Discernible cell surface protein variants of cd52 for use in cell therapy

Abstract

The present disclosure relates to the use of cells having discernible surface protein with engineered or naturally occurring mutation(s) but functional surface protein for use in therapy. The present invention also relates to the use of cells having discernible CD52 surface protein variants but functional surface protein for use in therapy, in particular adoptive cell therapy.

Description

[0001] DISCERNIBLE CELL SURFACE PROTEIN VARIANTS OF CD52 FOR

[0002] USE IN CELL THERAPY

[0003] FIELD OF THE INVENTION

[0004] The present disclosure relates to the use of cells having discernible surface protein with engineered or naturally occurring mutation(s) but functional surface protein for use in therapy. The present invention also relates to the use of cells having discernible CD52 surface protein variants but functional surface protein for use in therapy, in particular adoptive cell therapy.

[0005] BACKGROUND OF THE INVENTION

[0006] Cell based immunotherapy is emerging as the third pillar of medicine after small molecule therapy and treatments based on biologies such as recombinant proteins including antibodies. Cellular therapy can be used in oncology for treating hematopoietic malignant diseases, but also other applications such as the treatment of genetic diseases, solid organ tumors and autoimmune diseases are under development.

[0007] CD52-directed CAR T cells

[0008] Targeted therapies, which include cell-based therapies such as CAR cells (e.g. CART-cells, CAR NK cells or CAR macrophages), eliminate all cells expressing the target molecule. In those cases where the target is also expressed on the CAR T cells, this will result in fratricide, the CAR-T-mediated eradication of CAR-Ts.

[0009] More specifically, CAR T cells can be directed against CD52 and used to eliminate oncogenic cells in CD52-positive lymphoproliferative disorders, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), T-cell large granular lymphocyte leukemia (T-cell LGL), T-cell prolymphocytic leukemia (T-PLL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma (SMZL), high-grade B-cell lymphomas (HGBCL), non-Hodgkin lymphomas (NHL), angioimmunoblastic T-cell lymphomas (AITL), peripheral T-cell lymphomas (PTCL), hepatosplenic T cell lymphomas (HSTCL), T-prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphomas (ATLL), cutaneous T cell lymphomas (CTCL) and diffuse large B-cell lymphomas (DLBCL). More specifically, increased CD52 expression was shown in patients in remission or after relapse.

[0010] Such CAR T cells targeting CD52 can be generated based on alemtuzumab or other anti- CD52 antibodies, e.g., gatralimab.

[0011] Allogeneic CAR T cells ('off the shelf)

[0012] Prior to the administration of allogeneic CAR T cells, it is important to deplete the host's lymphocytes to prevent allograft rejection of the donor CAR T cells. This lymphodepletion is often performed using the anti-CD52 antibody alemtuzumab, which is marketed under the brand names Campath or Lemtrada. Currently, allogeneic CAR T cells are generated with a genetic knock-out of CD52 (Kim et al. 2018. Cell. 173:1439-53). However, this also abolishes the modulatory function of this receptor, which might result in undesired side effects.

[0013] Reduced intensity and targeted conditioning

[0014] In other cases, mild, targeted conditioning before a hematopoietic stem cell transplant is performed through a combination of specific agents, which are known to generate a niche for the transplanted CD34+ donor cells to engraft. Especially reduced intensity and targeted conditioning regimens can contain anti-CD52 antibody Campath / alemtuzumab (reviewed in Griffin et al. (2022) J Hematology & Oncology).

[0015] All these applications require that the lymphodepleting antibody such as alemtuzumab cannot bind to and therefore deplete, (1) the anti-CD52 CAR T cells or (2) the allogeneic 'off the shelf' CAR T cells or (3) lymphocytes originating from the donor-derived stem cells, during the targeting conditioning regimen, which includes alemtuzumab or another anti- CD52 antibody.

[0016] Targeted gene knock-out can be used to make cells resistant to a depleting agent such as an antibody or a CAR T cell, to prevent side effects or to allow more efficacious therapy. For example, CD33 CAR T cell resistant hematopoietic cells are being engineered by knocking out the entire CD33 gene (Kim et al. 2018. Cell. 173:1439-53), allowing for the normal hematopoiesis to regenerate during CD33-CAR T cell-mediated depletion.

[0017] In a similar way, CD52 is knocked out in allogeneic CAR T cells to withstand lymphodepletion by alemtuzumab (e.g., reviewed in Caldwell KJ et al., 2021, Front. Immunol.). CD52 is an inhibitory ligand of immune cells. Accordingly, it was shown that CD52 negative or CD52low T-cells exhibit elevated killing, auto-reactivity and increased cytokine release (Umeda et al., 2018, Clinical Immunology. Kinsella et al. 2021, Transplantation and Cellular Therapy). Therefore, the knock-out of CD52 on CAR T cells could result in undesired site effects like increased cytokine release (in worst case cytokine storm) or T cell exhaustion.

[0018] Recently it was shown that SIGLEC-10 mediates an anti-phagocytic, "don't eat me," signal capable of protecting cells from attack by Siglec-10-expressing macrophages (Barkal AA., 2019, Nature). In 2013 it was reported that SIGLEC-10 is an interaction partner of CD52 (Bandala-Sanchez E et al., 2013, Nat Immunol.), however, the same group published later that this might not be a general principle to explain the CD52 functional activity (Rashidi M et al., 2018, Cell Death Differ). If CD52 is indeed a ligand of SIGLEC-10 the CD52 - SIGLEC-10 axis might also be of relevance in blocking phagocytosis. A genetic knock-out of CD52 might therefore make lymphoid cells susceptible to phagocytosis and thus reduce their in vivo persistence.

[0019] The inventors in previous patent applications showed that a single amino acid difference in surface protein variants can be genetically engineered into hematopoietic cells to change the antigenicity and be discriminated by specific and selective antibodies (WO2017 / 186718, W02018 / 083071). Contrary to the approach where a surface protein is removed (KO cells), the surface protein variants in these cells retain their normal expression and function and enable to target surface proteins with important non- redundant functions.

[0020] CD52, also known as CAMPATH-1 antigen, CDw52 or HE5, is a membrane-associated protein expressed on most cells of the lymphoid lineage, such as T, B and NK cells but also on monocytes and macrophages. Beyond that, CD52 is also a major antigen on male reproductive tissue, including mature sperm cells (Koyama, Komori et al., 2009). Mature CD52 is a very small glycoprotein with a sequence of only 12 amino acids that is processed from a 61 amino acid precursor. Mature CD52 is heavily glycosylated at several residues and is linked at its C-terminus to a GPI membrane anchor, connecting it to the cell membrane. The mature peptide can be released from the cell surface by, e.g., phospholipase C cleavage and thus soluble CD52 is found in serum, enabling its use as a diagnostic marker for CD52-positive tumors (Albitar, Keating et al., 2004).

[0021] The function of CD52 is not fully understood, but several datasets show that CD52 exhibits mainly an inhibitory function on T, B cells, monocytes, macrophages and dendritic cells (Rashidi, Harrison et al., 2018). Accordingly, it was shown that CD52 will interact with the inhibitory sialic acid-binding immunoglobulin-like lectin 10 (SigleclO) to inhibit phosphorylation of the T cell receptor-associated kinases as well as T cell proliferation and activation (Bandala-Sanchez et al., 2013. Nature Immunol). B cells lacking surface CD52 expression are hyperresponsive to B cell receptor signaling, suggesting that CD52 has also an inhibitory role on B cells (Bhamidipati et al., 2021. Front. Immunol.). Indirect evidence from autoimmune patients and patients suffering from graft-versus-host- disease (GvHD) indicates also an inhibitory function of CD52, thus preventing autoreactivity and maybe suppressing GvHD (Kinsella FA et al., 2021, Transplantation and Cellular Therapy; Sato T. et al, 2016, Abstract 2883, 2016 ACR / ARHP Annual Meeting). Very recently, the macrophage-expressed CD24 (and potentially CD52) ligand SIGLEC-10 was reported to present a don't eat me signal, preventing the phagocytosis of CD24 positive cells, such as cancer cells (Barkal AA., 2019, Nature). In a similar way, CD52 could inhibit the phagocytosis of lymphocytes. The present disclosure aimed to identify amino acid residues of CD52 that are located in the mature 12 amino acid peptide exposed at the cell surface and that can be substituted in a manner such that a) the function of CD52 is not, or not substantially, altered, i.e. the variant of CD52 is functionally indistinguishable from the wild type version of CD52, and b) a moiety, such as an antibody or a CAR T cell, can bind to the wild type version of CD52, but shows a substantially decreased or no binding to the altered version of CD52, i.e. the variant of CD52 is immunologically distinguishable from the wild type version of CD52. Most single amino acid substitution in any given target protein will only affect the binding of a moiety, if the amino acid substitution is part of, or is close to, the epitope of the binding moiety. As also will be appreciated, single amino acid substitutions that do affect binding of a binding moiety to a target antigen can also impact the functionality of the target antigen. It is therefore a highly sophisticated and unpredictable task to identify those amino acid substitutions that fulfill both requirements that do affect binding a moiety to a target antigen and which at the same time do not, or not substantially, affect its function.

[0022] The present disclosure also aimed to identify amino acid residues of CD52 that are located in the mature 12 amino acid peptide and exposed on the cell surface and that can be substituted in a manner such that a) the expression of CD52 is not, or at least not substantially, altered, i.e. the level of CD52 expression is indistinguishable or substantially indistinguishable from the wild type version of CD52, and b) a moiety, such as an antibody or a CAR T cell, can bind to the wild type version of CD52, but shows a substantially decreased or no binding to the altered version of CD52, i.e. the variant of CD52 is immunologically distinguishable from the wild type version of CD52.

[0023] Several anti-CD52 moieties are known in the art. CD52 is the target of alemtuzumab (Campath, Lemtrada), a humanized IgGl for the treatment of chronic lymphocytic leukemia (CLL) and multiple sclerosis, which was developed by Bayer and is marketed by Genzyme / Sanofi. The activity of alemtuzumab seems to be mainly mediated by antibodydependent cellular cytotoxicity and complement-dependent cytotoxicity and depletes CD52highcells, mainly T and B cells. CD52 is also the target of gatralimab (GZ402668), a humanized IgGl developed by Genzyme / Sanofi. Its development was discontinued in 2018.

[0024] SUMMARY OF THE INVENTION

[0025] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52.

[0026] In certain embodiments, said first and second isoform are substantially functionally identical, are expressed at the same or substantially the same level, bind to SIGLEC-10 with the same of substantially the same affinity, suppress cells expressing SIGLEC-10 to the same orsubstantially the same degree, are phosphorylated upon binding of sialic acids to the same or substantially the same degree, decrease phosphorylation of kinases in immune cells to the same or substantially the same degree, inhibit phagocytosis to the same or substantially the same degree and / or have the same or substantially the same N- and O-glycosylation pattern.

[0027] In certain embodiments, said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent comprising at least a first antigen-binding region that binds specifically to said second isoform of CD52 to specifically deplete patient cells expressing said second isoform of CD52. Preferably, said medical treatment restores normal hematopoiesis after immunotherapy in the treatment of hematopoietic disease. Also preferably, said medical treatment is the treatment of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic syndrome (MDS), T-cell large granular lymphocyte leukemia (T-cell LGL), T-cell prolymphocytic leukemia (T-PLL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma (SMZL), high-grade B-cell lymphomas (HGBCL), non-Hodgkin lymphomas (NHL), angioimmunoblastic T-cell lymphomas (AITL), peripheral T-cell lymphomas (PTCL), hepatosplenic T cell lymphomas (HSTCL), T-prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphomas (ATLL), cutaneous T cell lymphomas (CTCL) and diffuse large B-cell lymphomas (DLBCL). Preferably, said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of CD52 to avoid eventual severe side effects such as graft-versus-host disease due to the transplantation.

[0028] In certain embodiments, said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof in combination with a therapeutically efficient amount of a depleting agent comprising at least a second antigen-binding region that binds specifically to said first isoform of CD52 to specifically deplete transferred cells expressing said first isoform of CD52. Preferably said medical treatment is the use in adoptive cell transfer therapy.

[0029] In certain embodiments, said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid at position Q31, T32, S33, S34, P35 or S36 of SEQ ID NO: 1.

[0030] In certain embodiments, said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10. In certain embodiments, said substitution is at amino acid position S33, S34 or P35 of SEQ ID NO: 1. In certain embodiments, amino acid S33 is substituted with A, C, D, E, F, H, I, K, P, R, V, or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Ch R, T, V, or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y. In certain embodiments, amino acid S33 is substituted with A, N, G or L, amino acid S34 is substituted with T, N, A, G or L, and / or amino acid P35 is substituted with T, S, N, A, G or L. In certain embodiments, the amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

[0031] In certain embodiments, said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18. In certain embodiments, said substitution is at amino acid position Q31, T32, S33, S34, P35 or S36 of SEQ ID NO: 1. In certain embodiments, amino acid Q31 is substituted with V, L or T, wherein amino acid T32 is substituted with I or P, wherein amino acid S33 is substituted with A, C, D, E, F, H, I, K, L, N, P, R, T, V, W or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y. In certain embodiments, amino acid S33 is substituted with N, T or L, amino acid S34 is substituted with G, L, or N, and / or amino acid P35 is substituted with A, G, L or S. In certain embodiments, amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

[0032] In certain embodiments, said depleting agent is an antibody, antibody-drug conjugate or an immune cell, preferably a T-cell bearing a chimeric antigen receptor (CAR) comprising a first antigen-binding region which binds specifically to said second isoform and does not bind or binds substantially weaker to said first isoform. In certain embodiments, said first isoform of CD52 is obtained by in vivo or ex vivo modifying the nucleic acid sequence encoding said first isoform of CD52 by gene editing, preferably by introducing into a cell a gene editing enzyme capable of inducing sitespecific mutations(s) within a target sequence encoding a surface protein region involved in the binding of agent comprising at least a first antigen-binding region.

[0033] In certain embodiments, said cell or population of cells is an immune cell, preferably a T-cell, bearing a chimeric antigen receptor (CAR).

[0034] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising aforementioned mammalian cells, preferably a hematopoietic stem cell or an immune cell such as T-cell.

[0035] In certain embodiments, the present disclosure relates to a depleting agent for use in selectively depleting the host cells in a patient in need thereof wherein said patient's native cells express a second isoform of CD52 and wherein said depleting agent comprises at least a first antigen-binding region which binds specifically to said second isoform of CD52, wherein said first antigen-binding region of said depleting binds specifically to an epitope including the amino acids S33, S34 and / or P35 of SEQ ID NO: 1, and wherein said first antigen-binding region comprises an antigen binding region, which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10.

[0036] In certain embodiments, the present disclosure relates to a depleting agent for use in selectively depleting the host cells in a patient in need thereof wherein said patient's native cells express a second isoform of CD52 and wherein said depleting agent comprises at least a first antigen-binding region which binds specifically to said second isoform of CD52, wherein said first antigen-binding region of said depleting binds specifically to an epitope including the amino acids Q31, T32, S33, S34 and / or P35 of SEQ ID NO: 1, and wherein said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18.

[0037] One of the objectives of the present disclosure is to develop a method to allow lymphodepletion, e.g., by alemtuzumab for allogeneic CAR T cells administration, reduced intensity / targeted conditioning using alemtuzumab, and provided CAR T cells directed against CD52, which are not subject to fratricide. The inventors thus sought variations of the surface-associated protein CD52, which are immunologically distinguishable while retaining or substantially retaining normal function and / or expression, and where amino acid changes originate from a single or multiple amino acid or nucleotide variations. In particular, the inventors identified variants of CD52, which change the antigenicity of CD52 to a specific antibody while retaining its normal expression and function, such as its inhibitory activity on B and T cells. This was achieved via a sophisticated campaign involving a screening project, a rational design approach and a comparison to naturally occurring polymorphisms.

[0038] The present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of CD52 and preferably wherein said first and second isoform are functional. Alternatively, said first isoform is generated via RNA editing.

[0039] The present disclosure also relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of CD52 and preferably wherein said first and second isoforms are expressed at the same level or substantially the same level.

[0040] In a particular embodiment, the present disclosure relates to the mammalian cell or population of cells, preferably CAR T cells wherein said medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent comprising at least a first antigenbinding region that binds specifically to said second isoform of CD52 to specifically deplete patient cells expressing said second isoform of CD52, preferably to enable efficient lymphodepletion to prevent rejection while not affecting the allogeneic CAR T cells expressing said first isoform of CD52.

[0041] In other embodiments the medical treatment relates to the mammalian cell or population of cells, preferably CAR T cells wherein said medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 as well as a chimeric antigen receptor directed against CD52 to said patient in need thereof to deplete patient cells expressing said second isoform of CD52, preferably to deplete CD52-positive cells in lymphoproliferative disorders, e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), T-cell large granular lymphocyte leukemia (T-cell LGL), T-cell prolymphocytic leukemia (T-PLL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma (SMZL), high-grade B-cell lymphomas (HGBCL), non-Hodgkin lymphomas (NHL), angioimmunoblastic T-cell lymphomas (AITL), peripheral T-cell lymphomas (PTCL), hepatosplenic T cell lymphomas (HSTCL), T-prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphomas (ATLL), cutaneous T cell lymphomas (CTCL) and diffuse large B-cell lymphomas (DLBCL).

[0042] In other embodiments, the present disclosure relates to the mammalian cell or population of cells, preferably hematopoietic stem cells for use in a medical treatment in a patient in need thereof wherein said medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent comprising at least a first antigen-binding region that binds specifically to said second isoform of CD52 to specifically deplete patient cells expressing said second isoform of CD52, preferably to more rapidly restore normal hematopoiesis after lymphodepletion in combination with mild / targeted conditioning in the treatment of hematopoietic disease.

[0043] In other embodiments the medical treatment relates to the restoration of the normal function in genetic diseases that are not originating in the hematopoietic and immune system but that can be treated by use of modified hematopoietic cells.

[0044] In another particular embodiment, the present disclosure relates to the mammalian cell or population of cells for use in a medical treatment in a patient in need thereof, wherein said medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform to said patient in need thereof in combination with a therapeutically efficient amount of a depleting agent comprising at least a second antigen-binding region that binds specifically to said first isoform to specifically deplete transferred cells expressing first isoform, preferably for use in adoptive cell transfer therapy, more preferably for the treatment of malignant hematopoietic disease acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), T-cell large granular lymphocyte leukemia (T- cell LGL), T-cell prolymphocytic leukemia (T-PLL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma (SMZL), high-grade B-cell lymphomas (HGBCL), non-Hodgkin lymphomas (NHL), angioimmunoblastic T-cell lymphomas (AITL), peripheral T-cell lymphomas (PTCL), hepatosplenic T cell lymphomas (HSTCL), T-prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphomas (ATLL), cutaneous T cell lymphomas (CTCL) and diffuse large B-cell lymphomas (DLBCL) and other lymphoproliferative neoplasms, again more preferably wherein said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of surface protein to avoid eventual severe side effects such as graft-versus-host disease due to the transplantation.

[0045] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a mammalian cell, preferably a hematopoietic stem cell or an immune cell such as a T cell, or a CAR T cell as described above and preferably a depleting agent and a pharmaceutically acceptable carrier.

[0046] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52. In certain embodiments said depleting agents binds substantially weaker to said second isoform of CD52.

[0047] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform are substantially functionally identical. In certain embodiments said depleting agent binds substantially weaker to said second isoform of CD52. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform are expressed at substantially the same level. In certain embodiments said depleting agent binds substantially weaker to said second isoform of CD52.

[0048] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform act as a inhibitory molecules on B and T cells.

[0049] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform lead to essentially the same modulation B and T cell function.

[0050] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform lead to the normal differentiation of hematopoietic cells. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform carry the GPI anchor at position S36.

[0051] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, and wherein said first and second isoform are N- and O-glycosylated.

[0052] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, wherein said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid in position Q31, T32, S33, S34, P35 and / or S36 of SEQ ID NO: 1, preferably an amino acid at position S33, S34 and / or P35 of SEQ ID NO: 1. In certain embodiments said depleting agents bind substantially weaker to said second isoform of CD52.

[0053] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, wherein said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid in position Q31, T32, S33, S34, P35 and / or S36. Among these substitutions, substitutions S33, S34 and P35 are particularly preferred. In certain embodiments said depleting agents binds substantially weaker to said second isoform of CD52.

[0054] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52, wherein said patient's native cells express a second isoform of CD52, and wherein said depleting agent comprises at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind to said second isoform of CD52, wherein residue Q31 is substituted with C, D, E, F, H, I, K, L, T, V, W or Y, residue T32 is substituted with I or P, residue S33 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, or Y, preferably A, G, L, N, or T, residue S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, preferably A, G. L, N or T, P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y, preferably A, G, L, N, S or T, and / or residue S36 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y.

[0055] The present disclosure also relates to a method for improving engraftment of hematopoietic stem cell transplants. Conditioning (depletion of HSCs) prior to hematopoietic stem cell transplantation (HSCT) is used to promote engraftment. Indeed, conditioning efficacy is associated with improved engraftment. Avoiding toxic conditioning is an important goal that can be achieved with the present disclosure. Current methods for conditioning involve the use of intravenous busulfan. Busulfan is a DNA alkylating drug originally designed to treat hematologic diseases, such as acute myeloid leukemia (AML). However, busulfan carries the risk of significant side effects, including sterility, primary or secondary malignancy, and additional acute and chronic toxicities.

[0056] The present disclosure also relates to a method to prevent fratricide of CAR T cells directed against CD52 in lymphoproliferative disorders.

[0057] The present disclosure also relates to a method to generate allogeneic CAR T-cells expressing functional CD52 and allowing CD52-directed lymphodepletion. The present disclosure also relates to a method to prevent complexation of soluble CD52 with alemtuzumab, increasing the fraction of free effective alemtuzumab and potentially overall efficacy of this lymphodepleting antibody.

[0058] FIGURE LEGENDS

[0059] Figure 1 shows the binding of full-length anti-CD52 Refmab #1 and Refmab #3 antibody in Mab format to HEK293-T cells transfected with human wildtype CD52 (SEQ ID No. 1) or with an empty vector. Both Refmabs bind to human wildtype CD52 in a concentration dependent manner. Cell transfected with the empty vector did not show significant binding to anti-human CD52 antibodies.

[0060] Figure 2 shows the binding of anti-CD52 alemtuzumab Fab fragments to HEK293-T cells transfected with human wildtype CD52 (SEQ ID No. 1) or with an empty vector. The alemtuzumab Fab fragment binds to human CD52 in a concentration dependent manner. Cells transfected with the empty vector did not show any binding to alemtuzumab.

[0061] Figures 3A-3F show the comprehensive substitution scan analyzing the binding of anti- CD52 antibodies RefMabtl and RefMab#2 to synthetic peptides representing the human mature 12-mer CD52 with a C-terminal extension of two amino acids (alanine-aspartic acid), where each amino acid was exchanged for all other 19 natural amino acids.

[0062] Figure 4 shows an alignment of the amino acid sequence of human mature CD52 with the corresponding residues from monkey CD52. Grey: Potential N-linked glycosylation sites. Black: Serine residue GPI anchor.

[0063] Figure 5 shows binding of RefmAbftl and RefMab#2 to processed, GPI+ and purified wildtype and variant CD52 expressed in and released from Expi293 PGAP2 knockout cells as determined by biolayer interferometry. Figure 6: A) shows flow cytometry assessment of the binding of MBP-CD52-WT or an MBP- CD52-variant (S34G / S34N / T32I) on HEK293 stable cell lines at 3 days post-transfection to 4 pg / mL of AF647-conjugated RefMabtl (left Y axis) or 4 pg / mL of anti-MBPtag antibody (right Y axis). B) shows flow cytometry assessment of the binding of MBP-CD52-WT or an MBP-CD52-variant (S34G / S34N / T32I) on HEK293 stable cell lines at 21 days posttransfection to increasing concentrations (0.01 - 100 pg / mL) of AF647-conjugated RefMabtl (left Y axis).

[0064] Figure 7 shows antibody-mediated FcgammaRllla activation of Jurkat / FcyRllla / NFAT-Luc CD52 KO cells upon co-cultivation with RefMabffl or REF001 and HEK293 cells overexpressing MBP-CD52 WT, S34G, S34N or T32I as determined by luminescence intensity (Y-axis; RLU).

[0065] Figure 8 shows the base editing efficiencies for variant CD52 S34G, S34G and CD52 KO as measured via next-generation sequencing in Jeko-1 cells. The intended edits represent only reads which were correctly edited for the respective variant or knock-out. Sequencing reads containing either amplification or sequencing errors or additionally to the intended edit any bystander edits are summarized as unintended edit.

[0066] Figure 9 A) shows flow cytometry assessment of Refmabtl binding to JEKO-1 cells expressing wildtype CD52 or CD52 shielding variants. CD52 shielding variants(S34G / S34N) and knock-outs were generated as described in Example 19. Cells were stained with 4 ug / mL AF647-conjugated RefMabtfl (left Y axis) or 4 ug / mL of anti-CD52 4C8 antibody (control antibody) (right Y axis). The results demonstrate that both shielding variants loose Refmabtl binding while expression of the variants is maintained. However, the S34G variant shows lower expression levels compared to wildtype CD52. B) shows flow cytometry assessment of the binding of WT CD52 or a CD52 variant (S34G / S34N) expressed on base edited JEKO-1 cells, or the lack of binding of CD52 KO edited JEKO-1 cells, to increasing concentrations (0.01 - 100 pg / mL) of AF647-conjugated RefMabtfl (left Y axis). Figure 10 shows antibody-mediated FcgammaRllla activation of Jurkat / FcyRHIa / NFAT-Liic CD52 KO cells upon co-cultivation with RefMabtl or REF001 and base edited JEKO-1 cells expressing CD52 WT, S34G, S34N or not expressing CD52 (KO) as determined by luminescence intensity (Y-axis; RLU). CD52 variant cells and CD52 knock-out cells caused no FcgammaRllla activation above background (effector cells only).

[0067] Figures 11 show the fitness and gene editing efficiency of T cells from 3 different donors after 2 days of expansion and activation post-thawing, then electroporation (EP) and subsequent culture for 5-6 additional days. (A) Cell viability is >80% at 2 days post-EP (D4) and >90% 5-6 days post-EP (D7-8). (B) The percentage of CD4 and CD8 T cells after thawing (Donorl=30%, Donor2=50%, Donor3=20%) changes during the process with generally an increase of CD4 T cells to approx. 50% at D7-8. (C) Proliferation rate varies depending on the donor but confirms that T cells are still able to proliferate post-editing between D2 to D7 or D8. (D) Percentage of shielding of CD52 evaluated as the loss of binding of Refmabtl antibody by flow cytometry at D4 and D7-8 is similar for the 3 donors, with >90% shielding for S34G variant while S34N variant and KO show approx. 70% shielding at D7-8. (E) The results obtained by flow cytometry are confirmed by genomic analysis by Sanger and NGS sequencing. (F) Bystander analysis with EditR confirms the high specificity of the sgRNA used to edit CD52 with only 2% bystander edit in position A12forS34G variant (ABE editor; on-target is A4) and 3.5% bystander edit in position C9 for S34N variant (CBE editor; on- target is C6).

[0068] Figures 12 show the fitness and gene editing efficiency of HSC cells from 1 donor after 2 days of pre-stimulation post-thawing, then electroporation and subsequent culture for 5 additional days. (A) Cell viability is approx. 80% immediately post-EP (D2), then increases above 90% 5 days post-EP (D7). (B) The percentage of LT-HSC for this donor is 30% after thawing and decrease to approx. 10% at D4 for all conditions. (C) The results obtained by genomic analysis by Sanger and NGS sequencing are similar: 30-40% editing at D4 for S34N variant and up to 60% for S34G variant. (D) Bystander analysis with EditR confirms the high specificity of the sgRNA used to edit CD52 with only 0.5% bystander edit in position A12

[0069] 19

[0070] RECTIFIED SHEET (RULE 91) ISA / EP for S34G variant (ABE editor; on-target is A4) and 1.0% bystander edit in position C9 for S34N variant (CBE editor; on-target is C6).

[0071] Figure 13 A) shows flow cytometry assessment of the binding of WT CD52 or a CD52 variant (S34G / S34N) expressed on base edited human T cells, or the lack of binding of CD52 KO edited T cells, to 4 pg / mL AF647-conjugated RefMabtl (left Y axis) or 4 pg / mL of anti-CD52 4C8 antibody (right Y axis). B) shows flow cytometry assessment of the binding of WT CD52 or a CD52 variant (S34G / S34N) expressed on base edited T cells, or the lack of binding of CD52 KO edited T cells, to increasing concentrations (0.01 - 50 pg / mL) of AF647-conjugated RefMabtl (left Y axis).

[0072] Figure 14 shows A) (left) Quantification of the percentage of phRodo+ cells within the human macrophage (CDllb-i- cells) population, (right) Median fluorescence intensity of phRodo dye in the macrophage (CDllb+) population. Both measurements were obtained by flow cytometry analysis after 2 hours of incubation with CD52 WT, KO, S34G or S34N edited T cells previously stained with phRodo dye. B) Quantification of phRodo-i- counts per image by Incucyte analysis after 2 hours incubation of human macrophages with CD52 WT, KO, S34G or S34N edited T cells previously stained with phRodo dye.

[0073] Figure 15 shows glycosylation assessment of GPI- and purified_CD52 wildtype and variants by weak anion exchange chromatography. The relative area under the peaks at given retention times is depicted and compared to the wildtype protein.

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] Immunotherapy is a promising therapy to treat cancer, genetic and autoimmune diseases. Immunodepleting agent such as antibodies or engineered immune cells directed to tumor antigen are administered into a patient to target and kill tumor cells. However, as tumor surface proteins are also expressed at the surface of normal cells including hematopoietic cells, this strategy can induce severe side effects to the patients, e.g., by altering hematopoiesis. To restore hematopoiesis in the patient, hematopoietic cells can be subsequently transplanted into the patient. However, the binding of the depleting agent not only to the diseased cells but also to the newly transplanted healthy cells can limit the maximal tolerated dose or limit the use to treatment before transplantation of healthy cells. Alternatively, transplanted cells need to be resistant to said immunodepleting agent in order not to be targeted and eliminated by it. One approach is therefore to select cells resistant to said immunodepleting agent used in immunotherapy while retaining their function to restore normal hematopoiesis in the patient or to provide a normal T cell compartment.

[0076] In another approach, depleting agents are used to eradicate lymphoid cells before administering allogeneic cell therapy such as CAR T cells to prevent rejection of the engineered cells. Binding of the depleting agent to the host lymphocytes as well as the cell therapy would result in depletion of the engineered cells in addition to the host cells. Another approach is therefore to generate allogeneic cells for therapy resistant to said immunodepleting agent used in immunotherapy while retaining their function to retain full functionality of the cell therapy in the patient.

[0077] The inventors develop a method to identify functional allelic variants in the genetic sequence encoding the surface protein region responsible for the binding of a specific 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. Said first isoform of a surface protein encoded by a nucleic acid with said polymorphism is not recognized by a specific depleting agent. This variant allele particularly does not alter or does not substantially alter the function of the surface protein and / or is expressed at the same or substantially the same level. Thus, said depleting agent can be used to bind specifically to the one isoform and not, or not substantially, the other isoform thereby depleting specifically cells expressing one isoform. For example, if the depleting agent binds specifically to the second isoform, but not the first isoform, said depleting agent will specifically deplete cells expressing said second isoform. In another embodiment, said first isoform can be recognized by a second agent and thus this second agent can be used to deplete specifically cells expressing the first isoform, but not second isoform. The cells expressing the first isoform of the surface protein encoded by at least one variant allele is advantageously used in medical treatment in a patient having cells expressing a second isoform, in particularfor depleting specifically transplanted or patient cells by using a second or first agent respectively.

[0078] It is impossible to predict, which mutation in a surface antigen can be used in such an approach. First, the mutations need to lie on a surface exposed stretch of the surface antigen that is accessible for the depleting agent. Second, the depleting agent needs to bind to this stretch on the exposed area of the surface antigen. Third, binding needs to be affected sufficiently enough so that the depleting agent can discriminate the first isoform from the second isoform. Residual binding to the other isoform should be minimal or, better, be completely absent. Fourth, the mutation should not affect, or only marginally affect, the function of the surface antigen. The mutated isoform should fulfill its biological function at least to an extent that is tolerable in a given therapeutic setting. Although certain tools exist to predict three-dimensional protein structure and post-translational modifications, only experimental testing can prove the usefulness of any given mutation.

[0079] Depleting agent

[0080] The present disclosure relates to an agent comprising an antigen binding region which binds specifically to one isoform of CD52 on a cell and does not bind or binds substantially weaker to another isoform of CD52. Such agent is referred to herein as "depleting agent". Both isoforms of CD52 are functional, i.e., CD52 is functional with respect to at least one relevant property. Preferably both isoforms of CD52 have that same function, i.e., they are functionally indistinguishable, or both isoforms are expressed at the same level or substantially the same level.

[0081] The two isoforms of CD52 differ however with respect to binding to the depleting agent. The depleting agent only binds specifically to one of the isoforms of CD52. The isoforms can therefore be described as functionally identical (or functionally substantially identical), but immunologically distinguishable. The first isoform and the second isoform of CD52 may be polymorphic alleles. Preferably, the first isoform and the second isoform of CD52 are naturally occurring polymorphic alleles. Also preferably, the first isoform and the second isoform of CD52 are single nucleotide polymorphism (SNP) alleles.

[0082] The first isoform and the second isoform of CD52 may also be genetically engineered alleles. Preferably the first isoform and the second isoform of CD52 differ by one, two, three, four or five amino acids. Most preferably the first isoform and the second isoform of CD52 differ by one amino acid.

[0083] Various methods can be used to determine the mutation that is to be introduced into CD52 to generate the second isoform. For example, mutations can be randomly inserted, followed by the functional and immunological screening of the variants generated. Alternatively, mutations can be rationally designed, for example by analysis of the secondary or tertiary protein structure of CD52.

[0084] The depleting agent comprises an antigen binding region, which binds specifically to one isoform of CD52 on a cell and does not bind or binds substantially weaker to another isoform. The depleting agent of the present disclosure can be divided into two main categories.

[0085] First, the depleting agent can be a polypeptide comprising an antigen binding region. Said polypeptide may consist of one or more polypeptide chains. Preferably said polypeptide comprising an antigen binding region is an antibody. Said polypeptide comprising an antigen binding region may 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, ankyrins, centyrins, domain antibodies, lipocalins, small modular immuno- pharmaceuticals, maxybodies, Protein A and affilins.

[0086] Said depleting agent can also be coupled to chemical drug, such as a cytotoxic payload or a proteolysis targeting chimera (PROTAC). Said polypeptide comprising an antigen binding region may also be a bispecific, biparatopic or multispecific antibody. Such molecules may also contain additional functional domains. For example, said polypeptide comprising an antigen binding region may be a T cell engager, for example a BiTE. Said polypeptide comprising an antigen binding region may also be fused to a cytokine or a chemokine, a toxin or to the extracellular domain of a cell surface receptor.

[0087] Alternatively, the depleting agent can be a cell comprising an antigen binding region. For example, the depleting agent can be a chimeric antigen receptor (CAR). In certain embodiments of the present disclosure, said cell comprising an antigen binding region are CAR T-cells, CAR NK cells, CAR monocytes or CAR macrophages. In a preferred embodiment of the present disclosure said cell comprising an antigen binding region is a CAR T-cell. In another preferred embodiment of the present disclosure said cell comprising an antigen binding region is a primary T cell comprising a CAR.

[0088] The depleting agent binds specifically to one isoform of CD52, but not the second isoform and thus specifically depletes cells expressing one isoform.

[0089] In certain embodiments, the present disclosure relates to an agent comprising a first antigen binding region which binds specifically to a second isoform of CD52 and does not bind a first isoform. In other embodiments, the present disclosure also relates to an agent comprising a second antigen binding region which binds specifically to the first isoform of CD52 and does not bind a second isoform. In certain embodiments said agents binds substantially weaker to said second isoform of CD52.

[0090] The first and the second isoform of CD52 may differ from each other by only one amino acid substitution. Said one amino acid difference between the first and the second isoform may also be the result of the presence of a single nucleotide polymorphism, such as a naturally occurring single nucleotide polymorphism. The first and the second isoform of CD52 may also differ from each other by more than one amino acid, such as by two, by three or by more than three amino acids. The first and the second isoform of CD52 may also differ from each other in that one of the isoforms has an insertion of one, of two, of three or of more than three amino acids compared to the other isoform. The first and the second isoform of CD52 may also differ from each other in that one of the isoforms has a deletion of one, of two, of three or of more than three amino acids compared to the other isoform. The two isoforms may also differ from each other by combinations of amino acid substitutions, insertions and / or deletions. In a preferred embodiment, said depleting agent is an antibody or an antigen-binding fragment. If the two isoforms of CD52 differ by more than one amino acid, then the amino acids changed may be adjacent to each other, i.e., direct neighboring amino acids, or they may be separated.

[0091] 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 an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies.

[0092] In natural antibodies of rodents and primates, 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 (A) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. In typical IgG antibodies, the light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes four domains, a variable domain (VH) and three constant domains (CHI, CH2 and CH3, collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR).

[0093] The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain variable region. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FR) can participate in the antibody binding site or influence the overall domain structure and hence the combining site. Complementarity Determining Regions or CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L- CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDRs set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. Accordingly, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3- CDR3-FR4.

[0094] The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in 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 in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system. In specific embodiments, an antibody provided herein is an antibody fragment, and more particularly any protein including an antigen-binding domain of an antibody as disclosed herein. The antigen-binding domain may also be integrated 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, diabodies, single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, IgNAR, bis-scFv, camelid antibodies, ankyrins, centyrins, domain antibodies, lipocalins, small modular immunopharmaceuticals, maxybodies, Protein A and affilins.

[0095] As used herein, an "antigen binding region" or "antigen-binding fragment of an antibody" means a part of an antibody, i.e. a molecule corresponding to a portion of the structure of the antibody, that exhibits antigen-binding capacity for a specific antigen, possibly in its native form; such fragment especially exhibits the same or substantially the same antigen-binding specificity for said antigen compared to the antigen-binding specificity of the corresponding four-chain antibody. The antigen-binding capacity can be determined by measuring the affinity between the antibody and the target fragment. This antigen-binding region may also be designated as "functional fragments" of antibodies.

[0096] The agents of the disclosure comprise antibodies and fragments thereof but also comprise artificial proteins with the capacity to bind antigens mimicking that of antibodies, also termed herein antigen-binding antibody mimetic. Antigen-binding antibody mimetics are organic compounds that specifically bind antigens, but that are not structurally related to antibodies. They are usually artificial peptides or small proteins with a molar mass of about 3 to 20 kDa.

[0097] The phrases "an antigen binding region recognizing an antigen" and "an antigen binding region having specificity for an antigen" are used interchangeably herein with the term "an antigen binding region which binds specifically to an antigen". As used herein, the term "specificity" refers to the ability of an agent comprising an antigen binding region such as an antibody to detectably bind an epitope presented on an antigen. "Specific binding" or "specifically bind to" includes binding with a monovalent affinity of about 10'8M (KD) or stronger. Preferably, binding is considered specific when the binding affinity is between 10'8M (KD) and 10'12M (KD), optionally between 10'8M (KD) and 1010M (KD), in particular at least 10’8M (KD). The affinity can be determined by various methods well known by the one 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 tested readily by, inter alia, comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than the target protein.

[0098] As used herein, the term "epitope" means the part of an antigen to which the antibody or antigen binding region thereof binds. The epitopes of protein antigens can be divided into two categories, conformational epitope and linear epitope. A conformational epitope corresponds to discontinuous sections of the antigen's amino acid sequence. A linear epitope corresponds to a continuous sequence of amino acids from the antigen.

[0099] In another aspect, it is further disclosed herein bispecific or multispecific molecules, such as bispecific antibodies or multispecific antibodies. For example, an antibody can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., another antibody or ligand for a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibody may in fact be derivatized or linked to more than one other functional molecule to generate multispecific molecules that bind to more than two different binding sites and / or target molecules; such multi-specific molecules are also intended to be encompassed by the terms "bispecific molecule", "bispecific antibody", "biparatopic molecule", "biparatopic antibody", "multispecific molecule" and "multispecific antibody" as used herein. To create a bispecific molecule, an antibody of the disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, disulfide bonds, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, cytokine, chemokine, toxin, PROTAC or a receptor extracellular domain, such that a bispecific molecule results. Specific bispecific and multispecific molecules contemplated by the present disclosure are T cell engagers, such as bispecific T cell engager, for example a BiTE.

[0100] As used herein, an agent which does not bind or binds substantially weaker to a particular isoform of CD52 includes an agent which is not able to bind to cells expressing said particular isoform. For experimental testing said agent may be labelled with a fluorescent marker or may be detected with a secondary antibody directed against said agent, and the percentage of cells presenting said fluorescent marker or said secondary antibody is determined by FACS analysis. Typically testing is done in cell lines expressing the recombinant target protein, i.e. CD52. The target protein may be expressed in its entirety. Alternatively, a truncated version may be used, wherein said truncated version at a minimum need to include the extra cellular domain or the regions of the extracellular domain containing the respective antibody epitope. To monitor the expression of the variant isoforms, cells may be stained with two agents simultaneously, one binding the epitope where variants were introduced and a second one that binds an epitope that is different from the one bound by the first agent, or alternatively an introduced extracellular terminal tag. The second epitope or tag remains unaltered and thus this staining serves as an expression control. As a non-binding control, cells are used that do not express the protein of interest or express only low endogenous levels of the protein of interest. As a reference binding control, cells that normally do not express the protein of interest or express only low endogenous levels of the protein of interest are transfected with the wildtype isoform. Different cell lines have different expression levels, but the expression is controlled through endogenous control elements such as promoters. Such cell lines can also be used to study the mode-of-action of a depleting agent, the effective shielding against a different mode-of-action, to test cytotoxicity and shielding / resistance from cytotoxicity or to test the function of the engineered receptors. Western Blot, ELISA or FACS can be used to analyze phosphorylation of signaling molecules. Proliferation assays can be used to assess the normal function of signaling molecules. Analysis of gene expression changes can serve to analyze gene expression compared to normal function. Cells can also be used to demonstrate the feasibility of editing a specific variant via different approaches, e.g., homology directed repair (H DR), base editing or prime editing.

[0101] Binding of said agent can result in depletion of the cell expressing the first isoform of CD52. Various mechanisms can lead to cell depletion. Antibody dependent cellular cytotoxicity (ADCC) results from binding of the agent to a target protein and activation of NK and other immune cells through the Fc part on the agent bound by an FcR expressed, e.g., by NK cells. The Fc part of an immunoglobulin refers to the C-terminal region of an immunoglobulin heavy chain. The Fc part can be wildtype or engineered. Mutations of enhanced, engineered Fc parts are known in the art. For certain therapeutic situations, it is desirable to reduce or abolish the normal binding of the wild-type Fc region of an antibody, such as of a wild-type IgG Fc region to one or more or all of Fc receptors and / or binding to a complement component, such as Cl q to reduce or abolish the ability of the antibody to induce effector function. For instance, it may be desirable to reduce or abolish the binding of the Fc region of an antibody to one or more or all the Fey receptors, such as: FcyRI, FcyRlla, FcyRllb, FcyRllla. Effector function can include, but is not limited to, one or more of the following: complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, 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 inducing apoptosis, crosslinking of target-bound antibodies, dendritic cell maturation, orT cell priming. Binding of said agent may also lead to the blocking of binding of the natural receptor ligand and thereby result in cell death and apoptosis without cell-mediated depletion.

[0102] A reduced or abolished binding of an Fc region to an Fc receptor and / or to Clq is typically achieved by mutating a wild-type Fc region, such as of an IgGl Fc region, more particular a human IgGl Fc region, resulting in a variant or engineered Fc region of said wild-type Fc region, e.g., a variant human IgGl Fc region. Substitutions that result in reduced binding can be useful. For reducing or abolishing the binding properties of an Fc region to an Fc receptor, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties and / or charges, are preferred.

[0103] In certain embodiments of the present disclosure the Fc region of the antibody is of the IgGl isotype, carrying mutations, i.e. the constant region carries a L234A, a L235A and a P329G mutation or PG-LALA mutations, i.e. the constant region carries a L234A, a L235A and a P329A mutation or PA-LALA mutations, or i.e. the constant region carries a L234A, a L235E, G237A, A330S and a P331S mutation or AEASS mutations. The skilled person will be aware of possibilities to engineer the Fc region to obtain a desired effect.

[0104] Surrogate ADCC assays constitute an industry standard to quantitate an agent's potency to mediate ADCC as described in the experimental part. Engineered Jurkat reporter cells carry an NFAT-responsive luciferase gene and an Fc receptor, such as human FcgRI, FcgRIla or FcgRI I la. Binding ofthe Fc receptorto bound antibody results in NFAT induction through receptor clustering and therefore a luciferase signal. Absence of binding and therefore clustering does not result in a luciferase signal. Cells expressing either no target protein (e.g. HEK293, CHO or chicken DF-1 cells) or human lymphoid cancer cells such as Raji, JEKO-1, or GDM1 cells engineered to be CD52-deficient (e.g. a CD52 knock-out), or expressing the wildtype protein (e.g. HEK293-CD52 or Raji, JEKO-1, or GDM1) or expressing individual variants (e.g. CD52 variants) were incubated with the test agent (e.g. antibody RefMabtfl or RefMab#2) and mixed with the ADCC reporter cells. Then luciferase was measured to quantify the ADCC signal. The luciferase luminescence signals were normalized to the maximal signal observed in HEK-CD52 or the corresponding myeloid or T cell cancer cell line. ADCC was measured with an ADCC Reporter Assay (Promega, Cat. No. G7015).

[0105] Complement-dependent cytotoxicity (CDC) assays are used to quantitate an agent's potency to mediate CDC as described in the experimental part. Cells expressing either no target protein (e.g., HEK293 or chicken DF-1 cells) or human lymphoid cancer cells such as Raji, JeKo-1, or GDM1 cells engineered to be CD52 -deficient (e.g. CD52 knock-out cells), or expressing the wildtype protein (e.g., DF1-CD52, or Raji, JeKo-1, or GDM1 cell lines) or expressing individual variants (e.g. CD52 variants) were incubated with the test agent (e.g. antibody RefMabffl or RefMab#2) and mixed with human or rabbit complement sera. After incubation the live cells are quantified by using, e.g., a luminescent dye measuring the relative number of dead cells in the cell suspension. Alternatively, the live cells can be quantified in the suspension.

[0106] The depleting agent according to the present disclosure binds specifically to one isoform of CD52 and allows the depletion of cells expressing said isoform.

[0107] More preferably, in specific embodiments, said depleting agent according to the present disclosure does not bind or binds substantially weaker to a first isoform of CD52 but binds specifically to a second isoform of CD52 and allows the depletion of said cells expressing said second isoform of CD52, in particular in methods of use as disclosed herein. In particular, said depleting agent which does not bind or binds substantially weaker to a first isoform of CD52 but binds specifically to a second isoform of CD52 expressed in patient's cell is used to deplete patient's cells but not hematopoietic stem cells or their progeny or T cells or CAR T cells expressing said first isoform of CD52 transplanted to restore hematopoiesis in said patient.

[0108] In another specific embodiments, said depleting agent according to the present disclosure does not bind or binds substantially weaker to a second isoform of CD52 but binds specifically to a first isoform of CD52 and allows the depletion of cells expressing said first isoform of CD52, in particular in methods of use as disclosed herein. In particular, said depleting agent which does not bind or binds substantially weaker to a second isoform of CD52 but binds specifically to a first isoform of CD52 expressed in transplanted cells is used to deplete specifically transplanted cells to avoid eventual severe side effects such as graft-versus-host disease due to transplantation.

[0109] Selective depletion of cells expressing a specific isoform of CD52 can be achieved without limitation by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).

[0110] In certain embodiments, the antigen binding region is coupled to an effector compound such as a drug or a 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, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, maytansinoids, calicheamicins, indolinobenzodiazepines, pyrrolobenzodiazepines, pyrridinobenzodiazepines, camptothecins, topotecan, irinotecan, belotecan, deruxtecan, alpha-amanitin, microcystins, auristatins and puromycin and analogs or homologs thereof.

[0111] In another particular embodiment, said depleting agent is an immune cell harboring an antigen receptor such as a chimeric antigen receptor (CAR). See for example Myburgh et al. Leukemia (2020) 34: 2688-703. Said immune cell may express a recombinant antigen binding region, also named antigen receptor on its cell surface. By "recombinant" is meant an antigen binding region which is not encoded by the cell in its native state, i.e., it is heterologous, non-endogenous. Expression of the recombinant antigen binding region can thus be seen to introduce a new antigen specificity to the immune cell, causing the cell to recognise and bind a previously unrecognised antigen. The antigen receptor may be isolated from any useful source. In certain embodiments of the present disclosure said cell comprising an antigen binding region is a CAR T-cell, a CAR NK cell, CAR Treg, CAR monocyte or a CAR macrophage. In a preferred embodiment of the present disclosure said cell comprising an antigen binding region is a CAR T-cell. In another preferred embodiment of the present disclosure said cell comprising an antigen binding region is a primary T cell comprising a CAR.

[0112] In a particular embodiment, said recombinant antigen receptor is a chimeric antigen receptor (CAR). CARs are fusion proteins comprising an antigen-binding region, typically derived from an antibody, linked to the signaling domain of the TCR complex. CARs can be used to direct immune cells such T-cells or NK cells against a target antigen, if a suitable antigen-binding region is selected. The antigen-binding region of a CAR is typically based on a scFv (single chain variable fragment) derived from an antibody. In addition to an N-terminal, extracellular antibodybinding region, CARs typically may comprise a hinge domain, which functions as a spacer to extend the antigen-binding region away from the plasma membrane of the immune effector cell on which it is expressed, a transmembrane (TM) domain, an intracellular signaling domain (e.g. the signaling domain from the zeta chain of the CD3 molecule (CD3<) of the TCR complex, or an equivalent) and optionally one or more co- stimulatory domains which may assist in signaling or functionality of the cell expressing the CAR. Signaling domains from co-stimulatory 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.

[0113] The skilled person is able to select an appropriate antigen binding region as described above with which to redirect an immune cell to be used according to the disclosure. In a particular embodiment, the immune cell for use in the method of the disclosure is a redirected T-cell, e.g., a redirected CD8+ T-cell or a redirected CD4+ T-cell, or a redirected NK cell.

[0114] Methods by which immune cells can be genetically modified to express a recombinant antigen binding region are well known in the art. A nucleic acid molecule encoding the antigen receptor may be introduced into the cell in the form of, e.g., a vector, or any other suitable nucleic acid construct, or by inserting the nucleic acid molecule into the genome using genome editing technologies. Vectors, and their required components, are well known in the art. Nucleic acid molecules encoding antigen binding region can be generated using any method known in the art, e.g., molecular cloning using PCR. Antigen binding region sequences can be modified using commonly used methods, such as site- directed mutagenesis.

[0115] CD52 CD52 (UniProt: P31358 also known as CAMPATH-1 antigen, CDw52, Cambridge pathology 1 antigen, epididymal secretory protein E5, human epididymis-specific protein 5 or He5), is an extracellularglycopeptide attached to the cell surface by a GPI anchor. The processed, mature protein consists of only 12 amino acids and is heavily glycosylated, with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O- glycosylation serine / threonine sites. It is present on the cell surface of the vast majority of lymphoid cells and many other hematopoietic cells but not on stem cells, with the highest expression on T lymphocytes. CD52 is a potential ligand for the lectin SIGLEC-10.

[0116] As CD52 seems to be an inhibitory ligand for lymphocytes, high expression of CD52 is a marker of antigen-activated T cells. These were shown to release soluble CD52, which might then bind to the inhibitory receptor Siglec-10. The CD52-SIGLEC-10 interaction is a possible suppressor mechanism involved in T cell homeostasis and the immune suppression mediated by CD52 seems to be driven by the glycan moieties, especially sialylated glycans.

[0117] Human immature CD52 has the following amino acid sequence (SEQ ID No. 1):

[0118] MKRFLFLLLTI SLLVMVQIQTGLSGQNDTSQTSSPSASSNI SGGIFLFFVANAI IHLFCF S

[0119] Processed, mature human CD52 consists of amino acids 25-36 of the full-length protein and has the following amino acid sequence (SEQ ID No. 2):

[0120] GQNDTSQTSSPS

[0121] In a particular embodiment, said surface protein is CD52. In other embodiments said surface protein is CD52 comprising the amino acid sequence of SEQ ID No. 1. In other embodiments said surface protein is CD52 consisting of the amino acid sequence of SEQ ID No. 1. In other embodiments said surface protein is CD52 comprising the amino acid sequence of SEQ ID No. 2. In other embodiments said surface protein is CD52 consisting of the amino acid sequence of SEQ ID No. 2. In certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein and preferably wherein said first and second isoform are functional.

[0122] In certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein, wherein said first and second isoform are functional.

[0123] In certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein, wherein said first and second isoform are substantially functionally identical.

[0124] Knowledge about the exact function of CD52 is still limited, although certain activities of CD52 have been described. In certain embodiments the present disclosure related to a first and a second isoform of CD52 wherein both isoforms are functional. In certain embodiments the present disclosure related to a first and a second isoform of CD52 wherein both isoforms are functionally indistinguishable. In the present invention "functionally indistinguishable" refers to a first and a second isoform of CD52 that are equally capable of performing the same function within a cell without significant impairment. In other words, the first and the second isoform are functionally largely indistinguishable. A slight functional impairment may be acceptable. In a preferred embodiment, said first isoform of CD52 remains functional and retain the capacity of performing the same function as the corresponding wildtype isoform within a cell without significant impairment.

[0125] CD52 suppresses cells, e.g., lymphocytes. Therefore, in certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein and wherein said first and said second isoform are suppressing the activity of immune cells

[0126] CD52 binding can also trigger a cascade that results in decreased phosphorylation of kinases in immune cells, such as B and T lymphocytes. Therefore, in certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein and wherein binding of said first and said second isoform can decrease phosphorylation of kinases in immune cells. In certain embodiments, said first and said second isoform decrease phosphorylation of BTK, SYK, AKT, LYN or PLC-A2. Ligand binding to macrophage expressed SIGLEC-10 inhibits phagocytosis through presenting a don't eat me signal. Therefore, in certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein and wherein said first and said second isoform are inhibiting phagocytosis.

[0127] Especially the N- and O-linked glycosylation of CD52 is important for the activity of CD52, mediating e.g. its binding to the lectin SIGLEC-10 (Bandala-Sanchez E et al., (2018) PNAS). Accordingly, AM Shathili et al (Front. Immunol. 2019) showed that specific sialoforms are required for the immune suppressive activity of human soluble CD52. Therefore, it will be important to retain N- and O-glycosylation on the CD52 isoforms.

[0128] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform are substantially functionally identical.

[0129] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform are expressed at the same or substantially the same level. In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform are expressed at the same or substantially the same level.

[0130] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform suppress cells expressing SIGLEC-10 to the same or substantially the same degree.

[0131] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform are phosphorylated upon binding of sialic acids to the same or substantially the same degree.

[0132] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform decrease phosphorylation of kinases in immune cells to the same or substantially the same degree. In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform inhibit phagocytosis to the same or substantially the same degree.

[0133] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first and second isoform have the same or substantially the same N- and O-glycosylation pattern.

[0134] In certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the surface protein CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said surface protein and wherein said first and said second isoform are expressed substantially to the same degree. In certain embodiments, said first and said second isoform are expressed to the same degree.

[0135] In line with the present disclosure, it is also possible to combine additional variants or isoforms of CD52 within the methods and compositions of the present disclosure. Such isoforms may for example include double mutants. Such isoforms may for example also include single and double mutants. The methods and compositions of the present disclosure may also be combined with cells carrying a CD52 knock out, e.g., a permanent knock out or a temporarily knock out (e.g. via CRISPRoff). The methods and compositions of the present disclosure may also be used in the depletion of lymphocytes to prevent graft rejection.

[0136] The methods and compositions of the present disclosure may also comprise cells expressing first isoform of CD52 and other surface protein variants such as CD117 variants, CD123 variants, CD33 variants, DLL-1 variants, CD45 variants, CD47 variants, CD7 variants, CLEC12A (CD371) variants, CD44 variants, FLT3 (CD135) variants, CD300LF variants, EVI2B variants, TPO variants, EPOR variants and any combination thereof.

[0137] Polymorphism of CD52

[0138] The cell expressing the first isoform of CD52 according to the present disclosure comprises genomic DNA with at least one polymorphic allele in the nucleic acid encoding said CD52 isoform. In particular, said polymorphism induces at least one mutation involved in the binding of a specific agent in comparison to said second isoform.

[0139] Said polymorphism is preferably within a nucleic acid sequence encoding the surface protein region of CD52 involved in binding of the first agent, preferably located in the extracellular portion of CD52, in particular, in a solvent-exposed secondary structure element. More particularly, said polymorphism is within a nucleic acid sequence encoding at least one specific amino acid residue involved in binding of the first agent. Said polymorphism can be a mutation such as a deletion, a substitution, an insertion, or a combination thereof of at least 1, 2, 3 or 4 nucleotides. In a particular embodiment, said polymorphism is a single nucleotide polymorphism.

[0140] The term "isoform" refers to a variant of a protein, which differs from another variant of the same protein by at least one amino acid difference. In the context of the present disclosure such difference may be a substitution of a single amino acid, but such differences may also be double, triple or multiple amino acid substitutions, or insertions or deletions. Also naturally occurring SNPs are isoforms.

[0141] The difference in the sequence of the two isoforms may also be genetically introduced. Also, here the sequence difference is preferably within a nucleic acid sequence encoding the mature CD52 region involved in binding of the first agent, preferably located in the extracellular portion of said surface protein, in particular, in a solvent-exposed secondary structure element. More particularly, said sequence difference is within a nucleic acid sequence encoding at least one specific amino acid residue involved in binding of the first agent. Said sequence difference can be a mutation such as a deletion, a substitution, an insertion or a combination thereof of at least 1, 2, 3 or 4 nucleotides. In a particular embodiment, said sequence difference is a single point mutation.

[0142] The present disclosure provides polymorphisms in CD52, including in particular polymorphisms including substitution of the residues Q31, T32, S33, S34, P35 or S36. Preferred polymorphisms include substitution of the residues Q31, T32, S33, S34 and / or P35. More preferred polymorphisms include substitution of the residues S33, S34 and / or P35.

[0143] Particular preferred polymorphisms include substitutions of the residue Q31, wherein Q31 is substituted with C, D, E, F, H, I, K, L, T, V, W or Y. Other preferred polymorphisms include substitutions of the residue Q31, wherein Q31 is substituted with L, T, or V. Other preferred polymorphisms include substitutions of the residue T32, wherein T32 is substituted with I or P. Other preferred polymorphisms include substitutions of the residue S33, wherein S33 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y. Other preferred polymorphisms include substitutions of the residue S33, wherein S33 is substituted with A, G, L, or N. Other preferred polymorphisms include substitutions of the residue S33, wherein S33 is substituted with L, N or T. Other preferred polymorphisms include substitutions of the residue S34, wherein S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y. Other preferred polymorphisms include substitutions of the residue S34, wherein S34 is substituted with A, G, L, N or T. Other preferred polymorphisms include substitutions of the residue S34, wherein S34 is substituted with A, G or N. Other preferred polymorphisms include substitutions of the residue S34, wherein S34 is substituted with L. Other preferred polymorphisms include substitutions of the residue P35, wherein P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y. Other preferred polymorphisms include substitutions of the residue P35, wherein P35 is substituted with A, G, L, N, S or T. Other preferred polymorphisms include substitutions of the residue P35, wherein P35 is substituted with L or S. Other preferred polymorphisms include substitutions of the residue P35, wherein P35 is substituted with G or L. Other preferred polymorphisms include substitutions of the residue S36, wherein S36 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y.

[0144] In certain embodiments the present disclosure relates to a variant CD52 polypeptide, wherein said variant CD52 polypeptide comprises at least one mutation in an amino selected from Q31, T32, S33, S34, P35 or S36 of wild type human CD52. In certain embodiments the present disclosure relates to a variant CD52 polypeptide, wherein said variant CD52 polypeptide comprises at least one mutation in an amino selected from N20, Q31, T32, S33, S34 and / or P35 of wild type human CD52.ln certain embodiments the present disclosure relates to a variant CD52 polypeptide, wherein said variant CD52 polypeptide comprises at least one mutation in an amino selected from N20, S33, S34 and / or P35 of wild type human CD52.

[0145] It will be appreciated that amino acid may be designated by the 3-letter code or the 1- letter code, which both are familiar to the skilled person.

[0146] Table 1 shows the 20 natural occurring amino acids:

[0147] Amino acid Three letter code One letter code alanine Ala A arginine Arg R asparagine Asn N aspartic acid Asp D cysteine Cys C glutamic acid Glu E glutamine Gin Q glycine Gly G histidine His H isoleucine lie 1 leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Vai V

[0148] In the experiments of the present disclosure certain variants of specific residues were identified. For practical reasons it is impossible to test any and all possible variants. It will however be understood that an identified variant may be substituted with a similar amino acid residue. For example, an acidic amino acid can be replaced by another acidic amino acid, since it can be expected to have the same effect. Likewise, a charged amino acid can be replaced by another charged amino acid. As an example, S34E is expected to be equivalent to S34D, since both, D and E are acidic amino acids. Natural polymorphism

[0149] In a particular embodiment, said cell according to the present disclosure is selected from a subject comprising native genomic DNA with at least one natural polymorphism allele, preferably single nucleotide polymorphism (SNP) in the nucleic acid encoding said isoform. In a particular embodiment, cells are selected from a subject that comprises native genomic DNA with at least one natural polymorphism allele, in particular SNP, in a nucleic acid sequence encoding CD52 region involved in anti-CD52 agent binding, preferably located in the extracellular portion of said surface protein, more preferably in a solvent- exposed secondary structure element.

[0150] Certain naturally occurring SNPs are described in the literature. These naturally occurring SNPs may be used within the spirit of the present disclosure with a respective binding agent which is able to discriminate such SNP from another isoform of CD52. A list of natural occurring SNPs of CD52 can be found in any respective database, such as gnomAD (https: / / gnomad.broadinstitute.org / ), dbSNP

[0151] (https: / / www.ncbi. nlm.nih.gov / snp / =) or GeneCards (https: / / www.genecards.org / ).

[0152] Gene editing

[0153] In another particular embodiment, said cell expressing the first isoform of CD52 according to the present disclosure is obtained by gene editing, preferably by changing the sequence encoding said surface protein in the patient's native genomic DNA.

[0154] The cell can be genetically engineered by introducing into the cell a gene editing system to induce said polymorphism resulting in insertion, deletion and / or substitution of amino acids of the surface protein. Said gene editing modality targets a nucleic acid sequence, named herein target sequence encoding surface protein region involved in first agent binding as described above. In particular, when said surface protein is CD52, said gene editing modality targets a nucleic acid encoding at least one amino acid residue in position Q31, T32, S33, S34, P35 or S36 of SEQ ID NO: 1. Preferably amino acid residue Q31 is substituted with C, D, E, F, H, I, K, L, T, V, W or Y, and / or residue T32 is substituted with I or P, and / or residue S33 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or residue S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or residue P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W or Y, and / or residue S36 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y. The term "nuclease" refers to a wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of phosphodiester bonds between nucleotides of a nucleic acid (DNA or RNA) molecule, preferably a DNA molecule. By "cleavage" is intended a double-strand break or a single-strand break event.

[0155] The term "sequence-specific nuclease" refers to a nuclease which cleaves nucleic acid in a sequence-specific manner. Different types of site-specific nucleases can be used, such as Meganucleases, TAL-nucleases (TALEN), Zing-finger nucleases (ZFN), or RNA / DNA guided endonucleases like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system 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).

[0156] According to the present disclosure, the nuclease generates a DNA cleavage within a target sequence, said target sequence encodes a surface protein region involved in first agent binding as described above. In particular embodiments, the inventors use the CRISPR system to induce a cleavage within a target sequence encoding surface protein region recognized by first agent as described above.

[0157] By "target sequence", it is intended targeting a part of the sequence encoding the region on CD52 involved in first agent binding as described as described above and / or sequences adjacent to said region on CD52 involved in first agent binding, in particular at least one (one or two) sequence of up to 50 nucleotides adjacent to said region on CD52 involved in first agent binding, preferably 20, 15, 10, 9, 8, 7, 6 or 5 nucleotides adjacent to said agent binding site.

[0158] CRISPR system involves two or more components, Cas protein (CRISPR-associated protein) and a guide RNA. The guide RNA can be a single guide RNA or a dual guide RNA. Cas protein is a DNA endonuclease that uses guide RNA sequence as a guide to recognize and generate double-strand cleavage in DNA that is complementary to the target sequence. Cas systems that generate single strand breaks require only one nuclease domain. Cas systems that generate double strand breaks require two nuclease domains. Cas protein may comprise two active cutting sites, such as HNH nuclease domain and RuvC-like nuclease domain.

[0159] By Cas protein is also meant an engineered endonuclease, homologue or orthologue of Cas 9 which is capable of cleaving target nucleic acid sequence. In particular embodiments, Cas protein may induce a cleavage in the nucleic acid target sequence which can correspond to either a double-stranded break or a single- stranded break. Cas protein variant may be a Cas endonuclease that does not naturally exist in nature and that is obtained by protein engineering or by random mutagenesis. The Cas protein can be one type of the Cas proteins known in the art. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), SaCas9, Casl2, Casl2a (Cpfl), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cmrl , Cmr3, Cmr4, Cmr5, Cnrr6, Csbl , Csb2, Csb3, Csxl7, CsxM, Csx IO, Cs 16, CsaX, Csx3, Cs I, Csxl5, Csfl, Csf2, CsO, Csf4, homologs, orthologs thereof, or modified versions thereof. Preferably Cas protein is Streptococcus pyogenes Cas 9 protein.

[0160] Cas is contacted with a guide RNA (gRNA) designed to comprise a complementary sequence to the target sequence to specifically induce DNA cleavage within said target sequence, in particular according to the present disclosure a complementary sequence of a part of the target sequence encoding the surface protein region recognized by agent as described above.

[0161] As used herein, a "guide RNA", "gRNA", "sgRNA" or "single guide RNA" refers to a nucleic acid that promotes the specific targeting or homing of a gRNA / Cas complex to a target nucleic acid.

[0162] In particular, gRNA refers to RNA that comprises a transactivating crRNA (tracrRNA) and a crRNA. Preferably, said guide RNA corresponds to a crRNA and tracrRNA which can be used separately or fused together to generate a single guide RNA. The complementary sequence pairing with the target sequence recruits Cas to bind and cleave the DNA at the target sequence. According to the present disclosure, crRNA is engineered to comprise a complementary sequence to a part of a target sequence as described above encoding surface protein region recognized by agent, such that it is capable of targeting said region. In a preferred embodiment sgRNA is used to target the binding site of the said binding agent. In another preferred embodiment, the guide RNA contains chemically modifications known to the person skilled in the art.

[0163] In a particular embodiment, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 nucleotides which is complementary to the target sequence. As used herein, the terms "complementary sequence" refers to the sequence part of a polynucleotide (e.g., part of crRNA or tracRNA) that can hybridize to another part of polynucleotides under standard low stringent conditions. Preferentially, the sequences are complementary to each other pursuant to the complementarity between two nucleic acid strands relying on Watson-Crick base pairing between the strands, i.e., the inherent base pairing between adenine and thymine (A-T) nucleotides and guanine and cytosine (G-C) nucleotides. Said gRNA can be designed by any methods known by one of skill in the art in view of the present disclosure.

[0164] According to the present disclosure said target sequence encodes surface protein region on CD52 involved in first agent binding, preferably located in the extracellular portion of mature CD52, more preferably in an extracellular loop in comparison to said second isoform, again more preferably comprising amino acid residues involved in agent binding.

[0165] In a preferred embodiment, when surface protein is CD52, said target sequence encodes a CD52 region involved in binding of a first agent, such as anti-CD52 agent binding as disclosed above. Preferably said target sequence encodes at least one residue in position Q31, T32, S33, S34, P35 and / or S36 of SEQ ID NO: 1.

[0166] The DNA strand break that is introduced by the nuclease according to the disclosure can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) which often results in small insertions and / or deletions or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR). In a preferred embodiment, said polymorphism within nucleic acid encoding the isoform of CD52 is induced via HDR repair following the DNA cleavage and the introduction of an exogeneous nucleotide sequence, named herein HDR template.

[0167] HDR template comprises a first and a second portion of sequence which are homologous to regions 5' and 3' of the target sequence, respectively and a middle sequence portion comprising 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 exogeneous sequence.

[0168] Preferably, homologous sequences of at least 20 bp, preferably more than 30 bp, more preferably more than 50 bp and most preferably less than 200 bp are used. Homologous sequences may be dsDNA or ssDNA. Preferably the homologous sequences are ds DNA. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the exogeneous sequence to be introduced should be located between the two arms. The flanking sequences may be symmetrical or asymmetrical. Both strands of the target nucleic acid, i.e., the plus strand or the minus strand, may be targeted. Optionally, a PAM sequence may be used, which may be silenced to improve HDR.

[0169] In a preferred embodiment, the cell according to the present disclosure is genetically engineered by introducing into said cell said site-specific nuclease which targets the sequence encoding the region on CD52 recognized by said first agent as described above and a HDR template.

[0170] In another particular embodiment, said 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). Base editor or prime editor can be used to introduce mutations at specific sites in the target sequence.

[0171] According to the present disclosure, the base editor or prime editor generates a mutation within the target sequence by sequence-specific targeting of the sequence encoding the region on CD52 involved in first agent binding.

[0172] In particular, said base editor or prime editor are CRISPR base or prime editors. Said CRISPR base or prime editor may comprise as catalytically inactive sequence specific nuclease a dead Cas protein (dCas). It may also comprise Cas9 with a mutated nuclease domain. dCas refers to a modified Cas nuclease which lacks endonucleolytic activity. Nuclease activity can be inhibited or prevented in dCas proteins by one or more mutations and / or one or more deletions in the HNH and / or RuvC-like catalytic domains of the Cas protein. The resulting dCas protein lacks nuclease activity but binds to a guide RNA (gRNA)-DNA complex with high specificity and efficiency to specific target sequence. In particular embodiment, said dCas may be a Cas nickase wherein one catalytic domain of the Cas is inhibited or prevented.

[0173] Said base editor is complexed with a guide RNA (gRNA) designed to comprise a complementary sequence of the target nucleic acid sequence to specifically bind said target sequence as described above.

[0174] Said gRNA can be designed by any methods known by one of skill in the art in view of the present disclosure. In a particular embodiment, said gRNA may target the sequence encoding the region on CD52 recognized by said first agent as described above.

[0175] As non-limiting examples said base editor is a nucleotide deaminase domain fused to a dead Cas protein, in particular Cas nickase. Said nucleotide deaminase may be an adenosine deaminase or cytidine deaminase. Said nucleotide deaminase may be natural or engineered deaminase.

[0176] In a particular embodiment, said base editor may be as non-limiting examples 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, casl2a- 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.

[0177] Said prime editor consists of a fusion of a catalytically inactive sequence specific nuclease as described above, particularly a Cas nickase and a catalytically active engineered reverse transcriptase (RT) enzyme. Said fusion protein is used in combination with a prime editing guide RNA (pegRNA) which contains the complementary sequence to the target sequence as described above, particularly when surface protein is CD52 comprises one of the sequences described herein and also an additional sequence comprising a sequence that binds to the primer binding site region on the DNA. In particular embodiment, said reverse transcriptase enzyme is a Maloney murine leukemia virus RT enzyme and variants thereof. Said prime editor may be as non-limiting examples selected from the group consisting of: PEI, 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.

[0178] Anti-CD52 agents

[0179] Several anti-CD52 moieties are known in the art. Alemtuzumab is approved for treatment of CLL and multiple sclerosis and marketed as Campath or Lemtrada. Gatralimab, also known as GZ-402668 or GLD52) was developed by developed by Sanofi for the treatment of progressive multiple sclerosis but was discontinued after phase 1 clinical trials.

[0180] These and other anti-CD52 moieties may be used in the context of the present disclosure. Anti-CD52 antibody alemtuzumab was generated in the present disclosure, in full length antibody format, with human IgGl isotype as well as half-life reduced human lgGl-H435A. Details are provided in Example 1. Research-grade Gatralimab was acquired from Proteogenix (WPX-TA1589). Alemtuzumab is referred to herein as Refmab #1. Gatralimab is referred to herein as Refmab #2.

[0181] In a particular embodiment, said depleting agent, which binds to said second isoform of CD52 and does not bind or binds substantially weaker to said first isoform of CD52 as described above binds specifically to an epitope including the amino acids Q31, T32, S33, S34, P35 or S36 of SEQ ID NO: 1. In certain preferred embodiments said depleting agents binds specifically to an epitope including the amino acids Q31, T32, S33, S34 or P35 of SEQ ID NO: 1. In certain preferred embodiments said depleting agents binds specifically to an epitope including the amino acids S33, S34 or P35 of SEQ ID NO: 1. In other preferred embodiments said depleting agents binds specifically to an epitope including the amino acids S34 or P35 of SEQ ID NO: 1.

[0182] In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the 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.

[0183] In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region derived from and retaining the binding specificity of an antibody binding comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the 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. In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region competing with an antibody binding comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the 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.

[0184] In another preferred embodiment, said anti-CD52 agent comprises an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the variable heavy chain of SEQ ID NO: 3; and b) an antibody light chain variable domain (VL) comprising variable light chain of SEQ ID NO: 4.

[0185] In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17 and VLCDR3 is SEQ ID NO: 18.

[0186] In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region derived from and retaining the binding specificity of an antibody binding comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17 and VLCDR3 is SEQ ID NO: 18.

[0187] In a preferred embodiment, said anti-CD52 agent comprises an antigen binding region competing with an antibody binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17 and VLCDR3 is SEQ ID NO: 18.

[0188] In another preferred embodiment, said anti-CD52 agent comprises an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the variable heavy chain of SEQ ID NO: 11; and b) an antibody light chain variable domain (VL) comprising variable light chain of SEQ ID NO: 12.

[0189] In another preferred embodiment, said anti-CD52 agent is an antibody selected from RefMabtfl and RefMab#2.

[0190] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34, P35 and / or S36 of SEQ ID NO: 1.

[0191] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34, and / or P35 of SEQ ID NO: 1.

[0192] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34, and / or P35 of SEQ ID NO: 1, and wherein amino acid S33 is substituted with A, C, D, E, F, H, I, K, P, R, V, W or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y.

[0193] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid S33 of SEQ ID NO: 1, and wherein said mutation is elected from S33A, S33N, S33G and S33L.

[0194] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid S34 of SEQ ID NO: 1, and wherein said mutation is elected from S34A, S34N, S34G, S34L and S34T.

[0195] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid P35 of SEQ ID NO: 1, and wherein said mutation is elected from P35A, P35N, P35G, P35L, P35S and PS35T.

[0196] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid P35 of SEQ ID NO: 1, and wherein said mutation is elected from P35A, P35N, P35G, P35L, P35S and PS35T.

[0197] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34, and / or P35 of SEQ ID NO: 1, and wherein said amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

[0198] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in amino acids S34 of SEQ ID NO: 1, and wherein said mutation is S34G or S34N.

[0199] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in amino acid P35 of SEQ ID NO: 1, and wherein said mutation is P35S or P35L.

[0200] In certain preferred embodiments, said depleting agent comprises an antigen binding region derived from and retaining the binding specificity of an antibody binding comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S34 and / or P35 of SEQ ID NO: 1.

[0201] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from Q31, T32, S33, S34, P35 and / or S36 of SEQ ID NO: 1.

[0202] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from Q31, T32, S33, S34 and / or P35 of SEQ ID NO: 1.

[0203] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from Q31, T32, S33, S34 and / or P35 of SEQ ID NO: 1, and wherein amino acid Q31 is substituted with V, L or T, wherein amino acid T32 is substituted with I or P, wherein amino acid S33 is substituted with A, C, D, E, F, H, I, K, L, N, P, R, T, V, W or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y. In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34 and / or P35 of SEQ ID NO: 1, wherein amino acid S33 is substituted with N, T or L, amino acid S34 is substituted with G, L, or N, and / or amino acid P35 is substituted with A, G, L or S.

[0204] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid S33 of SEQ ID NO: 1, wherein said mutation is elected from S33N, S33T and S33L. In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid S34 of SEQ ID NO: 1, wherein said mutation is S34L.

[0205] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation amino acid P35 of SEQ ID NO: 1, wherein said mutation is selected from P35G and P35L.

[0206] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in an amino acid selected from S33, S34 and / or P35 of SEQ ID NO: 1, and wherein said amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

[0207] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in amino acid P35 of SEQ ID NO: 1, and wherein said mutation is P35L.

[0208] In certain preferred embodiments, said depleting agent comprises an antigen binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18, wherein said depleting agent binds to one isoform of CD52 but not, or substantially weaker, to a second isoform of CD52, wherein one of said isoform is wild type CD52 and the other isoform has a mutation in amino acid Q31 of SEQ ID NO: 1, and wherein said mutation is Q31V, Q31L or Q31T.

[0209] It is further contemplated that the antigen-binding region of the anti-CD52 antibody may be further screened or optimized for their binding properties as above defined. In particular, it is contemplated that said antigen binding region thereof may have 1, 2, 3, 4, 5, 6, or more alterations in the amino acid sequence of 1, 2, 3, 4, 5, or 6 CDRs of monoclonal antibodies provided herein. It is contemplated that the amino acid in 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 or heavy variable region of antigen binding region may have an insertion, deletion, or substitution with a conserved or non-conserved amino acid. Such amino acids that can either be substituted or constitute the substitution are disclosed above.

[0210] In some embodiments, the amino acid differences are conservative substitutions, i.e., substitutions of one amino acid with another having similar chemical or physical properties (size, charge or polarity), which substitution generally does not adversely affect the biochemical, biophysical and / or biological properties of the antibody. In particular, the substitution does not disrupt the interaction of the antibody with the CD52 antigen. Said conservative substitution(s) are advantageously chosen within one of 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, nonpolar residues (M, L, I, V, C); and Group 5-large, aromatic residues (F, Y, W).

[0211] In certain embodiments, said first antigen-binding region comprises a heavy chain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences of SEQ ID NO: 3 and a light chain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences of SEQ ID NO: 4.

[0212] In certain embodiments, said first antigen-binding region comprises a heavy chain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences of SEQ ID NO: 11 and a light chain having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequences of SEQ ID NO: 12.

[0213] In certain embodiments, said anti-CD52 agent is an antibody, antibody-drug conjugate or an immune cell, preferably a T-cell bearing a chimeric antigen receptor (CAR) comprising a first antigen-binding region which binds specifically to said second isoform and does not bind or binds substantially weaker to said first isoform.

[0214] In a particular embodiment, said anti-CD52 agent can be a bispecific CD52 antibody, comprising at least one first binding specificity for CD52, for example, one antigen-binding region of anti-CD52 as described herein and a second binding specificity for a second target epitope or target antigen.

[0215] According to the present disclosure, said anti-CD52 agent can be an immune cell harboring an antigen receptor targeting CD52, such as a CAR targeting CD52, said antigen receptor comprising an antigen binding region as defined herein above.

[0216] In specific embodiments, said immune cell (e.g. T cell) harboring a CAR targeting CD52 recognizes a second isoform of CD52 as expressed in a patient in need thereof, and does not recognize a first isoform of CD52. In particular said immune cell may bind specifically to an epitope including the amino acids Q31, T32, S33, S23, P35 and / or 536 of SEQ ID NO: 1. In other particular said immune cell may bind specifically to an epitope including the amino acids S33, S34, and / or P35 of SEQ ID NO: 1.

[0217] In specific embodiments, said anti-CD52 agent can be an immune cell (e.g. T cell) harboring a CAR, said CAR comprising or derived from an antigen-binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the 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.

[0218] In a more particular embodiment, said anti-CD52 agent can be an immune cell (e.g. T cell) harboring a CAR comprising said first antigen-binding region e.g. scFv comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 4. (alemtuzumab)

[0219] In specific embodiments, said anti-CD52 agent can be an immune cell (e.g. T cell) harboring a CAR, said CAR comprising or derived from an antigen-binding region comprising a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17 and VLCDR3 is SEQ ID NO: 18. In a more particular embodiment, said anti-CD52 agent can be an immune cell (e.g. T cell) harboring a CAR comprising said first antigen-binding region e.g. scFv comprising a heavy chain variable domain comprising or consisting of an amino acid sequence of SEQ ID NO: 11 and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12.

[0220] In another preferred embodiment said anti-CD52 agent can be an immune cell harboring a CAR targeting a specific isoform of CD52 as described herein.

[0221] In particular, the disclosure also relates to depleting anti-CD52 agents as disclosed above (for example CAR cell composition or antibodies) comprising a first or a second antigen binding region for use in selectively depleting the host cells or transferred cells respectively, in a subject in need thereof.

[0222] Cells expressing a first isoform of CD52

[0223] The present disclosure relates to a mammalian cell, preferably a hematopoietic cell, or a population of cells expressing a first isoform of CD52 wherein said cell or population of cells express a first isoform of CD52, wherein said first isoform is not recognized by the depleting agent comprising a first antigen binding region as described herein.

[0224] The present disclosure also relates to a mammalian cell, preferably a hematopoietic cell, or a population of cells expressing a first isoform of CD52 wherein said cell or population of cells express a first isoform of CD52 comprising at least one polymorphic allele in the nucleic acid encoding said first isoform, and wherein said first isoform is not recognized by the depleting agent comprising a first antigen binding region as described herein.

[0225] The cell expressing said first isoform of CD52 which is not recognized by the depleting agent comprising a first antigen binding region as described herein may not necessarily comprise said polymorphism or genetically engineered allele in the genomic DNA. Said first isoform of CD52 may also be transiently expressed in said cell by any methodology known to the person skilled in the art.

[0226] Said first isoform of CD52 may also be generated in said cell by in vivo editing by any appropriate means known to the person skilled in the art.

[0227] Said cell or population of cells are particularly useful in medical treatment in a patient expressing a second isoform of CD52.

[0228] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said first isoform of CD52 is obtained by in vivo or ex vivo modifying the nucleic acid sequence encoding said first isoform of CD52 by gene editing, preferably by introducing into a cell a gene editing enzyme capable of inducing site-specific mutations(s) within a target sequence encoding a surface protein region involved in the binding of agent comprising at least a first antigen-binding region.

[0229] In a particular embodiment, said cells (e.g. hematopoietic stem cell) encoding or expressing said first isoform of CD52 not recognized by a depleting agent (e.g. hematopoietic cells) are particularly useful in medical treatment to restore normal hematopoiesis after immunotherapy, such as adoptive cell transfer in a patient expressing said second isoform, in particular wherein the treatment comprises administering a therapeutically efficient amount of said hematopoietic cells expressing said first isoform of CD52 in combination with a therapeutically efficient amount of a depleting agent targeting said second isoform of CD52. In particular, said hematopoietic cells, preferably hematopoietic stem cells are administered subsequently to said depleting agent. In another particular embodiment, said hematopoietic cells, preferably hematopoietic stem cells can be administered before or concurrently to said depleting agent.

[0230] In another particular embodiment, said cells expressing said first isoform of CD52 specifically recognized by depleting agent which does not bind or binds substantially weaker second isoform of CD52 are particularly useful in medical treatment in a patient expressing said second isoform of CD52, in particular to avoid severe side-effects related to transplanted cells carrying the first isoform (safety switch), wherein the treatment comprises administering a therapeutically efficient amount of a depleting agent targeting said first isoform of CD52. In particular, said hematopoietic cells, preferably immune cells harboring a CAR are administered prior to said depleting agent.

[0231] As used herein, the term cell relates to mammalian cells, preferably human cells.

[0232] In a particular embodiment, said cells are hematopoietic cells. Hematopoietic cells comprise immune 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 (DC) and plasmacytoid dendritic cells (pDCs).

[0233] In a preferred embodiment, said immune cells are T cells. In another preferred embodiment, said immune cells are primary T cells. As used herein, the term "T cell" includes cells bearing a T cell receptor (TCR) or a cell derived from a T cell bearing a TCR. T-cells according to the disclosure can be selected from the group consisting of inflammatory T-lymphocytes, cytotoxic T-lymphocytes, regulatory T-lymphocytes, memory T-lymphocytes, tumor infiltrating lymphocytes or helper T- lymphocytes included both type 1 and 2 helper T cells and Thl7 helper cells. In another embodiment, said cell can be derived from the group consisting of CD4+ T- lymphocytes 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-like T cells.

[0234] T-cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person. Alternatively, T cells can be differentiated from IPS cells.

[0235] In another preferred embodiment, said hematopoietic cells are hematopoietic stem cells. The stem cells can be adult stem cells, embryonic stem cells, more particularly nonhuman stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34+cells. Hematopoietic stem cells can be differentiated from iPS cells or can be harvested from umbilical cord blood, from bone marrow or from mobilized or not mobilized peripheral blood.

[0236] In certain embodiments, the cell is an allogeneic cell which refers to a cell derived from a donor that presents with an HLA genotype that is identical, similar or different to the HLA genotype of the person receiving the cell. The donor may be a related or unrelated person. In certain embodiments, the cell is an autologous cell which refers to a cell derived from the same person that is receiving the cell.

[0237] Said cells may originate from a healthy donor or from a patient, in particular from a patient diagnosed with cancer, genetic disease or an auto-immune disease or from a patient diagnosed with an infection. Hematopoietic cells can be extracted from blood, bone marrow or derived from stem cells. HSC's can for example be derived from iPS (induced pluripotent stem cells.

[0238] A person skilled in the art will choose the more appropriate cells according to the patient or subject to be transplanted.

[0239] The disclosure further relates to a composition of cells or a population of cells for use in the therapy as disclosed herein.

[0240] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of CD52, and wherein said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid in position Q31, T32, S33, S23, P35 and / or S36 of SEQ ID NO: 1. In certain preferred embodiments, said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid in position S33, S34, and / or P35 of SEQ ID NO: 1.

[0241] In other preferred embodiments, said polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid in position S33, S34 and P35 of SEQ ID NO: 1. In yet other preferred embodiments, said polymorphic or genetically engineered allele is characterized by a substitution of amino acid in position S34 or P35 of SEQ ID NO: 1.

[0242] CAR

[0243] For use in adoptive cell transfer therapy, said cell expressing first isoform of CD52 according to the present disclosure may be modified to display desired specificities and enhanced functionalities. In a particular embodiment, said cell may express a recombinant antigen binding region, also named antigen receptor on its cell surface as described above. In a particular embodiment, said recombinant antigen receptor is a chimeric antigen receptor (CAR). According to the present disclosure, said immune cell expressing a first isoform of CD52 and a CAR can be specifically depleted by the administration of a therapeutically efficient amount of an agent which comprises a second antigen binding region which specifically binds to said first isoform of CD52 but not to the second isoform of CD52, thereby avoiding eventual severe side effects due to transplantation of said immune cells or depletion by a lymphodepleting agent.

[0244] In a particular embodiment, the immune cell is redirected against a cancer antigen. By cancer antigen" is meant any antigen (i.e., a molecule capable of inducing an immune response) which is associated with cancer. An antigen as defined herein may be any type of molecule which induces an immune response, e.g., it may be a polysaccharide or a lipid, but most preferably it is a peptide (or protein). Human cancer antigens may be human or human derived. A cancer antigen may be a tumor-specific antigen, by which is meant an antigen which is not found in healthy cells. Tumor-specific antigens generally result from mutations, in particular frame-shift mutations which generate a wholly new amino acid sequence not found in the healthy human proteome.

[0245] Cancer antigens also include tumor-associated antigens, which are antigens whose expression or production is associated with, but not limited to, tumor cells. Examples of tumor-associated antigens include for instance 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), CD33, CD34, CD38, CD45, CD99, CD117, CD123, CD300LF, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross 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, neurofilament, neuron- specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2- PK), BCMA (TNFRSF17, CD269), CD19, CD22, CD45, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvlll (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six- transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor-specific antigen is integrin av03 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

[0246] In a particular embodiment, for use in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), the immune cell according to the present disclosure expresses a recombinant antigen binding region such as a CAR targeting CD52. Said cell expressing the first isoform and expressing the CAR (e.g. CAR-CD52) can be further specifically depleted by administering a depleting agent comprising a second antigenbinding region which binds specifically to the first isoform of CD52, but does not bind or binds substantially weaker to the second isoform of CD52, thereby avoiding eventual severe side effects such as graft-versus-host disease due to the transplantation.

[0247] In a particular embodiment, for use in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), the immune cell according to the present disclosure expresses a recombinant antigen binding region such as a CAR targeting CD52. Said cell expressing the first isoform and expressing the CAR (e.g. CAR-CD52) can be protected from depletion by administering a depleting agent comprising a second antigen-binding region which binds specifically to the second isoform of CD52, but does not bind or binds substantially weaker to the first isoform of CD52, thereby avoiding eventual severe side effects such as graft-versus-host disease by lymphodepletion of the host lymphocytes while not depleting the CAR cells.

[0248] In specific embodiments, said immune cell (e.g. T cell) expressing the first isoform harbors a CAR targeting CD52, said CAR comprising an antigen-binding region, e.g. scFv, comprising an antigen-binding region which binds specifically to an epitope of CD52 located within the mature protein, or within the polypeptide including the amino acids Q31, T32, S33, S34, P35 and / or S36 of SEQ ID NO: 1.

[0249] In particular, said immune cell (e.g., T cell) expressing first isoform harbors a CAR targeting CD52 comprising an antigen-binding region, e.g. scFv, comprising or derived from a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10, more preferably comprising an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 3 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 4.

[0250] In particular, said immune cell (e.g., T cell) expressing first isoform harbors a CAR targeting CD52 comprising an antigen-binding region, e.g. scFv, comprising or derived from a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17 and VLCDR3 is SEQ ID NO: 18, more preferably comprising an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 12.

[0251] In vitro methods for preparing cell expressing first isoform

[0252] The cell expressing the first isoform of CD52 according to the present disclosure can be genetically engineered by introducing into said cell a nucleic acid construct (e.g., mRNA) encoding at least one gene editing enzyme or ribonucleoprotein complex comprising gene editing enzyme and / or HDR template as described above. Alternatively, the gene editing system is transduced into said cells via a viral system, such as an adenoviral system. Said cell can also be genetically engineered by further introducing into said cell a nucleic acid construct encoding a CAR as described above. In particular, said method is an ex vivo method performed on a culture of cells.

[0253] 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- or double-stranded, which has been modified to contain segments of nucleic acid sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a "vector", i.e., a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell.

[0254] Preferably, the nucleic acid construct comprises said gene editing enzyme, gRNA, HDR template and / or CAR, operably linked to one or more control sequences. Said control sequences may be a ubiquitous, tissue-specific or inducible promoter which is functional in cells of target organs (i.e., hematopoietic cell). Such sequences which are well-known in the art include in particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, such as without limitation, enhancer, terminator, intron, silencer.

[0255] The nucleic acid construct as described above may be contained in an expression vector. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.

[0256] Examples of appropriate vectors include, but are not limited to, recombinant integrating, or non-integrating viral vectors and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA. Preferably, the vector is a recombinant integrating or non-integrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from herpes virus, retroviruses, lentivirus, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma viruses.

[0257] The present disclosure relates to a method for expressing a first isoform of a cell surface protein in a cell by introducing into said cell a nucleic acid construct (e.g. mRNA) encoding the gene editing enzyme or ribonucleoprotein complex comprising gene editing enzyme and / or HDR template as described above. Said method may further comprise a step of introducing into said cell a nucleic acid construct encoding a CAR. Said method involves introducing gene editing enzyme such as Cas protein, base editor or prime editor and guide RNA (crRNA, tracrRNA, or fusion guide RNA or pegRNA) into a cell. In particular, said gene editing enzyme is CRISPR / Cas gene editing enzyme as described above. In a more particular embodiment, said gene editing enzyme is a site-specific nuclease, more preferably CRISPR / Cas nuclease comprising a guide RNA and Cas protein, wherein said guide RNA in combination with Cas protein cleaves and induces cleavage within said target sequence comprising a nucleic acid encoding surface protein region involved in agent binding as described above.

[0258] Said Cas nuclease may be a high-fidelity Cas nuclease such as a high fidelity Cas9 nuclease.

[0259] Said gene editing enzyme, preferably guide RNA and / or Cas protein, base editor or prime editor as described above may be synthesized in situ in the cell as a result of the introduction of nucleic acid construct, preferably expression vector encoding said gene editing enzyme such as guide RNA and / or Cas protein, base editor or prime editor as described above into the cell. Alternatively, said gene editing enzyme such as guide RNA and / or Cas protein, base editor or prime editor may be produced outside the cell and then introduced thereto.

[0260] Said nucleic acid construct or expression vector can be introduced into cell by any methods known in the art and include, as non-limiting examples, stable transduction methods in which the nucleic acid construct or expression vector is integrated into the cell genome, 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.

[0261] In vivo editing

[0262] The cell expressing the first isoform of CD52 according to the present disclosure may also be edited in vivo. Various technologies exist that enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles and virus-like particles (see for example Cell (2022) 185: 2806-27), and also engineered / artificial viral capsids or protein nanocages (Levasseur et al (2021), ACS Chem. Biol.). The molecular machinery to convert CD52 into a first isoform of CD52 which is not recognized by the depleting agent can be accomplished by any of these methods.

[0263] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a depleting agent and / or a molecular machinery capable of in vivo editing a gene, wherein said molecular machinery capable of in vivo editing a gene comprises all components required to introduce a point mutation of wild type CD52 in a target cell into an isoform of CD52, and wherein said depleting agent binds to wild type CD52, but not to said isoform of CD52 for use in a medical treatment in a patient in need thereof.

[0264] Pharmaceutical composition and therapeutic use In a further aspect, the present disclosure also provides a pharmaceutical composition comprising cells or a population of cells expressing a first isoform of CD52 as described above with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0265] In a particular embodiment, said cell expressing the first isoform of CD52 is a hematopoietic stem cell.

[0266] In another particular embodiment, said cell expressing the first isoform of CD52 is an immune cell, preferably a T cell or a B cell.

[0267] In another particular embodiment, said cell expressing said first isoform of CD52 is an immune cell, preferably a T-cell, more preferably a primary T cell, bearing a chimeric antigen receptor (CAR), preferably a CAR which targets the second isoform of CD52 expressed by said patient's cells as described above.

[0268] The pharmaceutical composition may further comprise a depleting agent comprising a first or second antigen binding region as described above.

[0269] The pharmaceutical composition is formulated in a pharmaceutically acceptable carrier according to the route of administration. Preferably, the composition is formulated to be administered by intravenous injection. Pharmaceutical compositions suitable for such administration may comprise the cells expressing first isoform as described above, in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents.

[0270] Optionally, the composition comprising cells expressing first isoform of CD52 may be frozen for storage at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about -20° C, -80° C or any other appropriate temperature. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing. Alternatively, the cells may also be maintained at room temperature of refrigerated, e.g., at about 4° C.

[0271] The present disclosure relates to the cell or population of cells expressing a first isoform of CD52 as described above for use as a medicament, in particular for use in immunotherapy such as adoptive cell transfer therapy in a patient.

[0272] According to the present disclosure, said cell or population of cells (e.g., hematopoietic cells) expressing a first isoform of CD52 as described above, is used in a medical treatment in a patient in need thereof, wherein said medical treatment comprises administering a therapeutically efficient amount of cells or population of cells expressing said first isoform of CD52, in combination with a therapeutically efficient amount of a depleting agent (e.g. a CAR cell or antibody) that binds specifically to the second isoform or first isoform of CD52 to specifically depleting the patients or the transplanted cells, respectively.

[0273] As used herein, the term "in combination" or "in combination therapy" means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery". In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. In one embodiment, a depleting agent that binds to a second isoform or a first isoform of CD52 is administered at a dose and / or dosing schedule described herein, and the cells expressing the first isoform are administered at a dose and / or a dosing schedule described herein. In some embodiments, "in combination with," is not intended to imply that the depleting agent targeting the second (e.g. CAR cells or antibody recognizing a second isoform of CD52) or the first isoform of CD52 and compositions of cells expressing said first isoform of CD52, must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. The depleting agent (e.g. CAR cells or antibody targeting a second isoform of CD52) can be administered concurrently with, prior to or subsequent to a dose of the hematopoietic stem cells expressing the first isoform of CD52. In certain embodiments, each agent will be administered at a dose and / or on a time schedule determined for that particular agent.

[0274] Adoptive cell transfer therapy according to the disclosure can be used to treat patients diagnosed with cancer, genetic disease, autoimmune disease, infectious disease, a disease requiring a hematopoietic stem cell transplantation (HSCT), the prevention of organ rejection, the tumor conditioning regimen, tumor maintenance treatment, minimal residual disease treatment, the prevention of relapse.

[0275] The present disclosure also relates to the use of cells expressing a first isoform of CD52 as described above in the manufacture of a medicament for adoptive transfer cell therapy in a patient.

[0276] As used herein, the term "subject", or "patient" refers to an animal, preferably to a mammal in which an immune response can be elicited including human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat. More preferably, the patient is a human, including adult, child and human at the prenatal stage.

[0277] As used herein, the term "treatment", "treat" or "treating" refers to any act intended to ameliorate the health status of patients such as therapy, prevention, prophylaxis, and retardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0278] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas including relapses and treatment-related tumors e.g. secondary malignancies after use of cytotoxic therapy and hematopoietic stem cell transplantation (HSCT)) or may comprise solid tumors.

[0279] The term "autoimmune disease" as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen.

[0280] Infectious disease is a disease caused by pathogenic microorganism such as bacteria, viruses, parasites or fungi. In particular embodiments, infections according to the disclosure occur in immunosuppressed patients, such as patients after HSCT or patients who received a solid organ transplantation.

[0281] In a preferred embodiment, the present disclosure relates to a cell expressing first isoform of CD52 as described above for use in hematological cancer, preferably leukemia, lymphoma, myeloma, and myelodysplastic syndromes (MDS). Said diseases can be selected from the group consisting of CD52-positive hematological cancers such as acute lymphoblastic leukemia (ALL), CD52-positive subsets of acute myeloid leukemia (AML), T- cell large granular lymphocyte leukemia (T-cell LGL), T-cell prolymphocytic leukemia (T- PLL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), splenic marginal zone lymphoma (SMZL), high-grade B-cell lymphomas (HGBCL), non-Hodgkin lymphomas (NHL), angioimmunoblastic T-cell lymphomas (AITL), peripheral T-cell lymphomas (PTCL), hepatosplenic T cell lymphomas (HSTCL), T-prolymphocytic leukemia (T-PLL), adult T cell leukemia / lymphomas (ATLL), cutaneous T cell lymphomas (CTCL), Burkitt lymphoma and diffuse large B-cell lymphomas (DLBCL) or from mainly CD52- negative hematological cancers such as multiple myeloma (MM), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), Hodgkin lymphoma, CD52- negative subsets of acute myeloid leukemia (AML) as well as other hematolymphoid tumors.

[0282] In another particular embodiment, said cell or population of cells (e.g., hematopoietic cells) expressing a first isoform of CD52 as described above can be used for the treatment of autoimmune disease such as lupus, multiple sclerosis, scleroderma or systemic sclerosis.

[0283] The disclosure also relates to depleting agents (for example CAR cell composition or antibodies) comprising a first or a second antigen binding region for use in selectively depleting the host cells or transferred cells respectively, in a subject in need thereof.

[0284] Method for depleting specifically patient cells and not transplanted cells

[0285] According to the present disclosure, said cell or population of cells (e.g. hematopoietic cells) expressing a first isoform of CD52 as described above, is used in a medical treatment in a patient in need thereof, wherein said medical treatment comprises administering a therapeutically efficient amount of said cells or population of cells expressing said first isoform of CD52, in combination with a therapeutically efficient amount of a depleting agent (e.g. a CAR cell or antibody) that binds specifically to a second isoform of CD52.

[0286] Indeed, during immunotherapy, immunodepleting agent, such as a CAR expressing immune cells directed to CD52, can be administered to a patient to target and kill tumoral cells. However, as tumoral surface proteins are also expressed at the surface of normal hematopoietic cells, this strategy can induce severe side effects to the patients by altering hematopoiesis. To restore hematopoiesis in the patient, hematopoietic cells can be subsequently transplanted into the patient. However, these cells need to be resistant to said agent, i.e., the depleting agent for CD52 expressing cells, in order not to be targeted by it.

[0287] Alternatively, during immunotherapy, immunodepleting agent, such as an antibody directed to CD52, can be administered to a patient to target and kill host lymphocytes in order to prevent rejection of an allogeneic cell therapy. However, as surface proteins are expressed both at the surface of the host hematopoietic cells as well as the cells of the allogeneic therapeutic cells, this strategy can reduce the activity of the cell therapy by depleting the engineered allogeneic cells. To lymphodeplete the host lymphocytes while retaining numbers and activity of the allogeneic cells in the patient, the cells of the cell therapy need to be resistant to said agent, i.e., the depleting agent for CD52 expressing cells, in order not to be targeted by it.

[0288] Thus, alternatively, according to the present disclosure, the depleting agent comprising a first antigen binding region which binds specifically to a second isoform of CD52 can be administered to ablate specifically patient cells expressing said second isoform of CD52 and not transplanted cells expressing said first isoform of CD52. The selective depletion of patient cells, but not transplanted cells, allows to reconstitute the patient with a healthy hematopoietic system which will no longer be depleted by immunodepleting agent. Thus, according to the present therapeutic use, the patients have a functional immune system ratherthan go through a prolonged phase of immunodepression. The use of cells according to the present disclosure eliminates infections as a major complication of current HSC transplantation. Alternatively, the selective depletion of patient cells can also prevent the rejection of an allogeneic cell therapy while not depleting the engineered cells.

[0289] 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 a second isoform of CD52 comprising:

[0290] (i) administering an effective amount of a cell (e.g. hematopoietic stem cells) expressing a first isoform of CD52 wherein said cell expressing said first isoform of CD52 comprises genomic DNA with at least one polymorphic allele, preferably single nucleotide polymorphism (SNP) allele, or a genetically engineered allele in the nucleic acid encoding said first isoform and wherein said polymorphism is not present in the genome of the patient having cells expressing said second isoform of CD52 or a pharmaceutical composition thereof; and

[0291] (ii) administering a therapeutically efficient amount of an agent comprising at least a first antigen-binding region which binds specifically to said second isoform of CD52 and does not bind or binds substantially weaker to said first isoform of CD52 to deplete specifically cells expressing said second isoform of CD52 (patient's cells). In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, wherein said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent comprising at least a first antigen-binding region that binds specifically to said second isoform of CD52 to specifically deplete patient cells expressing said second isoform of CD52. In certain embodiment, said medical treatment restore normal hematopoiesis after immunotherapy in the treatment of hematopoietic disease. In certain embodiment, said medical treatment is the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other myeloproliferative neoplasms. In certain embodiments, said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of CD52 to avoid eventual severe side effects such as graft-versus-host disease due to the transplantation.

[0292] Said cells expressing the first isoform of CD52 or pharmaceutical compositions thereof are administered to a subject in combination with (e.g., before, simultaneously or following) an agent comprising a first antigen binding region as described above.

[0293] In a preferred embodiment, the depleting agent (e.g., CAR cells or antibody targeting a second isoform of CD52 is administered prior to (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 before), or subsequent to (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 after) a dose of the hematopoietic stem cells expressing a first isoform of said surface protein (e.g., a first isoform of CD52).

[0294] By a "therapeutically efficient amount" or "effective amount" is intended a number of cells, in particular hematopoietic stem cells expressing the first isoform of CD52 as described above administered to a subject that is sufficient to constitute a treatment as defined above, in particular restoration of normal hematopoiesis in a patient.

[0295] The administration of the cell or pharmaceutical composition according to the present disclosure may be carried out in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermal, intratumorally, intranodally, intramedullary, intramuscularly, by 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 injected directly into a tumor, lymph node, or site of infection.

[0296] The administration of the cells or population of cells can consist of the administration of 104-109cells per kg body weight, preferably 105to 107cells / kg body weight, more preferably 2xl06-5xl06cells per kg body weight including all integer values of cell numbers within those ranges. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. The cells or population of cells can be administrated in one or more doses. Timing of administration is within the judgment of managing 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 optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. In particular, the disclosure also relates to depleting anti-CD52 agents as disclosed above (for example CAR cell composition or antibodies) comprising a first antigen binding region for use in selectively depleting the host cells in a subject in need thereof.

[0297] Method for depleting specifically transplanted cells and not patient cells (safety switch).

[0298] According to the present disclosure, said cell or population of cells (e.g. hematopoietic cells) expressing a first isoform of CD52 as described above, is used in a medical treatment in a patient in need thereof, wherein said medical treatment comprises administering a therapeutically efficient amount of a cell or a population of cells expressing said first isoform of CD52, in combination with a therapeutically efficient amount of a depleting agent (e.g. a CAR cell or antibody) that binds specifically to said first isoform CD52.

[0299] The cell or population of cells, preferably immune cells expressing the first isoform of CD52 of the present disclosure is particularly used in adoptive transfer cell transfer therapy into a patient. Said transplanted cell expressing said first isoform of CD52 can be further depleted in patients by administering a therapeutically efficient amount of a depleting agent comprising a second antigen binding region, which specifically binds to the first isoform of CD52 particularly and does not bind or binds substantially weaker to the second isoform of CD52 expressed by patient's cells to avoid eventual severe side effects such as graft-versus-host disease due to the transplantation. In this case, said agent comprising a second antigen-binding region, which binds specifically to said first isoform of CD52 (expressed by transplanted cell) is administered to deplete specifically transplanted cells and not patient cells. Selective depletion of the transplanted cells constitutes an important safety feature by providing a "safety switch".

[0300] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, wherein said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof in combination with a therapeutically efficient amount of a depleting agent comprising at least a second antigen-binding region that binds specifically to said first isoform of CD52 to specifically deplete transferred cells expressing said first isoform of CD52. In certain embodiments said medical treatment is the use in adoptive cell transfer therapy.

[0301] Graft-versus-host disease (GvHD) relates to a medical complication following the receipt of transplanted tissue from a genetically different person. Immune cells in the donated tissue (the graft) recognize the recipient (the host) as foreign. In certain embodiments, the medical condition is graft-versus-host disease caused by hematopoietic stem cell transplantation or adoptive cell transfer therapy wherein immune cells are transferred into patient.

[0302] Said side effects can also occur when transplanted cells, particularly immune cells harboring a CAR have severe side effects such as cytokine release syndrome and / or neurotoxicity. In this case, the transplanted cells expressing the first isoform of CD52 can be eliminated when said cells become malignant or cause any type of unwanted on-target or off-target damage as a safety switch.

[0303] The present disclosure relates to a method for adoptive cell transfer therapy in a patient having cells expressing a second isoform of CD52 comprising:

[0304] (i) administering an effective amount of a cell expressing a first isoform of CD52 wherein said cell expressing said first isoform of CD52 comprises genomic DNA with at least one polymorphism allele, preferably single nucleotide polymorphism (SNP) allele, or a genetically engineered allele in the nucleic acid encoding said first isoform CD52 and wherein said polymorphism is not present in the genome of the patient having cells expressing said second isoform of CD52 or a pharmaceutical composition thereof; and

[0305] (ii) administering a therapeutically efficient amount of an agent comprising at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind or binds substantially weaker to said second isoform of CD52 to deplete specifically cells expressing said first isoform of CD52.

[0306] Said cells expressing the first isoform of CD52 or pharmaceutical compositions thereof are administered to a subject in combination with (e.g., before, simultaneously or following) an agent comprising a second antigen binding region as described above.

[0307] In a preferred embodiment, the depleting agent (e.g. CAR cells or antibody targeting a second isoform of CD52) is administered prior to (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 before), or subsequent to (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 after) a dose of the hematopoietic stem cells expressing a first isoform of CD52.

[0308] The administration of the cells or pharmaceutical composition according to the present disclosure may be carried out in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermal, intratumorally, intranodally, intramedullary, intramuscularly, by 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 injected directly into a tumor, lymph node, or site of infection.

[0309] The administration of the cells or population of cells can consist of the administration of 104-109cells per kg body weight, preferably 105to 107cells / kg body weight including all integer values of cell numbers within those ranges. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. The cells or population of cells can be administrated in one or more doses. Timing of administration is within the judgment of managing 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 optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art.

[0310] Accordingly, in specific embodiments, the disclosure relates to a depleting agent (e.g. a CAR cell or an antibody) for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of CD52 as described above, wherein said patient have native cells expressing a second isoform of CD52, and wherein said depleting agent comprising at least a second antigen-binding region which binds specifically to said first isoform of CD52 and does not bind or binds substantially weaker to said second isoform of CD52.

[0311] In another aspect, the present disclosure relates to a kit for expressing a first isoform CD52 as describe above into a cell, said kit comprising a gene editing enzyme, such as guide RNA in combination with a Cas protein, base editor or prime editor, nucleic acid construct, expression vector as described above or isolated cell according to the present disclosure.

[0312] EXAMPLES

[0313] Example 1: Generation of anti-CD52 MAbs and Fabs

[0314] Anti-CD52 antibody alemtuzumab (RefMabtl) was generated in Fab and Mab format based on publicly available sequence information. Gatralimab (RefMab#2) was bought from Proteogenix (WPX-TA1589). EPR23855-41 (RefMab#3) was bought from Abeam (#ab259794). Variable chains and CDRs (Kabat) of the antibodies (RefMabUl and RefMab#2) are shown in Table 2.

[0315] Table 2:

[0316] RefMabtl

[0317] SEQ ID No. Comment Sequence

[0318] 3 VH QVQLQESGPGLVRPSQTLSLTCTVSGFTFTDFYMNWVRQPPGR

[0319] GLEWIGFIRDKAKGYTTEYNPSVKGRVTMLVDTSKNQFSLRLSSV

[0320] TAADTAVYYCAREGHTAAPFDYWGQGSLVTVSS

[0321] 4 VL DIQMTQSPSSLSASVGDRVTITCKASQNIDKYLNWYQQKPGKAP

[0322] KLLIYNTNNLQTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCLQ

[0323] HISRPRTFGQGTKVEIK

[0324] 5 HCDR1 DFYMN

[0325] 6 HCDR2 FIRDKAKGYTTEYNPSVKG

[0326] 7 HCDR3 EGHTAAPFDY

[0327] 8 LCDR1 KASQNIDKYLN

[0328] 9 LCDR2 NTNNLQT

[0329] 10 LCDR3 LQHISRPRT

[0330] RefMab#2

[0331] 11 VH EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQAPG

[0332] KGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKNSLYLQMN

[0333] SLKTEDTAVYYCTPIDYWGQGTTVTVSS

[0334] 12 VL DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWVLQKP

[0335] GQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRVEAEDVGV

[0336] YYCVQGSHFHTFGQGTKLEIK 13 HCDR1 NYWMN

[0337] 14 HCDR2 WVRQAPGKGLEWVG

[0338] 15 HCDR3 IDY

[0339] 16 LCDR1 KSSQSLLYSNGKTYLN

[0340] 17 LCDR2 LVSKLDS

[0341] 18 LCDR3 VQGSHFHT

[0342] Additional features of the antibodies used, as well as the formats and isotypes of the full-length antibodies are shown in Table 3.

[0343] Table 3:

[0344] Antibody Format / isotype Source

[0345] RefMabtfl Alemtuzumab humanized IgGl V-region sequences taken from

[0346] Tabs Therapeutic Antibody Database

[0347] RefMabtl- Alemtuzumab humanized IgGl- V-region sequences taken from

[0348] H435A H435A Tabs Therapeutic Antibody

[0349] Database

[0350] RefMab#2 Gatralimab humanized IgGl Proteogenix, WPX-TA1589

[0351] RefMab#3 EPR23855-41 Rabbit IgG Abeam, #ab259794

[0352] Example 2: Binding of MAbs to cell-surface expressed CD52 and optimization of assay conditions

[0353] HEK-293T cells were transfected with a construct containing wild-type CD52 (SEQ ID No. 1) or with an empty vector. Binding of the antibodies to the transfected cells and the optimal assay conditions were evaluated in 384-well format. Detection of cellular expression was measured via high-throughput flow cytometry. Serial dilutions of each antibody were tested for immunoreactivity against cells expressing CD52 or vector alone. The optimal screening concentration for each antibody was determined based on the raw signal values and signal-to-background calculations. Results are shown in Figure 1. Each point represents the mean of four replicates. Both antibodies in Mab format did bind to human CD52 in a concentration dependent manner. Cell transfected with the empty vector did not show significant binding to antihuman CD52 antibodies.

[0354] Optimized assay conditions for flow cytometry are shown in Table 4 for RefMabtfl (Alemtuzumab) and RefMab#3 (EPR23855-41). Table 4:

[0355] Example 3: Binding of Fab to cell-surface expressed CD52 and optimization of assay conditions

[0356] Experiments were performed similar 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 CD52 or vector alone. The optimal screening concentration for the Fabs was determined based on the raw signal values and signal-to-background calculations. Results are shown in Figure 2. Each point represents the mean of four replicates.

[0357] The tested antibody in Fab format did bind to human CD52 in a concentration dependent manner. Cells transfected with the empty vector did not show any binding to anti-human CD52 antibodies. Optimized assay conditions for high throughput flow cytometry are shown in Table 5 for RefMabtfl (Alemtuzumab)

[0358] Table 5:

[0359] Example 4: Alanine scanning (epitope mapping) by flow cytometry

[0360] An alanine scan on human CD52 was performed to determine the residues on CD52 that are involved in binding to the antibodies investigated. The alanine scan was performed via shotgun mutagenesis epitope mapping (Integral Molecular, Philadelphia / PA, USA) as described in Immunology (2014) 143, 13-20. Briefly, a mutation library of CD52 was created by high-throughput, site-directed mutagenesis. Each residue was individually mutated to alanine, with alanine codons mutated to serine. The mutant library was arrayed in 384-well microplates and transiently transfected into HEK293-T. Following transfection, cells were incubated with the indicated antibodies (IgG or Fab) at concentrations pre-determined using an independent immunofluorescence titration curve on wild type CD52. Antibodies were detected using an Alexa Fluor 488-conjugated secondary antibody and mean cellular fluorescence was determined using Intellicyt iQue flow cytometry platform ( I nte 11 icyt / Sa rtorius) . Normally, mutated residues were identified as being critical to the antibody epitope if they did not support the reactivity of the test antibody but did support the reactivity of the control antibody, which was another anti- CD52 antibody (RefMab #3). This counterscreen strategy facilitates the exclusion of mutants that are locally misfolded or that have an expression defect. However, it turned out that both, RefMabWl and the control antibody Refmabtfl have overlapping epitopes. Binding of each antibody to each mutant clone was determined in duplicates. For each point, background fluorescence was subtracted from the raw data, which were then normalized to antibody reactivity with wild type CD52.

[0361] Since library screens of very high-affinity antibodies sometimes fail to yield critical residues for antibody binding, RefMabtl was converted into Fab format to weaken binding sufficiently to allow identification of critical residues for binding. However, RefMabffl Fab screening under standard conditions was still insufficient to identify critical residues for binding, therefore, high stringency conditions were implemented. These conditions include combinations of increased pH, increased salinity, increased temperature, and / or increased wash time (denoted with 'HS').

[0362] Example 5: Analysis and comparison of the identified variants from the alanine scan

[0363] The result of the alanine scan is shown in Table 6 below. Mean binding reactivities (and ranges) are listed for all identified critical residues. Critical residues for antibody binding (bold and underlined) were residues whose mutations led to a binding of the test Abs of less than 20%. An additional secondary residue (bold) was identified that did not meet the threshold guidelines but led to decreased binding and proximity to critical residues suggested that they may be part of the antibody epitope.

[0364] Table 6:

[0365] Critical residues whose mutation gave the lowest reactivities with RefMabtl are underlined in Table 7 below. Validated critical residues represent amino acids whose side chains make the highest energetic contributions to the antibody-epitope interaction (Bogan and Thorn, 1998; Lo Conte et al., 1999); therefore, the highlighted residues are likely the major energetic contributors to binding.

[0366] Table ?:

[0367] It is important to note that binding of both antibodies, RefMabtfl and #3, to these residues is decreased to below 20% as compared to wild type CD52. This could be due to loss of expression of these CD52 variants or indicate overlapping epitopes and might therefore not be an indication that the changes in amino acid positions are relevant for binding to the tested antibodies but instead result from reduced target expression. However, during the course of the project, it turned out that RefMabtl and Refmab#3 have indeed overlapping epitopes, explaining this observation and indicating that the residues listed in Table 7 indeed represent the epitope of RefMabtl.

[0368] Example 6: Comprehensive mutational analysis by high-throughput flow cytometry Since the mature human CD52 antigen is only a 12aa peptide, comprehensive mutational analysis was performed in parallel to the alanine scanning. Each of the 12 amino acids of human CD52 was exchanged to any of the 20 amino acids (except cysteine, the natural amino acid, and alanine, except for position S34 where Cysteine was also assessed). This screening was performed to confirm the residues on CD52 involved in binding identified above (Example 5) and to determine which exchanges in amino acids would abolish binding of the tested antibodies.

[0369] In short, binding of each test MAb to each mutant clone in the comprehensive library was determined, in duplicate (for some clones in quadruplicates), by high-throughput flow cytometry. For each mutation, background fluorescence was subtracted from the raw data, which were then normalized to MAb reactivity with WT target protein. Mean binding reactivities and ranges are listed in Table 8 below for all mutant clones. Mutations that reduced binding of RefMabtl to below 25% are highlighted (bold and underlined).

[0370] Table 8:

[0371]

[0372] Example 7: Analysis and comparison of the identified variants from the comprehensive analysis

[0373] Table 9 summarizes the variants that reduce binding of RefMab#! to below 20%. Table 9:

[0374] Residue Mutation

[0375] Q31 P, T, V

[0376] T32 E, F, G, 1, L, M, N, P, Q, R, V, W, Y

[0377] S33 A, D, E, F, 1, K, L, N, P, Q, R, V, W, Y

[0378] S34 C, D, E, F, G, H, 1, K, L, N, P, Q, R, T, V, W, Y

[0379] P35 D, E, G, H, 1, L, M, Q, R, S, T, W, Y

[0380] S36 D, E, F, G, H, 1, K, L, M, N, P, Q, T, W, Y

[0381] To further characterize respective variants and to be able to discriminate non-binding

[0382] CD52 variants from non-expressed variants, a comprehensive scanning peptide array was performed next (Example 8).

[0383] Example 8: Epitope Substitution Scans of RefMab#! and #2

[0384] The substitution scan of wild type CD52 peptide SGQNDTSQTSSPSAD (SEQ ID No. 19) was based on an exchange of all amino acid positions for the 20 standard amino acids (except the natural amino acid). The two C-terminal amino acids (AD) are not part of the natural, mature CD52 sequence but are required to enable binding of RefMab#l as they represent a mimotope for the GPI anchor (Hale G, 1995, Immunotechnology). Accordingly, the first N-terminal amino acid Ser was added to produce the required 15mer length for the peptide to allow standard synthesis and analysis. The resulting peptide microarray contained 300 different peptide variants of the wildtype peptide printed in triplicate (900 or 1,800 peptide spots per array) and were framed by additional HA (YPYDVPDYAG, 80 or 96 spots per array) control peptides (https: / / www.pepperprint.com / products- services / pepperchip-peptide-microarrays / custom-peptide-microarrays). Binding of RefMabtfl and #2 was assessed on the 300 peptides. Primary antibody concentrations were 1 pg / ml, 10 |ig / ml and 100 pg / ml in incubation buffer. Secondary antibody: Goat antihuman IgG (H+L) DyLight680 (0.2 pg / ml).

[0385] Microarray image analysis was done with PepSlide® Analyzer and a software algorithm breaks down fluorescence intensities of each spot into raw, foreground and background signal. This calculates averaged median foreground intensities and spot-to-spot deviations of spot triplicates. The data sets were analyzed to identify conserved and variable amino acid positions of the wild type peptides.

[0386] The results from the peptide microarrays are shown in Figures 3A-3F.

[0387] Example 9: Analysis and comparison of the identified variants from the alanine scan

[0388] The following Table 10 lists the compiled results from the comprehensive flow cytometry screen as well as the peptide microarray screening of binding of RefMabtl and #2 to CD52 variants. Major loss of binding is defined by values <2500 in the peptide microarray as well as (if available) <10% residual binding in the flow cytometry based comprehensive screen.

[0389] Table 10:

[0390] Residue Mutation Major loss of binding by antibody

[0391] Q31 C, D, E, F, H, 1 K, L, T, V, W, Y RefMab#2

[0392] T32 1 RefMab#2 S33 C, D, E, F, H, 1, K, P, R, V, W, Y RefMabffl

[0393] C, D, E, F, H, 1, K, L, M, N, P, R, V, W, Y RefMab#2

[0394] S34 A, C, D, E, F, G, H, 1, K, L, M, N, P, Q, R, T, V, W, Y RefMabtl

[0395] D, E, F, H, 1, K, L, M, P, Q, R, W, Y RefMab#2

[0396] P35 A, C, D, E, F, G, H, 1, K, L, M, N, Q, R, S, T, V, W, Y RefMabtfl

[0397] C, D, E, G, H, 1, K, L, M, Q, R, V, W RefMab#2

[0398] S36 A, C, D, E, F, G, H, 1, K, L, M, N, P, Q, R, T, V, W, Y RefMabtl

[0399] Example 10: Sequence analyses of human and monkey CD52 and selection of preferred variants for analysis

[0400] Published data show that the C-terminal Ser (S36) of human CD52 is required for the attachment of the GPI anchor and thus the connection of CD52 to the cell surface (Treumann et al (1995). J Biol Chem). In addition, CD52 carries an N-linked glycosylation at position N27 and O-linked glycans seem to be located at positions T32, S34 and / or S36 (Shathili et al. (2019). Frontiers Immunol). These posttranslational modifications are important forthe functional activity of CD52 and should be retained in the variant proteins.

[0401] Alignments of processed, mature CD52 from human and various monkey species show that positions 31 through 36 are conserved, with mainly amino acids with polar uncharged side chains (S, T, N, and at position 32 also Q) and small hydrophobic side chains (A, L, P) dominating in this region. No charged or bulky hydrophobic amino acids are found at these positions in any of the monkey species, which were assessed. However, sequence variants indicate that some sequence flexibility is tolerated (Figure 4).

[0402] The analysis of natural sequence variations showed that S34C, P35S and P35L occur in the human population

[0403] (https: / / gnomad. broadinstitute. org / gene / ENSG00000169442?dataset=gnomad_r2_l), again indicating the preference for uncharged, relatively small amino acids. As S36 needs to remain invariant in order to retain GPI anchoring, it was decided to focus on positions S33, S34 and P35 for our further analyses, especially the exchanges S34A, S34G, S34N, P35A, P35L, P35S and P35T.

[0404] In silico analysis indicates that the following variants could be amenable to base editing (Table 11):

[0405] Table 11:

[0406] WT_aa WT_codon Base editor type Variant_codon Variant aa

[0407] S33 AGC CBE AAC N33

[0408] All these variants are either small and hydrophobic or have polar uncharged side chains and are therefore expected to not dramatically change the position of the GPI anchor and the general glycosylation pattern.

[0409] Example 11: In silico prediction of GPI anchor position

[0410] NetGPI-1.1 (https: / / services.healthtech.dtu.dk / services / NetGPI-l.lZ) was used to predict the likelihood and position of GPI anchors. NetGPI is based on a deep learning approach, which relies on recurrent neural networks and incorporates an attention mechanism to indicate potential w-sites (= anchoring positions) for GPI anchors. Full-length human wildtype and variant CD52 amino acid sequences were entered into the prediction tool in FASTA format. The following variants have the highest likelihood to retain their co-site at position 36 (identical to wt CD52): S34A, S34G, S34N, P35T and P35L. In P35F, P35S and P35A, the w-site was predicted to be shifted to other proximal Ser residues (Gislason et al (2021). biorXiv). Also, when the natural, wildtype co-site S36 was mutated to P the GPI anchoring position was shifted.

[0411] Example 12: Affinity determination of RefMabtfl on chemically synthesized variants of CD52

[0412] The peptides in Table 12 were synthesized, representing non-glycosylated mimotopes of wildtype and variant CD52. Peptides contained an N-terminal biotin, a linker and the peptide sequence. At the C-terminus the AD dipeptide (Leo C James et al (1999). Journal of molecular biology) mimics the ethanolamine phosphate which is part of the RefMab#l epitope. The binding to RefMabtfl and an isotype control (RefOOl hlgGl) was performed on an Octet R8 (Sartorius) at 25 °C and 1,000 rpm using a lx kinetic buffer (Sartorius). The biotinylated CD52 peptides (0.2 ug / mL) were immobilized on streptavidin biosensors (Sartorius) for 300 s. The association of RefMabtfl (200 nM) as well as its dissociation was monitored by BLI for 300 s. Due to avidity effects, the binding was assessed qualitatively. Each interaction was assigned to one of 4 groups according to the nm shift at the end of the association (300 s), high binding (>4.5 nm), moderate binding (2.5-4.5 nm), low binding (0.5 -2.5 nm) and no binding (<0.5 nm). Results are shown in Table 12.

[0413] Table 12:

[0414] Mutation Peptide sequence RefMabtl binding

[0415] WT Biotin-linker-GQNDTSQTSSPSAD (SEQ ID No. 20) High

[0416] T32A Biotin-linker-GQNDTSQASSPSAD (SEQ ID No. 21) High

[0417] T32I Biotin-linker-GQNDTSQISSPSAD (SEQ ID No. 22) Moderate

[0418] S33R Biotin-linker-GQNDTSQTRSPSAD (SEQ ID No. 23) High

[0419] S33D Biotin-linker-GQNDTSQTDSPSAD (SEQ ID No. 24) Low

[0420] S33A Biotin-linker-GQNDTSQTASPSAD (SEQ ID No. 25) Moderate S33Q Biotin-linker-GQNDTSQTQSPSAD (SEQ ID No. 26) Moderate

[0421] S34G Biotin-linker-GQNDTSQTSGPSAD (SEQ ID No. 27) No binding

[0422] S34E Biotin-linker-GQNDTSQTSEPSAD (SEQ ID No. 28) No binding

[0423] S34D Biotin-linker-GQNDTSQTSDPSAD (SEQ ID No. 29) No binding

[0424] S34T Biotin-linker-GQNDTSQTSTPSAD (SEQ ID No. 30) No binding

[0425] S34V Biotin-linker-GQNDTSQTSVPSAD (SEQ ID No. 31) No binding

[0426] S34A Biotin-linker-GQNDTSQTSAPSAD (SEQ ID No. 32) No binding

[0427] S34N Biotin-linker-GQNDTSQTSNPSAD (SEQ ID No. 33) No binding

[0428] S34I Biotin-linker-GQNDTSQTSIPSAD (SEQ ID No. 34) No binding

[0429] P35S Biotin-linker-GQNDTSQTSSSSAD (SEQ ID No. 35) No binding

[0430] P35L Biotin-linker-GQNDTSQTSSLSAD (SEQ ID No. 36) No binding

[0431] P35T Biotin-linker-GQNDTSQTSSTSAD (SEQ ID No. 37) No binding

[0432] P35D Biotin-linker-GQNDTSQTSSDSAD (SEQ ID No. 38) No binding

[0433] P35E Biotin-linker-GQNDTSQTSSESAD (SEQ ID No. 39) No binding

[0434] P35H Biotin-linker-GQNDTSQTSSHSAD (SEQ ID No. 40) No binding

[0435] P35I Biotin-linker-GQNDTSQTSSISAD (SEQ ID No. 41) No binding

[0436] P35Q Biotin-linker-GQNDTSQTSSQSAD (SEQ ID No. 42) No binding

[0437] P35A Biotin-linker-GQNDTSQTSSASAD (SEQ ID No. 43) No binding

[0438] P35F Biotin-linker-GQNDTSQTSSFSAD (SEQ ID No. 44) No binding

[0439] S36A Biotin-linker-GQNDTSQTSSPAAD (SEQ ID No. 45) No binding

[0440] Example 13: Production and purification of fully processed (GPI-containing), glycosylated wildtype and variant CD52

[0441] In order to generate CD52 antigens that contain post-translational modifications and resemble the native protein more closely, PGAP2 KO Expi293F human cells were transfected with CD52 plasmids. PGAP2 is a transmembrane protein that catalyzes the addition of stearic acid to the lipid portion of the GPI anchor and cells deficient in PGAP2 lack stable surface expression and in consequence release the GPI anchored protein from the cell membrane into the supernatant. All CD52 constructs were designed to contain an N-terminal fusion to a maltose-binding protein (MBP) that serves to stabilize the CD52 protein and aid with the purification. After the transient transfection of Expi293F PGAP2 KO with full length CD52 constructs (MBP-CD52-wt / variant-GPI), proteins were expressed for 4 days at 37C 8% CO2, 75% humidity. After the harvest, supernatant containing CD52 proteins was incubated with amylose resin (E8021L, NEB). The bound protein was eluted in PBS containing 20mM maltose. Two separate aggregates from monomeric CD52 species, size exclusion chromatography was used. Monomeric peaks were concentrated and the purity of CD52 proteins was verified by SDS-PAGE (Table 13).

[0442] Table 13:

[0443] Example 14: Production and purification of secreted (non-GPI containing), glycosylated wildtype and variant CD52

[0444] The generation of secreted, non-GPI containing CD52 proteins (Table 14) followed the same procedure as for the GPI-containing CD52 proteins, except the DNA plasmids used for transfection lacked the GPI attachment signal sequence (MBP-CD52 wt / variant secreted), therefore naturally releasing the CD52 protein into the supernatant. Table 14:

[0445] Example 15: Affinity determination of RefMabfll and RefMab#2 using recombinant glycosylated wildtype and variant CD52

[0446] Binding to the elected variants was performed with antibodies RefMabtfl and RefMab#2. Binding of the antibodies to CD52 wildtype and variants was measured on an Octet system R8 (Sartorius) at 25 °C with shaking at 1,000 rpm using lx kinetic buffer (Sartorius). The elected variants were screened for their ability to bind Refmabtl or Refmab#2 using different concentrations of CD52 (wild type or variants). RefMAbtl and #2 were captured using Anti-Human Fc capture biosensor generation 2 (AHC2) (Sartorius, PN: 18-5143) for 300 s at 1-5 ug / mL. Human MBP-CD52-WT-GPI and variants, which were produced in and isolated from Expi293 pGap2 knockout cell line, were used as analyte at 7 different concentrations (1000-1 nM, 1:2 dilution). Association of the analytes to the antibody was monitored for 600 s and dissociation of the analytes from the antibody was monitored for 600 s. Reference subtraction was performed against buffer only wells. AHC2 tips were regenerated using 10 mM Gly-HCI pH 1.7. Data were analyzed with Octet Data Analysis software HT. Data were fitted to a 1:1 binding model. Kinetic rates ka and kd and response signal were globally fitted. To calculate the %bi nd ing of the variants compared to WT, the nm shift at the end of the association (600 s) after buffer subtraction was used. This value of each Refmab#l / Refmab#2-variant interaction was divided by the value of Refmab#l / Refmab#2 binding to the WT. Results are shown in Table 15 and Figure 5. T32I was measured in duplicates, the standard deviation is shown in brackets.

[0447] Table 15:

[0448] Example 16: Avidity determination of RefMab#! using recombinant glycosylated wildtype and variant CD52

[0449] Binding to the elected variants was performed with antibodies Refmab #1. Binding of the antibodies to CD52 wildtype and variants was measured in an Octet system R8 (Sartorius, SA-030159 and SA-030308) at 25 °C with shaking at 1,000 rpm using lx kinetic buffer (Sartorius, PN: 18-1105). The elected variants were screened for their ability to bind Refmab #1 and #2.

[0450] MBP-CD52-WT-GPI and variants were produced in and isolated from Expi293F PGAP2 knockout cell line and were biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin, No-Weigh (Thermo Fisher, A39257). Biotinylated CD52 wild type and variants were captured on streptavidin biosensor (SA) (Sartorius, PN 18-5019) for 300 s at 2ug / mL. As an analyte, Refmab #1 was used at 500 nM. Association of the analyte was monitored for 300 s and dissociation was monitored for 300 s. Data were analyzed using the Octet Data Analysis software HT 12.0. Binding level of each CD52 variants to Refmab #1 was calculated by subtracting the response signal at 1250 s by the loading, this value was compared to the wild type and shown as percentage. Results are shown in Table 16. Table 16: Example 17: Overexpression of recombinant wildtype and variant CD52 in mammalian cell lines

[0451] HEK293 mammalian cells, which show no expression of endogenous human CD52, were used for the overexpression of recombinant human CD52 wildtype or mutant CD52 variant peptide. Cells were transfected with a plasmid, including also the pcDNA3.1 backbone with neomycine resistance cassette and respective MBP-G3-CD52 open reading frame, expressing wildtype CD52 or expressing mutant CD52 variants. Due to the small size of the processed CD52 and heavily overlapping antibody epitopes it is difficult to find antibodies binding independently of RefMabtl and #2, which complicates assessment of expression levels of the respective CD52 variants. Therefore, the CD52 gene was genetically fused via a G3 linker with a maltose binding protein tag at its N-terminus to use an antibody against MBP as expression control.

[0452] Two days after transfection of cells via the Lipofectamine3000 system according to manufacturer's recommendations, selective pressure was applied with antibiotics (500 pg / mL of G418 / Neomycin; Gibco) for 21 days to generate stable cell lines. Mock transfected HEK293 cells were used as negative control for antibiotic selection. Stably transfected HEK293 cells were stained using two different antibodies, AF647-conjugated RefMabtl targeting human CD52 and anti-MBPtag targeting the MBP tag (see Example 18). Using this antibody combination, it is possible to evaluate the loss of binding of the antibody of interest to a specific, paired CD52 mutation. The retention of binding of the anti-MBP antibody is important to show that the mutant CD52 is still expressed by the cells and to normalize anti-CD52 binding to target expression.

[0453] Example 18: Recombinant CD52 variants render mammalian cells resistant to RefMabtl antibody binding, antibody-mediated Fcgamma activation and killing

[0454] To assess whether HEK293 cells that overexpress recombinant CD52 variant or wildtype CD52 are shielded from antibody binding, at comparable CD52 expression levels, 150000 cells were collected 3 days after transfection, per staining condition, per stable cell line as well as non-transfected HEK293 cells as negative control, and stained with increasing concentrations (0.01, 0.1, 1, 1, 50, 100 pg / mL) of the appropriate fluorophore-conjugated Refmabtl (ab269823 AlexaFluor Lightning-Link conjugation kit). Additionally, 150000 cells were collected per stable cell line as well as non-transfected HEK293 cells as negative control, per staining condition, and stained with 4 pg / mL of anti-MBP tag antibody (NovusBio; NB100-66609A F488) to assess expression levels of CD52 variants and wild-type. The stained cell suspensions were analyzed using a cell analyzer (Agilent Novocyte Quanteon). Edited and shielded cells do not bind the Refmabtl antibody paired with the shielding mutation, while wildtype, unedited cells do bind the Refmab #1 antibody. Simultaneously, CD52 expression levels on HEK293 cells were determined to be marginally lowered for variants (S34G, S34N, T32I) compared to WT (Figure 6).

[0455] Antibody-dependent cellular cytotoxicity (ADCC) of RefMabtl on wildtype and variant CD52 expressing cells was extrapolated from an FcgammaRllla activation assay performed with the ADCC Reporter Bioassay, V Variant (Promega, ref G7015). On day 1, Jurkat / FcyRII la / N FAT-Luc CD52 KO ADCC effector cells were diluted in ADCC buffer (Gibco RPMI-1640 plus 0.5% of Hyclone low-IgG FBS) to a cell density of 12 E6 cells / mL and 25 pL was added per well in a white 96 well-plate (Corning costar #3197). Then 25 pL (25000 cells) was added per well of the HEK293 stable cells, overexpressing wild-type and variants (S34G, S34N, T32I) respectively. Then 25 pL of RefMab#l antibody diluted into ADCC buffer at 3X the final concentration (final concentration ranging from 0 to 50 pg / mL) was added to the HEK293 stable cell conditions. The plate was then incubated for 22h at 37°C with 5% CO2. Luciferase activity is measured using the Bio-GloTM Luciferase Assay Reagent (G77941, Promega). On day 2, 75 pL of Reagent was added and cells were incubated for 10 min at RT in the dark with agitation. Luminescence is read with a plate reader. RefMab#l induced significant FcgammaRllla activation on the effector cells when co-cultivated with WT CD52 HEK293 cells. CD52 variant cells and CD52 knock-out cells show no FcgammaRllla activation above background (effector cells only), indicating that cells expressing CD52 variants and knock-out are not susceptible to RefMab#l mediated ADCC (Figure 7).

[0456] To measure complement-dependent cytotoxicity (CDC) of RefMab#l, wildtype and variant CD52 cells are plated in a microtiter plate. Different concentrations of RefMab#l as well as human complement sera are added to the cells. The mixture is incubated for 6 hrs at 37°C 5%CO2. CytoTox-Glo reagent (Promega) is used to quantify the lysed cells. RefMab#l induces significant complement-mediated cell lysis when cultivated with wt CD52 cell lines. CD52 variant and knock-out cells show no or only strongly reduced complement-mediated cell killing indicating that these cells are not susceptible to RefMabtl mediated CDC. Digitonin and Raji cells incubated with lug / ml of rituximab were used as positive controls.

[0457] Example 19: Generation of CD52 variants by base editing in human cancer cell lines

[0458] CD52 variants were generated via base editing. Alternatively, CD52 was knocked out via double strand induced NHEJ-mediated editing (Table 17). Base editing single guide RNAs (sgRNAs; Synthego) with SpCas9-SpRY or SpCas9-NG PAM flexibility (NRN / NYN or NG, respectively) (Walton et al (2020). Science) (Richter et al (2020). Nat Biotech), were designed to cover the target amino acids S33, S34, P35, S36 to enable an A-to-G (ABE) or C- to-T transition (CBE), respectively, that would change the wild-type amino acid to a variant. Per base editing condition, single guide RNA (sgRNA), or control scrambled sgRNA, was mixed with adenosine base editor (ABE) mRNA such as ABE8e-SpRY or ABE8e-NG or cytosine base editor (CBE) mRNA such as CBE4max-SprRY or CBE4max-NG mRNA (synthesized by TriLink) guided by the planned nucleotide transition (see Table 11), as well as with wildtype human cancer cells. More specifically, with Raji or JEKO-1 cells. Per base editing condition, a final concentration of 5 pM sgRNA was mixed with a final concentration of 10 or 20 ng / mL of the corresponding base editing mRNA, as well as with wildtype JEKO- 1 cells. Gene editing was assessed in JEKO-1 cells for the two most promising variants S34G and S34N based on an initial evaluation of the editing efficiency of all designed guides. Per condition, the mix of cells in electroporation buffer with gene editing reagents was then electroporated using the 4D Amaxa nucleofector (Lonza) following the manufacturer's recommendations. Electroporated cells were expanded for 5 days with medium renewal twice per week. Genomic DNA was isolated from bulk cells, the edited region was PCR amplified and the amplicons were sequenced via Next-generation Amplicon sequencing (150nt paired end sequencing, Illumina MiniSeq) 5 days post editing using standard protocols. Base editing efficiency, regarding variant generation as well as bystander events, was assessed for Next-generation sequencing reads via CRISPRESSO2 (Clement et al (2019). Nat Biotech). A CD52 KO was generated by disrupting the open reading frame in exon 1. The results of the editing, pre- and post-sorting, are shown in Figure 8. CD52 edited cells are sorted by flow cytometry to generate homogenously edited pools or single cell clones.

[0459] Base editing can infrequently lead to insertions or deletions at the target site, which could prevent Refmab binding like the anticipated edit, leading to these cells being sorted along with the cells that received the anticipated shielding edit. Therefore, sorted cells were assessed for frequency of alleles with the anticipated edit and alleles with an unwanted edit, such as insertion or deletion, via Sanger sequencing or Next-generation sequencing. Table 17:

[0460] Example 20: Gene editing of CD52+ human cell lines render human cells resistant to RefMabffl antibody binding and antibody-mediated Fcgamma activation

[0461] Genetically engineered JEKO-1 cells were generated as described in Example 19. Five days post-editing, JEKO-1 cells are sorted by flow cytometry to generate homogenously edited pools or single cells clones. Gene edited JEKO-1 cells are collected, stained with the appropriate fluorophore-labeled Refmabtl, to then undergo cell sorting for RefMabtfl non-binders (or binders for CD52 WT cells).

[0462] To assess post-sort whether gene edited JEKO-1 cells that express CD52 variant (S34G / S34N) or wildtype CD52 are shielded from antibody binding at equivalent CD52 expression levels, compared to JEKO-1 CD52 KO cells where CD52 is not expressed, 150000 cells were collected per staining condition, per stable cell line, and stained with increasing concentrations (0.01, 0.1, 1, 1, 50 pg / mL) of the appropriate fluorophore-conjugated Refmabtl (ab269823 AlexaFluor Lightning-Link conjugation kit). Additionally, 150000 cells were collected per stable cell line, per staining condition, and stained with 4 pg / mL of anti- CD52 4C8 antibody (NovusBio; NB100-66609A F488) to assess expression levels of CD52 variants and wild-type in comparison also to CD52 KO. The stained cell suspensions were analyzed using a cell analyzer (Agilent Novocyte Quanteon). Edited and shielded cells do not bind the Refmabtfl antibody paired with the shielding mutation, while wildtype, unedited cells do bind the Refmab #1 antibody. Simultaneously, CD52 expression levels on gene edited JEKO-1 cells were determined to be lower for variant S34G, but not S34N, compared to WT (Figure 9).

[0463] Antibody-dependent cellular cytotoxicity (ADCC) of RefMabtl on wildtype and variant CD52 expressing cells was extrapolated from an FcgammaRllla activation assay performed with the ADCC Reporter Bioassay, V Variant (Promega, ref G7015). On day 1, Jurkat / FcyRllla / NFAT-Luc CD52 KO ADCC effector cells were diluted in ADCC buffer (Gibco RPMI-1640 plus 0.5% of Hyclone low-IgG FBS) to a cell density of 6 E6 cells / mL and 25 pl_ was added per well in a white 96 well-plate (Corning costar #3197). Then 25 pL (25000 cells) was added per well of the gene edited post-sort JEKO-1 cells, expressing wild-type and variants (S34G, S34N) or no CD52 (KO) respectively. Then 25 pL of RefMab#l antibody diluted into ADCC buffer at 3X the final concentration (final concentration ranging from 0 to 50 pg / mL) was added to the respective conditions. The plate was then incubated for 22h at 37°C with 5% CO2. Luciferase activity is measured using the Bio-GloTM Luciferase Assay Reagent (G77941, Promega). On day 2, 75 pL of Reagent was added and cells were incubated for 10 min at RT in the dark with agitation. Luminescence is read with a plate reader. RefMab#l induced significant FcgammaRllla activation on the effector cells when co-cultivated with WT CD52 JEKO-1 cells. CD52 variant cells and CD52 knock-out cells caused no FcgammaRllla activation above background (effector cells only), indicating that cells expressing CD52 variants and knock-out are not susceptible to RefMabtl mediated ADCC (Figure 10) and thus shielding.

[0464] Example 21: Generation of CD52 variants by base editing in primary human T cells

[0465] Primary human T cells are isolated from leukopaks (CytoCare) via the TCT program employing the CD4 CD8 ab TS 520 kit, on the CiiniMacs Prodigy (Miltenyi). Alternatively, primary human T cells are isolated from the hCD34+ HSPC negative fraction subsequently to the hCD34+ HSPC isolation process (CiiniMacs Prodigy, TS310 kit, LP-34 program), employing the CiiniMacs Prodigy CD4 CD8 ab TS 520 and TCT program. Another approach includes isolation of primary human T cells from the hCD34+ HSPC negative fraction subsequently to the hCD34+ HSPC isolation process on the CiiniMacs Prodigy (TS310 kit, LP-34 program) according to manufacturer's recommendations on the autoMacs separator (Miltenyi). Isolated T cells are cryopreserved in CS5 or CS10 (StemCell).

[0466] T cells are thawed and resuspended in pre-warmed TexMACS (Miltenyi) medium containing TransAct and IL-2, as well as potentially IL-7 and IL-15 depending on the application. The T cells are cultured for 48 hours days prior to electroporation for gene editing purposes.

[0467] Certain CD52 variants are generated via base editing. Base editing single guide RNAs (sgRNAs; Synthego) with SpCas9-SpRY or SpCas9-NG PAM flexibility (NRN / NYN or NG, respectively) (Walton et al (2020). Science) (Richter et al (2020). Nat Biotech), are designed to cover the target amino acids S33, S34, P35, S36 to enable an A-to-G (ABE) or C-to-T transition (CBE), respectively, that would change the wild-type amino acid to an intended variant. Per base editing condition, single guide RNA (sgRNA), or control scrambled sgRNA, are mixed with adenosine base editor (ABE) mRNA such as ABE8e-SpRY or ABE8e-NG or cytosine base editor (CBE) mRNA such as CBE4max-SpRY or CBE4max-NG mRNA ( TriLink) guided by the planned nucleotide transition, as well as with wild-type human T cells. The T cells mixed with gene editing reagents are then electroporated using the GTx nucleofector (Maxcyte) following the manufacturer's recommendations. Electroporated cells are expanded for 4-5 or 8-10 days. At both time points, cell count and viability are measured, and phenotype is assessed via flow cytometry by staining for markers such as CD45, CD3, CD4, CD8, CD69, CD25. Genomic DNA is isolated from bulk cells, the edited region is PCR amplified and the amplicons are sequenced via Sanger sequencing (Microsynth) or Next-generation sequencing (in-house Illumina Mini-seq). Base editing efficiency, regarding variant generation as well as bystander events, is assessed for Sanger sequencing reads via Ed it R (Kluesner et al (2018). CRISPR Journal) and for Next-generation sequencing via CRISPRESSO2 (Clement et al (2019). Nat Biotech). CD52 variants which cannot be introduced by base editing are generated by homology-directed repair (HDR) and / or prime editing and / or other appropriate gene editing tools. CD52 knock-out cells are generated as well via gene editing by introducing a frameshift in the first coding exon. Our results show that edited T cells carrying respective CD52 variants had similar cell fitness compared to WT in term of cell viability, proportion of CD4 / CD8 T cells and fold expansion in culture (Figure 11A-B-C). The percentage of shielding evaluated by flow cytometry and genomic analysis demonstrate higher editing efficiency for the S34G variant then S34N or even KO (Figure 11D and E) with very low bystander activity on As and Cs adjacent to the targeted position (Figure 11F).

[0468] Example 22: Generation of CD52 variants by base editing in primary human CD34+ cells hCD34+ HSPCs are isolated from immobilized leukopaks (CytoCare) via the LP-34 process on the CiiniMacs Prodigy (Miltenyi). HCD34+ HSPCs are thawed and resuspended in prewarmed StemSpan AOF medium containing hSCF, hFlt3L, hTPO and potentially hlL-3.

[0469] CD52 variants are generated via base editing. Base editing single guide RNAs (sgRNAs; Synthego) with SpCas9-SpRY or SpCas9-NG PAM flexibility (NRN / NYN or NG, respectively) (Walton et al (2020). Science) (Richter et al (2020). Nat Biotech), were designed to cover the target amino acids S33, S34, P35, S36 to enable an A-to-G (ABE) or C-to-T transition (CBE), respectively, that would change the wild-type amino acid to a variant. Per base editing condition, single guide RNA (sgRNA), or control scrambled sgRNA, is mixed with adenosine base editor (ABE) mRNA such as ABE8eSpRY or ABE8e-NG or cytosine base editor (CBE) mRNA such as CBE4max-Spry or CBE4max-NG mRNA (TriLink) guided by the planned nucleotide transition, as well as with HSPCs. The HSPCs mixed with gene editing reagents are then electroporated using the GTx nucleofector (Maxcyte) following the manufacturer's recommendations. Electroporated cells are incubated at 37 degrees, 5% CO2 to recover. Two days post-electroporation, gene editing is assessed via flow cytometry using a fluorophore-coupled Refmabtfl as well as fluorescent antibodies targeting HSPC markers such as hematopoietic stem cell markers such as CD34, CD38, CD45RA, CD90, ITGA3 or EPCR. Also, genomic DNA is isolated from bulk cells, the edited region is PCR amplified and the amplicons are sequenced via Sanger sequencing (Microsynth) or Nextgeneration sequencing (in-house Mini-seq). Base editing efficiency, regarding variant generation as well as bystander events, is assessed for Sanger sequencing reads via EditR (Kluesner et al (2018). CRISPR Journal) and for Next-generation sequencing via CRISPRESSO2 (Clement et al (2019). Nat Biotech). CD52 variants which cannot be introduced by base editing may be generated by homology-directed repair (HDR) and / or prime editing and / or other appropriate gene editing tools. CD52 knock-out cells are generated as well via gene editing by introducing a frameshift in the first coding exon. Our results show that edited HSC cells with variants had similar cell fitness compared to WT in term of cell viability and proportion of CD34 subsets {Figure 12A and B). The percentage of editing measured by genomic analysis demonstrates higher editing efficiency for the S34G variant than S34N (Figure 12C) with very low bystander activity on As and Cs adjacent to the targeted position (Figure 12D).

[0470] Example 23: Gene editing of primary human T cells shields from RefMabtl antibody binding, antibody-mediated Fcgamma activation and killing

[0471] Primary human T cells were genetically engineered for CD52 variants as described in Example 21. Six to 12 days post-editing, T cells are sorted by flow cytometry to generate homogenously edited pools or single cells clones. To assess if gene edited T cells are shielded from antibody binding, cells are collected, stained with the appropriate fluorophore-labeled Refmabtl, as well as for T cell markers as previously mentioned, to then undergo cell sorting.

[0472] To assess whether T cells that express CD52 variant or wildtype CD52 or no CD52 (KO) are shielded from RefMabtl binding, at comparable CD52 expression levels, 150000 cells were collected per stable cell line, per staining condition, and stained with increasing concentrations (0.01, 0.1, 1, 1, 50, 50 pg / mL) of the appropriate fluorophore-conjugated Refmabtl (ab269823 AlexaFluor Lightning-Link conjugation kit). Additionally, 150000 cells were collected per editing condition (variants wildtype or KO), and stained with 4 pg / mL of anti-CD52 (4C8) antibody to assess expression levels of CD52 variants and wild type. The stained cell suspensions were analyzed using a cell analyzer (Agilent Novocyte Quanteon). Edited and shielded cells do not bind the Refmabtl antibody paired with the shielding mutation, while wildtype, unedited cells do bind the Refmab tl antibody (Figure 13). Simultaneously, CD52 expression levels were determined to be similar for CD52 variants (S34G, S34N) compared to CD52 WT (Figure 13A and B). Antibody-dependent cellular cytotoxicity (ADCC) of RefMabffl on wildtype and variant CD52 expressing cells is extrapolated from an FcgammaRllla activation assay performed with the ADCC Reporter Bioassay, V Variant (Promega, ref G7015). On day 1, Jurkat / FcyRllla / NFAT-Luc CD52 KO ADCC effector cells are diluted in ADCC buffer (Gibco RPMI-1640 plus 0.5% of Hyclone low-IgG FBS) to a cell density of 6 E6 cells / mL and 25 pL is added per well in a white 96 well-plate (Corning costar #3197). Then 25 pL (25000 cells) is added per well of the gene edited post-sort T cells, expressing wild-type and variants (S34G, S34N) or no CD52 (KO) respectively. Then 25 pL of RefMab#l antibody diluted into ADCC buffer at 3X the final concentration (final concentration ranging from 0 to 50 pg / mL) is added to the respective conditions. The plate is then incubated for 22h at 37°C with 5% CO2. Luciferase activity is measured using the Bio-GloTM Luciferase Assay Reagent (G77941, Promega). On day 2, 75 pL of Reagent is added and cells are incubated for 10 min at RT in the dark with agitation. Luminescence is read with a plate reader. RefMab#l induces significant FcgammaRllla activation on the effector cells when co-cultivated with WT CD52 T cells. CD52 variant cells and CD52 knock-out cells cause no FcgammaRllla activation above background (effector cells only), indicating that cells expressing CD52 variants and knock-out are not susceptible to RefMab#l mediated ADCC and thus shielding.

[0473] To measure complement-dependent cytotoxicity (CDC) of RefMab#l, wildtype and variant CD52 cells are plated in a microtiter plate. Different concentrations of RefMab#l as well as human complement sera are added to the cells. The mixture is incubated for 6 hrs at 37°C 5%CO2. CytoTox-Glo reagent (Promega) is used to quantify the lysed cells. RefMab#l induces significant complement-mediated cell lysis when cultivated with wt CD52 cell lines. CD52 variant and knock-out cells show no or only strongly reduced complement-mediated cell killing indicating that these cells are not susceptible to RefMab#l mediated CDC. Digitonin and Raji cells incubated with lug / ml of rituximab are used as positive controls.

[0474] Example 24: Wildtype and variant soluble CD52 suppress the activation and proliferation of CD4+ T cells in response to TCR activation PBMCs, alternatively pan-T cells or CD4+ T cells are incubated for up to 48 hrs with soluble anti-CD3 (+ / - anti-CD28) in the presence of wildtype soluble CD52 or CD52 variants or the negative control. Expression of the activation markers CD69 and CD25 is assessed on CD4+ T cells via flow cytometry, and absolute CD4+ numbers are quantified by flow cytometry. As compared to the negative control, wildtype and variant CD52 suppress T cell activation, thereby inhibiting CD4+ T cell proliferation after activation and block the increase in cell surface CD69 and CD25.

[0475] Example 25: Wildtype and variant soluble CD52 suppress B cell receptor (BCR) signaling in JeKo-1 mantle cell lymphoma cells

[0476] JeKo-1 cells are base edited to carry variant CD52. CD52 knock-out JeKo-1 cells are used as negative control, wildtype CD52 JeKo-1 cells as positive control. To address the effect of wildtype and variant CD52 on BCR-signaling, the phosphorylation levels of signaling molecules downstream of the BCR is measured, including phospho-BTK, phospho-AKT, phospho-SYK, phospho-PLC-y2 and phospho-Lyn following BCR engagement. Whereas there are significantly increased levels of phospho-proteins in the CD52 knock-out cells, wt and variant CD52 cells retain their suppressive function and exhibit significantly lower phospho-protein levels.

[0477] Example 26: Wildtype and variant CD52 inhibit phagocytosis

[0478] To address the effect of the various CD52 receptors, CD52 wildtype, variants or knock-out on the phagocytosis rate of T cells (target cells) by human macrophages (effector cells), in vitro phagocytosis assays were set up and performed with two independent T cell donors at 1:2 effector to target cell ratio. To this end, human primary T cells isolated from immobilized leukopaks (CytoCare) were base edited to carry variant CD52. CD52 knock-out T cells were used as negative control, corresponding wildtype CD52 cells as positive control. First, edited T cells were fluorescently labeled (e.g., with phRodo Cell Labeling kit from Sartorius) for lh at 37°C. After incubation, stained T cells were washed and prepared for co-cultivation with THP-1 derived human macrophages in cell culture medium, previously seeded in 96 well flat bottom plates at density of 20.000cells / well and differentiated for 3- 4 days in the presence of phorbol 12-myristate-13-acetate (PMA) at lOOng / mL. After a short spin, plates were incubated for 2 hours in the live imaging system Incucyte (Sartorius). 20x resolution images of the cells were taken every 10-15 min for brightfield and red fluorescent signal. Red fluorescent signal was observed when target cells were phagocytosed and reached an acidic (low pH) compartment like the lysosome of the macrophages. At the end of the incubation time, plates were treated with Accutase for lOmin at 37°C to detach macrophages from the plate and stained with anti-CDllb PE-Cy7 antibody to differentiate macrophages from T cells by flow cytometry. The percentage of phagocytosis was calculated as percentage of CDllb+ macrophages that are also positive for phRodo Red dye quantified by flow cytometry. In addition, the median fluorescence intensity of phRodo Red dye (PE channel) for the macrophage CDllb-i- population was also calculated. Results are shown in Figure 14A. From the Incucyte analysis, the number of phRodo+ counts after 2 hours of incubation per image was calculated per well and shown in Figure 14B. Overall, these results demonstrate that CD52 knock-out T cells are more efficiently phagocytized by human macrophages compared to T cells expressing CD52 wildtype or variants.

[0479] Example 27: Glycosylation status of wildtype and variant soluble and processed CD52

[0480] Weak anion exchange chromatography (WAX) was performed using Agilent Bio WAX column (Agilent Bio WAX NP3 SS 3um, non prous, 4.6x50 mm) on an Agilent 1260 II HPLC system. Non-GPI containing CD52 protein (generated as described in Example 14) was injected at a concentration of 0.25-1 mg / mL. The different species were separated according to their pl using a flow rate of 0.5-1 mL / min and a gradient from 0% to 100% 0.5 M sodium chloride in 20 mM sodium phosphate buffer pH 6.0 over 14 min. The separation was monitored via absorbance at 220 and 280 nm. The peaks were grouped and integrated using manual integration in the Agilent software. The relative area at 220 nm of the samples is depicted in Figure 15. Smaller or absent peaks between 7.0-7.8 min were observed for variants S34N and S34G, while the peak at 6.4 min increased compared to the wildtype protein.

[0481] Example 28: In vivo depletion of wildtype CD52 and protection of variant CD52 cells

[0482] Human CD34+ HSPCs are isolated from immobilized leukopaks (CytoCare) via the LP-34 process on the CiiniMacs Prodigy (Miltenyi). The human CD34+ HSPCs are base edited to introduce CD52 variants as described above. Electroporation-only / mock-edited HSPCs are used as wildtype CD52 control cells. Two days after editing ca. 1 mio HSPCs are transplanted each into immunodeficient mice (e.g., strain NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tmlWjl / ThomJ), NSG (NOD.Cg-Prkdc scid Il2rg tmlWjl / SzJ), NSG-SGM3 (NOD.Cg- Prkdcscid H2rgtmlWjl Tg(CMV-IL3,CSF2,KITLG)lEav / MloySzJ) or NSG-SGM3-IL-15 (NOD.Cg- Prkdcscid H2rgtmlWjl Tg(CMV-IL3,CSF2,KITLG)lEav Tg(IL15)lSz / J). Ca 14-20 weeks after engraftment alemtuzumab and / or alemtuzumab-ADC is administered to the mice. Depletion and shielding of CD52 wildtype and variant human cells are monitored in blood, bone marrow and spleen using flow cytometry and next generation sequencing. The differentiation of CD52-edited HSPCs into lymphocytes are assessed by quantifying human B- and T-cells in the humanized mice.

[0483] Alternatively, human pan-T-cells are isolated from buffy coats. The primary T-cells are base edited to introduce CD52 variants, and a CAR construct directed against CD123, CD20 or CD33 is transduced through lentiviral particles. Immunodeficient mice (strains see above) are humanized with ca. 1 mio CD34+ human HSPCs (+ / - base editing to introduce variants of CD52). About 14-20 weeks after engraftment up to 10 mio CAR T cells per mouse are administered. Depletion and shielding of CD52 wildtype and variant human cells are monitored in blood, bone marrow and spleen using flow cytometry and next generation sequencing.

[0484] Example 29: Fc engineering of RefMabtl to decrease in vivo half-life

[0485] Alemtuzumab is known to result in long-term depletion of especially ! cells (Trzonkowski et al. (2008) Am J Transplantation; Li et al. (2018), Clinical & Experimental Immunology), which results in severe immune suppression and increased infection rates. This might be a result of the terminal half-life of alemtuzumab, which was measured to be between 2 and 3 weeks (Rebello et al. (2001) Cytotherapy) and taking up to 30 days until the antibody becomes undetectable in serum (Li et al. (2018), Clinical & Experimental Immunology). This results in deep depletion of T lymphocytes. To allow faster recovery of T lymphocytes after lymphodepletion especially in transplant patients a derivative of alemtuzumab / RefMabftl was generated with reduced FcRn binding and thus with a reduced in vivo half-life.

[0486] This is shown by an in vivo pharmacokinetic study in human FcRn transgenic SCID mice (strain: B6. Cg-FcgrttmlDcrPrkdc5CldTg(FCGRT)32Dcr / DcrJ mice; The Jackson Laboratory). Alemtuzumab with human wildtype IgGl isotype and lgGl-H435A is administered at a single dose of 10 mg / kg to mice aged 8-10 weeks. Each cohort consists of 5 mice. Mice are bled retro-orbitally at specified timepoints (5min, 6h, 24h, 48h, 3d, 7d, 14d, 21d, 28d and 35d post-test article administration) and the blood is processed to serum. The serum is used for human IgG quantification by ELISA.

Claims

CLAIMS1. A mammalian cell or a population of cells expressing a first isoform of CD52 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD52, wherein said cell expressing said first isoform comprises a polymorphism or genetically engineered allele that is not present in the genome of the patient having cells expressing said second isoform of CD52, and which is characterized by at least one substitution of an amino acid at position Q31, T32, S33, S34 or P35 of SEQ ID NO: 1.

2. The mammalian cell or population of cells for use according to claim 1 wherein said first and second isoform are substantially functionally identical, are expressed at the same or substantially the same level, are phosphorylated upon binding of sialic acids to the same or substantially the same degree, decrease phosphorylation of kinases in immune cells to the same or substantially the same degree, inhibit phagocytosis to the same or substantially the same degree and / or have the same or substantially the same N- and O-glycosylation pattern.

3. The mammalian cell or population of cells, preferably hematopoietic stem cells for use according to any one of the preceding claims wherein said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent comprising at least a first antigen-binding region that binds specifically to said second isoform of CD52 to specifically deplete patient cells expressing said second isoform of CD52, preferably to restore normal hematopoiesis after immunotherapy in the treatment of hematopoietic disease, and preferably in the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF),blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other myeloproliferative neoplasms, more preferably wherein said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of CD52 to avoid eventual severe side effects such as graft-versus-host disease due to the transplantation.

4. The mammalian cell or population of cells, preferably hematopoietic stem cells for use according to any one of the preceding claims, wherein said medical treatment comprises administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of CD52 to said patient in need thereof in combination with a therapeutically efficient amount of a depleting agent comprising at least a second antigen-binding region that binds specifically to said first isoform of CD52 to specifically deplete transferred cells expressing said first isoform of CD52, preferably for use in adoptive cell transfer therapy.

5. The mammalian cell or population of cells for use according to claim 3 or 4, wherein said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10.

6. The mammalian cell or population of cells for use according to any one of the preceding claims, wherein said substitution is at amino acid position S33, S34 or P35 of SEQ ID NO: 1, preferably wherein amino acid S33 is substituted with A, C, D, E, F, G, H, I, K, L, N, P, R, V, W or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N,Q, R, S, T, V, W, Y, preferably wherein amino acid S33 is substituted with A, N, G or L, amino acid S34 is substituted with T, N, A, G or L, and / or amino acid P35 is substituted with T, S, N, A, G or L, and most preferably, wherein said amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

7. The mammalian cell or population of cells for use according to claim 3 or 4, wherein said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 is SEQ ID NO: 14 and VHCDR3 is SEO ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO: 17, VLCDR3 is SEQ ID NO: 18.

8. The mammalian cell or population of cells for use according to claim 7, wherein said substitution is at amino acid position Q31, T32, S33, S34 or P35 of SEQ ID NO: 1, preferably, wherein amino acid Q31 is substituted with V, L or T, wherein amino acid T32 is substituted with I or P, wherein amino acid S33 is substituted with A, C, D, E, F, H, I, K, L, N, P, R, T, V, W or Y, amino acid S34 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y, and / or amino acid P35 is substituted with A, C, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y, preferably wherein amino acid S33 is substituted with N, T or L, amino acid S34 is substituted with G, L, or N, and / or amino acid P35 is substituted with A, G, L or S, and most preferably wherein said amino acid substitution is selected from S33N, S34G, S34N, P35S, P35A and P35L.

9. The mammalian cell or population of cells for use according to any one of claims 3-8, wherein said depleting agent is an antibody, antibody-drug conjugate or an immune cell, preferably a T-cell bearing a chimeric antigen receptor (CAR) comprising a first antigenbinding region which binds specifically to said second isoform and does not bind or binds substantially weaker to said first isoform.

10. The mammalian cell or population of cells for use according to any one of the preceding claims, wherein said first isoform of CD52 is obtained by in vivo or ex vivo modifying the nucleic acid sequence encoding said first isoform of CD52 by gene editing, preferably by introducing into a cell a gene editing enzyme capable of inducing site-specific mutations(s) within a target sequence encoding a surface protein region involved in the binding of agent comprising at least a first antigen-binding region.

11. The mammalian cell or population of cells for use according to any one of the preceding claims, wherein said cell or population of cells is an immune cell, preferably a T-cell, bearing a chimeric antigen receptor (CAR).

12. A pharmaceutical composition comprising a mammalian cell, preferably a hematopoietic stem cell or an immune cell such as T-cell, as defined in any one of claims 1 to 11, and preferably a depleting agent as defined in any one of claims 3 to 11 and a pharmaceutically acceptable carrier.

13. A depleting agent for use in selectively depleting the host cells in a patient in need thereof wherein said patient's native cells express a second isoform of CD52 and wherein said depleting agent comprises at least a first antigen-binding region which binds specifically to said second isoform of CD52, wherein said first antigen-binding region of said depleting binds specifically to an epitope including the amino acids S33, S34 and / or P35 of SEQ ID NO: 1, and wherein said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 5, VHCDR2 is SEQ ID NO: 6 and VHCDR3 is SEQ ID NO: 7; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 8, VLCDR2 is SEQ ID NO: 9, VLCDR3 is SEQ ID NO: 10.

14. A depleting agent for use in selectively depleting the host cells in a patient in need thereof wherein said patient's native cells express a second isoform of CD52 and wherein said depleting agent comprises at least a first antigen-binding region which binds specifically to said second isoform of CD52, wherein said first antigen-binding region of said depleting binds specifically to an epitope including the amino acids Q31, T32, S33, S34 and / or P35 of SEQ ID NO: 1, and wherein said first antigen-binding region comprises an antigen binding region which has the same epitope specificity as an antigen binding region comprising: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3 wherein VHCDR1 is SEQ ID NO: 13, VHCDR2 isSEQ ID NO: 14 and VHCDR3 is SEQ ID NO: 15; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1,VLCDR2 and VLCDR3 wherein VLCDR1 is SEQ ID NO: 16, VLCDR2 is SEQ ID NO:17, VLCDR3 is SEQ ID NO: 18.

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