated cytolysis of control UCART 22.

Demonstration of effective depletion of UCART 22 after treatment with rituximab (RTX)

UCART 22 are allogeneic T cells and therefore must be eliminated upon detection by the patient's immune system. In addition, UCART 22 was engineered to co-express CD22 CAR and RQR8, a short membrane protein having two epitopes that are bound by rituximab (RTX), a therapeutic monoclonal antibody specific for human CD20. Thus, RQR8 allowed UCART 22 to be depleted by rituximab treatment.

Studied the possibility of elimination of cells UCART 22 by complement-dependent cytotoxicity (CDC) after treatment with RTX. Frozen UCART 22 (from 5 different batches) were thawed and co-cultured with CD22+ Raji cells (Raji cells) at a ratio of 1:0.25 for 3 days. After the indicated reactivation period, a CDC assay was performed (cells were incubated with RTX (100 μg/ml) for 2 h with or without baby rabbit complement (BRC). The results demonstrated that ~85% of CAR+ cells were efficiently eliminated in vitro in the presence of both RTX and complement.

Another mechanism of RTX action by which depletion of RQR8+ cells can be achieved is antibody-mediated cell-dependent cytotoxicity (ADCC) (Seidel et al., 2013). In this case, effector cells of the immune system (mainly natural killer (NK) cells) can actively lyse target cells whose membrane surface antigens are specifically bound by the antibody.

Additional experiments published by Martin Pule at UCL (Philip et al., 2014) demonstrate the in vitro efficacy of mediated elimination of RQR8+ T cells by CDC and ADCC (primary human T cells transduced with a bicilateral retroviral vector (SFG.RQR8.IRES. eGFP) that encodes RQR8 and GFP and selected using Miltenyi QBEND-10 beads). RTX dose dynamics and titration studies have demonstrated that CDC is highly effective at RTX concentrations of 25 µg/mL and above, with cytolysis occurring within 30 minutes. Similarly, sensitivity to mediated ADCC has been demonstrated for RQR8+ T cells.

In vivo studies

Rationale for choosing an animal species/model

Since UCART 22 is specific to humans, studies in standard immunocompetent animal models are unacceptable due to the rapid targeting and elimination of human UCART 22 cells as a result of immune responses to xenogeneic antigens.

Thus, two approaches to in vivo modeling of UCART 22 activity can be considered: syngeneic animal models and models of human tumor xenografts created on immunodeficient animals.

The approach based on the use of syngeneic animal models should include the re-creation/re-development of a species-specific version of UCART 22 for the model organism, followed by the evaluation of the functional properties of this species-specific UCART 22 analog product when transplanted into a syngeneic host. This approach is not considered relevant due to the fact that the properties of individual CARs vary significantly, and since species-specific differences in immune function, physiology and genetics significantly reduce the ability to extrapolate data on the activity of UCART 22 when applied to humans.

An immunocompromised mouse transplant model was chosen because it allows engraftment of both UCART 22 and human CD22+ tumor cells (B-ALL cell lines or primary samples). This animal model has been widely used to evaluate the in vivo activity of CAR T cell therapies. Because these models are subject to the same limitations as the above immunocompetent animal models in terms of tumor-targeted/non-targeted cytotoxicity and non-targeted cytotoxicity and completely eliminate any role of MHC mismatch, they are likely to are very valuable for studying the in vivo antitumor efficacy of CAR T cells. They act as a de facto standard for evaluating the in vivo activity of CAR T cell products (see, e.g., Carpenito et al., 2009; Gade et al., 2005; Gill et al., 2014; Hudecek et al. et al., 2010; Kenderian et al., 2015; Mardiros et al., 2013; Zhou et al., 2013 among many published articles). They have been shown to be able to discriminate between the antitumor activity of different CARs, establish their uptake capacity, and semi-quantify T-cell functions of value for use in humans in vivo (see, for example, Milone et al., 2009). Thus, human CD22+ tumor cells were transplanted into immunocompromised mice (NSG mice) followed by UCART 22 administration, serially assessing tumor burden and lifespan as an indicator of antitumor activity.

Selected Tumor Models

Using tumor xenografts, UCART 22 activity in vivo was evaluated against Daudi cells (Daudi), the CD22+ B-ALL (CCL-213, ATCC) cell line. The cell line was modified to express firefly luciferase (and GFP) by transduction with a lentiviral vector (amsbio LVP438-PBS) to track tumor burden by in vivo imaging (Daudi-Luc-GFP cells).

Selected lines of mice

The following lines of mice were used for in vivo preclinical trials:

1) Highly immunodeficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; Jackson Laboratory strain No. 5557) lacking mature T cells, B cells and functional NK cells to demonstrate antitumor activity.

2) Immunocompetent mouse model (C57BL/6 x Balb/c (F1) mice) to demonstrate the efficiency of the RQR8 or R2 depletion mechanism of the CD22 CAR.

Analysis Methods

The activity of UCART 22 in vivo was assessed using:

- measurements of bioluminescence of treated mice relative to the control group (for a xenograft model obtained using cell lines expressing luciferase),

- clinical signs,

- overall survival of mice.

Route of administration

The route of administration of UCART 22 in animals was by intravenous injection (single undivided dose). In humans, UCART 19 and UCART 22 were also injected intravenously (undivided dose), one, two or three, or four times individually or one after the other according to the following sequence 19. 22 19 22 19 or 22 19 22 19, or 19/22 19 /22.

Demonstration of antitumor activity of UCART 22 in vivo

UCART 22 activity has been demonstrated in immunodeficient mice transplanted with human Daudi-Luc-GFP tumor cells in combination with several batches of UCART 22 generated. An example of the test is shown in FIG. 8. In addition, a confirmatory study was continued to evaluate the in vivo activity of a GMP-created batch of UCART 22.

In general, the method was as follows: NSG mice were injected intravenously with 0.5×10 ⁶ Daudi-Luc-GFP cells on day day -7 and treated on day 0 with UCART 22 (IV injection, 2 doses: 3×10 ⁶ and 10 ×10 ⁶ UCART 22 cells/mouse, 5 mice/group) or left untreated (vehicle injection). Non-transduced double KO TRAC and CD52 T cells (NTD DKO) were injected as controls. The administered doses of UCART 22 were determined in accordance with the doses of CAR+ T cells, the use of which is known from the literature to demonstrate the antitumor activity in vivo of other CD22 CAR T cells (Haso et al., 2013). Treatment with UCART 22 resulted in elimination of tumor cells, as demonstrated by in vivo imaging and extended lifespan, with all treated mice alive at the end of the experiment (80 days post-treatment with UCART 22). These results indicate the antitumor activity of UCART 22.

CD52 cells are resistant to alemtuzumab in vivo and CD52-CAR+ T cells are active in vivo in the presence of alemtuzumab

Experiments in the development of UCART 19, another UCART product containing the same TRAC/CD52 double knockout, demonstrated that CD52 ^- β-T cells are resistant to alemtuzumab in vivo (Poirot et al., 2015).

In addition, the activity of UCART 19 cells in the presence of alemtuzumab was demonstrated in an NSG mouse tumor xenograft model. All mice treated with tumor cells but not treated with UCART 19 cells showed tumor progression leading to the need to kill them 13 days after injection. In 5 of 7 mice engrafted with tumors and treated with UCART 19 cells, tumors were completely controlled by day 13, and two remaining mice showed partial responses. In contrast, alemtuzumab treatment only slowed tumor progression in the absence of UCART 19 cell infusion (6/6 mice). In mice treated with alemtuzumab in combination with UCART 19 cells, tumor cells were eliminated from the bone marrow, as determined by luminometry or flow cytometry of cell suspensions obtained from bone marrow isolated 13 days after injection.

In conclusion, mice treated with alemtuzumab 2 days prior to UCART 19 cell infusion showed antitumor efficacy of UCART 19 cells in the presence of alemtuzumab in vivo. In addition, data from day 13 spleen transplants demonstrated that CD52 ^- β-T cells are resistant to alemtuzumab in vivo.

Demonstration of the efficacy of rituximab-induced depletion of RQR8+ cells in vivo

The sensitivity of RQR8+ UCART 22 to RTX-induced depletion has been previously demonstrated in vitro. In addition, researchers from the Martin Pule group found that RTX had the ability to eliminate RQRS+ cells in an immunocompetent mouse model using RTX, a functional equivalent of human IgGI (mRtx-IgG2a) reengineered to produce mouse IgG2a (Philip et al., 2014). The antibody is adapted for use in murine systems by transferring RTX binding sites to the murine Fc domain, which allows binding to murine Fc receptors with approximately the same affinity with which RTX binds to human Fc receptors. The study was performed using an immunocompetent haploidentical adoptive transfer model using RQR8-transduced C57BL/6 mouse splenocytes transferred into recipients that were non-lethally dosed C57BL/6 x Balb/c crosses (F1). This model allowed for high levels of engraftment in all lymphoid tissue maintained by allogeneic stimulation, but also with preservation of endogenous lymphocytes.

On day 1, 1.5×10 ⁶ RQR8+ donor splenocytes from C57BL/6 mice (splenocytes transduced with a retrovirus encoding the RQR8-2A-GD2CAR construct and purified using Miltenyi CD34 beads) were intravenously injected into F1 mice (C57BL/6 x Balb/c) 4 hours after preconditioning by X-ray irradiation with a dose of 5 Gy. On day 7, engraftment of donor cells was confirmed by flow cytometry in peripheral blood. Mice were treated three times with "murinized" RTX (ritux-mIgG2a 150 μg, intravenous injection in the tail vein on day 7, day 10 and day 12) or PBS (untreated mice). Each group consisted of 5 mice. Animals were sacrificed on day 14 for analysis by flow cytometry of the spleen, bone marrow, blood and lymph nodes. RTX-treated mice showed 50%, 60%, and 70% depletion of RQR8+ cells in spleen, bone marrow, and blood, respectively, within 6 hours of ritux-mIgG2a administration to mice.

A similar study was also performed using anti-CD19 CAR, which was used as a product of UCART 19. UCART 19 is also a product in the form of allogeneic engineered CAR T cells containing the same RQR8 construct as UCART 22.

Repetitive dosing (redosing)

In this experiment, the antitumor activity of a single injection or multiple injections of UCART 22 was investigated. The B-ALL tumor cell line expressing CD22 (Daudi cells expressing high levels of CD22) was used. Cells were transduced with a lentiviral vector to express GFP and firefly luciferase.

Tumor cells (0.5×10 ⁶ Daudi-Luc-GFP) were intravenously injected on day -7 to NSG mice (NOD.Cg-Prkdcscid I12rgtm1Wjl/SzJ, line 005557, The Jackson Laboratory) via the tail vein. On day 0, mice were randomly divided into 11 groups of 6 mice depending on the bioluminescent signal measured on day -1 and body weight.

Mice were treated 1, 2 or 3 times with UCART 22 (using 1 or 3 million CAR+ cells/mouse). UCART 22 was intravenously injected into mice on day 0 +/- day 10 +/- day 20.

The antitumor activity of UCART 22 was assessed by tumor burden determined by bioluminescent imaging on day -1, day 7, day 14, day 21, day 28 and day 35, using a survey of clinical signs and survival of mice.

Daudi model. treatment 1×10 ⁶ UCART 22 cells/mouse

Mice treated 2 or 3 times with 1×10 ⁶ UCART 22 showed better control of tumor progression and longer survival compared to mice treated with 1×10 ⁶ UCART 22 once (80% survival at day 60 using 3 doses compared to 0% when using 1 dose).

In one experiment, UCART 19 was intravenously injected into mice on day 30. In this case, the mice remained alive, and the level of cancer cells was below that set on day 90.

conclusions

UCART 22 activity has been demonstrated in vitro and in vivo against B-ALL cell lines and in vitro against primary B-ALL samples.

UCART 22 are allogeneic T cells and should therefore be eliminated after the patient's immune system has recovered. In addition, UCART 22 was designed to co-express RQR8, a short membrane protein having two epitopes that are bound by the monoclonal antibody rituximab. Thus, RQR8 allows RQR8+ depletion of UCART 22 with rituximab treatment in case of unmanaged UCART 22 related toxicity such as cytokine release syndrome (CRS) or GvHD prior to allo-HSCT (Allogeneic Hematopoietic Stem Cell Transplantation). The efficacy of rituximab in eliminating RQR8+ cells in vitro has been demonstrated using the CDC and ADCC assays. In addition, the efficacy of rituximab-induced depletion of RQR8+ cells has been demonstrated in vivo in blood, spleen, bone marrow, and lymph nodes in an immunocompetent mouse model and using a "murinized" version of rituximab.

Similar results were obtained using anti-CD22 CAR R2 or QR3 expressed in TCR deficient T cells, MHC class I deficient.

Resistance to alemtuzumab in CD52 ^- cells has been demonstrated in vitro and in vivo, which allows the potential use of alemtuzumab in the lymphodepletion mode.

Finally, resistance to CD8+ T cell-mediated destruction was found with R2, an anti-CD22 CAR expressed in TCR-deficient, MHC class I-deficient T cells.

Additional studies that were performed to assess the various risks are summarized in Table I and Table K and are detailed in the section below.

The potential risk of GvHD was assessed in immunocompromised mice earlier in the development of UCART products. The use of this animal model allowed efficient engraftment of human T cells and, as previously demonstrated, this model stably developed xenogenic GvHD after injection of human PBMCs or T cells (Ali et al., 2012; Schroeder and DiPersio, 2011).

Off-target CD22 CAR activity

The CD22 specificity of CD22 CAR was assessed using a GLP-compliant tissue cross-reactivity study demonstrating no unexpected off-target binding of the CD22 CAR scFv component to human tissues, and screening is ongoing using Retrogenix technology. . In addition, published data from a clinical study in a patient treated with m971-derived CAR-expressing T cells reported no off-target toxicity (Fry et al., 2017).

Because CD22 is expressed on healthy B cells, the risk of B cell aplasia resulting from/but not tumor-targeted activity of UCART 22 is possible with prolonged presence of UCART 22.

GVHD linked by UCART 22 CAR

The main safety risk of the UCART 22 product associated with the potential ability of UCART 22 to mediate a GvH response was eliminated by disrupting the TRAC gene and clearing TCRap cells. This risk was assessed earlier during the development of UCART products (UCART 19 and UCART 123) in preclinical in vivo trials. Signs of GvHD were found in all NSG mice injected with unmodified T cells, with more severe symptoms found at the highest dose, but no treatment-related changes were found in mice injected with UCART. To date, in a clinical trial setting, of 14 patients treated with UCART 19, 4 developed mild to moderate cutaneous GvHD (three scores of 1 and one score of 2) (Qasim et al., 2017), and these data are presented in ASH (American Hematological Society) convention in 2017 R. Benjamin and W. Qasim), and she did not require systemic treatment.

CRS

Other potential safety risks associated with the administration of immunotherapeutic products and the disease for which they are used have been identified: infusion-associated reaction, cytokine release syndrome (CRS), tumor lysis syndrome, infections, and neurotoxicity. CRS is a common adverse event with immunotherapy and especially with CAR T cells. In clinical trials of autologous anti-CD19 CAR T cells, it was found that the intensity of cytokine release depends on a combination of several parameters such as dose of injected CAR T cells, CAR signaling domains (4-1BB/CD28), level of proliferation of CAR T cells, and tumor burden of the patient on the day of treatment. These risks were assessed in vivo in a model corresponding to the recently described (Taraseviciute A., Kean L. and Jensen M.C. Creation of the First Non-Human Primate Model That Faithfully Recapitulates Chimeric Antigen Receptor (CAR) T Cell-Mediated Cytokine Release Syndrome (CRS) and Neurologic Toxicity Following B Cell-Directed CAR-T Cell Therapy Blood, 128(22), 2016, p.651 Accessed March 28, 2018 Retrieved from http://www.bloodjournal.org/content/ 128/22/651).

Initial dose recommendation for FIH studies

Standard methods for determining acceptable doses for clinical trials are not suitable for CAR T cell based immunotherapy due to the limited relevance of the animal model(s), the biodynamic nature of the product, and the immunoadaptive mechanism of action.

Therefore, the selection of the initial clinical dose is mainly based on (i) existing experience with the use of CAR T cells in humans (autologous CAR T cell literature); (II) cases in which care is needed and data obtained for patients who are included in the ongoing UCART 19 clinical trials at University College London (UCL) and King's College London (KCL) (ref. to (Qasim et al., 2017) , and presentations by R. Benjamin and W. Qasim at the 2017 ASH convention) and (III) doses currently explored or approved in studies of other autologous CARs for the treatment of B-ALL. This approach is described in the section on clinical trials.

Early results from clinical trials demonstrated a strong reduction in tumor mass with both assays (>80%) in patients treated with UCART 22. In addition, data demonstrated that even a short but effective treatment (two doses) could interrupt the development of malignant-associated B- disease cells and cleared (73% remission after 150 days), primarily after repeated dosing and/or use of UCART 22 and 19.

Claims (22)

1. Engineered human T-cell receptor knockout (TCR-KO) cell expressing on its surface a chimeric cassette-of-differentiation-specific antigen receptor (CAR) 22 (CD22) (UCART 22) and a safety switch , for preventing or treating a patient suffering from CD22+ mediated cancer or CD22+ mediated inflammatory disease, wherein said anti-CD22 CAR (CD22 CAR) comprises: I) an extracellular domain containing: - hinge domain from CD8alpha and - antigen-binding domain specific for CD22, ii) a transmembrane domain from CD8alpha, and III) intracellular signaling domain; wherein said CD22 CAR has a polypeptide sequence at least 80% identical to SEQ ID NO: 15, and said security switch contains: - RQR8 temporally linked to CD22 CAR by peptide 2A, or - two rituximab monoclonal antibody specific epitopes, or three rituximab monoclonal antibody specific epitopes, or three rituximab monoclonal antibody specific epitopes and QBEND-10 monoclonal antibody specific epitopes associated with CD22 CAR. 2. UCART 22 according to claim 1, wherein said extracellular domain further comprises a leader sequence. 3. UCART 22 according to claim 1 or 2, in which the polynucleotide sequence is at least 80% identical to SEQ ID NO: 11, is integrated into the genome and additionally contains an inactivated gene encoding the alpha constant of the T-cell receptor (TRAC) with an insertion , a deletion or mutation in SEQ ID NO: 18, with an undetectable level of T cell receptor (TCR) on the cell surface as determined by flow cytometry and an undetectable level of off-target events as determined by using guide sequence technology. 4. UCART 22 according to claim 3, wherein said anti-CD22 CAR is inserted preferably into the TRAC gene, preferably having SEQ ID NO: 18. 5. UCART 22 according to paragraphs. 1-3 containing another inactivated gene selected from the deoxytidine kinase (dCK) gene, the beta2 microglobulin (B2M) gene, the cluster of differentiation 52 (CD52) gene, preferably the CD52 gene. 6. UCART 22 according to one of paragraphs. 1-5, in which at least one additional gene is inactivated, where the specified gene is selected from the gene encoding aryl hydrocarbon receptor (AHR), transforming growth factor receptor (TGF receptor beta), interleukin 10 receptor (IL-10 R), protein 1 programmed cell death, their combinations. 7. UCART 22 according to one of paragraphs. 1-6, containing an additional single-chain variable fragment (scfv) specific for one of the following tumor-associated surface antigens, which are selected from differentiation cluster 19 (CD19), differentiation cluster 20 (CD20), differentiation cluster 30 (CD30), a major molecule histocompatibility complex (MCHC), immunoglobulin (Ig), differentiation cluster 3 (CD3), differentiation cluster 5 (CD5), differentiation cluster 34 (CD34), differentiation cluster 79 (CD79), preferably from differentiation cluster 79b (CD79b), differentiation cluster 138 (CD138), B7-1 differentiation cluster 80 (CD80), B-cell maturation antigen (BCMA CD269 or TNFRSF 17), Fms-like tyrosine kinase 3 (FLT-3) or paired box protein (PAX5), preferably CD19. 8. UCART 22 according to one of paragraphs. 1-7, wherein said CD22 CAR further comprises an antigen-binding domain specific for CD19, or said UCART 22 further comprises a CD19 CAR, preferably a CD19 CAR, at least 80% identical to SEQ ID NO: 25 or SEQ ID NO: 26 . 9. The population of cells for the prevention or treatment of a patient suffering from CD22+ mediated cancer or CD22+ mediated inflammatory disease, where the specified population contains UCART 22 according to one of paragraphs. 1-8. 10. Pharmaceutical composition for the prevention or treatment of a patient suffering from CD22+ mediated cancer or CD22+ mediated inflammatory disease, containing UCART 22 according to one of paragraphs. 1-8 or a cell population according to claim 9 and a pharmaceutically acceptable excipient. 11. Pharmaceutical composition according to claim 10, additionally containing bryostatin, preferably bryostatin-1. 12. Pharmaceutical composition according to one of paragraphs. 10 or 11 for the treatment of CD22-mediated hematologic cancer selected from lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, multiple myeloma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma, acute lymphocytic cancer, acute myeloid leukemia, relapsed or refractory CD22-expressing hematologic cancer, and an aggressive form of said CD22-associated hematologic cancer. 13. Pharmaceutical composition according to one of paragraphs. 11 or 12 for the treatment of relapsed or refractory B-cell expressing acute lymphocytic leukemia, preferably in pediatrics. 14. Pharmaceutical composition for use according to claim 12 or 13, where the treatment of the patient includes the introduction of at least one monoclonal antibody (MAb), preferably QBEND-10 and / or rituximab, to the specified patient at a dose that ensures contact of the specified UCART 22 at least with one specific MAT.

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