Identifiable cell surface protein variants of CD45 for use in cell therapy - Patent Application 20070123333

Abstract

The present disclosure relates to the use of cells having identifiable surface proteins that have engineered or naturally occurring mutations but functional surface proteins for use in therapy. The present invention also relates to the use of cells having identifiable CD45 surface protein variants but functional surface proteins for use in therapy, particularly adoptive cell therapy.

Description

[Technical field]

[0001] The present disclosure relates to the use of cells having identifiable surface proteins that have engineered or naturally occurring mutations but functional surface proteins for use in therapy. The present invention also relates to the use of cells having identifiable CD45 surface protein variants but functional surface proteins for use in therapy, particularly adoptive cell therapy.

[0002] Description of funds The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no. 818806). [Background technology]

[0003] Cell-based immunotherapy has emerged as the third pillar of small molecule therapy and post-treatment medicine based on biologics such as recombinant proteins, including antibodies. Cell therapy can be used in oncology to treat hematopoietic malignancies, but other applications such as the treatment of genetic diseases, solid organ tumors and autoimmune diseases are also under development. However, cell therapy can be associated with severe undesirable side effects. Indeed, cancer immunotherapy using chimeric antigen receptor (CAR) T cells has been successful in targeting and eradicating malignant cells expressing specific antigens, but it often does not distinguish between normal and malignant cells, thus inducing the destruction of the normal hematopoietic system. Targeted therapies include antibody-based therapies, such as conventional monoclonal antibodies, multispecific antibodies, such as T cell engagers (e.g., BiTEs), and cell therapies, such as CAR cells (e.g., CAR T cells, CAR NK cells or CAR macrophages), which eliminate all cells expressing the target molecule. However, most cancer cell surface antigens are shared with normal hematopoietic cells or other cells. Therefore, identifying targets that kill diseased cells, including tumors, while avoiding damage to healthy cells is a major challenge for targeted therapy (Perna et al., Cancer Cell (2017) 32: 506-519). In particular, in myeloid diseases, including myeloid malignancies such as myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) or blastic plasmacytoid dendritic cell neoplasms (BPDCN), cell surface antigens such as CD117, CD33 or CD123 are shared with normal myeloid progenitor cells. Thus, immunotherapy targeting CD117, CD33 or CD123 antigens for MDS, AML or BPDCN may be associated with depletion of normal hematopoietic cells in addition to malignant cells in patients (Gill SIBest practice & Research Clinical Hematology, 2019). As a result, targeted immunotherapies, including mAbs, T cell engagers or CAR T, have largely been challenging, in part due to the lack of truly disease-specific surface antigens (Gill SIBest practice & Research Clinical Hematology, 2019).

[0004] To regenerate normal hematopoiesis depleted by CD33-CAR T cell transfer, CD33 CAR T cell-tolerant hematopoietic cells have been engineered to have the entire CD33 gene knocked out (Kim et al., 2018. Cell. 173:1439-53). However, CD33 has a constitutive inhibitory effect on myeloid cells through its immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domain. Thus, it remains unclear how well loss of CD33 can be tolerated (Wiβfeld et al. Glia (2021) 69:1393-1412). CD33 knockout (CD33 KO) engineered cells transplanted into patients may show long-term functional defects (WO 2018 / 160768, Kim et al. 2018. Cell. 173:1439-53, Borot et al. 2019. PNAS. 116:11978-87, Humbert et al. 2019. Leukemia. 33:762-808). Indeed, the frequency of CD33 KO cells was reduced in two monkeys where long-term observation was reported. This may indicate impaired functionality of CD33 KO cells, for example, due to reduced engraftment of CD33 KO long-term repopulating HSCs (LT-HSCs) or due to competitive disadvantage (Kim et al. 2018. Cell. 173:1439-53). Moreover, the number of cell surface antigens with essential functions is very limited, and loss of said redundant cell surface antigens may induce antigen-negative relapse. CD19-negative relapse is observed in approximately 30% of patients receiving CD19-targeted CAR T therapy (Orlando et al. 2018 Nat Med 24:1504-6). Dual targeting of CD19 and CD123 can prevent relapse of antigen loss (Ruella et al. 2016 J Clin Invest 126:3814-26).

[0005] In previous patent applications, we have shown that single amino acid differences in surface protein variants can be engineered into hematopoietic cells to alter their antigenicity and be recognized by specific and selective antibodies (WO 2017 / 186718, WO 2018 / 083071). In contrast to approaches in which surface proteins are deleted (KO cells), the surface protein variants in these cells retain their normal expression and function, making it possible to target surface proteins with important non-redundant functions.

[0006] CD45 is also known as protein tyrosine phosphatase receptor type C (PTPRC) and is an enzyme encoded by the PTPRC gene (Kaplan et al., PNAS 87:7000-7004 (1990)). CD45 is a member of the protein tyrosine phosphatase (PTP) family, which includes signaling molecules that regulate various cellular processes including cell growth, differentiation, the mitotic cycle, and oncogenic transformation. CD45 contains an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and thus belongs to the receptor-type PTP family. CD45 is a type I transmembrane protein that exists in various isoforms on differentiated hematopoietic cells (except for erythrocytes, for example) (Holmes, Immunology 7:145-55 (2006)). CD45 has been shown to be a regulator of T and B cell antigen receptor signaling. It functions by direct interaction with components of the antigen receptor complex through its extracellular domain, or by activating various Src family kinases (SFKs), such as Lck, which are required for antigen receptor signaling through its cytoplasmic domain. CD45 also inhibits JAK kinases and thus functions as a negative regulator of cytokine receptor signaling.

[0007] CD45 is present on the surface of hematopoietic cells, including HSCs (hematopoietic stem cells), leukocytes and osteoclasts of hematopoietic origin (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations in CD45 in humans are associated with severe immune deficiency. This is primarily due to the absence of CD45 on T cells, where it is typically abundant and necessary for regulating SFK activity during antigen responses. CD45 deficiency (CD45 - / - ) Mouse bone marrow contains normal numbers of hematopoietic cells, but the numbers of the most primitive HSCs are reduced and their mobilization in response to G-CSF is impaired. In part, this defect is specific to HSCs. Without CD45-mediated downregulation of SFK activity, integrin-mediated adhesion is high and HSCs are more likely to remain in the stem cell niche. CD45 - / - HSCs also have defects in G-CSF-stimulated mobilization and homing to the chemokine CXCL12 / SDF-1, which adversely affects cell engraftment after transplantation. These defects can be rescued by supplementation with SFK inhibitors, indicating that this role is normally performed by CD45. Similarly, CD45 - / - Recipients also showed incomplete engraftment and subsequent mobilization of normal HSCs, indicating a role for CD45 in the stem cell niche and HSCs (Shivtiel et al., J Exp Med 205:2381 (2008)). Because CD45 is expressed, for example, on HSCs and leukocytes, it presents a target for therapy, including conditioning therapy, immune reset, and treatment of disease.

[0008] The present disclosure aimed to identify amino acid residues of CD45 that are exposed on the cell surface and can be substituted such that a) the function of CD45 is not altered or at least not substantially altered, i.e., the mutant of CD45 is functionally indistinguishable from the wild-type version of CD45, and b) that bind to the wild-type version of CD45, such as an antibody or CAR T cell, but show substantially reduced or no binding to the modified version of CD45, i.e., the mutant of CD45 is immunologically distinguishable from the wild-type version of CD45. Most single amino acid substitutions in any given target protein only affect the binding of the moiety if the amino acid substitution is part of or close to the epitope of the binding moiety. As will be appreciated, single amino acid substitutions that affect the binding of the binding moiety to the target antigen may also affect the functionality of the target antigen. Thus, it is a highly sophisticated and unpredictable challenge to identify amino acid substitutions that satisfy both requirements of affecting the binding of the moiety to the target antigen and at the same time not affecting or not substantially affecting its function.

[0009] CD45 is expressed on all nucleated hematopoietic cells and is therefore a target with a wide range of therapeutic applications. This includes HSC depletion, as autoreactive lymphocytes (B cells and T cells) are actively depleted, as well as autoimmune diseases. Anti-CD45 therapy is also useful for treating antigen-negative relapses of most targeted therapies.

[0010] Some anti-CD45 moieties are known in the art. QA17A19 (Biolegend, #393411) and HI30 (Biolegend, #304001) are commercially available mouse anti-human CD45 antibodies. Various anti-CD45 antibodies from Magenta Therapeutics are in development, most of which are developed as antibody-drug conjugates (e.g., WO2017219025, WO2020092654). BC8 is a commercially available mouse hybridoma antibody from IchorBio (#ICH1155). The BC8 antibody is the basis of the anti-CD45 antibody-radioconjugate developed by Actinium Pharmaceuticals (WO2017155937, WO2019084258, WO2020159656). Other anti-CD45 antibodies are disclosed in WO2016016442, WO2019115791 and WO2020058495 (INSERM), WO2017009473 (UCB), WO2019129178 (Shanghai Baize Medical Laboratory), WO2020018580 (Fred Hutchinson) and WO2020170254 (Ramot At Tel Aviv University). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 2018 / 160768 [Patent Document 2] International Publication No. 2017 / 186718 [Patent Document 3] International Publication No. 2018 / 083071 [Patent Document 4] International Publication No. 2017219025 [Patent Document 5] International Publication No. 2020092654 [Patent Document 6] International Publication No. 2017155937 [Patent Document 7] International Publication No. 2019084258 [Patent Document 8] International Publication No. 2020159656 [Patent Document 9] International Publication No. 2016016442 [Patent Document 10] International Publication No. 2019115791 [Patent Document 11] International Publication No. 2020058495 [Patent Document 12] International Publication No. 2017009473 [Patent Document 13] International Publication No. 2019129178 [Patent Document 14] International Publication No. 2020018580 [Patent Document 15] International Publication No. 2020170254 [Non-patent literature]

[0012] [Non-Patent Document 1] Perna et al., Cancer Cell (2017) 32:506-519) [Non-Patent Document 2] Gill SIBest practice&Research Clinical Hematology,2019 [Non-Patent Document 3] Kim et al.,2018.Cell.173:1439-53 [Non-Patent Document 4] Wiβfeld et al. Glia(2021)69:1393-1412 [Non-Patent Document 5] Borot et al.2019.PNAS.116:11978-87 [Non-Patent Document 6] Humbert et al.2019.Leukemia.33:762-808 [Non-Patent Document 7] Orlando et al.2018 Nat Med 24:1504-6) [Non-Patent Document 8] Ruella et al.2016 J Clin Invest 126:3814-26 [Non-Patent Document 9] Kaplan et al., PNAS 87:7000-7004(1990) [Non-Patent Document 10] Holmes, Immunology 7:145-55(2006) [Non-Patent Document 11] Shivtiel et al., J Exp Med 205:2381(2008) [Non-Patent Document 12] Ramot at Tel Aviv University Summary of the Invention

[0013] One of the objectives of the present disclosure is to develop safer methods for treating malignancies, particularly cancer, hematological malignancies, and myeloid diseases. Thus, the inventors sought mutations of the surface protein CD45 that are immunologically distinguishable while retaining or substantially retaining normal function, and in which the amino acid changes are derived from single or multiple amino acid or nucleotide mutations. In particular, the inventors identified rationally designed naturally occurring variants of CD45 and showed that these mutations alter the antigenicity of CD45 to certain antibodies while retaining its normal expression and function, that its intracellular domain mediates the dephosphorylation of target proteins, e.g., the tyrosine kinase Lck, and that its extracellular region interferes with the immune synapse, thereby regulating, e.g., T and B cell function. CD45 is also involved in the in vivo development of hematopoietic cells, which can be tested, e.g., in humanized mice. Similarly, the structure of CD45 is important, especially as an extracellular spacer.

[0014] The present disclosure relates to a mammalian cell or population of cells expressing a first isoform of CD45 for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of CD45, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of said patient having cells expressing said second isoform of CD45, and said first and second isoforms are functional. Alternatively, said first isoform is generated via RNA editing.

[0015] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells, preferably hematopoietic stem cells, for use in medical treatment in a patient in need thereof, said medical treatment comprising administering to said patient in need thereof a therapeutically effective amount of said cell or population of cells expressing a first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising at least a first antigen binding region that specifically binds to a second isoform of CD45 to specifically deplete the patient's cells expressing said second isoform of CD45, preferably to restore normal hematopoiesis after immunotherapy in the treatment of a hematopoietic disease, preferably a malignant hematopoietic disease, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), T-cell non-Hodgkin's lymphoma (T-NHL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma (NHL) or follicular lymphoma (FL).

[0016] In other embodiments, the medical treatment relates to restoration of hematopoietic or immune function in genetic disorders of the hematopoietic or immune system, such as severe combined immunodeficiency syndrome (SCID), sickle cell disease (SCD), beta thalassemia, Fanconi anemia, or Diamond-Blackfan anemia.

[0017] In other embodiments, medical treatment relates to the restoration of normal function in genetic diseases that do not originate from the hematopoietic and immune systems, but that can be treated by the use of modified hematopoietic cells.

[0018] In other embodiments, the medical treatment relates to restoration of normal immune function in autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) or multiple sclerosis (MS).

[0019] In another particular embodiment, the disclosure relates to a mammalian cell or population of cells for use in medical treatment in a patient in need thereof, said medical treatment being directed to a mammalian cell or population of cells for use in medical treatment in a patient in need thereof, preferably for use in adoptive cell transfer therapy, more preferably for use in the treatment of malignant hematopoietic diseases, such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), T-cell non-Hodgkin's lymphoma (T-NHL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma (NHL ... non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), non-Hodgkin's lymphoma (NHL), chronic myelogenous leukemia (CML), and administering to said patient in need thereof a therapeutically effective amount of said cell or population of cells expressing said first isoform in combination with a therapeutically effective amount of a depleting agent comprising at least a second antigen binding region that specifically binds to said first isoform for the treatment of non-human leukemia (NHL) or follicular lymphoma (FL), again more preferably said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of a surface protein to avoid eventual serious side effects such as graft-versus-host disease upon transplantation.

[0020] In another aspect, the present disclosure relates to a pharmaceutical composition comprising mammalian cells, preferably hematopoietic stem cells or immune cells, such as T cells as described above, and preferably a depleting agent, and a pharma- ceutically acceptable carrier.

[0021] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD45.

[0022] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, wherein the first and second isoforms are substantially functionally identical. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD45.

[0023] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, and wherein the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and wherein the first and second isoforms result in dephosphorylation of a target protein.

[0024] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's natural cells express a second isoform of CD45, and wherein the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and wherein the first and second isoforms result in dephosphorylation of the tyrosine kinase Lck.

[0025] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, and wherein the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and wherein the first and second isoforms result in essentially the same modulation of T cell function and / or B cell function.

[0026] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, and wherein the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and wherein the first and second isoforms result in normal differentiation of hematopoietic cells.

[0027] The present disclosure also provides a depletion agent for use in preventing or reducing the risk of serious side effects in a patient administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, the depletion agent comprising at least a second antigen binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and the polymorphism or engineered allele is a sequence The depleting agent is characterized by at least one substitution of an amino acid at positions E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO: 1, preferably at least one substitution of an amino acid at positions E230, N257, E259, F331, K352 or E353 of SEQ ID NO: 1. In a particular embodiment, the depleting agent binds substantially weaker to the second isoform of CD45.

[0028] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient receiving cells expressing a first isoform of CD45, wherein the patient's natural cells express a second isoform of CD45, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind to the second isoform of CD45, and the polymorphism or engineered allele is characterized by at least one substitution of amino acids at positions I328, N255, E360, E259, E364 and E269. Among these substitutions, substitutions I328V, N255G, E360G, E259G, E364K and E269G are particularly preferred. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD45.

[0029] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient administered cells expressing a first isoform of CD45, wherein the patient's native cells express a second isoform of CD45, the depleting agent comprising at least a second antigen-binding region that specifically binds to said first isoform of CD45 and does not bind to said second isoform of CD45, wherein residue E230 is substituted with K, and / or residue N257 is substituted with A, D, E, H, K, R, S, T or V, and / or residue E259 is substituted with G, N, T or Q, and and / or T264 is substituted by D or E, preferably by D, and / or N267 is substituted by A, H, S or V, and / or residue N286 is substituted by G, L or R, and / or E329 is substituted by A, and / or residue F331 is substituted by A or G, and / or Y340 is substituted by A, G, N, Q or S, and / or residue K352 is substituted by A, D, E, G, H, I, L, M, N, Q, S, T or Y, and / or residue E353 is substituted by A, H, I, K, L, S, T or R, preferably by A, I, K, L, S, T or R.

[0030] The present disclosure also relates to a method for improving the engraftment of hematopoietic stem cell transplantation. Conditioning (HSC depletion) prior to hematopoietic stem cell transplantation (HSCT) is used to promote engraftment. In fact, the conditioning effect is associated with improved engraftment. Avoiding toxic conditioning is an important goal that can be achieved with the present disclosure. Current methods for conditioning include the use of intravenous busulfan. Busulfan is a DNA alkylating drug that was initially designed to treat hematological diseases such as acute myeloid leukemia (AML). However, busulfan carries significant risks of side effects, including sterility, primary or secondary malignancies, and additional acute and chronic toxicities.

[0031] The present disclosure also relates to a human cell or population of human cells expressing a first isoform of CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD45, the human cell expressing the first isoform comprises genomic DNA having at least one polymorphic or engineered allele; the polymorphism or engineered allele is absent from the genome of a patient whose cells express the second isoform of CD45; Said polymorphic or engineered allele is characterized by at least one substitution of an amino acid at positions E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO: 1. Preferably, said first and said second isoforms are substantially functionally identical.

[0032] In certain embodiments, said first and said second isoforms dephosphorylate target proteins of CD45, activate the TCR signaling cascade, resulting in increased cytokine production and / or increased proliferation of T cells.

[0033] In a particular embodiment, said first and said second isoforms of CD45 dephosphorylate the tyrosine kinase Lck.

[0034] In certain embodiments, the medical treatment comprises administering to the patient in need thereof a therapeutically effective amount of the cell or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising an antigen-binding region that specifically binds to the second isoform of CD45, to specifically deplete the patient's cells expressing the second isoform of CD45.

[0035] In certain embodiments, said polymorphism or engineered allele is characterized by at least one substitution of an amino acid at position F331, K352 or E353 of SEQ ID NO: 1. In certain embodiments, said polymorphism or engineered allele is characterized by a substitution of an amino acid at position K352 of SEQ ID NO: 1. In certain embodiments, said substitution at position K352 is selected from K352E, K352H, K352I, K352L, K352M, K352N, K352Q, K352S and K352T, preferably K352D, K352E and K352H, more preferably said substitution is K352E.

[0036] In certain embodiments, the depleting agent used in conjunction with the amino acid substitution is at position N286, F331, K352 or E353 of SEQ ID NO:1 and is selected from the following: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and b) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:58 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:59.

[0037] In certain embodiments, the polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position E230, Y232, N257 or E259 of SEQ ID NO:1.

[0038] In a particular embodiment, said polymorphism or engineered allele is characterized by a substitution of an amino acid at position N257 of SEQ ID NO: 1. In a particular embodiment, said substitution at position N257 is selected from N257E, N257K, N257R and N257T, preferably N257R. In a particular embodiment, said polymorphism or engineered allele is characterized by a substitution of an amino acid at position E259 of SEQ ID NO: 1. In a particular embodiment, said substitution at position E259 is selected from E259N, E259Q, E259V and E259G, preferably E259V. In a particular embodiment, said polymorphism or engineered allele is characterized by a substitution of an amino acid at position Y232 of SEQ ID NO: 1, preferably Y232C.

[0039] In certain embodiments, the depleting agent used in conjunction with the amino acid substitution is at position E230, Y232, N257 or E259 of SEQ ID NO:1, an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO:20, VHCDR2 is SEQ ID NO:21 and VHCDR3 is SEQ ID NO:22; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO:23, VLCDR2 is SEQ ID NO:24 and VLCDR3 is SEQ ID NO:25; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:18 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:19.

[0040] In certain embodiments, said polymorphism or engineered allele is characterized by a substitution of the amino acid at position N257 of SEQ ID NO: 1. In certain embodiments, said polymorphism or engineered allele is characterized by a substitution of the amino acid at position N257 of SEQ ID NO: 1. In certain embodiments, said substitution at position N257 is selected from N257E, N257K, N257R and N257T, preferably N257R.

[0041] In certain embodiments, the depleting agent used in conjunction with a substitution of the amino acid at position N257 of SEQ ID NO:1 comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; and b) an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17; Preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:10 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:11.

[0042] In a particular embodiment, said polymorphism or engineered allele is characterized by at least one substitution of an amino acid at positions E230, N257, E259, F331, K352 or E353 of SEQ ID NO: 1. Said substitution may be selected from a CD45 variant, wherein said residue E230 is substituted with K, and / or residue N257 is substituted with A, D, E, H, K, R, S, T or V, and / or residue E259 is substituted with G, H, K, N, R, T or Q, and / or T264 is substituted with D or E, preferably with D, and / or N267 is substituted with A, H, L, S or V, and and / or residue N286 is substituted with G, L or R, and / or E329 is substituted with A, and / or residue F331 is substituted with A or G, and / or Y340 is substituted with A, G, N, Q or S, and / or residue K352 is substituted with A, D, E, G, H, I, L, M, N, Q, S, T or Y, and / or residue E353 is substituted with A, H, I, K, L, S, T or R.

[0043] The present disclosure also relates to a human cell or population of human cells for the uses disclosed herein, wherein said cell expressing said first isoform of CD45 is selected from a subject that comprises naturally occurring genomic DNA having at least one naturally occurring polymorphic allele in a nucleic acid encoding said first isoform.

[0044] The present disclosure also relates to a human cell or a population of human cells for a use as disclosed herein, wherein said first isoform of CD45 is obtained by modifying ex vivo a nucleic acid sequence encoding said first isoform of CD45 by gene editing, preferably by introducing into a human cell a gene editing enzyme capable of inducing site-specific mutations in a target sequence encoding a surface protein region involved in binding of an agent comprising at least a first antigen binding region.

[0045] The present disclosure also relates to a human cell or a population of human cells for use as disclosed herein, wherein said medical treatment comprises administering to said patient in need thereof a therapeutically effective amount of a human cell or a population of human cells expressing said first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising at least a first antigen binding region that specifically binds to said second isoform of CD45 to specifically deplete patient cells expressing said second isoform of CD45, preferably to restore normal hematopoiesis after immunotherapy in the treatment of hematopoietic disorders, preferably in the treatment of malignant hematopoietic disorders such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), T-cell non-Hodgkin's lymphoma (T-NHL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma (NHL) or follicular lymphoma (FL).

[0046] The present disclosure also relates to a human cell or population of human cells for the uses disclosed herein, wherein said depleting agent is an antibody, an antibody-drug conjugate or an immune cell, preferably a T cell, having a chimeric antigen receptor (CAR) comprising a first antigen-binding region that specifically binds to said second isoform and does not bind, or binds substantially weaker to, said first isoform.

[0047] The present disclosure also relates to a pharmaceutical composition comprising a human cell as disclosed herein, preferably a hematopoietic stem cell or an immune cell such as a T cell, preferably a depletion agent as disclosed herein, and a pharmaceutically acceptable carrier. [Brief description of the drawings]

[0048] [Figure 1] Binding of anti-CD45 antibodies to DF-1 cells transfected with human wild-type CD45 or empty vector is shown. Serial dilutions of each antibody were tested for immunoreactivity by flow cytometry. All five antibodies in MAb format bind to human CD45 positive cells in a concentration-dependent manner. Cells transfected with empty vector showed no binding of anti-CD45 antibodies. [Diagram 2] Binding of anti-CD45 Fab fragments to DF-1 cells transfected with human wild-type CD45 or empty vector is shown. Similar to the full-length antibody, the Fab fragments bind to human CD45 in a concentration-dependent manner. Cells transfected with empty vector did not show binding of either anti-CD45 antibody. [Diagram 3] The results of an alanine scan against human CD45 for the four antibodies tested are shown. For each mutant clone, the average binding value determined by flow cytometry was plotted as a function of expression. Clones with CD45 Ala mutants identified as important are circled. Secondary clones, i.e. clones that did not meet the initially set threshold but whose reduced binding activity and proximity to critical residues suggested that the mutant residue may be part of the antibody epitope, are squared. [Figure 4] The locations of identified key positions on the 3D structure of human CD45 are shown diagrammatically. [Diagram 5] Binding of the tested antibodies to the mutants identified for Refmab#1, i.e. mutants for which Refmab#1 shows less than 20% binding compared to wild type CD45, is shown. [Figure 6] Binding of the tested antibodies to the mutants identified for Refmab#2, i.e. mutants for which Refmab#2 shows less than 20% binding compared to wild type CD45, is shown. [Figure 7] Binding of the tested antibodies to mutants identified for Refmab#4, i.e. mutants for which Refmab#4 shows less than 20% binding compared to wild type CD45, is shown. [Figure 8] Figure 1 shows the in silico mutagenesis of selected variants of the present disclosure. The bars show the predicted Provean score (y-axis) for each variant (x-axis), predicting whether the protein sequence mutation may affect protein function. The dashed horizontal line indicates a predefined threshold (-2.5). All variants are predicted to be neutral, except for E259G (-2.570), whose predicted score is just below the threshold. [Figure 9]FIG. 1 shows the editing efficiency of CD45 using various base editors and sgRNA concentrations. [Figure 10] Human T cells engineered using base editing to express CD45 E259G are shown. Cells were then incubated with increasing concentrations of antibody-drug-conjugate, resulting in selective depletion of unedited cells but persistence of edited cells as measured by flow cytometry (A). Results were confirmed by Sanger sequencing (B). [Figure 11] It shows that the mutation F331del results in a loss of binding of Refmab#1, whereas the cells are still reactive with Refmab#3. [Figure 12] It shows that cells expressing wild-type CD45 are killed by the Refmab#1-antibody drug conjugate. In contrast, CD45 knockout cells, as well as cells expressing the CD45 E259G mutant, are protected from killing by the Refmab#1-antibody drug conjugate. [Figure 13] Results are shown from an experiment in which wild type CD45 was knocked out and cells were transfected with mutant CD45 isoforms. The CD45RO (wild type) form of the protein bound to both antibodies, whereas loss of binding was observed with CD45 mutant Rekmab#1. All mutants retained binding to Rekmab#3, demonstrating that the proteins were expressed by electroporated cells. CD45 knockout is shown in the top left. Wild type CD45 is shown in the top center. Top right: Mut8 (22 aa deletion). Bottom (left to right): Mut9 (F331 deletion), Mut12 (F331S) and Mut 13 (F331I). [Figure 14] Figure 1 shows CD45 expression in gene-edited CD34+ cells. Dot plots show data obtained after staining of cells electroporated without gene editing reagent (EP-RNP), in the presence of RNP complex alone (EP+RNP) or with F331del HDR template (EP+RNP+HDRT). [Figure 15]Representative chromatograms of size-exclusion chromatography (Panel A) and SDS-PAGE (Panel B) of recombinant purified human CD45 D1-D2 wild-type protein are shown. [Figure 16] Shown is the phosphorylation of Lck at position Tyr505 detected using the AlphaLISA assay. 10.000-50.000 Jurkat wild type or Jurkat CD45 knockout cells were incubated for 20 min in plates coated with anti-CD3 antibody before cell lysis and detection of phosphorylated Lck. Activation of Jurkat cells with anti-CD3 antibody leads to CD45 activation which in turn dephosphorylates Lck. Jurkat CD45 knockout cells are unable to dephosphorylate Lck upon activation. The figure shows the acceptor signal (counts) and is representative of one biological experiment with two technical replicates. [Figure 17] Thermal unfolding curves of recombinant purified wild-type and mutant human CD45 D1-D2 proteins using DSF and Sypro Orange. Data are expressed as relative fluorescence units (RFU, top) and the first derivative of RFU with respect to temperature (d(RFU) / dT, bottom). [Figure 18] Conformational / thermal stability data of recombinant purified wild-type and mutant human CD45 D1-D2 proteins measured by DSF. A) Illustrates the onset temperature, the temperature at which the protein begins to unfold, for selected hCD45 D1-2 deglycosylated wild-type and mutants, and hCD45 D1-2 glycosylated wild-type. The dotted line indicates the onset temperature of hCD45 D1-2 deglycosylated wild-type. B) The melting temperatures of hCD45 D1-2 deglycosylated wild-type and mutants, and hCD45 D1-2 glycosylated wild-type. The dotted line indicates the melting temperature of hCD45 D1-2 deglycosylated wild-type. [Figure 19]Figure 2 shows the binding of Rekmab#1 and Rekmab#4 to DF-1 cells expressing wt or mutant CD45 as measured by flow cytometry. The data show that mutation of residue K352 results in loss of binding of Rekmab#1 and mutation of residue N257 results in loss of binding of Rekmab#4. [Figure 20] Binding of hCD45 D1-2 recombinant, purified and deglycosylated wild type and mutant CD45 D1-D2 proteins to RefmAbs is shown. A) % binding to Refmab#1 is shown. B) % binding to Refmab#2 is shown. C) % binding to RefmAb#4 is shown. % binding was calculated by dividing the nm shift of hCD45 D1-2 mutant by the nm shift of hCD45 D1-2 wt. Antibody binding to wt CD45 binding was set to 100%. For this calculation, the nm shift of 500 nM of analyte (hCD45 D1-2 mutant and wt) at the end of the association was used. Marked with an asterisk are calculations using a nm shift of 50 nM instead of 500 nM of analyte (hCD45 D1-2 mutant and wt). ND stands for not determined and NA stands for not analyzed. [Figure 21] Figure 2 shows the binding of Refmab#1 and Refmab#4 to DF-1 cells expressing wt or mutant CD45 as measured by flow cytometry. The data show that mutation of residues K352 and E353 results in loss of binding of Refmab#1. Mutation of residue N257 results in loss of binding of Refmab#4, and mutation of residue E259 results in a strong reduction in binding of Refmab#4. [Figure 22] Figure 2 shows the binding of Refmab#1 and Refmab#4 to DF-1 cells expressing wt or mutant CD45 as measured by flow cytometry. The data show that mutation of residues K352 and E353 results in loss of binding of Refmab#1. Mutation of residue N257 results in loss of binding of Refmab#4, and mutation of residue E259 results in a strong reduction in binding of Refmab#4. [Diagram 23]Figure 3 shows human CD34+ HSPCs engineered using base editing to express CD45 K352E. A) Base editing with ABE8e-NG and B) Base editing with ABE8e-SpRY and various gRNAs. Repositioning the ABE8e base editor to a more favorable editing window increased editing efficiency to >30% shielded HSPCs as measured by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Immunotherapy is a promising therapeutic approach for treating cancer, genetic and autoimmune diseases. Immuno-depleting agents, such as antibodies or engineered immune cells directed against tumor antigens, are administered to patients to target and kill tumor cells. However, because tumor surface proteins are also expressed on the surface of normal cells, including hematopoietic cells, this strategy can induce severe side effects in patients, for example by altering hematopoiesis. To restore hematopoiesis in patients, hematopoietic cells can be subsequently transplanted into the patient. However, binding of the depleting agent to diseased cells as well as to newly transplanted healthy cells may limit the maximum tolerated dose or restrict the use of healthy cells for treatment prior to transplantation. Alternatively, transplanted cells need to be resistant to the immuno-depleting agent so as not to be targeted and eliminated by the agent. Our approach is therefore to select cells that are resistant to the immuno-depleting agent used in immunotherapy while retaining their function to restore normal hematopoiesis in patients.

[0050] The inventors develop a method to identify functional allelic variants of gene sequences encoding surface protein regions involved in binding of a particular depleting agent. Such variants can be naturally occurring polymorphisms and / or designed and engineered variants. Different isoforms of surface proteins can be selected or generated. The first isoform of surface protein encoded by the polymorphic nucleic acid is not recognized by a particular depleting agent. This variant allele does not change or does not substantially change the function of the particular surface protein. Thus, the depleting agent can be used to specifically deplete cells expressing one isoform by specifically binding to one isoform and not or does not substantially bind to the other isoform. For example, if the depleting agent specifically binds to the second isoform but not the first isoform, the depleting agent specifically depletes cells expressing the second isoform. In another embodiment, the first isoform can be recognized by a second agent, and thus the second agent can be used to specifically deplete cells expressing the first isoform but not the second isoform. Cells expressing a first isoform of a surface protein encoded by at least one mutant allele are advantageously used in medical treatment in patients having cells expressing the second isoform, in particular to specifically deplete transplanted cells or patient cells by using a second or first agent, respectively.

[0051] In such an approach, it is not possible to predict which mutations of the surface antigen can be used. First, the mutation must be present in a surface-exposed stretch of the surface antigen that is accessible to the depleting agent. Second, the depleting agent must bind to this stretch on the exposed region of the surface antigen. Third, binding must be sufficiently affected that the depleting agent can distinguish the first isoform from the second. Residual binding to other isoforms should be minimal or, better, completely absent. Fourth, the mutation should not affect or only slightly affect the function of the surface antigen. The mutant isoform should perform its biological function, at least to an extent that is acceptable in a given therapeutic situation. Although certain tools exist for predicting three-dimensional protein structures, only experimental testing can prove the usefulness of any given mutation.

[0052] Depleting agent The present disclosure relates to an agent that comprises an antigen-binding region that specifically binds to one isoform of CD45 on cells and does not bind or binds substantially weaker to another isoform of CD45. Such an agent is referred to herein as a "depleting agent". Both isoforms of CD45 are functional, i.e., CD45 is functional with respect to at least one relevant property. Preferably, both isoforms of CD45 have the same function, i.e., are functionally indistinguishable.

[0053] However, the two isoforms of CD45 differ in terms of binding to depleting agents. Depleting agents specifically bind to only one of the isoforms of CD45. Therefore, the isoforms can be described as functionally identical (or functionally substantially identical) but immunologically distinguishable.

[0054] In certain embodiments, the first and second isoforms of CD45 have substantially the same biophysical properties. In certain embodiments, the first and second isoforms of CD45 have substantially the same stability. In certain embodiments, the first and second isoforms of CD45 have the same stability. In certain embodiments, the first and second isoforms of CD45 have substantially the same melting temperature. In certain embodiments, the first and second isoforms of CD45 have the same melting temperature. In certain embodiments, the first and second isoforms of CD45 have substantially the same aggregation tendency. In certain embodiments, the first and second isoforms of CD45 have the same aggregation tendency. In certain embodiments, the first and second isoforms of CD45 have substantially the same tendency to form dimers. In certain embodiments, the first and second isoforms of CD45 have the same tendency to form dimers.

[0055] The first and second isoforms of CD45 can be polymorphic alleles.Preferably, the first and second isoforms of CD45 are naturally occurring polymorphic alleles.Also preferably, the first and second isoforms of CD45 are single nucleotide polymorphism (SNP) alleles.

[0056] The first and second isoforms of CD45 can also be genetically engineered alleles.Preferably, the first and second isoforms of CD45 differ by 1, 2, 3, 4 or 5 amino acids.Most preferably, the first and second isoforms of CD45 differ by one amino acid.

[0057] A variety of methods can be used to determine the mutations to be introduced into CD45 to generate the second isoform. For example, mutations can be randomly inserted into the surface protein, followed by functional and immunological screening of the generated mutants. Alternatively, mutations can be rationally designed, for example, by analysis of the secondary or tertiary protein structure of CD45.

[0058] The depleting agents contain an antigen-binding region that specifically binds to one isoform of CD45 on a cell and does not bind, or binds substantially weaker to, another isoform. The depleting agents of the present disclosure can be divided into two main categories.

[0059] First, the depleting agent can be a polypeptide comprising an antigen-binding region. Said polypeptide can be composed of one or more polypeptide chains. Preferably, said polypeptide comprising an antigen-binding region is an antibody. Said polypeptide comprising an antigen-binding region can also be an antibody fragment, an antibody drug conjugate, or another variant of an antibody or scaffold. Exemplary antibody fragments and scaffolds include single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, igNAR, bis-scFv, camelid antibodies, ankyrin, centrin, domain antibodies, lipocalin, small modular immunopharmaceuticals, maxibodies, protein A and affilin.

[0060] The polypeptide comprising the antigen-binding region may be a bispecific, bispecific or multispecific antibody. Such molecules may also contain additional functional domains. For example, the polypeptide comprising the antigen-binding region may be a T cell engager, such as a BiTE. The polypeptide comprising the antigen-binding region may also be fused to the extracellular domain of a cytokine or chemokine, a toxin or a cell surface receptor.

[0061] Alternatively, the depleting agent can be a cell that comprises an antigen-binding region. For example, the depleting agent can be a chimeric antigen receptor (CAR). In a specific embodiment of the present disclosure, the cell that comprises an antigen-binding region is a CAR T cell, a CAR NK cell or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell that comprises an antigen-binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell that comprises an antigen-binding region is a primary T cell that comprises a CAR.

[0062] The depleting agent specifically binds to one isoform of CD45 but not to a second isoform, thus specifically depleting cells expressing one isoform.

[0063] In certain embodiments, the present disclosure relates to an agent comprising a first antigen-binding region that specifically binds to a second isoform of CD45 and does not bind to the first isoform. In other embodiments, the present disclosure also relates to an agent comprising a second antigen-binding region that specifically binds to a first isoform of CD45 and does not bind to the second isoform. In certain embodiments, the agent binds substantially weaker to the second isoform of CD45.

[0064] The first and second isoforms of CD45 may differ from each other by only one amino acid substitution. The difference of said one amino acid between the first and second isoforms may also be the result of the presence of a single nucleotide polymorphism, such as a naturally occurring single nucleotide polymorphism. The first and second isoforms of CD45 may also differ from each other by more than one amino acid, for example, two, three, or more than three amino acids. The first and second isoforms of CD45 may also differ from each other in that one of the isoforms has an insertion of one, two, three, or more than three amino acids compared to the other isoform. The first and second isoforms of CD45 may also differ from each other in that one of the isoforms has a deletion of one, two, three, or more than three amino acids compared to the other isoform. The two isoforms may also differ from each other by a combination of amino acid substitutions, insertions, and / or deletions. In a preferred embodiment, the depleting agent is an antibody or an antigen-binding fragment. When two isoforms of CD45 differ by more than one amino acid, the altered amino acids may be adjacent to each other, i.e., directly adjacent amino acids, or they may be separated.

[0065] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and antibody variants (including derivatives).

[0066] In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). Both the light chain variable region (VL) and the heavy chain variable region (VH) determine the binding recognition and specificity to the antigen. The light chain constant region domain (CL) and the heavy chain constant region domain (CH) confer important biological properties such as antibody chain association, secretion, transplacental transport, complement fixation, and binding to Fc receptors (FcR).

[0067] Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. The specificity of an antibody lies in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is composed of residues mainly from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or may affect the overall domain structure and thus the binding site. Complementarity determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, which are called L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence intervening between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0068] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO:. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, which corresponds to the truncation or insertion of structural components, whether in the framework or complementarity determining regions (CDRs) of the basic variable domain structure. The exact Kabat numbering of residues can be determined for a given antibody by alignment of the homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), residues 50 to 65 (H-CDR2) and residues 95 to 102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24 to 34 (L-CDR1), residues 50 to 56 (L-CDR2) and residues 89 to 97 (L-CDR3) according to the Kabat numbering system.

[0069] In certain embodiments, the antibody provided herein is any protein that comprises an antibody fragment, more specifically the antigen-binding domain of the antibody disclosed herein.The antigen-binding domain can also be incorporated into another protein scaffold.Antibody fragments and scaffolds include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, diabody, single domain antibody, maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR, IgNAR, bis-scFv, camelid antibody, ankyrin, centrin, domain antibody, lipocalin, small modular immunopharmaceutical, maxibody, protein A and affilin.

[0070] As used herein, "antigen-binding region" or "antigen-binding fragment of an antibody" refers to a portion of an antibody, possibly in its native form, that exhibits antigen-binding ability for a particular antigen, i.e., a molecule corresponding to a portion of the antibody's structure. Such a fragment exhibits the same or substantially the same antigen-binding specificity for said antigen, in particular compared to the antigen-binding specificity of the corresponding four-chain antibody. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. This antigen-binding region is sometimes referred to as the "functional fragment" of an antibody.

[0071] The agents of the present disclosure include antibodies and fragments thereof, but also include artificial proteins that mimic the antigen of an antibody and have the ability to bind to an antigen, also referred to herein as antigen-binding antibody mimics. Antigen-binding antibody mimics are organic compounds that specifically bind to an antigen but are not structurally related to antibodies. They are artificial peptides or small proteins, usually with a molar mass of about 3 kDa to 20 kDa.

[0072] The phrases "antigen-binding region that recognizes an antigen" and "antigen-binding region that has specificity for an antigen" are used interchangeably herein with the term "antigen-binding region that specifically binds to an antigen." As used herein, the term "specificity" refers to the ability of an agent that includes an antigen-binding region, such as an antibody, to detectably bind to an epitope presented in the antigen.

[0073] "Specific binding" or "specifically binding" refers to a -8 M(KD) or stronger. Preferably, the binding affinity is 10 -8 M(KD) to 10 -12 M(KD), optional 10 -8 M(KD) to 10 -10 M(KD), especially at least 10 -8 If M(KD), the binding is considered to be specific. Affinity can be determined by various methods well known to those skilled in the art. These methods include, but are not limited to, surface plasmon resonance (SPR), biolayer interferometry (BLI), microscale thermophoresis (MST) and Scatchard plot. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reaction of said binding domain with a target protein or antigen to the reaction of said binding domain with a protein or antigen other than the target protein.

[0074] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody or its antigen-binding region binds. Epitopes of protein antigens can be divided into two categories: conformational epitopes and linear epitopes. Conformational epitopes correspond to discontinuous portions of the amino acid sequence of an antigen. Linear epitopes correspond to a continuous sequence of amino acids from an antigen.

[0075] In another embodiment, bispecific or multispecific molecules, such as bispecific or multispecific antibodies, are further disclosed herein. For example, an antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds at least two different binding sites or target molecules. An antibody can actually be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds more than two different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the terms "bispecific molecule," "bispecific antibody," "bispecific molecule," "bispecific antibody," "multispecific molecule," and "multispecific antibody" used herein. To create a bispecific molecule, an antibody of the present disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, disulfide bond, non-covalent bond or other method) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, cytokine, chemokine, toxin, or receptor extracellular domain, so that a bispecific molecule is generated. Certain bispecific and multispecific molecules contemplated by the present disclosure are T cell engagers, such as bispecific T cell engagers, e.g., BiTEs.

[0076] As used herein, an agent that does not bind or binds substantially weaker to a particular isoform of CD45 includes an agent that cannot bind to cells expressing said particular isoform. For experimental testing, said agent may be labeled with a fluorescent marker or detected with a secondary antibody against said agent, and the percentage of cells presenting said fluorescent marker or said secondary antibody is determined by FACS analysis. Typically, testing is performed on a cell line expressing a recombinant target protein, i.e., CD45. The target protein may be expressed in its entirety. Alternatively, a truncated form may be used, which must at least include the extracellular domain or a region of the extracellular domain that contains the respective antibody epitope. To monitor the expression of mutant isoforms, cells may be stained simultaneously with two agents, one that binds to the epitope where the mutant was introduced and the second that binds to a different epitope than that bound by the first agent. The second epitope remains unchanged, and therefore this staining serves as a control for expression. As a non-binding control, cells that do not express the protein of interest are used. As a maximal binding control, cells that do not normally express the protein of interest are transfected with the wild-type isoform. Different cell lines have different expression levels, but expression is controlled through endogenous control elements such as promoters. Such cell lines can also be used to study the mode of action of depleting agents, effective shielding against different modes of action, to test cytotoxicity and shielding / resistance from cytotoxicity, or to test the function of engineered receptors. Western blot, ELISA or FACS can be used to analyze phosphorylation of signaling molecules. Analysis of gene expression changes can help to analyze gene expression compared to normal function. Cells can also be used to demonstrate the feasibility of editing specific mutants via different approaches, e.g. homology-directed repair (HDR), base editing or prime editing.

[0077] Binding of the agent may result in depletion of cells expressing the first isoform of CD45. Various mechanisms may result in cell depletion. Antibody-dependent cellular cytotoxicity (ADCC) results from binding of the agent to a target protein and activation of NK cells via the Fc portion to the agent bound by the FcR expressed by the NK cells. The Fc portion of an immunoglobulin refers to the C-terminal region of the heavy chain of the immunoglobulin. The Fc portion may be wild-type or engineered. Enhanced and engineered Fc portion mutations are known in the art. In certain therapeutic situations, it is desirable to reduce or abolish the normal binding of a wild-type Fc region of an antibody, such as a wild-type IgG Fc region, to one or more or all of the Fc receptors and / or binding to a complement component, such as C1q, in order to reduce or abolish the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or abolish the binding of an Fc region of an antibody to one or more or all of the Fcy receptors, such as FcyRI, FcyRIla, FcyRIIb, FcyRIIIa. Effector functions may include, but are not limited to, one or more of the following: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), secretion of cytokines, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-bound antibodies, maturation of dendritic cells, or T-cell priming. Binding of the agent may also result in blocking of binding of natural receptor ligands, thereby resulting in cell death and apoptosis without cell-mediated depletion.

[0078] The reduced or abolished binding of the Fc region to the Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more particularly a human IgG1 Fc region, to produce a mutated or engineered Fc region of said wild-type Fc region, such as a mutant human IgG1 Fc region. Substitutions that result in reduced binding may be useful. In order to reduce or abolish the binding properties of the Fc region to the Fc receptor, non-conservative amino acid substitutions, i.e., replacing an amino acid with another amino acid having different structural and / or chemical properties and / or charge, are preferred.

[0079] In certain embodiments of the present disclosure, the Fc region of the antibody is of IgG1 isotype carrying LALA or PG-LALA mutations, i.e. the constant region carries L234A, L235A and P329G mutations or PA-LALA mutations, i.e. the constant region carries L234A, L235A and P329A mutations, or AEASS, i.e. the constant region carries L234A, L235A and P329A mutations, or L234A, L235E, G237A, A330S and P331S mutations. The skilled artisan will recognize the possibility of engineering the Fc region to obtain a desired effect.

[0080] The surrogate ADCC assay, as described in the experimental part, constitutes the industry standard for quantifying the potency of drugs mediating ADCC. Engineered Jurkat reporter cells have an NFAT-responsive luciferase gene and an Fc receptor, e.g., human FcgRIIIa. Binding of the Fc receptor with the binding antibody results in NFAT induction by receptor clustering and thus luciferase signal. Lack of binding, and thus clustering, does not result in luciferase signal. Cells that do not express the target protein (e.g., HEK or DF-1 cells, or human hematopoietic cancer cells such as TF-1, KG-1, KASUMI-1, K562 or Jurkat engineered to CD45 deficiency (e.g., human T-cell cancer cells such as Jurkat cells with CD45 knockout or CD45 knockout), wild-type protein (e.g., HEK-CD45 or DF-1-CD45, or TF-1, KG-1, KASUMI-1 or Jurkat cell lines) or individual mutants. (e.g., CD45 variants) were incubated with a test agent (e.g., antibody Refmab#1) and mixed with ADCC reporter cells. Luciferase was then measured to quantify the ADCC signal. Luciferase luminescence signals were normalized to the maximum signal observed in HEK-CD45, DF-1-CD45 or corresponding myeloid or T-cell cancer cell lines. ADCC was measured using the ADCC reporter assay (Promega, Cat. No. G7015).

[0081] Other potential modes of action are possible in line with the present disclosure. These include antibody-mediated displacement of the ligand of CD45, or antibody internalization combined with the use of antibody-drug conjugates. Another method of depleting target cells is through the use of T cell engager molecules. For example, a bispecific T cell engager using CD45 binding sites and CD3 (OKT3) binding sites from antibody Refmab#1 may be used. The same target cells were used as those used for the ADCC assay. Primary human T cells and bispecific T cell engagers are added. Activation of human T cells was quantified by FACS by determining the frequency of CD69 upregulation and / or cytokine release.

[0082] The depleting agent according to the present disclosure specifically binds to one isoform of CD45 and allows the depletion of cells expressing said isoform.

[0083] More preferably, in certain embodiments, the depleting agent according to the present disclosure does not bind or binds substantially weaker to the first isoform of CD45, but specifically binds to the second isoform of CD45, allowing depletion of the cells expressing the second isoform of CD45, particularly in the method of use as disclosed herein. In particular, the depleting agent that does not bind or binds substantially weaker to the first isoform of cellular CD45, but specifically binds to the second isoform of CD45 expressed in the patient's cells, is used to deplete the patient's cells, but does not deplete hematopoietic stem cells or their passages expressing the first isoform of CD45 that are transplanted to restore hematopoiesis in the patient.

[0084] In another specific embodiment, the depleting agent according to the present disclosure does not bind or binds substantially weaker to the second isoform of CD45, but specifically binds to the first isoform of CD45, allowing depletion of the cells expressing the first isoform of CD45, particularly in the method of use as disclosed herein. In particular, the depleting agent that does not bind or binds substantially weaker to the second isoform of CD45, but specifically binds to the first isoform of CD45 expressed in transplanted cells, is used to specifically deplete transplanted cells to avoid eventual severe side effects such as graft-versus-host disease due to transplantation.

[0085] Selective depletion of cells expressing specific isoforms of CD45 can be achieved without limitation by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).

[0086] In certain embodiments, the antigen-binding region is coupled to an effector compound, such as a drug or toxin. Such conjugates are referred to herein as "immunoconjugates," "antibody-drug conjugates," or "ADCs." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, maytansinoids, calicheamicin, indolinobenzodiazepines, pyrrolobenzodiazepines, pyridinobenzodiazepines, camptothecin, topotecan, irinotecan, belotecan, deltecan, alpha-amanitin, microcystin, auristatin, and puromycin and analogs or homologs thereof.

[0087] In another particular embodiment, the depleting agent is an immune cell having an antigen receptor, such as a chimeric antigen receptor (CAR). See, for example, Myburgh et al. Leukemia (2020) 34:2688-703. The immune cell may express a recombinant antigen-binding region, also called an antigen receptor, on its cell surface. By "recombinant" is meant an antigen-binding region that is not encoded by the cell in its native state, i.e., heterologous, non-endogenous. Thus, it can be seen that expression of a recombinant antigen-binding region introduces new antigen specificity to the immune cell, allowing the cell to recognize and bind a previously unrecognized antigen. The antigen receptor may be isolated from any useful source. In a particular embodiment of the present disclosure, the cell comprising the antigen-binding region is a CAR T cell, a CAR NK cell, a CAR Treg, or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell comprising the antigen-binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell comprising the antigen-binding region is a primary T cell comprising a CAR.

[0088] In certain embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR).CAR is a fusion protein that comprises an antigen-binding region, typically derived from an antibody, linked to the signaling domain of a TCR complex.CAR can be used to direct immune cells, such as T cells or NK cells, to target antigens when appropriate antigen-binding regions are selected.

[0089] The antigen-binding region of a CAR is typically based on an scFv (single-chain variable fragment) derived from an antibody. In addition to the N-terminal extracellular antibody-binding region, a CAR typically includes a hinge domain that serves as a spacer to extend the antigen-binding region away from the plasma membrane of the immune effector cell in which it is expressed, a transmembrane (TM) domain, an intracellular signaling domain (e.g., a signaling domain from the zeta chain (CD3ζ) of the CD3 molecule of the TCR complex, or equivalent), and optionally one or more costimulatory domains that may assist in signaling or functionality of the cell expressing the CAR. Signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), CD27, ICOS, and 4-1BB (CD137), can be added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR-modified immune cells.

[0090] Those skilled in the art can select the appropriate antigen-binding region for redirecting immune cells to be used according to the present disclosure. In certain embodiments, the immune cells for use in the methods of the present disclosure are redirected T cells, such as redirected CD8+ T cells or redirected CD4+ T cells, or redirected NK cells.

[0091] The method of genetically modifying immune cells to express recombinant antigen-binding regions is well known in the art.The nucleic acid molecule encoding an antigen receptor can be introduced into cells, for example, in the form of a vector or any other suitable nucleic acid construct, or by inserting the nucleic acid molecule into the genome using genome editing technology.Vectors and their necessary components are well known in the art.The nucleic acid molecule encoding an antigen-binding region can be generated using any method known in the art, for example, molecular cloning using PCR.The antigen-binding region sequence can be modified using commonly used methods, such as site-directed mutagenesis.

[0092] CD45 CD45 (UniProt: P08575; also known as protein tyrosine phosphatase receptor type C, PTPRC, LCA, B220 or LY5) is a member of the protein tyrosine phosphatase (PTP) family, which comprises signaling molecules that regulate a variety of cellular processes, including cell growth, differentiation, the mitotic cycle, and oncogenic transformation.

[0093] Human CD45 has the following amino acid sequence (SEQ ID NO:1): MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSSDPLPTHTTAFSPA STFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSVQTPHLPTHADSQTPSA GTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTASTFPTDPVSPLTTTLSLAHHSSAAL PARTSNTTITANTSDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYL YNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVP PGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLE PEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFH NFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTT KSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFR VKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYK IYDLHKKRSCNLDEQQELVERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSI PRVFSKFPIKEARKPFNQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPR KYIAAQGPRDETVDDFWRMIWEQKATVIVMVTRCEEGNRNKCAEYWPSMEEGTRAFGDVV VKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLKLRRRVNA FSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEA QYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYRSWRTQ HIGNQEENKSKNRNSNVIPYDYNRVPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINA SFIMSYWKPEVMIAAQGPLKETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGK QTYGDIEVDLKDTDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISM IQVVKQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVDIFQV VKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEFDNEVDKVKQDAN CVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASPALNQGS

[0094] In certain embodiments, the surface protein is CD45. In other embodiments, the surface protein is CD45 comprising the amino acid sequence of SEQ ID NO: 1. In other embodiments, the surface protein is CD45 consisting of the amino acid sequence of SEQ ID NO: 1.

[0095] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD45, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and preferably said first and second isoforms are functional. Preferably, said mammalian cells are human cells.

[0096] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD45, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and said first and second isoforms are functional. Preferably, said mammalian cells are human cells.

[0097] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and said first and second isoforms being substantially functionally identical. Preferably, said mammalian cells are human cells.

[0098] Several functions have been reported for CD45. In certain embodiments, the present disclosure relates to the first and second isoforms of CD45, both of which are functional. In certain embodiments, the present disclosure relates to the first and second isoforms of CD45, both of which are functionally indistinguishable. In the present invention, "functionally indistinguishable" refers to the first and second isoforms of CD45 that can equally perform the same function in cells without significant impairment. In other words, the first and second isoforms are functionally almost indistinguishable. Slight functional impairment can be tolerated. In a preferred embodiment, said first isoform of CD45 remains functional and retains the ability to perform the same function in cells as the corresponding wild-type isoform without significant impairment.

[0099] One function of CD45 is inclusion in immune synapse, thereby preventing T cell receptor engagement and T cell activation. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of surface protein, CD45, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprise genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, said first and second isoforms prevent T cell receptor engagement upon inclusion in immune synapse. Preferably, said mammalian cell is a human cell.

[0100] One function of CD45 is dephosphorylation of target protein.Therefore, in certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of surface protein, CD45, for use in medical treatment in a patient in need thereof, said patient having a cell expressing a second isoform of said surface protein, said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele is not present in the genome of the patient having a cell expressing said second isoform of said surface protein, and said first and said second isoforms dephosphorylate target protein of CD45.Preferably, said mammalian cell is a human cell.

[0101] One function of CD45 is the dephosphorylation of the tyrosine kinase Lck. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD45, for use in medical treatment in a patient in need thereof, said patient having a cell expressing a second isoform of said surface protein, said cell expressing said first isoform comprises genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele is absent in the genome of the patient having a cell expressing said second isoform of said surface protein, said first and said second isoforms dephosphorylate the tyrosine kinase Lck. Lck (UniProt: P06239) is a member of the Src family of protein tyrosine kinases (PTKs). Lck is a key signaling molecule in the selection and maturation of developing T cells. Preferably, said mammalian cell is a human cell.

[0102] One function of CD45 is the dephosphorylation of the tyrosine kinase Lck at position Y505. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD45, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, said first and said second isoforms dephosphorylate the tyrosine kinase Lck at position Y505. Preferably, said mammalian cell is a human cell.

[0103] One function of CD45 is its exclusion from the immunological synapse, thereby allowing the activation of the TCR signaling cascade. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of surface protein CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, and said first and said second isoforms activate the TCR signaling cascade. Preferably, said mammalian cell is a human cell.

[0104] One function of CD45 is to increase cytokine production.Thus, in certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of surface protein CD45 for use in medical treatment in a patient in need thereof, said patient having a cell expressing a second isoform of said surface protein, said cell expressing said first isoform comprises genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele is absent in the genome of the patient having a cell expressing said second isoform of said surface protein, and said first and said second isoforms increase cytokine production.Preferably, said mammalian cell is a human cell.

[0105] One function of CD45 is to increase T cell proliferation. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of surface protein CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprise genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele is absent in the genome of the patient having cells expressing said second isoform of said surface protein, and said first and said second isoforms increase T cell proliferation. Preferably, said mammalian cell is a human cell.

[0106] One function of CD45 is the normal differentiation of hematopoietic cells. This can be tested, for example, in humanized mice, for example, by transplanting engineered HSCs into humanized mice. Thus, in a particular embodiment, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of surface protein CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, and said first and said second isoforms result in normal differentiation of hematopoietic cells. Preferably, said mammalian cell is a human cell.

[0107] In line with the present disclosure, it is also possible to combine additional variants or isoforms of CD45 in the methods and compositions of the present disclosure. Such isoforms can include, for example, double mutants. Such isoforms can also include, for example, single mutants and double mutants. The methods and compositions of the present disclosure can also be combined with cells that have CD45 knockout, for example, permanent knockout or temporary knockout (e.g., via CRISPRoff). The methods and compositions of the present disclosure can also be used in the depletion of bone marrow cells in solid tumors to enhance tumor response.

[0108] The methods and compositions of the present disclosure can also be combined with combinations of cells, particularly where the surface protein is CD45 with knockout of other targets such as CD117, CD123, DLL-1, CD33, CD7, CLEC12A, CD44, Flt, CD300F, EVI2B, TPO and combinations thereof.

[0109] The disclosed methods and compositions may also include cells expressing the first isoform of CD45 and other surface protein variants, such as CD117 variants, CD123 variants, DLL-1 variants, CD33 variants, CD7 variants, CLEC12A (CD371) variants, CD44 variants Flt (CD135) variants, CD300F variants, EVI2B variants, TPO variants, and any combination thereof.

[0110] CD45 polymorphisms A cell expressing a first isoform of CD45 according to the present disclosure comprises genomic DNA having at least one polymorphic allele in a nucleic acid encoding said CD45, in particular said polymorphism induces at least one mutation involved in binding of a particular drug compared to said second isoform.

[0111] Said polymorphism is preferably located within the nucleic acid sequence encoding the surface protein region of CD45 involved in the binding of the first agent, preferably located in the extracellular portion of CD45, particularly in the solvent-exposed secondary structure element. More specifically, said polymorphism is located within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first agent. Said polymorphism may be a mutation, such as deletion, substitution, insertion or combination thereof, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides. In a particular embodiment, said polymorphism is a single nucleotide polymorphism.

[0112] The term "isoform" refers to a variant of a protein that differs from another variant of the same protein by at least one amino acid difference. In the context of this disclosure, such a difference may be a single amino acid substitution, but such a difference may also be a double, triple or multiple amino acid substitution, or an insertion or deletion. Naturally occurring SNPs are also isoforms.

[0113] The sequence difference between the two isoforms can also be genetically introduced. Also, the sequence difference is preferably within the nucleic acid sequence encoding the CD45 region involved in the binding of the first agent, and is preferably located in the extracellular part of the surface protein, particularly in the solvent-exposed secondary structure element. More specifically, the sequence difference is within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first agent. The sequence difference can be a mutation, such as a deletion, substitution, and / or insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides. In a particular embodiment, the sequence difference is a single point mutation.

[0114] The present disclosure provides polymorphisms of CD45, particularly including polymorphisms comprising substitutions of residues E230, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, E329, T330, F331, D334, Y340, K352, E353, E360, E364, Y372, or Y373. In certain embodiments, the present disclosure provides polymorphisms of CD45, particularly including polymorphisms comprising substitutions of residues E230, N257, E259, F331, K352, or E353. Particularly preferred polymorphisms include substitutions of residue E230, where E230 is replaced with K. Other preferred polymorphisms include substitutions of residue N257, where N257 is substituted with D, E, H, K, R, S, T, or V. Other preferred polymorphisms include substitutions of residue F331, where F331 is substituted with G. Other preferred polymorphisms include substitutions of residue K352, where K352 is substituted with H, I, L, M, N, Q, S, or T. Yet other preferred polymorphisms include substitutions of residue E353, where E353 is substituted with K or R.

[0115] In certain embodiments, the present disclosure relates to mutant CD45 polypeptides, said mutant CD45 polypeptides comprising at least one mutation at an amino acid selected from E230, N257, E259, T264, N267, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, Y372 and Y373 of wild-type human CD45. In preferred embodiments, said mutation is selected from E230, N257, E259, T264, N267, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, Y372 and Y373.

[0116] It will be understood that amino acids may be designated by either the three letter code or the one letter code, all of which are familiar to those of skill in the art.

[0117] Table 1 shows the 20 naturally occurring amino acids: [Table 1]

[0118] In the experiments of the present disclosure, specific mutants of specific residues were identified. For practical reasons, it is not possible to test every possible variation. However, it will be understood that the identified mutants can be replaced with similar amino acid residues. For example, acidic amino acids can be replaced with other acidic amino acids, since they are expected to have the same effect. Similarly, charged amino acids can be replaced with other charged amino acids. As an example, T264E is expected to be equivalent to T264D, since both D and E are acidic amino acids.

[0119] Natural polymorphism In certain embodiments, the cells according to the present disclosure are selected from a subject that contains naturally occurring genomic DNA having at least one naturally occurring polymorphic allele, preferably a single nucleotide polymorphism (SNP), in a nucleic acid encoding the isoform.

[0120] In certain embodiments, the cells are selected from a subject comprising naturally occurring genomic DNA having at least one naturally occurring polymorphic allele, in particular a SNP, in the nucleic acid sequence encoding a CD45 region involved in anti-CD45 agent binding, preferably located in the extracellular portion of said surface protein, more preferably in a solvent-exposed secondary structure element.

[0121] Certain naturally occurring SNPs have been described in the literature and may be used within the spirit of the present disclosure, along with respective binding agents capable of distinguishing such SNPs from other isoforms of CD45.

[0122] Some naturally occurring SNPs in human CD45 are shown in Table 2. The list of naturally occurring SNPs can also be found here: https: / / gnomad.broadinstitute.org / gene / ENSG 00000081237?dataset=gnomad_r2_1 [Table 2]

[0123] Gene editing In another particular embodiment, said cells expressing the first isoform of CD45 according to the present disclosure are obtained by gene editing, preferably by modifying the sequence encoding said surface protein in the patient's native genomic DNA.

[0124] The cell can be genetically engineered by introducing a gene editing system into the cell, which induces the polymorphism resulting in the insertion, deletion and / or substitution of amino acids in the surface protein.The gene editing modality targets the nucleic acid sequence, referred to herein as the target sequence, that codes for the surface protein region involved in the first drug binding.In particular, when the surface protein is CD45, the gene editing modality targets the nucleic acid that codes for at least one amino acid residue at positions E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO:1. Preferably, amino acid residue E230 is substituted by K, and / or residue Y232 is substituted by C, and / or residue N257 is substituted by A, D, E, H, K, R, S, T or V, and / or residue E259 is substituted by H, K, N, V, G, R, T or Q, and / or residue T264 is substituted by D or E, and / or residue N267 is substituted by A, H, L, S or V, and / or Residue N286 is substituted with D, G, L or R, and / or residue E329 is substituted with A, and / or residue F331 is substituted with A or G, and / or residue Y340 is substituted with A, G, N, Q or S, and / or residue K352 is substituted with A, D, E, G, H, I, L, M, N, Q, S, T or Y, and / or residue E353 is substituted with A, H, I, K, L, S, T or R. The gene editing enzyme can be a sequence specific nuclease, a base editor, a prime editor or a CRISPR transposon based system.

[0125] The term "nuclease" refers to a wild-type or mutant enzyme capable of catalyzing the hydrolysis (cleavage) of phosphodiester bonds between nucleotides of nucleic acid (DNA or RNA) molecules, preferably DNA molecules. "Cleavage" refers to a double-strand or single-strand break event.

[0126] The term "sequence-specific nuclease" refers to a nuclease that cleaves nucleic acid in a sequence-specific manner. Different types of site-specific nucleases can be used, such as meganucleases, TAL nucleases (TALENs), zinc finger nucleases (ZFNs), or RNA / DNA-guided endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems and Argonaute (Review in Li et al., Nature Signal transduction and targeted Therapy, 5, 2020; Guha et al., Computational and Structural Biotechnology Journal, 2017, 15, 146-160).

[0127] According to the present disclosure, the nuclease generates a DNA break in the target sequence, which encodes the surface protein region involved in the first agent binding as described above. In a particular embodiment, the inventors use the CRISPR system to induce a break in the target sequence that encodes the surface protein region recognized by the first agent as described above.

[0128] By "target sequence" it is intended to target a portion of the sequence encoding the region of CD45 involved in first drug binding as described above and / or at least one (one or two) sequences adjacent to said region of CD45 involved in first drug binding, in particular up to 50 nucleotides adjacent to said region of CD45 involved in first drug binding, preferably 20, 15, 10, 9, 8, 7, 6 or 5 nucleotides adjacent to said drug binding site.

[0129] CRISPR system includes two or more components, Cas protein (CRISPR-associated protein) and guide RNA. Guide RNA can be single guide RNA or dual guide RNA. Cas protein is a DNA endonuclease that recognizes and generates double-stranded breaks in DNA complementary to the target sequence using guide RNA sequence as a guide. Cas system that generates single-stranded breaks requires only one nuclease domain. Cas system that generates double-stranded breaks requires two nuclease domains. Cas protein can include two active cleavage sites, for example, HNH nuclease domain and RuvC-like nuclease domain.

[0130] Cas protein also refers to engineered endonucleases, homologs or orthologs of Cas 9 that can cleave a target nucleic acid sequence. In certain embodiments, the Cas protein can induce cleavage of a nucleic acid target sequence, which can correspond to either a double-stranded or single-stranded break. The Cas protein mutant can be a Cas endonuclease that does not occur in nature and is obtained by protein engineering or random mutagenesis. The Cas protein can be one type of Cas protein known in the art. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), SaCas9, Cas12, Cas12a (Cpf1), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csxl ... The Cas protein may be sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr3, Cmr4, Cmr5, Cnrr6, Csbl, Csb2, Csb3, Csxl7, CsxM, CsxlO, Csl6, CsaX, Csx3, Csl, Csxl5, Csfl, Csf2, CsO, Csf4, or a homolog, ortholog, or modified form thereof.Preferably, the Cas protein is the Streptococcus pyogenes Cas9 protein.

[0131] The Cas is contacted with a guide RNA (gRNA) designed to contain a sequence complementary to a target sequence to specifically induce DNA cleavage within said target sequence, particularly the complementary sequence of a portion of the target sequence that encodes a surface protein region recognized by the above-mentioned drug, according to the present disclosure.

[0132] As used herein, "guide RNA," "gRNA," "sgRNA," or "single guide RNA" refers to a nucleic acid that facilitates specific targeting or homing of a gRNA / Cas complex to a target nucleic acid.

[0133] In particular, gRNA refers to RNA comprising transactivating crRNA (tracrRNA) and crRNA. Preferably, said guide RNA corresponds to crRNA and tracrRNA, which can be used separately or fused together to generate a single guide RNA. Complementary sequence pairing with the target sequence recruits Cas to bind and cleave DNA at the target sequence.

[0134] According to the present disclosure, the crRNA is engineered to contain a sequence complementary to a portion of the above target sequence that encodes the surface protein region recognized by the drug, so that the crRNA can target the surface protein region recognized by the drug. In a preferred embodiment, the sgRNA is used to target the binding site of the binding agent. In another preferred embodiment, the guide RNA contains a chemical modification known to those skilled in the art.

[0135] In certain embodiments, the crRNA comprises a sequence of 5-50 nucleotides, preferably 15-30 nucleotides, more preferably 20 nucleotides, that is complementary to the target sequence. As used herein, the term "complementary sequence" refers to a sequence portion of a polynucleotide (e.g., a portion of a crRNA or a tracRNA) that can hybridize to another portion of the polynucleotide under standard low stringency conditions. Preferably, the sequences are complementary to each other according to interstrand Watson-Crick base pairing, i.e., complementarity between two nucleic acid strands that relies on inherent base pairing between adenine-thymine (AT) and guanine-cytosine (GC) nucleotides. The gRNA can be designed by any method known to the skilled artisan in view of the present disclosure.

[0136] According to the present disclosure, the target sequence encodes a surface protein region of CD45 involved in first drug binding, preferably located in the extracellular portion of CD45, more preferably located in an extracellular loop compared to the second isoform, and again more preferably includes amino acid residues involved in drug binding.

[0137] In a preferred embodiment, where the surface protein is CD45, the target sequence encodes a region of CD45 involved in binding of a first agent, such as anti-CD45 agent binding as disclosed above. Preferably, the target sequence encodes at least one residue at positions E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO:1.

[0138] DNA strand breaks introduced by nucleases according to the present disclosure can result in mutations of the DNA at the break site via non-homologous end joining (NHEJ), which frequently results in small insertions and / or deletions or replacements of the DNA surrounding the break site via homology-directed repair (HDR).

[0139] In a preferred embodiment, said polymorphism within a nucleic acid encoding an isoform of CD45 is induced via HDR repair following DNA breakage and introduction of an exogenous nucleotide sequence, referred to herein as an HDR template.

[0140] The HDR template comprises a first and a second portion of the sequence that are homologous to the 5' and 3' regions of the target sequence, respectively, and a middle sequence portion that contains a polymorphism. Following cleavage of the target sequence, a homologous recombination event is achieved between the genome containing the target sequence and the HDR template, and the genomic sequence containing the target sequence is replaced by the exogenous sequence.

[0141] Preferably, a homologous sequence of at least 20bp, preferably more than 30bp, preferably more than 50bp, more preferably less than 200bp is used. The homologous sequence can be dsDNA or ssDNA. Preferably, the homologous sequence is ds DNA. In fact, the shared DNA homology is located in the adjacent regions upstream and downstream of the cleavage site, and the exogenous sequence to be introduced should be located between the two arms. The adjacent sequences can be symmetric or asymmetric. Both strands of the target nucleic acid, i.e., the positive strand or the negative strand, can be targeted. Optionally, a PAM sequence can be used that can be silenced to improve HDR.

[0142] In a preferred embodiment, a cell according to the present disclosure is genetically engineered by introducing into said cell a sequence encoding a region of CD45 recognized by said first agent as described above and said site-specific nuclease targeted to the HDR template.

[0143] In another particular embodiment, the gene editing enzyme is a DNA base editor as described in Komor et al., Nature 533, 420-424, and in Rees HA, Liu DR. Nat Rev Genet. 2018; 19: 770-788, or a prime editor as described in Anzalone et al. Nature, 2019, 576: 149-157, Matsoukas et al., Front Genet. (2020) 11: 528, Chen et al. Cell (2021) 184: 5635-52, Koblan et al, Nat Biotechnol (2021) 39: 1414-25 and Kantor A. et al. Int. J. Mol. Sci. 2020, 21 (6240). The base editor or prime editor can be used to introduce mutations at specific sites in the target sequence.

[0144] According to the present disclosure, base editors or prime editors generate mutations within a target sequence by sequence-specific targeting of the sequence encoding the region of CD45 involved in first drug binding.

[0145] In particular, said base editor or prime editor is a CRISPR-based or prime editor. Said CRISPR base or prime editor may comprise a dead Cas protein (dCas) as a catalytically inactive sequence-specific nuclease. It may also comprise Cas9 with a mutated nuclease domain. dCas refers to a modified Cas nuclease that lacks endonuclease activity. Nuclease activity may be inhibited or prevented in dCas protein by one or more mutations and / or one or more deletions in the HNH and / or RuvC-like catalytic domains of Cas protein. The resulting dCas protein lacks nuclease activity but binds to guide RNA (gRNA)-DNA complexes with high specificity and efficiency to specific target sequences. In certain embodiments, said dCas may be a Cas nickase in which one catalytic domain of Cas is inhibited or prevented.

[0146] The base editor is complexed with a guide RNA (gRNA) designed to include a complementary sequence of a target nucleic acid sequence and specifically binds to the target sequence as described above.

[0147] The gRNA can be designed by any method known to those skilled in the art in view of the present disclosure. In certain embodiments, the gRNA can target a sequence that encodes a region on CD45 that is recognized by the first agent as described above.

[0148] As a non-limiting example, the base editor is a nucleotide deaminase domain fused to a dead Cas protein, in particular a Cas nickase. The nucleotide deaminase can be an adenosine deaminase or a cytidine deaminase. The nucleotide deaminase can be a natural or engineered deaminase.

[0149] In certain embodiments, the base editor may be a non-limiting example selected from the group consisting of BE1, BE2, BE3, BE4, HF-BE3, Sa-BE3, Sa-BE4, BE4-Gam, saBE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, SaKKH-BE3, cas12a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRIPS-X, ABE7.9, ABE7.10, ABE7.10*xABE, ABESa, ABEmax, ABE8e, VQR-ABE, VRER-ABE, and SaKKH-ABE.

[0150] The prime editor consists of a fusion of a catalytically inactive sequence-specific nuclease, particularly a Cas nickase, as described above, with a catalytically active engineered reverse transcriptase (RT) enzyme. The fusion protein, particularly when the surface protein is CD45, is used in conjunction with a prime editing guide RNA (pegRNA) that comprises a sequence complementary to the target sequence described above, and comprises one of the sequences listed in Table 13, and an additional sequence that also comprises a sequence that binds to the primer binding site region of DNA. In a particular embodiment, the reverse transcriptase is the Maloney Murine Leukemia Virus RT enzyme and mutants thereof. The prime editor may be a non-limiting example selected from the group consisting of PE1, PE2, PE3 and PE3b, or any of the prime editors described in Chen et al. Cell (2021) 184:5635-52 or Koblan et al, Nat Biotechnol (2021) 39:1414-25.

[0151] Anti-CD45 agents Several anti-CD45 moieties are known in the art, some of which are currently under development. QA17A19 (Biolegend, #393411) and HI30 (Biolegend, #304001) are commercially available mouse anti-human CD45 antibodies. Various anti-CD45s from Magenta are mostly under development as antibody drug conjugates (e.g., WO2017219025, WO2020092654). BC8 is a mouse hybridoma antibody commercially available from IchorBio (#ICH1155). The BC8 antibody is the basis of the anti-CD45 antibody-radioactive conjugates developed by Actinium (WO2017155937, WO2019084258, WO2020159656). Other anti-CD45 antibodies are disclosed in WO2016016442, WO2019115791 and WO2020058495 (INSERM), WO2017009473 (UCB), WO2019129178 (Shanghai Baize Medical Laboratory), WO2020018580 (Fred Hutchinson) and WO2020170254 (Ramot At Tel Aviv University). These and other anti-CD45 moieties can be used in the context of this disclosure. Some anti-CD45 antibodies have also been made in this disclosure in full-length antibody format, as well as in Fab format. Details are provided in Example 1.

[0152] In certain embodiments, said depleting agent that binds to said second isoform of CD45 and does not bind or binds substantially weakly to said first isoform of CD45 as described above specifically binds to an epitope comprising amino acids E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 and / or Y373 of SEQ ID NO: 1. More preferably, said depleting agent specifically binds to an epitope comprising amino acids N286, F331, K352 and / or E353 of SEQ ID NO: 1. In another preferred embodiment, the depleting agent specifically binds to an epitope comprising amino acids E230, Y232, N257 and / or E259 of SEQ ID NO:1.

[0153] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9.

[0154] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9.

[0155] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region derived from an antibody and retains the binding specificity of the antibody, and comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9.

[0156] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region derived from an antibody and retains the binding specificity of the antibody, and comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9.

[0157] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region that competes with an antibody comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9.

[0158] In a preferred embodiment, the anti-CD45 agent comprises an antigen-binding region that competes with an antibody comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9.

[0159] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO:2; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO:3.

[0160] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 58; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO:59.

[0161] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17.

[0162] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 10; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO:11.

[0163] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 20, VHCDR2 is SEQ ID NO: 21 and VHCDR3 is SEQ ID NO: 22; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 24 and VLCDR3 is SEQ ID NO: 25.

[0164] In another preferred embodiment, the anti-CD45 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 18; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 19.

[0165] In another preferred embodiment, the anti-CD45 agent is an antibody selected from Refmab#1, Refmab#2, Refmab#3, Refmab#4 and Refmab#5. In another preferred embodiment, the anti-CD45 agent is an antibody selected from QA17A19 (Biolegend, #393411), BC8 (IchorBio; #ICH1155), AbA (WO2020092654, SEQ ID NOs: 1 and 5), HI30 (Biolegend, #304001) and 2D1 (R&D Systems, #MAB1430). In another preferred embodiment, the anti-CD45 agent is an antibody comprising the CDRs of an antibody selected from QA17A19 (Biolegend, #393411), BC8 (IchorBio; #ICH1155), AbA (WO2020092654, SEQ ID NOs: 1 and 5), HI30 (Biolegend, #304001) and 2D1 (R&D Systems, #MAB1430). In another preferred embodiment, said anti-CD45 agent is an antibody that competes for binding to CD45 with an antibody selected from QA17A19 (Biolegend, #393411), BC8 (IchorBio; #ICH1155), AbA (WO2020092654, SEQ ID NOs: 1 and 5), HI30 (Biolegend, #304001) and 2D1 (R&D Systems, #MAB1430).

[0166] In a particular preferred embodiment, said depleting agent is or is derived from QA17A19 (Biolegend, #393411; Refmab#1), said depleting agent binds to one isoform of CD45 but does not bind or binds substantially weaker to a second isoform of CD45, one of said isoforms being wild type CD45 and the other isoform having a mutation in at least one of the following amino acid residues of CD45: F331, K352 and E353. Preferably, said mutations include one or more of F331G, K352H, K352E, K352D, K352I, K352L, K352M, K352N, K352Q, K352S, K352T, E353K or E353R.

[0167] In certain preferred embodiments, the depleting agent is or is derived from AbA (WO2020092654, SEQ ID NOs: 1 and 5; Refmab#2), and the depleting agent binds to one isoform of CD45 but does not bind, or binds substantially weaker, to a second isoform of CD45, one of which is wild-type CD45 and the other isoform has a mutation at amino acid residue N257 of CD45. Preferably, the mutation is N257D, N257E, N257K, N257R or N257T.

[0168] In certain preferred embodiments, the depleting agent is or is derived from BC8 (IchorBio; #ICH1155; Refmab#4), and the depleting agent binds to one isoform of CD45 but does not bind, or binds substantially weakly, to a second isoform of CD45, one of the isoforms being wild-type CD45 and the other isoform having a mutation in at least one of the following amino acid residues of CD45: E230, N257, E259, T264, N267, N286, S287, D292, F331, D334, Y340, K352, E353 and Y373. Preferably, the mutations include one or more of the following: E230K, N257T, N257H, N257R, N257S, N257V, E259G, E259N or E259Q.

[0169] It is further contemplated that the antigen-binding region of anti-CD45 antibody can be further screened or optimized for their binding properties as defined above. In particular, it is contemplated that said antigen-binding region can have 1, 2, 3, 4, 5, 6 or more changes in the amino acid sequence of 1, 2, 3, 4, 5 or 6 CDRs of the monoclonal antibody provided herein. It is contemplated that the amino acid at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 of the VJ or VDJ region of the light chain variable region or heavy chain variable region of the antigen-binding region can have insertion, deletion or substitution with conserved or non-conserved amino acids. Such amino acids that can be substituted or constitute substitutions are disclosed above.

[0170] In some embodiments, the amino acid difference is a conservative substitution, i.e., a substitution of one amino acid with another amino acid having similar chemical or physical properties (size, charge, or polarity), which generally does not adversely affect the biochemical, biophysical, and / or biological properties of the CD45 protein. In particular, the substitution does not disrupt the interaction of the antibody with the CD45 antigen. Said conservative substitution is advantageously selected among the following five groups: Group 1 - small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2 - polar, negatively charged residues and their amides (D, N, E, Q); Group 3 - polar, positively charged residues (H, R, K); Group 4 - large aliphatic, non-polar residues (M, L, I, V, C); and Group 5 - large, aromatic residues (F, Y, W).

[0171] In a more specific embodiment, the first antigen-binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 2, 10, 18, and 58, and / or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 3, 11, 19, and 59.

[0172] Said first antigen-binding region having an amino acid sequence having at least 90%, such as at least 95%, 96%, 97%, 98% or 99% identity with any one of the above defined amino acid sequences is also part of the present disclosure, typically said first antigen-binding region having at least equal or higher binding activity than said first antigen-binding region consisting of a heavy chain consisting of any one of the amino acid sequences selected from SEQ ID NOs: 2, 10, 18 and 58 and / or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 3, 11, 19 and 59.

[0173] In certain embodiments, the anti-CD45 agent may be a bispecific CD45 antibody comprising at least one first binding specificity for CD45, e.g., one antigen-binding region of an anti-CD45 antibody described herein, and a second binding specificity for a second target epitope or target antigen.

[0174] According to the present disclosure, the anti-CD45 agent can be an immune cell having a CD45-targeted antigen receptor, e.g., a CD45-targeted CAR, wherein the antigen receptor comprises an antigen-binding region as described above.

[0175] In certain embodiments, the immune cells (e.g., T cells) carrying CD45-targeted CAR recognize the second isoform of CD45 expressed in a patient in need thereof, and do not recognize the first isoform of CD45. In particular, the immune cells can specifically bind to an epitope comprising amino acids E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO:1. More preferably, the immune cells specifically bind to an epitope comprising amino acids N286, F331, K352 or E353 of SEQ ID NO:1.

[0176] In certain embodiments, the anti-CD45 agent can be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR comprises an antigen-binding region, e.g., an scFv, wherein the antigen-binding region comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9 and VLCDR3 is SEQ ID NO: 10.

[0177] In certain embodiments, the anti-CD45 agent can be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR comprises an antigen-binding region, e.g., an scFv, wherein the antigen-binding region comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9.

[0178] In a more specific embodiment, the anti-CD45 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:2, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:3.

[0179] In another more specific embodiment, said anti-CD45 agent may be an immune cell (e.g., a T cell) bearing a CAR comprising said first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:58, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:59.

[0180] According to the present disclosure, the anti-CD45 agent can be an immune cell having an antigen receptor that targets CD45, e.g., a CAR that targets a specific isoform of CD45, wherein the antigen receptor comprises an antigen-binding region as described above, and the immune cell does not express CD45 or expresses an isoform of CD45 that is not recognized by the CAR.

[0181] In certain embodiments, the anti-CD45 agent can be an immune cell (e.g., a T cell) bearing a CAR, the CAR targeting a specific isoform of CD45 comprising an antigen binding region, such as an scFv, the antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17; Additionally, the immune cells either do not express CD45 or express an isoform of CD45 that is not recognized by the CAR.

[0182] In a more specific embodiment, the anti-CD45 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 10, and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and the immune cell expresses an isoform of CD45 that is not recognized by the CAR.

[0183] In a more preferred embodiment, said anti-CD45 agent is the Refmab #1 antibody as described in the Examples.

[0184] In another preferred embodiment, said anti-CD45 agent may be an immune cell bearing a CAR targeting a specific isoform of CD45 as described in the examples.

[0185] In particular, the present disclosure also relates to depleting anti-CD45 agents (e.g., CAR cell compositions or antibodies) as described above that comprise a first or second antigen-binding region for use in selectively depleting host cells or transferred cells, respectively, in a subject in need thereof.

[0186] Cells expressing the first isoform of CD45 The present disclosure relates to a mammalian cell, preferably a hematopoietic cell, or a population of cells expressing a first isoform of CD45, said cell or population of cells expressing a first isoform of CD45 comprising at least one polymorphic allele in a nucleic acid encoding said first isoform, said first isoform not being recognized by a depleting agent comprising a first antigen-binding region as described herein. Preferably, said mammalian cell is a human cell.

[0187] The cell or population of cells is particularly useful for medical treatment in patients expressing the second isoform of CD45.

[0188] In a particular embodiment, the cells (e.g., hematopoietic stem cells) encoding or expressing the first isoform of CD45 that is not recognized by the depleting agent (e.g., hematopoietic cells) are particularly useful in medical treatments for restoring normal hematopoiesis after immunotherapy, such as adoptive cell transfer in patients expressing the second isoform, specifically, the treatments include administering a therapeutically effective amount of the hematopoietic cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent targeting the second isoform of CD45. In particular, the hematopoietic cells, preferably hematopoietic stem cells, are administered subsequent to the depleting agent. In another particular embodiment, the hematopoietic cells, preferably hematopoietic stem cells, may be administered prior to or simultaneously with the depleting agent.

[0189] In another particular embodiment, said cells expressing a first isoform of CD45 that is specifically recognized by a depleting agent that does not bind or binds substantially weaker to a second isoform of CD45 are particularly useful in medical treatment in patients expressing the second isoform of CD45, in particular to avoid serious side effects associated with transplanted cells bearing the first isoform (safety switch), said treatment comprising administering a therapeutically effective amount of a depleting agent that targets the first isoform of CD45. In particular, said hematopoietic cells, preferably immune cells bearing a CAR, are administered prior to said depleting agent.

[0190] As used herein, the term cell relates to a mammalian cell, preferably a human cell.

[0191] In certain embodiments, the cells are hematopoietic cells, including lymphocytes such as B cells and T cells, natural killer cells, myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes, dendritic cells (DCs) and platelet dendritic cells (pDCs), including immune cells.

[0192] In a preferred embodiment, the immune cell is a T cell. In another preferred embodiment, the immune cell is a primary T cell. As used herein, the term "T cell" includes cells bearing a T cell receptor (TCR) or cells derived from T cells bearing a TCR. The T cell according to the present disclosure can be selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, memory T lymphocytes, tumor-infiltrating lymphocytes, and helper T lymphocytes, including both type 1 and type 2 helper T cells and Th17 helper cells. In another embodiment, the cell can be derived from the group consisting of CD4+ and CD8+ T lymphocytes or non-classical T cells, such as MR1-restricted T cells, MAIT cells, NKT cells, gamma delta T cells, or innate immune-like T cells.

[0193] T cells can be obtained from multiple sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor.In certain embodiments, T cells can be obtained from a unit of blood taken from a subject using any number of techniques known to those skilled in the art.Alternatively, T cells can be differentiated from iPS cells.

[0194] In another preferred embodiment, the hematopoietic cells are hematopoietic stem cells. Stem cells can be adult stem cells, embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human stem cells are CD34 + Hematopoietic stem cells can be differentiated from iPS cells or can be harvested from umbilical cord blood, from bone marrow, or from mobilized or non-mobilized peripheral blood.

[0195] In certain embodiments, the cells are allogeneic, which refers to cells derived from a donor that exhibits the same, similar or different HLA genotype as the person receiving the cells. The donor may be related or unrelated. In certain embodiments, the cells are autologous, which refers to cells derived from the same person receiving the cells.

[0196] The cells can be derived from healthy donors or patients, particularly from patients diagnosed with cancer, genetic or autoimmune diseases, or from patients diagnosed with infectious diseases. Hematopoietic cells can be extracted from blood, bone marrow, or derived from stem cells. HSCs can be derived, for example, from iPS (induced pluripotent stem cells).

[0197] Those skilled in the art will be able to select the more appropriate cells depending on the patient or subject to be transplanted.

[0198] The present disclosure further relates to compositions of cells or populations of cells for use in the treatments disclosed herein.

[0199] CAR For use in adoptive cell transfer therapy, the cells expressing the first isoform of CD45 according to the present disclosure can be modified to exhibit desired specificity and enhanced functionality. In certain embodiments, the cells can express a recombinant antigen-binding region, also referred to as an antigen receptor, on their cell surface as described above. In certain embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). According to the present disclosure, the immune cells expressing the first isoform of CD45 and CAR can be specifically depleted by administration of a therapeutically effective amount of a drug comprising a second antigen-binding region that specifically binds to the first isoform of CD45 but not to the second isoform of CD45, thereby avoiding eventual severe side effects resulting from the transplantation of the immune cells.

[0200] In certain embodiments, immune cells are reoriented against cancer antigens. By "cancer antigen" is meant any antigen (i.e., a molecule capable of inducing an immune response) associated with cancer. An antigen as defined herein can be any type of molecule that induces an immune response, e.g., a polysaccharide or lipid, but most preferably a peptide (or protein). Human cancer antigens can be human or derived from humans. Cancer antigens can be tumor-specific antigens, meaning antigens that are not found in healthy cells. Tumor-specific antigens generally result from mutations, particularly frameshift mutations, that generate entirely new amino acid sequences not found in the proteome of healthy humans.

[0201] Cancer antigens also include tumor-associated antigens, which are antigens whose expression or production is associated with tumor cells, but are not limited to these. Examples of tumor-associated antigens include, for example, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, CD123, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), total cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin, nerve fiber, and the like. These include fiber, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD33, CD123, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor gamma chain alternative reading frame protein), Trp-p8, STEAP1 (prostate six transmembrane epithelial antigen 1), abnormal ras protein or abnormal p53 protein. In another particular embodiment, said tumor-associated or tumor-specific antigen is integrin ανβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

[0202] In certain embodiments, for use in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic diseases such as acute myeloid leukemia (AML) or B-acute lymphoblastic leukemia (B-ALL), immune cells according to the present disclosure express a recombinant antigen-binding region, such as a CD45-targeted CAR. The cells expressing a first isoform and expressing a CAR (e.g., CAR-CD45) can be further specifically depleted by administering a depleting agent comprising a second antigen-binding region that specifically binds to the first isoform of CD45 but not to the second isoform of CD45, thereby avoiding eventual severe side effects such as graft-versus-host disease resulting from transplantation.

[0203] In certain embodiments, the immune cells (e.g., T cells) expressing the first isoform have a CD45-targeted CAR, wherein the CAR comprises an antigen binding region, e.g., an scFv, that specifically binds to an epitope of CD45 located within the N-terminal domain or within a polypeptide comprising amino acids E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 and / or Y373 of SEQ ID NO:1, more preferably within amino acids E230, N257, E259, F331, K352 and / or E353 of SEQ ID NO:1.

[0204] In particular, the immune cell (e.g., T cell) expressing the first isoform has an antigen-binding region, e.g., a CD45-targeted CAR comprising an scFv, the antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 20, VHCDR2 is SEQ ID NO: 21 and VHCDR3 is SEQ ID NO: 22; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 24 and VLCDR3 is SEQ ID NO: 25; More preferably, it comprises an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:18 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:19.

[0205] In vitro method for preparing cells expressing the first isoform The cell expressing the first isoform of CD45 according to the present disclosure can be genetically engineered by introducing a nucleic acid construct (e.g., mRNA) encoding at least one gene editing enzyme or ribonucleoprotein complex comprising the above-mentioned gene editing enzyme and / or HDR template into said cell. Alternatively, the gene editing system is transduced into said cell via a viral system, such as an adenoviral system. The cell can also be genetically engineered by further introducing a nucleic acid construct encoding the above-mentioned CAR into said cell. In particular, the method is an ex vivo method carried out on a culture of cells.

[0206] The term "nucleic acid construct" as used herein refers to a nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that is modified to contain segments of nucleic acid sequences, assembled and juxtaposed in a manner that does not normally occur in nature. A nucleic acid construct is usually a "vector", a nucleic acid molecule that is used to deliver exogenously produced DNA into a host cell.

[0207] Preferably, the nucleic acid construct comprises said gene editing enzyme, HDR template and / or CAR operably linked to one or more control sequences.Said control sequence can be a ubiquitous, tissue-specific or inducible promoter that is functional in the cells of target organ (i.e., hematopoietic cells).Such sequences well known in the art include in particular promoters and further regulatory sequences that can further control the expression of transgene, such as but not limited to enhancers, terminators, introns, silencers.

[0208] The nucleic acid construct as described above may be included in an expression vector.The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome.The vector may include any means for ensuring self-replication.Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated.

[0209] Examples of suitable vectors include, but are not limited to, recombinant integrative or non-integrative virus vectors, and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.Preferably, vector is recombinant integrative or non-integrative virus vectors.Examples of recombinant virus vectors include, but are not limited to, vectors derived from herpesvirus, retrovirus, lentivirus, vaccinia virus, adenovirus, adeno-associated virus or bovine papilloma virus.

[0210] The present disclosure relates to a method for expressing a first isoform of a cell surface protein in a cell by introducing a nucleic acid construct (e.g., mRNA) encoding a gene editing enzyme or a ribonucleoprotein complex comprising the above-mentioned gene editing enzyme and / or HDR template into the cell. The method may further comprise a step of introducing a nucleic acid construct encoding a CAR into the cell. The method comprises introducing a gene editing enzyme, such as a Cas protein, a base editor or a prime editor and a guide RNA (crRNA, tracrRNa, or fusion guide RNA or pegRNA) into the cell. In particular, the gene editing enzyme is a CRISPR / Cas gene editing enzyme as described above. In a more particular embodiment, the gene editing enzyme is a site-specific nuclease, more preferably a CRISPR / Cas nuclease, comprising a guide RNA and a Cas protein, and the guide RNA in combination with a Cas protein cleaves and induces cleavage within the target sequence, comprising a nucleic acid encoding a surface protein region involved in drug binding as described above.

[0211] The Cas nuclease can be a high-fidelity Cas nuclease, such as a high-fidelity Cas9 nuclease.

[0212] Said gene editing enzyme, preferably a guide RNA and / or a Cas protein, a base editor or a prime editor as described above, may be synthesized in situ in a cell as a result of the introduction of a nucleic acid construct, preferably an expression vector encoding said gene editing enzyme, such as a guide RNA and / or a Cas protein, a base editor or a prime editor as described above, into the cell. Alternatively, said gene editing enzyme, such as a guide RNA and / or a Cas protein, a base editor or a prime editor, may be produced outside the cell and then introduced therein.

[0213] Said nucleic acid construct or expression vector can be introduced into the cell by any method known in the art, including, but not limited to, stable transduction methods in which the nucleic acid construct or expression vector is integrated into the genome of the cell, transient transfection methods in which the nucleic acid construct or expression vector is not integrated into the genome of the cell, and virus-mediated methods. For example, transient transformation methods include, for example, microinjection, electroporation, cell squeezing, particle bombardment or in vivo targeting approaches.

[0214] In vivo editing The cells expressing the first isoform of CD45 according to the present disclosure can also be edited in vivo.There are various technologies that allow therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles and virus-like particles (see, for example, Cell (2022) 185: 2806-27).The molecular mechanism of converting CD45 to the first isoform of CD45 that is not recognized by depleting agents can be achieved by any of these methods.

[0215] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a molecular machinery capable of editing a gene in vivo and a depletion agent, the molecular machinery capable of in vivo gene editing comprises all the components required to introduce a point mutation in a wild-type CD45 isoform in a target cell, The depleting agent binds to wild-type CD45 but not to the isoforms of CD45. It is intended for use in the medical treatment of a patient in need thereof.

[0216] Preferably, said isoform of CD45 is characterized by a substitution of the lysine at position 352 of wild-type CD45 with glutamic acid. Alternatively, said isoform of CD45 is characterized by a substitution of the lysine at position 352 of wild-type CD45 with aspartic acid. Alternatively, said isoform of CD45 is characterized by a substitution of the lysine at position 352 of wild-type CD45 with histidine. Alternatively, said isoform of CD45 is characterized by a substitution of the lysine at position 353 of wild-type CD45 with lysine. Alternatively, said isoform of CD45 is characterized by a substitution of the lysine at position 353 of wild-type CD45 with arginine.

[0217] Also preferably, the depleting agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9.

[0218] Alternatively, the depletion agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9.

[0219] Also preferably, said isoform of CD45 is characterized by a substitution of asparagine at position 257 of wild-type CD45 with glutamic acid. Alternatively, said isoform of CD45 is characterized by a substitution of asparagine at position 257 of wild-type CD45 with lysine. Alternatively, said isoform of CD45 is characterized by a substitution of asparagine at position 257 of wild-type CD45 with arginine. Alternatively, said isoform of CD45 is characterized by a substitution of asparagine at position 257 of wild-type CD45 with threonine. Alternatively, said isoform of CD45 is characterized by a substitution of glutamic acid at position 259 of wild-type CD45 with valine. Alternatively, said isoform of CD45 is characterized by a substitution of glutamic acid at position 259 of wild-type CD45 with glycine. Alternatively, said isoform of CD45 is characterized by a substitution of tyrosine at position 232 of wild-type CD45 with cysteine. Alternatively, said isoform of CD45 is characterized by a substitution of asparagine at position 286 of wild-type CD45 with aspartic acid.

[0220] Also preferably, the depleting agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 20, VHCDR2 is SEQ ID NO: 21 and VHCDR3 is SEQ ID NO: 22, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 24 and VLCDR3 is SEQ ID NO: 25.

[0221] Also preferably, the depleting agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17.

[0222] In certain embodiments, the present disclosure relates to a human cell or population of human cells expressing a first isoform of CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD45, the human cell expressing the first isoform comprises genomic DNA having at least one polymorphic or engineered allele; the polymorphism or engineered allele is absent from the genome of a patient whose cells express the second isoform of CD45; The polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position E230, Y232, N255, N257, E259, T264, N267, E269, N286, S287, D292, I328, T330, F331, D334, Y340, K352, E353, E360, E364, Y372 or Y373 of SEQ ID NO: 1. In certain embodiments, the medical treatment comprises administering to the patient in need thereof a therapeutically effective amount of the cell or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising an antigen-binding region that specifically binds to the second isoform of CD45 to specifically deplete the patient's cells expressing the second isoform of CD45.

[0223] In certain embodiments, the polymorphism or engineered allele is characterized by at least one substitution of an amino acid at position N286, F331, K352 or E353 of SEQ ID NO:1.

[0224] In certain embodiments, the polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position N286, F331, K352 or E353 of SEQ ID NO:1, and the depletor comprises an antigen-binding region and is selected from the following: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or b) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:58 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:59.

[0225] In certain embodiments, the polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position F331, K352 or E353 of SEQ ID NO:1, and the depletor binds to the same epitope as the antigen-binding region and is selected from: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or b) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:58 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:59.

[0226] In a preferred embodiment, said polymorphic or engineered allele is characterized by a substitution of the amino acid at position K352 of SEQ ID NO: 1. In a particular embodiment, said substitution of the amino acid at position K352 of SEQ ID NO: 1 is selected from K352E, K352H, K352I, K352L, K352M, K352N, K352Q, K352S and K352T, preferably said substitution is K352D, K352E and K352H, more preferably said substitution is K352E.

[0227] In certain embodiments, the polymorphism or engineered allele is characterized by at least one substitution of an amino acid at position E230, Y232, N257, E259 or N286 of SEQ ID NO:1.

[0228] In certain embodiments, the polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position E230, Y232, N257, E259 or N286 of SEQ ID NO:1, and the depletor comprises an antigen-binding region and is selected from the following: an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO:20, VHCDR2 is SEQ ID NO:21 and VHCDR3 is SEQ ID NO:22; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO:23, VLCDR2 is SEQ ID NO:24 and VLCDR3 is SEQ ID NO:25; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:18 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:19.

[0229] In certain embodiments, the polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position E230, Y232, N257, E259 or N286 of SEQ ID NO:1, and the depletor binds to the same epitope as an antigen-binding region comprising: an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO:20, VHCDR2 is SEQ ID NO:21 and VHCDR3 is SEQ ID NO:22; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO:23, VLCDR2 is SEQ ID NO:24 and VLCDR3 is SEQ ID NO:25; More preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:18 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:19.

[0230] In a preferred embodiment, said polymorphism or engineered allele is characterized by a substitution of the amino acid at position N257 of SEQ ID NO: 1. In a particular embodiment, said substitution of the amino acid at position N257 of SEQ ID NO: 1 is an N257E, N257K, N257R or N257T substitution, preferably said substitution is an N257R substitution.

[0231] In a preferred embodiment, said polymorphic or engineered allele is characterized by a substitution of the amino acid at position E259 of SEQ ID NO: 1. In a particular embodiment, said substitution of the amino acid at position E259 of SEQ ID NO: 1 is an E259N, E259Q, E259V or E259G substitution, preferably said substitution is an E259V substitution.

[0232] In a preferred embodiment, said polymorphism or engineered allele is characterized by an amino acid substitution at position Y232 of SEQ ID NO:1, preferably said substitution is a Y232C substitution.

[0233] In a preferred embodiment, said polymorphic or engineered allele is characterized by a substitution of the amino acid at position N286 of SEQ ID NO: 1, preferably said substitution is N286D.

[0234] In certain embodiments, the polymorphic allele or engineered allele is characterized by a substitution of an amino acid at position N257 of SEQ ID NO: 1. In certain embodiments, the polymorphic allele or engineered allele is characterized by a substitution of an amino acid at position N257 of SEQ ID NO: 1, and the depleting agent comprises an antigen-binding region and is selected from the following: an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17; Preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:10 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:11.

[0235] In certain embodiments, the polymorphic or engineered allele is characterized by an amino acid substitution at position N257 of SEQ ID NO:1, and the depletor binds to the same epitope as an antigen-binding region selected from: an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; and an antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17; Preferably, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:10 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:11.

[0236] In a preferred embodiment, said polymorphism or engineered allele is characterized by a substitution of the amino acid at position N257 of SEQ ID NO: 1. In a particular embodiment, said substitution of the amino acid at position N257 of SEQ ID NO: 1 is an N257E, N257K, N257R or N257T substitution, preferably said substitution is an N257R substitution.

[0237] Pharmaceutical Compositions and Therapeutic Uses In a further aspect, the present disclosure also provides a pharmaceutical composition comprising a cell or population of cells expressing the first isoform of CD45 described above, together with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients.

[0238] In a particular embodiment, said cells expressing the first isoform of CD45 are hematopoietic stem cells.

[0239] In another particular embodiment, said cell expressing a first isoform of CD45 is an immune cell, preferably a T cell, more preferably a primary T cell, bearing a chimeric antigen receptor (CAR), preferably a CAR that targets a second isoform of CD45 expressed by a cell of said patient as described above.

[0240] The pharmaceutical composition may further comprise a depleting agent comprising the first or second antigen-binding region as described above.

[0241] Pharmaceutical compositions are formulated in pharmaceutically acceptable carriers according to the route of administration.Preferably, the compositions are formulated to be administered by intravenous injection.The pharmaceutical compositions suitable for such administration can comprise the cells expressing the above-mentioned first isoform in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection or dispersion immediately before use, which can comprise antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickening agents.

[0242] Optionally, the composition comprising the cells expressing the first isoform of CD45 can be frozen for storage at any temperature suitable for cell storage.For example, the cells can be frozen at about -20°C, -80°C or any other suitable temperature.The cryogenically frozen cells can be stored and prepared for storage in a suitable container to reduce the risk of damage to the cells and maximize the chance that the cells survive after thawing.Alternatively, the cells can be maintained at refrigerated room temperature, for example, about 4°C.

[0243] The present disclosure relates to a cell or population of cells expressing the first isoform of CD45 as described above for use as a medicament, in particular for use in immunotherapy such as adoptive cell transfer therapy in a patient.

[0244] According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD45 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the second isoform or the first isoform of CD45 to specifically deplete the patient or transplant cells, respectively.

[0245] As used herein, the term "in combination" or "in combination therapy" means that two (or more) different therapies are delivered to a subject during the course of the subject's being afflicted by a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatments are discontinued for other reasons. In some embodiments, there is an overlap in administration, as the delivery of one treatment is still occurring when the delivery of the second treatment begins. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment has ended before the delivery of the other treatment begins. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. In one embodiment, a depleting agent that binds to the second isoform or the first isoform of CD45 is administered at a dose and / or dosing schedule described herein, and cells expressing the first isoform are administered at a dose and / or dosing schedule described herein. In some embodiments, "in combination with" is not intended to mean that the depleting agent targeting the second isoform of CD45 (e.g., CAR cells or antibodies that recognize the second isoform of CD45) or the first isoform and the composition of cells expressing the first isoform of CD45 (e.g., the first isoform of CD45) must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of this disclosure. The depleting agent (e.g., CAR cells or antibodies that target the second isoform of CD45) can be administered simultaneously with, before, or after a dose of hematopoietic stem cells expressing the first isoform of CD45. In certain embodiments, each agent is administered at a dose and / or time schedule determined for that particular agent.

[0246] Adoptive cell transfer therapy according to the present disclosure can be used to treat patients diagnosed with cancer, genetic diseases, autoimmune diseases, infectious diseases, diseases requiring hematopoietic stem cell transplantation (HSCT), prevention of organ rejection, tumor transplant conditioning, tumor maintenance therapy, minimal residual disease, and prevention of relapse.

[0247] The present disclosure also relates to the use of the above-described cells expressing the first isoform of CD45 in the manufacture of a medicament for adoptive transfer cell therapy in a patient.

[0248] As used herein, the term "subject" or "patient" refers to an animal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat, preferably a mammal in which an immune response can be elicited. More preferably, the patient is a human, including an adult, a child and a human in the prenatal stage.

[0249] As used herein, the terms "treatment," "treat," or "treating" refer to any action intended to improve the health status of a patient, such as the treatment, prevention and prophylaxis, and delay of disease. In certain embodiments, such terms refer to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, the terms refer to minimizing the progression or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0250] The cancer that can be treated includes tumor that is not vascularized or not yet substantially vascularized, and vascularized tumor.Cancer can include non-solid tumor (e.g., hematological tumor, e.g., leukemia and lymphoma, e.g., relapse and therapy-related tumor, e.g., secondary malignant tumor after cytotoxic therapy and hematopoietic stem cell transplantation (HSCT)) or can include solid tumor.

[0251] The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens.

[0252] An infectious disease is a disease caused by a pathogenic microorganism, such as a bacterium, a virus, a parasite, or a fungus. In certain embodiments, an infectious disease according to the present disclosure occurs in an immunosuppressed patient, such as a patient after HSCT or a patient who has received a solid organ transplant.

[0253] In a preferred embodiment, the present disclosure relates to a cell expressing the first isoform of CD45 as described above for use in treating a hematological cancer, preferably a leukemia or a lymphoproliferative disorder. Said leukemia may be selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), myeloproliferative neoplasms (MPN) including chronic myelogenous leukemia (CML), myelodysplastic syndrome / myeloproliferative neoplasm (MDS / MPN) overlap syndromes including chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), B and T cell non-Hodgkin's lymphoma, acute biphenotypic leukemia, hairy cell leukemia, interleukin-3 receptor subunit alpha positive leukemia, B cell acute lymphoblastic leukemia (B-ALL), T cell acute lymphoblastic leukemia (T-ALL), Hodgkin's lymphoma (HL), systemic mastocytosis and preferably MDS, preferably AML or BPDCN.

[0254] In certain embodiments, said cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD45 can be used for the treatment of solid tumors, particularly for selective depletion of bone marrow cells of a solid tumor in a patient, allowing immunotherapeutic agents such as immune checkpoint inhibitors, CAR T cells or tumor-infiltrating lymphocytes to access the tumor, since the bone marrow cells of the tumor may be immunosuppressive. In this situation, said cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD45 as described above can help replenish the hematopoietic system that may be affected by the treatment intended to deplete the bone marrow cells of the solid tumor.

[0255] In another specific embodiment, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD45 described above can be used for the treatment of an autoimmune disease such as lupus, multiple sclerosis, scleroderma or systemic sclerosis.

[0256] The present disclosure also relates to a depleting agent (e.g., a CAR cell composition or an antibody) comprising the first or second antigen-binding region for use in selectively depleting host cells or transferred cells, respectively, in a subject in need thereof.

[0257] A method to specifically deplete the patient's cells but not the transplanted cells According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing a first isoform of CD45 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the second isoform of CD45.

[0258] In fact, during immunotherapy, immune depleting agents, such as CAR-expressing immune cells targeting CD45, can be administered to patients to target and kill tumor cells. However, because tumor surface proteins are also expressed on the surface of normal hematopoietic cells, this strategy can induce severe side effects in patients by altering hematopoiesis. To restore hematopoiesis in patients, hematopoietic cells can be subsequently transplanted into patients. However, these cells need to be resistant to the agent (i.e., the depleting agent for CD45-expressing cells) so as not to be targeted by the agent.

[0259] Thus, alternatively, according to the present disclosure, a depleting agent comprising a first antigen-binding region that specifically binds to a second isoform of CD45 can be administered to specifically ablate the patient's cells expressing said second isoform of CD45 but not the transplanted cells expressing said first isoform of CD45. Selective depletion of the patient's cells but not the transplanted cells allows the patient to be reconstituted with a healthy hematopoietic system that is no longer depleted by the immune depleting agent. Thus, according to this therapeutic use, the patient has a functional immune system rather than undergoing long-term immunosuppression. The use of cells according to the present disclosure eliminates infection as a major complication of current HSC transplantation.

[0260] In another embodiment, the present disclosure relates to a method for adoptive cell transfer therapy, preferably for hematopoietic stem cell transplantation, to restore normal hematopoiesis in a patient having cells expressing the second isoform of CD45, comprising: (i) administering an effective amount of cells (e.g., hematopoietic stem cells) expressing a first isoform of CD45, wherein the cells expressing the first isoform of CD45 comprise genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically engineered allele, in a nucleic acid encoding the first isoform, wherein the polymorphism is not present in the genome of a patient having cells expressing the second isoform of CD45, or a pharmaceutical composition thereof; and (ii) administering a therapeutically effective amount of an agent comprising at least a first antigen-binding region that specifically binds to said second isoform of CD45 and does not bind or binds substantially weaker to said first isoform of CD45 to specifically deplete cells (the patient's cells) expressing said second isoform of CD45.

[0261] The cells expressing the first isoform of CD45 or a pharmaceutical composition thereof are administered to a subject in combination (e.g., before, simultaneously with, or after) with an agent comprising the first antigen-binding region as described above.

[0262] In a preferred embodiment, the depleting agent (e.g., a CAR cell or an antibody targeting the second isoform of CD45) is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells expressing the first isoform of said surface protein (e.g., the first isoform of CD45).

[0263] By "therapeutically effective amount" or "effective amount" is intended a number of cells, particularly hematopoietic stem cells, expressing the first isoform of CD45 as described above, administered to a subject sufficient to constitute a treatment as defined above, particularly the restoration of normal hematopoiesis in the patient.

[0264] Administration of the cells or pharmaceutical compositions according to the present disclosure may be performed in any convenient manner, including injection, transfusion, or implantation, transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In another embodiment, the cells or pharmaceutical compositions of the present disclosure are preferably administered by intravenous injection. The cells or pharmaceutical compositions of the present disclosure may be directly injected into a tumor, lymph node, or site of infection.

[0265] Administration of cells or populations of cells may be in any number from 10 to 100, including all integer values ​​of cell number within those ranges. 4 From 10 9 Cells / kg body weight, preferably 10 5 From 10 7 cells / kg body weight, more preferably 2×10 6 From 5×10 6The administration of cells / kg body weight may consist of administration of cells / kg body weight. The dosage administered will depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. The cells or population of cells may be administered in one or more doses. The timing of administration is within the judgment of the supervising physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art.

[0266] In particular, the present disclosure also relates to depleting anti-CD45 agents (e.g., CAR cell compositions or antibodies) described above that comprise a first antigen-binding region for use in selectively depleting host cells in a subject in need thereof.

[0267] A method to specifically deplete the transplanted cells but not the patient's cells (safety switch). According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD45 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the first isoform of CD45.

[0268] The cell or population of cells expressing the first isoform of CD45 of the present disclosure, preferably immune cells, is particularly used in adoptive cell transfer therapy into patients. The transplanted cells expressing the first isoform of CD45 can be further depleted in the patient by administering a therapeutically effective amount of a depleting agent, in particular comprising a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind or binds substantially weaker to the second isoform of CD45 expressed by the patient's cells, in order to avoid eventual severe side effects such as graft-versus-host disease due to transplantation. In this case, the agent comprising a second antigen-binding region that specifically binds to the first isoform of CD45 (expressed by the transplanted cells) is administered to deplete specifically the transplanted cells and not the patient's cells. The selective depletion of transplanted cells constitutes an important safety feature by providing a "safety switch".

[0269] Graft-versus-host disease (GvHD) refers to a medical complication following the receipt of transplanted tissue from a genetically different individual. The immune cells of the donated tissue (graft) recognize the recipient (host) as foreign cells. In certain embodiments, the medical condition is graft-versus-host disease caused by hematopoietic stem cell transplantation or adoptive cell transfer therapy, in which immune cells are transferred to the patient.

[0270] Said side effects may also occur when transplanted cells, especially immune cells bearing CAR, have severe side effects such as cytokine release syndrome and / or neurotoxicity.In this case, transplanted cells expressing the first isoform of CD45 can be eliminated when said cells become malignant or cause any type of undesired on-target or off-target damage as a safety switch.

[0271] The present disclosure relates to a method for adoptive cell transfer therapy in a patient having cells expressing a second isoform of CD45, the method comprising: (i) administering an effective amount of cells expressing a first isoform of CD45, wherein the cells expressing the first isoform of CD45 comprise genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically engineered allele, in a nucleic acid encoding the first isoform of CD45, wherein the polymorphism is not present in the genome of a patient having cells expressing the second isoform of CD45, or a pharmaceutical composition thereof; and (ii) administering a therapeutically effective amount of an agent that comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind or binds substantially weaker to the second isoform of CD45 to specifically deplete cells that express the first isoform of CD45.

[0272] The cells expressing the first isoform of CD45 or a pharmaceutical composition thereof are administered to a subject in combination (e.g., before, simultaneously with, or after) with an agent comprising a second antigen-binding region as described above.

[0273] In a preferred embodiment, the depleting agent (e.g., a CAR cell or an antibody targeting the second isoform of CD45) is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells expressing the first isoform of CD45.

[0274] Administration of the cells or pharmaceutical compositions according to the present disclosure may be performed in any convenient manner, including injection, transfusion, or implantation, transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In another embodiment, the cells or pharmaceutical compositions of the present disclosure are preferably administered by intravenous injection. The cells or pharmaceutical compositions of the present disclosure may be directly injected into a tumor, lymph node, or site of infection.

[0275] Administration of cells or populations of cells may be in any number from 10 to 100, including all integer values ​​of cell number within those ranges. 4 From 10 9 Cells / kg body weight, preferably 10 5 From 10 7 The administration of cells / kg body weight may consist of administration of cells / kg body weight. The dosage administered will depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. The cells or population of cells may be administered in one or more doses. The timing of administration is within the judgment of the supervising physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art.

[0276] Thus, in certain embodiments, the present disclosure relates to a depleting agent (e.g., a CAR cell or an antibody) for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD45 as described above, wherein the patient has naturally occurring cells expressing a second isoform of CD45, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD45 and does not bind or binds more weakly to the second isoform of CD45.

[0277] In another aspect, the present disclosure relates to a kit for expressing the first isoform of CD45 in a cell, comprising a gene editing enzyme such as a Cas protein, a base editor or a prime editor, a nucleic acid construct, a guide RNA in combination with an expression vector as described above, or an isolated cell according to the present disclosure.

[0278] In another aspect, the disclosure relates to a human cell or a population of human cells according to the disclosure for medical use, said medical use comprising administering to said patient in need thereof a therapeutically effective amount of said human cell or population of human cells expressing said first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising at least a second antigen binding region that specifically binds to said first isoform of CD45, thereby specifically depleting transferred cells expressing said first isoform of CD45, preferably for use in adoptive cell transfer therapy. More preferably, it is used for the treatment of malignant hematopoietic diseases such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), T-cell non-Hodgkin's lymphoma (T-NHL), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell acute lymphoblastic leukemia (T-ALL), non-Hodgkin's lymphoma (NHL) or follicular lymphoma (FL), and even more preferably, said depleting agent is subsequently administered to said human cell or a population of human cells expressing said first isoform of CD45 to avoid eventual severe side effects such as graft-versus-host disease due to transplantation. In a particular embodiment, said human cell or a population of human cells expressing said first isoform are immune cells, preferably T cells, bearing a chimeric antigen receptor (CAR). EXAMPLES

[0279] Example 1: Generation of anti-CD45 Fab and MAb Five different anti-CD45 antibodies were generated in Fab and MAb format based on publicly available sequence information or sources. The variable chains and CDRs (Kabat) of the antibodies (Refmab #1, #2 and #5) are shown in Table 5. Refmab #3 (HI30) is a mouse hybridoma antibody available from Biolegend (#304001). Refmab #5 (2D1) is a mouse hybridoma antibody available from R&D Systems (#MAB1430). [Table 5] JPEG2025500288000004.jpg202170

[0280] Further antibody characteristics, as well as the format and isotype of the full length antibodies, are shown in Table 6. [Table 6]

[0281] Example 2: Binding of MAbs to CD45 and optimization of assay conditions DF-1 cells (ATCC number CRL-12203) were transfected with constructs containing wild-type CD45 (SEQ ID NO: 1) or with an empty vector. Antibody binding to transfected cells and optimal assay conditions were evaluated in a 384-well format. Detection of cell expression was measured by high-throughput flow cytometry. Serial dilutions of each antibody were tested for immunoreactivity on cells expressing CD45 or vector alone. Based on raw signal values ​​and signal-background calculations, optimal screening concentrations of each antibody were determined. Results are shown in Figure 1. Each point represents the average of four replicates.

[0282] All five antibodies in Mab format bind to human CD45 in a concentration-dependent manner. Cells transfected with empty vector showed no binding to anti-human CD45 antibodies.

[0283] Optimized assay conditions for flow cytometry are shown in Table 7. [Table 7]

[0284] Example 3: Binding of Fab to CD45 and optimization of assay conditions A similar experiment was performed as described in Example 2, except that Fab fragments were tested instead of full-length antibodies. Serial dilutions of each Fab were tested for immunoreactivity against cells expressing wild-type CD45 or vector alone. Based on raw signal values ​​and signal-to-background calculations, optimal screening concentrations of Fab were determined. Results are shown in Figure 2. Each point represents the average of four replicates.

[0285] All four antibodies in Mab format bind to human CD45 in a concentration-dependent manner. Cells transfected with empty vector showed no binding to the anti-human CD45 antibody.

[0286] Optimized assay conditions for high-throughput flow cytometry are shown in Table 8. [Table 8]

[0287] Example 4: Alanine Scan Alanine scanning of human CD45 was performed to determine the residues of CD45 involved in binding to the investigated antibodies. Alanine scanning was performed by shotgun mutagenesis epitope mapping (Integral Molecular, Philadelphia / PA, USA) as described in Immunology (2014) 143, 13-20. Briefly, a mutant library of CD45 was created by high-throughput site-directed mutagenesis. Each residue was individually mutated to alanine and the alanine codon was mutated to serine. The mutant library was arrayed in 384-well microplates and transiently transfected into DF-1 cells. After transfection, cells were incubated with the indicated antibodies (IgG or Fab) at predetermined concentrations using independent immunofluorescence titration curves of wild-type CD45. Antibodies were detected using Alexa Fluor 488-conjugated secondary antibodies, and mean cell fluorescence was determined using the Intellicyt iQue flow cytometry platform (Intellicyt / Sartorius). Mutated residues were identified as important for the antibody epitope if they did not support the reactivity of the test antibody but did support the reactivity of a control antibody, which in each case was used as a control antibody for another anti-CD45 RefMab, e.g., RefMab#1, RefMab#2, RefMab#3, RefMab#4. This counterscreening strategy facilitates the elimination of variants that are locally misfolded or have expression defects. The binding of each antibody to each mutant clone was determined in duplicate. For each point, background fluorescence was subtracted from the raw data, then normalized to the antibody reactivity with wild-type CD45.

[0288] Library screening of very high affinity antibodies may not yield critical residues for antibody binding, so high affinity antibodies were converted to a Fab format to weaken binding sufficiently to allow identification of critical residues for binding. If Fab screening under standard conditions was still insufficient to identify critical residues for binding, high stringency conditions were implemented. These conditions included combinations of increasing pH, increasing salt concentration, increasing temperature, and / or increasing wash times. Antibodies that required high stringency conditions are denoted "HS".

[0289] For each mutated clone, the average binding value was plotted as a function of expression (represented by the reactivity of the control). See Figure 3. A threshold of >70% wild type binding to the control antibody and <20% wild type binding to the test antibody (dashed line) was applied to identify preliminary primary critical clones (circled). For clones that did not meet the set threshold, but whose reduced binding activity and proximity to critical residues suggested that the mutated residues may be part of the antibody epitope, secondary clones (squares) are highlighted.

[0290] The results of the alanine scan are shown in Table 9. The average binding reactivity (and range) is given for all critical residues identified. Critical residues for antibody binding (outlined in dark grey) were residues whose mutations were negative for binding to the test Ab but positive for binding to the control antibody. Additional secondary residues (outlined in light grey) were identified that did not meet the threshold guidelines, but whose reduced binding activity and proximity to the critical residues suggested they may be part of the antibody epitope. [Table 9] JPEG2025500288000009.jpg253170JPEG2025500288000010.jpg253170JPEG2025500288000011.jpg253170 JPEG2025500288000012.jpg253170JPEG2025500288000013.jpg253170JPEG2025500288000014.jpg253170 JPEG2025500288000015.jpg253170JPEG2025500288000016.jpg253170JPEG2025500288000017.jpg253170 JPEG2025500288000018.jpg253170JPEG2025500288000019.jpg253170JPEG2025500288000020.jpg144170

[0291] Table 10 summarizes the critical residues of each antibody tested. Residues whose mutations caused the least reactivity with the specific antibody are highlighted in bold and underlined. The critical residues examined represent amino acids whose side chains make the highest energy contribution to the antibody-epitope interaction (J. Mol. Biol. (1998) 280, 1-9; J. Mol. Biol. (1999) 285, 2177-2198); therefore, the highlighted residues are likely to be the major energy contributors to binding. [Table 10]

[0292] Example 5: Comprehensive mutation analysis The critical residues identified in Example 4 were investigated in more detail. First, a validation step of the identified critical residues was performed, taking into account the reproducibility of binding activity, surface accessibility, structural localization and distance to other critical sites, as well as the nature and biochemical properties of the substituted amino acid (e.g., cysteine ​​forming disulfide bridges or post-translational modification sites). After validation, each critical residue was subjected to comprehensive mutagenesis against biophysically relevant non-alanine amino acids selected based on the sequence and structure-related properties of the substituted amino acid as well as the newly introduced amino acid.

[0293] Antibodies were screened for binding to human CD45 mutants in IgG format. As in Example 4, the binding of each test antibody to each mutant clone in the comprehensive library was determined in duplicate by high-throughput flow cytometry. For each mutation, background fluorescence was subtracted from the raw data and then normalized to the antibody reactivity with wild-type CD45. For all mutant clones, the average binding reactivity and range are listed in Table 11. Mutations that caused less than 20% binding are highlighted in gray. [Table 11] JPEG2025500288000023.jpg253170JPEG2025500288000024.jpg253170JPEG2025500288000025.jpg253170JPEG2025500288000026.jpg138170

[0294] Table 12 summarizes the mutations that reduce the binding of the tested antibodies by less than 20%. [Table 12]

[0295] Example 6: Analysis and comparison of identified mutants FIG. 4 is a schematic showing the location of the identified variants on the 3D structure of human CD45.

[0296] Refmab#1 binds to a region distinct from that of the other tested antibodies, with the key mutants identified located between positions 328 and 373. Specifically, mutants at the following positions were identified where Refmab#1 showed less than 20% binding compared to wild-type CD45: residues E259, N286, I328, T330, F331, D334, Y340, K352, E353, Y372 and Y373. A comparison of the binding of the tested antibodies to some of these mutants is shown in Figure 5. Of the mutants identified, some appear to be less favorable, for example F331G, which is part of a non-conserved loop with a low accessible side chain. Particularly preferred mutants are the mutant F331, in particular F331G, the mutant K352, in particular the mutants K352E, K352H, K352I, K352L, K352M, K352N, K352Q, K352S and K352T, and the mutant K353, in particular the mutants E353H, E353K and E353R.

[0297] Four mutants, N257, E259, Y340 and Y372, were identified that inhibited the binding of Refmab#2 by less than 20% compared to CD45 wild type. Comparison of the binding of the tested antibodies to some of these mutants is shown in Figure 6. Mutant N257, especially mutants N257D, N257E, N257R and N257T, are particularly preferred mutants. Similarly, mutant N257K, which was not tested, is also a preferred mutant.

[0298] Various mutants were identified, E230, N257, E259, T264, N267, N286, S287, D292, F331, D334, Y340, K352, E353 and Y373, which inhibited the binding of Refmab#4 by less than 20% compared to CD45 wild type. Comparison of the binding of the tested antibodies to some of these mutants is shown in Figure 7. Mutants E230, in particular mutants E230K and E230R, mutant E259, in particular mutants E259N and E259Q, and mutant N257, in particular mutants N257M, N257P, N257T, N257H, N257R, N257S and N257V are particularly preferred mutants.

[0299] The data obtained so far were further analyzed by in silico mutagenesis. The aim was to analyze whether the protein sequence mutations affect the protein function. To do so, PROVEAN scores (PLoS ONE (2012); 7(10): e46688; Choy (2012), In Proceedings of the ACM Conference on Bioinformatics, Computational Biology and Biomedicine (BCB '12). ACM, New York, NY, USA, 414-417) were generated for candidate single amino acid substitutions at selected positions of CD45. First, a delta alignment score is calculated for each sequence that belongs to the top cluster of closely related sequences, i.e., the supporting sequence set. The delta scores are then averaged within and across clusters to generate the final PROVEAN score. If the predicted PROVEAN score is below (above) a given threshold (-2.5), the protein variant is predicted to have a deleterious (neutral) effect on the protein function.

[0300] The results are shown in Figure 8. Most of the experimentally identified mutants could be confirmed in silico. Only the mutant E259G is below the -2.5 threshold.

[0301] Example 7: Generation of CD45 mutants by base editing Base editing was used to test and validate that human CD45 is amenable to base-editing mutations.

[0302] To that end, we screened multiple sgRNAs with NG (N) protospacer adjacent motifs designed to target selected regions of CD45 against several base editors (ABEmax-SpG, xCas9(3.7)-BE4, CBE4max-SpCas9-NG, SPACE-NG, ABEmax-SpRY, CBE4max-SpG and ABE8e-NG). For each screening entry, 2 million K562 cells (ATCC CCL-243) were co-electroporated with 5ug of plasmid encoding the base editor and 1.5ug of sgRNA plasmid using a custom program: 1450V, 10ms, 3 pulses, using the Neon Transfection System 100μL Kit (ThermoFisher Scientific) and its proprietary T buffer (Invitrogen Ref:MPK10096Tb). 24 hours after co-electroporation, all conditions were sorted for GFP-positive cells utilizing the GFP cassette of the base editor plasmid using a BD FACS Aria III Cell Sorter (BD Bioscience). GFP-positive cells were then grown for an additional 2 days in 1 mL of RPMI-1640 (Sigma-Aldrich Ref: R8758-500ML) supplemented with 10% FCS and 100X GlutaMAX (ThermoFisher Scientific Ref: 35050061) and penicillin-streptomycin (1 / 1000). 72 hours after the first co-electroporation, we extracted gDNA for each condition where we performed PCR of the corresponding screened exons (9, 10 and 11). The PCR products were then sent for Sanger sequencing using the correct forward primer.

[0303] Primer pairs for PCR / sequencing of CD45 exons of interest: hCD45_Exon9_For = ACAAGCTGAGGTCCTTGTTAG (SEQ ID NO: 26) hCD45_Exon9_Rev = AGCAGAAAGTTCACCCACTTG (SEQ ID NO: 27) hCD45_Exon10_For = CCATAGCAATCTCAATCCTTGCC (SEQ ID NO: 28) hCD45_Exon10_Rev = TGCCTGTGTATAACAATTGCCAAG (SEQ ID NO: 29) hCD45_Exon11_For = TGACCTCAAGCTATGTATATGAGG (SEQ ID NO: 30) hCD45_Exon11_Rev = GAGACTGTTACCTCACACCATATAC (SEQ ID NO: 31)

[0304] Table 13 and Figure 9 show the most interesting hits from the screen (single amino acid changes and several other related mutant candidates).

[0305] The generated mutants were selected by computer-aided rational design. The following mutants were generated using each of the base editors and sg RNAs mentioned: [Table 13]

[0306] The results are shown in Figure 9. In summary, human CD45 is confirmed to be suitable for base editing. In particular, the mutants I328V, N255G, E360G, E259G, E364K, and E269G can be successfully edited by base editing. These residues can be efficiently edited in K562 cells and human T cells while maintaining the function of CD45. Therefore, this gene editing technology is compatible with the mutants identified in this disclosure and can be used in the respective clinical settings, for example, for safety switching or shielding.

[0307] Example 8: Base editing can shield human T cells from antibody-drug conjugate-mediated killing in vitro Human primary T cells were isolated from donor PBMCs using the EasySep Human T Cell Isolation Kit (Stemcell Technologies Ref.17951) according to the manufacturer's recommendations. The isolated human T cells were then incubated for 24 hours in 200 μL of human medium in 96-well plates (1.5e6 cells / mL). The next day, cells were activated at a concentration of 1.5e6 cells / mL by adding IL-2 (150U / mL), Il-7 (5ng / mL), IL-15 (5ng / mL) and Dynabead Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Gibco Ref:11132D) according to the manufacturer's recommendations (1:1 ratio of beads:cells). After 48 hours of incubation, cells were de-beaded and prepared for electroporation.

[0308] One million activated human T cells were electroporated with 7.5 μg of ABE8e-NG mRNA (TriLink) and 7.5 μg of sg7-E259G (SEQ ID NO: 33) or sg44-I283M+H285R+N286D (ATATCTCATAATTCATGTAC; SEQ ID NO: 64; Synthego) using the P3 Primary Cell 4D-Nucleofector X Kit L (Lonza) according to the manufacturer's recommendations. Electroporated cells were grown in 48-well plates in 1 mL of human medium supplemented with 500 U / mL IL-2 for 5 days, with medium refreshed every 48 hours.

[0309] To test whether base-edited human activated T cells are shielded from antibody-toxin complexes, 5,000 bulk base-edited T cells were incubated for 3 days in 100 μL of human medium supplemented with different concentrations of Rekmab#4-biotin-streptavidin-saporin (1:1 Rekmab#4-biotin:saporin-streptavidin; pre-incubated for 30 minutes at room temperature before adding to the wells). After 3 days of incubation, all cells from each condition were harvested, stained with Rekmab#4-Ax647, Rekmab#1-Ax488, and for viability, and resuspended in 200 uL of FACS buffer for flow cytometry analysis. The entire resuspension volume of each condition was then analyzed using a BD FACSAria III Cell Sorter (BD Biosciences). Live cells were sorted and sent for Sanger sequencing to assess enrichment of base edits, which correlated with increasing concentrations of Refmab#4-biotin-streptavidin-saporin.

[0310] The results are shown in Figure 10. PBS alone and unconjugated saporin (SAP) were used as negative controls. Approximately one-third of the base-edited cells lost binding to Refmab#4 (edited cells). Increasing the concentration of Refmab#4-biotin:saporin-streptavidin increased the depletion of unedited cells. At the highest concentration (1 nM), complete depletion of unedited cells (Refmab#4+ cells) was observed when antibody-toxin was added while edited cells persisted (Figure 10A, Refmab#4 low cells). This was confirmed by Sanger sequencing: increasing the concentration of Refmab#4-biotin:saporin-streptavidin increased the percentage of cells with A4→G4 base editing, resulting in the amino acid change E259G (Figure 10B). Similar results were obtained with sg44 (SEQ ID NO: 64), which yielded I283M+H285R+N286D. Thus, base editing in human T cells can shield cells from antibody-drug-conjugates resulting in enrichment of edited cells, as demonstrated for two independent examples.

[0311] Example 9: HDR-based gene editing renders human T cells resistant to killing by toxin-conjugated Refcab#1 Human T cells were isolated from PBMCs (peripheral blood mononuclear cells) by negative selection using EasySep™ Human T Cell Isolation Kit (Stemcell Technologies; Catalog No. 17951). Cells were left at 37°C for 12 hours and then activated with Dynabeads Human-T cells Activator CD23 / CD28 (Thermo Fisher; Catalog No. 111.31D) at a 1:1 ratio supplemented with IL2, IL7 and IL15 for 2 days. Activated T cells were then electroporated with 60 pmol of Cas9 conjugated to 120 pmol of sgRNA using a Nucleofector 4D unit (Lonza) in P3 buffer and pulsed EH115. PGA was added to the RNP at a ratio of gRNA:PGA:Cas9=1:0.8:1. For knock-in, the RNP mixture was supplemented with 50 pmol of homology-directed repair (HDR) template. Four days after electroporation, cells were screened by FACS for knock-in efficiency analysis. Cells were stained with anti-CD4 (OKT4), anti-CD8 (RTPA-8), anti-CD45 (Refmab#3) and anti-CD45 (Refmab#1).

[0312] The mutant used in this experiment is F331del, a CD45 mutant lacking a phenylalanine residue at position 331. Electroporation of cells with RNP alone resulted in knockout of CD45 as indicated by loss of binding of Rekmab #1 and #3 (Figure 11). Cells transfected with the HDR template encoding the point mutation lost binding to Rekmab #1 but remained reactive to Rekmab #3.

[0313] gRNA used in this experiment to edit CD45: CTTACCACACTGAAATCTGT (SEQ ID NO: 51) The HDRTs used for human T cell engineering are shown in Table 14: [Table 14]

[0314] Then, 5000 engineered and sorted human T cells were distributed in 96-well plates in 100ul of medium supplemented with 50U / ml IL2. Biotinylated Rekmab#1 was conjugated to streptavidin-conjugated ZAP in 1:1 ratio in PBS. Cells were incubated with 50nM Rekmab#1-ZAP mixture at 37°C for 3 days. At the end of incubation, 100ul of CellTiter Glow (Promega Catalog Number: G9241) was added to each well. Luminescence was read with an integration time of 1 second.

[0315] The results are shown in Figure 12. Wild type cells exhibited low luminescence, indicating killing by the antibody drug conjugate. In contrast, CD45 knockout cells, as well as cells expressing the CD45 mutant, are protected from killing.

[0316] Similarly, CD34+ HSPCs were engineered to express F331del. The cells were then incubated with the antibody Optimus Prime-tesirine (see Example 18). Unedited cells were depleted and CD45 KO and CD45 F331del cells were protected.

[0317] Example 10: Knockout of CD45 in cells and re-expression of CD45 mutants in cell lines K562 cells (ATCC CCL-243) were electroporated with RNP targeting CD45 using a Nucleofector 4D unit (Lonza). The gRNA was the same as that used in Example 9 (SEQ ID NO: 51). Four days after electroporation, cells were selected for CD45KO and isolated via limiting dilution to obtain single clones. Clones were grown and sequenced. Clones with all alleles showing indels in the CD45 gene were selected.

[0318] Cells expanded from selected clones were electroporated with the Neon transfection system (Thermo Fisher). 6.5ug of plasmid encoding a mutant form of CD45 was mixed with 2 million cells. Plasmids encoding the following mutants were used: [Table 15]

[0319] 24 hours after electroporation, cells were stained for FACS with anti-CD45 (Refmab#3) and anti-CD45 (Refmab#1) antibodies. The CD45RO (wild type) form of the protein bound to both antibodies, whereas loss of binding was observed with the CD45 mutant Refmab#1. All mutants retained binding to Refmab#3, demonstrating that the protein was expressed by electroporated cells. The results are shown in Figure 13.

[0320] The same experiment was also performed using Jurkat cells with essentially the same results.

[0321] Example 11: Technique for masking CD45 variants into human CD34+ HSPCs using HDR Human CD34 cells were isolated from G-CSF-mobilized healthy donors using CliniMACS Prodigy (Miltenyi Biotec) according to the manufacturer's recommendations. The isolated human CD34 cells were then pre-stimulated in culture for 2 days (HSC Brew GMP medium supplemented with 100ng / mL rhSCF, rhFlt3L, rhTPO and 60ng / mL rhIL3) and electroporated with CRISPR / Cas9 gene editing reagents (SpyFi Cas9 protein + gRNA = RNP complex, and HDR template) using a CliniMACS Prodigy Electroporator. The HDR template had the following sequence: TTTAAAATGGAAAAATATTGAAACCACTTGcGAcACtCAAaAAcATcACaTAtAGATTTCAGTGTGGTAAGAATATAACATTGACCAGAGAATTTTTTTTTGTGG (SEQ ID NO: 56).

[0322] Electroporated cells were grown for 7 days and analyzed by FACS. Cells were stained with two different CD45 antibodies, one binding to the region of the mutation (Refmab#1) and a second antibody binding to a different region (Refmab#3). Results show the presence of 50% knockout and 5-6% HDR-mediated knock-in cells (identified by loss of binding of the antibody targeting the mutated epitope, but retaining binding of the second antibody). See Figure 14.

[0323] Example 12: Expression of mutants in DF-1 cells DF-1 cells (ATCC number CRL-12203) show no staining upon incubation with an antibody against human CD45. They are therefore suitable cell lines for expressing human wild-type or mutant CD45 variants. DF-1 cells were transfected with constructs containing wild-type CD45 (SEQ ID NO: 1) or mutant CD45 variants.

[0324] DF-1 cells were transfected with selected CD45 mutants (K352E, K352H, N257R or N257T) or wild-type CD45 using Lipofectamine Lipofectamine™ 3000 Transfection Reagent (Thermo Fischer Scientific, Cat. No. L3000008). After 72 hours, transfection efficiency and CD45 expression were analyzed by FACS using antibodies Rekmab#4 directly labeled to AlexaFluor 647 (APC) and Rekmab#1 directly labeled to AlexaFluor 488 (FITC). Rekmab#1 and Rekmab#4 bind to different regions of CD45. Using this antibody combination, it is possible to assess loss of binding of an antibody of interest to a specific mutation of CD45 while measuring retention of binding of a second antibody, indicating that the mutant CD45 is still expressed and structurally folded by the cells.

[0325] The results are shown in Figure 19 (Panel A: wild type, Panel B: K352E, Panel C: K352H, Panel D: N257R, Panel E: N257T). The wild type protein binds to both antibodies, Refmab#1 and Refmab#4, whereas the K352E, K352H mutants are only detected by Refmab#4 and the N257R and N257T mutants are only detected by Refmab#1. This confirms that the mutation of residue K352 leads to loss of binding for Refmab#1 and the mutation of residue N257 leads to loss of binding for Refmab#4.

[0326] The same experiment was repeated and additional experiments were also tested. Mutants K352E, K352H, K352S, K352T, N257D, N257R, N257S, N257T, E353K, E259G, E259Q and E259N were tested. The results are shown in Figures 21 (K352E, K352H, K352S, K352T, N257D, N257R, respectively, panels A-F) and 22 (N257S, N257T, E353K, E259G, E259Q, E259N, respectively, panels A-F).

[0327] Example 13: Expression and purification of CD45 D1-D2 fragments and variants For accurate antibody-protein affinity measurements and structural characterization, the ectodomains of wild type and mutant d1-d2 CD45 were generated. The protein sequence (residues 225-394) is histidine tagged at the C-terminus and contains few N- and C-terminal additional amino acids important for crystal packing (full wt sequence ETGIEGRKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEGTKHHHHHH). Expi293F GnTI cells (Thermo Fisher; #A39240), which lack N-acetylglucosaminyltransferase I (GnTI) activity and therefore lack complex N-glycans, were used for protein expression. After harvesting, proteins were purified using Ni-NTA chromatography, followed by digestion of high mannose glycans with endoglycosidase H (EndoHf (New England BioLabs, P0703S)) overnight at 37 °C. EndoHf was removed from the protein solution containing amylose resin, and CD45 protein was further purified by size-exclusion chromatography in buffer 150 mM NaCl, 20 mM Hepes, pH 7.4. Peak monomer (c7-c10) and dimer fractions (if required) were concentrated using 10 kDa cut-off Amicon centrifugal filters, and protein aliquots were flash frozen in liquid nitrogen before storage at -150 °C. Monomeric CD45 D1-D2 wild-type protein was produced. Mutant CD45 proteins are produced using the same experimental procedure. The % monomer content of each protein was obtained from size-exclusion chromatograms (fractions c5-c11). Figure 15 shows representative chromatograms of size-exclusion chromatography (panel A) and SDS-PAGE (panel B) of purified non-glycosylated wt protein.

[0328] Example 14: Binding of CD45 variants to Refcam Analysis of binding to the selected variants was performed with antibodies Refmab#1, Refmab#2 and Refmab#4. Binding of antibodies to CD45 wild type and variants was measured at 25°C with shaking at 1,000 rpm using 1x dynamic buffer (Sartorius, PN:18-1105) in Octet systems RED96e or R8. Selected variants were screened for their ability to bind to Refmab#1 and Refmab#2 using different concentrations of CD45 (wild type or variants). Antibodies were captured by anti-human Fc capture biosensor (AHC) (Sartorius, PN:18-5060) at 0.5 to 1ug / mL for 300 seconds. As analytes, human CD45 wt and variants containing only domains 1, 2 (CD45 D1-2) were titrated at 3 to 7 different concentrations (2000nM to 1nM). Association of analyte to antibody was monitored for 300 or 600 seconds, and dissociation of analyte from antibody was monitored for 900 or 1800 seconds. Reference subtraction was performed against buffer-only wells. AHC chips were regenerated using 10 mM Gly-HCl pH 1.7. Data were analyzed using Octet Data Analysis software HT 12.0. Data were fitted to a 1:1 binding model. The kinetic rate k a and k d was fitted globally or a steady-state analysis was performed.

[0329] To analyze binding to Refmab#4, streptavidin (SA) biosensors (Sartorius, PN:18-5020) were first coated with CaptureSelect™ biotin anti-LC-κ (mouse) conjugate (Thermo Scientific, PN:7103152100) at 1ug / mL for 600 seconds. Refmab#4 was then captured by the coated SA biosensor at 0.5-1.0ug / mL for 300 seconds. Analyte titration, association and dissociation were performed similarly to Refmab#1 and #2.

[0330] The results are shown in Table 16 and Figure 20. The % binding of Refmab was calculated by dividing the nm shift of the hCD45 variant by hCD45 wt. The nm shift used for this calculation was relative to the highest hCD45 concentration (500 nM) at the end of the association (300 seconds). Marked with an asterisk are the % binding results calculated by dividing the nm shift of the hCD45 variant at 50 nM (instead of 500 nM) by the nm shift of hCD45 wt at 50 nM (instead of 500 nM). ND stands for not determined. NA stands for not analyzed. Mutations at positions 230, 257, 259 and 267 did not substantially affect binding to Rekmab#1, but no binding was observed (up to analyte concentration of 500nM) for K352D, K352E, K352H, K352I, K352L, K352N, K352T, E353K, E353R and double mutant N351D K352E. No binding was observed when positions T330 and F331 were deleted. Lower Rekmab#1 binding was observed for K352S and F331A.

[0331] The single point mutations E230K, E259N, H285R, N286D and the triple mutation I283M H285R N286D reduced binding to Rekmab#4, whereas no binding to Rekmab#4 was observed with hCD45 D1-2 mutants (N257D, N257E, N257K, N257R, N257G, N257T, E259G, E259Q, E259V, N267S and the double mutant H285R N286D) up to analyte concentrations of 500 nM.

[0332] Low Refmab#2 binding was observed with N257D and N257T, and no Refmab#2 binding was observed with N257E, N257K, N257R, and N257G (up to analyte concentrations of 500 nM). [Table 16]

[0333] Example 15: Characterization of CD45 mutants by nanoDSF The thermal stability of CD45 D1-D2 mutants was analyzed by differential scanning fluorimetry and monitoring tryptophan fluorescence using a Nanotemper Prometheus NT.48 NanoDSF (NanoTemper Technologies). Tryptophan fluorescence was measured using CD45 D1-D2 wild type and mutants at 0.25-1.0 mg / mL in 150 mM NaCl, 20 mM Hepes pH 7.4, increasing the temperature from 20 °C to 95 °C. Melting temperatures were determined as the inflection point of the sigmoidal curve and compared to CD45 D1-2 wt. The results are shown in Figure 17 and Figure 18.

[0334] Example 16: Functional Assays Dephosphorylation of target proteins by CD45 can be tested by the skilled artisan by any commonly used assay such as AlphaLISA immunoassay (Perkin Elmer). Cells expressing CD45 wild type or isoforms are activated with anti-CD3 antibodies at different time points (5-20 min) before cell lysis. Phosphorylation of Lck at position Tyr505 or total Lck is detected and read out by a plate reader.

[0335] Dephosphorylation of Lck at position Tyr505 by CD45 or total Lck was measured using AlphaLISA assay. Cells, e.g. Jurkat cells (wild type or its mutants), are harvested and pre-incubated with HBSS medium for 2 hours at 37°C. Assay plates (96-well plates) are coated with 10ug / mL of anti-CD3 antibody in 100uL of medium per well for 2 hours at 37°C or overnight at 4°C. Assay plates are washed twice with sterile PBS and cells are seeded at a concentration of 10.000-50.000 cells / well in 80uL of HBSS medium. The rest of the assay was performed according to the assay kit protocol by the manufacturer (PerkinElmer) including all necessary reagents (PerkinElmer AlphaLISA #ALSU-PLCK-A-HV and #ALSU-TLCK-A-HC). After 5-30 min, add 20 uL of 5x Lysis Buffer to the wells and incubate for 10 min at 350 rpm on a plate shaker. Transfer 5 uL of lysate to a 384 white well plate and add 5 uL of Acceptor Mix (according to manufacturer's protocol) to the wells, seal with foil, incubate for 2 min on a plate shaker, and incubate at room temperature for at least 1 h. Add 5 uL of Donor Mix (according to manufacturer's protocol) to the wells and mix the foil sealed plate for 2 min on a plate shaker and incubate at room temperature for at least 1 h. The plate is then read on an Envision plate reader (Perkin Elmer) to detect total and phosphorylated Lck.

[0336] The results of an assay measuring phosphorylation of Lck at position Tyr505 via AlphaLISA assay are shown in Figure 16. 10.000-50.000 Jurkat wild type or Jurkat CD45 knockout cells were incubated for 20 min in plates coated with anti-CD3 antibodies before cell lysis and detection of phosphorylated Lck. Activation of Jurkat cells with anti-CD3 antibodies leads to CD45 activation which in turn dephosphorylates Lck. Jurkat CD45 knockout cells are unable to dephosphorylate Lck upon activation. The figure shows the acceptor signal (counts) and is representative of one biological experiment with two technical replicates.

[0337] Example 17: Internalization of antibodies into human cells Antibody internalization can be tested by those skilled in the art by any commonly used assay, such as FACS. Cells expressing CD45 are incubated for different time points (1-24 hours) with an antibody labeled with a fluorophore, e.g., Alexa Fluor 488 (AF488), followed by washing and quenching with anti-AF488 antibody for 1 hour. The internalized antibody can give a signal in the FACS reading, whereas the signal of the antibody bound to the cell surface is quenched and cannot be detected.

[0338] Antibody internalization into TF-1 or Jurkat cells expressing CD45 or its variants is measured by FACS. TF-1 cells are seeded at a concentration of 1 million cells / mL in 0.1 mL of medium (TF1 cells: RPMI1640 supplemented with GlutaMAX+10% heat-inactivated FBS+2 ng / mL GM-CSF; Jurkat cells: RPMI ATCC modified+10% heat-inactivated FBS) in 96-well plates. The next day, cells are treated with 2-20 μg / mL of antibodies labeled with AlexaFluor 488 (AF488; Alexa Fluor® 488 Conjugation Kit (Fast)-Lightning-Link®, Abcam) for 1-24 h at 37 °C or 4 °C, after which cells are harvested and washed with ice-cold PBS. Cells are resuspended in ice-cold PBS containing 20-200ug / mL of anti-AF488 antibody (Alexa Fluor 488 polyclonal antibody, ThermoFisher Scientific) for 1 hour, followed by data acquisition on a FACS instrument (NovoCyte, Agilent). The signal of internalized antibody is measured by FACS, and the signal of extracellular antibody is quenched. The percentage of antibody internalization is calculated by dividing the signal of cells incubated with the quencher by the signal of cells incubated without the quencher.

[0339] Example 18: Improved Refmab Antibodies An improved version of the antibody Refmab#1 was generated. The amino acid sequence of an exemplary improved binder, antibody Optimus Prime, is shown in Table 17. [Table 17]

[0340] The antibody Optimus Prime was demonstrated to have a binding specificity identical to that of Refcmab#1.

[0341] Example 19: Masking HSCs by base editing To test the shielding of human HSCs engineered by base editing to express K352E, one million hCD 34+ HSPCs were electroporated 48 hours post-thaw with 7.5 μg of base editor mRNA (Trilink) and 13.6 μg of sgRNA (Synthego) (1:100 BE:sgRNA molar ratio) using the P3 primary cell 4D-Nucleofector X Kit L (Lonza) according to the manufacturer's recommendations with the Lonza CA-137 pulse program. Electroporated CD34+ HSPCs were continued to be cultured at 0.5e6 cells / mL in stem cell medium (StemSpan SFEM II (StemCell #09655) + 1 uL / mL hSCF (Miltenyi #130-096-695) + 1 mL / mL hFlt3-Ligand (Miltenyi #130-096-479) + 1 uL / mL hTPO (Miltenyi #130-095-752) refreshed every 5 days in 6-well flat bottom plates (Corning #3516). Editing was analyzed 5 days after electroporation using flow cytometry.

[0342] K352E can be engineered into CD 34+ HSPCs by base editing using ABE8e-NG mRNA (Trilink) and sgRNA-49 (SEQ ID NO: 33; Synthego). This resulted in <1% Refmab#1 unstained CD 34+ HSPCs (Figure 23A). To increase the editing rate, we attempted to reposition ABE8e to a more favorable position within the base editing window of the protospacer. To this end, we took advantage of the less restrictive PAM recognition ability of the SpRY Cas9 mutant (Science (2020) 368: 190-6). We tiled gRNAs within the region of interest, each shifting by 1 nt. This significantly increased the desired editing activity. Flow cytometry analysis showed that ABE8e-SpRY mRNA combined with sgRNA-49.3 (SEQ ID NO: 62) or sgRNA-49.4 (SEQ ID NO: 63) resulted in a population of Refmab#1 negative cells of 33% and 26%, respectively, without altering Refmab#4 binding (Figure 23B). Similar to ABE8e-NG+sgRNA-49, combining ABE8e-SpRY+sgRNA-49 resulted in less than 1% shielded cells. Sanger sequencing of bulk base-edited CD34+ HSPCs confirmed the improved K352E base editing by sgRNA-49.3 and sgRNA-49.4.

[0343] sgRNA-49 sequence: TGGAATGTGGAAACAATACT (SEQ ID NO: 33; can be targeted by both ABE8e-NG and ABE8e-SpRY) sgRNA-49.3 sequence: GGAATGTGGAAACAATACTA (SEQ ID NO:62; targetable only by ABE8e-SpRY) sgRNA-49.4 sequence: GAATGTGGAAACAATACTAG (SEQ ID NO:63; targetable only by ABE8e-SpRY)

Claims

1. A human cell or population of human cells expressing a first isoform of CD45 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD45; the cells expressing the first isoform comprise genomic DNA having at least one polymorphism or engineered allele; the polymorphism or engineered allele is absent from the genome of the patient whose cells express the second isoform of CD45; the polymorphism or engineered allele is characterized by at least one substitution of an amino acid at position N257, E259, N267, N286, K352 or E353 of SEQ ID NO: 1; The medical treatment comprises administering to a patient in need thereof a therapeutically effective amount of the cell or population of cells expressing the first isoform of CD45 in combination with a therapeutically effective amount of a depleting agent comprising an antigen-binding region that specifically binds to the second isoform of CD45, to specifically deplete the patient's cells that express the second isoform of CD45.

2. 2. The human cell or population of human cells of claim 1, wherein the first and second isoforms are substantially functionally identical, preferably the first and second isoforms have substantially identical biophysical properties, have substantially identical stability, have substantially identical melting temperatures, have substantially identical aggregation tendencies, and / or have substantially identical tendencies to form dimers.

3. 2. The human cell or population of human cells of claim 1, wherein the first and second isoforms dephosphorylate CD45 target proteins, activate the TCR signaling cascade, result in increased cytokine production, and / or result in increased T cell proliferation.

4. 2. The human cell or population of human cells of claim 1, wherein the first and second isoforms of CD45 dephosphorylate the tyrosine kinase Lck.

5. The polymorphic or engineered allele is characterized by at least one substitution of an amino acid at position K352 or E353 of SEQ ID NO: 1; 2. The human cell or population of human cells of claim 1.

6. The polymorphism or genetically engineered allele is characterized by an amino acid substitution at position K352 of SEQ ID NO: 1 selected from K352E, K352G, K352H, K352I, K352L, K352N, K352S and K352T, preferably wherein the substitution is K352E, or 6. The human cell or population of human cells of claim 5, wherein the polymorphism or genetically engineered allele is characterized by at least one substitution of an amino acid at position E353 of SEQ ID NO: 1 selected from E353K and E353R.

7. The depletor binds to the same epitope as an antigen-binding region selected from: a) an antigen-binding region comprising an antibody heavy chain variable domain (VH) comprising the three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising the three CDRs, VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8 and VLCDR3 is SEQ ID NO: 9; Preferably, the antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and b) an antigen-binding region comprising an antibody heavy chain variable domain (VH) comprising the three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 4, VHCDR2 is SEQ ID NO: 5 and VHCDR3 is SEQ ID NO: 6; and an antibody light chain variable domain (VL) comprising the three CDRs, VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 60, VLCDR2 is SEQ ID NO: 61 and VLCDR3 is SEQ ID NO: 9; Preferably, the antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 58 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:

59. A human cell or population of human cells according to claim 5.

8. 2. The human cell or population of human cells of claim 1, wherein the polymorphism or genetically engineered allele is characterized by at least one substitution of an amino acid at position N257, E259 or N267 of SEQ ID NO:

1.

9. 9. The human cell or population of human cells of claim 8, wherein the substitution at amino acid position N257 of SEQ ID NO: 1 is an N257E, N257K, N257R or N257T substitution, the substitution at amino acid position N259 of SEQ ID NO: 1 is an N259G substitution, or the substitution at amino acid position N267 of SEQ ID NO: 1 is N267S.

10. The depletor binds to the same epitope as an antigen-binding region comprising: an antibody heavy chain variable domain (VH) comprising the three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 20, VHCDR2 is SEQ ID NO: 21 and VHCDR3 is SEQ ID NO: 22; an antibody light chain variable domain (VL) comprising the three CDRs, VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 24 and VLCDR3 is SEQ ID NO: 25; More preferably, the antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 18 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:

19.

11. 2. The human cell or population of human cells of claim 1, wherein the polymorphism or genetically engineered allele is characterized by a substitution of the amino acid at position N257 of SEQ ID NO:

1.

12. 12. The human cell or population of human cells of claim 11, wherein the substitution of the amino acid at position N257 of SEQ ID NO: 1 is an N257E, N257K, N257R, N257G or N257D substitution.

13. The depletor binds to the same epitope as an antigen-binding region comprising: an antibody heavy chain variable domain (VH) comprising the three CDRs, VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 12, VHCDR2 is SEQ ID NO: 13 and VHCDR3 is SEQ ID NO: 14; an antibody light chain variable domain (VL) comprising the three CDRs, VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 15, VLCDR2 is SEQ ID NO: 16 and VLCDR3 is SEQ ID NO: 17; More preferably, the antigen-binding region comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 10 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:

11.

14. 2. The human cell or population of human cells of claim 1, wherein the first isoform of CD45 is obtained by modifying a nucleic acid sequence encoding the first isoform of CD45 ex vivo by gene editing, preferably by introducing into the human cell a gene editing enzyme capable of inducing site-specific mutations in a target sequence encoding a surface protein region involved in drug binding comprising at least a first antigen-binding region.

15. A pharmaceutical composition comprising human cells, preferably hematopoietic stem cells or immune cells, such as T cells as defined in any one of claims 1 to 14, a depletion agent as defined in any one of claims 1 to 14, and a pharmaceutically acceptable carrier.

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