WO2024121385A1

WO2024121385A1 - Two-dose regimen in immunotherapy

Info

Publication number: WO2024121385A1
Application number: PCT/EP2023/084892
Authority: WO
Inventors: Mark Gerard FRATTINI; Kathryn Jean NEWHALL; Ana Beatriz Corrêa KORNGOLD
Original assignee: Cellectis S.A.
Priority date: 2022-12-09
Filing date: 2023-12-08
Publication date: 2024-06-13

Abstract

This document relates to methods of immunotherapy comprising administering CAR-T cells to a subject in need thereof, in particular a 2-dose regimen of CAR-T therapy, and materials and compositions to carry out such methods.

Description

TWO-DOSE REGIMEN IN IMMUNOTHERAPY

FIELD OF THE INVENTION

The present document generally relates to the field of cancer, in particular, cell therapies and immunotherapies for the treatment of solid tumors or haematological cancers in patients.

BACKGROUND

Adoptive cell therapy, also known as cellular immunotherapy, is a form of treatment that uses the cells of the immune system to eliminate pathological cells, such as infected or malignant cells. Some of these approaches involve directly isolating a person’s own immune cells and simply expanding their numbers, whereas others involve genetically engineering immune cells from patients (autologous approach) or donors (allogeneic approach) to boost and/or redirect them towards specific target tissues. In the case of cancer, immune cells, especially immune cytolytic and helper T-lymphocytes, Natural Killers and Macrophages, are particularly powerful against cancer, due to their ability to bind to markers known as antigens on the surface of cancer cells. Cellular immunotherapies take advantage of this natural ability and can be deployed in different ways: Tumor-Infiltrating Lymphocyte (TIL) therapy, Engineered T Cell Receptor (TCR) cell therapy, Chimeric Antigen Receptor (“CAR”) immune cell therapy, and Natural Killer (NK) cell therapy.

Chimeric antigen receptors-expressing immune cells are cells that have been genetically engineered to express chimeric antigen receptors (CARs) usually designed to recognize specific tumor antigens and kill cancer cells that express said tumor antigen(s). These are generally T-cells expressing CARs (“CAR-T cells”), Natural Killer cells expressing CARs (“CAR-NK cells”), or macrophages expressing CARs.

CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signalling domains in a single or multiple fusion molecule(s). In general, the binding moiety of a CAR can include an antigen-binding domain of a single-chain antibody (“scFv”), comprising the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signalling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta (or CD3Q or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signalling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS, and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T-cells. CARs have successfully allowed T-cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Doth et al. Blood (2010) 116(7):1035-44).

Adoptive immunotherapy, which involves the transfer of autologous or allogeneic antigen-specific T-cells generated ex vivo, is a promising strategy to treat viral infections and cancer as confirmed by the increase in the number of clinical trials involving CAR-T cells.

Despite recent technical advances, the treatment of cancer remains a great challenge in healthcare. For instance, so far, CAR-Ts have been administered according to a single dose strategy, possibly repeated if the first dose was not sufficient to lead to a complete patient’s response, or according to a split dosing strategy to reduce toxic effects.

What is needed are new modes of treatment of cancers which improve the efficacy of cell-therapies, in particular CAR-T based therapies, while limiting or controlling their toxicity in the patients.

This background information is provided for informational purposes only. No admission is necessarily intended, nor should it be constmed, that any of the preceding information constitutes prior art against the present invention.

SUMMARY

This document provides methods and materials for treating cancer. For example, this document provides a new regimen of administration of CAR-T cells that improves the efficacy of the treatment. It is to be understood that both the foregoing general description of the embodiments and the following detailed description are exemplary, and thus do not restrict the scope of the embodiments.

The methods and materials provided herein are particularly suited for treatment of cancers characterized by the presence of CD 123 in the tumor. The methods and materials provided herein also are particularly suited to achieve a “universal” treatment, where the components of the treatment can be used in many unrelated patients.

In general, one aspect of this document features a method of treating a subject having a cancer, the method comprising administering successively, to the subject in need thereof, a first and second doses of engineered T-cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CARs targeting specifically an antigen associated with the cancer,

- wherein the second dose of CAR-T cells is administered between 10 and 17 days after the first dose of CAR-T cells, such as after 10, 11, 12, 13, 14, 15, 16 or 17 days;

- wherein, prior to the administration of the first dose of CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood;

- wherein no lymphodepleting treatment is administered between the first and second doses of CAR-T cells or concomitantly to the second dose of CAR-T cells; and/or for 28 days after the administration of the first dose of CAR-T cells.

Another aspect relates to a pharmaceutical composition comprising one dose of CAR-T cells for use in the treatment of a subject having a cancer, according to a method as described herewith comprising administering successively a first and second doses of CAR-T cells, wherein said CAR targets specifically an antigen associated with the patient’s cancer.

A still other aspect relates to a combination of a pharmaceutical composition comprising a first dose of CAR-T cells and a pharmaceutical composition comprising a second dose of CAR-T cells, said CARs targeting, respectively, specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer, according to the method described above.

In some aspects of this document, said methods and compositions for use refer to a pre-conditioning lymphodepleting treatment comprising a CD52-antibody such as alemtuzumab.

In some aspects of this document, said methods and compositions for use make use of engineered allogeneic CAR-T cells which are CD52 negative and TCR negative.

In particular aspects of this document, are provided methods involving specific chimeric antigen receptor (“CD123CAR” or “CAR”) expressed at the cell surface of T- cells, more particularly TCR-negative- and CD52 negative- T-cells, to be administered according to a 2-dose regimen after a pre-conditioning lymphodepleting treatment as disclosed herewith that is carried out in patients having a cancer, in particular AML.

Main embodiments

Embodiment 1. A method of treating a subject having a cancer, the method comprising administering successively, to the subject in need thereof, a first and second doses of engineered T-cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CARs targeting specifically an antigen associated with the cancer,

- wherein no lymphodepleting treatment is administered between the first and second doses of CAR-T cells or concomitantly to the second dose of CAR-T cells; and/or for 28 days after the administration of the first dose of CAR-T cells;

- optionally, wherein said CAR-T cells are CD52 negative. Embodiment 2. The method according to embodiment 1, wherein said lymphodepleting treatment comprises administration of an anti-CD52 therapeutic antibody such as alemtuzumab.

Embodiment 3. The method according to any one of embodiments 1 to 2, wherein said lymphodepleting treatment comprises administration of fludarabine or cyclophosphamide.

Embodiment 4. The method according to any one of embodiments 1 to 3, wherein said lymphodepleting treatment comprises administration of fludarabine and cyclophosphamide.

Embodiment 5. The method according to embodiment 4, wherein said lymphodepleting treatment comprises administration of fludarabine, cyclophosphamide and alemtuzumab.

Embodiment 6. The method according to embodiment 5, wherein said lymphodepleting treatment comprises administration of:

- fludarabine at between about 1 and 100 mg/m²/day, between about 10 and 75 mg/m²/day, between about 15 and 50 mg/m²/day, between about 20 and 40 mg/m²/day, or about 25, 30 or 40 mg/m²/day, for 1, 2, 3, 4, or 5 days;

- cyclophosphamide at between about 50 and 10000 mg/m²/day, between 50 and 5 000 mg/m²/day, between 50 and 1000 mg/m²/day, between 100 and 1000 mg/m²/day, between 500 and 1000 mg/m²/day, between 600 and 800 mg/m²/day, or about 600, 650, 700, 750 or 800 mg/m²/day, for 1, 2, 3, or 4 days;

- alemtuzumab at between about 5 and 50 mg/day, between 10 and 40 mg/day, between 10 and 20 mg/day, or at 10, 11, 12, 13, 14, or 15 mg/day, for 1, 2, 3, 4, or 5 days.

Embodiment 7. The method according to embodiment 5 or embodiment 6, wherein said lymphodepleting treatment comprises administration of:

(i) fludarabine at 30 mg/m²/day given over 15 to 30 minutes for 4 days, with a maximum daily dose of 60 mg,

(ii) cyclophosphamide at 750 mg/m²/day given over 1 hour for 3 days, with a maximum daily dose of 1.33 g, and

(iii) alemtuzumab at 12 mg/day over 4 to 6 hours for 4 days. Embodiment 8. The method according to any one of embodiments 1 to 7, wherein:

- the first dose of CAR-T cells comprises between about 10⁴ to about 10⁸ CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CAR-T cells, or between about 10⁵ to 10⁹ total CAR-T cells;

- the second dose of CAR-T cells comprises between about 10⁴ to about 10⁸ CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CAR-T cells, or between about 10⁵ to 10⁹ total CAR-T cells;

- wherein the number of CAR-T cells in the first dose and the number of CAR-T cells in the second dose are identical or different;

- wherein the antigen targeted by the CAR expressed by the CAR-T cells comprised in the first dose is identical or different from the antigen targeted by the CAR-T cells comprised in the second dose.

Embodiment 9. The method according to any one of embodiments 1 to 8, wherein said T- cells are primary T-cells.

Embodiment 10. The method according to embodiment 9, wherein said T-cells are cytotoxic T-lymphocyte and/or a helper T-lymphocytes.

Embodiment 11. The method according to any one of embodiments 9 to 10, wherein said primary T-cells originate from a human.

Embodiment 12. The method according to any one of embodiments 1 to 11, wherein said CAR-T cells are autologous with respect to the subject to be treated.

Embodiment 13. The method according to any one of embodiments 1 to 11, wherein said CAR-T cells are allogeneic with respect to the subject to be treated.

Embodiment 14. The method according to any one of embodiments 1 to 13, wherein said CAR-T cells have been genetically modified to confer resistance to at least one immune suppressive or chemotherapy drug.

Embodiment 15. The method according to any one of embodiments 1 to 14, wherein said CAR-T cells have been genetically modified to confer resistance to alemtuzumab.

Embodiment 16. The method according to embodiment 15, wherein said CAR-T cells have been genetically modified to suppress or repress expression of CD52 at the surface of said CAR-T cells. Embodiment 17. The method according to any one of embodiments 1 to 16, wherein said CAR-T cells have been genetically modified to suppress or repress expression of at least one component of a T-Cell Receptor (TCR) at the surface of said CAR-T cell.

Embodiment 18. The method according to any one of embodiments 1 to 17, wherein said CAR-T cells have been genetically modified to suppress or repress expression of a TCRa gene and/or a TCR0 gene.

Embodiment 19. The method according to any one of embodiments 1 to 18, wherein said CAR-T cells have at least one gene encoding TCR alpha, TCR beta, and/or CD3 that has been inactivated.

Embodiment 20. The method according to any one of embodiments 1 to 19, wherein said CAR-T cells have been genetically modified to suppress or repress expression of at least one gene encoding a MHC-I protein, such as 02m and HLA.

Embodiment 21. The method according to any one of embodiments 1 to 20, wherein said CAR-T cells have a 02m gene that has been inactivated and have, integrated in their genome, an exogenous sequence encoding a NK inhibitor such as a HLA-E peptide fusion protein.

Embodiment 22. The method according to any one of embodiments 1 to 21, wherein said CAR-T cells have been genetically modified to suppress or repress expression of a gene encoding an immune checkpoint protein and/or a receptor thereof.

Embodiment 23. The method according to any one of embodiments 1 to 22, wherein said CAR-T cells have been genetically modified to comprise a suicide gene.

Embodiment 24. The method according to any one of embodiments 1 to 23, wherein said CAR-T cells are one or more of: CD52 negative, TCR negative, B2M negative, PDCD1 negative.

Embodiment 25. The method according to any one of embodiments 1 to 24, wherein said CAR-T cells are at least CD52 negative or at least CD52 and TCR negative.

Embodiment 26. The method according to any one of embodiments 1 to 25, wherein said cancer is an haematological cancer or a solid cancer.

Embodiment 27. The method according to any one of embodiments 1 to 26, wherein said cancer is a leukemia such as leukemia selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

Embodiment 28. The method according to any one of embodiments 1 to 27, wherein said cancer is acute myelogenous leukemia (AML).

Embodiment 29. The method according to any one of embodiments 1 to 28, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting an antigen selected from the group consisting of CD 123, CD38, CLL1, FLT3, CD7, FRbeta, CD33, LEY.

Embodiment 30. The method according to any one of embodiments 1 to 29, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123.

Embodiment 31. The method according to any one of embodiments 1 to 30, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123 having a polypeptide structure comprising an extra cellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, a CD8a hinge, a CD8a transmembrane domain, and a cytoplasmic domain including a CD3 signaling domain and a co -stimulatory domain from 4-1BB, wherein said VH comprises the CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and said VL comprises the CDR sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

Embodiment 32. The method according to embodiment 31, wherein said VH has at least 80% identity with SEQ ID NO: 11 and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 15, and said VL has at least 80% identity with SEQ ID NO: 12 and comprises the CDRs of amino acid sequences SEQ ID NO: 16 to SEQ ID NO: 18.

Embodiment 32. The method according to embodiment 31 or embodiment 32, wherein said VH comprises the amino acid sequence of SEQ ID NO: 11 and said VL comprises the amino acid sequence of SEQ ID NO: 12.

Embodiment 33. The method according to any one of embodiments 31 to 33, wherein said CAR targeting CD 123 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 19, and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to 18.

DESCRIPTION OF THE FIGURES

The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

Figure 1 : schematic representation of immunotherapy protocols following a single dose regimen (A) or a 2-dose regimen (B) as described herewith. DLT: Dose Limiting Toxicity, LD: Lymphodepletion, EOT: End Of Treatment.

Figure 2: Absolute lymphocyte counts measured by a complete cell count and differential (A), CAR-T expansion measured by qPCR (vector copy number/microgram DNA) in whole blood (B), percentage of blasts in bone marrow measured by flow cytometry (C), after a first dose of CAR-T cells administration following a preconditioning lymphodepleting treatment comprising fludarabine and cyclophosphamide.

Figure 3: Absolute lymphocyte counts measured by a complete cell count and differential (A), CAR-T expansion measured by qPCR (vector copy number/microgram DNA) in whole blood (B), percentage of blasts in bone marrow measured by flow cytometry (C), after a first dose of CAR-T cells administration following a preconditioning lymphodepleting treatment comprising fludarabine, cyclophosphamide and alemtuzumab.

Figure 4: Cytokines secretion in patients having received a first dose of CAR-T cells after a preconditioning lymphodepleting treatment comprising fludarabine, cyclophosphamide and alemtuzumab.

DETAILED DESCRIPTION

This document provides methods and materials for use in a 2-dose regimen immunotherapy treatment, in particular a CAR-T based treatment of cancer, that enhances the efficacy of the treatment while avoiding additional toxicities.

This document provides methods for treating a cancer comprising administering successively two doses of engineered T-cells expressing a CAR targeting a cancer to a pre- conditioned subject who received a lymphodepleting treatment that allows depletion of the patient’s immune cells for at least 20 days, or at least 28 days, from the start of lymphodepletion treatment. It also provides compositions comprising a dose of CAR-T cells and combinations of compositions comprising a dose of CAR-T cells for use in said methods for treating a cancer.

It is contemplated that the 2-dose regimen as described herewith could be advantageous, in terms of efficacy and toxicity, for treating patients having a cancer, over a single-dose regimen, possibly repeated 2 times, or a split-dosing regimen. Without willing to be bound by theory, the effects of a 2-dose regimen in a method as described herewith could be analyzed as follows: after a preconditioning lymphodepleting treatment, the first dose of CAR-T cells has a debulking effect on the tumor and creates a microenvironment favorable for CAR-T cells expansion and activity, and the second dose of CAR-T cells, administered in the time-window when these favorable conditions are present and the patient’s own immune cells have not yet recovered, pursues the anti -tumor activity and removes the residual tumor so as to have more chances to achieve a complete response or stable disease in cancer patients treated according to the 2-dose regimen as described herewith.

For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of “or” means “and/or” unless stated otherwise. As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of’ and/or “consisting of’.

As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used.

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

The practice of the present invention will employ, unless otherwise indicated, techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, gene editing, and immunology, which belong to the knowledge of the skilled in the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.l54 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-FV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, immunology, cancer, molecular biology, and gene editing. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia ofMolecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).

As used herein, a "recipient" is a patient that receives a transplant, such as a transplant containing a population of engineered immune cells, e.g. T-cells. The transplanted cells administered to a recipient may be, e.g. autologous, syngeneic, or allogeneic cells.

As used herein, a "donor" is a mammal (e.g. a human) from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient. The one or more cells may be, e.g. a population of immune cells or hematopoietic stem cells to be engineered, expanded, enriched, or maintained according to the methods described herewith prior to administration of the cells or the progeny thereof into a recipient. In the allogeneic setting contemplated herewith, a “donor” is not the patient to be treated.

"Expansion" in the context of cells refers to the increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from expansion.

"Cell population" includes eukaryotic cells, such as mammalian, e.g. human, cells isolated from biological sources, for example, blood product or tissues. A cell population can derive from more than one cell.

As used herein, the term "pharmaceutical composition" refers to the active ingredient in combination with a pharmaceutically acceptable carrier and/or excipient e.g. a carrier and/or excipient commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, such as human beings, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term "administering," refers to the placement of a compound, cell, or population of cells as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Examples of possible routes of administration includes by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. For instance, the compositions described herein may be administered to a patient subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.

Pharmaceutical compositions comprising the compounds or cells disclosed herein can be administered by any appropriate route which results in an effective treatment in the patient. In some cases, the compositions are administered by intravenous injection.

The patient who can be treated with the materials and methods disclosed herewith can be a mammal, including a human and a non-human primate.

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

The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally-occurring amino acids.

As used herein, the terms "treat," "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal (e.g. a human), and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, e.g. causing regression of the disease, e.g. to completely or partially remove symptoms of the disease.

The term "subject" or "patient" as used herein includes mammals including nonhuman primates and humans.

An "effective amount" or "therapeutically effective amount" refers to that amount of a composition described herein which, when administered to a subject (e.g. human), is sufficient to aid in treating a disease. The amount of a composition that constitutes a "therapeutically effective amount" will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. When referring to an individual active ingredient or composition, administered alone, a therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients, compositions or both that result in the therapeutic effect, whether administered concurrently, simultaneously, or sequentially. By "vector" is meant a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A "vector" can include, but is not limited to, a viral vector, a plasmid, an oligonucleotide, a RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adenoassociated viruses (AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g. influenza virus), rhabdovirus (e.g. rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g. Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g. vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) into a genome. The term "locus" can refer to the specific physical location of a rare-cutting endonuclease target sequence on a chromosome. Such a locus can comprise a target sequence that is recognized and/or cleaved by a sequence-specific endonuclease as described herein. It is understood that the locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e. in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples.

The term "cleavage" when used in reference to nucleic acid refers to the breakage of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. Double stranded DNA, RNA, or DNA RNA hybrid cleavage can result in the production of either blunt ends or staggered ends.

"Sequence identity" refers to the identity between two nucleic acid molecules or polypeptides. It refers to the residues in the two sequences which are the same when the sequences are aligned for maximum correspondence. When a position in the compared sequence is occupied by the same base (or amino acid), then the molecules are identical at that position. A degree of identity between nucleic acid sequences (or amino acid sequences) is a function of the number of identical or matching nucleotides (or amino acids) at positions shared by the aligned nucleic acid sequences (or amino acid sequences). Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g. default setting. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated.

In one aspect, this document provides a method of treating a subject having a cancer, the method comprising administering successively, to the subject in need thereof, a first and second doses of engineered T-cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CARs targeting specifically an antigen associated with the cancer,

- wherein, prior to the administration of the first dose of CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment to eliminate, at least partially, the subject’s own immune cells to have, for at least 20 consecutive days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood; - wherein no lymphodepleting treatment is administered between the first and second doses of CAR-T cells or concomitantly to the second dose of CAR-T cells; and/or for 28 days after the administration of the first dose of CAR-T cells;

- optionally, wherein said CAR-T cells are CD52 negative.

In some cases of said methods, prior to the administration of the first dose of CAR- T cells, the subject has been preconditioned with a lymphodepleting treatment to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood.

The materials and steps useful in the methods described herewith are further detailed below.

1. Engineered immune cells

1.2 Source and type of cells

The methods and compositions described herewith can use any type of immune cells, including T-cells, NK-cells, and macrophages.

The source of the engineered immune-cells (e.g. T-cells) useful in the methods described herewith is not particularly limiting. In some cases, the engineered immune cells originate from a healthy donor or from a patient diagnosed with cancer.

In some cases, the source of the immune cells to be engineered are primary cells, and by “primary cell(s)” are intended cells taken directly from living tissue (e.g. biopsy material) and established for growth in vitro for a limited amount of time, meaning that they can undergo a limited number of population doublings. Primary cells are opposed to continuous tumorigenic or artificially immortalized cell lines.

Primary immune cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and from tumors, such as tumor infiltrating lymphocytes. Primary immune cells are collected from donors or patients through a variety of methods known in the art, as for instance by leukapheresis techniques as reviewed by Schwartz et al. (J. Clin. Apher. (2013), 28(3):145-284).

In some cases, said immune cells, e.g. T-cells, are part of a mixed population of immune cells which present different phenotypic characteristics, such as comprising CD4, CD8 and/or CD56 positive cells.

In some cases, the immune cells can derive from stem cells. The stem cells can be adult stem cells, embryonic stem cells, such as 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.

In the present application, the immune cells derived from stem cells, such as those deriving from induced pluripotent stem cells (iPSCs) (Yamanaka et al. (2008) Science. 322 (5903): 949-53) are also regarded as primary immune cells. Lentiviral expression of reprogramming factors has been used to induce multipotent cells from human peripheral blood cells (Staerk et al. (2010) Cell stem cell. 7 (1): 20-4 ; Loh et al. (2010) Cell stem cell. 7 (1): 15-9).

According to some cases, the immune cells can be derived from human embryonic stem cells by techniques well known in the art that do not involve the destruction of human embryos (Chung et al. (2008) Cell Stem Cell 2(2): 113-117).

In some cases, the engineered immune cells are T-cells and can be cytotoxic T- lymphocytes or helper T-lymphocytes.

In some cases, the immune-cells, e.g. T-cells, to be engineered are allogeneic. By “allogeneic” is meant that the cells originate from a donor or are produced and/or differentiated from stem cells in view of being infused into patients having a different haplotype. Such immune cells are generally engineered to be less allor eactive and/or become more persistent with respect to their patient host. More specifically, the method of engineering the allogeneic cells can comprise the step of reducing or inactivating TCR expression into T-cells, or into the stem cells to be derived into T-cells. This can be obtained by different sequence-specific reagents, such as by gene silencing or gene editing techniques by using for instance nucleases, base editing techniques, shRNA and RNAi as non-limited examples. In contrast, cells originating from the patient who will receive the engineered cells as part of an immunotherapy treatment are said to be “autologous” with respect to said patient.

1.2 Genetic engineering

The engineered immune-cells useful in the methods and compositions described herewith can be engineered ex-vivo to modify their immune specificity in order to perform adoptive immunotherapy, improve their anti-tumoral activity, reduce their immunogenicity, increase their persistence, and/or suppress their activity if necessitated by serious side-effects or threat to the patient’s life.

The cells can be genetically modified by using viral vectors and/or transient expression of rare-cutting endonucleases to introduce transgenes or inactivate endogenous genes as further described in the present specification. These techniques have been extensively reviewed in the art, like for instance by Maeder et al (Molecular Therapy (2016) 24(3): 430-446).

For instance, a gene can be inactivated or repressed by using rare-cutting endonuclease able to selectively inactivate by DNA cleavage that gene locus. Such rare- cutting endonuclease may be a TALE-nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease (such as Cas9).

A gene’s expression can also be inhibited or repressed by using (e.g. introducing into the T-cell) a nucleic acid molecule that specifically hybridizes (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA corresponding to that gene, thereby inhibiting transcription and/or translation of the gene. In some cases, the inhibition of expression a gene can be achieved by using (e.g. introducing into the T-cell) an antisense oligonucleotide, small interfering (siRNA), or short hairpin RNA (shRNA).

In some cases, the cells can be genetically engineered by using methylases, exonucleases, histone deacetylases, end-processing enzymes such as exonucleases, and more particularly cytidine deaminases such as those coupled with the CRISPR/cas9 system to perform base editing (i.e. nucleotide substitution) without necessarily resorting to cleavage by nucleases as described for instance by Hess et al. (Mol Cell. (2017) 68(1): 26- 43) and Rees et al. (Nat. Rev. Genet. (2018) 19, 770-788). In some cases, gene editing can be performed using a sequence-specific nuclease reagent, such as a sequence-specific endonuclease like a rare-cutting endonuclease like TALE Nuclease, or a RNA guide coupled with a guided endonuclease like CRISPR.

The terms “sequence-specific nuclease reagent” include reagents that have nickase or endonuclease activity. The sequence-specific nuclease reagent can be a chimeric polypeptide comprising a DNA binding domain and another domain displaying catalytic activity. Such catalytic activity can be for instance a nuclease to perform gene inactivation, or nickase or double nickase to preferentially perform gene insertion by creating cohesive ends to facilitate gene integration by homologous recombination.

The term “endonuclease” generally refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, a DNA molecule. Endonucleases (and, thus, sequence-specific endonucleases) do not cleave the DNA or RNA molecule irrespective of its sequence but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as “target sequences” or “target sites”. Endonucleases can be classified as rare-cutting endonucleases when having typically a polynucleotide recognition site greater than 10 base pairs (bp) in length, or of 14-55 bp. Rare-cutting endonucleases significantly increase homologous recombination by inducing DNA double-strand breaks (DSBs) at a defined locus thereby allowing gene repair or gene insertion therapies (Pingoud and Silva (2007) Nat. Biotechnol. 25(7): 743-4).

In some cases, the sequence specific-reagent can be a base editor able to perform base editing as described for instance inKomor eta/. (Nature (2019) 533(7603), 420-424) and in Mok et al. (Nature (2020) 583:631-637).

The term “base editor”, as used herein, refers to a catalytic domain capable of making a modification to a base ( e.g. A, T, C, G, or U) within a nucleic acid sequence by converting one base to another (e.g. A to G, A to C, A to T, C to T C to G, C to A, G to A, G to C, G to T, T to A, T to C, T to G). Base editors can include cytidine deaminases that convert target C/G to T/A and adenine base editors that convert target A/T to G/C. Adenosine deaminase can be, for instance, TadA or its variant TadA7.10 as described by Jeong etal. (Nat Biotechnol (2021) 39, 1426-1433). Different members of Apolipoprotein B mRNA editing enzyme (APOBEC) family can be used to convert cytidines to thymidines, such as the murine rAPOBECl and the human APOBEC3G as developed by Lee et al. (Science Advances (2020) 6(29)).

The sequence specific-reagents as defined herewith include TALE-base editors (BE), which can be generated by the fusion of transcription activator-like effector array proteins (TALE) with a base editor catalytic domain. The base editor catalytic domain can be a double-stranded DNA deaminase (“DddA”) that precisely makes nucleotide changes and/or corrects pathogenic mutations, rather than destroying DNA by double-strand breaks (DSBs). For instance, Mok et al. (Nature (2020) 583:631-637) recently developed TALE base editor by using the bacterial cytidine deaminase toxin DddAtox, from Burkohlderia cenocepacia, that has been split into non-toxic halves which have been fused to the C- terminus of paired (left and right) TALE binding domains, respectively, to form heterodimeric TALE base editors. In such setting, the deaminase DddAtox becomes active when its two halves, linked to their respective TALE binding domains, co-localize at a predetermined genomic locus. The split “DddA-N half and “DddA-C half’ can be obtained by cleaving the full DddAtox protein (SEQ ID NO: 87) at positions 1333 or 1397.

In some cases, such TALE-base editors can also comprise a domain that inhibits uracil glycosylase referred to as “UGI”, and/or a nuclear localization signal. The term “uracil glycosylase inhibitor” or “UGI,” as used herein, refers to a protein that is capable of inhibiting an uracil-DNA glycosylase base-excision repair enzyme. In some cases, a UGI domain can comprise a wild-type UGI or a canonical UGI. In some cases, the UGI proteins can include fragments of UGI and proteins homologous to a UGI or a UGI fragment, which are useful to improve the specificity of base editing performed at a predetermined locus.

1.2.1 expression of an antigen-targeting receptor

The engineered immune cells useful in the methods described herewith comprise an exogenous nucleic acid sequence encoding a recombinant TCR or a chimeric antigen receptor (CAR) targeting specifically an antigen associated with a tumor or cancer (also referred herewith as “tumor antigen”). Thus, the engineered immune cells can express a recombinant TCR or a chimeric antigen receptor (CAR) specific for a tumor antigen, such as a tumor antigen associated with AML. In some cases, the engineered immune cells useful in the methods and compositions for use described herewith express a CAR at their cell surface and comprise an exogenous sequence, stably inserted into their genome, encoding said CAR.

By “recombinant TCR” is meant an artificially modified T-cell receptor in which at least one of the TCR components is obtained by expression of an exogenous polynucleotide. The intracellular signalling domain of a recombinant TCR can be derived from the cytoplasmic part of a membrane bound receptor to induce cellular activation, e.g., the Fc epsilon RI receptor gamma-chain or the CD3 zeta-chain. By use of this type of recombinant receptor, one can combine the advantages of MHC-independent, antibodybased antigen binding with efficient T-cell activation upon specific binding to the receptor ligand. This approach can be regarded as an alternative to CARs for the engineering of antigen-specific T-cells for immunotherapy (Hornbach et al. (2002) Curr Gene Ther. 2(2):211-26). After introduction of the exogenous polynucleotide encoding a TCR in an immune cell, the recombinant TCR partially or completely replaces the expression of the endogenous TCR in said cell. In some cases, a recombinant TCR recognizes a tumor- expressed peptide/MHC complex. In some cases, a recombinant TCR a/p comprises an extracellular ligand binding domain and a transmembrane domain without stimulatory and/or co -stimulatory domain. Indeed, typically, recombinant TCR a/p do not contain activation or costimulation domains as they depend on endogenous CD3 chains for activation and endogenous CD28 for costimulation.

By “chimeric antigen receptor” or “CAR” is generally meant a synthetic receptor comprising a targeting moiety (also called “binding moiety”) that is associated with one or more signaling domains in a single fusion molecule. As defined herein, the term “chimeric antigen receptor” covers single chain CARs as well as multi-chain CARs. In some cases, the binding moiety of a CAR can comprise an antigen-binding domain of a single-chain antibody (scFv), comprising light chain and heavy chain variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti -tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX- 40 (CD134), and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T-cells. CARs are not necessarily only single chain polypeptides, as multi-chain CARs are also possible. According to the multi-chain CAR architecture, for instance as described in WO 2014/039523, the signalling domains and co-stimulatory domains are located on different polypeptide chains. Such multi-chain CARs can be derived from FcsRI, by replacing the high affinity IgE binding domain of FcsRI alpha chain by an extracellular ligand-binding domain such as scFv, whereas the N- and/or C-termini tails of FcsRI beta and/or gamma chains are fused to signal transducing domains and co- stimulatory domains, respectively. The extracellular ligand binding domain has the role of redirecting the immune cell (e.g. T-cell) specificity towards cell targets, while the signal transducing domains activate the immune cell response. CARs are generally expressed in effector immune cells to redirect their immune activity against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors. A component of a CAR is any functional subunit of a CAR that is encoded by an exogenous polynucleotide sequence introduced into the cell. For instance, this component can help the interaction with the target antigen, the stability or the localization of the CAR into the cell.

While the CARs expressed in the immune cells useful in the methods herein are not limited to a specific CAR structure, a nucleic acid that can be used to engineer the immune cells generally encodes a CAR comprising: an extracellular antigen -binding domain that binds to a tumor antigen, a hinge, a transmembrane domain, and an intracellular domain comprising a stimulatory domain and/or a primary signalling domain. Generally, the extracellular antigen-binding domain is a scFv comprising a Heavy variable chain (VH) and a Light variable chain (VL) of an antibody binding to a tumor antigen connected via a Linker. The extracellular antigen-binding domain can also derive from a single domain antibody (e.g. a nanobody) or from an ankyrin repeat domain. Thus, the extracellular antigen-binding domain can comprise one Heavy variable chain and no Light variable chain. Walser et al. (Viruses (2022): 14, 2242) describe ankyrin repeat domains. The transmembrane domain can be, for example, a CD8a transmembrane domain, a CD28 transmembrane domain, or a 4- IBB transmembrane domain. The co-stimulatory domain can be, for example, the 4-1 BB co-stimulatory domain or CD28 co-stimulatory domain. The primary signalling domain can be, for example, the CD3^ signalling domain.

The CARs expressed by the engineered immune cells described herewith can also comprise a signal peptide to direct the nascent protein to the endoplasmic reticulum and subsequent expression at the engineered cell’s surface. The signal peptide is cleaved after addressing the CAR to the cell surface.

Table 1 presents sequences of different domains typically present in a CAR.

Table 1: Sequences of some CAR components

A tumor-CAR, as well as a tumor-TCR, comprises an extracellular ligand (or antigen) binding domain that recognizes a tumor antigen. Hence, a tumor-CAR and a tumor-TCR as described herewith comprise an extracellular tumor antigen-binding domain.

The term “extracellular antigen binding domain” or “extracellular ligand binding domain” as used herein generally refers to an oligopeptide or polypeptide that is capable of binding a specific antigen, such as a tumor antigen. In some cases, the domain will be capable of interacting with a cell surface molecule, such as a ligand. For example, in some cases, an extracellular antigen-binding domain can be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In some cases, said extracellular antigen-binding domain can comprise a single chain antibody fragment (scFv) comprising the heavy (VH) and the light (VL) variable fragments of a target-antigen-specific monoclonal antibody joined by a flexible linker. The antigen binding domain of a CAR expressed on the cell surface of the engineered cells described herein can be any domain that binds to the target antigen and that derives from, for example, a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof.

As used herewith the term “tumor antigen” is meant to cover “tumor-specific antigen”, “tumor associated antigen” and “antigen associated with a cancer”. Tumor- Specific Antigens (TSA) are generally present only on tumor cells and not on any other cell, while Tumor- Associated Antigens (TAA) are present on some tumor cells and also present on some normal cells. “Tumor antigen,” as meant herein, also refers to mutated forms of a protein, which only appears in that form in tumors, while the non-mutated form is observed in non-tumoral tissues. A tumor antigen can be an antigen specific of, or associated with, a tumor or cancer, including haematological cancers and solid tumors.

In some cases, said engineered immune cells express a chimeric antigen receptor (CAR), or recombinant TCR, specific for a tumor antigen selected from the group consisting of CD7, CD25, CD30, CD33, CD34, CD37, CD38, CD47, CD56, CD98, CD117, CD 123, CD 133, CD 157, FLT3, CLL1, c-kit, MUC1, CXCR4, VEGF, NKG2D F, folate receptor beta (FR beta), hepatocyte growth factor (HGF), HLA-A2 and Lewis Y.

In some cases, said engineered immune cells express a chimeric antigen receptor (CAR), or recombinant TCR, specific for a tumor antigen selected from the group consisting of CD123, CD38, CLL1, FLT3, CD7, FRbeta, CD33, LEY.

In some cases, the tumor antigen is selected from the group consisting of CD 123, CD22, and CD 19.

According to some embodiments said engineered immune cells, e.g. T-cells, express a chimeric antigen receptor (CAR) specific for CD 123, referred herewith as “CD123CAR” or “CAR123”.

In some cases, a CD123CAR can comprise an extra cellular ligand binding-domain comprising VH and VL from a monoclonal anti-CD123 antibody, a hinge, a transmembrane domain, a cytoplasmic domain including a CD3 zeta signaling domain and a co -stimulatory domain from 4-1 BB.

In some cases, a CD123CAR can be one described in W02020/007593.

In some cases, a CAR targeting CD 123 can have a polypeptide structure comprising an extra cellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, a CD8a hinge, a CD8a transmembrane domain, and a cytoplasmic domain including a CD3 signaling domain and a co-stimulatory domain from 4- IBB, wherein said VH comprises the CDRs of amino acid sequences SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and said VL comprises the CDRs of amino acid sequences SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some cases, a CD123CAR can have VH and VL having at least 80%, 85%, 90%, 95%, or at least 99% identity with SEQ ID NO: 11 and SEQ ID NO: 12, respectively, and comprising the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 15 and SEQ ID NO: 16 to SEQ ID NO: 18, respectively.

In some cases, said VH comprises the amino acid sequence of SEQ ID NO: 11 and said VL comprises the amino acid sequence of SEQ ID NO: 12.

In some cases, said CAR targeting CD 123 can comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, or at least 99% identity with SEQ ID NO: 19, and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 18.

The CARs expressed by the engineered T-cells described herewith can comprise humanized versions of the VH and VL regions from murine monoclonal antibodies.

In some cases, said CD8a hinge comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 4.

In some cases, said CD8a transmembrane comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 6.

In some cases, said CD3^ signaling domain comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 9.

In some cases, said co -stimulatory domain from 4-1 BB comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 8.

In one example, the sequence of the CAR targeting CD 123 that can be expressed by the engineered CAR-T cells useful in the methods and compositions described herewith is as follows:

MALPVTALLLPLALLLHAARPEVKLVESGGGLVQPGGSLSLSCAASGFTFTDYY MSWVRQPPGKALEWLALIRSKADGYTTEYSASVKGRFTLSRDDSQSILYLQMN ALRPEDSATYYCARDAAYYSYYSPEGAMDYWGQGTSVTVSSGGGGSGGGGSG GGGSMADYI<DIVMTQSHI<FMSTSVGDRVNITCT<ASQNVDSAVAWYQQI<PGQS PKALIYSASYRYSGVPDRFTGRGSGTDFTLTISSVQAEDLAVYYCQQYYSTPWTF GGGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR (SEQ ID NO: 19) Once expressed and presented at the cell surface, said CAR specific for CD 123 loses its signal sequence (e.g. SEQ ID NO: 1) and its amino acid sequence becomes as follows:

EVKLVESGGGLVQPGGSLSLSCAASGFTFTDYYMSWVRQPPGKALEWL ALIRSKADGYTTEYSASVKGRFTLSRDDSQSILYLQMNALRPEDSATYYCARDA AYYSYYSPEGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSMADYKDIVMTQS HKFMSTSVGDRVNITCKASQNVDSAVAWYQQKPGQSPKALIYSASYRYSGVPD RFTGRGSGTDFTLTISSVQAEDLAVYYCQQYYSTPWTFGGGTKLEIKRTTTPAPR PPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO: 20).

In some instances, said CAR targeting CD123 can comprise an additional sequence comprising 2 epitopes specifically recognized by Rituximab (referred as R2) allowing the immune depletion of the engineered CAR positive immune cells as described for instance in W02016120216.

1.2.2 Further gene editing

In some cases, the engineered immune cells, e.g. T-cells, that have been modified to express a CAR or recombinant TCR targeting an antigen associated with a cancer can have one or more additional modifications.

In some cases, for instance when the immune cells are allogeneic with respect to the subject to be treated, the engineered T-cells can comprise an inactivated T-cell receptor (TCR) and can have been modified by inactivating at least one component of the TCR, e.g. by using a sequence-specific endonuclease such as a RNA guided endonuclease associated with a specific guide RNA, or using other gene editing approaches such as TALE-nucleases.

T cell receptors (TCR) are cell surface receptors that participate in the activation of T-cells in response to the presentation of antigen. The TCR is generally made from two chains, alpha and beta, which assemble to form a heterodimer and associates with the CD3- transducing subunits to form the T-cell receptor complex present on the cell surface. Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the alpha and beta chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T-cells. However, in contrast to immunoglobulins that recognize intact antigen, T-cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T-cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T-cell proliferation and the potential development of GvHD. It has been shown that normal surface expression of the TCR depends on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner (1990) Annu. Rev. Immunol. 8:139-67). The inactivation of TRAC (encoding TCRalpha constant domain) or TRBC (encoding TCRbeta constant domain) can result in the elimination of the TCR from the surface of T-cells preventing recognition of alloantigen and thus GVHD.

In some cases, at least 50%, at least 70%, at least 90%, or at least 95% of the engineered T-cells in the population useful in the methods described herewith are mutated in their TRAC, TRBC and/or CD3 alleles.

In some cases, the TCR can be inactivated by using specific TALE-nucleases, better known under the trademark TALEN® (Cellectis, 8, rue de la Croix Jarry, 75013 PARIS). This method has proven to be highly efficient in primary cells using RNA transfection as part of a platform allowing the mass production of allogeneic T-cells. See, e.g. WO 2013/176915, which is incorporated by reference herein in its entirety.

In some cases, the TCR can be inactivated using an RNA guided endonuclease associated with a specific guide RNA. U.S. Patent No. 10,870,864 describes methods for inactivating a TCR in cells using such methods, which is incorporated by reference herein. Engraftment of allogeneic T-cells is possible by inactivating at least one gene encoding a TCR component. In some cases, the TCR is rendered not functional in the cells by inactivating a TRAC gene and/or a TCRB gene. TCR inactivation in allogeneic T-cells aims to prevent or reduce GvHD. In some cases, the TCR gene can be inactivated by inserting into the TRAC locus of the cell’s genome at least one exogenous polynucleotide encoding a tumor-CAR. For instance, the TCR gene can be inactivated as described in Example 1 of WO 2020/007593, by electroporating the cells with mRNA encoding the two halves of the TALE-nuclease targeting the TRAC region of SEQ ID NO: 28, where the 2 half-nucleases have the amino acid sequence of SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

The characteristics of a TALE-nuclease that can be used to inactivate the CD52 gene in the cells are provided in Table 2.

Table 2: TALE-nucleases targeting TCRalpha gene

By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form and/or not presented at the cell’s surface in the case of membrane protein.

In some cases, the engineered immune cells, e.g. T-cells, can be modified to confer resistance to at least one immune suppressive and/or chemotherapy drug that may be used as pre-conditioning treatment. This aims at improving cancer therapy and selective engraftment of the transferred cells.

For instance, if the immunosuppressive treatment comprises a humanized antibody targeting CD52 antigen, the engineered cells are advantageously genetically modified to have their CD52 inactivated or repressed, as described for instance in WO 2013/176915. The CD52 gene can be inactivated for instance as described in Example 2 of WO 2020/007593, by electroporating the cells with mRNA encoding the two halves of the TALE-nuclease targeting the CD52 region of SEQ ID NO: 29, where the 2 half-nucleases have the amino acid sequence of SEQ ID NO: 26 and SEQ ID NO: 27, respectively.

The characteristics of a TALE-nuclease that can be used to inactivate the CD 52 gene in the cells are provided in Table 3.

Table 3: TALE-nucl eases targeting CD52 gene

If the immunosuppressive treatment comprises FK506 also known as Tacrolimus or fujimycin, the engineered cells are advantageously genetically modified to have a FKBP family gene member such as FKBP 12 or a variant thereof inactivated or repressed.

If the immunosuppressive treatment comprises cyclosporine, the engineered cells are advantageously genetically modified to have a cyclophilin family gene member or a variant thereof inactivated or repressed.

In some cases, the engineered immune cell can be further modified to confer resistance to a chemotherapy drug, such as a purine analogue drug, for example by inactivating DCK as described in WO 2015/75195.

Cytokine Release Syndrome (CRS) is the most common adverse event of CAR-T cell therapy. CRS is defined as a clinical syndrome that may occur after cell therapy due to the release of cytokines (substances secreted by immune cells) into the body’s blood stream. It has been shown that inactivation of Granulocyte-macrophage colonystimulating factor (GM-CSF) can prevent monocyte-dependent release of key cytokine release syndrome mediators (Sachdeva et al. (2019) J. Biol. Chem. 294(14) 5430-5437). Thus, the engineered immune cells useful in the methods and compositions as described herewith can be genetically modified to suppress expression, or cell surface presentation, of GM-CSF.

In some cases, in particular for allogeneic cells, the engineered cells can be modified to improve their persistence or lifespan into the patient, for instance by inactivating a gene encoding MHC-I component(s) such as HLA or B2M, such as described in WO 2015/136001 or by Liu et al. (2017, Cell Res 27:154-157).

Beta-2 microglobulin, also known as 02m, is the light chain of MHC class I molecules, and as such an integral part of the major histocompatibility complex. In human, 02m is encoded by the B2M gene.

In some cases, inhibition of expression of B2M can be achieved by a genome modification, such as through the expression in the cell of a rare-cutting endonuclease able to selectively inactivate by DNA cleavage the B2M gene (e.g. human B2M gene, NCBI Reference Sequence: NG 012920.1). Such rare-cutting endonuclease may be a TALE- nuclease, meganuclease, zing-finger nuclease (ZFN), or RNA guided endonuclease (such as Cas9). For instance, the B2M gene can be inactivated using TALE-nucleases as described in Example 3 of WO 2020/007593.

In some cases, inhibition of expression of B2M can be achieved by using (e.g. introducing into the T-cell) a nucleic acid molecule that specifically hybridizes (e.g. binds) under cellular conditions with the cellular mRNA and/or genomic DNA encoding 02m, thereby inhibiting transcription and/or translation of the gene. In some cases, the inhibition of expression of B2M can be achieved by using (e.g. introducing into the T-cell) an antisense oligonucleotide, ribozyme or interfering RNA (RNAi) molecule. In some cases, such nucleic acid molecule can comprise at least 10 consecutive nucleotides of the complement of the mRNA encoding human 02m.

Useful in the methods and compositions described herewith are engineered immune cells, e.g. T-cells, which have been modified to suppress or repress expression of HLA in said cells. The class I HLA gene cluster in humans comprises three major loci, B, C and A, as well as several minor loci. The class II HLA cluster also comprises three major loci, DP, DQ and DR, and both the class I and class II gene clusters are polymorphic, in that there are several different alleles of both the class I and II genes within the population. Useful in the methods and compositions described herewith are engineered immune cells, e.g. T-cells, have been modified to suppress or repress expression of CIITA in said cells. CIITA is the gene encoding class II major histocompatibility complex transactivator protein.

Useful in the methods and compositions described herewith are engineered immune cells, e.g. T-cells, which have been inactivated in at least one gene selected from the group consisting of RFXANK, RFX5, RFXAP, TAPI, TAP2, ZXDA, ZXDB and ZXDC. Inactivation may, for instance, be achieved by using a genome modification, such as through the expression in the cell of a rare-cutting endonuclease able to selectively inactivate, by DNA cleavage, a gene selected from the group consisting of RFXANK, RFX5, RFXAP, TAPI, TAP2, ZXDA, ZXDB and ZXDC. Such modifications can permit the engineered immune cells to be less alloreactive when infused into patients.

Useful in the methods and compositions described herewith are engineered immune cells (e.g. T-cells) which have a 02m gene that has been inactivated and have, integrated in their genome, an exogenous sequence encoding a NK inhibitor such as a HLA-E peptide fusion protein, for instance as disclosed in example 5 of WO 2020/007593.

Useful in the methods and compositions described herewith are engineered immune cells, e.g. T-cells, which have been genetically modified to suppress or repress expression of an immune checkpoint protein and/or the receptor thereof, in said cells, such as PDCD1 or CTLA4 as described in WO 2014/184744.

Useful in the methods and compositions described herewith are engineered immune cells, e.g. T-cells, which are one or more of: CD52 negative, TCR negative, B2M negative, CIITA negative, PDCD1 negative, GM-CSF negative; CTLA4 negative, dCK negative.

For instance, the engineered immune cells, e.g. T-cells, can be at least CD52- negative and TCR negative, or at least CD52-negative and B2M-negative.

In some cases, to reduce fratricide effect, the engineered immune cells, e.g. T-cells, as described herewith do not present at their cell surface the antigen targeted by the tumor- CAR.

In some cases, the engineered immune cells, e.g. T-cells, can be modified to comprise a suicide gene and/or a marker for cell sorting. In some instances, the engineered cells useful in the methods and compositions described herewith can comprise at their cell surface a CAR as described herewith, and at least one suicide domain (R)n (where R is a CD20 epitope recognized by Rituximab, and n is comprised between, and including, 0 to 10) and/or (Q)m (where Q is a CD34 epitope recognized by QBEN10, and m is comprised between, and including, 0 to 10), such as a region comprising 2 epitopes recognized by Rituximab and 1 epitope recognized by QBEN10 (referred to as “RQR8”) for cell depletion and/or cell sorting.

CD20 epitope recognized by Rituximab: CPYSNPSLC (SEQ ID NO: 21).

CD34 epitope recognized by QBEND10: ELPTQGTFSNVSTNVSPAKPTTTA (SEQ ID NO: 22).

In some cases, the engineered cells useful in the methods and compositions described herewith can comprise at their cell surface a CAR as described herewith, and a suicide domain having an amino acid sequence comprising SEQ ID NO: 23.

In some cases, the engineered cells comprise a polynucleotide encoding a CAR as described herewith and a suicide domain arranged in a polycistronic arrangement where both of the 2 transcription units are controlled by a unique promoter, have the same direction of transcription and are separated by a “self-cleaving” peptide such as a 2A peptide (e.g. P2A, T2A, E2A, F2A).

2. Methods of treatment comprising administration of CAR-T cells according to a 2- dose regimen

2.1 Preconditioning with a lymphodepleting treatment

Lymphodepleting conditioning regimen prior to adoptive cell transfer dramatically improves the efficacy of T-cell therapy. Without willing to be bound by theory, it is believed that lymphodepleting conditioning regimen works by multiple mechanisms, including eliminating sinks for homeostatic cytokines (such as IL-2, IL-7, and IL- 15), eradicating immunosuppressive elements (such as regulatory T cells and myeloid-derived suppressor cells), inducing costimulatory molecules and downregulating indoleamine 2,3- dioxygenase in tumor cells, and promoting expansion, function, and persistence of adoptively transferred T cells (Muranski et al. (2006) Nat. Clin. Pract. Oncol. 3(12):668- 681 ; Gattinoni et al. (2005) J. Exp. Med. 202(7): 907 -912 ; Ninomiya et al. (2015) Blood. 125(25):3905-3916).

Therefore, in general, before administering cells-based immunotherapy (in particular, CAR-engineered T-cells), patients receive pre-conditioning lymphodepleting agents, also called immunosuppressive agents, to reduce or eliminate the patient’s immune cells and improve CAR T-cell expansion and persistence.

In some cases of the methods described herewith, the pre-conditioning lymphodepleting treatment can be combined with a pre-conditioning chemotherapeutic agent, for example, to reduce tumor burden prior to administration of the engineered T- cells. In some cases, the lymphodepleting agent has the effect of both a lymphodepleting agent and a chemotherapeutic agent, i.e. by reducing the patient’s immune cells number and by, at least partially, reducing tumor burden.

In some embodiments of the methods described herewith, the lymphodepleting treatment aims to eliminate, at least partially, the subject’s own immune cells to have, for at least 20 consecutive days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood. In some cases, the lymphodepleting treatment allows to have less than about 200, or less than about 100, or less than about 10, or about 0 absolute lymphocyte counts per pl of subject’s whole blood, for at least 20 consecutive days.

In some embodiments, the lymphodepleting treatment referred to in the methods described herewith eliminates, at least partially, the subject’s own immune cells to have less than 500 absolute lymphocyte counts per pl of subject’s whole blood, for at least 20, at least 25, at least 26, at least 27, or at least 28 consecutive days.

In some embodiments, the lymphodepleting treatment referred to in the methods described herewith eliminates, at least partially, the subject’s own immune cells to have less than about 200, or less than about 100, or less than about 10, or about 0 absolute lymphocyte counts per pl of subject’s whole blood, for at least 20, at least 25, at least 26, at least 27, or at least 28 consecutive days.

In some embodiments of the methods described herewith, the lymphodepleting treatment aims to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood. In some cases, the lymphodepleting treatment allows to have absolute lymphocyte counts per pl of subject’s whole blood of less than about 200, or of less than about 100, or of about 0 for at least 20 days.

Absolute lymphocyte counts in a subject’s whole blood sample are measured by a complete cell count and differential as, for instance, described by George-Gay et al. (J Perianesth Nurs, 2003, 18(2):96-114).

In some cases, the lymphodepleting treatment comprises fludarabine, alone or in combination with another immunosuppressive agent.

In some cases, the lymphodepleting treatment comprises cyclophosphamide, alone or in combination with another immunosuppressive agent.

In some cases, the lymphodepleting treatment comprises an anti-CD52 antibody, such as alemtuzumab, alone or in combination with another immunosuppressive agent.

In some cases, the lymphodepleting treatment comprises an anti-CD52 antibody, such as alemtuzumab, in combination with fludarabine.

In some cases, the lymphodepleting treatment comprises an anti-CD52 antibody, such as alemtuzumab, in combination with cyclophosphamide.

In some cases, the lymphodepleting treatment comprises an anti-CD52 antibody, such as alemtuzumab, in combination with cyclophosphamide and fludarabine.

In some further cases, the lymphodepleting treatment comprises administration of fludarabine, cyclophosphamide and alemtuzumab.

As shown in the Example section, a lymphodepleting treatment comprising administration of fludarabine, cyclophosphamide and alemtuzumab generally allows a sustained and prolonged low level (inferior or equal to about 500 absolute lymphocyte counts per pl of subject’s whole blood) of lymphocytes in the patient’s whole blood, for at least 20, at least 25, or at least 28 consecutive days.

In some cases, the lymphodepletion treatment can comprise bendamustine.

In some cases, the lymphodepletion treatment can comprise bendamustine and fludarabine, for instance bendamustine at about 70 mg/m²/day and fludarabine at about 30 mg/m²/day, for 3 days.

In some cases, the lymphodepleting treatment can comprise bendamustine and alemtuzumab. In some cases, the lymphodepleting treatment can comprise a calcineurin inhibitor, a target of rapamycin, an interleukin-2alpha-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid such as dexamethasone, cyclosporine, or an immunosuppressive antimetabolite.

In some cases, the lymphodepleting agent can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days.

The lymphodepleting treatment may, for instance, comprise administration of fludarabine once daily for 1, 2, 3, 4, or 5 days, administration of cyclophosphamide once daily for 1, 2, 3, or 4 days, and administration of alemtuzumab once daily for 1, 2, 3, 4, or 5 days. For instance, the lymphodepleting treatment can comprise administration of fludarabine at between about 1 and 100 mg/m²/day, between about 10 and 75 mg/m²/day, between about 15 and 50 mg/m²/day, between about 20 and 40 mg/m²/day, or about 25, 30 or 40 mg/m²/day; administration of cyclophosphamide at between about 50 and 10 000 mg/m²/day, between 100 and 1000 mg/m²/day, between 500 and 1000 mg/m²/day, between 600 and 800 mg/m²/day, or about 600, 650, 700, 750 or 800 mg/m²/day; and/or administration of alemtuzumab at between about 0.1 to 0.5 mg/kg/day, between 0.15 and 0.2 mg/kg/day, or about 0.15 mg/kg/day. In some cases, alemtuzumab is administered at a fixed dose comprised between about 5 and 50 mg/day, between 10 and 40 mg/day, between 10 and 20 mg/day, or at 10, 11, 12, 13, 14, or 15 mg/day.

In some cases, the maximum daily dose of fludarabine is from 50 to 70 mg, e.g. about 60 mg, the maximum daily dose of cyclophosphamide is from 1 to 1.5 g, from 1.3 to 1.4 g, e.g. about 1.33 g, the maximum daily dose of alemtuzumab is from 10 to 15 mg, e.g. about 12 or 13 mg.

In some cases, the lymphodepleting treatment comprises administration of:

- cyclophosphamide at between about 50 and 10000 mg/ m²/day, between 50 and 5 000 mg/ m²/day, between 50 and 1000 mg/ m²/day, between 100 and 1000 mg/m²/day, between 500 and 1000 mg/ m²/day, between 600 and 800 mg/m²/day, or about 600, 650, 700, 750 or 800 mg/m²/day, for 1, 2, 3, or 4 days;

In some cases, the lymphodepleting treatment comprises administration of:

(i) fludarabine at 30 mg/m²/day (given for instance over 15 to 30 minutes) for 4 days, such as from Day -5 to Day -2, with a maximum daily dose of 60 mg,

(ii) cyclophosphamide at 750 mg/m²/day (given for instance over 1 hour) for 3 days, such as from Day -4 to Day -2, with a maximum daily dose of 1.33 g, and

(iii) alemtuzumab at 12 mg/day (given for instance over 4 to 6 hours) for 4 days, such as Day -5 to Day -2.

“Day -n” or “D-n” represents “n” days before Day 0, where Day 0 is the day the immunotherapy treatment is initially planned to start (i.e. the day when the injection of the first dose of engineered CAR-T cells is planned). Day 0 can fluctuate by 1, 2, or 3 days if this would not be recommended to start the immunotherapy that exact day, for instance due to the patient having fever, etc. In that case, the injection of the first dose of CAR-T cells can be postponed by a couple of days, compared to the initially planned Day 0, till the patient is fit to start immunotherapy.

As used herewith, “Day n” or “Dn” represents “n” days after Day 0, where Day 0 is the day the immunotherapy treatment is initially planned to start as detailed above.

In some instances, the lymphodepleting treatment starts between 2 and 8 days before the administration of the first dose of engineered T-cells as described herewith, such as between 2 and 5 days before, such as 2, 3, 4, or 5 days before.

In some cases, the lymphodepleting treatment is completed before immunotherapy starts (i.e. before the administration of the first dose of engineered T-cells as described herewith), and the compounds like fludarabine, cyclophosphamide and alemtuzumab, are administered intravenously (IV).

According to the methods described herewith, the lymphodepletion treatment is followed by a cell-immunotherapy treatment using engineered immune cells, which are administered in 2 successive doses, referred herein as a 2-dose regimen. According to the methods described herewith, the lymphodepleting treatment is administered prior to the administration of the first dose of CAR-T cells, and there is no further lymphodepleting treatment between the administration of the first dose and the administration of the second dose of CAR-T cells, concomitantly to the second dose of CAR-T cells, and/or for 28 days following the administration of the first dose of CAR-T cells.

In some cases, a preconditioning lymphodepleting treatment is completed on or shortly before the administration of the first dose of CAR-T cells, such as within the 1, 2, 3, 4, or 5 days preceding the administration of the first dose, and there is no lymphodepleting treatment before at least 28 days after the administration of the first dose of CAR-T cells.

A further preconditioning lymphodepleting treatment can take place after 28 days, and up to months or years after the previous lymphodepleting treatment, as a preconditioning lymphodepleting treatment in a repeat of the method of treatment as described herewith, for a subject whose cancer relapsed or progressed, for instance, after having been treated according to the method described herewith.

According to the methods described herewith, the preconditioning lymphodepletion treatment takes place only before the administration of the first dose of CAR-T cells in a 2-dose regimen.

2.1 Administration of successive doses of CAR-T cells

The methods of treating a subject having a cancer as disclosed herewith comprise administering successively, to the subject in need thereof, a first and second doses of engineered T-cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CARs targeting specifically an antigen associated with the cancer.

In some cases, the first dose of CAR-T cells is administered within about 3 and 8 days after the start of the pre-conditioning lymphodepleting treatment, such as 3, 4, 5, 6, 7, or 8 days after the start of the pre-conditioning lymphodepleting treatment, and/or between about 0 and 5 days after the completion of the pre-conditioning lymphodepleting treatment, such as on the day or 1, 2, 3, 4 or 5 days after the completion of the preconditioning lymphodepleting treatment. In some cases, the second dose of CAR-T cells is administered between about 10 and 20 days after the first dose of CAR-T cells, or between 10 and 17 days, such as after 10, 11, 12, 13, 14, 15, 16 or 17 days.

In some cases, the second dose of CAR-T cells is administered 13, 14, or 15 days after the first dose of CAR-T cells.

In some cases, the second dose of CAR-T cells is administered in the time-window when cytokine secretion measured in a patient’s sample (e.g. blood, biopsy) is between 10 and 1000 folds, or between 10 and 100 folds, higher than before the administration of the first dose of CAR-T cells, such as in the range of 10, 50, 100, 500, or 1000 folds higher.

In some cases, the second dose of CAR-T cells is administered in the time-window when cytokine secretion, e.g. IL-6, as measured in the patient’s blood, is between 10 and 1000 folds, or between 10 and 100 folds, higher than before the administration of the first dose of CAR-T cells, such as in the range of 10, 50, 100, 500, or 1000 folds higher.

In some cases, the second dose of CAR-T cells is administered at least 3, 4, 5 days after the day when the number of CAR-T cells from the first dose reached a peak.

In some cases, the first dose of CAR-T cells comprises between about 10⁴ to about 10⁸ CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CAR-T cells, or between about 10⁵ to 10⁹ total CAR-T cells, and the second dose of CAR-T cells comprises between about 10⁴ to about 10⁸ CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CAR-T cells, or between about 10⁵ to 10⁹ total CAR-T cells.

In some cases, the number of CAR-T cells in first dose is between about IxlO⁵ and 1x10⁷ cells/kg, between about 5 xlO⁵ and 5 xlO⁶ cells/kg, between about 6x10⁵ and 3x10⁶ cells/kg, between about 6x10⁵ and 2x10⁶ cells/kg, and the number of CAR-T cells in the second dose is between about 1x10⁵ and 1x10⁷ cells/kg, between about 5 xlO⁵ and 5 xlO⁶ cells/kg, between about 6x10⁵ and 3x10⁶ cells/kg, between about 6x10⁵ and 2x10⁶ cells/kg.

In some cases, the number of CAR-T cells in the first dose and the number of CAR- T cells in the second dose are identical.

In some cases, the number of CAR-T cells in the first dose and the number of CAR- T cells in the second dose are different. In some cases, the number of CAR-T cells in the second dose is higher than the number of CAR-T cells in the first dose, such as between about 1.5 and 3 times higher, such as 1.5, 2, 2.5, or 3 times higher. In some cases, the number of CAR-T cells in the second dose is lower than the number of CAR-T cells in the first dose, such as between about 1.5 and 3 times lower, such as 1.5, 2, 2.5, or 3 times lower.

In some cases, the number of CAR-T cells in the first dose is between about 5x10⁵ and 5xl0⁶ cells/kg, between about 5xlO⁵ and 1x10⁶ cells/kg, such as about 6 xlO⁵ cells/kg, 6.2 xlO⁵ cells/kg, 6.25 xlO⁵ cells/kg, 6.5 xlO⁵ cells/kg, 7 xlO⁵ cells/kg, and the number of CAR-T cells in the second dose is between about 1x10⁶ and 5x10⁶ cells/kg, between about 1.5xl0⁶ and 4xl0⁶ cells/kg, such as about 1.5xl0⁶ cells/kg, 2xl0⁶ cells/kg, 3xl0⁶ cells/kg, 3.3xl0⁶ cells/kg, 3.5xl0⁶ cells/kg, 4xl0⁶ cells/kg.

In some cases, the antigen targeted by the CAR expressed by the CAR-T cells comprised in the first dose is identical to the antigen targeted by the CAR-T cells comprised in the second dose.

In some cases, the antigen targeted by the CAR expressed by the CAR-T cells comprised in the first dose is different from the antigen targeted by the CAR-T cells comprised in the second dose.

In some cases, the first and second doses of CAR-T cells express a CAR targeting the same antigen.

In some cases, the first and second doses of CAR-T cells express a CAR targeting the same epitope of the same antigen.

In some cases, the first and second doses of CAR-T cells express a CAR targeting different epitopes of the same antigen.

In some cases, the first dose and/or second dose of CAR-T cells express a CD 123 CAR as described herewith.

In some cases, the first dose and/or second dose of CAR-T cells express a CAR as described herewith.

In some cases, the first dose and/or second dose of CAR-T cells comprise CAR-T cells which have the genetic modifications described herewith.

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to suppress or repress expression of at least one component of a T-Cell Receptor (TCR) at the surface of said CAR-T cells. In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to suppress or repress expression of at least one gene encoding a MHC-I protein, such as 02m and HLA.

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to suppress or repress expression of a gene encoding an immune checkpoint protein and/or a receptor thereof.

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to confer resistance to at least one immune suppressive or chemotherapy drug

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to be CD52 negative.

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to be at least CD52 and TCR negative

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to be at least one of CD52 negative, TCR negative, B2M negative, and PDCD1 negative.

In some cases, the first dose and/or the second dose of CAR-T cells comprise CAR- T cells which have been genetically modified to comprise a suicide gene.

In some cases, the first dose and/or the second dose of CAR-T cells comprise, integrated in their genome, an exogenous nucleic acid sequence encoding a suicide domain such as QBEN10 having an amino acid sequence comprising SEQ ID NO: 23.

It is not excluded that one or more additional doses of CAR-T cells can be administered after the second dose of CAR-T cells as described herewith, in the timewindow comprised between the day following the administration of the second dose and 28 days following the administration of the first dose, e.g. between about D14 and D28. In some cases, the administration of these one or more additional doses of CAR-T cells can occur without prior lymphodepleting treatment (except for the one that occurred before the administration of the first dose).

In some cases, these one or more additional doses of CAR-T cells are characterized by similar features in respect to the concentration of CAR-T cells, the phenotypic and genetic characteristics of the CAR-T cells, as described herewith for the first and second doses.

2.3 Additional optional treatments

The methods described herewith can also comprise the administration of a drug to prevent or treat Cytokine Release Syndrom (CRS), such as an anti-IL6 therapy such as anti-IL6 antibody or an anti-IL6R antibody.

The drug for preventing CRS can be administered before, at the same time, or after administration of the CAR-T cells.

In some cases, the administration of the drug for preventing CRS is administered before the CAR-T cells, and is, optionally, completed at least 1 hour before the administration of the CAR-T cells.

In some cases, the methods described herewith comprise administering an anti-IL6 or anti-IL6R antibody on the same day as the first dose of CAR-T cells and/or on the same day as the second dose of CAR-T cells.

In some cases, the drug for preventing CRS, such as an anti-IL6 antibody or anti- IL6R antibody, is administered the same day as the CAR-T cells.

Tocilizumab is a humanized anti-IL-6R monoclonal antibody recommended by the ASTCT (Lee etal. (2019) Biol Blood Marrow Transplant. 25, 625-638) and has marketing authorization for the treatment of CRS

In some additional cases, the methods described herewith comprise administering tocilizumab on the same day as the first dose of CAR-T cells and/or on the same day as the second dose of CAR-T cells.

For instance, Tocilizumab is administered intravenously at a dose comprised between 5 and 100 mg/kg, between 10 and 50 mg/kg, or at about 7, 8, 9, or 10 mg/kg, optionally at a maximum total dose of 800 mg. For instance, the administration of tocilizumab is completed at least 1 hour before the administration of the first and second doses of CAR-T cells.

In some cases, the methods described herewith is followed by a bone marrow transplant in the patient. In some cases, the methods described herewith are carried out in view of a bone marrow transplant in the patient having a cancer.

3. Patients and cancers

The methods and compositions described herewith could be used for treating a patient having any cancer including solid tumors and haematological cancers.

In some cases, said cancer is a pre-malignant or malignant cancer condition characterized by an overabundance of CD 123 -expressing cells.

In some cases, said cancer is a haematological cancer, such as a leukemia or a malignant lymphoproliferative disorder.

In some cases, said leukemia is selected from the group consisting of acute myelogenous leukemia (AML), chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia (ALL), chronic lymphoid leukemia, and myelodysplastic syndrome.

In some cases, said leukemia is acute myelogenous leukemia (AML).

In some cases, said hematological cancer is a malignant lymphoproliferative disorder.

In some cases, said malignant lymphoproliferative disorder is a lymphoma.

In some cases, said lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).

In some cases, the methods and compositions described herewith are for use in the treatment of cancers selected from the group consisting of acute myeloid leukemia (AML), Acute lymphocytic leukemia (ALL), and Non-Hodgkin Lymphoma (NHL).

AML patients with complex cytogenetic abnormalities and/or TP53 mutations (i.e. classified into the ELN Adverse genetic risk group (Dbhner etal., 2017, Blood. 129, 424- 447) specifically fall into the category of urgent unmet medical need, as these patients have especially dismal outcomes with all existing treatment modalities, including allogeneic transplantation. The methods and compositions for use described herewith could be used for treating patients with adverse genetic risk AML or as pre-treatment for patients with adverse genetic risk AML in view of obtaining more successful bone marrow transplant.

Adverse genetic risk is defined as per ELN guidelines (Dbhner et al., 2017, Blood. 129, 424-447) by any of the following genetic signatures: a. t(6;9)(p23;q34.1); DEK-NUP214; or b . t(v; 11 q23.3 ) ; KMT2 A rearranged; or c. t(9;22)(q34.1;ql l.2); BCR-ABL1 ; or d. inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM(EVIl); or e. -5 or del(5q); -7; -17/abn(17p) or Complex karyotype comprising three or more unrelated chromosome abnormalities in the absence of one of the World Health Organization-designated recurring translocations or inversions, i.e., t(8;21), inv(16) or t(16;16), t(9;l 1), t(v;ll)(v;q23.3), t(6;9), inv(3) or t(3;3); AML with BCR- ABL1 ; or f. Monosomal karyotype comprising presence of one single monosomy (excluding loss of X or Y) in association with at least one additional monosomy or structural chromosome abnormality (excluding core-binding factor AML); or h. Wild-type NPM1 and FLT3-ITD high or i. Mutated RUNX1 (except if co-occur with favorable-risk AML subtypes) or j. Mutated ASXL1 (except if co-occur with favorable-risk AML subtypes) or k. Mutated TP53.

Thus, in some cases, the methods and compositions described herewith are for use in the treatment of AML in a patient who has at least one genetic marker selected from:

- t(6;9)(p23;q34.1); DEK-NUP214; or

- t(v;l lq23.3); KMT2A rearranged; or

- t(9;22)(q34.1 ;ql l.2); BCR-ABL1 ; or

- inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM(EVIl); or

- -5 or del(5q); -7; -17/abn(17p) or

- Complex karyotype: Three or more unrelated chromosome abnormalities in the absence of one of the World Health Organization-designated recurring translocations or inversions, i.e., t(8;21), inv(16) or t(16;16), t(9;l l), t(v;l l)(v;q23.3), t(6;9), inv(3) or t(3;3); AML with BCR-ABL1; or

- Monosomal karyotype presenting one single monosomy (excluding loss of X or Y) in association with at least one additional monosomy or structural chromosome abnormality (excluding core-binding factor AML); or

- Wild-type NPM1 and FLT3-ITD high; or

- Mutated RUNX1 (except if co-occur with favourable-risk AML subtypes); or

- Mutated ASXL1 (except if co-occur with favourable-risk AML subtypes); or

- Mutated TP53.

In some cases, the methods and compositions described herewith are for use in the treatment of AML in a patient who has at least one genetic marker that is Mutated TP53.

4. Pharmaceutical compositions and uses thereof

Described herewith are pharmaceutical compositions comprising engineered T- cells expressing a CAR or recombinant TCR, useful in the methods described herewith, and a pharmaceutical acceptable excipient.

Also provided are pharmaceutical compositions comprising engineered CAR-T cells useful in the methods described herewith, and a pharmaceutical acceptable excipient.

Also described herewith are pharmaceutical compositions comprising a population of T-cells comprising the engineered CAR-T cells useful in the methods described herewith, and a pharmaceutical acceptable excipient.

Provided herewith are pharmaceutical compositions comprising a first or second dose of engineered T-cells expressing a CAR (CAR-T cells), said CAR targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer, according to the methods of treatment described herewith.

In some cases, is provided a pharmaceutical composition comprising a first or second dose of engineered T-cells expressing a CAR (CAR-T cells), said CAR targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer; - wherein said treatment comprises administering the first and second doses of said CAR-T cells successively;

- wherein the second dose of CAR-T cells is administered between 10 and 17 days after the first dose of CAR-T cells;

- wherein no lymphodepleting treatment is administered between the first and second doses of CAR-T cells, concomitantly to the second dose of CAR-T cells, and/or on any one of the at least 20 days following the administration of the first dose of CAR- T cells;

- optionally, wherein said engineered CAR-T cells are CD 52 negative.

Also provided herewith is a pharmaceutical composition comprising one dose of CAR-T cells, said CAR targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer; wherein said treatment comprises administering a first dose of CAR-T cells and a second dose of CAR-T cells successively; wherein the second dose of CAR-T cells is administered between 10 and 17 days after the first dose of CAR-T cells; wherein, prior to the administration of the first dose of CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood; wherein no lymphodepleting treatment is administered between the first and second doses of CAR-T cells, concomitantly to the second dose of CAR-T cells, and/or on any one of the at least 20 days following the administration of the first dose of CAR-T cells; optionally, wherein said engineered CAR-T cells are CD 52 negative; wherein said dose comprised in said pharmaceutical composition is the first or the second dose.

Also disclosed herewith is a combination of a pharmaceutical composition comprising a first dose of engineered T-cells expressing a CAR (CAR-T cells) and a pharmaceutical composition comprising a second dose of engineered T-cells expressing a CAR (CAR-T cells), said CARs targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer;

- wherein said treatment comprises administering the first and second doses of said CAR-T cells successively;

- optionally, wherein said engineered CAR-T cells are CD 52 negative.

Provided herewith is a pharmaceutical composition for use in the treatment of a cancer as mentioned above, wherein said T cells and CAR-T cells are as described in the present document, said lymphodepleting treatment is as described in the present document, said first and second doses of CAR-T cells are as described in the present document, and said cancer is as described in the present document. Provided herewith is a combination of pharmaceutical compositions for use in the treatment of a cancer as mentioned above, wherein said T cells and CAR-T cells are as described in the present document, said lymphodepleting treatment is as described in the present document, said first and second doses of CAR-T cells are as described in the present document, and said cancer is as described in the present document.

Also provided herewith is a pharmaceutical composition comprising a first or a second dose of engineered T-cells expressing a CAR specific for CD 123 (CD 123 CAR-T cells), for use in the treatment of a subject having a cancer characterized by CD 123- expressing cells;

- wherein said treatment comprises administering successively said first and second doses of said CD123CAR-T cells;

- wherein the second dose of CD123CAR-T cells is administered between 10 and 17 days after the first dose of CAR-T cells, such as after 10, 11, 12, 13, 14, 15, 16 or 17 days;

- wherein, prior to the administration of the first dose of CD 123 CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment comprising fludarabine, cyclophosphamide and alemtuzumab to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood;

- wherein no lymphodepleting treatment is administered between the first and second doses of CD123CAR-T cells, concomitantly to the second dose of CD123CAR-T cells, and/or on any one of the at least 20 days following the administration of the first dose of CD123CAR-T cells;

- wherein at least one of said first and second doses of CD 123 CAR-T cells comprises between about 10⁴ to about 10⁸ CD123CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CD 123 CAR-T cells, or between about 10⁵ to 10⁹ total CD123CAR-T cells;

- wherein the number of CAR-T cells in the first dose and the number of CAR-T cells in the second dose are identical or different; wherein said CD123CAR-T cells are CD52 negative, and, optionally, TCR negative.

Also provided herewith is a combination of a pharmaceutical composition comprising a first dose of CD123CAR-T cells and a pharmaceutical composition comprising a second dose of CD123CAR-T cells, for use in the treatment of a subject having a cancer characterized by CD 123 -expressing cells;

- wherein the second dose of CD123CAR-T cells is administered between 10 and 17 days after the first dose of CD123CAR-T cells, such as after 10, 11, 12, 13, 14, 15, 16, or 17 days;

- wherein, prior to the administration of the first dose of CD123CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment comprising fludarabine, cyclophosphamide and alemtuzumab to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood;

- wherein said CD123CAR-T cells are CD52 negative, and, optionally, TCR negative. In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said CD123CAR-T cells are CD52 negative and TCR negative.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said CD123CAR-T cells have, integrated in their genome, an exogenous sequence encoding a suicide domain having an amino acid sequence comprising SEQ ID NO: 23.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said lymphodepleting treatment comprises administration of:

- cyclophosphamide at between about 50 and 10 000 mg/m2/day, between 50 and 5 000 mg/m²/day, between 50 and 1000 mg/m²/day, between 100 and 1000 mg/m²/day, between 500 and 1000 mg/m²/day, between 600 and 800 mg/m²/day, or about 600, 650, 700, 750 or 800 mg/m²/day, for 1, 2, 3, or 4 days;

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said second dose of CD123CAR-T cells is administered 13, 14, or 15 days after the first dose of CD123CAR-T cells.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, the first dose and/or the second dose of CD123CAR-T cells comprises between about 10⁵ to about 10⁷ CD123CAR-T cells per kilogram body weight of the subject (cells/kg), such as from about 6.0 to 6.5 xlO⁵, from about 1.0 to 1.5 xlO⁶, from about 3.0 to 3.5 xlO⁶, or from about 5.0 to 5.5 xlO⁶CD123CAR- T cells/kg, and no more than 5.10⁸ total CD123CAR-T cells.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said first dose and/or second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123 having a polypeptide structure comprising an extra cellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, a CD8a hinge, a CD8a transmembrane domain, and a cytoplasmic domain including a CD3 signaling domain and a co-stimulatory domain from 4- IBB, wherein said VH comprises the CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and said VL comprises the CDR sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said VH has at least 80% identity with SEQ ID NO: 11 and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 15, and said VL has at least 80% identity with SEQ ID NO: 12 and comprises the CDRs of amino acid sequences SEQ ID NO: 16 to SEQ ID NO: 18.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said VH comprises the amino acid sequence of SEQ ID NO: 11 and said VL comprises the amino acid sequence of SEQ ID NO: 12.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said CAR targeting CD 123 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 19, and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to 18.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said CAR targeting CD 123 comprises the amino acid sequence of SEQ ID NO: 19.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said CD 123 CAR-T cells have, integrated in their genome, an exogenous sequence encoding a suicide domain having an amino acid sequence comprising SEQ ID NO: 23.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a pre-malignant or malignant cancer condition characterized by an overabundance of CD 123 -expressing cells. In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a haematological cancer.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is an haematological cancer that is a leukemia or a malignant lymphoproliferative disorder.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a leukemia selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a leukemia that is acute myelogenous leukemia (AML).

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is an hematological cancer that is a malignant lymphoproliferative disorder.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a malignant lymphoproliferative disorder that is a lymphoma.

In some cases of said pharmaceutical composition for use or of said combination of pharmaceutical compositions for use, said cancer is a lymphoma selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).

Although the methods and compositions of the invention are mainly described with T-cells expressing a CAR, they also apply to T-cells expressing a recombinant TCR, since in both cases the CAR and/or the TCR binds specifically to the antigen associated with the cancer from which the patient is affected.

The above written description provides a manner and process of making and using the invention such that any person skilled in the art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to limit the scope of the claimed invention.

EXAMPLES

The method of treatment of cancers based on the administration of CAR-T cells following a 2-dose regimen is illustrated herewith with a clinical trial of anti-CD123 allogeneic CAR-T cells (UCART123) in adult patients with relapsed or refractory (R/R) CD 123+ acute myeloid leukemia (AML).

Example 1: UCART123 product

The CD 123 CAR construct selected for use in Universal Chimeric Antigen Receptor T-cells targeting CD123 (UCART123) combines a scFv specific for CD123, the CD8 alpha hinge region and transmembrane domain, and a cytoplasmic tail composed of 4-1BB co -stimulatory and CD3 zeta signaling domains.

The lentiviral vector cassette that drives expression of the CD 123 CAR was designed to coexpress RQR8 (through a 2A peptide linker). RQR8 is a 136 amino acid artificial cell surface protein combining target epitopes from both human CD34 (to detect RQR8 using the QbendlO antibody) and human CD20 antigens. The expression of RQR8 on UCART123 cells permits targeted destruction of RQR8+ CD123 CAR+ T -cells through administration of rituximab.

UCART123 was additionally engineered to be TCRo.p negative through inactivation of the T-cell receptor alpha constant (TRAC) gene, using the Transcription Activator-Like Effector Nuclease (TALEN®) technology (see Table 2). TALENs® are artificially engineered nucleases that are capable of generating site-specific deoxyribonucleic acid (DNA) double-strand breaks at a desired target site leading to inactivation of the targeted gene.

The inactivation of the TRAC gene (encoding the TCRa subunit) results in the elimination of a functional TCRo.p at the T-cell surface. This is thought to circumvent the recognition of MHC disparities between donor and recipient through the donor cell’s TCR and to prevent the potential development of graft-versus-host disease (GvHD).

UCART123 cells are also engineered with a second TALEN® targeting the CD52 gene (see Table 3), in order to produce a mixture of CD52+ and CD52- cells. CD52- cells will be resistant to cell depletion by anti-CD52 therapies such as the monoclonal antibody alemtuzumab. This allows therefore the potential use of this monoclonal antibody in the lymphodepleting regimen prior to treatment withUCART123.

In brief, the UCART123 product comprises allogeneic engineered CAR-T cells which co-express a CAR targeting CD 123 and RQR8 and are knocked-out for TRAC and CD52 genes. The CAR expressed by the UCART123 comprises a VH of SEQ ID NO: 11, a VL of SEQ ID NO: 12, a CD8 alpha hinge of SEQ ID NO: 4, a CD8 alpha transmembrane of SEQ ID NO: 6, a CD3zeta stimulatory domain of SEQ ID NO: 9, and a 4-1 BB costimulatory domain of SEQ ID NO: 8. RQR8 comprises the amino acid sequence of SEQ ID NO: 23.

Example 2: Clinical trial of UCART123 in AML

This example briefly describes the schedule and conditions, as initially planned, of the on-going clinical trial of UCART123 in AML.

1. Generalities

This is a Phase I, first-in-human, open-label, dose-finding and expansion study of UCART123 administered intravenously to patients with AML. The study comprises a dose escalation phase with UCART123 in patients with relapsed or refractory (R/R) AML, and a dose-expansion phase in which additional patients with R/R AML will be enrolled to further evaluate the safety and preliminary efficacy of UCART123 at the dose/schedule and with the corresponding lymphodepletion regimen selected for further study. The dose escalation with UCART123 explores the doses of UCART123 ranging from 1.25xl0⁵ CD123CAR+ T-cells/kg to 5.05xl0⁶ CD123CAR+ T-cells/kg (including DL1 of 2.5xl0⁵, DL2 of 6.25xl0⁵, and DL3 of 3.30xl0⁶ CD123CAR+ T-cells/kg). Intermediate dose levels (e.g. DL2i of 1.5 xlO⁶ CD123CAR+ T-cells/kg) could also be tested. Table 4 summarizes the dose-levels tested.

Table 4.

The lymphodepleting regimen can be modified (either in composition or in doses) and adapted during the study upon safety, biological, and/or clinical activity observations.

In a first part of the dose-escalation of the clinical trial, in a single dose regimen, Patients received or will receive, prior to UCART123 administration, a lymphodepleting regimen consisting of either fludarabine and cyclophosphamide administered alone (Arm FC) or in combination with alemtuzumab (Arm FCA).

In a second part of the dose-escalation and in dose-expansion, Patients received or will receive a lymphodepleting regimen consisting of fludarabine and cyclophosphamide administered in combination with alemtuzumab (Arm FCA).

In the single regimen in the dose-escalation, Arms FC and FCA already started and progressed from DL1 through DL2 and DL2i, till up to, at least DL3.

Also, a two-dose regimen is added to the FCA Arm in the dose escalation to evaluate the safety and preliminary anti-leukemia activity of 2 infusions of UCART123 where the first infusion is preceded by a lymphodepletion, while the second infusion is not.

Figure 1 gives a schematic representation of a single-dose regimen (A) and a 2- dose regimen according to the methods described herewith (B). In brief, according to a single-regimen, one dose of CAR-T cells is administered after a preconditioning lymphodepleting treatment. This single-regimen may be repeated (i.e. administering another dose of CAR-T cells after another preconditioning lymphodepleting treatment) on a case-by-case basis after having completed the DLT observation period (i.e. after at least D28). According to the 2-dose regimen, after lymphodepletion, CAR T-cells are infused on DO and a second infusion of the equivalent, higher, or lower dose of CAR T-cells occurs around D14 with no additional lymphodepletion. In the 2-dose regimen protocol, the DLT observation period is 42 days starting from DO and can be extended to 8 weeks for patients with clinically significant cytopenias and/or bone marrow aplasia.

2. Lymphodepletion

The lymphodepleting regimen preceding UCART123 administration will consist of fludarabine and cyclophosphamide administered alone (Arm FC) or in combination with alemtuzumab (Arm FCA).

The lymphodepleting regimen will start as early as possible after enrollment. Patients must be hospitalized for lymphodepletion (beginning on Day -5).

Arm FC comprises administration of fludarabine and cyclophosphamide (FC) as follows: a) fludarabine 30 mg/m²/day IV given over 15 to 30 minutes for 4 days from Day -5 to Day -2 with a maximum daily dose of 60 mg b) cyclophosphamide 750 mg/m²/day IV given over 1 hour for 3 days from Day -4 to Day -2 with a maximum daily dose of 1.33 g

Arm FCA comprises administration of fludarabine, cyclophosphamide and alemtuzumab (FCA) as follows: a) fludarabine 30 mg/m²/day IV given over 15 to 30 minutes for 4 days (from Day -5 to Day -2) with a maximum daily dose of 60 mg b) cyclophosphamide 750 mg/m²/day IV given over 1 hour for 3 days (from Day -4 to Day -2) with a maximum daily dose of 1.33 g c) alemtuzumab 12 mg/day IV over 4 to 6 hours for 4 days (Day -5 to Day -2)

The doses and schedules of fludarabine, cyclophosphamide and alemtuzumab used in the lymphodepletion regimen may be changed during the study based on the ongoing review of the safety, activity, and translational data. Upon the review of the data, the total dose of alemtuzumab used in FCA regimen may be increased to a total of 60 mg if lymphodepletion observed is not sufficient to promote UCART123 expansion, proliferation, and persistence.

The lymphodepletion will take place from Day -5 to Day -2 during the week preceding UCART123 infusion at Day 0. Medical or other events may delay UCART123 infusion up to 2 days at the investigator’s discretion, with an appropriate documented rationale.

The dose expansion portion of this study will use the FCA lymphodepletion regimen.

3. Treatment by UCART123

The administration date of the initial UCART123 infusion is defined as Day 0 (DO). On Day 0, patients will receive an intravenous (IV) dose of UCARTT23 calculated based on the weight recorded at DO, as slow infusion.

3.1 Single dose regimen (comparative reference)

In the single dose regimen, administration of UCARTT23 at Day 0 is preceded by lymphodepletion as described above (FC or FCA).

UCART123 product is administered at one of the doses mentioned in Table 4.

After completing the Dose Limiting Toxicity (DLT) observation period (i.e. after 28 days from Day 0 of initial UCART123 administration) for subjects enrolled in the single dose regimen, administration of a second dose of UCART123 may be considered on a case-by-case basis. Administration of a second dose could be considered, for instance, if the patient has either stable disease or some level of reduction in bone marrow blasts and no safety complications related to the administration of the first dose of UCART123.

The patient may be retreated at the same, lower, or higher dose of UCART123 than previously received. The administration of this second dose of UCART123 will require a second period of lymphodepletion prior to administration of the second dose of UCART123, where this second lymphodepletion regimen may be the same or different than the one the patient previously received (i.e. FC, FCA or changes in dose and schedule of the agents).

In these conditions, as the administration of a second dose of UCART123 will be preceded or concomitant to a lymphodepletion treatment, this will constitute a repetition of classical single dose regimen preceded by a lymphodepleting, i.e. repeating the combination of a lymphodepletion treatment and a CAR-T infusion, after having observed that the first single dose regimen treatment was not sufficient to obtain a partial or complete response in the patient.

3.2 Two-dose regimen

In the two-dose regimen, the first administration of UCART123 at Day 0 is preceded by lymphodepletion as described above (FCA), followed by a second administration of UCART123 between Day 14 and 17 without carrying out another lymphodepletion treatment between DO and D28. The first and second infusions of UCART123 can be combined with the administration of Tocilizumab with or without concomitant corticosteroids as a prophylactic treatment of CAR-T cell-associated cytokine release syndrome.

If administered, Tocilizumab (8 mg/kg and maximum total dose 800 mg) will be administered on Day 0 intravenously over 1 hour and should be completed at least 1 hour before UCART123 infusion.

Tocilizumab is a humanized anti-IL-6R monoclonal antibody recommended by the ASTCT (Lee et al. (2019) Biol Blood Marrow Transplant. 25, 625-638) and has marketing authorization for the treatment of CRS.

UCART123 product will be administered at one of the doses mentioned in Table 4. UCART123 dose level 2 (6.25x 10⁵ cells/kg) is the first dose planned based on the safety data generated to date.

As indicated above, the day the patient receives the initial infusion of CAR-T cells defines Day 0. The second infusion of CAR-T cells is planned to be administered between Day 14 and 17, provided the patient meets the criteria for giving the second dose as outlined below.

Administration of a second dose of allogeneic UCART123 cells during this window will obviate the need for additional lymphodepletion which would not be advisable in the patient population due to potential for additional risks of prolonging myelosuppression or any other additional toxicities associated with administration of a second lymphodepletion.

3.3 Eligibility criterial for UCART123 administration

The eligibility criteria for initial UCART123 administration in FC and FC A arms are as follows (these criteria should be completed at Day 0, prior to any UCART123 administration):

1. No active uncontrolled infections;

2. Afebrile (<38°C per CTCAE v5.0);

3. Adequate organ function as defined in the trial inclusion criterion #5, and no new clinical or laboratory findings that, in the opinion of the investigator, might jeopardize the patient’s safety.

The eligibility criteria for second UCART123 administration are as follows:

1. No active uncontrolled infections on the day of infusion;

2. No CRS Grade > 2 on the day of infusion. If CRS Grade > 2 occurs at Day 14 then the second dose of UCART123 cells can be delayed until Day 17 to allow for resolution to Grade 1 or less;

3. Adequate organ function on the day of infusion, as defined in the trial inclusion criterion#5, and no new clinical or laboratory findings that, in the opinion of the investigator, might jeopardize the patient’s safety.

4. No Dose Limiting Toxicity (DLT) after the first UCART123 dose. Trial inclusion criterion #5 are as follows:

Adequate organ function, including renal and hepatic function based on the last assessment performed within the Screening Period, defined as: i) Creatinine clearance >60 mL/min (assessed as glomerular filtration rate using the Cockcroft & Gault formula or MDRD); ii) Alanine aminotransferase and aspartate aminotransferase <3 x upper limit of normal (ULN); iii) Total bilirubin <2 x ULN (except for patients with a history of Gilbert’s Syndrome confirmed by UGT1A1 mutation); iv) Left ventricular ejection fraction (LVEF) >50% as assessed by echocardiography or Multi Gated Acquisition Scan (MUGA); and v) Must have a minimum level of pulmonary reserve defined as Grade <2 dyspnea and pulse oxygenation >92% on room air.

4. Study Endpoints

Safety endpoints include Incidence, nature, and severity of adverse events and serious adverse events (SAEs) throughout the study (CTCAE v5.0; Lee et al. (Biol Blood Marrow Transplant. (2019) 25, 625-638) for CRS; Cairo and Bishop (Br. J. Haematol. (2004) 127, 3-11) for TLS, New consensus criteria 2016 (Harris et al (J. Am. Soc. Blood Marrow Transplant. (2016) 22, 4-10) for GvHD.

Efficacy endpoints include:

- Antileukemic activity, as measured by European Leukemia Net (ELN) Response Criteria in AML (Dohner et al., 2017). Response will be assessed following initial UCART123 administration at Day 14, Day 28, Day 56 and End of Treatment (EOT) then every 3 months until the end of Follow-up Period, and then as clinically relevant.

- Response assessment (CR, PR, etc.) as assessed by the investigator will be summarized. Summaries will be provided across all patients, by line of therapy. Duration of response (DoR) and PFS will be summarized. Alemtuzumab endpoints include:

- Monitoring of anti-CD52 (alemtuzumab) antibodies in serum pre- and post administration of alemtuzumab;

- Quantitation of alemtuzumab levels in serum post administration;

- Quantitation of T, B, and NK cells and total lymphocytes in peripheral blood post administration.

Exploratory endpoints include:

- Level of CD 123 CAR+ T-cells including CD4+ and CD8+ subsets measured by multiparameter flow cytometry in blood and bone marrow;

- Measurement of CD 123 CAR VCN per unit DNA by qPCR or ddPCR in blood and bone marrow;

- Monitoring of CD 123 CAR transcripts by digital polymerase chain reaction (ddPCR) or other methods in blood and bone marrow;

- Quantitation of soluble cytokines/growth factors and CRP levels in serum or plasma;

- Monitoring of human anti-mouse antibody (HAMA) production in serum or plasma;

- Monitoring of anti-HLA antibodies in serum or plasma;

- Monitoring patient leukemia cells in blood and bone marrow (including the potential impact of UCART123vl.2) by multiparameter flow cytometry (analysis of blast cell numbers and CD 123 antigen density);

- Monitoring of HSCs (CD34+ cells) in bone marrow by multiparameter flow cytometry;

- RCL testing by qPCR in blood;

- Monitoring lymphocyte subpopulations in blood samples by flow cytometry; and

- Evaluation of genomic DNA from bone marrow and/or peripheral blood by targeted next generation sequencing.

Example 3: Alemtuzumab prolongs lymphodepletion

Comparison of Figures 2A and 3A shows that absolute lymphocyte counts measured by a complete cell count and differential is about 0 for 10 days after FC, while it is about 0 for about at least 20 days after FCA pre-conditioning lymphodepleting regimen. Thus, addition of alemtuzumab to a lymphodepleting regimen comprising fludarabine and cyclophosphamide prolongs the depletion of patient’s immune cells by at least 10 more days.

Example 4: UCART123 expansion over time

As shown in Figure 2B (FC lymphodepleting regimen) and Figure 2C (FCA lymphodepleting regimen), after both types of preconditioning lymphodepleting regimens (FC or FC+ Alemtuzumab) followed by the administration of a first dose of UCART123 at DL2, UCART123 expansion, measured by qPCR from the patient’s whole blood sample, started after between about 5-10 days after the administration of UCART123 and reached a plateau at about 10-12 days. Depending on the patient, the peak of UCART123 was maintained for about 10 more days or dropped quickly in a couple of days.

Example 5: Cytokine secretion and UCART123 expansion are correlated

After lymphodepletion (FCA) and infusion of the first dose of UCART123, the profile of cytokines secretion measured in a sample of the patient’s serum by a Luminex panel is as follows (Figure 4):

IL-2 secretion peaked 1-2 days after 1^st dose UCART-123 infusion. IL-2 constitutes an early biomarker for UCART123 cell proliferation.

IL-6, IFN-gamma and TNF-alpha secretions were correlated with UCART123 cell expansion window.

For patients who experienced Grade 5 CRS, cytokines secretion was in the same range as patients with Grade 1 CRS.

The results of this example shows that the first dose of CAR-Ts generates a pro- inflammatory environment that could enhance the activity of a second dose of CAR-Ts.

Example 6: Anti-leukemic activity of UCART123

Anti-leukemic activity of UCART123 as measured by flow cytometry in the sample of a patient pretreated with either FC or FCA and then treated with a first dose of UCART123 (DL2) is represented in Figures 2C (FC) and 3C (FCA), respectively. Patient 114-229 (FCA, DL2): Achieved greater than 90% BMblast reduction (60% to 5%) at D28 (Stable Disease).

Patient 104-226 (FCA, DL2): Achieved CRi at D28 followed by MRD negative, Complete Response (CR) at D56.

In contrast, for some other patients, anti-leukemic activity of UCART123 did not allow a complete response or stable disease.

Conclusion: Results of Examples 4 to 6 show that UCART123 expansion correlated with reduction in tumor burden at DL2 (6.25 x 10⁵ cells/kg) but, at this dose, UCART123 cell function was not sufficient for sustained anti-leukemic activity in all patients. The leukemia debulking effect of the first dose of UCART123 could help restore bone marrow stromal function to support homeostasis of the immune system and increase local availability of cytokines that are necessary for C AR-T cells survival and expansion. Hence, a second dose of UCART123, administered between DIO to D17, could benefit from a favorable micro-environment and complete the reduction of the tumor and, thus, treat the patient’s cancer more efficiently.

Example 7 : Preliminary results of AMELI-01

AMELI-01 is a Phase 1 open-label dose-escalation trial evaluating the safety, tolerability, expansion and persistence of UCART123 given at escalating dose levels after lymphodepletion (LD) with either fludarabine and cyclophosphamide (FC) or FC with alemtuzumab (FCA) in patients with r/r AML. Alemtuzumab was added to the LD regimen to sustain host T-cell and Natural Killer (NK) cell depletion and to promote UCART123 - cell expansion and persistence.

Preliminary data from patients who received UCART123 at one of the following dose levels: dose level 1 (DL1) 2.5xl0⁵ cells/kg; dose level 2 (DL2) 6.25xl0⁵ cells/kg; intermediate dose level 2 (DL2i) 1.5xl0⁶ cells/kg; or dose level 3 (DL3) 3.30xl0⁶ cells/kg after lymphodepletion with FC or FCA are as follows:

17 patients received lymphodepletion and UCART123; 8 patients received UCART123 after FC at DL1 [n=2]; DL2 [n=3]; DL2i [n=2]; DL3 [n=l], and 9 patients received UCART123 after FCA at DL2 [n=8]; DL2i [n=l]. Enrolled patients were predominantly male [n=10], adult (median age 57 range [18-65]) and most were European Leukemia Net (ELN) adverse risk with high risk cytogenetic and molecular abnormalities including TP53 mutations. Additionally, enrolled patients were heavily pretreated with a median of 4 prior lines of therapy [range 3-9], 9 patients received prior hematopoietic stem cell transplantation (HSCT).

Safety Data

Adequate lymphodepletion was not achieved in the FC arm, as host lymphocyte recovery was observed in 7/8 patients prior to Day 28, and only 3/8 patients demonstrated UCART123 expansion.

In contrast, the FC A LD regimen resulted in robust lymphodepletion for greater than 28 days in all patients, and 7/9 patients demonstrated UCART123 expansion, with Cmax ranging from 13,177 to 330,530 copies/pg DNA, an almost 9-fold increase compared with FC LD, and a significant increase in AUC<o-28 days) (p=0.04; FC 10.2±15.7 vs. FCA 34.9±28.4).

Cytokine release syndrome (CRS) occurred in 8 patients in the FC arm and 9 patients in the FCA arm. In the FC arm, 1 patient experienced Grade 3 immune effector cell- associated neurotoxicity syndrome (ICANS) and 2 patients experienced Grade 4 protocol- defined dose limiting toxicities (DLTs). In the FCA arm, 2 patients experienced Grade 5 DLTs secondary to CRS.

Efficacy Data

Evidence of UC ART 123 anti -tumor activity was observed in 4 patients at DL2 or above with best overall responses in the FCA arm:

• a patient who failed 5 prior lines of therapy experienced a durable minimal residual disease (MRD) negative complete response (CR) with full count recovery at Day 56 that continues beyond 1 year

• a patient with stable disease achieved greater than 90% bone marrow blast reduction (60% to 5%) at Day 28.

The data show that adding alemtuzumab to the FC LD regimen was associated with sustained lymphodepletion and significantly higher UCART123 cell expansion, which correlated with improved anti-tumor activity. Overall, these data support the continued use of UCART123 after FCA lymphodepletion in patients with r/r AML.

General conclusion: The accumulated translational data to date demonstrates adequate lymphodepletion during the 28-day DLT period after one cycle of the current lymphodepletion regimen. Further, the current regimen and interval of lymphopenia has shown to correlate with UCART123 expansion and signs of clinical activity. A second dose of UCART123 cells given during the Day 10 -Day 17 window could allow for a second period of UCART123 expansion with the intent of improving the clinical activity without adding unnecessary toxicity of an additional UCART123 infusion as the overall disease burden will be reduced at the time of the second infusion.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having a cancer, the method comprising administering successively, to the subject in need thereof, a first and second doses of engineered T- cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CARs targeting specifically an antigen associated with the cancer,

- optionally, wherein said CAR-T cells are CD52 negative.

2. The method according to claim 1, wherein said lymphodepleting treatment comprises administration of an anti-CD52 therapeutic antibody such as alemtuzumab.

3. The method according to any one of claims 1 to 2, wherein said lymphodepleting treatment comprises administration of fludarabine or cyclophosphamide.

4. The method according to any one of claims 1 to 3, wherein said lymphodepleting treatment comprises administration of fludarabine and cyclophosphamide.

5. The method according to claim 4, wherein said lymphodepleting treatment comprises administration of fludarabine, cyclophosphamide and alemtuzumab.

6. The method according to claim 5, wherein said lymphodepleting treatment comprises administration of:

- fludarabine at between about 1 and 100 mg/m²/day, between about 10 and 75 mg/m²/day, between about 15 and 50 mg/m²/day, between about 20 and 40 mg/m²/day, or about 25, 30 or 40 mg/m²/day, for 1, 2, 3, 4, or 5 days; - cyclophosphamide at between about 50 and 10 000 mg/m²/day, between 50 and 5 000 mg/m²/day, between 50 and 1000 mg/m²/day, between 100 and 1000 mg/m²/day, between 500 and 1000 mg/m²/day, between 600 and 800 mg/m²/day, or about 600, 650, 700, 750 or 800 mg/m²/day, for 1, 2, 3, or 4 days;

- alemtuzumab at between about 5 and 50 mg/day, between 10 and 40 mg/day, between 10 and 20 mg/day, or at 10, 11, 12, 13, 14, or 15 mg/day, for 1, 2, 3, 4, or 5 days. The method according to claim 5 or claim 6, wherein said lymphodepleting treatment comprises administration of: fludarabine at 30 mg/m²/day given over 15 to 30 minutes for 4 days, with a maximum daily dose of 60 mg; cyclophosphamide at 750 mg/m²/day given over 1 hour for 3 days, with a maximum daily dose of 1.33 g; and alemtuzumab at 12 mg/day over 4 to 6 hours for 4 days. The method according to any one of claims 1 to 7, wherein:

- the first dose of CAR-T cells comprises between about 10⁴ to about 10⁸ CAR-T cells per kilogram body weight of the subject (cells/kg) and no more than 10⁹ total CAR- T cells, or between about 10⁵ to 10⁹ total CAR-T cells;

- wherein the antigen targeted by the CAR expressed by the CAR-T cells comprised in the first dose is identical or different from the antigen targeted by the CAR-T cells comprised in the second dose. The method according to any one of claims 1 to 8, wherein said T-cells are primary T- cells. The method according to claim 9, wherein said T -cells are cytotoxic T -lymphocyte and/or a helper T-lymphocytes.

11. The method according to any one of claims 9 to 10, wherein said primary T-cells originate from a human.

12. The method according to any one of claims 1 to 11, wherein said CAR-T cells are autologous with respect to the subject to be treated.

13. The method according to any one of claims 1 to 11, wherein said CAR-T cells are allogeneic with respect to the subject to be treated.

14. The method according to any one of claims 1 to 13, wherein said CAR-T cells have been genetically modified to confer resistance to at least one immune suppressive or chemotherapy drug.

15. The method according to any one of claims 1 to 14, wherein said CAR-T cells have been genetically modified to confer resistance to alemtuzumab.

16. The method according to claim 15, wherein said CAR-T cells have been genetically modified to suppress or repress expression of CD52 at the surface of said CAR-T cells.

17. The method according to any one of claims 1 to 16, wherein said CAR-T cells have been genetically modified to suppress or repress expression of at least one component of a T-Cell Receptor (TCR) at the surface of said CAR-T cell.

18. The method according to any one of claims 1 to 17, wherein said CAR-T cells have been genetically modified to suppress or repress expression of a TCRa gene and/or a TCR0 gene.

19. The method according to any one of claims 1 to 18, wherein said CAR-T cells have at least one gene encoding TCR alpha, TCR beta, and/or CD3 that has been inactivated.

20. The method according to any one of claims 1 to 19, wherein said CAR-T cells have been genetically modified to suppress or repress expression of at least one gene encoding a MHC-I protein, such as 02m and HLA.

21. The method according to any one of claims 1 to 20, wherein said CAR-T cells have a 02m gene that has been inactivated and have, integrated in their genome, an exogenous sequence encoding a NK inhibitor such as a HLA-E peptide fusion protein.

22. The method according to any one of claims 1 to 21 , wherein said CAR-T cells have been genetically modified to suppress or repress expression of a gene encoding an immune checkpoint protein and/or a receptor thereof.

23. The method according to any one of claims 1 to 22, wherein said CAR-T cells have been genetically modified to comprise a suicide gene.

24. The method according to any one of claims 1 to 23, wherein said CAR-T cells are one or more of: CD52 negative, TCR negative, B2M negative, PDCD1 negative.

25. The method according to any one of claims 1 to 24, wherein said CAR-T cells are at least CD52 negative or at least CD52 and TCR negative.

26. The method according to any one of claims 1 to 25, wherein said cancer is an haematological cancer or a solid cancer.

27. The method according to any one of claims 1 to 26, wherein said cancer is a leukemia such as leukemia selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome.

28. The method according to any one of claims 1 to 27, wherein said cancer is acute myelogenous leukemia (AML).

29. The method according to any one of claims 1 to 28, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting an antigen selected from the group consisting of CD123, CD38, CLL1, FLT3, CD7, FRbeta, CD33, LEY.

30. The method according to any one of claims 1 to 29, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123.

31. The method according to any one of claims 1 to 30, wherein at least one of said first dose and second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123 having a polypeptide structure comprising an extra cellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, a CD8a hinge, a CD8a transmembrane domain, and a cytoplasmic domain including a CD3 signaling domain and a co-stimulatory domain from 4-1BB, wherein said VH comprises the CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and said VL comprises the CDR sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

32. The method according to claim 31 , wherein said VH has at least 80% identity with SEQ ID NO: 11 and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 15, and said VL has at least 80% identity with SEQ ID NO: 12 and comprises the CDRs of amino acid sequences SEQ ID NO: 16 to SEQ ID NO: 18.

33. The method according to claim 31 or claim 32, wherein said VH comprises the amino acid sequence of SEQ ID NO: 11 and said VL comprises the amino acid sequence of SEQ ID NO: 12.

34. The method according to any one of claims 31 to 33, wherein said CAR targeting CD 123 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 19, and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to 18.

35. A pharmaceutical composition comprising a first or second dose of engineered T- cells expressing a chimeric antigen receptor (CAR) (CAR-T cells), said CAR targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer;

- optionally, wherein said engineered CAR-T cells are CD 52 negative.

36. A combination of a pharmaceutical composition comprising a first dose of engineered T-cells expressing a CAR (CAR-T cells) and a pharmaceutical composition comprising a second dose of engineered T-cells expressing a CAR (CAR-T cells), said CARs targeting specifically an antigen associated with a cancer, for use in the treatment of a subject having said cancer;

- optionally, wherein said engineered CAR-T cells are CD 52 negative. The pharmaceutical composition for use according to claim 35, wherein said T cells and CAR-T cells are as mentioned in any one of claims 9 to 25 and claims 29 to 34, said lymphodepleting treatment is as mentioned in any one of claims 2 to 7, said first and second doses of CAR-T cells are as mentioned in claim 8, and said cancer is as mentioned in any one of claims 26 to 28. The combination of pharmaceutical compositions for use according to claim 36, wherein said T cells and CAR-T cells are as mentioned in any one of claims 9 to 25 and claims 29 to 34, said lymphodepleting treatment is as mentioned in any one of claims 2 to 7, said first and second doses of CAR-T cells are as mentioned in claim 8, and said cancer is as mentioned in any one of claims 26 to 28. A pharmaceutical composition comprising a first or a second dose of engineered T-cells expressing a chimeric antigen receptor (CAR) (CAR-T cells) specific for CD 123 (CD 123 CAR-T cells), for use in the treatment of a subject having a cancer characterized by CD 123 -expressing cells; - wherein said treatment comprises administering successively said first and second doses of said CD123CAR-T cells;

- wherein the second dose of CD123CAR-T cells is administered between 10 and 17 days after the first dose of CD123CAR-T cells, such as after 10, 11, 12, 13, 14, 15,

16 or 17 days;

- wherein said CD123CAR-T cells are CD52 negative, and, optionally, TCR negative. A combination of a pharmaceutical composition comprising a first dose of CD 123 CAR-T cells and a pharmaceutical composition comprising a second dose of CD 123 CAR-T cells, for use in the treatment of a subject having a cancer characterized by CD 123 -expressing cells;

- wherein the second dose of CD 123 CAR-T cells is administered between 10 and 17 days after the first dose of CAR-T cells, such as after 10, 11, 12, 13, 14, 15, 16, or

17 days; - wherein, prior to the administration of the first dose of CD123CAR-T cells, the subject has been preconditioned with a lymphodepleting treatment comprising fludarabine, cyclophosphamide and alemtuzumab to eliminate, at least partially, the subject’s own immune cells to have, from at least the day of the administration of the first dose of CAR-T cells and for at least the 20 following days, less than 500 absolute lymphocyte counts per pl of subject’s whole blood;

- wherein said CD123CAR-T cells are CD52 negative, and, optionally, TCR negative. The pharmaceutical composition for use according to claim 39 or the combination of pharmaceutical compositions for use according to claim 40, wherein said lymphodepleting treatment is as mentioned in any one of claims 2 to 5 or 7, or wherein said lymphodepleting treatment comprises administration of:

- alemtuzumab at between about 5 and 50 mg/day, between 10 and 40 mg/day, between 10 and 20 mg/day, or at 10, 11, 12, 13, 14, or 15 mg/day, for 1, 2, 3, 4, or 5 days. The pharmaceutical composition for use according to any one of claims 39 or 41 or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41, wherein the first dose and/or the second dose of CD123CAR-T cells comprises between about 10⁵ to about 10⁷ CD123CAR-T cells per kilogram body weight of the subject (cells/kg), such as from about 6.0 to 6.5 xlO⁵, from about 1.0 to 1.5 xlO⁶, from about 3.0 to 3.5 xlO⁶, or from about 5.0 to 5.5 xlO⁶ CD123CAR-T cells/kg, and no more than 5.10⁸ total CD123CAR-T cells. The pharmaceutical composition for use according to any one of claims 39 or 41 to 42 or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 42, wherein said first dose and/or second dose of CAR-T cells comprises CAR-T cells expressing a CAR targeting CD 123 having a polypeptide structure comprising an extra cellular ligand binding-domain comprising a heavy chain variable region (VH) and a light chain variable region (VL) from a monoclonal anti-CD123 antibody, a CD8a hinge, a CD8a transmembrane domain, and a cytoplasmic domain including a CD3 signaling domain and a co -stimulatory domain from 4-1BB, wherein said VH comprises the CDR sequences of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 and said VL comprises the CDR sequences of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18. The pharmaceutical composition for use according to any one of claims 39 or 41 to 43 or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 43, wherein said VH has at least 80% identity with SEQ ID NO: 11 and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to SEQ ID NO: 15, and said VL has at least 80% identity with SEQ ID NO: 12 and comprises the CDRs of amino acid sequences SEQ ID NO: 16 to SEQ ID NO: 18. The pharmaceutical composition for use according to claim 43 or 44 or the combination of pharmaceutical compositions for use according to claim 43 or 44, wherein said VH comprises the amino acid sequence of SEQ ID NO: 11 and said VL comprises the amino acid sequence of SEQ ID NO: 12. The pharmaceutical composition for use according to any one of claims 43 to 45 or the combination of pharmaceutical compositions for use according to any one of claims 43 to 45, wherein said CAR targeting CD123 comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 19, and comprises the CDRs of amino acid sequences SEQ ID NO: 13 to 18. The pharmaceutical composition for use according to any one of claims 43 to 46 or the combination of pharmaceutical compositions for use according to any one of claims 43 to 46, wherein said CAR targeting CD 123 comprises the amino acid sequence of SEQ ID NO: 19. The pharmaceutical composition for use according to any one of claims 39 or 41 to 47 or the combination of pharmaceutical compositions for use according to claim 40 or 41 to 47, wherein said CD123CAR-T cells have, integrated in their genome, an exogenous sequence encoding a suicide domain having an amino acid sequence comprising SEQ ID NO: 23. The pharmaceutical composition for use according to any one of claims 39 or 41 to 48 or the combination of pharmaceutical compositions for use according to any one of claims any one of claims 40 or 41 to 48, wherein said cancer is a pre-malignant or malignant cancer condition characterized by an overabundance of CD 123 -expressing cells. The pharmaceutical composition for use according to any one of claims 39 or 41 to 49 or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 49, wherein said cancer is a haematological cancer. The pharmaceutical composition for use according to any one of claims 39 or 41 to 50 or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 50, wherein said haematological cancer is a leukemia or a malignant lymphoproliferative disorder. The pharmaceutical composition for use according to any one of claims 39 or 41 to 51, or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 51, wherein said leukemia is selected from the group consisting of acute myelogenous leukemia, chronic myelogenous leukemia, melodysplastic syndrome, acute lymphoid leukemia, chronic lymphoid leukemia, and myelodysplastic syndrome. The pharmaceutical composition for use according to any one of claims 39 or 41 to 52, or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 52, wherein said leukemia is acute myelogenous leukemia (AML). The pharmaceutical composition for use according to any one of claims 39 or 41 to 53, or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 54, wherein said hematological cancer is a malignant lymphoproliferative disorder. The pharmaceutical composition for use according to any one of claims 39 or 41 to 54, or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 54, wherein said malignant lymphoproliferative disorder is a lymphoma. The pharmaceutical composition for use according to any one of claims 39 or 41 to 55, or the combination of pharmaceutical compositions for use according to any one of claims 40 or 41 to 55, wherein said lymphoma is selected from the group consisting of multiple myeloma, non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell).

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