Identifiable cell surface protein variants of CD117 for use in cell therapy

Abstract

The present disclosure relates to the use of cells having identifiable surface proteins that have engineered or naturally occurring mutations but functional surface proteins for use in therapy. The present invention also relates to the use of cells having identifiable CD117 surface protein variants but functional surface proteins for use in therapy, particularly adoptive cell therapy.

Description

[Technical field]

[0001] summary The present disclosure relates to the use of cells having identifiable surface proteins that have engineered or naturally occurring mutations but functional surface proteins for use in therapy. The present invention also relates to the use of cells having identifiable CD117 surface protein variants but functional surface proteins for use in therapy, particularly adoptive cell therapy.

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

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

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

[0005] In previous patent applications, we have shown that single amino acid differences in surface protein variants can be engineered into hematopoietic cells to alter their antigenicity and be recognized by specific and selective antibodies (WO 2017 / 186718, WO 2018 / 083071). In contrast to CD33 KO cells, the surface protein variants in these cells retain their normal expression and function, making it possible to target surface proteins with important non-redundant functions.

[0006] CD117 (also called c-kit or stem cell factor receptor (SCRF)) is a single transmembrane receptor tyrosine kinase that binds to the ligand stem cell factor (SCF). SCF activates its tyrosine kinase activity and induces homodimerization of CD117, which signals through both the PI3K-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998 152(5):1 259).

[0007] CD117 was first discovered as an oncogene and has been studied in the field of oncology (see, for example, Stankov et al. (2014) Curr Pharm Des. 20:2849-80). CD117 is highly expressed on hematopoietic stem and progenitor cells (HSCs). This expression pattern makes CD117 a potential target for conditioning across a wide range of diseases (Russkamp et al. Exp. Hematol. (2021) 95:31-45; Czechowicz et al. Nat Commun (2019) 10:617). However, there remains a need for anti-CD117-based therapies that are effective in conditioning patients for transplants, such as bone marrow transplants.

[0008] The present disclosure aimed to identify amino acid residues of CD117 that are exposed on the cell surface and can be substituted such that a) the function of CD117 is not altered or at least not substantially altered, i.e., the mutants of CD117 are functionally indistinguishable from the wild-type version of CD117, and b) they bind to the wild-type version of CD117, such as antibodies or CAR T cells, but show substantially reduced or no binding to the modified version of CD117, i.e., the mutants of CD117 are immunologically distinguishable from the wild-type version of CD117. Most single amino acid substitutions in any given target protein only affect the binding of the moiety if the amino acid substitution is part of or close to the epitope of the binding moiety. As will be appreciated, many single amino acid substitutions that affect the binding of a binding moiety to a target antigen also affect the functionality of the target antigen. Thus, it is a highly sophisticated and unpredictable challenge to identify amino acid substitutions that satisfy both requirements of affecting the binding of a moiety to a target antigen and at the same time not affecting or not substantially affecting its function.

[0009] Several anti-CD117 moieties are known in the art, some of which are currently under development. Antibody SR-1 was first isolated from a hybridoma (WO1992017505). Humanized versions of SR-1 have been produced (WO2007127317; WO2020112687). Anti-CD117 drug conjugates are described in WO2016020791. Other anti-CD117 antibodies are described in WO2015050959 and WO2019084064. Certain anti-CD117 antibodies are also commercially available, such as antibody 104D2 Dianova (#117PE-100T). These and other anti-CD117 moieties can be used in the context of the present disclosure. WO2021041945 discloses genomic alterations in antigens, including CD117, generated by base editing. However, these antigenic alterations were not characterized, and in particular the biological function of the variants was not tested. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] International Publication No. 2018 / 160768 [Patent Document 2] International Publication No. 2017 / 186718 [Patent Document 3] International Publication No. 2018 / 083071 [Patent Document 4] International Publication No. 1992017505 [Patent Document 5] International Publication No. 2007127317 [Patent Document 6] International Publication No. 2020112687 [Patent Document 7] International Publication No. 2016020791 [Patent Document 8] International Publication No. 2015050959 [Patent Document 9] International Publication No. 2019084064 [Patent Document 10] International Publication No. 2021041945 [Non-patent literature]

[0011] [Non-Patent Document 1] Perna et al.,Cancer Cell(2017)32:506-519 [Non-Patent Document 2] Gill SIBest practice&Research Clinical Hematology,2019 [Non-Patent Document 3] Kim et al.2018.Cell.173:1439-53 [Non-Patent Document 4] Wiβfeld et al. Glia(2021)69:1393-1412 [Non-Patent Document 5] Borot et al.2019.PNAS.116:11978-87 [Non-Patent Document 6] Humbert et al.2019.Leukemia.33:762-808 [Non-Patent Document 7] Orlando et al.2018 Nat Med 24:1504-6) [Non-Patent Document 8] Ruella et al.2016 J Clin Invest 126:3814-26 [Non-Patent Document 9] Kindblom et al.,Am J.Path.1998 152(5):1 259) [Non-Patent Document 10] Stankov et al.(2014)Curr Pharm Des.20:2849-80 [Non-Patent Document 11] Russkamp et al.Exp.Hematol.(2021)95:31-45;Czechowicz et al.Nat Commun(2019)10:617 Summary of the Invention

[0012] One of the objectives of the present disclosure is to develop safer methods for treating malignancies, particularly cancer, hematological malignancies, and myeloid diseases. Thus, the inventors sought mutations of the surface protein CD117 that are immunologically distinguishable while retaining or substantially retaining normal function, and in which the amino acid changes are derived from single or multiple amino acid or nucleotide mutations. In particular, the inventors have identified rationally designed naturally occurring mutants of CD117 and shown that these mutations alter the antigenicity of CD117 to specific antibodies while retaining its normal expression and function, particularly binding to SCF, SCF-dependent proliferation, and / or SCF-dependent phosphorylation.

[0013] The present disclosure relates to a mammalian cell or population of cells expressing a first isoform of CD117 for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of CD117, said cells expressing said first isoform comprise genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of said patient having cells expressing said second isoform of CD117, and said first and second isoforms are functional. Alternatively, said first isoform is generated via RNA editing.

[0014] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells, preferably hematopoietic stem cells, for use in medical treatment in a patient in need thereof, said medical treatment comprising administering to said patient in need thereof a therapeutically effective amount of said cell or population of cells expressing a first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent comprising at least a first antigen binding region that specifically binds to a second isoform of CD117 to specifically deplete the patient's cells expressing the second isoform of CD117, preferably to restore normal hematopoiesis after immunotherapy in the treatment of a hematopoietic disease, preferably a malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), B-acute lymphoblastic leukemia (B-ALL), or mastocytosis.

[0015] In other embodiments, the medical treatment relates to restoration of hematopoietic or immune function in genetic disorders of the hematopoietic or immune system, such as severe combined immunodeficiency syndrome (SCID), sickle cell disease (SCD), beta thalassemia, Fanconi anemia, or Diamond-Blackfan anemia.

[0016] In other embodiments, medical treatment relates to the restoration of normal function in genetic diseases that do not originate from the hematopoietic and immune systems, but that can be treated by the use of modified hematopoietic cells.

[0017] In other embodiments, the medical treatment relates to restoration of normal immune function in autoimmune diseases such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) or multiple sclerosis (MS).

[0018] In another particular embodiment, the present disclosure relates to a mammalian cell or population of cells for use in medical treatment in a patient in need thereof, said medical treatment comprising administering to said patient in need thereof a therapeutically effective amount of said cell or population of cells expressing said first isoform in combination with a therapeutically effective amount of a depleting agent comprising at least a second antigen binding region that specifically binds to said first isoform, preferably for use in adoptive cell transfer therapy, more preferably for the treatment of a malignant hematopoietic disease, such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (CML), B-acute lymphoblastic leukemia (B-ALL) or systemic mastocytosis, again more preferably said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of a surface protein to avoid eventual severe side effects such as graft-versus-host disease from transplantation.

[0019] In another aspect, the present disclosure relates to a pharmaceutical composition comprising mammalian cells, preferably hematopoietic stem cells or immune cells, such as T cells as described above, and preferably a depleting agent, and a pharma- ceutically acceptable carrier.

[0020] The present disclosure relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD117, wherein the patient's native cells express a second isoform of CD117, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind to the second isoform of CD117. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD117.

[0021] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD117, wherein the patient's native cells express a second isoform of CD117, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind to the second isoform of CD117, wherein the first and second isoforms are substantially functionally identical. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD117.

[0022] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD117, wherein the patient's native cells express a second isoform of CD117, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind to the second isoform of CD117, and wherein the first and second isoforms bind SCF, resulting in SCF-dependent proliferation and / or resulting in SCF-dependent phosphorylation. In certain embodiments, the depleting agent binds substantially weaker to the second isoform of CD117.

[0023] The present disclosure also provides a depleting agent for use in preventing or reducing the risk of serious side effects in a patient administered cells expressing a first isoform of CD117, wherein the patient's native cells express a second isoform of CD117, and the depleting agent comprises at least a second antigen binding region that specifically binds to the first isoform of CD117 and does not bind to the second isoform of CD117, and wherein the polymorphism or engineered allele is located at positions E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y206, Y305, Y308, Y409, Y500, Y510, Y601, Y702, Y711, Y720, Y730, Y740, Y750, Y760, Y770, Y780, Y790, Y810, Y820, Y830, Y840, Y850, Y860, Y870, Y880, Y890, Y910, Y920, Y930, Y940, Y950, Y960, Y970, Y980, Y990, Y100, Y101, Y102, Y103, Y104, Y105, Y106, Y107, Y108, Y109, Y110, Y111, Y112, Y113, Y114, Y115, Y116, Y117, Y118, Y119, Y120, Y121, Y122, Y123, Y125, Y127, Y127, Y130, Y140, Y141, Y142, Y143 1, more preferably at least one substitution of an amino acid at positions E73, V120, D121, R122, S123, K127, K193, S239, Y259 or S261 of SEQ ID NO: 1, more preferably at least one substitution of an amino acid at positions E73, D121, R122, S123, S239, Y259 or S261 of SEQ ID NO: 1, most preferably at least one substitution of an amino acid at positions E73, D121 or S123 of SEQ ID NO: 1. In a particular embodiment, said depleting agent binds substantially weaker to said second isoform of CD117.

[0024] The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of serious side effects in a patient administered cells expressing a first isoform of CD117, wherein the patient's native cells express a second isoform of CD117, and wherein the depleting agent comprises at least a second antigen binding region that specifically binds to said first isoform of CD117 and does not bind to said second isoform of CD117, and wherein amino acid residue E73 is substituted with an amino acid selected from the group consisting of K, L, Q, G, A, Y, R, F, I, M, P and W, preferably selected from the group consisting of K, L, Q, G, A, Y and R, more preferably selected from K, Y, R, or residue E73 is deleted. and / or residue D121 is substituted with S, V, Y, H, K, R or T, more preferably with Y, H, K, R or T, most preferably with H or K, and / or residue S123 is substituted with V, I, L, P, F, Y, M, D, E, K or R, more preferably with P, F or K, most preferably with K, and / or residue S239 is substituted with an amino acid selected from the group consisting of H and K, and / or residue Y259 is substituted with an amino acid selected from the group consisting of E, A, G, P, C and H, preferably with P, A or G, most preferably with A, and / or residue K193 is substituted with an amino acid selected from the group consisting of G, T, M, D and E. In a particular embodiment, said depleting agent binds substantially weaker to said second isoform of CD117.

[0025] The present disclosure also relates to a method for improving the engraftment of hematopoietic stem cell transplantation. Conditioning (HSC depletion) prior to hematopoietic stem cell transplantation (HSCT) is used to promote engraftment. In fact, the conditioning effect is associated with improved engraftment. One drawback of myeloablative (toxic) conditioning is that it can result in increased levels of chimerism. Avoiding toxic conditioning is an important goal that can be achieved with the present disclosure. Current methods for conditioning include the use of intravenous busulfan. Busulfan is a DNA alkylating drug that was initially designed to treat blood diseases such as acute myeloid leukemia (AML). However, busulfan carries significant risks of side effects, including sterility, primary or secondary malignancies, and additional acute and chronic toxicity.

[0026] The present disclosure also relates to an anti-CD117 agent that competes for binding to CD117 or a variant of CD117 with an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22. In a particular embodiment, said antigen binding region comprises the following mutations: a) an asparagine to glutamic acid substitution in the VLCDR1 region (SEQ ID NO: 20); b) a substitution of aspartic acid with glutamic acid in the VLCDR3 region (SEQ ID NO: 22), and optionally c) The second asparagine in the VLCDR3 region (SEQ ID NO: 22) is substituted with a lysine. Preferably, the anti-CD117 agent does not bind to the E73K mutant of CD117. Also preferably, the anti-CD117 agent does not bind to the D121K mutant of CD117. Also preferably, the anti-CD117 agent does not bind to the S123K mutant of CD117.

[0027] The present disclosure also relates to an anti-CD117 agent that competes for binding to CD117 or a variant of CD117 with an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 25, VHCDR2 is SEQ ID NO: 26 and VHCDR3 is SEQ ID NO: 27; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 28, VLCDR2 is SEQ ID NO: 29 and VLCDR3 is SEQ ID NO: 30.

[0028] The present disclosure also relates to an anti-CD117 agent that competes for binding to CD117 or a variant of CD117 with an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 33, VHCDR2 is SEQ ID NO: 34 and VHCDR3 is SEQ ID NO: 35; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 36, VLCDR2 is SEQ ID NO: 37 and VLCDR3 is SEQ ID NO: 38. [Brief description of the drawings]

[0029] [Figure 1]Figure 1 shows binding of anti-CD117 antibodies to HEK-293T cells transfected with human wild-type CD117 or empty vector. Serial dilutions of each antibody were tested for immunoreactivity by flow cytometry. All six antibodies in MAb format bind to human CD117 in a concentration-dependent manner. CD117-negative HEK-293T cells (transfected with empty vector) did not show any antibody binding. [Diagram 2] Figure 2 shows binding of anti-CD117 Fab fragments to HEK-293T cells transfected with human wild-type CD117 or empty vector. Similar to the full-length antibody, the Fab fragments also bind to human CD117 in a concentration-dependent manner. CD117-negative HEK-293T cells (transfected with empty vector) did not show any antibody binding. [Diagram 3] The results of an alanine scan against the human CD117 extracellular domain for each of the six antibodies tested are shown. The mean binding values ​​determined by flow cytometry for each mutant CD117 clone were plotted as a function of expression. Clones identified as important for antibody binding are shown in grey circles. Secondary clones, i.e. clones that did not meet the initially set threshold but whose reduced binding activity and proximity to critical residues suggested that the mutant residues may be part of the antibody epitope, are shown in open circles. [Figure 4] Figure 1 shows the aggregation behavior of selected recombinant purified CD117 mutants. The line indicates the monomer content of wild-type CD117 recombinant purified protein. The monomer content was estimated from preparative size-exclusion chromatograms. The monomer content of most mutants was acceptable. ND stands for "non-determinable." [Diagram 5] Figure 1 shows the production yields obtained for selected recombinant purified CD117 mutants. The line indicates the yield of wild-type CD117. The production yields of all mutants except for a very few mutants were sufficiently high. [Figure 6]Figure 1 shows the t1 / 2 melting temperatures measured for selected recombinant purified CD117 mutants. The line indicates the melting temperature of wild type CD117. For one mutant, V120P, two melting temperatures were measured. [Figure 7] Figure 2 shows the binding of antibody Refmab#1 to selected recombinant purified variants of human CD117 by comparing the nm shift at the end of association with WT. [Figure 8] Figure 1 shows the binding affinity of antibody Refmab#1 to selected recombinant purified variants of human CD117. Panel A: The KD of all variants to Refmab#1 was determined. Filled circles indicate the measured KD determined from two individual measurements, open circles indicate a nm shift below 0.1 nm or no shift was observed, binding affinity higher than 2 μM. Error bars indicate standard deviation. Panel B: The nm shift at the end of association (600 s) is plotted against the antigen concentration used, which ranges from 2000 to 5 nM. Mutants D121K, S123K and S123E do not show any binding up to 2000 nM. Error bars indicate standard deviation of two individual measurements. [Figure 9] Binding of selected recombinant purified mutants of human CD117 to SCF is shown. The increase in KD of binding of SCF to wild-type human CD117 (measured using construct CD117 D1-2-3, which has domains 1, 2 and 3 with binding sites for SCF) is shown. "Not determinable" means that binding was observed but the KD could not be determined, and "No binding" indicates that no binding was observed. [Figure 10] Figure 1 shows the binding of recombinant purified selected mutants of human CD117 to SCF. The increase in KD for binding of SCF to wild-type human CD117 (measured with construct CD117 D1-2-3, which has domains 1, 2 and 3 with binding sites for SCF) is shown. Binding was analyzed by using seven concentrations of CD117 mutants. Error bars indicate the standard deviation from two individual measurements. A KD increase of less than 1 corresponds to stronger SCF binding of the mutant compared to CD117 WT, and a KD increase of more than 1 corresponds to weaker SCF binding of the mutant compared to CD117 WT. [Figure 11] We demonstrate that wild-type TF-1 cells, as well as cells carrying an E73K knock-in of CD117, proliferate when stimulated with 100ng / ml SCF. Increasing concentrations of Refmab#1 decrease proliferation in wild-type cells but not in E73K cells. Knock-in cells, designated "KI1" and "KI2," were obtained from two independent knock-in experiments and subsequently FACS-sorted to obtain pure E73K knock-in populations. [Figure 12] We demonstrate that SCF-induced phosphorylation can be blocked by an antibody (Refmab#1) in wild-type CD117 TF-1 cells, whereas SCF-induced phosphorylation is not affected by the same antibody in E73K mutant of CD117 TF-1 cells. CD117 knockout (ko) TF-1 cells do not show SCF-induced CD117 phosphorylation. [Figure 13] A map showing the binding of tested crRNAs to position E73 is shown. [Figure 14] A map showing binding of tested crRNAs to positions 120-123 is shown. [Figure 15] Qualitative analysis of the binding of antibodies Refmab#2 and Refmab#3 to selected recombinant purified variants of human CD117. "Not determinable" means that binding was observed but the KD could not be quantified due to biphasic behavior. [Figure 16] Figure 2 shows that RefMab#1 had a significant inhibitory effect on the proliferation of wild-type TF-1 cells, whereas proliferation of TF-1 cells with knock-ins of all CD117 mutants tested was not affected by RefMab#1. [Figure 17] Figure 2 shows that binding of Refmab#1 is completely abolished in TF-1 cells expressing CD117 mutants E73K, E73Y, D121K and S123K, whereas wild type TF-1 cells expressing wt CD117 bind. [Figure 18]Figure 2 shows SCF-dependent phosphorylation of CD117 at Tyr719 in two independently generated knock-in populations of wild-type TF-1 cells and the CD117 mutant E73K. The CD117 mutants D121K and S123K behaved similarly (data not shown). [Figure 19] We show that SCF-dependent CD117 phosphorylation in wild-type TF-1 cells is blocked by the antibodies Refmab#1-Ernie (referred to as "Ernie") and Refmab#1-Bert (referred to as "Bert"), whereas it is not affected in TF-1 cells carrying the D121K and S123K mutants of CD117 phosphorylation. The same is observed for the mutants E73K, E73Y and S123F (data not shown). [Figure 20] 1 shows that antibody Rekmab#1-Bert effectively depletes unedited wild-type human HSPCs from CD34+ humanized mice in vivo. [Figure 21] The antibody Remab#1-Bert shows that the antibody Remab#1-Bert depletes unedited wild-type CD117+ bone marrow cells and E73K edited cells in vivo, whereas the antibody Remab#1-Bert leaves E73K mutant CD117+ bone marrow cells.In the presence of the isotype control antibody, CD34+ human E73K mutant CD117 cells essentially engraft in immunodeficient mice, as well as unedited CD34+ human cells. [Figure 22] Figure 2 shows that wild-type DF-1 cells, but not those transfected with the D121K or S123K mutants of CD117, are depleted by the tested ADCs (Refmab#1-Ernie-teserine (A) and Refmab#1-Bert-teserine (B)). D121K A5 and D121K A6 represent two different batches of plasmid for the same mutant. [Diagram 23]Depletion of unedited CD34+ progenitor cells (gated as live / hCD45+ / CD34+ / CD38-) in mice receiving antibody Rekmab#1-Bert compared to mice receiving isotype control antibody is shown. In contrast, injection of Rekmab#1-Bert resulted in enrichment of E73K, D121K and S123K edited mutant cells compared to animals receiving isotype control antibody. Unedited and edited cells were identified by staining with CD117-specific antibody clones 104D2 and SR-1. Cells were classified as unedited (104D2+ and SR-1+) or edited (104D2+ but SR-1-). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] Immunotherapy is a promising therapeutic approach for treating cancer, genetic and autoimmune diseases. Immuno-depleting agents, such as antibodies or engineered immune cells directed against tumor antigens, are administered to patients to target and kill tumor cells. However, because tumor surface proteins are also expressed on the surface of normal cells, including hematopoietic cells, this strategy can induce severe side effects in patients, for example by altering hematopoiesis. To restore hematopoiesis in patients, hematopoietic cells can be subsequently transplanted into the patient. However, binding of the depleting agent to diseased cells as well as to newly transplanted healthy cells may limit the maximum tolerated dose or restrict the use of healthy cells for treatment prior to transplantation. Alternatively, transplanted cells need to be resistant to the immuno-depleting agent so as not to be targeted and eliminated by the agent. Our approach is therefore to select cells that are resistant to the immuno-depleting agent used in immunotherapy while retaining their function to restore normal hematopoiesis in patients.

[0031] The inventors develop a method to identify functional allelic variants of gene sequences encoding surface protein regions involved in binding of a particular depleting agent. Such variants can be naturally occurring polymorphisms and / or designed and engineered variants. Different isoforms of surface proteins can be selected or generated. The first isoform of surface protein encoded by the polymorphic nucleic acid is not recognized by a particular depleting agent. This variant allele does not change or does not substantially change the function of the particular surface protein. Thus, the depleting agent can be used to specifically deplete cells expressing one isoform by specifically binding to one isoform and not or does not substantially bind to the other isoform. For example, if the depleting agent specifically binds to the second isoform but not the first isoform, the depleting agent specifically depletes cells expressing the second isoform. In another embodiment, the first isoform can be recognized by a second agent, and thus the second agent can be used to specifically deplete cells expressing the first isoform but not the second isoform. Cells expressing a first isoform of a surface protein encoded by at least one mutant allele are advantageously used in medical treatment in patients having cells expressing the second isoform, in particular to specifically deplete transplanted cells or patient cells by using a second or first agent, respectively.

[0032] Without advanced in silico analysis, it is not possible to predict which mutations of a surface antigen may be used in such an approach. First, the mutation must reside in a surface-exposed stretch of the surface antigen that is accessible to the depleting agent. Second, the depleting agent must bind to this stretch on the exposed region of the surface antigen. Third, binding must be affected sufficiently that the depleting agent can distinguish the first isoform from the second. Residual binding to other isoforms should be minimal or, better, completely absent. Fourth, the mutation should not affect or only slightly affect the function of the surface antigen. The mutant isoform should perform its biological function, at least to an extent that is acceptable in a given therapeutic context. Although in silico methods can assist in identifying and prioritizing optimal candidates, experimental testing is required to validate the utility of any given mutation.

[0033] Depleting agent The present disclosure relates to an agent that comprises an antigen-binding region that specifically binds to one isoform of CD117 on cells and does not bind to another isoform of CD117 or binds substantially weaker.Such an agent is referred to herein as a "depleting agent".Both isoforms of CD117 are functional, i.e., CD117 is functional with respect to at least one relevant property.Preferably, both isoforms of CD117 have the same function, i.e., are functionally indistinguishable.

[0034] However, the two isoforms of CD117 differ in terms of binding to depleting agents. Depleting agents specifically bind only to one of the isoforms of CD117. Therefore, isoforms can be described as functionally identical (or functionally substantially identical) but immunologically distinguishable.

[0035] The first and second isoforms of CD117 can be polymorphic alleles.Preferably, the first and second isoforms of CD117 are naturally occurring polymorphic alleles.Also preferably, the first and second isoforms of CD117 are single nucleotide polymorphism (SNP) alleles.

[0036] The first and second isoforms of CD117 can also be genetically engineered alleles.Preferably, the first and second isoforms of CD117 differ by 1, 2, 3, 4 or 5 amino acids.Most preferably, the first and second isoforms of CD117 differ by one amino acid.

[0037] Various methods can be used to determine the mutations to be introduced into CD117 to generate the second isoform. For example, mutations can be randomly inserted into the surface protein, followed by functional and immunological screening of the generated mutants. Alternatively, mutations can be rationally designed, for example, by analysis of the secondary or tertiary protein structure of CD117.

[0038] The depleting agents contain an antigen-binding region that specifically binds to one isoform of CD117 on a cell and does not bind, or binds substantially weaker to, another isoform. The depleting agents of the present disclosure can be divided into two main categories.

[0039] First, the depleting agent can be a polypeptide comprising an antigen-binding region. Said polypeptide can be composed of one or more polypeptide chains. Preferably, said polypeptide comprising an antigen-binding region is an antibody. Said polypeptide comprising an antigen-binding region can also be an antibody fragment, an antibody drug conjugate, or another variant of an antibody or scaffold. Exemplary antibody fragments and scaffolds include single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, bis-scFv, camelid antibodies, ankyrin, centrin, domain antibodies, lipocalin, small modular immunopharmaceuticals, maxibodies, protein A and affilin.

[0040] The polypeptide comprising the antigen-binding region may be a bispecific, bispecific or multispecific antibody. Such molecules may also contain additional functional domains. For example, the polypeptide comprising the antigen-binding region may be a T cell engager, such as a BiTE. The polypeptide comprising the antigen-binding region may also be fused to the extracellular domain of a cytokine or chemokine, a toxin or a cell surface receptor.

[0041] Alternatively, the depleting agent can be a cell that comprises an antigen-binding region. For example, the depleting agent can be a chimeric antigen receptor (CAR). In a specific embodiment of the present disclosure, the cell that comprises an antigen-binding region is a CAR T cell, a CAR NK cell or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell that comprises an antigen-binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell that comprises an antigen-binding region is a primary T cell that comprises a CAR.

[0042] The depleting agent specifically binds to one isoform of CD117 but not to the second isoform, thus specifically depleting cells expressing one isoform.

[0043] In certain embodiments, the present disclosure relates to an agent comprising a first antigen-binding region that specifically binds to a second isoform of CD117 and does not bind to the first isoform. In other embodiments, the present disclosure also relates to an agent comprising a second antigen-binding region that specifically binds to a first isoform of CD117 and does not bind to the second isoform. In certain embodiments, the agent binds substantially weaker to the second isoform of CD117.

[0044] The first and second isoforms of CD117 may differ from each other by only one amino acid substitution. Said one amino acid difference between the first and second isoforms may also be the result of the presence of a single nucleotide polymorphism, such as a naturally occurring single nucleotide polymorphism. The first and second isoforms of CD117 may also differ from each other by more than one amino acid, for example, two, three, or more than three amino acids. The first and second isoforms of CD117 may also differ from each other in that one of the isoforms has an insertion of one, two, three, or more than three amino acids compared to the other isoform. The first and second isoforms of CD117 may also differ from each other in that one of the isoforms has a deletion of one, two, three, or more than three amino acids compared to the other isoform. The two isoforms may also differ from each other by a combination of amino acid substitutions, insertions, and / or deletions. In a preferred embodiment, said depleting agent is an antibody or an antigen-binding fragment. When two isoforms of CD117 differ by more than one amino acid, the altered amino acids may be adjacent to each other, i.e. directly adjacent amino acids, or they may be separated.

[0045] The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and antibody variants (including derivatives).

[0046] In natural rodent and primate antibodies, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain contains distinct sequence domains. In a typical IgG antibody, the light chain contains two domains, the variable domain (VL) and the constant domain (CL). The heavy chain contains four domains, the variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). Both the light chain variable region (VL) and the heavy chain variable region (VH) determine the binding recognition and specificity to the antigen. The light chain constant region domain (CL) and the heavy chain constant region domain (CH) confer important biological properties such as antibody chain association, secretion, transplacental transport, complement fixation, and binding to Fc receptors (FcR).

[0047] Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of one light chain and one heavy chain variable region. The specificity of an antibody lies in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is composed of residues mainly from hypervariable or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable or framework regions (FRs) may participate in the antibody binding site or may affect the overall domain structure and thus the binding site. Complementarity determining regions, or CDRs, refer to amino acid sequences that together define the binding affinity and specificity of the natural Fv region of a natural immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, which are called L-CDR1, L-CDR2, L-CDR3, and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Framework region (FR) refers to the amino acid sequence intervening between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs of the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0048] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO:. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering, which corresponds to the truncation or insertion of structural components, whether in the framework or complementarity determining regions (CDRs) of the basic variable domain structure. The exact Kabat numbering of residues can be determined for a given antibody by alignment of the homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), residues 50 to 65 (H-CDR2) and residues 95 to 102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24 to 34 (L-CDR1), residues 50 to 56 (L-CDR2) and residues 89 to 97 (L-CDR3) according to the Kabat numbering system.

[0049] In certain embodiments, the antibody provided herein is any protein that comprises an antibody fragment, more specifically the antigen-binding domain of the antibody disclosed herein.The antigen-binding domain can also be incorporated into another protein scaffold.Antibody fragments and scaffolds include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, diabody, single domain antibody, maxibody, minibody, intrabody, diabody, triabody, tetrabody, v-NAR, bis-scFv, camelid antibody, ankyrin, centrin, domain antibody, lipocalin, small modular immunopharmaceutical, maxibody, protein A and affilin.

[0050] As used herein, "antigen-binding region" or "antigen-binding fragment of an antibody" refers to a portion of an antibody, possibly in its native form, that exhibits antigen-binding ability for a particular antigen, i.e., a molecule corresponding to a portion of the antibody's structure. Such a fragment exhibits the same or substantially the same antigen-binding specificity for said antigen, in particular compared to the antigen-binding specificity of the corresponding four-chain antibody. Antigen-binding ability can be determined by measuring the affinity between the antibody and the target fragment. This antigen-binding region is sometimes referred to as the "functional fragment" of an antibody.

[0051] The agents of the present disclosure include antibodies and fragments thereof, but also include artificial proteins that mimic the antigen of an antibody and have the ability to bind to an antigen, also referred to herein as antigen-binding antibody mimics. Antigen-binding antibody mimics are organic compounds that specifically bind to an antigen but are not structurally related to antibodies. They are artificial peptides or small proteins, usually with a molar mass of about 3 kDa to 20 kDa.

[0052] The phrases "antigen-binding region that recognizes an antigen" and "antigen-binding region that has specificity for an antigen" are used interchangeably herein with the term "antigen-binding region that specifically binds to an antigen." As used herein, the term "specificity" refers to the ability of an agent that includes an antigen-binding region, such as an antibody, to detectably bind to an epitope presented in the antigen.

[0053] "Specific binding" or "specifically binding" refers to a -8 M(KD) or stronger. Preferably, the binding affinity is 10 -8 M(KD) to 10 -12 M(KD), optional 10 -8 M(KD) to 10 -10 M(KD), especially at least 10 -8 If M(KD), the binding is considered to be specific. Affinity can be determined by various methods well known to those skilled in the art. These methods include, but are not limited to, surface plasmon resonance (SPR), biolayer interferometry (BLI), microscale thermophoresis (MST) and Scatchard plot. Whether a binding domain specifically reacts with or binds to a target can be easily tested, inter alia, by comparing the reaction of said binding domain with a target protein or antigen to the reaction of said binding domain with a protein or antigen other than the target protein.

[0054] As used herein, the term "epitope" refers to the portion of an antigen to which an antibody or its antigen-binding region binds. Epitopes of protein antigens can be divided into two categories: conformational epitopes and linear epitopes. Conformational epitopes correspond to discontinuous portions of the amino acid sequence of an antigen. Linear epitopes correspond to a continuous sequence of amino acids from an antigen.

[0055] In another embodiment, bispecific or multispecific molecules, such as bispecific or multispecific antibodies, are further disclosed herein. For example, an antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand for a receptor), to generate a bispecific molecule that binds at least two different binding sites or target molecules. An antibody can actually be derivatized or linked to more than one other functional molecule to generate a multispecific molecule that binds more than two different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the terms "bispecific molecule," "bispecific antibody," "bispecific molecule," "bispecific antibody," "multispecific molecule," and "multispecific antibody" used herein. To create a bispecific molecule, an antibody of the present disclosure can be functionally linked (e.g., by chemical coupling, genetic fusion, disulfide bond, non-covalent bond or other method) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, cytokine, chemokine, toxin, or receptor extracellular domain, so that a bispecific molecule is generated. Certain bispecific and multispecific molecules contemplated by the present disclosure are T cell engagers, such as bispecific T cell engagers, e.g., BiTEs.

[0056] As used herein, an agent that does not bind or binds substantially weaker to a particular isoform of CD117 includes an agent that cannot bind to cells expressing said particular isoform. For experimental testing, said agent may be labeled with a fluorescent marker or detected with a secondary antibody against said agent, and the percentage of cells presenting said fluorescent marker or said secondary antibody is determined by FACS analysis. Typically, testing is performed in a cell line expressing a recombinant target protein, i.e., CD117. The target protein may be expressed in its entirety. Alternatively, a truncated form may be used, which must at least include the extracellular domain or a region of the extracellular domain that contains the respective antibody epitope. To monitor the expression of mutant isoforms, cells may be stained simultaneously with two agents, one that binds to the epitope where the mutant was introduced and the second that binds to a different epitope than that bound by the first agent. The second epitope remains unchanged, and therefore this staining serves as a control for expression. As a non-binding control, cells that do not express the protein of interest are used. As a maximal binding control, cells that do not normally express the protein of interest are transfected with the wild-type isoform. Different cell lines have different expression levels, but expression is controlled through endogenous control elements such as promoters. Such cell lines can also be used to study the mode of action of depleting agents, effective shielding against different modes of action, to test cytotoxicity and shielding / resistance from cytotoxicity, or to test the function of engineered receptors. Western blot, ELISA or FACS can be used to analyze phosphorylation of signaling molecules. Analysis of gene expression changes can help to analyze gene expression compared to normal function. FACS-based assays can be used to analyze binding of SCF to CD117 on cells. Cells can also be used to demonstrate the feasibility of editing specific mutants via different approaches, e.g. homology-directed repair (HDR), base editing or prime editing.

[0057] The binding of the agent may result in depletion of cells expressing the first isoform of CD117. Various mechanisms may result in cell depletion. Antibody-dependent cellular cytotoxicity (ADCC) results from the binding of the agent to a target protein and activation of NK cells via the Fc portion to the agent bound by FcR expressed by such NK cells. The Fc portion of an immunoglobulin refers to the C-terminal region of the heavy chain of the immunoglobulin. The Fc portion may be wild-type or engineered. Enhanced and engineered mutations of the Fc portion are known in the art. In certain therapeutic situations, it is desirable to reduce or abolish the normal binding of the wild-type Fc region of an antibody, such as the wild-type IgG Fc region, to one or more or all of the Fc receptors and / or to complement components, such as C1q, in order to reduce or abolish the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or abolish the binding of the Fc region of an antibody to one or more or all of the Fcy receptors, such as FcyRI, FcyRIla, FcyRIIb, FcyRIIIa, etc. Effector functions may include, but are not limited to, one or more of the following: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), 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 signal transduction to induce apoptosis, cross-linking of target-bound antibodies, maturation of dendritic cells, or T cell priming. Binding of the agent may also result in blocking the binding of natural receptor ligands, thereby resulting in cell death and apoptosis without cell-mediated depletion.

[0058] The reduced or abolished binding of the Fc region to the Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more particularly a human IgG1 Fc region, to produce a mutated or engineered Fc region of said wild-type Fc region, such as a mutant human IgG1 Fc region. Substitutions that result in reduced binding can be useful. In order to reduce or abolish the binding properties of the Fc region to the Fc receptor, non-conservative amino acid substitutions, i.e., replacing an amino acid with another amino acid having different structural and / or chemical properties and / or charge, are preferred.

[0059] In certain embodiments of the present disclosure, the Fc region of the antibody is of IgG1 isotype carrying LALA or PG-LALA mutations, i.e. the constant region carries L234A, L235A and P329G mutations or PA-LALA mutations, i.e. the constant region carries L234A, L235A and P329A mutations, or AEASS, i.e. the constant region carries L234A, L235A and P329A mutations, or L234A, L235E, G237A, A330S and P331S mutations. The skilled artisan will recognize the possibility of engineering the Fc region to obtain a desired effect.

[0060] The surrogate ADCC assay, as described in the experimental part, constitutes the industry standard for quantifying the efficacy of drugs that mediate ADCC. Engineered Jurkat reporter cells have an NFAT-responsive luciferase gene and an Fc receptor, e.g., human FcgRIIIa. Binding of the Fc receptor with the binding antibody results in NFAT induction by receptor clustering and thus luciferase signal. Lack of binding, and therefore clustering, results in no luciferase signal. Cells that do not express the target protein (e.g., HEK or DF-1 cells or human myeloid cancer cells such as TF-1, KG-1 or KASUMI-1 with CD117 knockout), cells that express wild-type protein (e.g., DF-1-CD117 or HEK-CD117 of TF-1, KG-1 or KASUMI-1 cell lines) or cells that express individual mutants (e.g., CD117 mutants) were incubated with the test agent (e.g., antibody Refmab#1) and mixed with the ADCC reporter cells. Luciferase was then measured to quantify ADCC signal. Luciferase luminescence signal was normalized to the maximum signal observed in HEK-CD117, DF-1-CD117 or corresponding myeloid cancer cell lines. ADCC was measured using ADCC reporter assay (Promega, Catalog No. G7015).

[0061] Other potential modes of action are possible in line with the present disclosure. These include antibody-mediated displacement of SCF, dimerization blockers, or antibody binding to the outside of either of these regions, for example, by using ADC. Another method of depleting target cells is through the use of T cell engager molecules. For example, a bispecific T cell engager using CD117 binding sites and CD3 (OKT3) binding sites from antibody Refmab#1 may be used. The same target cells are used as used for the ADCC assay. Primary human T cells and bispecific T cell engager are added. Activation of human T cells was quantified by FACS by determining the frequency of CD69 upregulation.

[0062] The depleting agent according to the present disclosure specifically binds to one isoform of CD117 and allows the depletion of cells expressing said isoform.

[0063] More preferably, in certain embodiments, the depleting agent according to the present disclosure does not bind or binds substantially weaker to the first isoform of CD117, but specifically binds to the second isoform of CD117, allowing depletion of the cells expressing the second isoform of CD117, particularly in the method of use as disclosed herein. In particular, the depleting agent that does not bind or binds substantially weaker to the first isoform of cellular CD117, but specifically binds to the second isoform of CD117 expressed in the patient's cells, is used to deplete the patient's cells, but does not deplete the hematopoietic stem cells or their passages expressing the first isoform of CD117 that are transplanted to restore hematopoiesis in the patient.

[0064] In another specific embodiment, the depleting agent according to the present disclosure does not bind or binds substantially weaker to the second isoform of CD117, but specifically binds to the first isoform of CD117, allowing depletion of the cells expressing the first isoform of CD117, particularly in the method of use as disclosed herein. In particular, the depleting agent that does not bind or binds substantially weaker to the second isoform of CD117, but specifically binds to the first isoform of CD117 expressed in transplanted cells, is used to specifically deplete transplanted cells to avoid eventual severe side effects such as graft-versus-host disease due to transplantation.

[0065] Selective depletion of cells expressing specific isoforms of CD117 can be achieved without limitation by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP).

[0066] In certain embodiments, the antigen-binding region is coupled to an effector compound, such as a drug or toxin. Such conjugates are referred to herein as "immunoconjugates," "antibody-drug conjugates," or "ADCs." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, maytansinoids, calicheamicin, indolinobenzodiazepines, pyrrolobenzodiazepines, pyridinobenzodiazepines, camptothecin, topotecan, irinotecan, belotecan, deltecan, alpha-amanitin, microcystin, auristatin, and puromycin and analogs or homologs thereof.

[0067] In another particular embodiment, the depleting agent is an immune cell having an antigen receptor, such as a chimeric antigen receptor (CAR). See, for example, Myburgh et al. Leukemia (2020) 34:2688-703. The immune cell may express a recombinant antigen-binding region, also called an antigen receptor, on its cell surface. By "recombinant" is meant an antigen-binding region that is not encoded by the cell in its native state, i.e., heterologous, non-endogenous. Thus, it can be seen that expression of a recombinant antigen-binding region introduces new antigen specificity to the immune cell, allowing the cell to recognize and bind a previously unrecognized antigen. The antigen receptor may be isolated from any useful source. In a particular embodiment of the present disclosure, the cell comprising the antigen-binding region is a CAR T cell, a CAR NK cell, a CAR Treg, or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell comprising the antigen-binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell comprising the antigen-binding region is a primary T cell comprising a CAR.

[0068] In certain embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR).CAR is a fusion protein that comprises an antigen-binding region, typically derived from an antibody, linked to the signaling domain of a TCR complex.CAR can be used to direct immune cells, such as T cells or NK cells, to target antigens when appropriate antigen-binding regions are selected.

[0069] The antigen-binding region of a CAR is typically based on an scFv (single-chain variable fragment) derived from an antibody. In addition to the N-terminal extracellular antibody-binding region, a CAR typically includes a hinge domain that serves as a spacer to extend the antigen-binding region away from the plasma membrane of the immune effector cell in which it is expressed, a transmembrane (TM) domain, an intracellular signaling domain (e.g., a signaling domain from the zeta chain (CD3ζ) of the CD3 molecule of the TCR complex, or equivalent), and optionally one or more costimulatory domains that may assist in signaling or functionality of the cell expressing the CAR. Signaling domains from costimulatory molecules, including CD28, OX-40 (CD134), 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.

[0070] Those skilled in the art can select the appropriate antigen-binding region for redirecting immune cells to be used according to the present disclosure. In certain embodiments, the immune cells for use in the methods of the present disclosure are redirected T cells, such as redirected CD8+ T cells or redirected CD4+ T cells, or redirected NK cells.

[0071] The method of genetically modifying immune cells to express recombinant antigen-binding regions is well known in the art.The nucleic acid molecule encoding an antigen receptor can be introduced into cells, for example, in the form of a vector or any other suitable nucleic acid construct, or by inserting the nucleic acid molecule into the genome using genome editing technology.Vectors and their necessary components are well known in the art.The nucleic acid molecule encoding an antigen-binding region can be generated using any method known in the art, for example, molecular cloning using PCR.The antigen-binding region sequence can be modified using commonly used methods, such as site-directed mutagenesis.

[0072] CD117 CD117 (UniProt: P10721; also known as KIT, c-Kit or SCFR) is a cytokine receptor expressed on the surface of hematopoietic stem cells as well as other cell types. CD117 is a receptor tyrosine kinase type III that binds to SCF (UniProt: P21583; also known as stem cell factor Kit ligand or mast cell growth factor). Binding of CD117 to SCF results in the formation of a dimer that activates tyrosine kinase activity, thereby activating signaling molecules that propagate signals into the cell. Signaling through CD117 plays a role in cell survival, proliferation, and differentiation.

[0073] Human CD117 has the following amino acid sequence (SEQ ID NO:1): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTD PGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLV DRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYH RLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSS SVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSAN VTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWE DYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDR LVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDS SAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAF GKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGAC TIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNE YMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGM AFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPES IFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMY DIMKTCWDADPLKRPTFKQIVQLIEKQISETNHIYSNLANCSPNRQKPVVDHSVRINSV GSTASSSQPLLVHDDV Human SCF has the following amino acid sequence (SEQ ID NO:2): MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNNVKDVTKLVANLPKDYMITLKYVPG MDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSS KDLKKSFKSPEPRLFTPEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVT KPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKR QPSLTRAVENIQINEEDNEISMLQEKEREFQEV

[0074] In certain embodiments, the surface protein is CD117. In other embodiments, the surface protein is CD117 comprising the amino acid sequence of SEQ ID NO: 1. In other embodiments, the surface protein is CD117 consisting of the amino acid sequence of SEQ ID NO: 1.

[0075] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and preferably said first and second isoforms are functional.

[0076] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprise genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and said first and second isoforms are functional.

[0077] In certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein CD117 for use in medical treatment in a patient in need thereof, wherein the patient has cells expressing a second isoform of the surface protein, the cells expressing the first isoform comprise genomic DNA having at least one polymorphism or engineered allele, wherein the polymorphism or engineered allele is absent in the genome of a patient having cells expressing the second isoform of the surface protein, and wherein the first and second isoforms are substantially functionally identical.

[0078] Several functions have been reported for CD117. In certain embodiments, the present disclosure relates to the first and second isoforms of CD117, both of which are functional. In certain embodiments, the present disclosure relates to the first and second isoforms of CD117, both of which are functionally indistinguishable. In the present invention, "functionally indistinguishable" refers to the first and second isoforms of CD117 that can equally perform the same function in cells without significant impairment. In other words, the first and second isoforms are functionally almost indistinguishable. Slight functional impairment can be tolerated. In a preferred embodiment, said first isoform of CD117 remains functional and retains the ability to perform the same function as the corresponding wild-type isoform in cells without significant impairment.

[0079] One function of CD117 is binding to SCF.Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprise genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, and said first and second isoforms bind to SCF.

[0080] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, and wherein said first and second isoforms bind to SCF to a substantially similar extent.

[0081] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, CD117, and wherein said first and second isoforms bind to a polypeptide comprising SEQ ID NO:2.

[0082] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, CD117, and wherein said first and second isoforms bind to a polypeptide consisting of SEQ ID NO:2.

[0083] In other embodiments, the disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, CD117, and wherein said first and second isoforms bind in a substantially similar manner to a polypeptide comprising SEQ ID NO: 2. In other embodiments, said polypeptide is a polypeptide consisting of SEQ ID NO: 2.

[0084] In other embodiments, the disclosure provides a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of said surface protein, CD117, said first and second isoforms differing by no more than 60%, no more than 50%, no more than 40%, preferably no more than 30%, more preferably no more than 20%, and even more preferably no more than 10%. D In another embodiment, the present invention relates to a mammalian cell or population of cells for use, which binds to a polypeptide comprising SEQ ID NO: 2 at a ribozyme level. In another embodiment, the polypeptide is a polypeptide consisting of SEQ ID NO: 2.

[0085] The binding of CD117 to SCF can be tested by those skilled in the art by any commonly used assay, such as a FACS assay that titrates and measures the binding of biotinylated SCF and fluorescently labeled streptavidin to cells. An example of such an assay is described in Example 8.

[0086] Another function of CD117 is SCF-dependent proliferation. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of the patient having cells expressing said second isoform of said surface protein, said first and second isoforms resulting in SCF-dependent proliferation. These cells show increased proliferation upon addition of SCF. This increased proliferation is measured after 2-5 days using an agent for quantifying cell viability, such as Cell Titer Glow (Promega).

[0087] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of a patient having cells expressing said second isoform of said surface protein, said first and second isoforms resulting in SCF-dependent proliferation in a substantially similar manner. This can be tested in an SCF-dependent cell line, such as TF-1 or HSC. An example of such an assay is described in Example 9. In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, and wherein said first and second isoforms differ by no more than 40%, preferably no more than 30%, more preferably no more than 20%, and even more preferably no more than 10% resulting in SCF-dependent growth.In other embodiments, the disclosure provides a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, said first and second isoforms confer SCF-dependent proliferation, and said first isoform has a binding affinity (K) that is no more than 4-fold, preferably no more than 3-fold, more preferably no more than 2-fold higher than the second isoform. D In another embodiment, the first isoform results in a loss of EC50 of 40% or less, more preferably 35% or less, and even more preferably 30% or less compared to the second isoform as measured by FACS.

[0088] SCF-dependent proliferation can be tested by those skilled in the art by any commonly used assays such as cell count, colony forming unit (CFU) assay, cell titer glow (CTG; Promega) and carboxyfluorescein succinimidyl ester (CFSE) cell proliferation assay. For cell count assay, non-modified HSCs cultured in liquid medium in the presence of anti-CD117 antibodies will show a strong decrease in cell number proliferation over time compared to genetically modified HSCs expressing isoforms of CD117. By 14-day CFU assay, the hematopoietic potential of HSCs, i.e., their ability to give rise to differentiated colonies, is evaluated in semi-solid medium. Non-modified HSCs cultured in the presence of anti-CD117 antibodies are unable to form hematopoietic colonies, whereas genetically modified HSCs are protected from anti-CD117 antibodies and give rise to colonies. When cultured in the absence of anti-CD117 antibodies, genetically modified HSCs show similar proliferation to non-modified HSCs in liquid or semi-solid culture, thus confirming the retention of normal function of CD117 isoforms.

[0089] In certain embodiments, the first isoform results in a loss of maximum proliferation of 40% or less, more preferably 35% or less, and even more preferably 30% or less compared to the second isoform as measured via EC50 by FACS.

[0090] Another function of CD117 is SCF-dependent phosphorylation. Thus, in certain embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA with at least one polymorphism or engineered allele, said polymorphism or engineered allele not present in the genome of the patient having cells expressing said second isoform of said surface protein, said first and second isoforms being phosphorylated upon addition of SCF to cell culture medium (SCF-dependent phosphorylation).

[0091] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, and wherein said first and second isoforms effect SCF-dependent phosphorylation in a substantially similar manner.

[0092] In other embodiments, the disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, said first and second isoforms resulting in SCF-dependent phosphorylation that differs by no more than 30%, more preferably no more than 20%, even more preferably no more than 10%. In other embodiments, the reduction in SCF-dependent phosphorylation of the first isoform is less than 50% compared to the second isoform.

[0093] SCF-dependent phosphorylation can be tested by the skilled artisan by any commonly used assay such as enzyme-linked immunoassay (ELISA), FACS or Western blot. Cells expressing CD117 are incubated with SCF for different time points (5-15 min) before cell harvest and freezing in cell lysis buffer. Upon cell lysis by flash freezing in liquid nitrogen and thawing at 37 °C, the amount of phosphorylated CD117 at position Tyr719 is assessed using ELISA or Western blot methods, which are specifically known to detect phosphorylated Tyr719 of CD117, as well as the total level of CD117 as a control.

[0094] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, and wherein the KD of the first isoform of CD117-SCF is less than 4-fold, preferably less than 3-fold, more preferably less than 2-fold, and most preferably less than 1.5-fold higher than the KD of the second isoform of CD117-SCF. Preferably, said second isoform is wild type CD117.

[0095] In other embodiments, the present disclosure relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, CD117, for use in medical treatment in a patient in need thereof, wherein said patient has cells expressing a second isoform of said surface protein, said cells expressing said first isoform comprising genomic DNA having at least one polymorphism or engineered allele, said polymorphism or engineered allele being absent in the genome of a patient having cells expressing said second isoform of CD117, and wherein the KD of the first isoform of CD117-SCF is less than 0.25-fold, preferably less than 0.33-fold, more preferably less than 0.5-fold, and most preferably less than 0.66-fold lower than the KD of the second isoform of CD117-SCF.

[0096] In line with the present disclosure, it is also possible to combine additional variants or isoforms of CD117 in the methods and compositions of the present disclosure. Such isoforms can include, for example, double mutants. Such isoforms can also include, for example, single mutants and double mutants. The methods and compositions of the present disclosure can also be combined with cells that have CD117 knockout, for example, permanent knockout or temporary knockout (e.g., via CRISPRoff). The methods and compositions of the present disclosure can also be used in the depletion of bone marrow cells in solid tumors to enhance tumor response.

[0097] The methods and compositions of the present disclosure can also be combined with combinations of cells, particularly where the surface protein is CD117 with knockout of other targets such as CD45, CD123, CD33, CD7, CLEC12A, CD44, FLT3, CD300F, EVI2B, TPO and combinations thereof.

[0098] The methods and compositions of the disclosure may also include cells expressing the first isoform of CD117 (CD117 variants) and other surface protein variants, such as CD123 variants, CD33 variants, CD7 variants, CLEC12A variants, CD45 variants, FLT3 variants, CD300F variants, EVI2B variants, TPO variants, and any combination thereof.

[0099] CD117 polymorphism A cell expressing a first isoform of CD117 according to the present disclosure comprises genomic DNA having at least one polymorphic allele in a nucleic acid encoding said CD117, in particular said polymorphism induces at least one mutation involved in the binding of a particular agent compared to said second isoform.

[0100] Said polymorphism is preferably located within the nucleic acid sequence encoding the surface protein region of CD117 involved in the binding of the first agent, and is preferably located in the extracellular portion of CD117, particularly in the solvent-exposed secondary structure element. More specifically, said polymorphism is located within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first agent. Said polymorphism can be a mutation, such as deletion, substitution, insertion or combination thereof, of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides. In a particular embodiment, said polymorphism is a single nucleotide polymorphism.

[0101] The sequence difference between the two isoforms can also be genetically introduced. Also, the sequence difference is preferably within the nucleic acid sequence encoding the CD117 region involved in the binding of the first agent, and is preferably located in the extracellular part of the surface protein, particularly in the solvent-exposed secondary structure element. More specifically, the sequence difference is within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first agent. The sequence difference can be a mutation, such as a deletion, substitution, and / or insertion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides. In a particular embodiment, the sequence difference is a single point mutation.

[0102] The present disclosure provides polymorphisms in CD117, including polymorphisms that include substitutions of residues E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y259, N260, S261, D266, Y269, or R271. In certain embodiments, the present disclosure provides polymorphisms in CD117, including specific polymorphisms that include substitutions of residues E73, V120, D121, R122, S123, K127, K193, S239, Y259, or S261. In certain embodiments, the present disclosure provides polymorphisms in CD117, including specific polymorphisms that include substitutions of residues E73, D121, R122, S123, S239, Y259, or S261. In other embodiments, the disclosure provides polymorphisms of CD117, particularly including polymorphisms comprising substitutions of residues E73, D121 or S123. Particularly preferred polymorphisms include substitutions of residue E73, where E73 is substituted with an amino acid selected from the group consisting of K, L, Q, G, Y and R. Preferably, said substitution is E73K, E73Y or E73R. Other preferred polymorphisms include substitutions of residue D121, where D121 is substituted with an amino acid selected from the group consisting of S, V, Y, H, K, R and T. Preferably, said substitution is D121Y, D121H, D121K, D121R or D121T. Most preferably, said substitution is D121H or D121K. Other preferred polymorphisms include substitutions of residue S123, where S123 is substituted with P, F or K. Most preferably, said substitution is S123K. Other preferred polymorphisms include substitutions of residue S239, where S239 is replaced with H or K. Other preferred polymorphisms include substitutions of residue K193, where K193 is replaced with G, T, M, D or E. Still other preferred polymorphisms include substitutions of residue Y259, where Y259 is replaced with an amino acid selected from the group consisting of E, A, G, P, C and H. Preferably, said substitution is Y259P, Y259A or Y259G. Most preferably, said substitution is Y259A.

[0103] In certain embodiments, the present disclosure provides a variant of CD117, wherein said variant of CD117 comprises the amino acid sequence of SEQ ID NO:1, wherein one or more of the amino acids are selected from the group consisting of E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y259, N260, S261, D266, Y269, and R271. Preferably, the amino acid is selected from the group consisting of E73, V120, D121, R122, S123, K127, K193, S239, Y259 and S261, more preferably selected from the group consisting of E73, D121, R122, S123, S239, Y259 and S261, most preferably selected from the group consisting of E73, D121 and S123. In certain preferred embodiments, the amino acid is E73. In other preferred embodiments, the amino acid is D121. In other preferred embodiments, the amino acid is S123. In other preferred embodiments, the amino acid is S239. In other preferred embodiments, the amino acid is Y259.

[0104] In certain embodiments, the disclosure provides a variant of CD117, wherein said variant of CD117 comprises the amino acid sequence of SEQ ID NO: 1, wherein residue E73 is substituted with an amino acid selected from the group consisting of K, L, Q, G, Y and R, preferably E73K, E73Y or E73R. In particular embodiments, the disclosure provides a variant of CD117, wherein said variant of CD117 comprises the amino acid sequence of SEQ ID NO: 1, wherein residue D121 is substituted with an amino acid selected from the group consisting of S, V, Y, H, K, R and T, preferably Y, H, K, R or T, most preferably H or K. In certain embodiments, the disclosure provides a variant of CD117, wherein said variant of CD117 comprises the amino acid sequence of SEQ ID NO: 1, wherein residue S123 is substituted with P, F or K, most preferably K. In certain embodiments, the disclosure provides a variant of CD117 comprising the amino acid sequence of SEQ ID NO: 1, wherein residue S239 is substituted with H or K. In certain embodiments, the disclosure provides a variant of CD117 comprising the amino acid sequence of SEQ ID NO: 1, wherein residue K193 is substituted with G, T, M, D or E. In particular embodiments, the disclosure provides a variant of CD117 comprising the amino acid sequence of SEQ ID NO: 1, wherein residue Y259 is substituted with an amino acid selected from the group consisting of E, A, G, P, C and H, preferably P, A or G, most preferably A.

[0105] In certain embodiments, the present disclosure relates to a mammalian cell or a population of cells that expresses one of the aforementioned variants of CD117.

[0106] It will be understood that amino acids may be designated by either the three letter code or the one letter code, all of which are familiar to those of skill in the art.

[0107] Table 1 shows the 20 naturally occurring amino acids.

[0108] [Table 1]

[0109] Natural polymorphism In certain embodiments, the cells according to the present disclosure are selected from a subject that contains naturally occurring genomic DNA having at least one naturally occurring polymorphic allele, preferably a single nucleotide polymorphism (SNP), in a nucleic acid encoding the isoform.

[0110] In certain embodiments, the cells are selected from subjects containing naturally occurring genomic DNA having at least one naturally occurring polymorphic allele, in particular a SNP, in the nucleic acid sequence encoding a CD117 region involved in anti-CD117 agent binding, preferably located in the extracellular portion of the surface protein, more preferably in a solvent-exposed secondary structural element.

[0111] Certain naturally occurring SNPs have been described in the literature and may be used within the spirit of the present disclosure, together with respective binding agents capable of distinguishing such SNPs from other isoforms of CD117.

[0112] Some naturally occurring SNPs of human CD117 are shown in Table 2. A list of naturally occurring SNPs can also be found in the gnomAD database: https: / / gnomad.broadinstitute.org / gene / ENSG00000157404?dataset=gnomad_r2_1.

[0113] [Table 2]

[0114] Gene editing In another particular embodiment, said cells expressing the first isoform of CD117 according to the present disclosure are obtained by gene editing, preferably by modifying the sequence encoding said surface protein in the patient's native genomic DNA.

[0115] The cell can be genetically engineered by introducing into the cell a gene editing system that induces said polymorphism resulting in insertion, deletion and / or substitution of amino acids in the surface protein. Said gene editing modality targets a nucleic acid sequence, herein referred to as a target sequence, that encodes a surface protein region involved in said first drug binding. In particular, when said surface protein is CD117, said gene editing modality targets a nucleic acid encoding at least one amino acid residue at positions E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y259, N260, S261, D266, Y269 or R271 of SEQ ID NO: 1. Preferably, amino acid residue E73 is replaced with an amino acid selected from the group consisting of K, L, Q, G, Y and R, preferably selected from the group consisting of K, Y and R. Also preferably, amino acid residue D121 is substituted with S, V, Y, H, K, R or T, preferably with Y, H, K, R or T, most preferably with H or K. Also preferably, amino acid residue S123 is substituted with P, F or K, most preferably with K. Also preferably, amino acid residue K193 is substituted with G, T, M, D or E. Also preferably, amino acid residue S239 is substituted with H or K. Also preferably, amino acid residue Y259 is substituted with E, A, G, P, C or H, more preferably with P, A or G, most preferably with A.

[0116] The gene editing enzyme can be a sequence-specific nuclease, a base editor, a prime editor or a CRISPR transposon-based system.

[0117] The term "nuclease" refers to a wild-type or mutant enzyme capable of catalyzing the hydrolysis (cleavage) of phosphodiester bonds between nucleotides of nucleic acid (DNA or RNA) molecules, preferably DNA molecules. "Cleavage" refers to a double-strand or single-strand break event.

[0118] The term "sequence-specific nuclease" refers to a nuclease that cleaves nucleic acid in a sequence-specific manner. Different types of site-specific nucleases can be used, such as meganucleases, TAL nucleases (TALENs), zinc finger nucleases (ZFNs), or RNA / DNA-guided endonucleases such as clustered regularly interspaced short palindromic repeats (CRISPR) / Cas systems and Argonaute (Review in Li et al., Nature Signal transduction and targeted Therapy, 5, 2020; Guha et al., Computational and Structural Biotechnology Journal, 2017, 15, 146-160).

[0119] According to the present disclosure, the nuclease generates a DNA break in the target sequence, which encodes the surface protein region involved in the first agent binding as described above. In a particular embodiment, the inventors use the CRISPR system to induce a break in the target sequence that encodes the surface protein region recognized by the first agent as described above.

[0120] By "target sequence" it is intended to target a portion of the sequence encoding the region of CD117 involved in the first drug binding as described above and / or at least one (one or two) sequences adjacent to said region of CD117 involved in the first drug binding, in particular up to 50 nucleotides adjacent to said region of CD117 involved in the first drug binding, preferably 20, 15, 10, 9, 8, 7, 6 or 5 nucleotides adjacent to said drug binding site.

[0121] CRISPR system includes two or more components, Cas protein (CRISPR-associated protein) and guide RNA. Guide RNA can be single guide RNA or dual guide RNA. Cas protein is a DNA endonuclease that recognizes and generates double-stranded breaks in DNA complementary to the target sequence using guide RNA sequence as a guide. Cas system that generates single-stranded breaks requires only one nuclease domain. Cas system that generates double-stranded breaks requires two nuclease domains. Cas protein can include two active cleavage sites, for example, HNH nuclease domain and RuvC-like nuclease domain.

[0122] Cas protein also refers to engineered endonucleases, homologs or orthologs of Cas 9 that can cleave a target nucleic acid sequence. In certain embodiments, the Cas protein can induce cleavage of a nucleic acid target sequence, which can correspond to either a double-stranded or single-stranded break. The Cas protein mutant can be a Cas endonuclease that does not occur in nature and is obtained by protein engineering or random mutagenesis. The Cas protein can be one type of Cas protein known in the art. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), SaCas9, Cas12, Cas12a (Cpf1), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csxl ... The Cas protein may be sa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr3, Cmr4, Cmr5, Cnrr6, Csbl, Csb2, Csb3, Csxl7, CsxM, CsxlO, Csl6, CsaX, Csx3, Csl, Csxl5, Csfl, Csf2, CsO, Csf4, or a homolog, or an ortholog, or a modified form thereof.Preferably, the Cas protein is the Streptococcus pyogenes Cas9 protein.

[0123] The Cas is contacted with a guide RNA (gRNA) designed to contain a sequence complementary to a target sequence to specifically induce DNA cleavage within said target sequence, particularly the complementary sequence of a portion of the target sequence that encodes a surface protein region recognized by the above-mentioned drug, according to the present disclosure.

[0124] As used herein, "guide RNA," "gRNA," "sgRNA," or "single guide RNA" refers to a nucleic acid that facilitates specific targeting or homing of a gRNA / Cas complex to a target nucleic acid.

[0125] In particular, gRNA refers to RNA comprising transactivating crRNA (tracrRNA) and crRNA. Preferably, said guide RNA corresponds to crRNA and tracrRNA, which can be used separately or fused together to generate a single guide RNA. Complementary sequence pairing with the target sequence recruits Cas to bind and cleave DNA at the target sequence.

[0126] According to the present disclosure, the crRNA is engineered to contain a sequence complementary to a portion of the above target sequence that encodes the surface protein region recognized by the drug, so that the crRNA can target the surface protein region recognized by the drug. In a preferred embodiment, the sgRNA is used to target the binding site of the binding agent. In another preferred embodiment, the guide RNA contains a chemical modification known to those skilled in the art.

[0127] In certain embodiments, the crRNA comprises a sequence of 5-50 nucleotides, preferably 15-30 nucleotides, more preferably 20 nucleotides, that is complementary to the target sequence. As used herein, the term "complementary sequence" refers to a sequence portion of a polynucleotide (e.g., a portion of a crRNA or a tracRNA) that can hybridize to another portion of the polynucleotide under standard low stringency conditions. Preferably, the sequences are complementary to each other according to interstrand Watson-Crick base pairing, i.e., complementarity between two nucleic acid strands that relies on inherent base pairing between adenine-thymine (AT) and guanine-cytosine (GC) nucleotides. The gRNA can be designed by any method known to the skilled artisan in view of the present disclosure.

[0128] According to the present disclosure, the target sequence encodes a surface protein region of CD117 involved in first drug binding, preferably located in the extracellular portion of CD117, more preferably located in an extracellular loop compared to the second isoform, and again more preferably includes amino acid residues involved in drug binding.

[0129] In a preferred embodiment, where the surface protein is CD117, said target sequence encodes a region of CD117 involved in binding of a first agent, such as anti-CD117 agent binding as disclosed above. Preferably, said target sequence encodes at least one residue at positions E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y259, N260, S261, D266, Y269 or R271 of SEQ ID NO:1.

[0130] In certain embodiments, said crRNA targets a sequence encoding a region of CD117 involved in binding of the first agent and may in particular comprise one of the sequences listed in Table 3 (crRNA sequence).

[0131] [Table 3]

[0132] In other words, when the surface protein is CD117, the nucleic acid construct may preferably comprise: - a gRNA sequence according to SEQ ID NO: 3 to 8 targeting a sequence encoding amino acid residue E73 according to SEQ ID NO: 1, or - a gRNA sequence selected from the group consisting of SEQ ID NOs: 9 to 14, which targets a sequence encoding amino acid residues V120, D121, R122, S123 of SEQ ID NO: 1.

[0133] In other specific embodiments, gene editing is performed via HDR, and the HDR template may include one of the sequences shown in Table 4.

[0134] [Table 4]

[0135] DNA strand breaks introduced by nucleases according to the present disclosure can result in mutations of the DNA at the break site via non-homologous end joining (NHEJ), which frequently results in small insertions and / or deletions or replacements of the DNA surrounding the break site via homology-directed repair (HDR).

[0136] In a preferred embodiment, said polymorphism within a nucleic acid encoding an isoform of CD117 is induced via HDR repair following DNA breakage and introduction of an exogenous nucleotide sequence, referred to herein as an HDR template.

[0137] The HDR template comprises a first and a second portion of the sequence that are homologous to the 5' and 3' regions of the target sequence, respectively, and a middle sequence portion that contains a polymorphism. Following cleavage of the target sequence, a homologous recombination event is achieved between the genome containing the target sequence and the HDR template, and the genomic sequence containing the target sequence is replaced by the exogenous sequence.

[0138] Preferably, a homologous sequence of at least 20bp, preferably more than 30bp, preferably more than 50bp, more preferably less than 200bp is used. The homologous sequence can be dsDNA or ssDNA. Preferably, the homologous sequence is ds DNA. In fact, the shared DNA homology is located in the adjacent regions upstream and downstream of the cleavage site, and the exogenous sequence to be introduced should be located between the two arms. The adjacent sequences can be symmetric or asymmetric. Both strands of the target nucleic acid, i.e., the positive strand or the negative strand, can be targeted. Optionally, a PAM sequence can be used that can be silenced to improve HDR.

[0139] In a preferred embodiment, a cell according to the present disclosure is genetically engineered by introducing into said cell a sequence encoding a region of CD117 recognized by said first agent as described above and said site-specific nuclease targeted to the HDR template.

[0140] In another particular embodiment, the gene editing enzyme is a DNA base editor as described in Komor et al., Nature 533, 420-424, and in Rees HA, Liu DR. Nat Rev Genet. 2018; 19: 770-788, or a prime editor as described in Anzalone et al. Nature, 2019, 576: 149-157, Matsoukas et al., Front Genet. (2020) 11: 528, Chen et al. Cell (2021) 184: 5635-52, Koblan et al, Nat Biotechnol (2021) 39: 1414-25 and Kantor A. et al. Int. J. Mol. Sci. 2020, 21 (6240). The base editor or prime editor can be used to introduce mutations at specific sites in the target sequence.

[0141] According to the present disclosure, base editors or prime editors generate mutations within a target sequence by sequence-specific targeting of the sequence encoding the region of CD117 involved in first drug binding.

[0142] In particular, said base editor or prime editor is a CRISPR-based or prime editor. Said CRISPR base or prime editor may comprise a dead Cas protein (dCas) as a catalytically inactive sequence-specific nuclease. It may also comprise Cas9 with a mutated nuclease domain. dCas refers to a modified Cas nuclease that lacks endonuclease activity. Nuclease activity may be inhibited or prevented in dCas protein by one or more mutations and / or one or more deletions in the HNH and / or RuvC-like catalytic domains of Cas protein. The resulting dCas protein lacks nuclease activity but binds to guide RNA (gRNA)-DNA complexes with high specificity and efficiency to specific target sequences. In certain embodiments, said dCas may be a Cas nickase in which one catalytic domain of Cas is inhibited or prevented.

[0143] The base editor is complexed with a guide RNA (gRNA) designed to include a complementary sequence of a target nucleic acid sequence and specifically binds to the target sequence as described above.

[0144] The gRNA can be designed by any method known to those skilled in the art in view of the present disclosure. In certain embodiments, the gRNA can target a sequence that encodes the region on CD117 that is recognized by the first agent as described above.

[0145] As a non-limiting example, the base editor is a nucleotide deaminase domain fused to a dead Cas protein, in particular a Cas nickase. The nucleotide deaminase can be an adenosine deaminase or a cytidine deaminase. The nucleotide deaminase can be a natural or engineered deaminase.

[0146] In certain embodiments, the base editor may be a non-limiting example selected from the group consisting of BE1, BE2, BE3, BE4, HF-BE3, Sa-BE3, Sa-BE4, BE4-Gam, saBE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, SaKKH-BE3, cas12a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRIPS-X, ABE7.9, ABE7.10, ABE7.10*xABE, ABESa, ABEmax, ABE8e, VQR-ABE, VRER-ABE, and SaKKH-ABE.

[0147] The prime editor consists of a fusion of a catalytically inactive sequence-specific nuclease, particularly a Cas nickase, as described above, with a catalytically active engineered reverse transcriptase (RT) enzyme. The fusion protein, particularly when the surface protein is CD117, is used in conjunction with a prime editing guide RNA (pegRNA) that comprises a sequence complementary to the target sequence described above, and includes one of the sequences listed in Table 3, and an additional sequence that also includes a sequence that binds to the primer binding site region of DNA. In a particular embodiment, the reverse transcriptase is the Maloney Murine Leukemia Virus RT enzyme and mutants thereof. The prime editor may be, as a non-limiting example, selected from the group consisting of PE1, PE2, PE3 and PE3b, or any of the prime editors described in Chen et al. Cell (2021) 184:5635-52 or Koblan et al, Nat Biotechnol (2021) 39:1414-25.

[0148] Anti-CD117 agents Several anti-CD117 moieties are known in the art, some of which are currently under development. Antibody SR-1 was first isolated from a hybridoma (WO1992017505). Humanized versions of SR-1 were made (WO2007127317; WO2020112687). Anti-CD117 drug conjugates are described in WO2016020791. Other anti-CD117 antibodies are described in WO2015050959 and WO2019084064. Certain anti-CD117 antibodies are also commercially available, such as antibody 104D2 Dianova (#117PE-100T). These and other anti-CD117 moieties can be used in the context of the present disclosure. Some anti-CD117 antibodies have also been made in the present disclosure in full-length antibody format, as well as in Fab format. Details are provided in Example 1.

[0149] In certain embodiments, said depleting agent that binds to said second isoform of CD117 and does not bind or binds substantially weakly to said first isoform of CD117 as described above specifically binds to an epitope comprising amino acids E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, Y259, N260, S261, D266, Y269 and / or R271 of SEQ ID NO: 1. More preferably, said depleting agent specifically binds to an epitope comprising amino acids E73, V120, D121, R122, S123, K127, K193, S239, Y259 and / or S261 of SEQ ID NO: 1. Even more preferably, the depleting agent specifically binds to an epitope comprising amino acids E73, D121, R122, S123, S239, Y259 and / or S261 of SEQ ID NO: 1. Most preferably, the depleting agent specifically binds to an epitope comprising amino acids E73, D121 and / or S123 of SEQ ID NO: 1.

[0150] In a preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22.

[0151] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22.

[0152] In another embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; The antigen binding region contains the following mutations: - an asparagine in the VLCDR1 region (SEQ ID NO: 20) is replaced with glutamic acid, - an aspartic acid in the VLCDR3 region (SEQ ID NO: 22) is replaced with a glutamic acid, and optionally - The second asparagine in the VLCDR3 region (SEQ ID NO: 22) is replaced by a lysine.

[0153] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22. The antigen binding region contains the following mutations: - an asparagine in the VLCDR1 region (SEQ ID NO: 20) is replaced with glutamic acid, - an aspartic acid in the VLCDR3 region (SEQ ID NO: 22) is replaced with a glutamic acid, and optionally - The second asparagine in the VLCDR3 region (SEQ ID NO: 22) is replaced by a lysine.

[0154] In another embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60.

[0155] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60.

[0156] In another embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61.

[0157] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61.

[0158] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 16.

[0159] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 16.

[0160] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 62.

[0161] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 62.

[0162] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 63.

[0163] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 15; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 63.

[0164] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 25, VHCDR2 is SEQ ID NO: 26 and VHCDR3 is SEQ ID NO: 27; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 28, VLCDR2 is SEQ ID NO: 29 and VLCDR3 is SEQ ID NO: 30.

[0165] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 25, VHCDR2 is SEQ ID NO: 26 and VHCDR3 is SEQ ID NO: 27; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 28, VLCDR2 is SEQ ID NO: 29 and VLCDR3 is SEQ ID NO: 30.

[0166] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 23; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 24.

[0167] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 23; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 24.

[0168] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 33, VHCDR2 is SEQ ID NO: 34 and VHCDR3 is SEQ ID NO: 35; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 36, VLCDR2 is SEQ ID NO: 37 and VLCDR3 is SEQ ID NO: 38.

[0169] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 33, VHCDR2 is SEQ ID NO: 34 and VHCDR3 is SEQ ID NO: 35; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 36, VLCDR2 is SEQ ID NO: 37 and VLCDR3 is SEQ ID NO: 38.

[0170] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 31; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 32.

[0171] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 31; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 32.

[0172] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 41, VHCDR2 is SEQ ID NO: 42 and VHCDR3 is SEQ ID NO: 43; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 44, VLCDR2 is SEQ ID NO: 45 and VLCDR3 is SEQ ID NO: 46.

[0173] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 41, VHCDR2 is SEQ ID NO: 42 and VHCDR3 is SEQ ID NO: 43; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 44, VLCDR2 is SEQ ID NO: 45 and VLCDR3 is SEQ ID NO: 46.

[0174] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 39; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 40.

[0175] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 39; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 40.

[0176] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 49, VHCDR2 is SEQ ID NO: 50 and VHCDR3 is SEQ ID NO: 51; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 52, VLCDR2 is SEQ ID NO: 53 and VLCDR3 is SEQ ID NO: 54.

[0177] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 49, VHCDR2 is SEQ ID NO: 50 and VHCDR3 is SEQ ID NO: 51; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 52, VLCDR2 is SEQ ID NO: 53 and VLCDR3 is SEQ ID NO: 54.

[0178] In another preferred embodiment, the anti-CD117 agent comprises an antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 47; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 48.

[0179] In another preferred embodiment, the anti-CD117 agent competes with an antigen-binding region for binding to CD117 or a variant of CD117, and the anti-CD117 agent comprises: a) an antibody heavy chain variable domain (VH) comprising a variable heavy chain of SEQ ID NO: 47; and b) An antibody light chain variable domain (VL) comprising the variable light chain of SEQ ID NO: 48.

[0180] It is further contemplated that the antigen-binding region of anti-CD117 can be further screened or optimized for their binding properties as defined above. In particular, it is contemplated that said antigen-binding region can have 1, 2, 3, 4, 5, 6 or more changes in the amino acid sequence of 1, 2, 3, 4, 5 or 6 CDRs of the monoclonal antibody provided herein. It is contemplated that the amino acid at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 of the VJ or VDJ region of the light or heavy chain variable region of the antigen-binding region can have insertion, deletion or substitution with conserved or non-conserved amino acids. Such amino acids that can be substituted or constitute substitutions are disclosed above.

[0181] In some embodiments, the amino acid difference is a conservative substitution, i.e., a substitution of one amino acid with another amino acid having similar chemical or physical properties (size, charge, or polarity), which generally does not adversely affect the biochemical, biophysical, and / or biological properties of the antibody. In particular, the substitution does not disrupt the interaction of the antibody with the CD117 antigen. Said conservative substitution is advantageously selected among the following five groups: Group 1 - small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2 - polar, negatively charged residues and their amides (D, N, E, Q); Group 3 - polar, positively charged residues (H, R, K); Group 4 - large aliphatic, non-polar residues (M, L, I, V, C); and Group 5 - large, aromatic residues (F, Y, W).

[0182] In a more particular embodiment, said first antigen-binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 15, 23, 31, 39 and 47, and / or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 16, 62, 63, 24, 32, 40 and 48.

[0183] Said first antigen-binding region having an amino acid sequence having at least 90%, such as at least 95%, 96%, 97%, 98% or 99% identity with any one of the above defined amino acid sequences is also part of the present disclosure, typically said first antigen-binding region having at least equal or higher binding activity than said first antigen-binding region consisting of a heavy chain consisting of any one of the amino acid sequences selected from SEQ ID NOs: 15, 23, 31, 39 and 47 and / or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 16, 62, 63, 24, 32, 40 and 48.

[0184] In certain embodiments, the anti-CD117 agent may be a bispecific CD117 antibody comprising at least one first binding specificity for CD117, e.g., one antigen-binding region of anti-CD117 described herein, and a second binding specificity for a second target epitope or target antigen.

[0185] According to the present disclosure, the anti-CD117 agent may be an immune cell having a CD117-targeted antigen receptor, such as a CD117-targeted CAR, wherein the antigen receptor comprises the antigen-binding region described above.

[0186] In certain embodiments, the immune cells (e.g., T cells) carrying CD117-targeted CAR recognize the second isoform of CD117 expressed in a patient in need thereof, and do not recognize the first isoform of CD117. In particular, the immune cells can specifically bind to an epitope comprising amino acids E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239 Y259, N260, S261, D266, Y269 and / or R271 of SEQ ID NO: 1. More preferably, the immune cells specifically bind to an epitope comprising amino acids E73, V120, D121, R122, S123, K127, K193, S239, Y259 and / or S261 of SEQ ID NO: 1. Even more preferably, said immune cells specifically bind to an epitope comprising amino acids E73, D121, R122, S123, S239, Y259 and / or S261 of SEQ ID NO: 1. Most preferably, said immune cells specifically bind to an epitope comprising amino acids E73, D121 and / or S123 of SEQ ID NO: 1.

[0187] In certain embodiments, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR comprises an antigen binding region, e.g., an scFv, wherein the antigen binding region comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22.

[0188] In certain embodiments, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR comprises an antigen binding region, e.g., an scFv, wherein the antigen binding region comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60.

[0189] In certain embodiments, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR comprises an antigen binding region, e.g., an scFv, wherein the antigen binding region comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61.

[0190] In a more specific embodiment, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16.

[0191] In a more specific embodiment, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 62.

[0192] In a more specific embodiment, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63.

[0193] According to the present disclosure, the anti-CD117 agent can be an immune cell having an antigen receptor that targets CD117, e.g., a CAR that targets a specific isoform of CD117, wherein the antigen receptor comprises an antigen-binding region as described above, and the immune cell does not express CD117 or expresses an isoform of CD117 that is not recognized by the CAR.

[0194] In certain embodiments, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR targets a specific isoform of CD117 and comprises an antigen-binding region, such as an scFv, which comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; Additionally, the immune cells either do not express CD117 or express an isoform of CD117 that is not recognized by the CAR.

[0195] In certain embodiments, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR targets a specific isoform of CD117 and comprises an antigen-binding region, such as an scFv, which comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; Additionally, the immune cells either do not express CD117 or express an isoform of CD117 that is not recognized by the CAR.

[0196] In certain embodiments, the anti-CD117 agent may be an immune cell (e.g., a T cell) bearing a CAR, wherein the CAR targets a specific isoform of CD117 and comprises an antigen-binding region, such as an scFv, which comprises: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; Additionally, the immune cells either do not express CD117 or express an isoform of CD117 that is not recognized by the CAR.

[0197] In a more specific embodiment, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16, and the immune cell expresses an isoform of CD117 that is not recognized by the CAR.

[0198] In another specific embodiment, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 62, and the immune cell expresses an isoform of CD117 that is not recognized by the CAR.

[0199] In another specific embodiment, the anti-CD117 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen-binding region, e.g., an scFv, which comprises a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, and a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63, and the immune cell expresses an isoform of CD117 that is not recognized by the CAR.

[0200] In a more preferred embodiment, said anti-CD117 agent is the Refmab #1 antibody as described in the Examples.

[0201] In another preferred embodiment, said anti-CD117 agent may be an immune cell bearing a CAR targeting a specific isoform of CD117 as described in the examples.

[0202] In particular, the present disclosure also relates to depleting the above-mentioned anti-CD117 agents (e.g., CAR cell compositions or antibodies) comprising a first or second antigen-binding region for use in selectively depleting host cells or transferred cells, respectively, in a subject in need thereof.

[0203] Cells expressing the first isoform of CD117 The present disclosure relates to a mammalian cell, preferably a hematopoietic cell, or a population of cells expressing a first isoform of CD117, wherein said cell or population of cells expresses a first isoform of CD117 comprising at least one polymorphic allele in a nucleic acid encoding said first isoform, and said first isoform is not recognized by a depletion agent comprising a first antigen-binding region described herein.

[0204] Said cell or population of cells is particularly useful for medical treatment in patients expressing the second isoform of CD117.

[0205] In a particular embodiment, the cells (e.g., hematopoietic stem cells) encoding or expressing the first isoform of CD117 that is not recognized by the depleting agent (e.g., hematopoietic cells) are particularly useful in medical treatments for restoring normal hematopoiesis after immunotherapy, such as adoptive cell transfer in patients expressing the second isoform, specifically, the treatments include administering a therapeutically effective amount of the hematopoietic cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent targeting the second isoform of CD117. In particular, the hematopoietic cells, preferably hematopoietic stem cells, are administered subsequent to the depleting agent. In another particular embodiment, the hematopoietic cells, preferably hematopoietic stem cells, may be administered prior to or simultaneously with the depleting agent.

[0206] In another particular embodiment, said cells expressing a first isoform of CD117 specifically recognized by a depleting agent that does not bind or binds substantially weaker to the second isoform of CD117 are particularly useful in medical treatment in patients expressing the second isoform of CD117, in particular to avoid serious side effects associated with transplanted cells bearing the first isoform (safety switch), said treatment comprising administering a therapeutically effective amount of a depleting agent targeting the first isoform of CD117. In particular, said hematopoietic cells, preferably immune cells bearing a CAR, are administered before said depleting agent.

[0207] As used herein, the term cell relates to a mammalian cell, preferably a human cell.

[0208] In certain embodiments, the cells are hematopoietic cells, including lymphocytes such as B cells and T cells, natural killer cells, myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes, dendritic cells (DCs) and platelet dendritic cells (pDCs), including immune cells.

[0209] In a preferred embodiment, the immune cell is a T cell. In another preferred embodiment, the immune cell is a primary T cell. As used herein, the term "T cell" includes a cell having a T cell receptor (TCR) or a cell derived from a T cell having a TCR. The T cell according to the present disclosure can be selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, memory T lymphocytes, tumor-infiltrating lymphocytes, or helper T lymphocytes, including both type 1 and type 2 helper T cells and Th17 helper cells. In another embodiment, the cell can be derived from the group consisting of CD4+ and CD8+ T lymphocytes or non-classical T cells, such as MR1-restricted T cells, MAIT cells, NKT cells, gamma delta T cells, or innate immune-like T cells.

[0210] T cells can be obtained from multiple sources, including but not limited to peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor.In certain embodiments, T cells can be obtained from a unit of blood taken from a subject using any number of techniques known to those skilled in the art.Alternatively, T cells can be differentiated from iPS cells.

[0211] In another preferred embodiment, the hematopoietic cells are hematopoietic stem cells. Stem cells can be adult stem cells, embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells. Representative human stem cells are CD34 + Hematopoietic stem cells can be differentiated from iPS cells or can be harvested from umbilical cord blood, from bone marrow, or from mobilized or non-mobilized peripheral blood.

[0212] In certain embodiments, the cells are allogeneic, which refers to cells derived from a donor that exhibits the same, similar or different HLA genotype as the person receiving the cells. The donor may be related or unrelated. In certain embodiments, the cells are autologous, which refers to cells derived from the same person receiving the cells.

[0213] The cells can be derived from healthy donors or patients, particularly from patients diagnosed with cancer, genetic or autoimmune diseases, or from patients diagnosed with infectious diseases. Hematopoietic cells can be extracted from blood, bone marrow, or derived from stem cells. HSCs can be derived, for example, from iPS (induced pluripotent stem cells).

[0214] Those skilled in the art will be able to select the more appropriate cells depending on the patient or subject to be transplanted.

[0215] The present disclosure further relates to compositions of cells or populations of cells for use in the treatments disclosed herein.

[0216] CAR For use in adoptive cell transfer therapy, the cells expressing the first isoform of CD117 according to the present disclosure can be modified to exhibit desired specificity and enhanced functionality. In certain embodiments, the cells can express a recombinant antigen-binding region, also referred to as an antigen receptor, on their cell surface as described above. In certain embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). According to the present disclosure, the immune cells expressing the first isoform of CD117 and CAR can be specifically depleted by administration of a therapeutically effective amount of a drug comprising a second antigen-binding region that specifically binds to the first isoform of CD117 but not to the second isoform of CD117, thereby avoiding eventual severe side effects resulting from the transplantation of the immune cells.

[0217] In certain embodiments, immune cells are reoriented against cancer antigens. By "cancer antigen" is meant any antigen (i.e., a molecule capable of inducing an immune response) associated with cancer. An antigen as defined herein can be any type of molecule that induces an immune response, e.g., a polysaccharide or lipid, but most preferably a peptide (or protein). Human cancer antigens can be human or derived from humans. Cancer antigens can be tumor-specific antigens, meaning antigens that are not found in healthy cells. Tumor-specific antigens generally result from mutations, particularly frameshift mutations, that generate entirely new amino acid sequences not found in the proteome of healthy humans.

[0218] Cancer antigens also include tumor-associated antigens, which are antigens whose expression or production is associated with tumor cells, but are not limited to these. Examples of tumor-associated antigens include, for example, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, CD123, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), total cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin, nerve fiber, and the like. These include fiber, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, dimeric form of pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD19, CD22, CD33, CD123, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T-cell receptor gamma chain alternative reading frame protein), Trp-p8, STEAP1 (prostate six transmembrane epithelial antigen 1), abnormal ras protein or abnormal p53 protein. In another specific embodiment, said tumor-associated or tumor-specific antigen is integrin ανβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

[0219] In certain embodiments, for use in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic diseases such as acute myeloid leukemia (AML) or B-acute lymphoblastic leukemia (B-ALL), immune cells according to the present disclosure express a recombinant antigen-binding region, such as a CD117-targeted CAR. The cells expressing a first isoform and expressing a CAR (e.g., CAR-CD117) can be further specifically depleted by administering a depleting agent comprising a second antigen-binding region that specifically binds to the first isoform of CD117 but does not bind, or binds substantially weaker, to the second isoform of CD117, thereby avoiding eventual severe side effects such as graft-versus-host disease resulting from transplantation.

[0220] In certain embodiments, the immune cells (e.g., T cells) expressing the first isoform have a CAR that targets CD117, and the CAR is targeted to a region within the N-terminal domain or amino acids E73, T74, V120, D121, R122, S123, Y125, K127, K193, I201, K203, S239, or 140. more preferably amino acids E73, V120, D121, R122, S123, K127, K193, S239, Y259 and / or S261 of SEQ ID NO:1, even more preferably amino acids E73, D121, R122, S123, S239, Y259 and / or S261 of SEQ ID NO:1, and most preferably amino acids E73, D121 and / or S123 of SEQ ID NO:1.

[0221] In particular, the immune cell (e.g., T cell) expressing the first isoform has an antigen-binding region, e.g., a CD117-targeted CAR comprising an scFv, the antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; More preferably, it comprises an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:15 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:16.

[0222] Alternatively, the immune cell (e.g., T cell) expressing the first isoform has an antigen-binding region, e.g., a CD117-targeted CAR comprising an scFv, the antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; More preferably, it comprises an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:15 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO:62.

[0223] Alternatively, the immune cell (e.g., T cell) expressing the first isoform has an antigen-binding region, e.g., a CD117-targeted CAR comprising an scFv, the antigen-binding region comprising: a) an antibody heavy chain variable domain (VH) comprising three CDRs, VHCD1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCD2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and b) an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; More preferably, it comprises an antigen-binding region comprising a heavy chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 63.

[0224] In vitro method for preparing cells expressing the first isoform The cell expressing the first isoform of CD117 according to the present disclosure can be genetically engineered by introducing a nucleic acid construct (e.g., mRNA) encoding at least one gene editing enzyme or ribonucleoprotein complex comprising the above-mentioned gene editing enzyme and / or HDR template into said cell. Alternatively, the gene editing system is transduced into said cell via a viral system, such as an adenoviral system. The cell can also be genetically engineered by further introducing a nucleic acid construct encoding the above-mentioned CAR into said cell. In particular, the method is an ex vivo method carried out on a culture of cells.

[0225] The term "nucleic acid construct" as used herein refers to a nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single-stranded or double-stranded, that is modified to contain segments of nucleic acid sequences, assembled and juxtaposed in a manner that does not normally occur in nature. A nucleic acid construct is usually a "vector", a nucleic acid molecule that is used to deliver exogenously produced DNA into a host cell.

[0226] Preferably, the nucleic acid construct comprises said gene editing enzyme, HDR template and / or CAR operably linked to one or more control sequences.Said control sequence can be a ubiquitous, tissue-specific or inducible promoter that is functional in the cells of target organ (i.e., hematopoietic cells).Such sequences well known in the art include in particular promoters and further regulatory sequences that can further control the expression of transgene, such as but not limited to enhancers, terminators, introns, silencers.

[0227] The nucleic acid construct as described above may be included in an expression vector.The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome.The vector may include any means for ensuring self-replication.Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated.

[0228] Examples of suitable vectors include, but are not limited to, recombinant integrative or non-integrative virus vectors, and vectors derived from recombinant bacteriophage DNA, plasmid DNA or cosmid DNA.Preferably, vector is recombinant integrative or non-integrative virus vectors.Examples of recombinant virus vectors include, but are not limited to, vectors derived from herpesvirus, retrovirus, lentivirus, vaccinia virus, adenovirus, adeno-associated virus or bovine papilloma virus.

[0229] The present disclosure relates to a method for expressing a first isoform of a cell surface protein in a cell by introducing a nucleic acid construct (e.g., mRNA) encoding a gene editing enzyme or a ribonucleoprotein complex comprising the above-mentioned gene editing enzyme and / or HDR template into the cell. The method may further comprise a step of introducing a nucleic acid construct encoding a CAR into the cell. The method comprises introducing a gene editing enzyme, such as a Cas protein, a base editor or a prime editor and a guide RNA (crRNA, tracrRNa, or fusion guide RNA or pegRNA) into the cell. In particular, the gene editing enzyme is a CRISPR / cas gene editing enzyme as described above. In a more particular embodiment, the gene editing enzyme is a site-specific nuclease, more preferably a CRISPR / Cas nuclease, comprising a guide RNA and a Cas protein, and the guide RNA in combination with a Cas protein cleaves and induces cleavage within the target sequence, comprising a nucleic acid encoding a surface protein region involved in drug binding as described above.

[0230] In a preferred embodiment, the nucleic acid construct comprises a CRISPR / Cas nuclease capable of targeting a nucleic acid sequence encoding a region of CD117 involved in binding to a depleting agent, preferably the nucleic acid construct comprises: - a gRNA sequence according to SEQ ID NO: 3 to 8 targeting a sequence encoding amino acid residue E73 according to SEQ ID NO: 1, or - a gRNA sequence selected from the group consisting of SEQ ID NOs: 9 to 14, which targets a sequence encoding amino acid residues V120, D121, R122, S123 of SEQ ID NO: 1.

[0231] The Cas nuclease can be a high-fidelity Cas nuclease, such as a high-fidelity Cas9 nuclease.

[0232] Said gene editing enzyme, preferably a guide RNA and / or a Cas protein, a base editor or a prime editor as described above, may be synthesized in situ in a cell as a result of the introduction of a nucleic acid construct, preferably an expression vector encoding said gene editing enzyme, such as a guide RNA and / or a Cas protein, a base editor or a prime editor as described above, into the cell. Alternatively, said gene editing enzyme, such as a guide RNA and / or a Cas protein, a base editor or a prime editor, may be produced outside the cell and then introduced therein.

[0233] Said nucleic acid construct or expression vector can be introduced into the cell by any method known in the art, including, but not limited to, stable transduction methods in which the nucleic acid construct or expression vector is integrated into the genome of the cell, transient transfection methods in which the nucleic acid construct or expression vector is not integrated into the genome of the cell, and virus-mediated methods. For example, transient transformation methods include, for example, microinjection, electroporation, cell squeezing, particle bombardment or in vivo targeting approaches.

[0234] In vivo editing The cells expressing the first isoform of CD117 according to the present disclosure can also be edited in vivo.There are various technologies that allow therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles and virus-like particles (see, for example, Cell (2022) 185: 2806-27).The molecular mechanism of converting CD117 to the first isoform of CD117 that is not recognized by depleting agents can be achieved by any of these methods.

[0235] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a molecular machinery capable of editing a gene in vivo and a depletion agent, the molecular machinery capable of in vivo gene editing comprises all the components required to introduce a point mutation in a wild-type CD117 isoform in a target cell, The depleting agent binds to wild-type CD117 but not to the isoforms of CD117. It is intended for use in the medical treatment of a patient in need thereof.

[0236] Preferably, said isoform of CD117 is characterized by a substitution of aspartic acid at position 121 of wild-type CD117 with lysine. Alternatively, said isoform of CD117 is characterized by a substitution of serine at position 123 of wild-type CD117 with lysine.

[0237] Also preferably, the depleting agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60.

[0238] Alternatively, the depletion agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61.

[0239] Alternatively, the depletion agent comprises: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. An antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22.

[0240] As used herein, the term "molecular mechanism capable of editing genes" refers to the arrangement of genes and / or nucleic acids required to edit target genes in vivo when delivered to target sites or cells via respective vehicles.Mechanisms also include respective delivery vehicles, such as viral vectors, lipid nanoparticles or virus-like particles.

[0241] Pharmaceutical Compositions and Therapeutic Uses In a further aspect, the present disclosure also provides a pharmaceutical composition comprising a cell or population of cells expressing the first isoform of CD117 described above, together with one or more pharma- ceutical or physiologically acceptable carriers, diluents or excipients.

[0242] In a particular embodiment, said cells expressing the first isoform of CD117 are hematopoietic stem cells.

[0243] In another particular embodiment, said cell expressing the first isoform of CD117 is an immune cell, preferably a T cell, more preferably a primary T cell, bearing a chimeric antigen receptor (CAR), preferably a CAR that targets the second isoform of CD117 expressed by the patient's cells as described above.

[0244] The pharmaceutical composition may further comprise a depleting agent comprising the first or second antigen-binding region as described above.

[0245] Pharmaceutical compositions are formulated in pharmaceutically acceptable carriers according to the route of administration.Preferably, the compositions are formulated to be administered by intravenous injection.The pharmaceutical compositions suitable for such administration can comprise the cells expressing the above-mentioned first isoform in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injection or dispersion immediately before use, which can comprise antioxidants, buffers, bacteriostatic agents, solutes or suspending agents or thickening agents.

[0246] Optionally, the composition comprising the cells expressing the first isoform of CD117 can be frozen for storage at any temperature suitable for cell storage.For example, the cells can be frozen at about -20°C, -80°C or any other suitable temperature.The cryogenically frozen cells can be stored and prepared for storage in a suitable container to reduce the risk of damage to the cells and maximize the chance that the cells survive after thawing.Alternatively, the cells can be maintained at refrigerated room temperature, for example, about 4°C.

[0247] The present disclosure relates to a cell or population of cells expressing the first isoform of CD117 as described above for use as a medicament, in particular for use in immunotherapy such as adoptive cell transfer therapy in a patient.

[0248] According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the second isoform or the first isoform of CD117 to specifically deplete patient or transplant cells, respectively.

[0249] As used herein, the term "in combination" or "in combination therapy" means that two (or more) different therapies are delivered to a subject during the course of the subject being afflicted by a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with a disorder and before the disorder is cured or eliminated, or before the treatments are discontinued for other reasons. In some embodiments, there is an overlap in administration, as the delivery of one treatment is still occurring when the delivery of the second treatment begins. This may be referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment has ended before the delivery of the other treatment begins. The delivery may be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered. In one embodiment, a depleting agent that binds to the second isoform or the first isoform of CD117 is administered at a dose and / or dosing schedule described herein, and cells expressing the first isoform are administered at a dose and / or dosing schedule described herein. In some embodiments, "in combination with" is not intended to mean that the depleting agent targeting the second isoform of CD117 (e.g., CAR cells or antibodies that recognize the second isoform of CD117) or the first isoform and the composition of cells expressing the first isoform of CD117 must be administered simultaneously and / or formulated for delivery together, although these delivery methods are within the scope of this disclosure. The depleting agent (e.g., CAR cells or antibodies that target the second isoform of CD117) can be administered simultaneously with, before, or after a dose of hematopoietic stem cells expressing the first isoform of CD117. In certain embodiments, each agent is administered at a dose and / or time schedule determined for that particular agent.

[0250] Adoptive cell transfer therapy according to the present disclosure can be used to treat patients diagnosed with cancer, genetic diseases, autoimmune diseases, infectious diseases, diseases requiring hematopoietic stem cell transplantation (HSCT), prevention of organ rejection, tumor transplant conditioning, tumor maintenance therapy, minimal residual disease, and prevention of relapse.

[0251] The present disclosure also relates to the use of the above-mentioned cells expressing the first isoform of CD117 in the manufacture of a medicament for adoptive transfer cell therapy in a patient.

[0252] As used herein, the term "subject" or "patient" refers to an animal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat, preferably a mammal in which an immune response can be elicited. More preferably, the patient is a human, including an adult, a child and a human in the prenatal stage.

[0253] As used herein, the terms "treatment," "treat," or "treating" refer to any action intended to improve the health status of a patient, such as the treatment, prevention and prophylaxis, and delay of disease. In certain embodiments, such terms refer to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, the terms refer to minimizing the progression or worsening of a disease resulting from the administration of one or more therapeutic agents to a subject with such a disease.

[0254] The cancer that can be treated includes tumor that is not vascularized or not yet substantially vascularized, and vascularized tumor.Cancer can include non-solid tumor (e.g., hematological tumor, e.g., leukemia and lymphoma, e.g., relapse and therapy-related tumor, e.g., secondary malignant tumor after cytotoxic therapy and hematopoietic stem cell transplantation (HSCT)) or can include solid tumor.

[0255] The term "autoimmune disease" as used herein is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens.

[0256] An infectious disease is a disease caused by a pathogenic microorganism, such as a bacterium, a virus, a parasite, or a fungus. In certain embodiments, an infectious disease according to the present disclosure occurs in an immunosuppressed patient, such as a patient after HSCT or a patient who has received a solid organ transplant.

[0257] In a preferred embodiment, the present disclosure relates to cells expressing the first isoform of CD117 as described above for use in hematological cancer, preferably leukemia, lymphoma, myeloma or other lymphoproliferative disorders. Said leukemia may be selected from the group consisting of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), blastic plasmacytoid dendritic cell neoplasm (BPDCN), myeloproliferative neoplasms (MPN) including chronic myelogenous leukemia (CML), myelodysplastic syndrome / myeloproliferative neoplasm (MDS / MPN) overlap syndromes including chronic myelomonocytic leukemia (CMML), chronic lymphocytic leukemia (CLL), B and T cell non-Hodgkin's lymphoma, acute biphenotypic leukemia, hairy cell leukemia, interleukin-3 receptor subunit alpha positive leukemia, B cell acute lymphoblastic leukemia (B-ALL), T cell acute lymphoblastic leukemia (T-ALL), Hodgkin's lymphoma (HL), systemic mastocytosis and preferably MDS, preferably AML or BPDCN.

[0258] In certain embodiments, said cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 can be used for the treatment of solid tumors, particularly for selective depletion of bone marrow cells of a solid tumor in a patient, allowing immunotherapeutic agents such as immune checkpoint inhibitors, CAR T cells or tumor-infiltrating lymphocytes to access the tumor, since the bone marrow cells of the tumor may be immunosuppressive. In this situation, said cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 as described above can help replenish the hematopoietic system that may be affected by the treatment intended to deplete the bone marrow cells of the solid tumor.

[0259] In another specific embodiment, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 described above can be used for the treatment of an autoimmune disease such as lupus, multiple sclerosis, scleroderma or systemic sclerosis.

[0260] The present disclosure also relates to a depleting agent (e.g., a CAR cell composition or an antibody) comprising the first or second antigen-binding region for use in selectively depleting host cells or transferred cells, respectively, in a subject in need thereof.

[0261] A method to specifically deplete the patient's cells but not the transplanted cells According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the second isoform of CD117.

[0262] In fact, during immunotherapy, immune depleting agents, such as CAR-expressing immune cells targeting CD117, can be administered to patients to target and kill tumor cells. However, because tumor surface proteins are also expressed on the surface of normal hematopoietic cells, this strategy can induce severe side effects in patients by altering hematopoiesis. To restore hematopoiesis in patients, hematopoietic cells can be subsequently transplanted into patients. However, these cells need to be resistant to the agent (i.e., the depleting agent for CD117-expressing cells) so as not to be targeted by the agent.

[0263] Thus, alternatively, according to the present disclosure, a depleting agent comprising a first antigen-binding region that specifically binds to a second isoform of CD117 can be administered to specifically ablate the patient's cells expressing said second isoform of CD117 but not the transplanted cells expressing said first isoform of CD117. Selective depletion of the patient's cells but not the transplanted cells allows the patient to be reconstituted with a healthy hematopoietic system that is no longer depleted by the immune depleting agent. Thus, according to this therapeutic use, the patient has a functional immune system rather than undergoing long-term immunosuppression. The use of cells according to the present disclosure eliminates infection as a major complication of current HSC transplantation.

[0264] In another embodiment, the present disclosure relates to a method for adoptive cell transfer therapy, preferably for hematopoietic stem cell transplantation, to restore normal hematopoiesis in a patient having cells expressing the second isoform of CD117, comprising: (i) administering an effective amount of cells (e.g., hematopoietic stem cells) expressing a first isoform of CD117, wherein the cells expressing the first isoform of CD117 comprise genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically engineered allele, in a nucleic acid encoding the first isoform, wherein the polymorphism is not present in the genome of a patient having cells expressing the second isoform of CD117, or a pharmaceutical composition thereof; and (ii) administering a therapeutically effective amount of an agent comprising at least a first antigen-binding region that specifically binds to the second isoform of CD117 and does not bind or binds substantially weaker to the first isoform of CD117 to specifically deplete cells (patient's cells) expressing the second isoform of CD117.

[0265] The cells expressing the first isoform of CD117 or a pharmaceutical composition thereof are administered to a subject in combination (e.g., before, simultaneously with, or after) with a drug comprising the first antigen-binding region as described above.

[0266] In a preferred embodiment, the depleting agent (e.g., a CAR cell or an antibody targeting the second isoform of CD117) is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells expressing the first isoform of the surface protein (e.g., the first isoform of CD117).

[0267] By "therapeutically effective amount" or "effective amount" is intended a number of cells, particularly hematopoietic stem cells, expressing the first isoform of CD117 as described above, administered to a subject sufficient to constitute a treatment as defined above, particularly restoration of normal hematopoiesis in the patient.

[0268] Administration of the cells or pharmaceutical compositions according to the present disclosure may be performed in any convenient manner, including injection, transfusion, or implantation, transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In another embodiment, the cells or pharmaceutical compositions of the present disclosure are preferably administered by intravenous injection. The cells or pharmaceutical compositions of the present disclosure may be directly injected into a tumor, lymph node, or site of infection.

[0269] Administration of cells or populations of cells may be in any number from 10 to 100, including all integer values ​​of cell number within those ranges. 4 From 10 9 Cells / kg body weight, preferably 10 5 From 10 7 cells / kg body weight, more preferably 2×10 6 From 5×10 6The administration of cells / kg body weight may consist of administration of cells / kg body weight. The dosage administered will depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. The cells or population of cells may be administered in one or more doses. The timing of administration is within the judgment of the supervising physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art.

[0270] In particular, the present disclosure also relates to depleting the above-mentioned anti-CD117 agents (e.g., CAR cell compositions or antibodies) comprising the first antigen-binding region for use in selectively depleting host cells in a subject in need thereof.

[0271] A method to specifically deplete the transplanted cells but not the patient's cells (safety switch). According to the present disclosure, the cells or population of cells (e.g., hematopoietic cells) expressing the first isoform of CD117 as described above are used for medical treatment of a patient in need thereof, the medical treatment comprising administering a therapeutically effective amount of the cells or population of cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or an antibody) that specifically binds to the first isoform of CD117 of the surface protein.

[0272] The cell or population of cells expressing the first isoform of CD117 of the present disclosure, preferably immune cells, is particularly used in adoptive cell transfer therapy into patients. The transplanted cells expressing the first isoform of CD117 can be further depleted in the patient by administering a therapeutically effective amount of a depleting agent that specifically binds to the first isoform of CD117 and does not bind or binds substantially weaker to the second isoform of CD117 expressed by the patient's cells, in order to avoid eventual serious side effects such as graft-versus-host disease due to transplantation. In this case, the agent that includes a second antigen-binding region that specifically binds to the first isoform of CD117 (expressed by the transplanted cells) is administered to deplete specifically the transplanted cells and not the patient's cells. The selective depletion of transplanted cells constitutes an important safety feature by providing a "safety switch".

[0273] Graft-versus-host disease (GvHD) refers to a medical complication following the receipt of transplanted tissue from a genetically different individual. The immune cells of the donated tissue (graft) recognize the recipient (host) as foreign cells. In certain embodiments, the medical condition is graft-versus-host disease caused by hematopoietic stem cell transplantation or adoptive cell transfer therapy, in which immune cells are transferred to the patient.

[0274] Said side effects may also occur when transplanted cells, especially immune cells bearing CAR, have severe side effects such as cytokine release syndrome and / or neurotoxicity.In this case, transplanted cells expressing the first isoform of CD117 can be eliminated when said cells become malignant or cause any type of undesired on-target or off-target damage as a safety switch.

[0275] The present disclosure relates to a method for adoptive cell transfer therapy in a patient having cells expressing a second isoform of CD117, the method comprising: (i) administering an effective amount of cells expressing a first isoform of CD117, wherein the cells expressing the first isoform of CD117 comprise genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically engineered allele, in a nucleic acid encoding the first isoform of CD117, wherein the polymorphism is not present in the genome of a patient having cells expressing the second isoform of CD117, or a pharmaceutical composition thereof; and (ii) administering a therapeutically effective amount of an agent comprising at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind or binds substantially weaker to the second isoform of CD117, thereby specifically depleting cells expressing the first isoform of CD117.

[0276] The cells expressing the first isoform of CD117 or a pharmaceutical composition thereof are administered to a subject in combination (e.g., before, simultaneously with, or after) with an agent comprising a second antigen-binding region as described above.

[0277] In a preferred embodiment, the depleting agent (e.g., CAR cells or antibodies targeting the second isoform of CD117) is administered before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) the dose of hematopoietic stem cells expressing the first isoform of CD117.

[0278] Administration of the cells or pharmaceutical compositions according to the present disclosure may be performed in any convenient manner, including injection, transfusion, or implantation, transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In another embodiment, the cells or pharmaceutical compositions of the present disclosure are preferably administered by intravenous injection. The cells or pharmaceutical compositions of the present disclosure may be directly injected into a tumor, lymph node, or site of infection.

[0279] Administration of cells or populations of cells may be in any number from 10 to 100, including all integer values ​​of cell number within those ranges. 4 From 10 9 Cells / kg body weight, preferably 10 5 From 10 7 The administration of cells / kg body weight may consist of administration of cells / kg body weight. The dosage administered will depend on the age, health and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment and the nature of the desired effect. The cells or population of cells may be administered in one or more doses. The timing of administration is within the judgment of the supervising physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of the optimal range of effective amounts of a given cell type for a particular disease or condition is within the skill of the art.

[0280] Thus, in certain embodiments, the present disclosure relates to a depleting agent (e.g., a CAR cell or an antibody) for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD117 as described above, wherein the patient has naturally occurring cells expressing a second isoform of CD117, and the depleting agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind or binds substantially weaker to the second isoform of CD117.

[0281] In another aspect, the present disclosure relates to a kit for expressing the first isoform of CD117 in a cell, comprising a gene editing enzyme such as a Cas protein, a base editor or a prime editor, a nucleic acid construct, a guide RNA in combination with an expression vector as described above, or an isolated cell according to the present disclosure.

[0282] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a substitution of aspartic acid at position 121 of wild-type CD117 with lysine, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 60; It is intended for use in the medical treatment of a patient in need thereof.

[0283] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a serine to lysine substitution at position 123 of wild-type CD117, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 60; It is intended for use in the medical treatment of a patient in need thereof.

[0284] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a substitution of aspartic acid at position 121 of wild-type CD117 with lysine, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 61; It is intended for use in the medical treatment of a patient in need thereof.

[0285] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a serine to lysine substitution at position 123 of wild-type CD117, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 61; It is intended for use in the medical treatment of a patient in need thereof.

[0286] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a substitution of aspartic acid at position 121 of wild-type CD117 with lysine, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 22; It is intended for use in the medical treatment of a patient in need thereof.

[0287] In certain embodiments, the present disclosure relates to the combination of: a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a serine to lysine substitution at position 123 of wild-type CD117, and b) a depleting agent, comprising: i. an antibody heavy chain variable domain (VH) comprising three CDRs, VHCDR1, VHCDR2 and VHCDR3, where VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19, and ii. an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21, and VLCDR3 is SEQ ID NO: 22; It is intended for use in the medical treatment of a patient in need thereof. EXAMPLES

[0288] Example 1: Generation of anti-CD117 Fab and MAb Six different anti-CD117 antibodies were generated in Fab and MAb format based on publicly available sequence information or sources. The variable chains and CDRs (Kabat) of five of the antibodies (Refmab #1-#5) are shown in Table 4. The sixth antibody (Refmab #6) is antibody 104D2, a commercially available anti-CD117 antibody from BioLegend (catalog number 313202; alternative supplier: Dianova (#117PE-100T)). Antibody YB5.B8 was used as an expression control (Invitrogen, catalog number 14-1179-82).

[0289] [Table 4] JPEG2024546996000006.jpg242166JPEG2024546996000007.jpg154166

[0290] Further characteristics of the antibodies, as well as the format and isotype of the full-length antibodies, are shown in Table 5.

[0291] [Table 5]

[0292] Example 2: Binding of MAbs to CD117 and optimization of assay conditions HEK-293T cells were transfected with constructs containing wild-type CD117 (SEQ ID NO: 1) or with an empty vector. Antibody binding to transfected cells and optimal assay conditions were evaluated in a 384-well format. Detection of cell expression was measured by high-throughput flow cytometry. Serial dilutions of each antibody were tested for immunoreactivity on cells expressing CD117 or vector alone. Based on raw signal values ​​and signal-background calculations, optimal screening concentrations of each antibody were determined. Results are shown in Figure 1. Each point represents the average of four replicates.

[0293] All six antibodies in Mab format bind to human CD117 in a concentration-dependent manner. Cells transfected with empty vector showed no binding to anti-human CD117 antibodies.

[0294] Optimized assay conditions for flow cytometry are shown in Table 6.

[0295] [Table 6]

[0296] Example 3: Binding of Fab to CD117 and optimization of assay conditions A similar experiment was performed as described in Example 2, except that Fab fragments were tested instead of full-length antibodies. Serial dilutions of each Fab were tested for immunoreactivity against cells expressing wild-type CD117 or vector alone. Based on raw signal values ​​and signal-to-background calculations, optimal screening concentrations for each Fab were determined. Results are shown in Figure 2. Each point represents the average of four replicates.

[0297] All six antibodies in Mab format bind to human CD117 in a concentration-dependent manner. Cells transfected with empty vector showed no binding to anti-human CD117 antibodies.

[0298] Optimized assay conditions for high-throughput flow cytometry are shown in Table 7.

[0299] [Table 7]

[0300] Example 4: Alanine Scan Alanine scanning of human CD117 was performed to determine the residues of CD117 involved in binding to the six antibodies investigated. Alanine scanning was performed by shotgun mutagenesis epitope mapping (Integral Molecular, Philadelphia / PA, USA) as described in Immunology (2014) 143, 13-20. Briefly, a mutation library of CD117 was created by high-throughput site-directed mutagenesis. Each residue was individually mutated to alanine and the alanine codon was mutated to serine. The mutant library was arrayed in 384-well microplates and transiently transfected into HEK293T cells. After transfection, cells were incubated with the indicated antibodies (IgG or Fab) at the given concentrations using independent immunofluorescence titration curves of wild-type CD117. Antibodies were detected using an Alexa Fluor 488-conjugated secondary antibody, and mean cell fluorescence was determined using the Intellicyt iQue flow cytometry platform (Intellicyt / Sartorius). Mutated residues were identified as important for the antibody epitope if they did not support the reactivity of the test antibody but supported the reactivity of the reference antibody (antibody YB5.B8 (Invitrogen, Cat. No. 14-1179-82)). This counterscreening strategy facilitates the elimination of mutants that are locally misfolded or have expression defects. Binding of each antibody to each mutant clone was determined in duplicate. For each point, background fluorescence was subtracted from the raw data, then normalized to antibody reactivity with wild-type CD117.

[0301] Library screening of very high affinity antibodies may not yield critical residues for antibody binding, so high affinity antibodies were converted to Fab format to weaken binding sufficiently to allow identification of critical residues for binding. If Fab screening under standard conditions was still insufficient to identify critical residues for binding, high stringency conditions were performed. These conditions included combinations of increasing pH, increasing salt concentration, increasing temperature, and / or increasing wash times. Antibodies that required high stringency conditions are denoted as "HS".

[0302] For each mutated clone, the average binding value was plotted as a function of expression (represented by the reactivity of the control). See Figure 3. A threshold of >70% wild-type binding to the control antibody and <20% wild-type binding to the test antibody (dashed line) was applied to identify preliminary primary significant clones (grey circles). For clones that did not meet the set threshold, but whose reduced binding activity and proximity to critical residues suggested that the mutated residues may be part of the antibody epitope, secondary clones (open circles) are highlighted.

[0303] The identified critical residues are summarized in Table 8. The average binding reactivity (and range) is given for all identified critical residues. Critical residues for antibody binding (grey and open) were residues whose mutations were negative for binding to the test Ab but positive for binding to the control antibody. Additional secondary residues (outlined as boxed cells) were identified that did not meet the threshold guidelines, but whose reduced binding activity and proximity to the critical residues suggested they may be part of the antibody epitope.

[0304] [Table 8]

[0305] Table 9 summarizes the critical residues for each of the six antibodies tested. Residues whose mutations caused the least reactivity with the specific antibody are highlighted in bold and underlined. The critical residues identified represent the amino acids whose side chains make the highest energy contribution to the antibody-epitope interaction (J. Mol. Biol. (1998) 280, 1-9; J. Mol. Biol. (1999) 285, 2177-2198); therefore, the highlighted residues are likely to be the major energy contributors to binding.

[0306] [Table 9]

[0307] Example 5: Comprehensive mutation analysis The critical residues for binding of each of the six antibodies to human CD117 identified in Example 4 were investigated in more detail. First, a validation step of the identified critical residues was performed, taking into account reproducibility of binding activity, surface accessibility, structural localization and distance to other critical sites, as well as the nature and biochemical properties of the substituted amino acid (e.g., cysteine ​​forming disulfide bridges or post-translational modification sites). After validation, each critical residue was subjected to comprehensive mutagenesis to biophysically relevant non-alanine amino acids selected based on the sequence- and structure-related properties of the substituted amino acid as well as the newly introduced amino acid.

[0308] Antibodies were screened for binding to human CD117 mutants in IgG format. As in Example 4, the binding of each test antibody to each mutant clone in the comprehensive library was determined in duplicate by high-throughput flow cytometry. For each mutation, background fluorescence was subtracted from the raw data and then normalized to the antibody reactivity with wild-type CD117. For all mutant clones, the average binding reactivity and range are listed in Table 10. Mutations that caused less than 25% binding are highlighted in gray.

[0309] [Table 10] JPEG2024546996000014.jpg245166JPEG2024546996000015.jpg245166JPEG2024546996000016.jpg245166JPEG2024546996000017.jpg158166

[0310] Based on these results, and taking into account the positions of the individual residues in the three-dimensional structure, as well as their substitution propensity inferred based on evolutionary analysis of related protein sequences, the residues shown in Table 11 were selected as the most promising candidates for follow-up studies.

[0311] [Table 11]

[0312] Example 6: Biophysical characterization of selected CD117 variants The biophysical properties of the selected mutants were tested. Aggregation of the selected CD117 mutants was determined by preparative size-exclusion chromatography. The yield of the selected CD117 mutants was determined by dividing the amount of protein after purification by the amount produced. The melting temperature was measured by differential scanning fluorimetry using Sypro Orange (Sigma, PN:S5692-50ul) and an RT-PCR device (C1000 Thermal Cycler, Biorad). Samples were measured in duplicate at 0.25-1.0 mg / mL in PBS buffer, and Sypro Orange was added at 5x final concentration. The temperature was increased sequentially from 25°C to 95°C (10 s by 0.5°C steps). The fluorescence increase was monitored as a function of temperature, and the melting temperature was determined as the inflection point of the sigmoidal curve and compared to wild-type CD117. The results are shown in Figure 4 (aggregation), Figure 5 (yield) and Figure 6 (melting temperature).

[0313] The monomer content of most mutants was acceptable, with only a few mutants (D121V, D121Y, D121T, S123V, S123I, S239K) showing less than 50% monomer content. Also, the production yields of all mutants except for a very few mutants (E73del, D121V, S239H) were sufficiently high. Similarly, the melting temperatures were within the acceptable range, except for mutant V120P, for which two melting points were measured, indicating structural differences compared to wild-type CD117.

[0314] Example 7: Binding of antibody Refmab#1 to selected mutants A more advanced analysis of binding to selected variants was performed with antibody Refmab#1. Binding of Refmab#1 to wt and variants (analytes) was measured at 25°C with 1x dynamic buffer (Sartorius, PN:18-1105) in Octet systems RED96e or R8 with shaking at 1,000 rpm. First, the selected variants were screened for their ability to bind to Refmab#1 using three different concentrations of analyte. Antibody Refmab#1 was captured by an anti-human Fc capture biosensor (AHC) (Sartorius, PN:18-5060) at 0.5 to 1ug / mL for 300 seconds. As analytes, human CD117 wt and variants containing only domains 1, 2 and 3 (CD117 D1-2-3) were titrated at 500nM, 50nM and 5nM. The association and dissociation of the analyte to Refmab#1 was monitored for 300 and 900 seconds, respectively. Reference subtraction was performed against buffer-only wells. The AHC chip was regenerated using 10 mM Gly-HCl pH 1.7. Data was analyzed using Octet Data Analysis software HT 12.0. Data was fitted to a 1:1 binding model (where possible). The kinetic rate k a and k d was fitted throughout. The binding levels of the selected mutants were compared to the wild type and are shown as percentages in Figure 7. For this qualitative analysis, the binding level of the top analyte concentration at the end of the association step was used.

[0315] The results are shown in Figure 7. Several CD117 mutants, including E73Y, E73R, D121Y, D121H, D121K, D121T, R122S, R122H, R122E, S123P, S123F, S123E and S123K, showed a particularly strong reduction in binding to Rekmab#1. Mutants E73K, D121R, S123V, S123I, S123M and S123Q also showed a strong reduction in binding to Rekmab#1. The results of the Octet system also correlated well with those observed in the FACS experiments.

[0316] To further characterize the binding of Refmab#1 to CD117 D1-2-3 mutants E73K, E73L, E73R, E73Y, D121H, D121K, S123K and S123F, the analyte was titrated at seven different concentrations (2000nM-5nM). Antibody Refmab#1 was captured by the AHC biosensor at 0.5 to 1 ug / mL for 300 seconds. Association and dissociation to Refmab#1 were extended to 600 and 1000 seconds, respectively. Steady-state analysis was performed.

[0317] The KD of antibody Refmab#1 against the E73K mutant of human CD117 was reduced 1200-fold compared to wild-type CD117 (1200 nM vs. 1 nM as determined by two independent experiments). The KD of antibody Refmab#1 against the E73L mutant of human CD117 was reduced approximately 500-fold compared to wild-type CD117 (485 nM vs. 1 nM, as determined by two individual experiments), the E73Y mutant of human CD117 was reduced approximately 700-fold compared to wild-type CD117 (691 nM vs. 1 nM, as determined by two individual experiments), the E73R mutant of human CD117 was reduced approximately 1000-fold compared to wild-type CD117 (958 nM vs. 1 nM, as determined by two individual experiments), and the S123F mutant of human CD117 was reduced approximately 1,500-fold compared to wild-type CD117 (1500 nM vs. 1 nM, as determined by two individual experiments). No binding of Rekmab#1 (nm shifts greater than 0.1 nm) was observed with 2000 nM of human CD117 mutants D121H, D121K and S123K. See Figure 8.

[0318] Example 8: SCF binding to selected CD117 mutants SCF binding is a function of CD117. SCF binding is important for the transduction of functional signals by CD117. Binding of selected mutants to SCF was measured with Octet systems RED96e or R8 at 25°C with shaking at 1000 rpm in 1x Kinetic Buffer (Sartorius, PN:18-1105). Biotinylated Avitag-hSCF (Acros Biosystems, PN:SCF-H82E1) was captured on a streptavidin (SA) biosensor (Sartorius, PN:18-5019) at 0.5–1 ug / mL for 600 seconds.

[0319] A construct carrying domains 1, 2 and 3 of wild-type CD117 (CD117 D1-2-3wt) and a construct carrying domains 4 and 5 of wild-type CD117 (CD117 D4-5wt) were used in this experiment. Domains 1, 2 and 3 contain the binding site for SCF. Domains 4 and 5 were used as negative non-binding controls.

[0320] CD117 D1-2-3wt and its variants are titrated at different concentrations from 1000nM to 5nM. Association was monitored for 300s and dissociation for 600s. Binding to human CD117 domains 4 and 5 (CD117 D4-5) was performed under the same conditions as the non-binding control. Reference subtraction was performed against buffer only wells. Biosensors were not regenerated and a new set of SA biosensors was used for each analyte. Data were analyzed using Octet Data Analysis software HT 12.0. Steady-state data analysis was performed due to the fast on / off nature of the interaction.

[0321] The results are shown in Figure 9.

[0322] The selected mutants showed different degrees of binding to SCF. In particular, the E73 mutants (E73L, E73Q, E73K, E73Y, E73R, and E73A) showed little loss of binding to SCF, and all mutants had a K of less than 2 compared to wild-type CD117. D This was also observed for the D121 mutants D121Y, D121H, D121K, D121R and D121T, the S123 mutants S123P, S123F, S123K, S123A, S123I, S123M and S123Q, the S239 mutant S239K, and the Y259 mutant Y259A.

[0323] The S123V, K127L and Y259E mutants also showed binding to SCF, albeit to a slightly lower extent. The KD of mutants E73L, E73Q, E73K, E73Y, E73R, E73A, D121Y, D121H, D121K, D121R, D121T, S123P, S123F, S123A, S123I, S123M, S123Q, S239K and Y259A was increased by less than two-fold compared to wild-type CD117. Mutants E73A, E73L, E73Q, E73K, E73Y, E73R, D121Y, D121H, D121K, D121R, D121T, S123V, S123P, S123F, S123K, S123A, S123I, S123M, S123Q, S123V, S239H, S239K, Y259E, Y259P and Y259A D was increased less than three-fold compared to wild-type CD117. D was increased less than 4-fold compared to wild-type CD117. Exemplary results are shown in FIG.

[0324] A cell-based assay was also set up to measure binding of SCF. A 1:1 mixture of biotinylated SCF and streptavidin PE was incubated for 45 minutes. During the incubation, TF-1 wild type cells are seeded at 1 million cells / ml. Fc blocking is performed for 15 minutes (Trustain Biolegend) and then 0.1-100ug / ml of either Ref001 (isotype control antibody with specificity for hen egg white lysozyme) or anti-CD117 antibody is added to the cells. After another 15 minutes and a washing step, a mixture of biotinylated SCF and streptavidin PE is added to the cells for 30 minutes. Finally, two more washing steps are performed and 7-AAD viability staining solution is added to the cells for 10 minutes. The plates are then measured via a Novocyte Quanteon flow cytometer.

[0325] Example 9: SCF-dependent proliferation of selected CD117 mutants SCF-dependent proliferation is another function of CD117. SCF-dependent proliferation was measured as follows: TF-1 cells were seeded at a cell concentration of 150,000 cells / ml in sterile leukocyte culture-treated plates in RPMI medium + 10% FCS. Cells were treated with 100 ng / ml SCF and titrated with various CD117 antibodies or different concentrations of isotype control antibodies. After 3 days of incubation, CellTiter-Glo 2.0 assay (Promega) was performed and luminescence was read in a luminometer (e.g., Envision or Phera Star plate reader).

[0326] CD117 wild type TF-1 cells were compared to TF1 cells with an E73K knock-in. The E73K knock-in in the clonal population differs in that KI1 was first treated with Alt-R HDR Enhancer V2 (IDT), resulting in 61% of alleles being restored via HDR-mediated editing observed via NGS, whereas KI2 was untreated, resulting in 39% HDR. Sorting the edited TF1 cells using a SONY MA900 and removing wild type and knock-out cells from the final subset yielded both KI1 and KI2, thus resulting in a highly pure knock-in.

[0327] The results are shown in Figure 11. Both wild type and E73K knock-in cells proliferated strongly, but knock-out cells did not proliferate. Treatment with increasing concentrations of Refmab#1 resulted in a decrease in proliferation of wild type cells, but not E73K cells. Cells treated with avapritinib also showed no proliferation for either type of cells (wild type, E73 and knock-out; data not shown).

[0328] In a separate experiment, the SCF-dependent proliferation of mutants E73K, E73Y, D121K, S123F and S123K was compared to wild-type CD117. TF-1 cells were seeded at a cell concentration of 150,000 cells / ml in RPMI+GlutaMax+10% FCS in sterile, clear-bottom, leukocyte culture-treated plates. Cells were treated with titrations of SCF. After 3 days of incubation, a CellTiter-Glo 2.0 assay (Promega) was performed, luminescence was read in a luminometer (Envision plate reader) and EC50 was determined. Two different experiments were performed in triplicate in each experiment.

[0329] The EC50 was similar for all constructs tested. Table 12

[0330] [Table 12]

[0331] Thus, binding of the mutant to SCF was unaffected compared to the wild type.

[0332] We then tested the proliferation of the mutants with increasing concentrations of RefMab#1 at a constant concentration of SCF (100ng / ml). TF-1 cells were seeded at a cell concentration of 150,000 cells / ml in sterile leukocyte culture-treated plates with clear bottom in RPMI+GlutaMax+10% FCS and a fixed concentration of SCF of 100ng / ml in the medium, and the cells were treated with different concentrations of RefMab#1. After 3 days of incubation, the CellTiter-Glo 2,0 assay (Promega) was performed and luminescence was read in a luminometer (Envision plate reader).

[0333] The results are shown in Figure 16. RefMab#1 had a significant effect on the proliferation of wild-type TF-1 cells, whereas the proliferation of all CD117 mutants tested was unaffected by RefMab#1.

[0334] For human CD34+ HSCs (different donors), cells are seeded in sterile leukocyte culture-treated plates at a cell density of 30,000 cells / ml in X-Vivo 20 medium containing 100ng / ml SCF, 50ng / ml TPO and 50ng / ml FLT-3 ligand. HSCs are then treated with various CD117 antibodies or different concentrations of isotype control antibodies and plates are incubated for 5 days. After 5 days, CellTiter-Glo 2.0 assay (Promega) is performed and luminescence is read in a luminometer (e.g., Envision or Phera Star plate reader).

[0335] Example 10: SCF-dependent phosphorylation of selected CD117 mutants SCF-dependent phosphorylation is another function of CD117. SCF-dependent phosphorylation was measured using TF-1 cells expressing CD117 (as shown in previous experiments). TF-1 cells were seeded at a concentration of 1 million cells / mL in 1 mL of medium (RPMI1640 supplemented with GlutaMAX+10% heat-inactivated FBS) in 6-well plates. Cells were treated with 0.5ug / mL of antibody or 500nM of avaplatinib. Cells were then treated with 100ng / mL of recombinant human SCF (1mg / mL of PeproGMP® recombinant human SCF) for 5 minutes before harvesting. After washing the cells with ice-cold PBS, the cells were resuspended in 250μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). Cells were flash frozen in liquid nitrogen and thawed twice at 37℃, then frozen at -80℃ until further use. Undiluted samples were thawed and processed according to the manufacturer's protocol for the PathScan® Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling). Absorbance (OD_450 nm) was assessed using Envision (Perkin Elmer).

[0336] Exemplary results are shown in Figure 12 for the E73K mutant of CD117. In wild-type cells, SCF-induced phosphorylation can be blocked with an antibody that competes with SCF for binding to CD117. SCF-induced phosphorylation is also blocked by avapritinib, a known inhibitor of CD117-SCF interaction. In contrast, SCF-induced phosphorylation is not blocked by an antibody against the E73K mutant of CD117. Avapritinib still blocks the SCF-induced phosphorylation in the E73K mutant.

[0337] Example 11: Selective blocking / depletion of cells expressing the first or second isoform The selected CD117 mutants are introduced into TF-1 cells by genome editing. The edited cells are then incubated with a toxin coupled to SR-1. The selective depletion of cells expressing the binding (wild-type) CD117 isoform can be monitored by FACS or viability assays. The assay can be performed with bulk edited cells. In the case of HDR, this results in a mixed population of cells including wild-type cells, KO cells and knock-in cells with correctly edited mutants. The three populations can be identified by FACS using SR-1 and 104D2. Alternatively, the three populations can be purified by FACS and subsequently incubated with a toxin coupled to SR-1.

[0338] Cells expressing wild-type CD117 die, but gene-edited cells expressing the mutant survive.

[0339] Instead of a toxin conjugated to SR-1, other modes of action can be used, including but not limited to ADCC or CAR T.

[0340] Instead of TF-1 cells, other cell lines that do not endogenously express CD117 (e.g., HEK293, DF-1) can be used. Transiently or stably recombinantly expressed wild-type or mutant CD117 can then be used as target cells for killing assays. Alternatively, primary human HSCs can be isolated as CD34+ cells. Gene-edited CD34+ HSCs can be transferred into immunodeficient mice (e.g., NSG, NSG-SGM3 or NBSGW) for engraftment studies. This allows for the engraftment and differentiation of progenitor cells to be monitored in vivo. Furthermore, depleting agents, e.g., toxin-conjugated SR-1, can be applied in vivo to monitor selective depletion of binding CD117 isoforms but not non-binding CD117 mutants.

[0341] Example 12: SR-1 binding to gene-edited mutants of CD117 Genetic editing of CD117 was performed in TF-1 cells using HDR. Twelve different crRNAs were tested, six of which targeted amino acid residue E73 (Table 3) and six of which targeted amino acid residues 120-123 (Table 3). Figure 13 shows a map depicting the binding of the tested crRNAs to position E73. Figure 14 shows a map depicting the binding of the tested crRNAs to positions 120-123.

[0342] Binding of antibody SR-1 to TF-1 cells was measured by FACS 7 days after electroporation with Cas9 protein, crRNA / tracrRNA or sgRNA and HDR template (Table 4). Loss of binding to gene-edited mutants compared to wild-type cells is shown in Table 13:

[0343] [Table 13]

[0344] Similar results were obtained with the anti-CD117 antibody 104D2.

[0345] HDR templates of SEQ ID NOs: 15-18 are ordered as Alt-R™ HDR Donor Oligos (Integrated DNA Technologies). The HDR templates have two different lengths, are in the plus or minus strand, and at least one base mutation introduces an E to K mutation. The HDR templates are used with crRNA KIT_E73_4 (SEQ ID NO: 6). The HDR templates may contain at least one silent mutation in the PAM to avoid recutting.

[0346] Example 13: Internalization of antibodies into CD117 mutants Antibody internalization can be tested by those skilled in the art by any commonly used assay, such as FACS. Cells expressing CD117 are incubated with antibodies labeled with a fluorophore, e.g., Alexa Fluor 488 (AF488), for various time points (0.5-6 h), followed by washing and quenching with anti-AF488 antibody for 1 h. The internalized antibodies can give a signal in the FACS reading, whereas the signal of the antibody bound to the cell surface is quenched and cannot be detected.

[0347] Antibody internalization into TF-1 cells expressing CD117 or its variants is measured by FACS. TF-1 cells are seeded at a concentration of 1 million cells / mL in 0.1 mL of medium (RPMI1640 supplemented with GlutaMAX+10% heat-inactivated FBS+2ng / mL GM-CSF) in 96-well plates. The next day, cells are treated with 2-20 μg / mL of antibodies labeled with Alexa Fluor 488 (AF488; Alexa Fluor® 488 Conjugation Kit (Fast)-Lightning-Link®, Abcam) for 30-360 min at 37 °C or 4 °C, after which cells are harvested and washed with ice-cold PBS. Cells are resuspended in ice-cold PBS containing 20-200ug / mL of anti-AF488 antibody (Alexa Fluor 488 polyclonal antibody, ThermoFisher Scientific) for 1 hour, followed by data acquisition on a FACS instrument (NovoCyte, Agilent). The signal of internalized antibody is measured by FACS, and the signal of extracellular antibody is quenched. The percentage of antibody internalization is calculated by dividing the signal of cells incubated with the quencher by the signal of cells incubated without the quencher.

[0348] Example 14: Binding of antibodies Refmab #2 and #3 to selected mutants A similar experiment to that in Example 7 was performed, but using Refmab #2 and #3, and the results are shown in Figure 15.

[0349] Refmab#2 showed a strong decrease in binding to mutants R122H, S239H and S239K compared to wild-type CD117. Refmab#3 showed a strong decrease in binding to mutants Y259A, Y259G, Y259P and S261V compared to wild-type CD117. A complete loss of binding was observed for Refmab#3 with mutants S261Q and S261E.

[0350] Example 15: Improved Refmab Antibodies Improved versions of antibody Refmab#1 were generated. The amino acid sequences of the improved binders, Refmab#1-Ernie and Refmab#1-Bert, are shown in Table 14.

[0351] [Table 14]

[0352] Refmab#1, Refmab#1-Ernie and Refmab#1-Bert were compared in various assays. Both derivatives have lower IC50s than the original Refmab#1 antibody on TF-1 cells (0.0176 and 0.0441 μg / ml compared to 0.2760 μg / ml) and HSPCs (0.00521 and 0.0313 μg / ml compared to 0.0623 μg / ml) in SCF-dependent proliferation assays. Both derivatives also inhibited SCF-dependent phosphorylation at least as strongly as the original Refmab#1 antibody. Both derivatives also inhibited binding of SCF to CD117. All antibodies also inhibit binding of SCF to CD117 in hematopoietic stem cells. All antibodies also deplete human HSCs in mice transplanted with human CD34+ HSPCs.

[0353] Example 16: Refcab#1 titration on engineered TF-1 cells The binding of Refmab#1 was evaluated by flow cytometry assay. TF-1 cells were seeded in FACS Buffer (PBS+2% FCS+1mM EDTA) at a cell concentration of 1 million cells / ml. After Fc blocking with 1:20 diluted TruStain (BioLegend, #422302) in FACS buffer for 15 min at 4°C, the cells were washed twice with FACS buffer. Subsequently, SR-1 titration was added to the cells using 5-0.00015μg / mL antibody for 30 min on a plate shaker at 4°C. The cells were washed twice with FACS buffer and the secondary labeled antibody (IgG(H+L) cross-adsorbed goat anti-human, Alexa Fluor® 488, Invitrogen, A11013) was added to the cells for 30 min on a plate shaker at 4°C. Cells were washed twice with FACS buffer, after which a 1:100 dilution of 7AAD stain (cell viability) in FACS buffer was added for several minutes and cells were analyzed using a Novocyte Quanteon (Agilent).

[0354] The results are shown in Figure 17. Binding of antibody Refmab#1 is completely abolished on TF-1 cells expressing the CD117 mutant E73K. Results of two independent TF-1 E73K clones are shown.

[0355] Example 17: SCF-dependent phosphorylation of CD117 TF-1 cells (wild type, knockout and mutants E73K, D121K and S123K of CD117) were seeded at a concentration of 1 million cells / mL in 2 mL of medium (RPMI1640 supplemented with GlutaMAX+10% heat-inactivated FBS) in 6-well plates. Cells were treated with 1–20 ng / mL recombinant human SCF (1 mg / mL PeproGMP recombinant human SCF) for 5 min before cell collection. After washing the cells with ice-cold PBS, the cells were resuspended in 500 μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). Cells were flash frozen in liquid nitrogen and thawed twice at 37 °C before freezing at -80 °C until further use. Undiluted samples were thawed and processed according to the manufacturer's protocol of the PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling). Absorbance (OD_450 nm) was assessed using Envision (Perkin Elmer).

[0356] Exemplary results for mutant E73K are shown in Figure 18. Wild-type TF-1 cells and E73K, D121K and S123K mutants of CD117 showed SCF-dependent phosphorylation of Tyr719 of CD117. Phosphorylation levels in knockout cells are at background levels.

[0357] Example 18: Conserved signaling of CD117 mutants in the presence of blocking antibodies TF-1 cells (wild type and mutants E73K, E73Y, D121K, S123F and S123K of CD117) were seeded in 2 mL of medium (RPMI1640 supplemented with GlutaMAX+10% heat-inactivated FBS) in 6-well plates at a concentration of 1 million cells / mL. Cells were pretreated with 0.5 μg / mL of antibody Refmab#1-Ernie or Refmab#1-Bert for 30 min, followed by the addition of 100 ng / mL of recombinant human SCF (1 mg / mL PeproGMP® recombinant human SCF) for 5 min. Cells were harvested, washed once with ice-cold PBS and resuspended in 500 μL of lysis buffer (lysis buffer from Signaling containing protease inhibitors and phosphatase inhibitors). Cells were then flash frozen in liquid nitrogen and thawed twice at 37° C. before being frozen at −80° C. until further use. Undiluted samples were thawed and processed according to the manufacturer's protocol for the PathScan Phospho-c-Kit (Tyr719) Sandwich ELISA Kit (Cell Signaling) or Human c-Kit (CD117) ELISA Kit (Abcam). Optical density (OD_450 nm) was assessed using Envision (Perkin Elmer).

[0358] Exemplary results of mutants D121K and S123K are shown in Figure 19. SCF-dependent CD117 phosphorylation in wild-type TF-1 cells is blocked by antibodies Refmab#1-Ernie and Refmab#1-Bert, but is not affected in TF-1 cells with D121K and S123K mutants of CD117 phosphorylation. The same is observed for mutants E73K, E73Y and S123F (data not shown).

[0359] Example 19: HSC depletion experiments Depletion of human HSCs in mice using various mAbs was adapted from Pang et al. (Blood (2019) 133:2069-78).

[0360] NBSGW mice (Jackson Laboratories) were injected with 1 Mio HSPCs. 8, 10, 12 and 14 days after cell injection, mice received antibody Refmab#1-Bert at 25 mg / kg iv per dose. After 16 weeks, mice were euthanized and blood, spleen and bone marrow were analyzed by FACS. Results of bone marrow HSC depletion are shown in Figure 20. The figure shows in vivo depletion of HSCs after injection of Refmab#1-Bert compared to animals that received an isotype control antibody. HSCs were identified by FACS as live / hCD45+ / CD34+ / CD38- / CD45RA- / CD90+.

[0361] In another experiment, NSG mice (Jackson Laboratories) were sublethally irradiated 1 day before injection with 1 Mio gene-edited HSPCs carrying the E73K mutant of CD117. 8, 10, 12 and 14 days after cell injection, mice were intravenously administered 25 mg / kg antibody Refmab#1-Bert or 25 mg / kg isotype control antibody per dose. After 16 weeks, mice were euthanized and blood, spleen and bone marrow were analyzed by FACS.

[0362] Bone marrow results are shown in Figure 21. CD117+ myeloid cells were identified by FACS as live / hCD45+ / CD33+ / CD117+. To identify CD117+ cells, antibody clone 104D2 was used, which does not interfere with the binding of Rekmab#1 and Rekmab#1-Bert to CD117. Clone 104D2 was used as an expression control. Based on double staining with anti-CD117 clone SR-1 and anti-CD117 clone 104D2, cells were classified as unedited (104D2+ and SR-1+) or E73K edited (104D2+ but SR-1-).

[0363] Figure 21 shows the depletion of unedited cells in mice administered with antibody Refmab#1-Bert compared to mice administered with isotype control antibody.In contrast, injection of Refmab#1-Bert resulted in enrichment of E73K mutant cells compared to animals receiving isotype control antibody.

[0364] Example 20: CD117 mutants are resistant to treatment with ADCs In this experiment, chicken DF-1 cells were used. DF-1 cells lack CD117. Cells were transfected with selected CD117 mutants (E73Y, D121K, S123K). Transfected cells were treated with ADCs (both antibodies coupled to tetherin, Rekmab#1-Ernie and Rekmab#1-Bert) at concentrations of 0.1, 1 and 10 μg / ml. After 48 hours, cells were analyzed by FACS.

[0365] The results are shown in Figure 22. D121K A5 and D121K A6 represent two different batches of plasmids for the same mutant. Both tested ADCs, Refmab#1-Ernie-teserine and Refmab#1-Bert-teserine, led to effective depletion of wild-type DF-1 cells, but not DF-1 cells transfected with the D121K or S123K mutants of CD117.

[0366] Example 21: Additional HSC depletion experiments Depletion of human HSCs in mice using various mAbs was adapted from Pang et al. (Blood (2019) 133:2069-78).

[0367] NBSGW mice (Jackson Laboratories) were injected with 1 Mio HSPCs from two different donors carrying E73K, S123K or D121K variants of CD117. A control group of mice received unedited electroporated control HSPCs. 7, 9, 11 and 12 days after cell injection, mice received 4 mg / kg of antibody Refmab#1-Bert iv per dose. After 16 weeks, mice were euthanized and blood, spleen and bone marrow were analyzed by FACS. Results for depletion of CD117 unedited cells and enrichment of CD117 edited cells are shown in Figure 23. CD117-specific antibody clones 104D2 and SR-1 were used to identify unedited and edited cells. Binding of 104D2 is not affected by gene editing, whereas SR-1 does not bind to edited variants of CD117. Based on double staining with both anti-CD117 clones, cells were classified as unedited (104D2+ and SR-1+) or edited (104D2+ but SR-1−).

[0368] Figure 23 shows the depletion of unedited CD34+ progenitor cells (gated as live / hCD45+ / CD34+ / CD38-) in mice receiving antibody Remab#1-Bert, compared to mice receiving isotype control antibody. In contrast, injection of Remab#1-Bert resulted in enrichment of E73K, D121K and S123K edited mutant cells, compared to animals receiving isotype control antibody.

Claims

1. A mammalian cell or population of cells expressing a first isoform of CD117 for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of CD117; the cells expressing the first isoform comprise genomic DNA having at least one polymorphism or engineered allele; the polymorphism or engineered allele is absent from the genome of the patient having cells expressing the second isoform of CD117; 10. A mammalian cell or population of cells, wherein said polymorphism or genetically engineered allele is characterized by at least one substitution of an amino acid at positions E73, D121, R122, S123, S239, Y259 and / or S261 of SEQ ID NO:

1.

2. The mammalian cell or population of cells described in claim 1, wherein the medical treatment comprises administering to a patient in need thereof a therapeutically effective amount of the cell or population of cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent comprising an antigen-binding region that specifically binds to the second isoform of CD117, in order to specifically deplete the patient's cells that express the second isoform of CD117.

3. wherein said first and second isoforms are substantially functionally identical, preferably the first and second isoforms bind to SCF and result in SCF-dependent proliferation and / or result in SCF-dependent phosphorylation, or the first and second isoforms of CD117 bind to SCF to a substantially similar extent, and preferably the KD of the first isoform of CD117 for SCF is less than 4-fold, preferably less than 3-fold, more preferably less than 2-fold, and most preferably less than 1.5-fold higher than the KD of the second isoform of CD117 for SCF; 2. The mammalian cell or population of cells of claim 1.

4. 2. The mammalian cell or population of cells of claim 1, wherein residue E73 is substituted with an amino acid selected from the group consisting of K, L, Y and R, and / or residue D121 is substituted with an amino acid selected from the group consisting of Y, H, K, R or T, most preferably H or K, and / or residue S123 is substituted with an amino acid selected from the group consisting of P, F or K, preferably K, and / or residue S239 is substituted with H or K, and / or residue Y259 is substituted with an amino acid selected from the group consisting of E, A, G, P, C and H, preferably P, A or G, most preferably A, and / or residue K193 is substituted with an amino acid selected from the group consisting of G, T, M, D and E.

5. The mammalian cell or population of cells of claim 2, wherein the first isoform of CD117 is obtained by modifying a nucleic acid sequence encoding the first isoform of CD117 by gene editing, preferably by introducing into the cell a gene editing enzyme capable of inducing site-specific mutations within a target sequence encoding a CD117 surface protein region involved in binding of the depleting agent, including at least a first antigen-binding region.

6. 2. The mammalian cell or population of cells of claim 1, wherein the medical treatment restores normal hematopoiesis after immunotherapy in the treatment of a hematopoietic disorder, preferably in the treatment of a malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), systemic mastocytosis, chronic myeloid leukemia (CML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), or B-acute lymphoblastic leukemia (B-ALL).

7. 3. The mammalian cell or population of cells of claim 2, wherein the depleting agent is an antibody, antibody-drug conjugate, or immune cell, preferably a T cell, having a chimeric antigen receptor (CAR) comprising a first antigen-binding region that specifically binds to the second isoform and does not bind or binds substantially weaker to the first isoform.

8. the first antigen-binding region comprises: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; or b) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; or c) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; 6. The mammalian cell or population of cells of claim 5, comprising:

9. the medical treatment comprises administering to the patient in need thereof a therapeutically effective amount of the cell or population of cells expressing the first isoform of CD117 in combination with a therapeutically effective amount of a depleting agent comprising an antigen-binding region that specifically binds to the first isoform of CD117, to specifically deplete transferred cells that express the first isoform of CD117; The use is in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic diseases such as acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN) or B-acute lymphoblastic leukemia (B-ALL), 2. The mammalian cell or population of cells of claim 1.

10. The mammalian cell or population of cells described in claim 9, wherein the depleting agent is subsequently administered to the cell or population of cells expressing the first isoform of CD117 to avoid eventual serious side effects such as graft-versus-host disease due to transplantation.

11. A pharmaceutical composition comprising a mammalian cell or population of cells according to any one of claims 1 to 10, optionally a depleting agent according to any one of claims 2 to 10 and a pharmaceutically acceptable carrier.

12. A depletion agent for use in preventing or reducing the risk of serious side effects in a patient who has been administered cells expressing a first isoform of CD117, wherein the patient's natural cells express a second isoform of CD117, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD117 and does not bind or binds substantially weaker to the second isoform of CD117.

13. 1. A depletion agent for use in selectively depleting host cells in a patient in need thereof, wherein native cells of the patient express a second isoform of CD117, and wherein the depletion agent comprises at least a first antigen-binding region that specifically binds to the second isoform of CD117, and wherein the first antigen-binding region of the depletion agent specifically binds to an epitope comprising amino acids E73, D121, R122, S123, S239, Y259 and / or S261 of SEQ ID NO: 1, more preferably an epitope comprising amino acids E73, S123, K127 and / or Y259 of SEQ ID NO: 1; Preferably, the first antigen-binding region comprises: a) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; or b) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; or c) an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; A depleting agent comprising:

14. It is a combination, a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 being characterized by a substitution of aspartic acid with lysine at position 121 of wild-type CD117; and b) a depleting agent, i. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; ii. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; or iii. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and a depleting agent comprising an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; A combination of.

15. It is a combination, a) a mammalian cell or population of cells expressing an isoform of CD117, said isoform of CD117 characterized by a serine to lysine substitution at position 123 of wild-type CD117; and b) a depleting agent, i. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 20, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 22; ii. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 60; or iii. an antibody heavy chain variable domain (VH) comprising the three CDRs VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO: 17, VHCDR2 is SEQ ID NO: 18 and VHCDR3 is SEQ ID NO: 19; and a depleting agent comprising an antibody light chain variable domain (VL) comprising the three CDRs VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 59, VLCDR2 is SEQ ID NO: 21 and VLCDR3 is SEQ ID NO: 61; A combination of.

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