CD33-identifiable surface protein variants for use in cell therapy

Abstract

This disclosure relates to the use of cells having identifiable surface proteins, which have engineered or naturally occurring mutations but are functional surface proteins, for therapeutic use. The present invention also relates to the use of cells having identifiable CD33 surface protein variants but are functional surface proteins, for therapeutic use, particularly in adoptive cell therapy.

Description

[Technical Field]

[0001] Description of financial support The project leading to this application was funded by the European Research Council (ERC) under the European Union's Horizon 2020 Research and Innovation Programme (Funding Agreement No. 818806). [Background technology]

[0002] Cell-based immunotherapy is emerging as the third pillar of post-treatment medicine, following small molecule therapies and biologics such as recombinant proteins containing antibodies. While cell therapy can be used in oncology to treat hematopoietic malignancies, other applications are under development, including the treatment of genetic disorders, solid organ tumors, and autoimmune diseases. However, cell therapy can be associated with severe and undesirable side effects. Indeed, cancer immunotherapy using chimeric antigen receptor (CAR) T cells has successfully targeted and eradicated malignant cells expressing specific antigens, but often fails to distinguish between normal and malignant cells, thus inducing the destruction of the normal hematopoietic system. Targeted therapies include antibody-based therapies, e.g., conventional monoclonal antibodies, multispecific antibodies, e.g., T cell engagers (e.g., BiTE), and cell therapies, e.g., 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 tumor cells, 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 syndrome (MDS), acute myeloid leukemia (AML), or chronic myeloid leukemia (CMML), cell surface antigens such as CD117, CD33, or CD123 are shared with normal myeloid progenitor cells and even hematopoietic stem cells. Thus, immunotherapy targeting CD117, CD33, or CD123 antigens for MDS, AML, or CMML may lead to depletion of normal hematopoietic cells in addition to malignant cells in the patient (Gill SIB Best practice & Research Clinical Hematology, 2019).As a result, targeted immunotherapies, including mAbs, T cell engagers, or CAR T cells, have often been challenging because some lack truly disease-specific surface antigens (Gill SIB Best practice & Research Clinical Hematology, 2019).

[0003] To regenerate normal hematopoiesis depleted by CD33-CAR T cell transfusion, CD33 CAR T cell-resistant 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 via the signaling domain of its immune receptor tyrosine-based inhibitory motif (ITIM). Therefore, it remains unclear to what extent CD33 loss can be well tolerated (Wissfeld et al. Glia (2021) 69:1393-1412). CD33 knockout (CD33 KO) cells transplanted into patients may exhibit long-term functional defects (International Publication No. 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). In fact, the frequency of CD33 KO cells decreased in two monkeys reported to undergo long-term observation. This may indicate dysfunction of CD33 KO cells, for example, due to reduced engraftment of CD33 KO long-term regrowth HSCs (LT-HSCs) or competitive disadvantage (Kim et al. 2018. Cell. 173:1439-53). Furthermore, the number of cell surface antigens with non-essential functions is very limited, and the loss of these redundant cell surface antigens can 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 antigen-loss relapse (Ruella et al. 2016 J Clin Invest 126:3814-26).

[0004] In previous patent applications, the inventors demonstrated that a single amino acid difference in surface protein variants can be genetically engineered into hematopoietic cells to alter their antigenicity, making them identifiable by specific and selective antibodies (International Publication No. 2017 / 186718, International Publication No. 2018 / 083071). In contrast to approaches in which surface proteins are removed (KO cells), surface protein variants in these cells retain their normal expression and function, allowing for the targeting of surface proteins with important non-redundant functions.

[0005] CD33, also known as SIGLEC3 or P67, is a transmembrane receptor expressed on myeloid cells, but can also be found on some lymphoid cells, such as a subset of NK cells. The extracellular portion of CD33 contains two immunoglobulin domains (one IgV and one IgC2 domain), and therefore CD33 belongs to the immunoglobulin superfamily. The intracellular portion of CD33 contains a specific motif that plays a role in inhibiting cell activity, the so-called immunoreceptor tyrosine-based inhibitory motif or ITIM.

[0006] CD33 can be stimulated by sialic acid residues, for example, found on glycoproteins or glycolipids. Upon sialic acid binding, the cytosolic portion of CD33 is phosphorylated, acting as a binding site for Src homology 2 (SH2) domain-containing proteins such as SHP phosphatases. This leads to a cascade of phagocytic inhibition in cells.

[0007] This disclosure aims to identify amino acid residues of CD33 exposed on the cell surface that can be substituted in a way that a) does not alter the function of CD33, or at least does not alter it substantially, i.e., the variant of CD33 is functionally indistinguishable from the wild-type version of CD33, and b) a portion such as an antibody or CAR T cell binds to the wild-type version of CD33, but substantially reduces or does not bind to the altered version of CD33, i.e., the variant of CD33 is immunologically distinguishable from the wild-type version of CD33. Most single amino acid substitutions in any given target protein affect the binding of a portion only if the amino acid substitution is part of or near the epitope of the binding portion. Similarly, as should be understood, a single amino acid substitution that affects the binding of a binding portion to a target antigen may also affect the functionality of the target antigen. Therefore, identifying amino acid substitutions that affect the binding of a portion to a target antigen while simultaneously not affecting or substantially not affecting its function is an extremely challenging and unpredictable task.

[0008] This disclosure also aims to identify amino acid residues of CD33 that are exposed on the cell surface and can be substituted in a way that a) CD33 expression is not altered, or at least substantially altered, i.e., the level of CD33 expression is indistinguishable from, or substantially indistinguishable from, the wild-type version of CD33, and b) a portion such as an antibody or CAR T cell binds to the wild-type version of CD33, but binding to the altered version is substantially reduced or absent, i.e., the CD33 variant can be substituted in a way that makes the CD33 variant immunologically distinguishable from the wild-type version of CD33.

[0009] Several anti-CD33 moieties are known in the art. CD33 is the target of gemtuzumab ozogamicin (Mylotarg), an antibody-drug conjugate developed by Pfizer / Wyeth-Ayerst for the treatment of acute myeloid leukemia (AML). CD33 is also the target of badastoximab platirine, an antibody-drug conjugate developed by Seagen Inc. Lintuzumab is a humanized antibody against CD33 developed by Seagen Inc. for the treatment of acute myeloid leukemia (AML). BI-836858 is a fully human IgG1 anti-CD33 monoclonal antibody with antineoplastic activity developed by Boehringer Ingelheim. AMG330 is a bispecific T-cell engager with specificity for CD33 and CD3, developed by Amgen for the treatment of AML. IMG779 is an antibody-drug conjugate against CD33 developed by ImmunoGen Inc. [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 [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] Wissfeld 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 [Summary of the Invention] [Problems to be Solved by the Invention]

[0012] One object of the present disclosure is to develop a safer method for treating malignant tumors, particularly cancer, hematological malignancies, and myeloid diseases. Therefore, the inventors sought variants of the surface protein CD33 that are immunologically distinguishable while retaining or substantially retaining normal function and / or expression, and whose amino acid changes are derived from single or multiple amino acid or nucleotide variants. In particular, the inventors identified variants of CD33 that change the antigenicity of CD33 to a specific antibody while retaining its normal expression and functions such as binding to sialic acid, stimulation by sialic acid, phosphorylation upon binding to sialic acid, the ability to act as a docking site for Src homology 2 (SH2) domain-containing proteins, and / or the ability to cause inhibition of phagocytosis. This was achieved by a sophisticated campaign involving screening projects, rational design approaches, and comparison with naturally occurring polymorphisms. [Means for Solving the Problems]

[0013] This disclosure relates to mammalian cells or cell populations expressing a first isoform of CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of CD33, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, and the polymorphic or genetically engineered allele is not present in the genome of the patient having cells expressing the second isoform of CD33, and preferably, the first and second isoforms are functional. Alternatively, the first isoform is generated via RNA editing. The disclosure also relates to mammalian cells or cell populations expressing a first isoform of CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of CD33, and the cells expressing the first isoform include genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of CD33, and preferably, the first and second isoforms are expressed at the same or substantially the same level.

[0014] In certain embodiments, the disclosure relates to mammalian cells or cell populations, preferably hematopoietic stem cells, for use in medical treatments in patients requiring such treatment, wherein the medical treatment is to specifically deplete patient cells expressing the second isoform of CD33, preferably to restore normal hematopoiesis after immunotherapy in the treatment of hematopoietic diseases, preferably acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and blastic plasmacytoid dendritic cell neoplasms (BPDCN). In the treatment of malignant hematopoietic disorders such as neoplasms and other myeloproliferative neoplasms, the treatment includes administering to a patient in need a therapeutically effective amount of cells or a population of cells expressing the first isoform of CD33, in combination with a therapeutically effective amount of a depleting agent containing at least a first antigen-binding region that specifically binds to the second isoform of CD33.

[0015] In other embodiments, the medical treatment relates to the 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, the medical treatment relates to the restoration of normal function in genetic disorders that do not originate from the hematopoietic and immune systems but can be treated by the use of modified hematopoietic cells.

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

[0018] In another specific embodiment, the disclosure relates to mammalian cells or cell populations for use in medical treatment in patients requiring medical treatment, wherein the medical treatment is to specifically deplete transferred cells expressing a first isoform, preferably for use in adoptive cell transfer therapy, preferably for acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and blastic plasmacytoid dendritic cell neoplasms (BPDCN). For the treatment of malignant hematopoietic disorders such as neoplasms and other myeloproliferative neoplasms, the treatment involves administering a therapeutically effective amount of cells or a population of cells expressing the first isoform to a patient in need, in combination with a therapeutically effective amount of a depleting agent containing at least a second antigen-binding region that specifically binds to the first isoform, wherein the depleting agent is administered subsequently to the cells or population of cells expressing the first isoform of the surface protein to avoid ultimate serious side effects such as graft-versus-host disease due to transplantation.

[0019] In another aspect, the disclosure relates to a pharmaceutical composition comprising mammalian cells, preferably hematopoietic stem cells or immune cells such as the myeloid cells or NK cells, and preferably a depleting agent and a pharmaceutically acceptable carrier.

[0020] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33. In certain embodiments, the depletion agent binds substantially weaker to the second isoform of CD33.

[0021] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms are substantially functionally identical. In certain embodiments, the depletion agent binds substantially weaker to the second isoform of CD33.

[0022] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms are expressed at substantially the same level. In certain embodiments, the depletion agent binds substantially weaker to the second isoform of CD33.

[0023] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in a patient who has received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms bind to sialic acid.

[0024] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms are stimulated by sialic acid.

[0025] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms act as docking sites for Src homology 2 (SH2) domain-containing proteins.

[0026] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms have the ability to cause phagocytic inhibition. The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms result in essentially the same modulation of myeloid cell function and / or NK cell function.

[0027] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms result in essentially the same regulation of microglial function.

[0028] The disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but does not bind to the second isoform of CD33, and the first and second isoforms result in normal differentiation of hematopoietic cells.

[0029] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but not to the second isoform of CD33, and the polymorphic allele or genetically engineered allele is characterized by at least one substitution of an amino acid at position N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of SEQ ID NO: 1, preferably at least one substitution of an amino acid at position N20, F21, W22 and / or Y50 of SEQ ID NO: 1. In certain embodiments, the depletion agent binds substantially weaker to the second isoform of CD33.

[0030] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 but not to the second isoform of CD33, and the polymorphic allele or genetically engineered allele is characterized by at least one amino acid substitution at positions N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50. Of these substitutions, substitutions N20, F21, W22 and Y50 are particularly preferred. In certain embodiments, the depletion agent binds substantially weaker to the second isoform of CD33.

[0031] This disclosure also relates to a depletion agent for use in preventing or reducing the risk of serious adverse events in patients who have received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 and does not bind to the second isoform of CD33, wherein residue N20 is G, Substances are substituted with S, D, E, K, V, R, or H; residue F21 is substituted with V, L, I, S, M, N, Q, or H; residue W22 is substituted with S, T, E, R, D, K, or H; Q24 is substituted with R; F43 is substituted with S; F44 is substituted with P or S; H45 is substituted with Y; residue P48 is substituted with F, D, or S; residue Y49 is substituted with A; and / or residue Y50 is substituted with S, L, R, D, E, A, K, or H.

[0032] This disclosure also relates to methods for improving engraftment in hematopoietic stem cell transplantation. Pre-hematopoietic stem cell transplantation (HSCT) conditioning (HSC depletion) is used to promote engraftment. In fact, the conditioning effect is associated with improved engraftment. Avoiding toxic conditioning is a key objective that can be achieved in this disclosure. Current methods for conditioning include the use of intravenous busulfan. Busulfan is a DNA alkylating agent originally designed to treat hematological disorders such as acute myeloid leukemia (AML). However, busulfan carries a risk of significant side effects, including infertility, primary or secondary malignancies, and further acute and chronic toxicity. [Brief explanation of the drawing]

[0033] [Figure 1] This shows the binding of anti-CD33 antibodies to HEK293-T cells transfected with human wild-type CD33 or an empty vector. Serial dilutions of each antibody were tested for immunoreactivity by flow cytometry. All eight antibodies in Mab format bound to human CD33 in a concentration-dependent manner. Cells transfected with the empty vector did not show binding to human CD33. [Figure 2] This shows the binding of the anti-CD33 Fab fragment to HEK293-T cells transfected with human wild-type CD33 or an empty vector. Similar to the full-length antibody, the Fab fragment also binds to human CD33 in a concentration-dependent manner. Cells transfected with the empty vector did not show binding to human CD33. [Figure 3]The results of alanine scans for human CD33 for the six antibodies tested are shown. For each mutant clone, the mean binding value determined by flow cytometry is plotted as a function of expression. CD33 clones with alanine substitutions identified as important are circled. Secondary clones, i.e., those that did not meet the initially set threshold but whose reduced binding activity and proximity to important residues suggested that the mutated residue might be part of the antibody epitope, are indicated by squares. [Figure 4] The 3D structures of CD33 with the residues identified by the alanine scan for the six antibodies tested are shown. [Figure 5] This shows an evaluation of the surface exposure of specific amino acid residues in CD33. [Figure 6] This shows antibody binding to a mutant that shows less than 10% binding to Refmab#1 compared to wild-type CD33. [Figure 7] The antibody shows binding to a mutant that exhibits less than 10% binding to Refmab#3 compared to wild-type CD33. [Figure 8] The antibody shows binding to a mutant that exhibits less than 10% binding to Refmab#4 compared to wild-type CD33. [Figure 9] This shows histograms of binding to ReFMab#3 after base editing of human CD34+HSPC by various sgRNAs. sgRNA-E showed the highest loss of binding to ReFMab#3, followed by sgRNA-S. [Figure 10] This shows the binding of edited HSPCs to ReFMab#3 as measured by flow cytometry. It can be seen that 95.8% of non-targeted control cells bound to ReFMab#3, while 89.0% of cells transfected with sgRNA-E did not. [Figure 11]We confirmed that the success of base editing is related to the loss of binding to ReFMab#3, as determined by NGS sequencing. Essentially all edited cells (A) showed a loss of binding to ReFMab#3, while essentially all unedited cells (C) remained reactive to ReFMab#3. Only half of the intermediate cell population (B) showed intended gene editing, consistent with heterozygous mutations resulting in only partial loss of binding. [Figure 12] In a colony-forming unit assay, edited cells differentiated into myeloid and erythrocyte cells to the same extent as unedited cells, demonstrating that gene editing did not affect cell differentiation. [Figure 13] Further confirmation that edited HSPCs are functionally indistinguishable from unedited HSPCs when measured using an in vitro differentiation assay. [Figure 14] This shows the engraftment of edited and unedited HSPCs in NBSGW mice after 13 weeks. Mouse HSPCs are almost completely replaced by human HSPCs (A). The absolute number of hCD45+ cells is equivalent between unedited and edited cells (B). The majority of sgRNA-E edited HSPCs lose binding to Reffmab#3 (loose) as a surrogate indicator of successful editing (C). [Figure 15] Peripheral blood at 13 weeks shows differentiation of edited and unedited HSPCs, with comparable numbers of myeloid cells (A), B lymphocytes and T lymphocytes (B, C), plasmacytoid dendritic cells and classical dendritic cells (D, E), and monocytes (F). [Figure 16] The experiment in Example 23 is outlined below. [Figure 17] The images show the luminescence of mice injected with tumor cells on day 0 (A: MOLM-14, B: OCI-AML2). After the start of treatment on day 10, rapid tumor growth was observed in animals treated with isotype controls or saline, but tumors shrank in animals treated with ReFMab#1 and ReFMab#3. The luminescence in these animals was comparable to that of control mice that did not receive tumor cell injections. [Figure 18] Flow cytometry of peripheral blood (A, B) and bone marrow 19 days after injection of tumor cells is shown. Refmab #1 and Refmab #3 eliminate both MOLM-14 (A, C) cells and OCI-AML (B, D) cells compared to isotype controls and saline. [Figure 19] This shows the binding of CD33 wild-type and mutant to sialic acid ligands. [Modes for carrying out the invention]

[0034] Immunotherapy is a promising treatment for cancer, hereditary, and autoimmune diseases. Immunodepletion agents, such as antibodies against tumor antigens or engineered immune cells, are administered to patients to target and kill tumor cells. However, since tumor surface proteins are also expressed on the surface of normal cells, including hematopoietic cells, this strategy can induce serious side effects in patients, for example, by altering hematopoiesis. To restore hematopoiesis in the patient, hematopoietic cells can then be transplanted. However, the binding of depletion agents to newly transplanted healthy cells, as well as diseased cells, may limit the maximum tolerated dose or restrict the use of healthy cells for pre-transplant therapy. Alternatively, the transplanted cells need to be resistant to the immunodepletion agent so that they are not targeted and eliminated by the agent. Therefore, one approach is to select cells that are resistant to the immunodepletion agent used in immunotherapy while retaining their function in restoring normal hematopoiesis in the patient.

[0035] The inventors have developed a method for identifying functional allele variants in gene sequences encoding surface protein regions involved in the binding of specific depletion agents. Such variants may be naturally occurring polymorphisms as well as / or designed and engineered variants. Different isoforms of the surface protein can be selected or generated. The first isoform of the surface protein encoded by the nucleic acid having the polymorphism is not recognized by the specific depletion agent. This variant allele does not alter or substantially alter the function of the surface protein, and / or is expressed at the same or substantially the same level. Thus, the depletion agent can be used to specifically deplete cells expressing one isoform by specifically binding to one isoform and not binding to or substantially binding to the other isoform. For example, if the depletion agent specifically binds to a second isoform rather than a first isoform, the depletion agent will specifically deplete cells expressing the second isoform. In another embodiment, the first isoform may be recognized by a second agent, and therefore the second agent may be used to specifically deplete cells expressing the first isoform, rather than the second isoform. Cells expressing the first isoform of the surface protein encoded by at least one mutant allele are advantageously used in medical procedures in patients having cells expressing the second isoform, particularly by using the second or first agent, respectively, to specifically deplete transplanted cells or patient cells.

[0036] This approach makes it impossible to predict which mutations of the surface antigen may be used. First, the mutation must be present in the surface-exposed stretch of the surface antigen accessible to the depletor. Second, the depletor must bind to this stretch on the exposed region of the surface antigen. Third, binding must be sufficient so that the depletor can distinguish the first isoform from the second isoform. Residual binding to other isoforms should be minimal or, better, completely absent. Fourth, the mutation must have little to no effect on the function of the surface antigen. The mutated isoform must perform its biological function to an acceptable degree, at least under a given therapeutic context. While specific tools exist for predicting three-dimensional protein structure, only experimental testing can demonstrate the usefulness of any given mutation.

[0037] Depletion agent This disclosure relates to agents comprising an antigen-binding domain that specifically binds to one isoform of CD33 on a cell and does not bind to or binds substantially less to another isoform of CD33. Such agents are referred to herein as “depletion agents.” Both isoforms of CD33 are functional, i.e., CD33 is functional with respect to at least one relevant property. Preferably, both isoforms of CD33 have the same function, i.e., they are functionally indistinguishable, or both isoforms are expressed at the same or substantially the same level.

[0038] However, the two isoforms of CD33 differ in their binding to depletion agents. Depletion agents specifically bind to only one of the CD33 isoforms. Therefore, the isoforms can be described as functionally identical (or functionally substantially identical) but immunologically distinct.

[0039] The first and second isoforms of CD33 may be polymorphic alleles. Preferably, the first and second isoforms of CD33 are naturally occurring polymorphic alleles. Also preferably, the first and second isoforms of CD33 are single nucleotide polymorphism (SNP) alleles.

[0040] The first and second isoforms of CD33 may also be genetically engineered alleles. Preferably, the first and second isoforms of CD33 differ by one, two, three, four, or five amino acids. Most preferably, the first and second isoforms of CD33 differ by one amino acid.

[0041] Various methods can be used to determine the mutations to be introduced into CD33 to generate a second isoform. For example, mutations can be randomly inserted, followed by functional and immunological screening of the resulting variants. Alternatively, mutations can be rationally designed, for example, by analyzing the secondary or tertiary protein structure of CD33.

[0042] Depletion agents include an antigen-binding region that specifically binds to one isoform of CD33 on a cell and does not bind to, or binds substantially weakly to, another isoform. The depletion agents of this disclosure can be divided into two main categories.

[0043] Firstly, the depletion agent may be a polypeptide containing an antigen-binding region. The polypeptide may consist of one or more polypeptide chains. Preferably, the polypeptide containing the antigen-binding region is an antibody. The polypeptide containing the antigen-binding region may also be an antibody fragment, an antibody-drug conjugate, or another variant of an antibody or scaffold. Exemplary antibody fragments and antibody scaffolds include single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, igNAR, bis-scFv, camelid antibodies, ankyrin, centinrin, domain antibodies, lipocalin, small-module immunotherapies, maxybodies, protein A, and affilin.

[0044] The depletion agent can also be coupled to chemical drugs, such as cytotoxic payloads or proteolytically targeted chimeric agents (PROTACs).

[0045] The polypeptide containing the antigen-binding domain may be a bispecific, biparatope, or multispecific antibody. Such molecules may also contain further functional domains. For example, the polypeptide containing the antigen-binding domain may be a T cell engager, such as BiTE. The polypeptide containing the antigen-binding domain may also be fused to the extracellular domain of a cytokine or chemokine, toxin, or cell surface receptor.

[0046] Alternatively, the depletion agent may be a cell containing an antigen-binding region. For example, the depletion agent may be a chimeric antigen receptor (CAR). In certain embodiments of this disclosure, the cell containing the antigen-binding region is a CAR T cell, a CAR NK cell, or a CAR macrophage. In preferred embodiments of this disclosure, the cell containing the antigen-binding region is a CAR T cell. In another preferred embodiment of this disclosure, the cell containing the antigen-binding region is a primary T cell containing a CAR.

[0047] The depletion agent specifically binds to one isoform of CD33 but not to the second isoform, and therefore specifically depletes cells expressing that one isoform.

[0048] In certain embodiments, the disclosure relates to a drug comprising a first antigen-binding region that specifically binds to a second isoform of CD33 but does not bind to a first isoform. In other embodiments, the disclosure also relates to a drug comprising a second antigen-binding region that specifically binds to a first isoform of CD33 but does not bind to a second isoform. In certain embodiments, the drug binds substantially weaker to the second isoform of CD33.

[0049] The first and second isoforms of CD33 may differ from each other by only one amino acid substitution. This 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 CD33 may also differ from each other by more than one amino acid, e.g., two, three, or more amino acids. The first and second isoforms of CD33 may also differ in that one isoform has one, two, three, or more amino acid insertions compared to the other isoform. The first and second isoforms of CD33 may also differ in that one isoform has one, two, three, or more amino acid deletions compared to the other isoform. The two isoforms may also differ from each other by combinations of amino acid substitutions, insertions, and / or deletions. In a preferred embodiment, the depletor is an antibody or antigen-binding fragment. If two isoforms of CD33 differ by more than one amino acid, the altered amino acids may be adjacent to each other, i.e., directly adjacent, or they may be separated.

[0050] As used herein, the term “antibody” refers to an immunoglobulin molecule and a molecule containing the immunologically active portion of an immunoglobulin molecule, i.e., an antigen-binding site that binds immunospecifically to an antigen. Thus, the term “antibody” encompasses not only the entire antibody molecule but also antibody fragments and variants (including derivatives) of antibodies.

[0051] In natural antibodies from rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by disulfide bonds. 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 antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains different sequence domains. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively called CH). The variable regions of both the light (VL) and heavy (VH) chains determine the binding recognition and specificity to the antigen. The constant region domains of the light chain (CL) and heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental migration, complement binding, and binding to the Fc receptor (FcR).

[0052] The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin, consisting of a variable region of one light chain and a variable region of one heavy chain. Antibody specificity lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is primarily composed of residues derived from the hypervariable region or complementarity-determining region (CDR). Occasionally, residues from the non-hypervariable region or framework region (FR) may be involved in the antibody binding site or may affect the entire domain structure, and therefore the binding site. The complementarity-determining region or CDR refers to an amino acid sequence that together defines the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light chain and heavy chain of an immunoglobulin each have three CDRs, called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, the antigen binding site typically contains six CDRs, including sets of CDRs derived from the heavy chain V region and the light chain V region, respectively. The framework region (FR) refers to the amino acid sequence interposed between CDRs. Therefore, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0053] Residues in antibody variable domains are traditionally numbered according to a system devised by Kabat et al. This system is described in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereafter “Kabat et al.”). This numbering system is used herein. Kabat residue notation does not necessarily directly correspond to the linear numbering of amino acid residues in the sequence number sequence. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to the shortening or insertion of structural components, regardless of the framework of the basic variable domain structure or the complementarity-determining region (CDR). The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with the “standard” Kabat numbering sequence. The CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR1), 50-65 (H-CDR2), and 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), 50-56 (L-CDR2), and 89-97 (L-CDR3) according to the Kabat numbering system.

[0054] In certain embodiments, the antibodies provided herein are antibody fragments, more particularly any protein comprising the antigen-binding domain of the antibodies disclosed herein. The antigen-binding domain may also be incorporated into another protein scaffold. Examples of antibody fragments and scaffolds include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, diabodies, single-domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, IgNAR, bis-scFv, camelid antibodies, ankyrin, centinrin, domain antibodies, lipocalin, small-module immunopharmaceuticals, maxybodies, protein A, and affilin.

[0055] As used herein, “antigen-binding region” or “antigen-binding fragment of an antibody” means, in some cases, a portion of an antibody that exhibits antigen-binding ability to a particular antigen, i.e., a molecule corresponding to a portion of the antibody's structure, which, in its native form, exhibits the same or substantially the same antigen-binding specificity to that 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 may also be called the “functional fragment” of the antibody.

[0056] The agents of this disclosure include antibodies and fragments thereof, but also include artificial proteins, also referred herein as antigen-binding antibody mimes, which have the ability to mimic the antigen-binding ability of antibodies. Antigen-binding antibody mimes are organic compounds that specifically bind to antigens but are not structurally related to antibodies. They are typically artificial peptides or small proteins with a molar mass of about 3 to 20 kDa.

[0057] The terms “antigen-recognizing antigen-binding region” and “antigen-binding region having specificity for the antigen” are used interchangeably herein with the term “antigen-binding region that specifically binds to the antigen.” As used herein, the term “specificity” refers to the ability of a drug containing an antigen-binding region, such as an antibody, to detectably bind to an epitope presented on an antigen.

[0058] "Specific binding" or "to bind specifically" is approximately 10 -8 Includes binding with monovalent affinity of M(KD) or higher. Preferably, the binding affinity is 10 -8 M(KD)~10 -12 M(KD), arbitrarily 10 -8 M(KD)~10 -10 M(KD), especially at least 10 -8 If the binding domain is M(KD), the binding is considered 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 plots. Whether a binding domain specifically reacts with or binds to a target can be easily tested, in particular, by comparing the reaction of the binding domain with the target protein or antigen with the reaction of the binding domain with proteins or antigens other than the target protein.

[0059] As used herein, the term “epitope” means the portion of an antigen to which an antibody or its antigen-binding region binds. Protein antigen epitopes can be divided into two categories: structural epitopes and linear epitopes. Structural epitopes correspond to discontinuous portions of the amino acid sequence of an antigen. Linear epitopes correspond to continuous sequences of amino acids from an antigen.

[0060] In another embodiment, bispecific or multispecific molecules, such as bispecific antibodies or multispecific antibodies, are further disclosed herein. For example, an antibody may be derivatized or ligated to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand against a receptor) to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Antibodies may, in practice, be derivatized or ligated to one or more other functional molecules to produce a multispecific molecule that binds to more than two different binding sites and / or target molecules, and such multispecific molecules are also intended to be encompassed in the terms “bispecific molecule,” “bispecific antibody,” “double paratope molecule,” “double paratope antibody,” “multispecific molecule,” and “multispecific antibody,” as used herein. To produce a bispecific molecule, the antibodies of this disclosure may be functionally ligated to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, cytokine, chemokine, toxin, PROTAC, or receptor extracellular domain, so that a bispecific molecule is produced (e.g., by chemical coupling, gene fusion, disulfide bond, non-covalent bond, or other means). The specific bispecific and multispecific molecules envisioned by this disclosure are T cell engagers, such as bispecific T cell engagers, such as BiTE.

[0061] As used herein, agents that do not bind to or substantially weakly bind to a particular isoform of CD33 include agents that cannot bind to cells expressing that particular isoform. For experimental testing, the agent may be labeled with a fluorescent marker or detected with a secondary antibody against the agent, and the percentage of cells displaying the fluorescent marker or secondary antibody is determined by FACS analysis. Typically, the test is performed on a cell line expressing a recombinant target protein, i.e., CD33. The target protein may be expressed in its entirety, or a cleaved form may be used, which must contain at least the extracellular domain or region of the extracellular domain containing the respective antibody epitope. To monitor the expression of the mutant isoform, cells may be stained simultaneously with two agents, one binding to the epitope into which the mutant is introduced, and the other binding to a different epitope than the one bound by the first agent. The second epitope remains unchanged and therefore this staining serves as an expression control. As a non-binding control, cells that do not express the protein of interest are used. As a maximum binding control, cells that do not normally express the protein of interest are transfected with a wild-type isoform. Different cell lines have different expression levels, but expression is controlled via endogenous regulatory elements such as promoters. Such cell lines can also be used to study the mode of action of depletion agents, to study effective shielding against different modes of action, to test cytotoxicity and shielding / resistance to cytotoxicity, or to test the function of engineered receptors. Phosphorylation of signaling molecules can be analyzed using Western blotting, ELISA, or FACS. Analysis of gene expression changes can be useful for analyzing gene expression in comparison to normal function. Cells can also be used to demonstrate the feasibility of editing specific mutants via different approaches, such as homologous recombination repair (HDR), base editing, or prime editing.

[0062] The binding of the drug in question may lead to the depletion of cells expressing the first isoform of CD33. Various mechanisms can lead to cell depletion. Antibody-dependent cellular cytotoxicity (ADCC) arises from the binding of the drug to a target protein and the activation of NK cells via the Fc portion on the drug, which is bound by FcR expressed by NK cells. The Fc portion of immunoglobulins refers to the C-terminal region of the immunoglobulin heavy chain. The Fc portion can be wild-type or engineered. Enhanced and engineered mutations in the Fc portion are known in the art. In certain therapeutic situations, it is desirable to reduce or eliminate 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 Fc receptors and / or to complement components such as C1 q, in order to reduce or eliminate the antibody's ability to induce effector function. For example, it may be desirable to reduce or eliminate the binding of the Fc region of antibodies against one or more Fcy receptors such as FcyRI, FcyRIla, FcyRIIb, and FcyRIIIa. Effector functions may include, but are not limited to, one or more of the following: complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling that induces apoptosis, crosslinking of target-binding antibodies, dendritic cell maturation, or T cell priming. The binding of the drug may also result in the blockage of binding to the intrinsic receptor ligand, thereby inducing cell death and apoptosis without cell-mediated depletion.

[0063] The reduction or loss of binding of an Fc region to the Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, e.g., the IgG1 Fc region, more specifically the human IgG1 Fc region, to result in a variant or manipulated Fc region, e.g., a mutant human IgG1 Fc region. Substitutions that result in reduced binding may be useful. To reduce or lose the binding properties of an Fc region to the Fc receptor, non-conservative amino acid substitutions, i.e., substituting one amino acid with another amino acid having different structural and / or chemical properties and / or charge, are preferred.

[0064] In certain embodiments of this disclosure, the Fc region of the antibody is of the IgG1 isotype and harbors mutations, i.e., the constant region harbors L234A, L235A and P329G mutations (PG-LALA mutations), i.e., the constant region harbors L234A, L235A and P329A mutations (PA-LALA mutations), or i.e., the constant region harbors L234A, L235E, G237A, A330S and P331S mutations (AEASS mutations). Those skilled in the art will recognize the possibility of manipulating the Fc region to obtain the desired effect.

[0065] The surrogate ADCC assay, as described in the Laboratory section, constitutes an industry standard for quantifying the efficacy of ADCC-mediated agents. Manipulated Jurkat reporter cells possess an NFAT-responsive luciferase gene and an Fc receptor such as human FcgRI, FcgRIIa, or FcgRIIIa. Binding of the Fc receptor to the bound antibody induces NFAT via receptor clustering, resulting in luciferase signaling. Lack of binding, and therefore clustering, does not result in luciferase signaling. Cells that do not express the target protein (e.g., HEK or chicken DF-1 cells), or human hematopoietic cancer cells such as TF-1, KG-1, KASUMI-1, U937, THP-1, or MV4-11 manipulated to be CD33-deficient (e.g., CD33 knockout), or cells expressing the wild-type protein (e.g., HEK-CD33 or DF-1-CD33, or TF-1, KG-1, KASUMI-1, U937, THP-1, or MV4-11 cell lines), or cells expressing individual mutants (e.g., CD33 mutants) were incubated with a test agent (e.g., antibodies Renmab#1, Renmab#3, or Renmab#4) and mixed with ADCC reporter cells. The ADCC signal was then quantified by measuring luciferase levels. Luciferase luminescence signaling was normalized to the maximum signal observed in HEK-CD33, DF-1-CD33, or the corresponding myeloid or T-cell cancer cell lines. ADCC was measured using the ADCC reporter assay (Promega, catalog no. G7015).

[0066] Other potential modes of action in line with this disclosure are also possible. These include antibody internalization combined with the use of antibody-drug conjugates. Another method of depleting target cells is by the use of T cell engager molecules. For example, a bispecific T cell engager with CD33 binding sites and CD3 (OKT3) binding sites derived from antibodies ReFMab#1, ReFMab#3, or ReFMab#4 may be used. Use the same target cells used in the ADCC assay. Add primary human T cells and the bispecific T cell engager. Human T cell activation is quantified by FACS, for example, by determining the upregulation of CD25 or CD69 and / or the frequency of cytokine release, or by determining T cell-mediated cell death.

[0067] The depletion agent described herein specifically binds to one isoform of CD33, enabling the depletion of cells expressing that isoform.

[0068] More preferably, in specific embodiments, the depletion agent according to the Disclosure does not bind to or binds substantially less weakly to the first isoform of CD33, but specifically binds to the second isoform of CD33, enabling the depletion of cells expressing the second isoform of CD33, in particular, in the manner of use disclosed herein. In particular, the depletion agent, which does not bind to or binds substantially less weakly to the first isoform of CD33, but specifically binds to the second isoform of CD33 expressed in the patient's cells, is used to deplete the patient's cells, but does not deplete hematopoietic stem cells expressing the first isoform of CD33 or their offspring that have been transplanted in the patient to restore hematopoiesis.

[0069] In another specific embodiment, the depletion agent according to the Disclosure does not bind to, or binds substantially less weakly to, a second isoform of CD33, but specifically binds to a first isoform of CD33, enabling the depletion of cells expressing the first isoform of CD33, particularly in the manner of use disclosed herein. In particular, the depletion agent, which does not bind to, or binds substantially less weakly to, a second isoform of CD33, but specifically binds to the first isoform of CD33 expressed in transplanted cells, is used to specifically deplete transplanted cells and avoid the ultimate serious side effects of transplantation, such as graft-versus-host disease.

[0070] Selective depletion of cells expressing specific isoforms of CD33 can be achieved without limitation by complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP).

[0071] In certain embodiments, the antigen-binding domain 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.” Cytotoxic or cytotoxic agents include any agent that is harmful to cells (e.g., killing them). Examples include Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, meitansinoids, calicheamicin, indolinobenzodiazepine, pyrrolobenzodiazepine, pyridinobenzodiazepine, camptothecin, topotecan, irinotecan, berotecan, deltecan, alpha-amanitin, microcystine, auristatin, and puromycin, as well as their analogues or homologues.

[0072] In certain embodiments, the antigen-binding domain is coupled to a PROTAC. As used herein, the term "PROTAC" refers to a proteorisis-targeted chimera. A PROTAC generally has three components: an E3 ubiquitin ligase-binding group (E3LB), a linker, and a protein-binding group. PROTACs and PROTAC-binding domains are known to those skilled in the art (see, for example, An et al, EBioMedicine. 2018 Oct;36:553-562).

[0073] In another specific 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.

[0074] "Recombinant" means an antigen-binding region that is not encoded by the cell in its native state, i.e., heterogeneous and non-endogenous. Thus, the expression of recombinant antigen-binding regions is seen as introducing new antigen specificity to immune cells, causing cells to recognize and bind to antigens that were not previously recognized. Antigen receptors can be isolated from any useful source. In certain embodiments of this disclosure, the cell containing the antigen-binding region is a CAR T cell, CAR NK cell, CAR Treg, or CAR macrophage. In preferred embodiments of this disclosure, the cell containing the antigen-binding region is a CAR T cell. In another preferred embodiment of this disclosure, the cell containing the antigen-binding region is a primary T cell containing a CAR.

[0075] In certain embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). A CAR is a fusion protein containing an antigen-binding region, typically derived from an antibody, linked to the signaling domain of a TCR complex. When an appropriate antigen-binding region is selected, a CAR can be used to direct immune cells, such as T cells or NK cells, towards a target antigen.

[0076] The antigen-binding region of a CAR is typically based on an antibody-derived scFv (single-stranded variable fragment). In addition to the N-terminal extracellular antibody-binding region, a CAR may typically include a hinge domain that acts as a spacer to extend the antigen-binding region away from the plasma membrane of the immune effector cell on which it is expressed, a transmembrane (TM) domain, an intracellular signaling domain (e.g., a signaling domain derived from the zeta chain of the CD3 molecule (CD3ζ) of the TCR complex, or an equivalent), and one or more costimulatory domains that may optionally support signaling or functionality of the cell expressing the CAR. Signaling domains from costimulatory molecules including CD28, OX-40 (CD134), CD27, ICOS, and 4-1BB (CD137) can be added alone (second generation) or in combination (third generation) to enhance the viability and increase proliferation of CAR-modified immune cells.

[0077] Those skilled in the art can select the above-mentioned appropriate antigen-binding regions for redirecting immune cells to be used in accordance with this disclosure. In certain embodiments, the immune cells to be used in the methods of this disclosure are redirected T cells, for example, redirected CD8+ T cells or redirected CD4+ T cells, or redirected NK cells.

[0078] Methods for genetically modifying immune cells to express recombinant antigen-binding regions are well known in the art. Nucleic acid molecules encoding antigen receptors can be introduced into cells, for example, in the form of vectors or any other suitable nucleic acid construct, or by inserting the nucleic acid molecule into the genome using genome editing techniques. Vectors and their necessary components are well known in the art. Nucleic acid molecules encoding antigen-binding regions can be generated using any method known in the art, for example, molecular cloning using PCR. Antigen-binding region sequences can be modified using commonly used methods such as site-directed mutagenesis.

[0079] CD33 CD33 (UniProt: P20138; also known as SIGLEC3, SIGLEC-3, P67, sialic acid-binding Ig-like lectin 3, FLJ00391, or Gp67) is a transmembrane receptor primarily expressed on myeloid cells. CD33 binds to sialic acid and is therefore a member of the SIGLEC family of lectins. The extracellular portion of this receptor contains two immunoglobulin domains (one IgV and one IgC2 domain), placing CD33 within the immunoglobulin superfamily. The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) involved in inhibiting cellular activity. CD33 can be stimulated by molecules containing sialic acid residues, such as glycoproteins or glycolipids. Upon binding, the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CD33, located in the cytosolic portion of the protein, is phosphorylated and acts as a docking site for Src homology 2 (SH2) domain-containing proteins such as SHP phosphatases. This leads to a cascade that inhibits phagocytosis in cells.

[0080] Human CD33 has the following amino acid sequence (SEQ ID NO: 1). MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYW FREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM ERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWL SAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTT GIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTH PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSE VRTQ In certain embodiments, the surface protein is CD33. In other embodiments, the surface protein is CD33 containing the amino acid sequence of SEQ ID NO: 1. In other embodiments, the surface protein is CD33 consisting of the amino acid sequence of SEQ ID NO: 1.

[0081] In certain embodiments, the Disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and preferably the first and second isoforms are functional.

[0082] In certain embodiments, the Disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms are functional.

[0083] In certain embodiments, the Disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms are substantially functionally identical.

[0084] While knowledge of the precise function of CD33 remains limited, certain activities of CD33 have been described. In certain embodiments, this disclosure relates to first and second isoforms of CD33 in which both isoforms are functional. In certain embodiments, this disclosure relates to first and second isoforms of CD33 in which both isoforms are functionally indistinguishable. In the present invention, “functionally indistinguishable” means first and second isoforms of CD33 in which they can perform the same function equally within the cell without significant impairment. In other words, the first and second isoforms are functionally almost indistinguishable. A slight functional impairment may be acceptable. In preferred embodiments, the first isoform of CD33 remains functional and retains the ability to perform the same function within the cell as the corresponding wild-type isoform without significant impairment.

[0085] One function of CD33 is sialic acid binding. Accordingly, in certain embodiments, the disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms bind to sialic acid.

[0086] CD33 is stimulated by molecules containing sialic acid. Accordingly, in certain embodiments, the disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms are stimulated by molecules containing sialic acid.

[0087] Upon binding of sialic acid, the immune receptor tyrosine-based inhibitory motif (ITIM) of CD33 is phosphorylated. Therefore, in certain embodiments, the disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical procedures in a patient requiring medical treatment, the patient having cells expressing a second isoform of the surface protein, the cells expressing the first isoform comprising genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms are phosphorylated upon binding of sialic acid. In certain embodiments, the phosphorylation of CD33 occurs at the immune receptor tyrosine-based inhibitory motif (ITIM) of CD33.

[0088] When phosphorylated, CD33 can act as a docking site for Src homology 2 (SH2) domain-containing proteins, such as SHP phosphatases. Therefore, in certain embodiments, the disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical procedures in patients requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and upon phosphorylation, the first and second isoforms can act as docking sites for Src homology 2 (SH2) domain-containing proteins. In certain embodiments, the Src homology 2 (SH2) domain-containing protein is an SHP phosphatase.

[0089] When phosphorylated, CD33 can also induce a cascade that inhibits phagocytosis. Therefore, in certain embodiments, the disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, the patient having cells expressing a second isoform of the surface protein, the cells expressing the first isoform comprising genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms can inhibit phagocytosis. In certain embodiments, phagocytosis is inhibited upon phosphorylation of CD33.

[0090] In certain embodiments, the Disclosure relates to a population of mammalian cells or cells expressing a first isoform of the surface protein CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of the surface protein, and the cells expressing the first isoform contain genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of the surface protein, and the first and second isoforms are expressed to substantially equal degrees. In certain embodiments, the first and second isoforms are expressed to equal degrees.

[0091] In accordance with this disclosure, it is also possible to combine further variants or isoforms of CD33 within the methods and compositions of this disclosure. Such isoforms may include, for example, a double mutant. Such isoforms may also include, for example, a single mutant and a double mutant. The methods and compositions of this disclosure may also be used in the depletion of myeloid cells in solid tumors to enhance the tumor response.

[0092] The methods and compositions of the present disclosure may also be used in combination with cell combinations, particularly when the surface protein is CD33 and other targets such as CD117, CD123, DLL-1, CD45, CD47, CD7, CLEC12A, CD44, FLT3, CD300LF, EVI2B, TPO, and combinations thereof are knocked out.

[0093] The methods and compositions of the present disclosure may also include cells expressing a first isoform of CD33, as well as other surface protein variants, such as CD117 variants, CD123 variants, DLL-1 variants, CD45 variants, CD47 variants, CD7 variants, CLEC12A (CD371) variants, CD44 variants, FFLT3 (CD135) variants, CD300LF variants, EVI2B variants, TPO variants, and any combination thereof.

[0094] CD33 polymorphism Cells expressing the first isoform of CD33 according to this disclosure contain genomic DNA having at least one polymorphic allele in the nucleic acid encoding the CD33 isoform. In particular, the polymorphism induces at least one mutation that is involved in the binding of a specific drug compared to the second isoform.

[0095] The polymorphism is preferably located within the nucleic acid sequence encoding the surface protein region of CD33 involved in the binding of the first drug, and more preferably within the extracellular portion of CD33, particularly within the secondary structural elements exposed to the solvent. More specifically, the polymorphism is located within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first drug. The polymorphism may be a mutation such as a 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 certain embodiments, the polymorphism is a single nucleotide polymorphism.

[0096] The term "isoform" refers to a variant of a protein that differs from another variant of the same protein in that at least one amino acid difference. In the context of this disclosure, such a difference may be a single amino acid substitution, but such a difference may also be a double, triple, or multiple amino acid substitution, or an insertion or deletion. Naturally occurring SNPs are also isoforms.

[0097] Sequence differences between the two isoforms can also be introduced genetically. Here, the sequence difference is preferably located within the nucleic acid sequence encoding the CD33 region involved in the binding of the first drug, and preferably located in the extracellular portion of the surface protein, particularly in the secondary structural elements exposed to the solvent. More specifically, the sequence difference is located within the nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first drug. The sequence difference may be a mutation such as a 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 certain embodiments, the sequence difference is a single-point mutation.

[0098] This disclosure provides polymorphisms of CD33, including polymorphisms that include substitutions of residues N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50. Particularly preferred polymorphisms include substitutions of residues N20, F21, W22 and / or Y50.

[0099] Particularly preferred polymorphisms include substitution of residue N20, where N20 is replaced with G, S, D, E, K, V, R, or H. Other preferred polymorphisms include substitution of residue F21, where F21 is replaced with V, L, I, S, M, N, Q, or H. Other preferred polymorphisms include substitution of residue W22, where W22 is replaced with S, T, E, R, D, K, or H. Other preferred polymorphisms include substitution of residue Q24, where Q24 is replaced with R. Other preferred polymorphisms include substitution of residue F43, where F43 is replaced with S. Other preferred polymorphisms include substitution of residue F44, where F44 is replaced with P or S. Other preferred polymorphisms include substitution of residue H45, where H45 is replaced with Y. Other preferred polymorphisms include substitution of residue P48, where P48 is replaced with F, D, or S. Other preferred polymorphisms include substitution of residue Y49, where Y49 is replaced with A. Other preferred polymorphisms include substitution of residue Y50, where Y50 is replaced with S, L, R, D, E, A, K, or H. Other preferred polymorphisms include substitutions of residues Y49 and Y50, where Y49 is replaced with A and Y50 is replaced with A.

[0100] In certain embodiments, the disclosure relates to a mutant CD33 polypeptide, which comprises at least one mutation in an amino acid selected from N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of wild-type human CD33.

[0101] It will be understood that each amino acid can be specified by a three-letter code or a one-letter code that is well known to those skilled in the art.

[0102] Table 1 shows 20 natural amino acids: [Table 1]

[0103] In the experiments described herein, specific variants of specific residues were identified. For practical reasons, it is impossible to test every possible variant. However, it will be understood that the identified variants can be substituted with similar amino acid residues. For example, acidic amino acids can be replaced with other acidic amino acids, as similar effects can be expected. Similarly, charged amino acids can be replaced with other charged amino acids. As an example, T264E is expected to be equivalent to T264D, since both D and E are acidic amino acids.

[0104] natural polymorphism In certain embodiments, the cells relating to this disclosure are selected from subjects comprising native genomic DNA having at least one innate polymorphic allele, preferably a single nucleotide polymorphism (SNP), in the nucleic acid encoding the isoform.

[0105] In certain embodiments, cells are selected from subjects that include native genomic DNA having at least one native polymorphic allele, particularly an SNP, in the nucleic acid sequence encoding a CD33 region involved in anti-CD33 agent binding, preferably located in the extracellular portion of the surface protein, more preferably in a secondary structural element exposed to the solvent.

[0106] Certain naturally occurring SNPs are documented in the literature. These naturally occurring SNPs, along with their respective binders that can distinguish such SNPs from other isoforms of CD33, may be used within the scope of this disclosure. Lists of naturally occurring SNPs of CD33 can be found in any of the following databases, such as gnomAD (https: / / gnomad.broadinstitute.org / ), dbSNP (https: / / www.ncbi.nlm.nih.gov / snp / =), or GeneCards (https: / / www.genecards.org / ).

[0107] gene editing In another specific embodiment, the cells expressing the first isoform of CD33 according to this disclosure are obtained by gene editing, preferably by altering the sequence encoding the surface protein in the patient's native genomic DNA.

[0108] Cells can be genetically engineered by introducing a gene editing system into the cell that induces the polymorphism resulting in the insertion, deletion, and / or substitution of amino acids in a surface protein. The gene editing mode targets a nucleic acid sequence referred to herein as a target sequence, which encodes the surface protein region involved in the first drug binding described above. In particular, if the surface protein is CD33, the gene editing mode targets a nucleic acid encoding at least one amino acid residue at positions N20, F21, W22, Q24, F43, F44, H45, P48, Y49, and / or Y50 of SEQ ID NO: 1. Preferably, amino acid residue N20 is substituted with G, S, D, E, K, V, R, or H, and / or residue F21 is substituted with V, L, I, S, M, NQ, or H, and / or residue W22 is substituted with S, T, E, R, D, K, or H, and / or residue Q24 is substituted with R, and / or residue F43 is substituted with S, and / or residue F44 is substituted with P or S, and / or residue H45 is substituted with Y, and / or residue P48 is substituted with F, D, or S, and / or residue Y49 is substituted with A or D, and / or residue Y50 is substituted with S, L, R, D, E, K, A, or H. The gene editing enzyme may be a sequence-specific nuclease, a base editor, a prime editor, or a CRISPR transposon-based system.

[0109] The term "nuclease" refers to a wild-type or mutant enzyme capable of catalyzing the hydrolysis (cleavage) of phosphodiester bonds between nucleotides in nucleic acid (DNA or RNA) molecules, preferably DNA molecules. "Cleavage" refers to a double-strand break or single-strand break event.

[0110] The term "sequence-specific nuclease" refers to a nuclease that cleaves nucleic acids 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 guide end nucleases like clustered and regularly arranged short palindromic sequence repeats (CRISPR) / Cas systems and Argonaut (Li et al., Nature Signal transduction and targeted Therapy, 5, 2020 review; Guha et al., Computational and Structural Biotechnology Journal, 2017, 15, 146-160).

[0111] According to this disclosure, a nuclease causes a DNA cleavage within a target sequence, which encodes a surface protein region involved in the binding of the first drug as described above. In certain embodiments, the inventors use a CRISPR system to induce a cleavage within a target sequence encoding the surface protein region recognized by the first drug as described above.

[0112] The “target sequence” is intended to target a portion of the sequence encoding the region on CD33 involved in the first drug binding as described above, and / or a sequence adjacent to said region on CD33 involved in the first drug binding, particularly up to 50 nucleotides adjacent to said region on CD33 involved in the first drug binding, preferably at least one (one or two) sequences of 20, 15, 10, 9, 8, 7, 6, or 5 nucleotides adjacent to said drug binding site.

[0113] The CRISPR system comprises two or more components: a Cas protein (CRISPR-associated protein) and a guide RNA. The guide RNA can be a single guide RNA or a double guide RNA. The Cas protein is a DNA endonuclease that uses the guide RNA sequence as a guide to recognize and generate double-strand breaks in DNA complementary to the target sequence. Cas systems that produce single-strand breaks require only one nuclease domain. Cas systems that produce double-strand breaks require two nuclease domains. The Cas protein may contain two active cleavage sites, such as an HNH nuclease domain and a RuvC-like nuclease domain.

[0114] The term Cas protein also refers to an engineered endonuclease, homolog, or orthologue of Cas9 that can cleave a target nucleic acid sequence. In certain embodiments, a Cas protein may induce cleavage of a nucleic acid target sequence that may correspond to either double-strand or single-strand breaks. Cas protein variants may be Cas endonucleases that do not exist in nature and are obtained by protein engineering or random mutagenesis. A Cas protein may 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, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cmrl, Cmr3, Cmr4, Cmr5, Cnrr6, Csbl, Csb2, Csb3, Csxl7, CsxM, Csx Examples include 1O, Csl6, CsaX, Csx3, Csl, Csxl5, Csfl, Csf2, CsO, Csf4, their homologs, orthologues, or modified versions thereof. Preferably, the Cas protein is the Streptococcus pyogenes Cas9 protein.

[0115] Cas is brought into contact with a guide RNA (gRNA) designed to contain a complementary sequence to the target sequence, thereby specifically inducing DNA cleavage within the target sequence, and in particular, within the complementary sequence of a portion of the target sequence that encodes a surface protein region recognized by the above-mentioned agent, according to this disclosure.

[0116] As used herein, “guide RNA,” “gRNA,” “sgRNA,” or “single guide RNA” refers to a nucleic acid that facilitates the specific targeting or homing of a gRNA / Cas complex to a target nucleic acid.

[0117] In particular, gRNA refers to RNA containing transactivating crRNA (tracrRNA) and crRNA. Preferably, the guide RNA corresponds to crRNA and tracrRNA that can be used separately or fused together to generate a single guide RNA. Complementary sequence pairing with the target sequence recruits Cas to bind to and cleave DNA at the target sequence.

[0118] According to this disclosure, the crRNA is engineered to include a complementary sequence to a portion of the target sequence encoding the surface protein region recognized by the drug, so that it 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 binder. In another preferred embodiment, the guide RNA includes chemical modifications known to those skilled in the art.

[0119] In certain embodiments, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 nucleotides, that is complementary to the target sequence. As used herein, the term “complementary sequence” refers to a segment of a polynucleotide (e.g., a portion of crRNA or tracRNA) that can hybridize to another portion of the polynucleotide under standard low-stringent conditions. Preferably, the sequences are complementary to each other according to the complementarity between two nucleic acid strands, which depends on inter-strand Watson-Crick base pairing, i.e., the intrinsic base pairings between adenine-thymine (AT) nucleotides and guanine-cytosine (GC) nucleotides. Such gRNA can be designed by any method known to those skilled in the art in consideration of this disclosure.

[0120] According to this disclosure, the target sequence encodes a surface protein region on CD33 involved in first drug binding, preferably located in the extracellular portion of CD33, more preferably located in the extracellular loop compared to the second isoform, and here again more preferably containing amino acid residues involved in drug binding.

[0121] In a preferred embodiment, if the surface protein is CD33, the target sequence encodes a CD33 region involved in the binding of a first agent, such as anti-CD33 agent binding as disclosed above. Preferably, the target sequence encodes at least one residue at positions N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of SEQ ID NO: 1.

[0122] DNA strand breaks introduced by nucleases according to this disclosure may result in DNA mutations at the break site via non-homologous end joining (NHEJ), often leading to small insertions and / or deletions, or replacement of DNA around the break site via homologous recombination repair (HDR).

[0123] In preferred embodiments, the polymorphism in the nucleic acid encoding the CD33 isoform is induced via post-DNA cleavage HDR repair and the introduction of an exogenous nucleotide sequence referred to herein as an HDR template.

[0124] The HDR template includes first and second portions of a sequence homologous to the 5' and 3' regions of the target sequence, respectively, and an intermediate sequence portion containing polymorphisms. Following the cleavage of the target sequence, a homologous recombination event is achieved between the genome containing the target sequence and the HDR template, and the genome sequence containing the target sequence is replaced by the exogenous sequence.

[0125] Preferably, homologous sequences of at least 20 bp, preferably greater than 30 bp, more preferably greater than 50 bp, and most preferably less than 200 bp are used. The homologous sequences may be dsDNA or ssDNA. Preferably, the homologous sequences are dsDNA. In fact, the shared DNA homology is located in the adjacent regions upstream and downstream of the cleavage site, and the introduced exogenous sequence should be located between the two arms. The adjacent sequences may be symmetric or asymmetric. Both strands of the target nucleic acid, i.e., the positive or negative strand, may be targeted. Optionally, PAM sequences that can be silenced to improve HDR may be used.

[0126] In a preferred embodiment, the cells according to this disclosure are genetically engineered by introducing a site-specific nuclease and HDR template that targets a sequence encoding a region on CD33 recognized by the first drug as described above.

[0127] In another specific embodiment, the gene editing enzyme is a DNA-based editor described in Komor et al., Nature 533, 420-424 and Rees HA, Liu DR. Nat Rev Genet. 2018;19:770-788, or a prime editor 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). Using either the base editor or the prime editor, mutations can be introduced at specific sites in the target sequence.

[0128] According to this disclosure, the base editor or prime editor generates mutations within the target sequence by sequence-specific targeting of the sequence encoding the region on CD33 involved in first drug binding.

[0129] In particular, the base editor or prime editor is a CRISPR-based editor or prime editor. The CRISPR-based editor or prime editor may contain an inactive (dead)Cas protein (dCas) as a catalytically inactive sequence-specific nuclease. It may also contain Cas9 having a mutant nuclease domain. dCas refers to a modified Cas nuclease lacking endonuclease activity. Nuclease activity can be inhibited or prevented in the dCas protein by one or more mutations and / or deletions in the HNH and / or RuvC-like catalytic domains of the Cas protein. The resulting dCas protein lacks nuclease activity but binds to the guide RNA (gRNA)-DNA complex with high specificity and efficiency to a specific target sequence. In certain embodiments, the dCas may be a Cas nickas in which one catalytic domain of Cas is inhibited or prevented.

[0130] The base editor forms a complex with a guide RNA (gRNA) designed to contain a complementary sequence to the target nucleic acid sequence, and then specifically binds to the target sequence as described above.

[0131] The gRNA may be designed by any method known to those skilled in the art in consideration of this disclosure. In certain embodiments, the gRNA may target a sequence encoding a region on CD33 recognized by the first drug as described above.

[0132] As a non-limiting example, the base editor is a nucleotide deaminase domain fused to an inactive (dead) Cas protein, particularly Cas nickase. The nucleotide deaminase may be an adenosine deaminase or a cytidine deaminase. The nucleotide deaminase may be a native or engineered deaminase.

[0133] 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.

[0134] The prime editor consists of a fusion of a catalytically inactive sequence-specific nuclease, particularly Cas nickase, with a catalytically active, engineered reverse transcriptase (RT) enzyme. The fusion protein is used in combination with a prime editing guide RNA (pegRNA) containing a sequence complementary to the target sequence, particularly when the surface protein is CD33, and also includes an additional sequence containing one of the sequences described herein and a sequence that binds to a primer-binding site region on DNA. In certain embodiments, the reverse transcriptase enzyme is Maloney mouse leukemia virus RT enzyme and its variants. The prime editor may be a non-limiting example selected from the group consisting of PE1, PE2, PE3, and PE3b, or any of the prime editors described in Chen et al. Cell (2021) 184:5635-52 or Koblan et al. Nat Biotechnol (2021) 39:1414-25.

[0135] Anti-CD33 agents Several anti-CD33 moieties are known in the art, and some are currently under development. WM53 (BioLegend; #303402) and 2337A (R&D Systems; #MAB11373-100) are commercially available research-grade monoclonal antibodies. Other anti-CD33 moieties include gemtuzumab ozogamicin (Mylotarg), an antibody-drug developed by Pfizer / Wyeth-Ayerst; badastoximab ptaririn, an antibody-drug conjugate developed by Seagen Inc.; lintuzumab, a humanized antibody developed by Seagen Inc.; BI-836858 and AMG330, fully human IgG1 anti-CD33 monoclonal antibodies developed by Boehringer Ingelheim; a bispecific T-cell engager with specificity for CD33 and CD3 developed by Amgen; and IMG779, an antibody-drug conjugate developed by ImmunoGen Inc. VCAR33 is a CD33-targeted CAR-T cell product developed by VOR Therapeutics. These and other anti-CD33 moieties may be used in the context of this disclosure. Several anti-CD33 antibodies were also prepared in full-length antibody format and, similarly, in Fab format in this disclosure. Details are provided in Example 1.

[0136] In certain embodiments, the depletion agent, which binds to the second isoform of CD33 and does not bind to the first isoform of CD33 as described above, or binds substantially weakly, specifically binds to epitopes containing amino acids N20, F21, W22, Q24, F43, F44, H45, P48, Y49, and / or Y50 of SEQ ID NO: 1. In certain preferred embodiments, the depletion agent specifically binds to epitopes containing amino acids N20, F21, W22, and / or Y50 of SEQ ID NO: 1. In other preferred embodiments, the depletion agent specifically binds to epitopes containing amino acids N20, F21, and W22 of SEQ ID NO: 1. In other preferred embodiments, the depletion agent specifically binds to epitopes containing amino acid Y50 of SEQ ID NO: 1.

[0137] In a preferred embodiment, the anti-CD33 agent is a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region.

[0138] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region that is derived from and maintains the binding specificity of antibody binding containing these.

[0139] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region that competes with antibody binding.

[0140] In another preferred embodiment, the anti-CD33 agent is as follows: a) The antibody heavy chain variable domain (VH) containing the variable heavy chain of Sequence ID No. 2, b) Includes an antibody light chain variable domain (VL) containing the variable light chain of Sequence ID No. 3, and an antigen-binding region containing the above.

[0141] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region.

[0142] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that maintains the binding specificity of antibody binding.

[0143] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that competes with the antibody-binding region.

[0144] In another preferred embodiment, the anti-CD33 agent is as follows: a) The antibody heavy chain variable domain (VH) containing the variable heavy chain of Sequence ID No. 18, b) Includes an antibody light chain variable domain (VL) containing the variable light chain of Sequence ID No. 19, and an antigen-binding region containing the above.

[0145] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region.

[0146] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region that maintains the binding specificity of antibody binding.

[0147] In a preferred embodiment, the anti-CD33 agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region that competes with the antibody-binding region.

[0148] In another preferred embodiment, the anti-CD33 agent is as follows: a) The antibody heavy chain variable domain (VH) containing the variable heavy chain of Sequence ID No. 25, b) Includes an antibody light chain variable domain (VL) containing the variable light chain of Sequence ID No. 26, and an antigen-binding region containing the above.

[0149] In another preferred embodiment, the anti-CD33 agent is an antibody selected from Refmab#1, Refmab#2, Refmab#3, Refmab#4, Refmab#5, Refmab#6, Refmab#7, and Refmab#8.

[0150] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but not to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform having an amino acid mutation selected from N20, F21 and / or W22 of SEQ ID NO: 1.

[0151] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid N20 of SEQ ID NO: 1, the mutation being N20K.

[0152] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21S.

[0153] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but not to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform having an amino acid mutation selected from N20, F21 and / or W22 of SEQ ID NO: 1.

[0154] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid N20 of SEQ ID NO: 1, the mutation being N20K.

[0155] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21S.

[0156] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but not to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has an amino acid mutation selected from N20, F21, W22, and / or Q24 of SEQ ID NO: 1.

[0157] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid N20 of SEQ ID NO: 1, the mutation being N20G.

[0158] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21L.

[0159] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21S.

[0160] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but not to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has an amino acid mutation selected from N20, F21, W22, and / or Q24 of SEQ ID NO: 1.

[0161] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid N20 of SEQ ID NO: 1, the mutation being N20G.

[0162] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21L.

[0163] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid F21 of SEQ ID NO: 1, the mutation being F21S.

[0164] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds to it substantially less weakly, with one isoform being wild-type CD33 and the other isoform having a mutation at amino acid Y50 of SEQ ID NO: 1.

[0165] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid Y50 of SEQ ID NO: 1, the mutation being Y50D.

[0166] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region, The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid Y50 of SEQ ID NO: 1, the mutation being Y50R.

[0167] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds to it substantially less weakly, with one isoform being wild-type CD33 and the other isoform having a mutation at amino acid Y50 of SEQ ID NO: 1.

[0168] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid Y50 of SEQ ID NO: 1, the mutation being Y50D.

[0169] In a particular preferred embodiment, the depletion agent is as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprises three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and includes an antigen-binding region that maintains the binding specificity of the antibody binding thereto. The depletion agent binds to one isoform of CD33 but does not bind to a second isoform of CD33, or binds substantially less weakly to it, one of which is wild-type CD33 and the other isoform has a mutation at amino acid Y50 of SEQ ID NO: 1, the mutation being Y50R.

[0170] The antigen-binding regions of anti-CD33 antibodies are further intended to be screened or optimized for their binding properties as defined above. In particular, the antigen-binding regions are intended to have 1, 2, 3, 4, 5, 6 or more variations in the amino acid sequences of 1, 2, 3, 4, 5, or 6 CDRs of the monoclonal antibodies provided herein. The amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the CDR1, CDR2, CDR3, CDR4, CDR5, or CDR6 of the light chain variable region or the VJ or VDJ region of the heavy chain variable region are intended to have insertions, deletions, or substitutions by conserved or non-conserved amino acids. Such amino acids that can be substituted or constitute substitutions are disclosed above.

[0171] In some embodiments, the amino acid difference is a conservative substitution, i.e., the substitution of one amino acid with another amino acid having similar chemical or physical properties (size, charge, or polarity), and this substitution generally does not adversely affect the biochemical, biophysical, and / or biological properties of the antibody. In particular, the substitution does not disrupt the interaction between the antibody and the CD33 antigen. The conservative substitution(s) are advantageously selected from one of the following five groups: Group 1 - small aliphatic, nonpolar, or slightly polar residues (A, S, T, P, G); Group 2 - polar, negatively charged residues and their amides (D, N, E, Q); Group 3 - polar, positively charged residues (H, R, K); Group 4 - large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5 - large aromatic residues (F, Y, W).

[0172] In more specific embodiments, the first antigen-binding region includes a heavy chain variable domain comprising or consisting of one amino acid sequence selected from SEQ ID NOs: 2, 10, 18, 25, 33, and 41, and / or a light chain variable domain comprising or consisting of one amino acid sequence selected from SEQ ID NOs: 3, 11, 19, 26, 34, and 42.

[0173] The first antigen-binding region having an amino acid sequence that is at least 90%, for example, at least 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences defined above is also part of the disclosure, and typically the first antigen-binding region has at least equal or higher binding activity than the first antigen-binding region comprising a heavy chain consisting of any one of the amino acid sequences selected from SEQ ID NOs: 2, 10, 18, 25, 33, and 41, and / or a light chain variable domain containing or consisting of any one of the amino acid sequences selected from SEQ ID NOs: 3, 11, 19, 26, 34, and 42.

[0174] In certain embodiments, the anti-CD33 agent may be a bispecific CD33 antibody comprising at least one first binding specificity to CD33, such as one antigen-binding region of the anti-CD33 described herein, and a second binding specificity to a second target epitope or target antigen.

[0175] According to this disclosure, the anti-CD33 agent may be an antigen receptor that targets CD33, for example, an immune cell having a CD33-targeting CAR, and the antigen receptor includes an antigen-binding domain as described herein.

[0176] In certain embodiments, immune cells (e.g., T cells) having a CD33-targeting CAR recognize a second isoform of CD33 expressed in patients requiring it, but do not recognize a first isoform of CD33. In particular, these immune cells may specifically bind to epitopes containing amino acids N20, F21, W22, Q24, F43, F44, H45, P48, Y49, and / or Y50 of SEQ ID NO: 1. More specifically, these immune cells may specifically bind to epitopes containing amino acids N20, F21, W22, and / or Y50 of SEQ ID NO: 1.

[0177] In certain embodiments, the anti-CD33 agent may be an immune cell having a CAR (e.g., a T cell), and the CAR is a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) The antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and the antigen-binding region comprising an three CDRs, VLCDR1, VLCDR2, and VLCDR3.

[0178] In a more specific embodiment, the anti-CD33 agent may be an immune cell (e.g., a T cell) having an scFv that includes a CAR containing the first antigen-binding region, for example, a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 2, and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 3.

[0179] In certain embodiments, the anti-CD33 agent may be an immune cell having a CAR (e.g., a T cell), and the CAR is a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) The antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and comprises or derives from an antigen-binding region.

[0180] In a more specific embodiment, the anti-CD33 agent may be an immune cell (e.g., a T cell) having an scFv that includes a CAR containing the first antigen-binding region, for example, a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 18, and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 19.

[0181] In certain embodiments, the anti-CD33 agent may be an immune cell having a CAR (e.g., a T cell), and the CAR is a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) The antibody light chain variable domain (VL) comprising three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and comprises or derives from an antigen-binding region.

[0182] In a more specific embodiment, the anti-CD33 agent may be an immune cell (e.g., a T cell) having an scFv that includes a CAR containing the first antigen-binding region, for example, a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 25, and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 26.

[0183] In another preferred embodiment, the anti-CD33 agent may be an immune cell having a CAR that targets a specific isoform of CD33, as described in the examples.

[0184] In particular, the disclosure also relates to the above-mentioned depletion anti-CD33 agents (e.g., CAR cell compositions or antibodies) comprising a first or second antigen-binding domain for use in selectively depleting host cells or transplanted cells, respectively, in subjects requiring selective depletion of host cells or transplanted cells.

[0185] Cells expressing the first isoform of CD33 This disclosure relates to mammalian cells, preferably hematopoietic cells, or a population of cells expressing a first isoform of CD33, wherein the cells or population of cells express a first isoform of CD33, and the first isoform is not recognized by a depleting agent comprising a first antigen-binding domain as described herein.

[0186] The disclosure also relates to mammalian cells, preferably hematopoietic cells, or a population of cells expressing a first isoform of CD33, wherein the cells or population of cells express a first isoform of CD33 comprising at least one polymorphic allele in the nucleic acid encoding the first isoform, and the first isoform is not recognized by a depleting agent comprising a first antigen-binding region as described herein.

[0187] Cells expressing the first isoform of CD33 that is not recognized by the depletion agent containing the first antigen-binding domain described herein do not necessarily have to contain the polymorphic or genetically modified allele in their genomic DNA.

[0188] The first isoform of CD33 may also be transiently expressed in such cells by any method known to those skilled in the art.

[0189] The first isoform of CD33 can also be generated in the cells by in vivo editing using any suitable means known to those skilled in the art.

[0190] These cells or cell populations are particularly useful in medical treatments for patients expressing the second isoform of CD33.

[0191] In certain embodiments, cells (e.g., hematopoietic stem cells) encoding or expressing the first isoform of CD33 that is not recognized by the depleting agent (e.g., hematopoietic cells) are particularly useful in medical procedures to restore normal hematopoiesis after immunotherapy, such as adoptive cell transfer in patients expressing the second isoform, in particular, the procedure comprising administering a therapeutically effective amount of the hematopoietic cells expressing the first isoform of CD33 in combination with a therapeutically effective amount of a depleting agent targeting the second isoform of CD33. In particular, the hematopoietic cells, preferably hematopoietic stem cells, are administered after the depleting agent. In another particular embodiment, the hematopoietic cells, preferably hematopoietic stem cells, may be administered before or concurrently with the depleting agent.

[0192] In another specific embodiment, cells expressing the first isoform of CD33, which is specifically recognized by a depletion agent that does not bind to or binds substantially less weakly to the second isoform of CD33, are particularly useful in medical treatment in patients expressing the second isoform of CD33, in particular to avoid serious side effects associated with transplanted cells having the first isoform (safety switch), the treatment comprising administering a therapeutically effective dose of a depletion agent targeting the first isoform of CD33. In particular, the hematopoietic cells, preferably immune cells having CAR, are administered before the depletion agent.

[0193] As used herein, the term "cell" refers to a mammalian cell, preferably a human cell.

[0194] In certain embodiments, the cells are hematopoietic cells. Hematopoietic cells include lymphocytes such as B cells and T cells, myeloid cells such as natural killer cells and monocytes, and immune cells including macrophages, eosinophils, mast cells, basophils, granulocytes, dendritic cells (DCs), and plasmacytoid dendritic cells (pDCs).

[0195] In certain embodiments, the immune cells are T cells. In another preferred embodiment, the immune cells are primary T cells. As used herein, the term “T cell” includes cells having a T cell receptor (TCR) or cells derived from T cells having a TCR. The T cells according to this disclosure may 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 cells may be derived from CD4+ T lymphocytes and CD8+ T lymphocytes or non-classical T cells, such as MR1-restricted T cells, MAIT cells, NKT cells, gamma delta T cells, or native-like T cells.

[0196] T cells can be obtained from a number of non-limited sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from infection sites, ascites, pleural fluid, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from units 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.

[0197] In another preferred embodiment, the hematopoietic cells are hematopoietic stem cells. The stem cells may 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. A typical human stem cell is CD34. + Hematopoietic stem cells are cells. They can be differentiated from iPS cells, or they can be collected from umbilical cord blood, bone marrow, or recruited or unrecruited peripheral blood.

[0198] In certain embodiments, cells are allogeneic cells, meaning cells derived from a donor whose HLA genotype is identical, similar, or different from that of the person receiving the cells. The donor may be a relative or an unrelated person. In certain embodiments, cells are autologous cells, meaning cells derived from the same person receiving the cells.

[0199] These cells may originate from healthy donors or patients, particularly those diagnosed with cancer, genetic disorders, or autoimmune diseases, or those diagnosed with infectious diseases. Hematopoietic cells may be extracted from blood, bone marrow, or derived from stem cells. HSCs may be derived, for example, from iPS cells (induced pluripotent stem cells).

[0200] Those skilled in the art will select more appropriate cells depending on the patient or subject to be transplanted.

[0201] This disclosure further relates to a composition or population of cells for use in the therapeutics disclosed herein.

[0202] In certain embodiments, the Disclosure relates to mammalian cells or cell populations expressing a first isoform of CD33 for use in medical treatment in a patient requiring medical treatment, wherein the patient has cells expressing a second isoform of CD33, and the cells expressing the first isoform include genomic DNA having at least one polymorphic or genetically engineered allele, the polymorphic or genetically engineered allele not present in the genome of the patient having cells expressing the second isoform of CD33, and the polymorphic or genetically engineered allele is characterized by at least one amino acid substitution at positions N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of SEQ ID NO: 1. In certain preferred embodiments, the polymorphic or genetically engineered allele is characterized by at least one amino acid substitution at positions N20, F21, W22 and / or Y50 of SEQ ID NO: 1.

[0203] In another preferred embodiment, the polymorphic allele or genetically engineered allele is characterized by at least one substitution at the amino acids at positions N20, F21, and W22 of SEQ ID NO: 1. In yet another preferred embodiment, the polymorphic allele or genetically engineered allele is characterized by a substitution at the amino acid at position Y50 of SEQ ID NO: 1.

[0204] CAR For use in adoptive cell transfer therapy, cells expressing the first isoform of CD33 according to this disclosure can be modified to exhibit desired specificity and enhanced functionality. In certain embodiments, the cells may express a recombinant antigen-binding region, also called 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 this disclosure, immune cells expressing the first isoform of CD33 and a CAR can be specifically depleted by administering a therapeutically effective dose of a drug containing a second antigen-binding region that specifically binds to the first isoform of CD33 but not to the second isoform of CD33, thereby avoiding the ultimate serious side effects resulting from the transplantation of such immune cells.

[0205] In certain embodiments, immune cells are redirected to cancer antigens. “Cancer antigen” means any antigen associated with cancer (i.e., a molecule capable of inducing an immune response). Antigens as defined herein can be any type of molecule that induces an immune response, for example, polysaccharides or lipids, but most preferably peptides (or proteins). Human cancer antigens can be human or of human origin. Cancer antigens can be tumor-specific antigens, meaning antigens not found in healthy cells. Tumor-specific antigens generally arise from mutations, particularly frameshift mutations that produce entirely new amino acid sequences not found in the healthy human proteome.

[0206] Cancer antigens include tumor-associated antigens, whose expression or production is associated with, but not limited to, tumor cells. Examples of tumor-associated antigens include 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 fiber acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin, and others. These include thyroid filaments, 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, CD45, CD123, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma surrogate reading frame protein), Trp-p8, STEAP1 (prostate 1 six-transmembrane epithelial antigen), abnormal ras protein, or abnormal p53 protein. In another specific embodiment, the tumor-associated antigen or tumor-specific antigen is integrin ανβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene), or Ral-B.

[0207] In certain embodiments, for use in adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic disorders such as acute myeloid leukemia (AML) or B-acute lymphoblastic leukemia (B-ALL), the immune cells according to this disclosure express a recombinant antigen-binding domain such as a CD33-targeting CAR. The cells expressing a first isoform and expressing a CAR (e.g., CAR-CD33) can be further specifically depleted by administering a depletion agent containing a second antigen-binding domain that specifically binds to the first isoform of CD33 but does not bind to, or substantially weaker than, the second isoform of CD33, thereby avoiding serious adverse events such as graft-versus-host disease resulting from transplantation.

[0208] In certain embodiments, the immune cells expressing the first isoform have a CD33-targeting CAR, the CAR comprising an antigen-binding region, e.g., scFv, which specifically binds to an epitope of CD33 located within the N-terminal domain or within a polypeptide comprising amino acids N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of SEQ ID NO: 1.

[0209] In particular, the immune cells expressing the first isoform are as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 4, VHCDR2 is sequence number 5, and VHCDR3 is sequence number 6, b) Having an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 7, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 9, and having an antigen-binding region, e.g., a CAR-targeted CD33 containing scFv, which comprises or is derived therefrom, More preferably, the antigen-binding region includes a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 3.

[0210] In particular, the immune cells expressing the first isoform are as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 20, VHCDR2 is sequence number 21, and VHCDR3 is sequence number 22. b) Having an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 23, VLCDR2 is sequence number 8, and VLCDR3 is sequence number 24, and an antigen-binding region, e.g., a CAR-targeted CD33 comprising scFv, which comprises or is derived therefrom, More preferably, the antigen-binding region includes a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 18 and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 19.

[0211] In particular, the immune cells expressing the first isoform are as follows: a) An antibody heavy chain variable domain (VH) containing three CDRs, VHCDR1, VHCDR2, and VHCDR3, where VHCDR1 is sequence number 27, VHCDR2 is sequence number 28, and VHCDR3 is sequence number 29. b) Having an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is sequence number 30, VLCDR2 is sequence number 31, and VLCDR3 is sequence number 32, and having an antigen-binding region, e.g., a CAR-targeted CD33 containing scFv, which comprises or is derived therefrom, More preferably, the antigen-binding region includes a heavy chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 25 and / or a light chain variable domain containing or comprising the amino acid sequence of SEQ ID NO: 26.

[0212] In vitro method for preparing cells expressing the first isoform Cells expressing the first isoform of CD33 according to this disclosure can be genetically engineered by introducing into the cells a nucleic acid construct (e.g., mRNA) encoding at least one gene editing enzyme or ribonucleoprotein complex, including the gene editing enzyme and / or HDR template described above. Alternatively, the gene editing system can be transduced into the cells via a viral system, such as an adenovirus system. The cells can also be genetically engineered by further introducing into them a nucleic acid construct encoding the CAR described above. In particular, the method is an ex vivo method performed on cell cultures.

[0213] As used herein, the term “nucleic acid construct” 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, modified to contain segments of nucleic acid sequences, and combined and juxtaposed in ways not normally found in nature. A nucleic acid construct is typically a “vector,” i.e., a nucleic acid molecule used to deliver exogenously produced DNA to a host cell.

[0214] Preferably, the nucleic acid construct comprises a gene editing enzyme, an HDR template, and / or a CAR operably linked to one or more regulatory sequences. The regulatory sequences may be ubiquitous, tissue-specific, or inducible promoters that function in cells of a target organ (i.e., hematopoietic cells). Such sequences known in the art include, in particular, promoters, and further regulatory sequences that can further control the expression of the transgene, such as, but not limited to, enhancers, terminators, introns, and silencers.

[0215] The nucleic acid constructs described above may be included in the expression vector. The vector may be an autonomous replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may include any means to ensure self-replication. Alternatively, the vector may, upon introduction into a host cell, be integrated into the genome and replicate along with the integrated chromosome(s).

[0216] Suitable vectors include, but are not limited to, recombinant embedded or non-embedded viral vectors, and vectors derived from recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA. Preferably, the vector is a recombinant embedded or non-embedded viral vector. Examples of recombinant viral vectors include, but are not limited to, vectors derived from herpesviruses, retroviruses, lentiviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, or bovine papillomaviruses.

[0217] This disclosure relates to a method for expressing a first isoform of a cell surface protein in cells by introducing a nucleic acid construct (e.g., mRNA) encoding a gene editing enzyme or ribonucleoprotein complex, including the gene editing enzyme and / or HDR template described above, into the cells. The method may further include the step of introducing a nucleic acid construct encoding a CAR into the cells. The method includes introducing a gene editing enzyme, such as a Cas protein, a base editor or prime editor, and a guide RNA (crRNA, tracrRNA, or fusion guide RNA or pegRNA) into the cells. In particular, the gene editing enzyme is the CRISPR / Cas gene editing enzyme described above. In more detailed embodiments, the gene editing enzyme is a site-specific nuclease comprising the guide RNA and the Cas protein, more preferably a CRISPR / Cas nuclease, wherein the guide RNA combined with the Cas protein cleaves and induces cleavage within the target sequence containing the nucleic acid encoding the surface protein region involved in drug binding as described above.

[0218] The Cas nuclease in question may be a high-fidelity Cas nuclease, such as a high-fidelity Cas9 nuclease.

[0219] The gene editing enzyme described above, preferably guide RNA and / or Cas protein, base editor, or prime editor, can be synthesized in situ within a cell as a result of introducing a nucleic acid construct, preferably an expression vector encoding the gene editing enzyme such as the guide RNA and / or Cas protein, base editor, or prime editor, into the cell. Alternatively, the gene editing enzyme such as the guide RNA and / or Cas protein, base editor, or prime editor can be produced extracellularly and then introduced into the cell.

[0220] The nucleic acid construct or expression vector can be introduced into cells by any method known in the art, and non-limiting examples include stable transduction methods in which the nucleic acid construct or expression vector is integrated into the cell genome, transient transfection methods in which the nucleic acid construct or expression vector is not integrated into the cell genome, and virus-mediated methods. For example, transient transformation methods include, for example, microinjection, electroporation, cell squeezing, particle bombardment or in vivo targeting approaches.

[0221] In vivo editing Cells expressing the first isoform of CD33 according to the present disclosure can also be edited in vivo. There are various techniques that enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles (see, e.g., Cell (2022) 185:2806-27). The molecular mechanism for converting CD33 to the first isoform of CD33 that is not recognized by the depletion agent can be achieved by any of these methods.

[0222] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising a molecular mechanism capable of editing genes and depletion agents in vivo for use in medical treatment in a patient in need of medical treatment. The molecular mechanism capable of editing genes in vivo includes all the components required to introduce point mutations of wild-type CD33 in target cells into isoforms of CD33. The depletion agent binds to wild-type CD33 but does not bind to the isoform of CD33.

[0223] Pharmaceutical composition and therapeutic use In a further aspect, the present disclosure also provides a pharmaceutical composition comprising a cell or cell population expressing the first isoform of CD33 as described above, together with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0224] In certain embodiments, the cells expressing the first isoform of CD33 are hematopoietic stem cells.

[0225] In another embodiment, the cell expressing the first isoform of CD33 is an immune cell having a chimeric antigen receptor (CAR), preferably a T cell, more preferably a primary T cell, that targets a second isoform of CD33 expressed by the patient's cells as described above.

[0226] The pharmaceutical composition may further include a depleting agent having the first or second antigen-binding region as described above.

[0227] The pharmaceutical composition is formulated in a pharmaceutically acceptable carrier according to the route of administration. Preferably, the composition is formulated to be administered by intravenous injection. A pharmaceutical composition suitable for such administration may contain cells expressing the first isoform described above, in combination with one or more pharmaceutically acceptable sterile isotonic or non-aqueous solutions (e.g., equilibrium salt solutions (BSS)), dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into a sterile injection solution or dispersion immediately before use, which may contain antioxidants, buffers, bacteriostatic agents, solutes or suspensions or thickeners.

[0228] Optionally, a composition containing cells expressing a first isoform of CD33 may be frozen for storage at any temperature suitable for cell preservation. For example, cells may be frozen at approximately -20°C, -80°C, or any other suitable temperature. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce the risk of cell damage and maximize the chances of cell survival after thawing. Alternatively, cells may be maintained at room temperature under refrigeration, for example, approximately 4°C.

[0229] This disclosure relates to cells or cell populations expressing the first isoform of CD33 described above, for use as pharmaceuticals, particularly for use in immunotherapies such as adoptive cell transfer therapy in patients.

[0230] According to this disclosure, cells or cell populations (e.g., hematopoietic cells) expressing the first isoform of CD33 as described above are used in medical treatment of patients requiring medical treatment, the medical treatment comprising administering a therapeutically effective amount of cells or cell populations expressing the first isoform of CD33 in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or antibodies) that specifically binds to a second isoform of CD33 or the first isoform, respectively, in order to specifically deplete the patient or transplanted cells.

[0231] As used herein, the terms “combined” or “in combination therapy” mean that two (or more) different treatments are delivered to a subject during the period in which the subject is suffering from the disorder, for example, that two or more treatments are delivered after the subject is diagnosed with the disorder but before the disorder is cured or eliminated or before treatment is discontinued for any other reason. In some embodiments, there is an overlap in administration, such that the delivery of one treatment is still taking place when the delivery of the second treatment has begun. This may be referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment is completed before the delivery of the other treatment begins. The delivery may be such that the effect of the first treatment being delivered is still detectable when the second treatment is delivered. In one embodiment, a depletion agent that binds to a second or first isoform of CD33 is administered in the doses and / or dosing schedule described herein, and cells expressing the first isoform are administered in the doses and / or dosing schedule described herein. In some embodiments, “in combination with” is not intended to imply that a depletion agent targeting a second isoform of CD33 (e.g., CAR cells or antibodies that recognize the second isoform of CD33) or a first isoform, and a composition of cells expressing the first isoform of CD33 must be administered simultaneously and / or formulated for joint delivery, although these delivery methods are within the scope of this disclosure. The depletion agent (e.g., CAR cells or antibodies that target the second isoform of CD33) may be administered simultaneously with, before, or after a dose of hematopoietic stem cells expressing the first isoform of CD33. In certain embodiments, each agent is administered in a dose and / or time schedule determined for that particular agent.

[0232] The adoptive cell transfer therapy described herein can be used to treat patients diagnosed with cancer, genetic disorders, autoimmune diseases, infectious diseases, diseases requiring hematopoietic stem cell transplantation (HSCT), prevention of organ rejection, tumor conditioning regimens, tumor maintenance therapy, minimal residual disease, and prevention of relapse.

[0233] This disclosure also relates to the use of cells expressing the first isoform of CD33 described above in the manufacture of pharmaceuticals for adoptive cell therapy in patients.

[0234] As used herein, the terms “subject” or “patient” refer to animals, including humans, pigs, chimpanzees, dogs, cats, cattle, mice, rabbits, or rats, preferably mammals in which an immune response can be induced. More preferably, the patient is human, including adult, child, and fetal human.

[0235] As used herein, the terms “treatment,” “to treat,” or “to treat” refer to any action intended to improve a patient’s health condition, such as treating, preventing, taking precautions against, or delaying a disease. In certain embodiments, such terms refer to the improvement or elimination of a disease or symptoms associated with a disease. In other embodiments, the terms refer to minimizing the spread or exacerbation of a disease resulting from the administration of one or more therapeutic agents to a subject having such a disease.

[0236] Cancers that can be treated include tumors that are not angiogenic or have not yet substantially angiogenic, as well as angiogenic tumors. Cancers may include non-solid tumors (e.g., hematological malignancies, e.g., leukemia and lymphoma (including relapsed and treatment-related tumors, e.g., secondary malignancies after the use of cytotoxic therapy and hematopoietic stem cell transplantation (HSCT))) or solid tumors.

[0237] As used herein, the term “autoimmune disease” is defined as a disorder resulting from an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to autoantigens.

[0238] Infectious diseases are illnesses caused by pathogenic microorganisms such as bacteria, viruses, parasites, or fungi. In certain embodiments, the infections described herein occur in immunocompromised patients, such as patients after HSCT or patients who have undergone solid organ transplantation.

[0239] In preferred embodiments, the present disclosure relates to cells expressing the first isoform of CD33 described above for use in hematological cancers, preferably leukemia or lymphoproliferative disorders. The leukemia may be selected from the group consisting of malignant hematopoietic diseases such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), blastic plasmacytoid dendritic cell neoplasms (BPDCN) and other myeloproliferative neoplasms, preferably MDS or preferably AML.

[0240] In certain embodiments, cells or cell populations expressing a first isoform of CD33 (e.g., hematopoietic cells) may be used for the treatment of solid tumors, particularly for the selective depletion of myeloid cells in solid tumors in patients, allowing immunotherapeutic agents such as immune checkpoint inhibitors, CAR T cells, or tumor-infiltrating lymphocytes to access the tumor, because myeloid cells in tumors can be immunosuppressive. In this context, cells or cell populations expressing the first isoform of CD33 as described above (e.g., hematopoietic cells) may be useful in replenishing hematopoiesis that may be affected by therapies intended to deplete myeloid cells in solid tumors.

[0241] In another specific embodiment, the cell or cell population (e.g., hematopoietic cells) expressing the first isoform of CD33 described above can be used for the treatment of autoimmune diseases such as lupus, multiple sclerosis, scleroderma or systemic sclerosis.

[0242] The present disclosure also relates to a depletion agent (e.g., CAR cell composition or antibody) comprising a first or second antigen-binding region for use in selectively depleting a host cell or transplanted cell, respectively, in a subject that requires selective depletion of the host cell or transplanted cell, respectively.

[0243] Method for specifically depleting patient cells and not depleting transplanted cells According to the present disclosure, the cell or cell population (e.g., hematopoietic cells) expressing the first isoform of CD33 as described above is used in a medical treatment in a patient requiring medical treatment, the medical treatment comprising administering a therapeutically effective amount of the cell or cell population expressing the first isoform of CD33 in combination with a therapeutically effective amount of a depletion agent (e.g., CAR cells or antibody) that specifically binds to the second isoform of CD33.

[0244] In fact, during immunotherapy, an immune depletion agent such as a CAR-expressing immune cell against CD33 can be administered to a patient to target and kill tumor cells. However, since 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 a patient, hematopoietic cells can then be transplanted into the patient. However, these cells need to be resistant to the agent, i.e., the depletion agent for CD33-expressing cells, since they are not targeted by the agent.

[0245] Therefore, or according to this disclosure, a depletion agent comprising a first antigen-binding domain that specifically binds to a second isoform of CD33 may be administered to specifically remove patient cells expressing the second isoform of CD33, without removing transplanted cells expressing the first isoform of CD33. Selectively depleting patient cells but not non-transplanted cells allows for the reconstruction of the patient with a healthy hematopoietic system that is no longer depleted by the immunodepletion agent. Thus, according to this therapeutic use, the patient has a functional immune system rather than undergoing prolonged immunosuppression. The use of cells according to this disclosure eliminates infection, which is a major complication of current HSC transplantation.

[0246] In another embodiment, the present disclosure relates to a method for adoptive cell transfer therapy, preferably for hematopoietic stem cell transplantation, for restoring normal hematopoiesis in a patient having cells expressing a second isoform of CD33. (i) Administering an effective amount of cells expressing a first isoform of CD33 (e.g., hematopoietic stem cells), wherein the cells expressing the first isoform of CD33 contain genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically modified allele in the nucleic acid encoding the first isoform, and the polymorphism is not present in the genome or pharmaceutical composition of a patient having cells expressing a second isoform of CD33. (ii) A method comprising administering a therapeutically effective amount of a drug containing at least a first antigen-binding region that specifically binds to the second isoform of CD33 and does not bind to or binds substantially less to the first isoform of CD33, thereby specifically depleting cells expressing the second isoform of CD33 (the patient's cells).

[0247] The cell expressing the first isoform of CD33 or its pharmaceutical composition is administered to a subject in combination with (e.g., before, simultaneously with, or after) an agent comprising the first antigen-binding region as described above.

[0248] In a preferred embodiment, the depleting agent (e.g., a CAR cell or an antibody targeting the second isoform of CD33) 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 before) or after (e.g., 5 minutes after, 15 minutes after, 30 minutes after, 45 minutes after, 1 hour after, 2 hours after, 4 hours after, 6 hours after, 12 hours after, 24 hours after, 48 hours after, 72 hours after, 96 hours after, 1 week after, 2 weeks after, 3 weeks after, 4 weeks after, 5 weeks after, 6 weeks after, 8 weeks after, 12 weeks after, or 16 weeks after) the dose of hematopoietic stem cells expressing the first isoform of the surface protein (e.g., the first isoform of CD33).

[0249] "Therapeutically effective amount" or "effective amount" is intended to mean a certain number of cells, particularly hematopoietic stem cells, expressing the first isoform of CD33 as described above, administered to a subject that is sufficient to constitute the treatment as defined above, particularly the restoration of normal hematopoiesis in a patient.

[0250] Administration of the cell or pharmaceutical composition according to the present disclosure can be performed in any convenient manner including injection, transfusion, transplantation or implantation. The compositions described herein can be administered to a patient by subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intravenous or intralymphatic injection, or intraperitoneally. In another embodiment, the cell or pharmaceutical composition of the present disclosure is preferably administered by intravenous injection. The cell or pharmaceutical composition of the present disclosure can be directly injected into a tumor, lymph node or site of infection.

[0251] Administration of the cells or cell population includes all integer values of the number of cells within their range, 10 4 ~10 9 cells / kg body weight, preferably 105 ~10 7 Cells / kg body weight, more preferably 2 × 10⁻⁶ 6 ~5×10 6 The administration may consist of cells per kg of body weight. The dosage administered depends on the recipient's age, health status, and weight, the type of concomitant therapy if any, the frequency of treatment, and the nature of the desired effect. Cells or cell populations may be administered in one or more doses. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the subject. Cells or cell populations may be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of effective doses of a given cell type for a specific disease or condition is within the scope of this art.

[0252] In particular, the disclosure also relates to the above-mentioned depletion anti-CD33 agent (e.g., a CAR cell composition or antibody) comprising a first antigen-binding domain for use in selectively depleting host cells in subjects requiring selective depletion of host cells.

[0253] A method (safety switch) that depletes the specifically transplanted cells, rather than the patient's own cells. According to this disclosure, cells or cell populations (e.g., hematopoietic cells) expressing the first isoform of CD33 as described above are used in medical treatments in patients requiring medical treatment, the medical treatments include administering a therapeutically effective amount of cells or cell populations expressing the first isoform of CD33 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 CD33.

[0254] Cells or cell populations expressing the first isoform of CD33 of this disclosure, preferably immune cells, are particularly used in adoptive cell transfer therapy to patients. The transplanted cells expressing the first isoform of CD33 may be further depleted in the patient by administering a therapeutically effective dose of a depletion agent containing a second antigen-binding region that specifically binds to the first isoform of CD33 expressed by the patient's cells, but does not bind to, or binds substantially less strongly to, the second isoform of CD33, in order to avoid serious adverse events such as graft-versus-host disease resulting from the transplant. In this case, the agent containing the second antigen-binding region that specifically binds to the first isoform of CD33 (expressed by the transplanted cells) is administered to deplete the transplanted cells specifically, rather than the patient's cells. Selective depletion of transplanted cells constitutes an important safety feature by providing a “safety switch”.

[0255] Graft-versus-host disease (GvHD) refers to medical complications following the receipt of a transplanted tissue from a genetically different person. Immune cells in 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.

[0256] These side effects can also occur if the transplanted cells, particularly immune cells with CARs, have serious side effects such as cytokine release syndrome and / or neurotoxicity. In this case, transplanted cells expressing the first isoform of CD33 can be eliminated if the cells become malignant or cause any type of undesirable on-target or off-target injury as a safety switch.

[0257] This disclosure relates to a method for adoptive cell transfer therapy in a patient having cells expressing a second isoform of CD33, (i) Administering an effective amount of cells expressing a first isoform of CD33, wherein the cells expressing the first isoform of CD33 contain genomic DNA having at least one polymorphic allele, preferably a single nucleotide polymorphism (SNP) allele, or a genetically engineered allele in the nucleic acid encoding the first isoform of CD33, and the polymorphism is not present in the genome or pharmaceutical composition of a patient having cells expressing a second isoform of CD33. (ii) A method comprising administering a therapeutically effective amount of a drug containing at least a second antigen-binding region that specifically binds to the first isoform of CD33 and does not bind to, or binds substantially less to, the second isoform of CD33, thereby specifically depleting cells expressing the first isoform of CD33.

[0258] The cells expressing the first isoform of CD33 or a pharmaceutical composition thereof are administered to the subject in combination with (for example, before, simultaneously with, or after) a drug containing the second antigen-binding domain as described above.

[0259] In preferred embodiments, the depletion agent (e.g., CAR cells or antibodies targeting a second isoform of CD33) 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 before) 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 after) the dose of hematopoietic stem cells expressing a first isoform of CD33.

[0260] The administration of cells or pharmaceutical compositions according to this disclosure may be carried out in any convenient manner, including by injection, blood transfusion, transfer, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous, or intralymphatic injection, or intraperitoneal. In another embodiment, the cells or pharmaceutical compositions of this disclosure are preferably administered by intravenous injection. The cells or pharmaceutical compositions of this disclosure may be injected directly into a tumor, lymph node, or site of infection.

[0261] The administration of cells or cell populations includes all integer values ​​of the number of cells within those ranges, 10 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 7 The administration may consist of cells per kg of body weight. The dose administered depends on the recipient's age, health status, and weight, the type of concomitant therapy if any, the frequency of treatment, and the nature of the desired effect. Cells or cell populations may be administered in one or more doses. The timing of administration is within the discretion of the attending physician and depends on the clinical condition of the subject. Cells or cell populations may be obtained from any source, such as a blood bank or donor. While individual needs vary, determining the optimal range of effective doses of a given cell type for a specific disease or condition is within the scope of this art.

[0262] Accordingly, in specific embodiments, the present disclosure relates to a depletion agent (e.g., CAR cells or an antibody) for use in preventing or reducing the risk of serious adverse events in a patient who has received cells expressing a first isoform of CD33 as described above, wherein the patient has intrinsic cells expressing a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 and does not bind to, or binds substantially less to, the second isoform of CD33.

[0263] In another aspect, the present disclosure relates to a kit for expressing the first isoform CD33 described above in cells, comprising a guide RNA combined with a Cas protein, a gene editing enzyme such as a base editor or prime editor, a nucleic acid construct, the expression vector described above, or isolated cells according to the present disclosure. [Examples]

[0264] Example 1: Preparation of anti-CD33 Fab and MAb Eight different anti-CD33 antibodies were prepared in Fab and MAb formats based on publicly available sequence information or sources.

[0265] Table 2 shows the variable chains and CDRs (Kabat) of the antibodies (Refmab#1~#6). Refmab#7 (WM53) is a mouse monoclonal antibody available from BioLegend (#303402). Refmab#8 (2337A) is a rabbit monoclonal antibody available from R&D Systems (#MAB11373-100).

[0266] [Table 2] JPEG2026516593000003.jpg188130JPEG2026516593000004.jpg29130

[0267] Further characteristics of the antibodies used, as well as the format and isotype of the full-length antibodies, are shown in Table 3.

[0268] [Table 3]

[0269] Example 2: Binding of MAb to CD33 and optimization of assay conditions HEK-293T cells were transfected with a construct containing wild-type CD33 (SEQ ID NO: 1) or an empty vector. Antibody binding to transfected cells and optimal assay conditions were evaluated in a 384-well format. Cell expression detection was measured by high-throughput flow cytometry. Serial dilutions of each antibody were tested for immunoreactivity against cells expressing only CD33 or the vector. The optimal screening concentration for each antibody was determined based on raw signal values ​​and signal-background calculations. The results are shown in Figure 1. Each point represents the average of four replicates.

[0270] Mab-format antibodies bind to human CD33 in a concentration-dependent manner. Cells transfected with an empty vector did not show significant binding to the anti-human CD33 antibody.

[0271] The optimized assay conditions for flow cytometry are shown in Table 4 for Refmab #1 to #4 and in Table 5 for Refmab #5 to #8.

[0272] [Table 4]

[0273] [Table 5]

[0274] Example 3: Binding of Fab to CD33 and optimization of assay conditions The experiment was conducted similarly to that described in Example 2, except that a Fab fragment was tested instead of a full-length antibody. Serial dilutions of each Fab were tested for immunoreactivity against cells expressing wild-type CD33 or only the vector. The optimal screening concentration of Fab was determined based on raw signal values ​​and signal-background calculations. The results are shown in Figure 2. Each point represents the average of four replicates.

[0275] All antibodies tested in Fab format bound to human CD33 in a concentration-dependent manner. Cells transfected with an empty vector showed no binding to the anti-human CD33 antibody.

[0276] Optimized assay conditions for high-throughput flow cytometry are shown in Table 6 for Refmab #1 to #3 and in Table 7 for Refmab #4 to #6.

[0277] [Table 6]

[0278] [Table 7]

[0279] Example 4: Alanine Scanning Alanine scanning was performed on human CD33 to determine the CD33 residues involved in binding to the antibodies under investigation. Alanine scanning was performed by shotgun mutagenesis epitope mapping (Integral Molecular, Philadelphia / PA, USA) as described in Immunology (2014) 143, 13-20. Briefly, a mutant library of CD33 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 sequenced in a 384-well microplate and transiently transfected with HEK293-T. After transfection, cells were incubated with antibodies (IgG or Fab) indicated at predetermined concentrations using independent immunofluorescence titration curves on wild-type CD33. Antibodies were detected using Alexa Fluor 488 conjugate secondary antibodies, and mean cell fluorescence was determined using the Intellicyt iQue flow cytometry platform (Intellicyt / Sartorius). Mutant residues were identified as important to the antibody epitope if they did not support the reactivity of the test antibody but supported the reactivity of the control antibody. In each case, another anti-CD33 RefMab was used as the control antibody; for example, RefMab #8 was used as the control antibody for RefMab #1, RefMab #7 for RefMab #2, #3, and #6, and RefMab #1 for RefMab #4 and #5. This counterscreening strategy facilitates the exclusion of mutants that are locally misfolded or have expression defects. The binding of each antibody to each mutant clone was determined in a double-delayed manner. For each point, background fluorescence was subtracted from the raw data and then normalized to antibody reactivity with wild-type CD33.

[0280] Library screening of very high-affinity antibodies may not yield the critical residues necessary for antibody binding. Therefore, high-affinity antibodies were converted to Fab format to sufficiently weaken binding and enable the identification of these critical residues. If Fab screening under standard conditions was still insufficient to identify the critical residues, high-stringency conditions were implemented. These conditions included combinations of increased pH, increased salinity, increased temperature, and / or increased washing time. Antibodies requiring high-stringency conditions are indicated as "HS".

[0281] For each mutant clone, the average binding value was plotted as a function of expression (represented by control reactivity). See Figure 3. To identify preliminary primary important clones (circled), thresholds (dashed lines) of wild-type binding above 70% to the control antibody and below 20% to the test antibody were applied. Secondary clones (squares) are highlighted for clones that did not meet the set thresholds but whose reduced binding activity and proximity to important residues suggested that the mutated residue could be part of the antibody epitope.

[0282] The results of the alanine scan are shown in Table 8. The average binding reactivity (and range) is listed for all identified critical residues. Critical residues for antibody binding (outlined in dark gray) were those that resulted in binding of test Ab with less than 20% mutation. Additional secondary residues (outlined in light gray) were identified that resulted in 20–40% binding of test Ab compared to wild-type CD33.

[0283] [Table 8] JPEG2026516593000011.jpg87136JPEG2026516593000012.jpg87136JPEG2026516593 000013.jpg87136JPEG2026516593000014.jpg87136JPEG2026516593000015.jpg87136 JPEG2026516593000016.jpg87136JPEG2026516593000017.jpg87136JPEG2026516593 000018.jpg87136JPEG2026516593000019.jpg87136JPEG2026516593000020.jpg84136

[0284] Example 5: Analysis and comparison of mutants identified from alanine scan Table 9 summarizes the residues that influence the binding of each tested antibody to the shown variants, based on the data presented in Table 8. All residues that resulted in less than 40% binding of the tested antibody compared to wild-type CD33 are shown. Remote residues that resulted in less than 20% binding are shown in bold and underlined.

[0285] [Table 9]

[0286] As shown in Table 9, certain mutants strongly block the binding of all antibodies. This is the case, for example, with mutants C41, W60, C101, Y116, F118, and D205. The binding of all eight antibodies to these mutants is reduced to less than 40% compared to wild-type CD33. This is likely due to significant structural disruption or loss of expression in these mutants, and does not indicate that changes in amino acid positions are related to binding to the tested antibodies. Therefore, these mutants were deselected.

[0287] The identified potentially useful residues were plotted on the 3D structure of CD33. See Figure 4. This analysis, along with further criteria such as the reproducibility of binding activity, surface accessibility, structural localization and distance to other important sites, and the nature and biochemical characteristics of substituted amino acids (e.g., cysteine-forming disulfide bridges or post-translational modification sites), were taken into consideration for selecting residues for the subsequent comprehensive mutational analysis.

[0288] Example 6: Comprehensive Mutation Analysis Based on the experimental data from Example 4 and the analysis from Example 5, specific amino acid residues were subjected to a more comprehensive mutational analysis. Here, each residue underwent comprehensive mutagenesis to selected biophysically appropriate non-alanine amino acids based on the sequence and structure-related characteristics of the substituted and newly introduced amino acids.

[0289] Antibodies were screened for binding to IgG-format human CD33 variants. Similar to Example 4, binding of each test antibody to each mutant clone in the comprehensive library was determined in two-column sequences by high-throughput flow cytometry. For each mutation, background fluorescence was subtracted from the raw data and then normalized to antibody reactivity with wild-type CD33. The average binding reactivity and range for all mutant clones are listed in Table 10. Mutations that resulted in less than 20% binding are highlighted in dark gray. Residues that resulted in 20-40% binding of test Ab compared to wild-type CD33 are highlighted in light gray.

[0290] [Table 10] JPEG2026516593000023.jpg86136JPEG2026516593000024.jpg86136JPEG2026516593 000025.jpg86136JPEG2026516593000026.jpg86136JPEG2026516593000027.jpg67136

[0291] Example 7: Analysis and comparison of mutants identified from comprehensive analysis Table 11 summarizes the variants that reduced the binding of the tested antibodies to less than 20%.

[0292] [Table 11] JPEG2026516593000029.jpg77129

[0293] This analysis was also subjected to additional validation, such as evaluation of the surface exposure of each residue. An exemplary analysis is shown in Figure 5. It can be seen that certain residues are buried and therefore inaccessible to the protein surface. Such residues, including amino acids at positions 25, 43, 120, 129, and 134, were deselected.

[0294] Figures 6-8 show important CD33 mutants identified as exhibiting reduced binding of ReFMab#1, ReFMab#3, and ReFMab#4, respectively, compared to wild-type CD33.

[0295] Example 8: Binding of CD33 mutant to Refmab Binding to selected mutants was analyzed using antibodies ReFMab#1, ReFMab#3, and ReFMab#4. Antibody binding to CD33 wild-type and mutants was measured at 25°C with shaking at 1,000 rpm using Octet system R8 (Sartorius, SA-030159 and SA-030308) with 1× reaction rate buffer (Sartorius, PN:18-1105). Selected mutants were screened for their ability to bind to ReFMab#1, ReFMab#3, and ReFMab#4 using different concentrations of CD33 (wild-type or mutant). Antibodies were captured at 0.7 ug / mL for 300 seconds using an anti-human Fc capture biosensor (AHC) (Sartorius, PN:18-5060). Human CD33 wt and mutants were used as analytes at three different concentrations (500, 50, and 5 nM). The association of the analyte with the antibody was monitored for 700 seconds, and the dissociation of the analyte from the antibody was monitored for 1200 seconds. Reference subtraction was performed on buffer-only wells. AHC chips were regenerated using 10 mM glycine-HCl pH 1.7. Data were analyzed using Octet Data Analysis software HT 12.0. Data were fitted to a 1:1 binding model. Kinetic velocities ka and kd, as well as response signals, were fitted overall. Binding levels of each CD33 mutant to ReFMab#1, ReFMab#3, and ReFMab#4 were calculated by normalizing the response at the end of association by loading, and these values ​​were compared to the wild type and expressed as percentages. The mutant N20R binds nonspecifically to the capture biosensor. Results are shown in Table 12. JPEG2026516593000030.jpg22153

[0296] [Table 12]

[0297] Example 9: Avidity binding of CD33 mutant to Refmab Binding to selected mutants was analyzed using antibodies ReFMab#1, ReFMab#3, and ReFMab#4. Antibody binding to CD33 wild-type and mutants was measured at 25°C with shaking at 1,000 rpm using Octet system R8 (Sartorius, SA-030159 and SA-030308) with 1× reaction rate buffer (Sartorius, PN:18-1105). The selected mutants were screened for their ability to bind to ReFMab#1, ReFMab#3, and ReFMab#4.

[0298] CD33 wild-type and mutant strains were biotinylated with EZ-Link® Sulfo-NHS-LC-Biotin, No-Weigh (Thermo Fisher, A39257). Biotinylated CD33 wild-type and mutant strains were captured on streptavidin biosensors (SA) (Sartorius, PN 18-5019) at concentrations of 2 to 16 ug / mL for 300 seconds. Refmab #1 was used as the analyte at 300 nM or 500 nM, and Refmab #3 and Refmab #4 were used at 500 nM. Analyte aggregation was monitored for 300 seconds, and dissociation for 900 seconds. Data were analyzed using Octet Data Analysis software HT 12.0. The binding levels of each CD33 mutant to ReFMab#1, ReFMab#3, and ReFMab#4 were calculated by subtracting the response signal during loading at 1080 seconds. These values ​​were then compared to the wild type and expressed as percentages. The results are shown in Table 13. JPEG2026516593000032.jpg16154

[0299] [Table 13]

[0300] Example 10: Generation of CD33 mutants by base editing in primary human T cells Specific CD33 variants were created by base editing. To do this, a publicly available base editor (Nat Biotech (2020) 38:883-91), ABE8e-NG, was used to target a selected region of CD33. The created variants and the sgRNAs used are shown in Table 14.

[0301] For base editing, 1.5 μg of base editor (ABE8e-NG mRNA; ordered from Trilink) and 1.5 μg of sgRNA (ordered from Synthego) were electroporated into 187,500 human activated T cells isolated from PBMCs using the EasySep Human T Cell Isolation Kit (Stemcell Technologies #17951). 24 hours after isolation, the human T cells were activated for 48 hours in 96-well plates at a concentration of 1.5 million cells / mL using IL-2 (150 U / mL), IL-7 (5 ng / mL), IL-17 (5 ng / mL), and Dynabead Human T-Activator CD3 / CD28 (Gibco #11132D), according to the manufacturer's recommendation (1:1 bead:cell ratio). Human activated T cells were debeaded, and for each condition, 187,500 cells were mixed in a small shuttle nucleocubette with 1.5 μg of ABE8e-NG mRNA, 1.5 μg of sgRNA, 8.2 μL of primary P3 cells, and 1.8 μL of supplement 1. Electroporation was performed using a Lonza 4D-Nucleofector with the EH-115 pulse program. Immediately after electroporation, 90 μL of pre-warmed human T cell medium (RPMI-1640 (Sigma-Aldrich #R8758-500ML) + 10% thermo-inactivated human serum (male, AB+) + 100× Na-pyruvate (Gibco #11360-039) + 100× GlutaMAX (ThermoFisher)) was added. (Scientific#35050061) + beta-mercaptoethanol (Gibco#31350-010) + HEPES (Sigma#H0887) + 100×MEM non-essential amino acids (Gibco#11140-050) + 1% penicillin + streptomycin (Gibco#15140-122) were directly added to the nucleocuvette and incubated at 37°C for 20 minutes to recover the cells. The electroporated conditions were then transferred to a 96-well flat-bottom plate supplemented with 500 U / mL of IL-2. The medium was refreshed every 48 hours. Five days after electroporation, bulk cells from each condition were spun down at 2200 RCF for 5 minutes.The pellet was resuspended in 30 μL of QuickExtract solution (Lucigen #QE09050) in a PCR tube and vortexed for 1 minute. The inventors incubated the mixture in a thermoblock at 60°C for 6 minutes. The lysate was thoroughly mixed again by vortexing for 1 minute and re-incubated at 98°C for 10 minutes. After concentration measurement, the final lysate was ready for PCR. The gDNA under each condition was subjected to the following two PCR reactions: (1) amplification across CD33 using primers Fwd-1 and Rev-1, and (2) amplification across the pseudogene SIGLEC22P using primers Fwd-2 and Rev-2 (Table 14). The pseudogene SIGLEC22P is located 13.5 kb upstream of CD33 and shares more than 90% sequence similarity with the first three exons of CD33. Therefore, we also evaluated whether base editing resulted in undesirable alterations to the SIGLEC22P pseudogene.

[0302] [Table 14]

[0303] The PCR products were purified using a column and then sequenced by Sanger sequencing (Microsynth) using primer Fwd-1 for the CD33 amplicon and primer Fwd-2 for the SIGLEC22P amplicon, respectively.

[0304] Base editing efficiency was quantified from Sanger sequencing reads using EditR (CRISPR J.(2018)1:239-250).

[0305] The results are summarized in Tables 15 and 16. No editing was observed for residues D18 and N20. Some sgRNAs yielded editing efficiencies of 10–50% (F21, F44_1), while others yielded over 80% (Q244, F44_2, H45, Y49, Y50). Some sgRNAs resulted in bystander modifications with varying efficiencies. Some sgRNAs (F44_1) resulted in silent bystanders (nucleotide changes did not alter the amino acid sequence). Some sgRNAs also enabled editing of SIGLEC22P. In summary, it can be said that most of the investigated mutants are suitable for base editing. Experimental conditions need to be optimized, which is something that can be addressed in routine experiments.

[0306] [Table 15]

[0307] [Table 16]

[0308] Example 11: Base-edited CD33 mutants in primary CD34-positive human cells exhibit loss of binding to anti-CD33 antibodies. For CD33 base editing, leukocyte packs were purchased from CytoCare, and hCD34+HSPCs were isolated using the LP-34 process with CliniMACS Prodigy (Miltenyi). hCD34+HSPC was thawed and resuspended in pre-warmed HSPC medium (StemSpan SFEM II (StemCell#09655) + 1 μL / mL hSCF (Miltenyi#130-096-695) + 1 μL / mL hFlt3-ligand (Miltenyi#130-096-479) + 1 μL / mL hTPO (Miltenyi#130-095-752)). After 2 days of incubation, 1 million hCD34+HSPC were subjected to 7.5 μg of SpRy Cas9 mutant (Science 2020 (368:190-6); TebuBio) and 13.6 μg of sgRNA (Synthego; 1:100) in large nucleocubets (Lonza#V4XP-3024) for each base editing condition. Mix 82 μL of primary P3 and 18 μL of supplement 1 (BE:sgRNA molar ratio) and use the Lonza CA-137 pulse program. Electroporation was performed using a 4D-Nucleofector. Alternatively, 200,000 hCD34+HSPC cells were used, with other components appropriately scaled. Nucleocubets (Lonza#V4XP-3032) were used for this scaling. Immediately after electroporation, 900 or 80 μL of pre-warmed stem cell medium was added directly to the nucleocubets, and the cells were incubated at 37°C for 20 minutes to recover. Electroporated hCD34+HSPCs were cultured at 500,000 cells / mL in 6-well or 48-well flat-bottom plates in stem cell medium supplemented with hSCF, hFLT3-ligand, and hTPO (see above), with the cultures refreshed every 5 days. gDNA from bulk cells was sequenced 5 days after electroporation to evaluate base editing efficiency.

[0309] Twenty-one different sgRNAs were tested to generate the CD33 W22R mutant by base editing.

[0310] Figure 9 shows a histogram of the binding of representative sgRNAs to ReFMab#3 after base editing of human CD34+HSPCs, compared to a non-target control (NTC). It can be seen that the majority of cells were edited with sgRNA-E. The sequences of the sgRNAs used are shown in Table 17:

[0311] [Table 17]

[0312] Edited human HSPCs were tested for binding to ReFMab#3 and a control antibody by flow cytometry. Cells were washed with PBS and then stained for viability with eFluor780 (dilution 1:1000, eBioscience, #65-0865-14). After further washing, cells were labeled with ReFMab#3 containing AF647 and FITC-HIM3-4 (dilution 1:25, BioLegend, #303304). Results for sgRNA-E are shown in Figure 10. 95.8% of untransformed control cells reacted with ReFMab#3, while 89.0% of cells transfected with sgRNA-E did not react with ReFMab#3. Binding to FITC-HIM3-4 was not affected by editing.

[0313] To further confirm that the success of base editing is related to the loss of binding to ReFMab#3, NGS was performed on the selected cells. The results are shown in Figure 11. Essentially, all cells that lost binding to ReFMab#3(A) showed homozygous editing (targeted editing / targeted editing), while all cells that were essentially reactive to ReFMab#3(C) showed homozygous wild-type sequences. The intermediate cell population (B) showed heterozygous editing (WT / targeted editing).

[0314] Example 12: Base-edited CD33 mutants of primary CD34-positive human cells are functionally indistinguishable from unedited cells. Human CD34-positive HSPCs edited with sgRNA NTC (control) and sgRNA-E (mutant W22R) were tested by colony-forming unit (CFU) assay as follows: HSCs edited with sgRNA-E and NTC (control) were washed, resuspended in IMDM 2% FBS medium (200 cells / well), and added to MethoCult (Stemcell #044349). Then, 1 ml of the suspension was carefully dispensed into a plate (Stemcell SmartDish®) and incubated at 37°C for 14 days. Images were then taken and colonies were quantified. The results are shown in Figure 12.

[0315] As can be seen, cells that differentiated to a similar degree into myeloid cells and erythrocytes demonstrate that gene editing did not affect cell differentiation.

[0316] Sanger sequencing showed that approximately 65% ​​of sgRNA-E edited HSPCs exhibited homozygous editing, while approximately 30% exhibited heterozygous editing. The remaining cells could not be clearly grouped.

[0317] To further confirm that gene editing did not affect the function of the edited HSPCs, HSPCs edited with sgRNA NTC (control) and sgRNA-E (mutant W22R) were tested in an in vitro differentiation assay. In this assay, 2000 cells were each treated with 1% L-glutamine, 1% penicillin, and a cytokine mix (Flt3 ligand (20 ng / μl, Miltenyi biotech#130-096-479), TPO (50 ng / μl, Miltenyi biotech#130-095-752), SCF 100 ng / μl (Miltenyi biotech#130-096-695), IL3 (10 ng / μl, Miltenyi biotech#130-095-071), IL6 (50 ng / μl, Miltenyi biotech#130-093-934), EPO (3 ng / μl, Stemcell Technologies#78007), IL2 (10 ng / μl, Miltenyi Cells were seeded in 96-well plates in 100 μl StemPor-34 SFM kits (Gibco, #10639-011) supplemented with biotech#130-097-743), IL7 (20 ng / μl, Milteny biotech#130-095-743), GM-CSF (20 ng / μl, Fisher#PHC2015), and human LDL (500 ng / ml, Stemcell Technologies, #2698). After 4 days, an additional 100 μl of cytokine medium was added, and cells were divided at 8 and 10 days. Cell differentiation was evaluated by FACS at 14 days. The results are shown in Figure 13. As can be seen, no difference in differentiation could be observed.

[0318] Example 13: Introduction of a CD33 mutant by gene editing makes human CD34+ cells resistant to death by ReFMab conjugated with a toxin. Human CD34+ cells are gene-edited as described above using base editing, prime editing, or HDR to introduce the relevant mutant. The edited CD34+ cells are then used as bulk cells or sorted to enrich edited cells with their respective CD33 mutants.

[0319] Approximately 10 3 ~10 pieces 4 Individual engineered and sorted human CD34+ cells are seeded into 96-well plates in 100 μl of medium supplemented with cytokines hSCF, hFLT3L, and hTPO. Refmab is directly conjugated to a toxic payload such as PBD / Tesirin or SN38. Cells are incubated at 37°C for 3–5 days with serial dilutions of the Refmab antibody-drug conjugate (ADC). At the end of the incubation period, 100 μl of CellTiter Glow (Promega catalog number: G9241) is added to each well. Luminescence is read at an integration time of 1 second. Unedited CD34+ cells expressing wild-type CD33 cells serve as a positive control for maximum cell death.

[0320] Alternatively, edited bulk CD34+ cells are incubated with serial dilutions of ReFMab antibody-drug conjugate (ADC) at 37°C for 3–5 days. After incubation, cells are harvested, stained with appropriate fluorescently labeled ReFMab, and resuspended in 200 μL of FACS buffer for flow cytometry analysis. The cell suspension is then analyzed using a flow cytometer. Wild-type CD33 cells bind to both CD33 antibodies, while edited shielded CD33 cells bind only to the unrelated antibody / Refmab. Compared to a PBS control (without ADC), depletion of ReFMab-positive cells is observed upon addition of antibody-toxin. Furthermore, ReFMab-negative cells persist and are shielded from ADC-mediated death.

[0321] Example 14: Gene editing of CD33+ human cell lines is shielded from antibody binding and cell death. For CD33 base editing, human myeloid cell lines such as U937, KG-1, or THP-1 are electroporated with ABE8e-NG mRNA (TriLink) and sgRNA (Synthego) according to the manufacturer's recommendations. The electroporated cells are grown for 5 days, and the culture medium is refreshed every 48 hours.

[0322] CD33 variants that cannot be introduced by base editing are generated by homology-directed repair (HDR) and / or prime editing of human CD34+ cells using appropriate tools. Binding and cell death assays are performed as described below.

[0323] CD33-edited U937 and THP-1 cells are sorted by flow cytometry to generate a uniformly edited pool or single-cell clone.

[0324] To assess whether gene-edited human cells are shielded from antibody-binding cells, cells are harvested, stained with appropriate fluorescently labeled ReFMab and anti-CD33 antibodies, and resuspended in 200 μL of FACS buffer for flow cytometry analysis. The cell suspension is analyzed using a cell analyzer (flow cytometer). Compared to isotype controls, edited and shielded cells bind to unrelated ReFMab / anti-CD33 antibodies but not to ReFMab paired with the shielding mutation. Wild-type, unedited cells bind to both antibodies. Unrelated ReFMab / CD33 antibodies serve as a control for CD33 expression.

[0325] To confirm whether gene-edited human cells are shielded from antibody-toxin conjugate depletion, gene-edited and sorted cells are incubated for 3–5 days in 100 μL of human medium supplemented with gradually increasing concentrations of ReFMab-based antibody-drug conjugate. At the end of the incubation period, 100 μL of CellTiter Glow (Promega catalog number: G9241) is added to each well. Luminescence is read at an integration time of 1 second. Unedited cells expressing wild-type CD33, as well as CD33-negative cells, serve as positive (maximum killing) and negative (background killing) controls.

[0326] Compared to a PBS control, depletion of ReFMab-positive cells is observed upon addition of antibody-toxin. Furthermore, ReFMab-negative cells remain present and are shielded from antibody-toxin-mediated cell death.

[0327] Mutants that cannot be introduced by base editing are generated by homologous recombination repair and / or prime editing using appropriate tools. Binding and death assays are performed as described above.

[0328] Example 15: Overexpression of recombinant CD33 mutant in cell lines Mammalian cell lines such as HEK293 cells or chicken DF-1 cells, which express endogenous CD33, are suitable for overexpression of recombinant human wild-type or mutant CD33 variants. Cells are transfected with a construct expressing wild-type CD33 or a construct expressing a mutant CD33 variant. Two to three days after transfection, cells are selected using an antibiotic (G418 / neomycin) to create a stable cell line. Alternatively, transient CD33-expressing cells can be used directly for FACS analysis. Transient cells or stable cell lines are stained using two different human CD33 antibodies. One antibody binds to the region of the mutation, and the second antibody binds to a different region. This antibody combination can be used to assess the loss of binding of the target antibody to a specific counter-CD33 mutation. Retention of binding of the second antibody is important to indicate that mutant CD33 is still being expressed by the cells.

[0329] Example 16: Binding of CD33 wild-type and mutant to sialic acid ligands The functionality of the selected mutants was evaluated by binding to sialic acid in an ELISA assay. MaxiSorp plates (Thermo Fisher 43954) were coated overnight with 30 ng / well of CD33 wild-type and CD33 mutants and blocked with 2% Bovin Serum Albumin. Biotin-conjugated polyacrylamide probes substituted with Neu5Aca3'Lac-Gly (Neu5Acα2-3Galβ1-4Glcβ-sp4-PAA-biot "5.3SLL", GlycoNZ, 0060-BP) or Neu5Aca6'Lac-C2 (Neu5Acα2-6Galβ1-4Glcβ-sp2-PAA-biot "5.6SLL", GlycoNZ, 0063a-BP) were incubated at 250 ng / well for 2 hours and washed three times. The binding signal was detected by adding 50 μL / well of streptavidin HRP (Biotechne DY998), diluted 200-fold, for 30 minutes, followed by 50 μL / well of 3,3',5,5'-tetramethylbenzidine (TMB, Thermo Fisher 002023), and incubating for 10 minutes. The reaction was stopped with stop solution (Thermo Fisher N600). The absorbance at 450 nm was measured for the binding signal, and the absorbance at 570 nm was measured for the background. Samples were compared using an unpaired t-test.

[0330] The results are shown in Figure 19. CD33 R119A does not bind to sialic acid, and since the R119 residue is conserved among the Siglec family and plays an important role in binding to sialic acid, this is a negative control mutant. CD33 wild-type and the W22R mutant bind to sialic acid, more strongly with Neu5Aca6'Lac-C2 than with Neu5Aca3'Lac-Gly, suggesting a preference for 5.6 binding over 5.3 binding. The W22R mutant does not significantly affect sialic acid binding compared to the wild type. The F21S mutant either does not bind to sialic acid or binds much more weakly than the wild type. The double mutant F21S_W22R binds weakly to sialic acid, more strongly than F21S and weaker than W22R.

[0331] Example 17: Inhibition of phagocytosis by CD33 wild-type and mutant. CD33 is described as an antiphagocytic receptor that restricts phagocytosis in cells such as human microglia and macrophages. As expected, knockout or functional blockade of human CD33 results in increased phagocytosis, and therefore the functional activity of CD33 mutants can be assessed by quantifying the phagocytic activity of wild-type CD33 versus mutant CD33 cells. Human myeloid cell lines such as THP-1 and U937 are used to evaluate CD33-mediated phagocytosis.

[0332] Therefore, the CD33 mutant is introduced into the genomes of THP-1 and U937 cells using the method described above, and then sorted to generate a uniformly edited pool or single-cell clone (see also above).

[0333] Prior to the assay, wild-type and genetically edited mutant CD33 THP-1 and U937 cells are cultured, harvested, centrifuged, resuspended in medium, and seeded in 96-well plates. Fluorescent cargoes such as fluorescent polystyrene beads, pHrodo bioparticles (E. coli, zymosan), tetramethylrhodamine isocyanate-dextran, pHrodo-labeled myelin, and fluorescently labeled amyloid beta are added to the cells, and incubated at 37°C for 30 minutes to 4 hours. For bead-based cargoes, the percentage of cells that take up at least one bead is used for quantification by flow cytometry; however, for non-bead-based cargoes, the degree of phagocytosis is determined by evaluating the median fluorescence intensity (MFI) of the fluorescence signal with background subtraction (cells without cargo). Alternatively, real-time monitoring and quantification of phagocytosis can be performed using an Incucyte instrument.

[0334] Example 18: CD33 mutant and Refmab#1 Among all the mutants tested, amino acid residues N20, F21, and W22 appear to be particularly important for the binding of ReFMab#1 to CD33. Mutations in these residues result in a strong decrease in binding. Of these three residues, N20 and F21 are particularly important.

[0335] Regarding N20, specific amino acid substitutions appear to be particularly useful in inhibiting the binding of ReFMab#1 to CD33. These include substitutions N20G, N20D, N20E, N20K, N20H, and N20R. Among these variants, N20G and N20K are preferred because they offer good production yields and their biophysical properties are essentially indistinguishable from wild-type CD33.

[0336] For F21 as well, specific amino acid substitutions appear to be particularly useful in inhibiting the binding of ReFMab#1 to CD33. These include substitutions F21V, F21L, F21I, F21S, F21M, F21N, F21Q, and F21H. Among these mutants, F21V, F21L, F21I, and F21S are preferred because they have good production yields and their biophysical properties are essentially indistinguishable from wild-type CD33. Mutants F21V, F21S, and F21I are most preferred due to their favorable aggregation behavior.

[0337] Example 19: CD33 mutant and Refmab#3 Among all the mutants tested, amino acid residues N20, F21, and W22 appear to be particularly important for the binding of ReFMab#3 to CD33. Mutations in these residues result in a strong decrease in binding. Of these three residues, N20 and F21 are particularly important.

[0338] With regard to N20, specific amino acid substitutions appear to be particularly useful in inhibiting the binding of ReFMab#3 to CD33. These include substitutions N20G, N20E, N20D, N20R, N20H, and N20K. Among these variants, N20G, N20K, and N20D are preferred because they offer good production yields and their biophysical properties are essentially indistinguishable from wild-type CD33. N20K and N20D are even more preferred due to their lower tendency to aggregate.

[0339] For F21 as well, specific amino acid substitutions appear to be particularly useful in inhibiting the binding of ReFMab#3 to CD33. These include the substituted F21V, F21L, F21I, F21M, F21N, F21Q, F21H, and F21S. Among these variants, F21I, F21V, F21L, and F21S are also preferred because they have good production yields and their biophysical properties are essentially indistinguishable from wild-type CD33. F21I, F21V, and F21S are even more preferred due to their lower tendency to aggregate.

[0340] With respect to W22, specific amino acid substitutions appear to be particularly useful in inhibiting the binding of ReFMab#3 to CD33. These include substitutions W22R, W22D, W22K, W22H, and W22T. Among these variants, W22R is preferred because it has good production yield and its biophysical properties are essentially indistinguishable from wild-type CD33. W22T has good production yield, biophysical properties, and shielding from ReFMab#3, but introduces a potential N-linked glycosylation site that can alter the glycosylation pattern of CD33, and is therefore undesirable.

[0341] Example 20: CD33 mutant and Refmab #4 Among all the mutants tested, amino acid residue Y50 appears to be particularly important for the binding of ReFMab#4 to CD33. Mutations in this residue result in a strong decrease in binding.

[0342] Certain amino acid substitutions appear particularly useful for inhibiting the binding of ReFMab#4 to CD33. These include substitutions Y50D, Y50E, Y50K, Y50H, and Y50R. Among these mutants, Y50D and Y50R are preferred because they offer good production yields and their biophysical properties are essentially indistinguishable from wild-type CD33.

[0343] Example 21: Biophysical characteristics of the CD33 mutant Low aggregation tendency and high melting temperature are characteristic of the structural integrity and stability of biomolecules such as the extracellular domains of protein receptors. Therefore, the extracellular domains of wild-type and mutant CD33 (amino acids D18-H259; Uniprot P20138-1) were expressed in 50-100 ml of CHOEBNALT85 cell line using an N-terminal His6 tag. Capture from the cell culture supernatant was performed using a 5 mL HisTrap Excel column, followed in some cases by gel filtration using Superdex 75 and subsequent sterile filtration. The extracellular domains of CD33 R119A and the double mutant F21S_W22R were purified using IMAC chromatography before changing the buffer to PBS pH 7.4. The recombinant purified CD33 extracellular domains were then tested using various biophysical assays to evaluate protein integrity and stability relative to the wild-type protein.

[0344] Selected mutants with relevant point mutations should not affect the structural integrity of the protein, or only slightly, and therefore retain the biophysical properties of the surface antigen. By comparing each parameter with wild-type CD33, it is ensured that properties close to those of non-mutant CD33 are retained.

[0345] To test CD33 mutants, CD33 wild-type and mutants were diluted to 1 mg / mL, combined with Sypro Orange (Sigma-Adrich, S5692), and exposed to a temperature gradient from 25°C to 90°C using an RT-PCR instrument (Biorad, C1000 thermal cycler). Fluorescence was monitored as a function of temperature. Initial fluorescence at 25°C was obtained from the raw data, and the first derivative curve provides the melting temperature in degrees Celsius.

[0346] After capture and purification, monomer content of all mutants was evaluated by HPLC size exclusion chromatography (Waters BioSuite 250 4um UHR SEC 4.6×300 mm or AdvanceBio SEC 300A 2.7um 4.6×300 mm). Protein peaks were monitored at 220 nm and 280 nm, and relative peak areas were measured as a percentage.

[0347] The results are shown in Table 18. [Table 18]

[0348] All CD33 variants except N20S, N20D, N20E, N20G, F21L, and F43S exhibit a low aggregation tendency similar to wild-type CD33, possess a monomer fraction of over 90%, and are therefore preferred variants in terms of aggregation.

[0349] All CD33 mutants except F43S and F44P exhibit high thermal stability as measured by their Tm(°C), showing similar protein stability to wild-type CD33, and are therefore preferred mutants in terms of thermal stability.

[0350] Example 22: Engraftment and differentiation of edited cells in mice Human HSPCs were edited using sgRNA-E as described in Example 11. Two days after electroporation, the cells were frozen in CryoStor CS10 medium (Stem Cell Technologies #07930). After thawing and washing, sgRNA-E HSPC (780,000 cells each) and NTC HSPCs (720,000 cells each) or saline solution were immediately injected into the tail vein of 4-week-old female NBSGW mice (Jackson Laboratory; stock #026622). After 13 weeks, peripheral blood and bone marrow of the mice were analyzed to confirm whether edited HSPCs differentiated or engrafted differently from unedited HSPCs. For this purpose, 0.2 ml of blood and both hind limbs were collected from each mouse after euthanasia. Cell suspensions were prepared, erythrocytes were lysed with ACK lysis buffer, and the cell suspensions were then filtered. All cells were stained with a survival dye (Zombie UV Fixable Viability Kit 1:1000, Biolegend #423108).

[0351] Bone marrow cells were then incubated with CD33 FITC (1:50, Biolegend #303304), CD34 PerCP-Cy5.5 (1:10, BD Biosciences #347222), CD14 PE (1:50, Biolegend #982508), CD10 PECF594 (1:50, BD Biosciences #562396), CD90 PE-Cy7(1:50, BD Biosciences#561558), CD33 APC(Refmab#3), CD38 AF700(Beckman Coulter#B23489), CD45 RA APC / Fire(1:50, Biolegend#304152), CD3 BV421(1:50, Biolegend#300434), CD45 V500(1:50, BD Samples were stained with Biosciences#560777), mCD45 BV605 (1:200, BD Biolegend#103155), CD123 BV650 (1:50, Biolegend#306020), and CD117 BV711 (1:50, Biolegend#313230), and FcR blocking agent human (1:200, Miltenyi#130-059-901) was added.

[0352] Peripheral blood cells were then treated with CD33 FITC (1:50, Biolegend #303304), CD34 PerCP-Cy5.5 (1:100, BD Biosciences #347222), CD335 PE (1:50, BD Biosciences #557991), CD11c PECF594 (1:50, BD Biosciences #562393), CD13 PE-Cy7(1:50, Biolegend#301712), CD33 APC(Refmab#3), mCD45 AF700(Biolegend#103128), CD14 APC-Cy7(1:50, Biolegend#301820), CD3 BV421(1:50, Biolegend#300434), CD45 Samples were stained with V500 (1:50, BD Biosciences#560777), CD123 BV650 (1:50, Biolegend#306020), CD117 BV711 (1:50, Biolegend#313230), and CD19 BV786 (1:50, BD Biosciences#363028), and FcR blocking agent human (1:200, Miltenyi#130-059-901) was added. Data were recorded using a BD SSRFortessa instrument with BD FACSDiva software and analyzed with FlowJo software.

[0353] The results can be seen in Figures 14 and 15. sgRNA-E HSPC and NTC HSPC almost completely replaces the mouse hematopoietic system. Similar frequencies of HSC (CD34+, CD38-) and LT-HSC (CD34+, CD38-, CD45 RA-, CD90+) are observed in both conditions (Figure 14D, E). After 13 weeks (Figure 14C) sgRNA-E Since the frequency of edited cells within HSPCs is comparable to the pre-injection distribution, unedited HSPCs do not offer a selective advantage. sgRNA-E HSPC and NTC The differentiation from HSPC is indistinguishable (Figure 15).

[0354] In conclusion, edited and unedited HSCPs were observed to exhibit indistinguishable differentiation profiles and comparable engraftment rates.

[0355] Example 23: In vivo efficacy of anti-CD33 ADC in mice transplanted with AML cell lines In this experiment, AML cell lines mCherry-luc labeled MOLM-14 (1 million cells; RRID: CVCL_7916) and mCherry-luc labeled OCI-AML2 cells (2 million cells; RRID: CVCL_1619) were transplanted into the tail veins of 18-week-old NBSGW mice (Jackson Laboratory; stock #026622). After 10 days, the mice were treated with physiological saline, ADC (Mylotarg, Repmab #3-ADC), or a control antibody against chicken lysozyme at 1 mg / kg body weight. The experimental overview is shown in Figure 16.

[0356] The proliferation of tumor cell lines was measured by repeated luminescence measurements after intraperitoneal injection of 100 μl of D-luciferin (BioSynth#L-8220) using Newton 7.0 imaging (Vilber). The results are shown in Figure 17. Tumor growth continued in mice treated with physiological saline or control ADC, but it was clearly observed that tumor growth was effectively eliminated in mice treated with antibodies ReFMab#1 or ReFMab#3. The same observations were made for both AML cell lines tested.

[0357] Tumor cells, as measured by positive staining for hCD45 cells, were quantified in blood and bone marrow at the experimental endpoint on day 19. After euthanasia of mice, 0.2 ml of blood and both hind limbs were collected from each mouse. Cell suspensions were prepared, erythrocytes were lysed with ACK lysis buffer, and the cell suspensions were then filtered. All cells were stained with a survival dye (Zombie UV Fixable Viability Kit 1:1000, Biolegend #423108).

[0358] Bone marrow cells as follows: CD33 FITC (1:50, Biolegend#303304), CD34 PerCP-Cy5.5 (1:10, BD Biosciences#347222), CD45 PE (1:200, Biolegend#393412), CD90 PE-Cy7 (1:50, BD Biosciences#561558), CD33 APC(Refmab#3), CD38 AF700(Beckman Coulter#B23489), CD45 RA APC / Fire(1:50, Biolegend#304152), CD3 BV421(1:50, Biolegend#300434), CD45 V500(1:50, BD Biosciences#560777), mCD45 Samples were stained with antibodies against BV605 (1:200, BD Biolegend#103155), CD123 BV650 (1:50, Biolegend#306020), and CD117 BV711 (1:50, Biolegend#313230), and then treated with human FcR blocking agent (1:200, Miltenyi#130-059-901).

[0359] Peripheral blood cells were then treated with CD33 FITC (1:50, Biolegend #303304), CD34 PerCP-Cy5.5 (1:100, BD Biosciences #347222), CD45 PE (1:200, Biolegend #393412), CD13 PE-Cy7 (1:50, Biolegend #301712), CD33 APC(Refmab#3), mCD45 AF700(Biolegend#103128), CD14 APC-Cy7(1:50, Biolegend#301820), CD3 BV421(1:50, Biolegend#300434), CD45 V500(1:50, BD Biosciences#560777), CD123 Samples were stained with BV650 (1:50, Biolegend#306020), CD117 BV711 (1:50, Biolegend#313230), and CD19 BV786 (1:50, BD Biosciences#363028), and FcR blocking agent human (1:200, Miltenyi#130-059-901) was added.

[0360] Data acquisition and analysis were performed as described in Example 22.

[0361] The results are shown in Figure 18. Treatment with Refmab#1 and Refmab#3 resulted in cell counts comparable to those of mice without tumor transplantation. ADCs of Refmab#1 and Refmab#3 effectively eliminated the AML cell lines MOLM-14 and OCI-AML2 in vivo.

[0362] Example 24: In vitro death assay sgRNA-E To demonstrate selective death of HSPCs, a death assay was performed starting 5 days after electroporation of HSCPs to allow for complete editing and expression of the CD33 mutant on edited cells. MOLM-14, NTC HSPC and sgRNA-EHSPC are incubated with gradually increasing concentrations of Refmab#3 ADC.

[0363] After 7 days, the cells were harvested and stained with a survival dye as well as ReFMab#3 and the control antibody CD33 HIM3-4. MOLM-14 and NTC HSPCs are killed, but sgRNA-EHSPCs are shielded from ADC toxicity due to the loss of Refmab#3 binding.

[0364] Example 25: In vivo experiment NTC In contrast to HSPC, sgRNA-E To demonstrate shielding of HSPC to ReFMab#3 targeted therapy, in vivo experiments will be conducted using humanized NBSGW mice. Stable engraftment will be confirmed by peripheral blood flow cytometry. The animals will then be treated with either ReFMab#3 ADC or CAR-T. Three weeks after treatment, the mice will be sacrificed, and the bone marrow, spleen, and peripheral blood will be evaluated for engraftment and differentiation.

[0365] Next, 50% of the bone marrow from the aforementioned experiment was used for secondary transplantation in NSG-SGM3 mice, demonstrating the success of editing LT-HSCs that could engraft in the secondary host.

[0366] This model can also be used to demonstrate sustained human hematopoiesis after treatment with ReFMab#3 in humanized NBSGW mice challenged by injection of tumor cells.

Claims

1. Mammalian cells or cell populations expressing a first isoform of CD33 for use in medical procedures in patients requiring medical treatment, wherein the patient has cells expressing a second isoform of CD33, The cells expressing the first isoform contain polymorphic or genetically modified alleles not present in the genome of the patient having cells expressing the second isoform of CD33, and the first and second isoforms are substantially functionally identical and / or expressed at the same or substantially the same level. Mammalian cells or cell populations in which the polymorphic allele or genetically engineered allele is characterized by at least one amino acid substitution at positions N20, F21, W22, Q24, F43, F44, H45, P48, Y49 and / or Y50 of SEQ ID NO:

1.

2. Mammalian cells or cell populations for use according to claim 1, wherein the polymorphic allele or genetically engineered allele is characterized by at least one amino acid substitution at positions N20, F21, W22 and / or Y50 of SEQ ID NO:

1.

3. Mammalian cells or cell populations for use according to claim 1 or 2, wherein the function is the ability to bind to sialic acid, stimulate by sialic acid, phosphorylation upon binding to sialic acid, act as a docking site for Src homology 2 (SH2) domain-containing proteins, and / or cause inhibition of phagocytosis.

4. Mammalian cells or cell populations for use according to claim 3, wherein residue N20 is substituted with G, S, D, E, K, V, R or H, and / or residue F21 is substituted with V, L, I, S, M, N, Q or H, and / or residue W22 is substituted with S, T, E, R, D, K or H, and / or Q24 is substituted with R, and / or F43 is substituted with S, and / or F44 is substituted with P or S, and / or H45 is substituted with Y, and / or residue P48 is substituted with F, D or S, and / or residue Y49 is substituted with A, and / or residue Y50 is substituted with S, L, R, D, E, A, K or H.

5. Mammalian cells or cell populations for use according to any one of claims 1 to 4, wherein the first isoform of CD33 is obtained by modifying the nucleic acid sequence encoding the first isoform of CD33 in vivo or ex vivo by introducing a gene editing enzyme into cells that can induce site-directed mutations (or more) in a target sequence encoding a surface protein region involved in drug binding, preferably including at least a first antigen-binding region, by gene editing.

6. Preferably, the medical procedure specifically depletes patient cells expressing the second isoform of CD33, and preferably, in the treatment of hematopoiesis, normal hematopoiesis is restored after immunotherapy, and preferably, in the treatment of malignant hematopoietic diseases such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), blastic plasmacytoid dendritic cell neoplasms (BPDCN), and other myeloproliferative neoplasms, at least one that specifically binds to the second isoform of CD33. The process involves administering to a patient in need a therapeutically effective amount of cells or a population of cells expressing the first isoform of CD33 in combination with a therapeutically effective amount of a depleting agent containing an antigen-binding domain, preferably the depleting agent being an antibody, an antibody-drug conjugate, or immune cells, preferably T cells having a chimeric antigen receptor (CAR) comprising a first antigen-binding domain that specifically binds to the second isoform and does not bind to, or binds substantially less to, the first isoform, for use according to any one of claims 1 to 5, preferably hematopoietic stem cells.

7. The first antigen-binding region of the depletion agent specifically binds to an epitope containing amino acids N20, F21 and / or W22 of SEQ ID NO: 1, and the first antigen-binding region is as follows: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (SEQ ID NO: 4), VHCDR2 (SEQ ID NO: 5), and VHCDR3 (SEQ ID NO: 6), b) A mammalian cell or cell population for use according to claim 6, comprising an antibody light chain variable domain (VL) comprising three CDRs VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8, and VLCDR3 is SEQ ID NO: 9, and an antigen-binding region having the same epitope specificity as the antigen-binding region comprising the antigen-binding region.

8. The first antigen-binding region of the depletion agent specifically binds to an epitope containing amino acids N20, F21 and / or W22 of SEQ ID NO: 1, and the first antigen-binding region is as follows: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (sequence number 20), VHCDR2 (sequence number 21), and VHCDR3 (sequence number 22), b) A mammalian cell or cell population for use according to claim 6, comprising an antigen-binding region having the same epitope specificity as the antigen-binding region, comprising three antibody light chain variable domains (VLs) comprising VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 8, and VLCDR3 is SEQ ID NO:

24.

9. The first antigen-binding region of the depletion agent specifically binds to an epitope containing amino acids P48 and / or Y50 of SEQ ID NO: 1, and the first antigen-binding region is as follows: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (SEQ ID NO: 27), VHCDR2 (SEQ ID NO: 28), and VHCDR3 (SEQ ID NO: 29), b) A mammalian cell or cell population for use according to claim 6, comprising an antigen-binding region having the same epitope specificity as the antigen-binding region, comprising three antibody light chain variable domains (VLs) comprising VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 31, and VLCDR3 is SEQ ID NO:

32.

10. A pharmaceutical composition comprising mammalian cells as defined in any one of claims 1 to 9, preferably immune cells such as hematopoietic stem cells or T cells, and preferably a depleting agent and a pharmaceutically acceptable carrier as defined in any one of claims 6 to 9.

11. A depletion agent for use in preventing or reducing the risk of serious adverse events in a patient who has received cells expressing a first isoform of CD33, wherein the patient's intrinsic cells express a second isoform of CD33, and the depletion agent comprises at least a second antigen-binding region that specifically binds to the first isoform of CD33 and does not bind to, or binds substantially less to, the second isoform of CD33.

12. A depletion agent for use in selectively depleting host cells in a patient requiring selective depletion of host cells, wherein the patient's intrinsic cells express a second isoform of CD33, the depletion agent comprises at least a first antigen-binding region that specifically binds to the second isoform of CD33, the first antigen-binding region of the depletion agent specifically binds to an epitope comprising amino acids N20, F21 and / or W22 of SEQ ID NO: 1, and the first antigen-binding region is: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (SEQ ID NO: 4), VHCDR2 (SEQ ID NO: 5), and VHCDR3 (SEQ ID NO: 6), b) A depletion agent comprising an antibody light chain variable domain (VL) containing three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 7, VLCDR2 is SEQ ID NO: 8, and VLCDR3 is SEQ ID NO: 9, and an antigen-binding region having the same epitope specificity as the antigen-binding region.

13. A depletion agent for use in selectively depleting host cells in a patient requiring selective depletion of host cells, wherein the patient's intrinsic cells express a second isoform of CD33, the depletion agent comprises at least a first antigen-binding region that specifically binds to the second isoform of CD33, the first antigen-binding region of the depletion agent specifically binds to an epitope comprising amino acids N20, F21 and / or W22 of SEQ ID NO: 1, and the first antigen-binding region is: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (sequence number 20), VHCDR2 (sequence number 21), and VHCDR3 (sequence number 22), b) A depletion agent comprising an antibody light chain variable domain (VL) containing three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 23, VLCDR2 is SEQ ID NO: 8, and VLCDR3 is SEQ ID NO: 24, and an antigen-binding region having the same epitope specificity as the antigen-binding region.

14. A depletion agent for use in selectively depleting host cells in a patient requiring selective depletion of host cells, wherein the patient's intrinsic cells express a second isoform of CD33, the depletion agent comprises at least a first antigen-binding region that specifically binds to the second isoform of CD33, the first antigen-binding region of the depletion agent specifically binds to an epitope containing amino acid Y50 of SEQ ID NO: 1, and the first antigen-binding region is as follows: a) A variable antibody heavy chain domain (VH) comprising three CDRs, VHCDR1 (SEQ ID NO: 27), VHCDR2 (SEQ ID NO: 28), and VHCDR3 (SEQ ID NO: 29), b) A depletion agent comprising an antibody light chain variable domain (VL) containing three CDRs, VLCDR1, VLCDR2, and VLCDR3, where VLCDR1 is SEQ ID NO: 30, VLCDR2 is SEQ ID NO: 31, and VLCDR3 is SEQ ID NO: 32, and an antigen-binding region having the same epitope specificity as the antigen-binding region.

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