CN117813110A

CN117813110A - Distinguishable cell surface protein variants for use in cell therapy

Info

Publication number: CN117813110A
Application number: CN202280054951.XA
Authority: CN
Inventors: 罗萨尔巴·勒波雷; 卢卡斯·杰克尔; 斯特凡尼·尤林格尔; 埃马纽埃尔勒·兰德曼; 亚历山德罗·西诺波利; 阿梅莉·维德克尔; 安娜·德沃; 安娜·卡穆斯; 安娜·海登; 罗米纳·马特-马罗内
Original assignee: Semio Treatment Co ltd; Universitaet Basel
Current assignee: Semio Treatment Co ltd; Universitaet Basel
Priority date: 2021-08-06
Filing date: 2022-08-05
Publication date: 2024-04-02

Abstract

The present invention relates to the use of cells having distinguishable surface proteins with engineered or naturally occurring mutations but still functional surface proteins in therapy. The invention also relates to the use of cells having a distinguishable CD123 surface protein variant but still a functional surface protein in therapy, in particular adoptive cell therapy.

Description

Distinguishable cell surface protein variants for use in cell therapy

Technical Field

The present invention relates to the use of cells having distinguishable surface proteins (discernible surface protein) having engineered or naturally occurring mutations but still functional surface proteins in therapy. The invention also relates to the use of cells having a distinguishable CD123 surface protein variant (but still a functional surface protein) in therapy, in particular adoptive cell therapy.

Statement regarding costs

The program leading to this application has accepted funds (dial agreement No. 818806) provided by the European Research Council (ERC) in accordance with the European Union Horizon 2020 (European Union's Horizon 2020) for research and innovation planning.

Background

Cell therapy is emerging as the third largest medical mainstay following small molecule therapy and biological agent-based therapies such as recombinant proteins including antibodies. Cell therapy is useful in oncology for the treatment of hematopoietic malignancies, and other applications, such as the treatment of genetic diseases, solid organ tumors, and autoimmune diseases are also under development. However, cell therapy may be accompanied by serious adverse side effects. In fact, although cancer immunotherapy with Chimeric Antigen Receptor (CAR) T cells has been successful in targeting and eradicating malignant cells that express a particular antigen, it generally does not distinguish between normal and malignant cells, thereby inducing disruption of the normal hematopoietic system. Targeted therapies, including antibody-based therapies such as conventional monoclonal antibodies, multispecific antibodies, such as T cell conjugates (e.g., biTE), and cell (such as CAR cells (e.g., CAR T cells, CAR NK cells, or CAR macrophages)) therapies will eliminate all cells expressing the target molecule. However, most cancer cell surface antigens are also common to normal hematopoietic cells or other cells. Thus, determining targets that kill diseased cells, including tumors, while avoiding damage to healthy cells is a major challenge for targeted therapies. In particular, in myeloid disorders including myeloid malignancies, such as myelodysplastic syndrome (MDS), acute Myeloid Leukemia (AML) or blast plasmacytoid dendritic cell tumor (BPDCN), cell surface antigens such as CD33 or CD123 are shared by normal myeloid progenitor cells. Thus, immunotherapy targeting CD33 or CD123 antigens against MDS, AML or PBDCN may be accompanied by the depletion of normal hematopoietic cells in patients other than malignant cells (Gill s.i. best practice & Research Clinical Hematology, 2019). Thus, targeted immunotherapy involving mabs, T cell adaptors or CAR T is mostly difficult to achieve, partly due to the lack of a true disease-specific surface antigen (Gill s.i. best practie & Research Clinical Hematology, 2019).

In order to regenerate normal hematopoiesis consumed by CD33-CAR T cell transfer, CD33-CART resistant hematopoietic cells are being engineered in such a way that the entire CD33 gene is knocked out (Kim et al 2018.Cell. 173:1439-53). However, CD33 has a constitutive inhibitory effect on myeloid lineage cells through its immunoreceptor tyrosine-based inhibitory motif (ITIM) signaling domain. Thus, it is not clear how well the loss of CD33 can be tolerated. CD33 knockout (CD 33 KO) engineered cells transplanted in patients may have long-term functional defects and very heterogeneous consequences (WO 2018/160768,Kim et al.2018.Cell.173:1439-53,Borot et al.2019.PNAS.116:11978-87,Humbert et al.2019.Leukemia.33:762-808). In fact, the frequency of CD33 KO cells was reduced in two monkeys, for which long-term observations were reported. This may indicate that the function of the cells is impaired, for example by reducing the engraftment of CD 33-KO-long term regenerative HSC (LT-HSC) or by competing for inferior CD33-KO (Kim et al 2018.Cell. 173:1439-53). Furthermore, the number of cell surface antigens with optional functions is very limited, and loss of the redundant cell surface antigen can induce antigen negative recurrence. CD19 negative recurrence was observed in approximately 30% of patients receiving CD19 targeted CAR T treatment (Orlando et al 2018Nat Med 24:1504-6). Dual targeting of CD19 and CD123 can prevent antigen loss recurrence (Ruella et al 2016J Clin Invest 126:3814-26).

WO2018/160768 describes a method in which hematopoietic cells are engineered in such a way that all epitopes on the surface antigen CD33 are deleted. It is expected that the antigen with this larger deletion does not have the same function as the corresponding wild-type surface antigen. WO2014/138805 discloses certain variants of CD123 that exhibit reduced binding to certain antibodies. Cell Reports (2014) 8:410-9 discloses certain amino acids involved in binding of CSL362 to CD 123. US20190185573 discloses antibody binding properties of certain variants of CD 123. None of these documents discloses the use of such variants as contemplated in the present disclosure. EP3769816 discloses antibody chains having homology to CDRs of certain molecules disclosed in the present disclosure. Blood (2017) 130 support 1:2625 discloses a method of treating cancer using anti-CD 123 CAR-T cells. Such treatment does not use any variant of CD 123.

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

Disclosure of Invention

It is one of the objects of the present disclosure to develop a safer method for treating malignant tumors, in particular cancer, hematological malignant tumors, myeloid disorders. Thus, the inventors sought variants of surface proteins that are immunologically distinguishable while maintaining normal function, and in which the amino acid changes result from single nucleotide or polynucleotide variations. In particular, the inventors identified rationally designed and naturally occurring variants of CD123 and suggested that these mutations altered the antigenicity of CD123 to a particular antibody while retaining its normal expression and function (specifically interleukin-3 (IL-3) binding).

The present invention relates to a mammalian cell or population of cells expressing a first isoform of a surface protein, preferably CD123, for use in medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said surface protein, wherein said cells expressing said first isoform comprise genomic DNA having at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of a patient having cells expressing said second isoform of said surface protein, and preferably wherein said first isoform and said second isoform are functional.

In a particular embodiment, the present invention relates to mammalian cells or cell populations, preferably hematopoietic stem cells, for use in medical treatment of a patient in need thereof, wherein the medical treatment comprises: administering to said patient in need thereof a therapeutically effective amount of said cell or cell population expressing said first isoform in combination with a therapeutically effective amount of a depleting agent, preferably to restore normal hematopoiesis following immunotherapy for treating a hematopoietic disorder, preferably a malignant hematopoietic disorder, said depleting agent comprising at least a first antigen binding region that specifically binds said second isoform to specifically deplete patient cells expressing the second isoform, such as Acute Myeloid Leukemia (AML), a blast plasmacytoid dendritic cell tumor (BPDCN) or B-acute lymphoblastic leukemia (B-ALL).

In another specific embodiment, the invention relates to a mammalian cell or cell population for use in medical treatment of a patient in need thereof, wherein the medical treatment comprises: administering to said patient in need thereof a therapeutically effective amount of said cell or cell population expressing said first isoform in combination with a therapeutically effective amount of a depleting agent, preferably for adoptive cell transfer therapy, more preferably for treating a malignant hematopoietic disorder comprising at least a second antigen binding region that specifically binds said first isoform to specifically deplete metastatic cells expressing the first isoform, such as Acute Myeloid Leukemia (AML), blast plasmacytoid dendritic cell tumor (BPDCN) or B-acute lymphoblastic leukemia (B-ALL), again more preferably wherein said depleting agent is subsequently administered to said cell or cell population expressing said first isoform of surface protein to avoid eventual serious side effects, such as graft versus host disease due to transplantation.

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

The invention also relates to a depleting agent for preventing or reducing the risk of serious side effects in a patient who has received cells expressing a first isoform of a surface protein, wherein the patient's primordial cells express a second isoform of a surface protein, and wherein the depleting agent comprises at least a second antigen binding region that specifically binds to the first isoform and not to the second isoform, preferably wherein the surface protein is CD123.

In another aspect, the invention relates to a depleting agent for selectively depleting host cells of a patient in need thereof, wherein the patient' S primordial cells express a second isoform of a surface protein, and wherein said depleting agent comprises at least a first antigen binding region that specifically binds to said second isoform, preferably wherein said surface protein is CD123, and wherein said first antigen binding region of said depleting agent specifically binds to an epitope comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1, more preferably wherein said first antigen binding region comprises:

a) An antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO 2, VHCD2 is SEQ ID NO 3 and VHCDR3 is SEQ ID NO 4; and

b) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7, VLCDR3 is SEQ ID NO 8, again more preferably wherein said first antigen binding region comprises a heavy chain variable domain comprising or consisting of any of the amino acid sequences selected from SEQ ID NO 9, 11 and 13, or any of the amino acid sequences selected from SEQ ID NO 9, 11 and 13, and/or a light chain variable domain comprising or consisting of any of the amino acid sequences selected from SEQ ID NO 10, 12 and 14.

Drawings

Fig. 1: a) Relative Solvent Accessibility (RSA) of each residue of CD 123. RSA was calculated for the CSL362 bound state and CSL362 free state based on the X-ray structure of the CD123-CSL362 complex (PDB ID:4 JZJ). Data for the N-terminal domain are shown. Arrows indicate RSA for residues E51, S59 and R84. B) 3D structure of CD123-CSL362 complex and selected amino acid variants. CD123 residues E51, S59 and R84 are shown as rods. The structure of CSL362 antibody is shown as a molecular surface.

Fig. 2: flow cytometry of CD123 variants stained with two monoclonal antibody clones MIRG123 and 6H6 and controls (HEK, HEK-CD 123). Variants are encoded according to their binding to the elimination (underlined), weak (circles) or strong (asterisks) of MIRG 123. Controls HEK and HEK-CD123 are gray.

Fig. 3: quantification of FACS plots in fig. 2. Control and% MIRG123 of all variants were plotted ⁺ 6H6 ⁺ Double positive (upper right quadrant) cells. The 6H6 binding was not altered (see fig. 2). Summary of 3 independent experiments. Mutant variants were identified as non-binders (underlined,<1％ MIRG123 ⁺ 6H6 ⁺ ) Weak binders (circles, 1-20% MIRG123 ⁺ 6H6) And a strong binding substance (asterisk,>20％ MIRG123 ⁺ 6H6)。

fig. 4: CD123 isoforms prevent fcgnriiia activation. Relative luminescence signal measured after co-culturing HEK, HEK-CD123 (wild type) and HEK CD123 variant isoforms with Jurkat/fcyriiia/NFAT-Luc reporter cells. RLU was normalized to the signal measured with HEK-CD123 (wild type). Mutant variants were identified as non-binders (underlined,<1％ MIRG123 ⁺ 6H6 ⁺ ) Weak binders (circles, 1-20% MIRG123 ⁺ 6H6) And a strong binding substance (asterisk,>20％ MIRG123 ⁺ 6H6)。

fig. 5: CD123 isoform variants prevent CD3/CSL362 BiTE-mediated T cell activation. Stable target cell lines HEK, HEK-CD123 and CD123 variants were co-cultured with human primary pan T cells in the presence of 300ng/mL CD3/CSL362 BiTE at a 10:1 E:T ratio. Shows the% CD69 measured after co-culture with HEK, HEK-CD123 or all CD123 variants ⁺ Summary of T cells. Data were normalized to% CD69 in the presence of HEK target cells ⁺ And (3) cells. Error bars show mean ± SD. Data represent 5 independent blood donors and experiments with 2 technical replicates per group.

Fig. 6: CD123 isotype variants are immune to CD3/CSL362 BiTE mediated killing of human T cells. Stable target cell lines HEK, HEK-CD123 and CD123 variants were co-cultured with human primary pan T cells in the presence of 300ng/mL CD3/CSL362 BiTE at a 10:1 E:T ratio. Specific BiTE-mediated killing (%) of HEK, HEK-CD123 and all CD123 variants after 72 hours of co-culture is shown. Error bars show mean ± SD. Data represent 5 independent blood donors and experiments with 2 technical replicates per group. CD123 variant expression was confirmed by FACS using monoclonal antibody 6H 6.

Fig. 7: CD 123-specific second generation CARs were non-viral HDR mediated integration into exon 1 of the TRAC locus using the design of CRISPR/Cas9 and CSL 362-based CAR constructs.

Fig. 8: results of FACS analysis of effector CAR T cell activation (CD 69) after one day of co-culture of effector CAR T cells with different target cells are represented. % CD69 after 24 hour co-culture of CAR T cells alone (effector T cells) or in the presence of HEK, HEK-CD123 or all CD123 variants ⁺ Summarizing. Data were normalized to% CD69 in the presence of HEK target cells ⁺ And (3) cells. Control T cells that did not express CAR were not activated.

Fig. 9: the quantized basis data of fig. 8. Quantification of HEK, HEK-CD123 and variants thereof by specific killing of CD123 specific CAR T cells as measured by flow cytometry on the first day of co-culture. Error bars show mean ± SD. Data were from 3 independent blood donors and experiments with 2 technical replicates per group.

Fig. 10: a) BLI sensorgrams of CD123 WT and E51T for CSL 362. B) At 280 seconds, the binding level of cd123_wt and variants to the captured CSL362 antibody (captured at the same level) was at the highest concentration of 50 nM. C) BLI-sensed profile of cd123_wt and E51T for control antibody 6H 6. D) Cd123_wt and variants bound to the captured 6H6 antibody at 250 seconds (normalized to capture/load level). E) BLI sensorgrams of E51Q and E51T for CSL 362. F) At 280 seconds, the cd123_variant at a higher concentration than B (highest concentration of 300 nM) had a binding level to the captured CSL362 antibody (captured at the same level). G) Cd123_wt at the highest concentration of 50nM and cd123_variant at the highest concentration of 300nM at 250 seconds were combined with the captured votuzumab (captured at the same level).

Fig. 11: a) Cd123_wt and E51T BLI sensorgrams binding to IL 3. B) Binding level of IL3 to captured biotinylated CD123 variants at 250 seconds (normalized to capture/load level).

Fig. 12: heat-induced unfolding was monitored using a SYPRO Orange. In the first derivative data inserted. Fluorescence was monitored in PBS buffer 5x Sypro orange dye. The protein concentration was 0.25mg/mL. The heating rate used was 1.5 ℃/min ramp, recorded from 25 to 95 ℃. A) Thermally induced unfolding of cd123_wt and CD123 variants (E51T, E51K, E51Q, S59P, S E and R84E). B) The heat-induced unfolding of CD123 variants (E51A, S59R, S59F, S Y, R Q and R84T).

Fig. 13: a large number of TF-1 cells were cultured as controls (no crRNA), KO (crRNA but no HDRT) or KI (crRNA+KIHDRT, E51K or E51T). Control cells cultured with IL-3 or GM-CSF maintained MIRG123+, 6H2+. KO cells cultured with GM-CSF largely retain MIRG123-, 6H2+. In contrast, in cultures containing KO cells gradually cultured with IL-3, MIRG123+, 6H2+ cell populations became detectable. On day 6, the mirg123+,6h6+ population predominated, indicating that cells expressing CD123 receptor have a competitive advantage in the presence of IL 3. In KI cells (E51K or E51T), the population of MIRG 123-6H26+ and the population of MIRG123+6H26+ gradually increased with IL 3. This is less pronounced in cells cultured with GM-CSF. Thus, KI cells (MIRG 123-6H2+) have functional receptors.

Fig. 14: TF-1 cells (wild-type cells, knockout cells, and E51K and E51T knockout cells) were tested for their ability to be stimulated by IL 3. TF-1 knock-in cells expressing E51K and E51T variants of CD123 can proliferate to a similar extent as TF-1 cells expressing wild-type CD123 upon addition of hIL 3. The knockdown cells showed only minimal response to hll 3.

Fig. 15: antibody MIRG123 was tested for its ability to bind to TF-1 cells (wild type cells, knockdown cells, and E51K and E51T knockin cells). MIRG123 resulted in dose-dependent proliferation blocking and apoptosis of IL3 stimulated wild-type TF-1 cells. In contrast, TF-1 knockin cells expressing E51K and E51T variants of CD123 were effectively protected from the blocking effect of MIRG 123.

Fig. 16: HSPC cells with E51K and E51T knockins can be successfully generated. Plotted are% of knocked-in cells 2 and 5 days after Electroporation (EP).

Fig. 17: HSCs with E51K and E51T knockins showed loss of binding to antibody MIRG123, but maintained CD123 expression as assessed by control antibody 6H 6.

Fig. 18: LT HSCs (long term hematopoietic stem cells) were also successfully edited, as well as MPP1 cells (cd34+cd38-CD 90-CD45 RA-) and MPP2 cells (cd34+cd38-CD 90-CD45 ra+).

Fig. 19 and 20: co-culture of human effector T cells with control or edited HSPC cells (E: T=3:1) in the presence of CD3/CSL362 BiTE resulted in a reduction of wild-type HSPC when treated with BiTE, as measured by quantification of flow cytometry patterns. In contrast, HSCs expressing CD 123E 51K or E51T variants are protected and enriched. Error bars show mean ± SD. Data were generated in the case of 2 independent donors.

Detailed Description

Immunotherapy is a promising therapy for the treatment of cancer, genetic diseases and autoimmune diseases. An immune depleting agent, such as an engineered immune cell directed against a tumor antigen, is administered to a patient to target and kill tumor cells. However, since tumor surface proteins are also expressed on the surface of normal cells including hematopoietic cells, such a strategy may have serious side effects on patients, for example, by altering hematopoiesis. To restore hematopoiesis in a patient, hematopoietic cells may then be transplanted into the patient. However, the depleting agent binds not only to diseased cells but also to newly transplanted healthy cells, which may limit the maximum tolerated dose or limit therapeutic use prior to transplanting healthy cells. Alternatively, the transplanted cells need to be resistant to the immune depleting agent in order not to be targeted and eliminated by it. Thus, the inventors selected cells that are resistant to the immune depleting agent used in immunotherapy, while retaining their function for restoring normal hematopoiesis in patients.

The present inventors developed a method for identifying functional allelic variants in genetic sequences encoding regions of a surface protein responsible for binding to specific depleting agents. Such variants may be naturally occurring polymorphisms and/or engineered variants. Different isoforms of the surface protein may be selected or produced. The first isoform of the surface protein encoded by the nucleic acid having the polymorphism is not recognized by a specific depleting agent. Such variant alleles in particular do not alter the function of the surface proteins. Thus, the depleting agent can be used to specifically bind one isoform and not the other, thereby specifically depleting cells expressing one isoform. For example, if the depleting agent specifically binds to the second isoform but does not bind to the first isoform, the depleting agent will specifically deplete cells expressing the second isoform. In another embodiment, the first isoform is recognizable by a second agent, and thus the second agent can be used to specifically deplete cells expressing the first isoform but not the second isoform. Cells expressing a first isoform of a surface protein encoded by at least one variant allele are advantageously used for medical treatment of patients having cells expressing a second isoform, in particular by depleting specific engrafted cells or patient cells using a second agent or a first agent, respectively.

Consumable agent

The present disclosure relates to an agent comprising an antigen binding region that specifically binds one isoform of a surface protein on a cell, but not the other isoform of the surface protein. Such formulations are referred to herein as "consumable agents". Both isoforms of the surface protein are functional, i.e. the surface protein is functional with respect to at least one relevant property of the surface protein. Preferably, the two isoforms of the surface protein have the same function, i.e. they are functionally indistinguishable.

However, the two isoforms of surface proteins differ in their binding to the depleting agent. The depleting agent binds specifically to only one of the isoforms of the surface protein. Thus, the isoforms may be described as functionally identical, but immunologically distinguishable.

The first isoform and the second isoform of the surface protein may be polymorphic alleles of the surface protein. Preferably, the first isoform and the second isoform of the surface protein are naturally occurring polymorphic alleles of the surface protein. Also preferably, the first isoform and the second isoform of the surface protein are Single Nucleotide Polymorphism (SNP) alleles.

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

Various methods can be used to determine mutations to be introduced into the surface protein to produce the second isoform. For example, mutations can be randomly inserted into a surface protein, and the resulting variants can then be subjected to functional and immunological screening. Alternatively, mutations can be rationally designed, for example, by analyzing the secondary or tertiary protein structure of the surface protein.

The depleting agent comprises an antigen binding region that specifically binds one isoform of a surface protein on a cell, but does not bind another isoform of the surface protein. The consumable agents of the present disclosure can be divided into two broad categories.

First, the depleting agent may be a polypeptide comprising an antigen binding region. The polypeptide may consist of one or more polypeptide chains. Preferably, the polypeptide comprising an antigen binding region is an antibody. The polypeptide comprising an antigen binding region may also be an antibody fragment, an antibody-drug conjugate or another variant of an antibody or scaffold. Exemplary antibody fragments and scaffolds include single domain antibodies, large antibodies, small antibodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NAR, diavs, camelid antibodies, ankyrin, centyrin domain antibodies, lipocalin (lipocalin), minimodular immunopharmaceuticals, maxiantibodies (maxyantibodies), protein A and affilin.

The polypeptide comprising an antigen binding region may also be a bispecific, bi-site or multi-specific antibody. Such molecules may also comprise additional functional domains. For example, the polypeptide comprising an antigen binding region may be a T cell adaptor, such as BiTE. The polypeptide comprising an antigen binding region may also be fused to a cytokine or chemokine, or to an extracellular domain of a cell surface receptor.

Alternatively, the depleting agent may be a cell comprising an antigen binding region. For example, the depleting agent can be a Chimeric Antigen Receptor (CAR). In certain embodiments of the present disclosure, the cell comprising the antigen binding region is a CAR T cell, a CAR NK cell, or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell comprising an antigen binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell comprising the antigen binding region is a primary T cell comprising a CAR.

The depleting agent specifically binds one of the isoforms of the surface protein, but does not bind the second isoform and thus specifically depletes cells expressing one isoform.

In certain embodiments, the disclosure relates to agents comprising a first antigen binding region that specifically binds a second isoform of a surface protein but does not bind the first isoform. In other embodiments, the disclosure also relates to an agent comprising a second antigen binding region that specifically binds the first isoform and not the second isoform.

The first isoform and the second isoform of the surface protein may differ from each other by only one amino acid substitution. The one amino acid difference between the first isoform and the second isoform may also be the result of the presence of a single nucleotide polymorphism, e.g., a naturally occurring single nucleotide polymorphism. The first isoform and the second isoform of the surface protein may also differ from each other by more than one amino acid, such as by two, three or more than three amino acids. The first isoform and the second isoform of the surface protein may also differ from each other in that one isoform has one, two, three or more amino acid insertions compared to the other isoform. The first isoform and the second isoform of the surface protein may also differ from each other in that one isoform has deletions of one, two, three or more than three amino acids as compared to the other isoform. In a preferred embodiment, the depleting agent is an antibody or antigen binding fragment.

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

In rodent and primate natural antibodies, two heavy chains are linked to each other by disulfide bonds, each heavy chain being linked to a light chain by disulfide bonds. There are two types of light chains, lambda and kappa. There are five major heavy chain classes (or isotypes) that determine the functional activity of an antibody molecule: igM, igD, igG, igA and IgE. Each chain comprises a different sequence domain. In a typical IgG antibody, the light chain comprises two domains, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, one variable domain (VH) and three constant domains (CH 1, CH2 and CH3, collectively referred to as CH). The variable regions of the light chain (VL) and heavy chain (VH) determine the binding recognition and specificity for an antigen. The constant region domains of the light Chain (CL) and heavy Chain (CH) confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding and binding to Fc receptors (FcR).

Fv fragments are the N-terminal part of the Fab fragments of immunoglobulins, consisting of a variable portion of one light chain and one heavy chain. The specificity of an antibody is due to the structural complementarity between the binding site of the antibody and the epitope. The antibody binding site consists of residues primarily from the hypervariable or Complementarity Determining Regions (CDRs). Occasionally, residues from non-hypervariable regions or Framework Regions (FR) may participate in the antibody binding site, or affect the overall domain structure, thereby affecting the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that collectively define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins have three CDRs designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, an antigen binding site typically comprises six CDRs comprising the respective sets of CDRs for the heavy chain V region and the light chain V region. The Framework Region (FR) refers to the amino acid sequence interposed between CDRs. Thus, the variable regions of the light and heavy chains typically comprise 4 framework regions and 3 CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Residues in the antibody variable domains are typically numbered according to the system designed by Kabat et al. This system is proposed in Kabat et al, 1987,in Sequences of Proteins of Immunological Interest,USDepartment of Health and Human Services,NIH,USA (Kabat et al, 1992, hereinafter "Kabat et al"). This numbering system is used in this specification. Kabat residue nomenclature does not always correspond directly to the linear numbering of amino acid residues in the SEQ ID sequence. The actual linear amino acid sequence, which corresponds to shortening or insertion of the structural components of the basic variable domain structure, whether framework or Complementarity Determining Regions (CDRs), may comprise fewer or additional amino acids than the strict Kabat numbering. For a given antibody, the correct Kabat coding of residues may be determined by aligning homologous residues in the antibody sequence with a "standard" Kabat numbering sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (H-CDR 1), residues 50-65 (H-CDR 2) and residues 95-102 (H-CDR 3). According to the Kabat numbering system, the CDRs of the light chain variable domain are located at residues 24-34 (L-CDR 1), residues 50-56 (L-CDR 2) and residues 89-97 (L-CDR 3).

In a specific embodiment, the antibodies provided herein are antibody fragments, and more particularly, are any proteins comprising an antigen binding domain of an antibody as disclosed herein. The antigen binding domain may also be integrated into another protein scaffold. Antibody fragments and scaffolds include, but are not limited to, fv, fab, F (ab ') 2, fab', dsFv, scFv, sc (Fv) 2, diabodies, single domain antibodies, large antibodies, small antibodies, intracellular antibodies, diabodies, triabodies, tetrabodies, v-NAR, diavs, camelid antibodies, ankyrin, centyrin, domain antibodies, lipocalins, small modular immunopharmaceuticals, maxiantibodies, protein a, and affilin.

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

Reagents of the present disclosure include antibodies and fragments thereof, but also include artificial proteins having the ability to bind antigen that mimics the ability of an antibody to bind antigen, also referred to herein as antigen-binding antibody mimics. Antigen-binding antibody mimetics are organic compounds that specifically bind an antigen, but are structurally independent of an antibody. They are generally artificial peptides or small proteins with a molar mass of about 3 to 20 kDa.

The phrases "antigen binding region that recognizes an antigen" and "antigen binding region that is specific for an antigen" are used interchangeably herein with the term "antigen-binding region that specifically binds an antigen". As used herein, the term "specific" refers to the ability of an agent comprising an antigen binding region (such as an antibody) to detectably bind to an epitope presented on an antigen.

"distinct affinity" or "specific binding" to … "includes having about 10 ^-8 M (KD) or greater affinity binding. Preferably, when the binding affinity is 10 ^-8 M (KD) to10 ^-12 M (KD), optionally 10 ^-8 M (KD) to 10 ^- ¹⁰ M (KD), in particular at least 10 ^-8 M (KD) 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), biological Layer Interferometry (BLI), microscale thermophoresis (MST) and Scatchard plot. Whether a binding domain specifically reacts with or binds to a target can be readily tested by specifically comparing the reaction of the binding domain with a target protein or antigen to the reaction of the binding domain with proteins or antigens other than the target protein.

As used herein, the term "epitope" means the portion of an antigen to which an antibody or antigen binding region thereof binds. Epitopes of protein antigens can be divided into two classes, conformational epitopes and linear epitopes. Conformational epitopes correspond to discrete portions of the antigen amino acid sequence. A linear epitope corresponds to a contiguous sequence of amino acids from an antigen.

In another aspect, further disclosed herein are bispecific or multispecific molecules, such as bispecific antibodies or multispecific antibodies. For example, an antibody may be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand to a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. Antibodies may in fact be derivatized or linked to more than one other functional molecule to produce multispecific molecules that bind to more than two different binding sites and/or target molecules; such multispecific molecules are also encompassed by the terms "bispecific molecule," "bispecific antibody," "dual site molecule," "dual site antibody," "multispecific molecule," and "multispecific antibody" as used herein. To produce a bispecific molecule, an antibody of the invention may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, cytokine, chemokine, or receptor extracellular domain, to produce a bispecific molecule. Specific bispecific and multispecific molecules contemplated by the present disclosure are T cell adaptors, such as bispecific T cell adaptors, e.g., biTE.

As used herein, an agent that does not bind to a particular isoform includes an agent that does not bind to cells expressing the particular isoform. In particular, the reagent is labeled with a fluorescent label or detected with a secondary antibody directed against the reagent, and the percentage of cells that present the fluorescent label or the secondary antibody staining on the surface detected by FACS analysis is determined as described in the experimental section. To monitor expression of the variant isoforms, cells are stained with two agents simultaneously, one agent binding to an epitope into which the variant is introduced (e.g., anti-CD 123 CSL 362) and a second agent binding to an epitope different from the epitope to which the first agent binds (e.g., anti-CD 123 clone 6H 6). The second epitope remained unchanged, so this staining was used as an expression control. As non-binding controls, cells that do not express the protein of interest (e.g., HEK cells) are used. As a maximum binding control, cells that normally do not express the protein of interest are transfected with a wild-type isoform (e.g., HEK-CD 123). Thus, in a specific embodiment, when the percentage of cells bound to an agent (e.g., an anti-CD 123 antibody) coupled to a fluorescent marker at the cell surface is detected by FACS analysis to be below 10%, preferably below 5%, more preferably below 1%, or below a detectable limit, the agent is not able to bind to cells expressing the particular isoform. Binding (i.e., binding to both the control reagent and the reagent of interest) was thereby measured by fluorescence in FACS in the upper right quadrant. The reduced binding also results in reduced fluorescence of the first reagent but not the second reagent.

In an alternative assay, binding of two agents (one agent binds to an epitope in which the variant is introduced (e.g., anti-CD 123 antibody CSL 362), while the second agent binds to a different epitope than the epitope bound by the first agent (e.g., anti-CD 123 clone 6H 6)) is performed label-free and real-time measurement by biolayer interferometry on the purified recombinant CD123 extracellular domain of the wild-type as well as the variant isotype. Thus, in a specific embodiment, the first agent is unable to bind to the recombinant CD123 variant isoform extracellular domain when no relevant signal above background is detected at an antibody concentration of 50 to 300 nM. The detectable binding of the second agent to the non-variant epitope at a concentration of 50 to 300nM serves as a binding and integrity control.

Binding of the agent may result in depletion of cells expressing the first isoform. Various mechanisms may lead to cell depletion. Antibody-dependent cellular cytotoxicity (ADCC) results from the binding of an agent to a target protein and activation of NK cells by an Fc portion on the agent that binds to FcR expressed by the NK cells. The Fc portion of an immunoglobulin refers to the C-terminal region of the heavy chain of an immunoglobulin. The Fc portion may be wild-type or engineered. Mutations in the enhanced engineered Fc portion are known in the art. For certain therapeutic conditions, it is desirable to reduce or eliminate the wild-type Fc region of an antibody, such as the wild-type IgG Fc region, from normal binding to one or more or all Fc receptors and/or from binding to complement components, e.g., C1q, thereby reducing or eliminating the ability of the antibody to induce effector function. For example, it may be desirable to reduce or eliminate the Fc region of an antibody from one or more or all Fcy receptors, such as: fcyRI, fcyRIla, fcyRIIb, fcyRIIIa. The effector functions may include, but are not limited to, one or more of the following: complement Dependent Cytotoxicity (CDC), antibody dependent cell mediated cytotoxicity (ADCC), antibody Dependent Cellular Phagocytosis (ADCP), cytokine secretion, immune complex mediated antigen uptake by antigen presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-bound antibodies, dendritic cell maturation or T cell priming.

Reducing or eliminating the binding of an Fc region to an Fc receptor and/or C1 q is typically achieved by mutating a wild-type Fc region, such as a lgG1 Fc region, more particularly a human lgG1 Fc region, to thereby produce a variant or engineered Fc region of the wild-type Fc region (e.g., a variant human lgG1 Fc region). Substitutions that result in reduced binding may be useful. To reduce or eliminate the binding properties of the Fc region to the Fc receptor, non-conservative amino acid substitutions, i.e., substitution of one amino acid with another amino acid having a different structure and/or chemical property, are preferred.

As described in the experimental section, proxy ADCC assays constitute an industry standard for quantifying the efficacy of agents that mediate ADCC. Engineered Jurkat report cells carry the NFAT-responsive luciferase gene and Fc receptor. Binding of the Fc receptor to the bound antibody results in NFAT induction and thus luciferase signal. No binding will result in no luciferase signal. Cells that do not express the target protein (e.g., HEK), wild-type protein (e.g., HEK-CD 123) or individual variant (e.g., CD123 variant) are incubated with a test agent (e.g., CSL362 or MIRG 123) and mixed with ADCC reporter cells. Luciferase was then measured to quantify ADCC signal. Luciferase luminescence signal was normalized to the maximum signal observed in HEK-CD 123. ADCC was measured using ADCC reporting assay (Promega, catalog No. G7015).

An alternative method of depleting target cells is by using T cell adapter molecules. The inventors constructed bispecific T cell adaptors using the CD123 binding site derived from CSL362 and the CD3 (OKT 3) binding site as described in Hutmacher, leuk Res, 2019. The same target cells used for ADCC assay were used. Primary human T cells and bispecific T cell adaptors are added. Activation of human T cells was quantified by FACS by determining the frequency of CD69 upregulation. Furthermore, specific killing was calculated as described in the methods.

The depleting agents according to the present disclosure specifically bind to one isoform of a surface protein and allow depletion of cells expressing the isoform.

More preferably, in particular embodiments, particularly in methods of use as disclosed herein, the depleting agent according to the present disclosure does not bind to a first isoform of a cell surface protein, but specifically binds to a second isoform of the cell surface protein, and allows depletion of the cells expressing the second isoform. In particular, the depleting agent that does not bind to the first isoform of the cell surface protein but specifically binds to the second isoform expressed in cells of the patient is used to deplete cells of the patient, but is not used to deplete hematopoietic stem cells expressing the first isoform or progeny thereof that are transplanted for restoring hematopoietic function in the patient.

In another specific embodiment, particularly in methods of use as disclosed herein, the depleting agent according to the present disclosure does not bind to the second isoform of the cell surface protein, but specifically binds to the first isoform of the cell surface protein and allows depletion of cells expressing the first isoform. In particular, the depleting agent that does not bind to the second isoform of the cell surface protein but specifically binds to the first isoform expressed in the transplanted cells is used to specifically deplete the transplanted cells to avoid eventual serious side effects such as graft versus host disease due to transplantation.

Selective depletion of cells expressing a particular isoform of a surface protein can be achieved without limitation by Complement Dependent Cytotoxicity (CDC), antibody Dependent Cellular Cytotoxicity (ADCC) or Antibody Dependent Cellular Phagocytosis (ADCP).

In certain embodiments, the antigen binding region is conjugated to an effector compound, such as a drug or toxin. Such conjugates are referred to herein as "immunoconjugates" or "antibody-drug conjugates" (ADCs). Cytotoxins or cytotoxic agents include any agent that is detrimental to (e.g., kills) cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecac, mitomycin, etoposide, teniposide (tenoposide), vincristine, vinblastine, colchicine (t.colchicin), doxorubicin, daunomycin, dihydroxyanthrax, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, maytansinoids, calicheamicin, indoline benzodiazepines, pyrrolobenzodiazepine, alpha-muscarinic peptides, microcystins, auristatins, and puromycin, and analogs or homologs thereof.

In another specific embodiment, the depleting agent is an immune cell carrying an antigen receptor, such as a Chimeric Antigen Receptor (CAR). The immune cells may express recombinant antigen binding regions, also known as antigen receptors, on their cell surfaces. By "recombinant" is meant an antigen binding region that is not encoded by a cell in its native state, i.e., it is heterologous, non-endogenous. Thus, it can be seen that expression of the recombinant antigen binding region introduces new antigen specificity into immune cells, resulting in the cell recognizing and binding to previously unrecognized antigen. The antigen receptor may be isolated from any useful source. In certain embodiments of the present disclosure, the cell comprising the antigen binding region is a CAR T cell, a CAR NK cell, or a CAR macrophage. In a preferred embodiment of the present disclosure, the cell comprising an antigen binding region is a CAR T cell. In another preferred embodiment of the present disclosure, the cell comprising the antigen binding region is a primary T cell comprising a CAR.

In a specific embodiment, the recombinant antigen receptor is a Chimeric Antigen Receptor (CAR). CARs are fusion proteins comprising an antigen binding region, typically derived from an antibody, linked to a signaling domain of a TCR complex. If an appropriate antigen binding region is selected, the CAR can be used to direct immune cells such as T cells or NK cells against the target antigen.

The antigen binding region of a CAR is typically based on scFv (single chain variable fragment) derived from an antibody. In addition to the N-terminal extracellular antibody binding region, CARs can generally comprise a hinge domain that functions as a spacer to extend the antigen binding region away from the plasma membrane of immune effector cells expressing it; a Transmembrane (TM) domain; an intracellular signaling domain (e.g., a signaling domain from the zeta chain of the CD3 molecule (cd3ζ) of the TCR complex, or equivalent); and optionally one or more co-stimulatory domains, which may assist in signaling or function of the CAR-expressing cell. The signaling domains from co-stimulatory molecules, including CD28, OX-40 (CD 134) and 4-1BB (CD 137), may be added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of the CAR modified immune cells.

The person skilled in the art is able to select a suitable antigen binding region as described above, with which to redirect immune cells to be used according to the invention. In a specific embodiment, the immune cells used in the methods of the invention are redirected T cells, e.g., redirected cd8+ T cells or redirected cd4+ T cells, or redirected NK cells.

The inventors generated CD123 specific CARs with the single chain variable fragment (scFv) of clone CSL362, the CD8A hinge and transmembrane domain (Gen CD8A ENSG 00000153563), the intracellular signaling moiety 4-1BB (Gen TNFRSF9 ENSG 00000049249) and CD3 ζ (Gen CD247 ENSG 00000198821). As described in the examples, specific killing can be measured by determining the number of living cells via flow cytometry. Calculating specific killing according to the indicated formula: (1-number of live target cells co-cultured with CAR T cells/number of live target cells co-cultured with control cells) ×100.

Methods by which immune cells can be genetically modified to express recombinant antigen binding regions are well known in the art. The nucleic acid molecule encoding the antigen receptor may be introduced into the cell in the form of a vector or any other suitable nucleic acid construct. Carriers and their required components are well known in the art. Nucleic acid molecules encoding the antigen binding region may be produced using any method known in the art, for example, molecular cloning using PCR. The antigen binding region sequence may be modified using conventional methods, such as site-directed mutagenesis.

anti-CD 123 agents

anti-CD 123 agents are known in the art. For example, tutocarlizumab (CSL 362) is a humanized anti-CD 123 antibody to CSL Limited, whereas tutocarlizumab is a bispecific T cell adapter developed by macrogenetics (Uy et al blood 137:751-62). Chongqing Precision Biotech developed an anti-CD 123/anti-CAR-T. MB-102 is an anti-CD 123/anti-CD 28 costimulatory cytokine developed by Mustang Bio. IMG-532 is an anti-CD 123-ADC developed by Immunogen. UCART-123 is an anti-CD 123 CAR-T developed by Cellofectis. Vickers is an anti-CD 123/anti-CD 3 bispecific antibody developed by Xencor. Other anti-CD 123 CAR-T antibodies were also under development, including those of GeMoaB monoclonal antibodies, novartis (JEZ-567), hrain Biotechnology (HRAIN-004) and Hebei Senlang Biotechnology. Bispecific molecules under development include APVO-436 of Aptevo Therapeutics and JNJ-63709178 of Johnson & Johnson. All of these molecules may be used primarily in the methods and compositions of the present disclosure, provided that they can distinguish between two isoforms of a cell surface protein according to the present disclosure.

Tatuzumab (CSL 362), fu Tuozhu mab and veltuzumab are derivatives of the same parent antibody, with their CDRs having 98-100% sequence identity. Thus, knowing the epitope of CSL362, we can assume that valtuzumab and veltuzumab will bind to the same epitope.

In a specific embodiment, when the surface protein is CD123, the depleting agent that binds to the second isoform and does not bind to the first isoform as described above specifically binds to an epitope comprising amino acids T48, D49, E51, A56, D57, Y58, S59, M60, P61, A62, V63, N64, T82, R84, V85, A86, N87, P89, F90, S91 of SEQ ID NO. 1.

In a preferred embodiment, the anti-CD 123 agent comprises an antigen binding region comprising:

a) An antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCDR1 is SEQ ID NO:2 (DYYMK), VHCDR2 is SEQ ID NO:3 (DIIPSNGATFYNQKFKG) and VHCDR3 is SEQ ID NO:4 (SHLLRASWWAY); and

b) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 (ESSQSLLNSGNQKNYLT) or SEQ ID NO 6 (KSSQSLLNSGNQKNYL), VLCDR2 is SEQ ID NO 7 (WASTRES) and VLCDR3 is SEQ ID NO 8 (QNDYSYPYT).

It is further contemplated that the antigen binding region may be further screened or optimized for its binding properties as defined above. In particular, it is envisaged that the antigen binding region thereof may have 1, 2, 3, 4, 5, 6 or more changes in the amino acid sequence of 1, 2, 3, 4, 5, 6 CDRs of a monoclonal antibody provided herein, in particular SEQ ID NOs 3-8. It is contemplated that amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of CDR1, CDR2, CDR3, CDR4, CDR5 or CDR6 of the VJ or VDJ region of the light or heavy variable region of the antigen binding region may have insertions, deletions or substitutions of conserved or non-conserved amino acids. Such amino acids which may be substituted or constitute substitutions are disclosed above.

In some embodiments, the amino acid differences are conservative substitutions, i.e., one amino acid is substituted for another amino acid having similar chemical or physical properties (size, charge, or polarity), which substitutions typically do not adversely affect the biochemical, biophysical, and/or biological properties of the antibody. In particular, the substitution does not disrupt the interaction of the antibody with the CD123 antigen. The conservative substitution is 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, non-polar residues (M, L, I, V, C); and group 5-large aromatic residues (F, Y, W).

In another preferred embodiment, the anti-CD 123 agent comprises an antigen binding region that binds to the same epitope as an antigen binding region comprising:

In another preferred embodiment, the anti-CD 123 agent comprises an antigen binding region having the same epitope specificity as an antigen binding region comprising:

In another preferred embodiment, the anti-CD 123 agent comprises an antigen binding region that is immunologically indistinguishable from an antigen binding region comprising:

In a more specific embodiment, the first antigen binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 10, 12 and 14.

In another specific embodiment, the first antigen binding region comprises a heavy chain variable domain and/or a light chain variable domain that binds to the same epitope as the antigen binding region: the heavy chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 9, 11 and 13, and the light chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 10, 12 and 14.

In another specific embodiment, the first antigen binding region has the same epitope specificity as the antigen binding region: the antigen binding region comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 9, 11 and 13 and/or comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 10, 12 and 14.

In another specific embodiment, the first antigen binding region is immunologically indistinguishable from an antigen binding region comprising: comprising or consisting of any one of the amino acid sequences selected from SEQ ID NO. 9, 11 and 13 and/or comprising or consisting of any one of the amino acid sequences selected from SEQ ID NO. 10, 12 and 14.

Also part of the present disclosure is the first antigen binding region thereof having an amino acid sequence having at least 90%, e.g., at least 95%,96%,97%,98% or 99% identity to any of the amino acid sequences defined above, typically a first antigen binding region having at least equal or higher binding activity than the first antigen binding region consisting of the heavy and light chains: the heavy chain consists of any one of the amino acid sequences selected from SEQ ID NO. 9, 11 and 13, and the light chain consists of any one of the amino acid sequences selected from SEQ ID NO. 10, 12 and 14.

In a specific embodiment, the anti-CD 123 agent can be a bispecific CD123 antibody having at least one first binding specificity for CD123, such as one antigen binding region of anti-CD 123 described herein, and a second binding specificity for a second target epitope or target antigen. In particular, the bispecific antibody is a bifunctional fusion anti-CD 123 and as described in Kuo s.r., et al protein eng.des.sel.2012;25:561-569,137; hussaini m., blood.2013; 122:360; chicili G.R., sci.Transl.Med.2015;7:289ra82 and Al-Hussaini M.blood.2016; 127:122-131.

According to the present disclosure, the anti-CD 123 agent may be an immune cell carrying a CD 123-targeting antigen receptor, such as a CD 123-targeting CAR, comprising an antigen binding region as described above.

In certain embodiments, the immune cells (e.g., T cells) bearing the CD 123-targeted CAR recognize a second isoform of CD123 as expressed in a patient in need thereof, and do not recognize a first isoform of CD 123. In particular, the immune cells can specifically bind to an epitope comprising amino acids T48, D49, E51, A56, D57, Y58, S59, M60, P61, A62, V63, N64, T82, R84, V85, A86, N87, P89, F90, S91 of SEQ ID NO. 1.

In particular embodiments, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR comprising an antigen binding region, e.g., an scFv, comprising:

b) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7 and VLCDR3 is SEQ ID NO 8.

In another particular embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises an antigen binding region, e.g., an scFv, that comprises an antigen binding region that binds to the same epitope as an antigen binding region comprising:

In another particular embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that includes an antigen binding region, e.g., an scFv, that has the same epitope specificity as an antigen binding region comprising:

In another particular embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises an antigen binding region, e.g., an scFv, that is immunologically indistinguishable from an antigen binding region comprising:

In a more specific embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises the first antigen binding region, e.g., an scFv, that comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 9, 11, and 13, and a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 10, 12, and 14.

In another specific embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises the first antigen binding region, e.g., an scFv, that binds to the same epitope as an antigen binding region comprising: a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOS: 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOS: 10, 12 and 14.

In another specific embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises the first antigen binding region, e.g., an scFv, that has the same epitope specificity as an antigen binding region comprising the heavy chain variable domain and/or the light chain variable domain: the heavy chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 9, 11 and 13, and the light chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NO. 10, 12 and 14.

In another specific embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that comprises the first antigen binding region, e.g., an scFv, that is immunologically indistinguishable from an antigen binding region comprising the following heavy chain variable domains and/or light chain variable domains: the heavy chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NOS: 9, 11 and 13, and the light chain variable domain comprises or consists of any one of the amino acid sequences selected from SEQ ID NOS: 10, 12 and 14.

In a preferred embodiment, the anti-CD 123 agent may be an immune cell bearing a CAR targeting a specific isoform of CD123 as described in the examples, typically the anti-CD 123CAR comprises a hinge domain, a CD8a transmembrane domain and an intracellular signaling domain from the zeta chain of the CD3 molecule and a co-stimulatory domain 4-1BB, preferably the anti-CD 123CAR comprises or consists of the sequence SEQ ID NO: 15.

According to the present disclosure, the anti-CD 123 agent may be an immune cell carrying an antigen receptor targeting CD123, such as a CAR targeting a specific isoform of CD123, the antigen receptor comprising an antigen binding region as described above, and the immune cell does not express CD123 or express an isoform of CD123 that is not recognized by the CAR.

In particular embodiments, the anti-CD 123 agent can be an immune cell (e.g., a T cell) that carries a CAR that targets a particular isotype of CD123, comprising an antigen binding region, e.g., an scFv, comprising:

b) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7, VLCDR3 is SEQ ID NO 8;

and the immune cells do not express CD123 or express an isoform of CD123 that is not recognized by the CAR.

In a more specific embodiment, the anti-CD 123 agent can be an immune cell (e.g., a T cell) bearing a CAR comprising the first antigen binding region, e.g., an scFv, comprising a heavy chain variable domain selected from or consisting of any of the amino acid sequences of SEQ ID NOs 9, 11 and 13, and/or a light chain variable domain selected from or consisting of any of the amino acid sequences of SEQ ID NOs 9, 11 and 13, comprising any of the amino acid sequences of SEQ ID NOs 10, 12 and 14, or consisting of any of the amino acid sequences of SEQ ID NOs 10, 12 and 14, and expressing an isoform of CD123 not recognized by the CAR.

In a more preferred embodiment, the anti-CD 123 agent is a CSL362 antibody as described in the examples.

In another preferred embodiment, the anti-CD 123 agent is an MIRG123 antibody as described in the examples.

In another preferred embodiment, the anti-CD 123 agent may be an immune cell bearing a CAR that targets a particular isoform of CD123 as described in the examples, typically the anti-CD 123CAR comprises a hinge domain, a CD8a transmembrane domain and an intracellular signaling domain from the zeta chain of the CD3 molecule, and a co-stimulatory domain 4-1BB, preferably the anti-CD 123CAR comprises or consists of the sequence SEQ ID NO: 15.

In particular, the disclosure also relates to depleting an anti-CD 123 agent (e.g., CAR cell composition or antibody) as described above comprising a first antigen binding region or a second antigen binding region for selectively depleting host cells or metastatic cells, respectively, in a subject in need thereof.

Cells or cell populations expressing a first isoform of a surface protein

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

The cell or cell population is particularly useful in the medical treatment of patients expressing a second isoform of the cell surface protein.

In a specific embodiment, the cells (e.g., hematopoietic cells) (e.g., hematopoietic stem cells) encoding or expressing the first isoform that are not recognized by a depleting agent are particularly useful in medical treatment that restores normal hematopoiesis following immunotherapy (such as adoptive cell transfer in a patient expressing the second isoform), particularly wherein the treatment comprises administering a therapeutically effective amount of the hematopoietic cells expressing the first isoform in combination with a therapeutically effective amount of a depleting agent that targets the second isoform. In particular, the hematopoietic cells, preferably hematopoietic stem cells, are administered after the depleting agent. In another specific embodiment, the hematopoietic cells, preferably hematopoietic stem cells, may be administered prior to or concurrently with the depleting agent.

In another specific embodiment, the cells expressing the first isoform, which are specifically recognized by a depleting agent that does not bind to the second isoform, are particularly useful in the medical treatment of patients expressing the second isoform, particularly for avoiding serious side effects (safety switches) associated with transplanted cells, wherein said treatment comprises administering a therapeutically effective amount of a depleting agent targeting the first isoform. In particular, the hematopoietic cells, preferably CAR-bearing immune cells, are administered prior to the depleting agent.

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

In a specific embodiment, the cell is a hematopoietic cell. Hematopoietic cells include immune cells including lymphocytes such as B cells and T cells, natural killer cells, bone marrow cells such as monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes, dendritic Cells (DCs), and plasmacytoid dendritic cells (pdcs).

In a preferred embodiment, the immune cell is a T cell. In another preferred embodiment, the immune cells are primary T cells. As used herein, the term "T cell" includes cells that carry a T Cell Receptor (TCR) or cells derived from a T cell that carries a TCR. T cells according to the present disclosure may be selected from the group consisting of inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, tumor-infiltrating lymphocytes, or helper T lymphocytes (including type 1 and type 2 helper T cells and Th17 helper cells). In another embodiment, the cells may be derived from the group consisting of cd4+ T lymphocytes and cd8+ T lymphocytes or non-classical T cells such as MR1 restricted T cells, MAIT cells, NKT cells, γδ T cells or congenital T cells.

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

In another preferred embodiment, the hematopoietic cells are hematopoietic stem cells. The stem cells may be adult stem cells, embryonic stem cells, more particularly non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34 ⁺ And (3) cells. Hematopoietic stem cells may differentiate from iPS cells, or may be harvested from mobilized or non-mobilized peripheral blood.

In certain embodiments, the cells are allogeneic cells, which refer to donor-derived cells that exhibit similar HLA, at least for some HLA's, to the person receiving the cells. The donor may be a related or unrelated person. In certain embodiments, the cell is an autologous cell, which refers to a cell derived from the same person that is receiving the cell.

The cells may be derived from healthy donors or patients, in particular from patients diagnosed with cancer or autoimmune diseases or from patients diagnosed with infection. Hematopoietic cells may be extracted from blood, or may be derived from stem cells.

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

The present disclosure further relates to a cell composition or cell population for use in therapy as disclosed herein.

Surface proteins

According to the present disclosure, the cells express a first isoform of a surface protein. Surface proteins according to the present disclosure are proteins that attach to cell membranes. The surface protein is also referred to herein as a cell surface antigen. In particular, in hematopoietic cells, the surface protein may be a cell surface marker selected from the group consisting of CD123, CD33, CD7, CD117, CD45, CD135, CLEC12a, CD44 and CD 70.

In a specific embodiment, the surface protein is CD123, which is encoded by the interleukin-3 receptor alpha subunit (IL 3 RA) gene (Gene ID: 3563). IL3RA is a specific subunit of heterodimeric cytokine receptors consisting of a ligand-specific alpha subunit and a signal transduction beta subunit shared by the receptors for interleukin 3 (IL 3), colony stimulating factor 2 (CSF 2/GM-CSF) and interleukin 5 (IL 5). IL-3 is a multipotent cytokine that promotes the development of hematopoietic progenitor cells into erythroid, myeloid and lymphoid cells. Splice transcript variants encoding different proteins have been found, type IL3RA 1 (NCBI reference number: NP-002174.1, 2021, day 1, month 10) (SEQ ID NO: 1) and type 2 (NCBI reference number: NP-001254642.1, 2021, day 1, month 10).

Additional variants or isoforms of CD123 may also be combined in the methods and compositions of the present disclosure according to the present disclosure. Such isoforms may, for example, include double mutants. Such isoforms may also include, for example, single mutants and double mutants. The methods and compositions of the present disclosure can also be combined with cells carrying a CD123 knockout, e.g., a permanent knockout or a temporary knockout (e.g., by CRISPRoff). The methods and compositions of the present disclosure may also be used to deplete myeloid cells in solid tumors to enhance tumor responses.

The methods and compositions of the present disclosure may also be combined with cellular compositions, particularly when the surface protein is CD123 with KO of other targets, i.e., CD33 KO, CD7 KO, CLEC12A, CD44 KO, and combinations thereof.

The methods and compositions of the present disclosure may also comprise cells expressing CD123 (CD 123 variants) and other surface protein variants such as CD33 variants, CD7 variants, and any combination thereof of the first isotype.

Polymorphism of surface protein isoforms

Cells expressing a first isoform of a surface protein according to the present disclosure comprise genomic DNA having at least one polymorphic allele in a nucleic acid encoding the surface protein. In particular, the polymorphism induces at least one mutation in a region of the surface protein involved in binding of a specific agent, as compared to the second isoform.

In a preferred embodiment, the first isoform of the surface protein retains functionality and retains the ability to perform the same function within the cell as the corresponding wild-type isoform without significant damage. In particular, the first isoform of CD123 has one or more of the following properties similar to the second isoform of CD 123:

expression on the surface of the cell, the expression on the surface of the cell,

the holding structure is a structure of a holding device,

-the binding of IL-3 to the substrate,

intracellular signaling capacity (e.g. STAT5 phosphorylation/signaling), in response to IL-3 inducing cell proliferation of cell lines, differentiation of humanized mice into multiple lineages/cell types, pDC function of pDC isolated from humanized mice, as measured in the functional assay described in the experimental section.

The first isoform of CD123 may have hematopoietic stem cell transplantation capability or edited hematopoietic stem cell colony forming capability similar to the second isoform of CD 123. In more detail, proliferation of TF-1 cells can be induced by IL-3, and this IL-3 dependent induction of proliferation is blocked by CSL362 or MIRG 123. Introduction of CD123 variant isoforms into TF-1 cells by a suitable gene editing method such as HDR allows to determine the proliferation rate of TF-1 cells in the case of each CD123 variant isoform and to directly compare it with IL-3 dependent proliferation of wild type TF-1 cells. The CD123 variant isoforms that result in the same proliferation rate as wild-type CD123 are considered functionally equivalent.

In particular, when the surface protein is CD123 (IL 3 RA), the first isoform of CD123 remains functional and is activated by IL-3, which is a cytokine produced by antigen-activated T cells, and can induce IL-3 signaling.

The polymorphism is preferably within a nucleic acid sequence encoding a surface protein region involved in the binding of the first agent, said surface protein region preferably being located in an extracellular part of the surface protein, in particular in a solvent-exposed secondary structural element. More specifically; the polymorphism is within a nucleic acid sequence encoding at least one specific amino acid residue involved in binding of the first agent. The polymorphism may be a mutation, such as a deletion, substitution and/or insertion of at least 1,2,3,4,5,6,7,8,9, 10, 12, 15 or 20 nucleotides. In a specific embodiment, the polymorphism is a single nucleotide polymorphism.

Sequence differences between the two isoforms may also be introduced genetically. Furthermore, the sequence differences here are preferably within the nucleic acid sequence encoding a surface protein region involved in the binding of the first agent, which surface protein region is preferably located in the extracellular part of the surface protein, in particular in the solvent-exposed secondary structural element. More specifically; the sequence differences are within a nucleic acid sequence encoding at least one specific amino acid residue involved in the binding of the first agent. The sequence differences may be mutations, such as deletions, substitutions and/or insertions of at least 1,2,3,4,5,6,7,8,9, 10, 12, 15 or 20 nucleotides. In a specific embodiment, the sequence difference is a single point mutation.

The inventors identified natural polymorphisms in the nucleic acid sequence encoding the amino acid residues involved in the binding of anti-CD 123 agents as described above, in particular polymorphisms that induce substitution of residues E51, S59 or R84, preferably E51 or S59, relative to the CD123 sequence (SEQ ID NO: 1). Thus, the present disclosure relates to cells expressing isoforms of CD123, wherein the isoforms have a substitution in at least one amino acid residue selected from E51, S59 and R84, preferably E51 or S59, of CD123 (SEQ ID NO: 1).

Natural polymorphism

In a specific embodiment, the cell according to the present disclosure is selected from a subject comprising natural genomic DNA having at least one natural polymorphic allele, preferably a Single Nucleotide Polymorphism (SNP) in a nucleic acid encoding the isoform.

In a specific embodiment, when the surface protein is CD123, the cell is selected from a subject comprising native genomic DNA having at least one native polymorphic allele, in particular a SNP, in a nucleic acid sequence encoding a CD123 region involved in the binding of an anti-CD 123 agent, said CD123 region preferably being located in an extracellular portion of said surface protein, more preferably in a solvent-exposed secondary structural element. More specifically, the polymorphic allele is within a nucleic acid sequence encoding residues E51, S59 and/or R84 of SEQ ID NO. 1, preferably E51 or S59 of SEQ ID NO. 1. In particular, the polymorphic allele causes a substitution of at least one amino acid residue selected from positions E51, S59 and/or R84 of SEQ ID NO. 1, preferably positions E51 or S59 of SEQ ID NO. 1. Preferably, amino acid residue E51 is substituted with an amino acid selected from the group consisting of K, N, T, S, Q, R, M, G and a, preferably K or T. Also preferably, amino residue S59 is selected from the group consisting of I, G, P, E, L, T, K, F, R and Y; preferably an amino acid substitution in the group consisting of P or E. Also preferably, amino acid residue R84 is substituted with an amino acid selected from the group consisting of T, K, S, Q, N, E, H, L and a.

Gene editing method

In another specific embodiment, the cells expressing the first isoform according to the disclosure are obtained by genetic editing, preferably by altering the sequence encoding the surface protein in the patient's native genomic DNA.

Cells can be genetically engineered by: introducing a gene editing enzyme into the cell to induce the polymorphism, thereby resulting in insertion, deletion and/or substitution of an amino acid of the surface protein. The gene editing enzyme targets a nucleic acid sequence encoding a region of the surface protein involved in the binding of the first agent as described above, which is designated herein as a target sequence. In particular, when the surface protein is CD123, the gene-editing enzyme preferably targets the nucleic acid encoding at least one residue at position E51, S59 and/or R84 of SEQ ID NO. 1 in such a way that at least one residue at position E51, S59 and/or R84 of SEQ ID NO. 1 is substituted. Preferably, amino residue E51 is substituted with an amino acid selected from the group consisting of K, N, T, S, Q, R, M, G and a, preferably K or T. Also preferably, amino acid residue S59 is selected from the group consisting of I, G, P, E, L, T, K, F, R and Y; preferably an amino acid substitution in the group consisting of P or E. Also preferably, amino acid residue R84 is substituted with an amino acid selected from the group consisting of T, K, S, Q, N, E, H, L and a.

The gene-editing enzyme may be a sequence-specific nuclease, a base or a primer editor.

The term "nuclease" refers to a wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of a nucleic acid (DNA or RNA) molecule, preferably a phosphodiester bond between nucleotides of a DNA molecule. By "cleavage" is meant a double strand break or single strand break event.

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 homing endonucleases, TAL nucleases (TALENs), zinc Finger Nucleases (ZFNs), or RNA/DNA guided endonucleases, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems and Argonaute (Review in Li et al Nature Signal transduction and targeted Therapy,5,2020; guha et al Computational and Structural Biotechnology Journal,2017,15,146-160).

According to the present disclosure, the nuclease produces DNA cleavage within a target sequence encoding a region of a surface protein involved in the binding of the first agent as described above. In a specific embodiment, the present inventors use a CRISPR system to induce cleavage within a target sequence encoding a region of a surface protein identified by a first agent as described above.

"target sequence" is intended to target a portion encoding: the sequence of the surface protein region involved in the binding of the first agent and/or the sequence adjacent to said surface protein region involved in the binding of the first agent as described above, in particular the sequence of at least one (one or two) up to 50 nucleotides adjacent to said surface protein region involved in the binding of the first agent, preferably 20, 15, 10,9,8,7,6 or 5 nucleotides adjacent to said repressor binding site.

The CRISPR system involves two or more components, a Cas protein (CRISPR-associated protein) and a single guide RNA. Cas protein is a DNA endonuclease that uses a guide RNA sequence as a guide to recognize and create a DNA double strand cutter that is complementary to a single guide RNA sequence. The Cas protein contains two active cleavage sites, namely HNH nuclease domain and RuvC-like nuclease domain.

Cas protein also means an engineered endonuclease or homolog of Cas9 that is capable of cleaving a target nucleic acid sequence. In particular embodiments, the Cas protein may induce cleavage in a nucleic acid target sequence, which may correspond to a double-strand break or a single-strand break. Cas protein variants may be Cas endonucleases obtained by protein engineering or random mutagenesis that do not occur naturally in nature. Cas proteins may be one type of Cas protein known in the art. Non-limiting examples of Cas proteins include Casl, caslB, cas, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csnl and Csxl 2), caslO, csyl, csy, csy3, csel, cse2, cscl, csc2, csa5, csn2, csm3, csm4, csm5, cmrl, cmr3, cmr4, cmr5, cnrr6, csbl, csb2, csb3, csxl7, csxM, csx lO, cs l6, csaX, csx3, cs l, csxl5, csfl, csf2, csO, csf4, homologs, orthologs, or modified versions thereof. Preferably, the Cas protein is a streptococcus pyogenes Cas9 protein.

Cas is contacted with a guide RNA (gRNA) designed to comprise a complement of a target sequence to specifically induce DNA cleavage within the complement of the target sequence, particularly a portion of the target sequence encoding a surface protein region recognized by an agent as described above in accordance with the present disclosure.

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

In particular, gRNA refers to RNA comprising transactivation crRNA (tracrRNA) and crRNA. Preferably, the guide RNA corresponds to crRNA and tracrRNA that can be used alone or fused together. The complementary sequence paired with the target sequence recruits Cas to bind DNA at the target sequence and cleave DNA at the target sequence.

According to the present disclosure, crrnas are engineered to comprise a complement of a portion of a target sequence encoding a surface protein region recognized by an agent as described above, such that they are capable of targeting the region.

In a specific embodiment, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 nucleotides, complementary to the target sequence. As used herein, the term "complementary sequence" refers to a portion of a sequence of a polynucleotide (e.g., a portion of crRNA or tracRNA) that can hybridize to another portion of the polynucleotide under standard low stringency conditions. Preferably, the sequences are complementary to each other according to complementarity between two nucleic acid strands that depend upon Watson-Crick base pairing between the strands, i.e., inherent base pairing between adenine and thymine (A-T) nucleotides and between guanine and cytosine (G-C) nucleotides. In view of the present disclosure, the gRNA may be designed by any method known to those skilled in the art.

According to the present disclosure, the target sequence encodes a surface protein region involved in the binding of the first agent, said surface protein region preferably being located in the extracellular portion of the surface protein, more preferably in the extracellular loop compared to the second isoform, again more preferably comprising amino acid residues involved in the binding of the agent.

In a preferred embodiment, when the surface protein is CD123, the target sequence encodes a CD123 region that is involved in binding of a first agent, such as an anti-CD 123 agent as described above. Preferably, the target sequence encodes position E51, S59 and/or R84 of SEQ ID NO. 1, preferably at least one residue at E51 or S59 of SEQ ID NO. 1.

In a specific embodiment, the gRNA can target a sequence encoding a CD123 region involved in binding of the first agent, and particularly comprises one of the sequences described in table 1 (the gRNA sequence).

TABLE 1 gRNA sequence

In other words, when the surface protein is CD123, the nucleic acid construct may preferably comprise:

a gRNA sequence of SEQ ID NO. 16 or 17, which targets a sequence encoding a substitution E51K relative to SEQ ID NO. 1,

-a gRNA sequence selected from the group consisting of SEQ ID NOs 18 to 20, which targets a sequence encoding a substitution S59P relative to SEQ ID No. 1, or

-a gRNA sequence selected from the group consisting of SEQ ID NOs 21 to 23, which targets a sequence encoding a substitution R84E relative to SEQ ID No. 1.

DNA strand breaks introduced by nucleases according to the present disclosure can lead to DNA mutations at the cleavage site through non-homologous end joining (NHEJ), which typically lead to small insertions and/or deletions or substitutions of DNA around the cleavage site through Homology Directed Repair (HDR).

In a preferred embodiment, the polymorphism within the nucleic acid encoding the surface protein isoform is induced by HDR repair after DNA cleavage and introduction of an exogenous nucleotide sequence (referred to herein as an HDR template).

The HDR template comprises first and second portions of sequences homologous to regions 5 'and 3', respectively, of the target sequence, and an exogenous sequence comprising a polymorphism. After cleavage of the target sequence, a homologous recombination event is effected between the genome comprising the target sequence and the HDR template, and the genomic sequence containing the target sequence is replaced with an exogenous sequence.

Preferably, homologous sequences of at least 20bp, preferably greater than 50bp, and more preferably less than 200bp are used. In fact, the common DNA homolog is located in the flanking region upstream and downstream of the cleavage site, and the foreign sequence to be introduced should be located between the two arms.

In a preferred embodiment, the cells according to the present disclosure are genetically engineered by introducing into the cells the site-specific nuclease and HDR template targeting the sequence encoding the surface protein region recognized by the first agent as described above.

In another specific embodiment, the gene editing enzyme is, for example, komor et al, nature533,420-424, doi:10.1038/aperture 17946 neutralizing Rees HA, liu DR.Nat Rev Genet.2018Dec;19 (12) DNA base editor as described in 770-788, or as in Anzalone AV.Etal. Nature,2019,576:149-157,Matsoukas IG.Front Genet.2020;11:528 and Kantor A.et al int.J.mol.Sci.2020,21 (6240). A base editor or primer editor may be used to introduce mutations at specific sites in the target sequence.

According to the present disclosure, a base editor or primer editor creates a mutation within a target sequence by sequence specific targeting of a sequence encoding a region of a surface protein involved in the binding of a first agent.

In particular, the base editor or primer editor is a CRISPR base editor or primer editor. The CRISPR base or primer editor comprises dead Cas protein (dCas) as a catalytically inactive sequence-specific nuclease. dCas refers to a modified Cas nuclease that lacks endonuclease activity. Nuclease activity in dCas protein can be inhibited or prevented by one or more mutations and/or one or more deletions in HNH and/or RuvC-like catalytic domains of Cas protein. The resulting dCas protein lacks nuclease activity, but binds to guide RNA (gRNA) -DNA complexes with high specificity and efficiency for specific target sequences. In particular embodiments, the dead Cas can be a Cas nickase in which one catalytic domain of Cas is inhibited or prevented.

The base editor is contacted with a guide RNA (gRNA) designed to contain a complementary sequence to a target nucleic acid sequence to specifically bind the target sequence, as described above.

In view of the present disclosure, the gRNA may be designed by any method known to those skilled in the art. In a specific embodiment, the gRNA can target a sequence encoding a region of a surface protein identified by the first agent as described above, and particularly when the surface protein is CD123, it comprises one of the sequences described in table 1 (a gRNA sequence).

As a non-limiting example, the base editor is a nucleotide deaminase domain fused to a dead Cas protein, particularly a Cas nickase. The nucleotide deaminase may be an adenosine deaminase or a cytidine deaminase. The nucleotide deaminase may be a natural or engineered deaminase.

In a specific embodiment, the base editor may be selected from the group consisting of, by way of non-limiting example: BE1, BE2, BE3, BE4, HF-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-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRIPS-X, ABE 7.9.9, ABE7.10 x xABE, ABESa, ABEmax, ABE e, VQR-ABE, VRER-ABE, and SaKKH-ABE.

The primer editor consists of a fusion of a catalytically inactive sequence-specific nuclease, in particular a Cas endonuclease and a catalytically active engineered Reverse Transcriptase (RT), as described above. The fusion protein is used in combination with a primer editing guide RNA (pegRNA) comprising a complementary sequence to the target sequence as described above, in particular when the surface protein is CD123, comprising one of the sequences described in table 1 and further comprising a further sequence comprising a sequence that binds to a primer binding site region on DNA. In a specific embodiment, the reverse transcriptase is a Maloney murine leukemia Virus RT enzyme and variants thereof. The primer editor may be selected from the group consisting of PE1, PE2, PE3, and PE3b as non-limiting examples.

CAR

For use in adoptive cell transfer therapy, the cells expressing the first isoform according to the present disclosure may be modified to exhibit the desired specificity and enhanced functionality. In particular, cells can be modified to target specific targets. In a specific embodiment, the cell may express a recombinant antigen binding region on its cell surface, also designated as an antigen receptor, as described above. In a specific embodiment, the recombinant antigen receptor is a Chimeric Antigen Receptor (CAR). According to the present disclosure, the immune cells expressing a first isoform of a cell surface protein and a CAR can be specifically depleted by administering a therapeutically effective amount of an agent comprising a second antigen-binding region that specifically binds the first isoform of the surface protein but not the second isoform, thereby avoiding eventual serious side effects due to transplantation of the immune cells.

In a specific embodiment, the immune cells are redirected against a cancer antigen. "cancer antigen" means any antigen associated with cancer (i.e., a molecule capable of inducing an immune response). An antigen as defined herein may be any type of molecule that induces an immune response, e.g. it may be a polysaccharide or a lipid, but most preferably it is a peptide (or protein). The human cancer antigen may be human or humanized. The cancer antigen may be a tumor-specific antigen, which means an antigen not found in healthy cells. Tumor-specific antigens are usually caused by mutations, in particular frame-shifting mutations that result in completely new amino acid sequences not found in the healthy human proteome.

Cancer antigens also include tumor-associated antigens, which are antigens whose expression or production is associated with tumor cells (but are not limited to). Examples of tumor-associated antigens include, for example, her2, prostate Stem Cell Antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calomelanin, MUC-1, epithelial membrane protein (EMA), epithelial Tumor Antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CDl17, CD123, chromogranin, cytokeratin, myotonin, glial Fibrillary Acidic Protein (GFAP), specific cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-a (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, myo-specific actin, neurofilament, neuronal Specific Enolase (NSE), placental alkaline phosphatase, synaptosin, thyroglobulin, thyroid transcription factor-1, the dimeric form of pyruvate kinase isozymes M2 (tumor M2-PK), CD19, CD22, CD33, CD123, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl 7), mesothelin, PAP (prostaacid phosphatase), prostaglandin, TARP (T cell receptor gamma alternate reading frame protein), trp-p8, STEAP1 (six transmembrane epithelial antigen 1) abnormal ras protein or abnormal p53 protein. In another specific embodiment, the tumor-associated antigen or tumor-specific antigen is integrin ανβ3 (CD 61), lactation hormone, K-Ras (V-Ki-Ras 2 Kirsten rat sarcoma viral oncogene) or Ral-B.

In a specific embodiment, for use in adoptive cell transfer therapy, preferably for treating a malignant hematopoietic disorder, such as Acute Myeloid Leukemia (AML) or B-acute lymphoblastic leukemia (B-ALL), an immune cell according to the present disclosure expresses a recombinant antigen binding region, such as a CD 123-targeted CAR. Cells expressing the first isoform and expressing a CAR (e.g., CAR-CD 123) can be further specifically depleted by administering an depleting agent comprising a second antigen binding region that specifically binds to the first isoform of CD123 but does not bind to the second isoform of CD123, thereby avoiding eventual serious side effects such as graft versus host disease due to transplantation.

In a particular embodiment, the immune cell (e.g., T cell) expressing the first isotype carries a CD 123-targeting CAR comprising an antigen binding region, e.g., scFv, comprising an epitope that specifically binds CD123 located within the N-terminal domain or comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1.

In particular, the immune cells (e.g., T cells) expressing the first isotype carry a CD 123-targeted CAR comprising an antigen binding region, e.g., scFv, comprising:

b) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7, VLCDR3 is SEQ ID NO 8,

more preferably comprises the following antigen binding regions: the antigen binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOS: 10, 12 and 14.

In vitro method for preparing cells expressing a first isoform

A cell expressing a first isoform according to the present disclosure may be genetically engineered by introducing into the cell a nucleic acid construct (e.g., mRNA) encoding at least one gene-editing enzyme or ribonucleoprotein complex comprising a gene-editing enzyme and/or HDR template as described above. The cell may also be genetically engineered by further introducing into the cell a nucleic acid construct encoding a CAR as described above. In particular, the method is an ex vivo method performed on a culture of cells.

The term "nucleic acid construct" as used herein refers to an artificial nucleic acid molecule produced by using recombinant DNA techniques. A nucleic acid construct is a single-or double-stranded nucleic acid molecule that has been modified to include fragments of a nucleic acid sequence that are combined and juxtaposed in a manner that would otherwise not exist in nature. A nucleic acid construct is typically a "vector", i.e., a nucleic acid molecule that is used to deliver exogenously produced DNA into a host cell.

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

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

Examples of suitable vectors include, but are not limited to, recombinant integrating or non-integrating viral vectors, and vectors derived from recombinant phage DNA, plasmid DNA, or cosmid DNA. Preferably, the vector is a recombinant integrative or non-integrative viral vector. Examples of recombinant viral vectors include, but are not limited to, vectors derived from herpes viruses, retroviruses, lentiviruses, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma viruses.

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

In a preferred embodiment, the nucleic acid construct comprises a CRISPR/Cas nuclease capable of targeting a nucleic acid sequence encoding a surface protein region involved in binding to a consuming agent. When the surface protein is CD123, the nucleic acid construct preferably comprises:

Since the nucleic acid constructs, preferably expression vectors, encoding said gene editing enzymes, such as guide RNA and/or Cas proteins, base editors and primer editors as described above are introduced into the cells, said gene editing enzymes, preferably guide RNA and/or Cas proteins, base editors or primer editors as described above can be synthesized in situ in the cells. Alternatively, the gene-editing enzyme, such as guide RNA and/or Cas protein, base editor or primer editor, may be produced extracellularly and then introduced into the cell.

The nucleic acid construct or expression vector may be introduced into the cell by any method known in the art, and as non-limiting examples, the method includes stable transduction methods in which the nucleic acid construct or expression vector is integrated into the genome of the cell, transient transfection methods in which the nucleic acid construct or expression vector is not integrated into the genome of the cell, and virus-mediated methods. For example, transient transformation methods include, for example, microinjection, electroporation, cell extrusion, or particle bombardment.

Pharmaceutical composition

In another aspect, the present disclosure also provides a pharmaceutical composition comprising a cell or cell population of a first isoform expressing a cell surface protein as described above, and one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

In a specific embodiment, the cell expressing the first isoform is an immune cell, preferably a T cell, more preferably a primary T cell, carrying a Chimeric Antigen Receptor (CAR), preferably a CAR targeting a second isoform of CD123 expressed by cells of the patient as described above.

In another specific embodiment, the cell expressing the first isoform of a cell surface protein is a hematopoietic stem cell.

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

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

Optionally, the composition comprising cells expressing the first isoform may be frozen for storage at any temperature suitable for cell storage. For example, the cells may be frozen at-20 ℃, -80 ℃ or any other suitable temperature. The cryogenically frozen cells may be stored in a suitable container and ready for storage to reduce the risk of cell damage and maximize the likelihood of cell survival after thawing. Alternatively, the cells may also be kept at room temperature or in a refrigerated state, for example at about 4 ℃.

Therapeutic use

The present disclosure relates to a cell or cell population expressing a first isotype as described above for use as a medicament, in particular for use in immunotherapy, such as adoptive cell transfer therapy of a patient.

According to the present disclosure, the cell or cell population (e.g., hematopoietic cells) expressing a first isoform of a cell surface protein (e.g., a first isoform of CD 123) as described above is used in medical treatment of a patient in need thereof, wherein the medical treatment comprises administering a therapeutically effective amount of the cell or cell population expressing the first isoform of a cell surface protein in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or antibodies) that specifically binds to a second isoform or first isoform of the cell surface protein (e.g., CD 123) to specifically deplete patient cells or transplanted cells, respectively.

As used herein, the term "combination" or "combination therapy" means that two (or more) different therapies are delivered to a subject during a period of time in which the subject has a disorder, e.g., the two or more therapies are delivered after the subject is diagnosed with the disorder and before the disorder has been cured or eliminated or the therapy has otherwise ceased. In some embodiments, when delivery of the second treatment is initiated, delivery of one treatment is still occurring, such that there is overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery. In other embodiments, the delivery of one therapy ends before the delivery of another therapy begins. The delivery may be such that when the second treatment is delivered, the effect of the first treatment delivered is still detectable. In one embodiment, the depleting agent that binds the second isoform or the first isoform of the cell surface protein is administered at a dose and/or dosing regimen described herein, and the cells expressing the first isoform are administered at a dose and/or dosing regimen described herein. In some embodiments, "in combination with …" is not intended to mean that a depleting agent that targets a second isoform of a cell surface protein (e.g., CAR cells or antibodies that recognize the second isoform of CD 123) or a first isoform and a composition of cells expressing the first isoform of the cell surface protein (e.g., the first isoform of CD 123) must be administered and/or formulated together for delivery, but such delivery methods are within the scope of the present disclosure. The depleting agent (e.g., CAR cells or antibodies that target the second isoform of CD 123) can be administered concurrently with a dose of hematopoietic stem cells that express the first isoform of cell surface protein (e.g., the first isoform of CD 123), or after a dose of hematopoietic stem cells that express the first isoform of cell surface protein (e.g., the first isoform of CD 123) (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 after a dose of hematopoietic stem cells that express the first isoform of cell surface protein (e.g., the first isoform of CD 123) (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 4 weeks, 3 weeks, 4 weeks, 8 weeks, 6 weeks, 8 weeks, 12 weeks). In certain embodiments, each agent will be administered at a dosage and/or schedule determined for that particular agent.

Adoptive cell transfer therapies according to the present disclosure are useful for treating patients diagnosed with cancer, autoimmune diseases, infectious diseases, diseases requiring Hematopoietic Stem Cell Transplantation (HSCT), for the prevention of organ rejection, tumor conditioning regimens, tumor maintenance therapy, minimal residual disease, prevention of recurrence.

The disclosure also relates to the use of a cell of a first isoform expressing a cell surface protein as described above for the manufacture of a medicament for adoptive transfer cell therapy of a patient.

As used herein, the term "subject" or "patient" refers to an animal, preferably a mammal, including a human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat, that can elicit an immune response. More preferably, the subject is a human, including adults, children and humans at the prenatal stage.

As used herein, the term "treatment", "treatment" or "treatment" refers to any action intended to improve the health of a patient, such as the treatment, prevention, prophylactic treatment and delay of a disease. In certain embodiments, such terms refer to the amelioration or eradication of a disease or symptom associated with a disease. In other embodiments, the term refers to minimizing the spread or exacerbation of a disease caused by the administration of one or more therapeutic agents to a subject suffering from such a disease.

Cancers that may be treated include non-vascularized or not substantially vascularized tumors, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas, including relapsed and treatment-related tumors, e.g., secondary malignant tumors following Hematopoietic Stem Cell Transplantation (HSCT)), or may include solid tumors.

The term "autoimmune disease" as used herein is defined as a condition caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens.

Infectious diseases are diseases caused by pathogenic microorganisms such as bacteria, viruses, parasites or fungi. In particular embodiments, infection according to the present disclosure occurs in immunosuppressed patients, such as patients following HSCT or patients who have received solid organ transplantation.

In a preferred embodiment, the present disclosure relates to a cell of a first isoform expressing CD123 as described above for use in hematological cancer, preferably leukemia or lymphoproliferative disease. The leukemia may be selected from the group consisting of Acute Myeloid Leukemia (AML), myelodysplastic syndrome (MDS), blast plasmacytoid dendritic cell tumor (BPDCN), chronic myeloid leukemia, chronic Lymphoid Leukemia (CLL), acute mixed leukemia, hairy cell leukemia, interleukin-3 receptor subunit alpha positive leukemia, B-acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), hodgkin Lymphoma (HL), systemic mastocytosis, preferably MDS, preferably AML or BPDCN.

In a specific embodiment, the cell or population of cells (e.g., hematopoietic cells) expressing a first isoform of a cell surface protein (e.g., a first isoform of CD 123) as described above can be used to treat a solid tumor, particularly for selectively depleting myeloid cells in a solid tumor in a patient, such that an immunotherapeutic agent, such as an immune checkpoint inhibitor, CAR T cells or TIL, enters the tumor, as the myeloid cells in the tumor can be immunosuppressive. In this case, the cell or population of cells (e.g., hematopoietic cells) expressing the first isoform of a cell surface protein (e.g., CD123 first isoform) as described above may be used to supplement the hematopoietic system that may be affected by therapies aimed at depleting myeloid cells in solid tumors.

In another specific embodiment, the cell or population of cells (e.g., hematopoietic cells) expressing a first isoform of a cell surface protein (e.g., a first isoform of CD 123) as described above can be used to treat an autoimmune disease, such as lupus, multiple sclerosis, scleroderma.

The disclosure also relates to a depleting agent (e.g., CAR cell composition or antibody) comprising a first antigen binding region or a second antigen binding region for selectively depleting host cells or metastatic cells, respectively, in a subject in need thereof.

Methods for specifically depleting patient cells, but not engrafted cells.

According to the present disclosure, the cell or cell population (e.g., hematopoietic cells) expressing a first isoform of a cell surface protein (e.g., CD123 first isoform) as described above is used in medical treatment of a patient in need thereof, wherein the medical treatment comprises administering a therapeutically effective amount of the cell or cell population expressing the first isoform of a cell surface protein in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or antibodies) that specifically binds a second isoform of a cell surface protein (e.g., CD 123).

Indeed, during immunotherapy, an immune depleting agent, such as CAR-expressing immune cells that are directed to a tumor antigen (e.g., CD 123), 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 may have serious adverse effects on patients by altering hematopoiesis. To restore hematopoiesis in a patient, hematopoietic cells may then be transplanted into the patient. However, these cells need to be resistant to the agent (e.g., the depleting agent of CD123 expressing cells) so as not to be targeted by it.

Thus, alternatively, in accordance with the present disclosure, depleting agents comprising a first antigen binding region that specifically binds a second isoform of a cell surface protein (e.g., CD 123) may be administered to ablate specific patient cells that express the second isoform of a cell surface protein (e.g., the second isoform of CD 123) instead of transplanted cells that express the first isoform (e.g., the first isoform of CD 123). Selective depletion of patient cells rather than transplanted cells allows for reconstitution of a patient with a healthy hematopoietic system that will no longer be consumed by the immune depleting agent. Thus, according to the therapeutic use of the present invention, the patient has a functional immune system, rather than undergoing a long period of immunosuppression. The use of cells according to the present disclosure eliminates infection as a major complication of current HSC transplantation.

In another embodiment, the disclosure relates to a method for adoptive cell transfer therapy, preferably for hematopoietic stem cell transplantation to restore normal hematopoiesis in a patient with cells of a second isoform that express a surface protein (e.g., CD 123), the method comprising:

(i) Administering an effective amount of cells (e.g., hematopoietic stem cells) that express a first isoform of the surface protein (e.g., CD 123), wherein the cells that express the first isoform (e.g., CD 123) comprise genomic DNA having at least one polymorphic allele, preferably a Single Nucleotide Polymorphism (SNP) allele, or a genetically engineered allele in a nucleic acid encoding the first isoform, and wherein the polymorphism is not present in the genome of a patient having cells of the second isoform or pharmaceutical composition thereof that express the surface protein (e.g., CD 123); and

(ii) Administering a therapeutically effective amount of an agent comprising at least a first antigen binding region that specifically binds the second isoform of the surface protein (e.g., a second isoform of CD 123) and does not bind the first isoform of the surface protein (e.g., a first isoform of CD 123) to specifically deplete cells (patient cells) expressing the second isoform of the surface protein.

The cells expressing the first isoform or a pharmaceutical composition thereof are administered to a subject in combination (e.g., prior to, concurrently with, or subsequent to) an agent comprising a first antigen-binding region as described above.

In a preferred embodiment, the depleting agent (e.g., CAR cells or antibodies that target the second isoform of CD 123) is administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells expressing the first isoform of surface protein (e.g., the first isoform of CD 123) or after a dose of hematopoietic stem cells expressing the first isoform of surface protein (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, 6 weeks, 12 weeks, 8 weeks, or 16 weeks).

A "therapeutically effective amount" or "effective amount" is a desired number of cells, particularly hematopoietic stem cells expressing a first isotype as described above, administered to a subject sufficient to constitute a treatment as defined above, particularly to restore normal hematopoietic function in a patient.

Administration of cells or pharmaceutical compositions according to the present disclosure may be performed in any convenient manner, including injection, infusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. In another embodiment, the cells or pharmaceutical compositions of the invention are preferably administered by intravenous injection. The cells or pharmaceutical compositions of the invention may be injected directly into a tumor, lymph node or infection site.

The administration of the cells or cell populations may include administration of 10 per kilogram of body weight ⁴ -10 ⁹ Individual cells, preferably 10 per kg body weight ⁵ To 10 ⁷ Individual cells, including all integer values of the number of cells in those ranges. The dose administered will depend on the age, health and weight of the recipient, as well as the type of treatment (if any), the frequency of treatment and the nature of the desired effect. The cell or cell population may be administered in one or more doses. The timing of administration is at the discretion of the attending physician and depends on the clinical condition of the subject. The cell or cell population may be obtained from any source, such as a blood bank or donor. Although individual needs vary, it is within the skill in the art to determine the optimal range of effective amounts for a given cell type for a particular disease or condition.

In particular, the disclosure also relates to an anti-CD 123 depleting agent (e.g., CAR cell composition or antibody) as disclosed above comprising a first antigen binding region for selectively depleting host cells in a subject in need thereof.

Methods for specific depletion of transplanted cells rather than patient cells (safety switch).

According to the present disclosure, the cell or cell population (e.g., hematopoietic cells) expressing a first isoform of the surface protein (e.g., a first isoform of CD 123) as described above is used in the medical treatment of a patient in need thereof, wherein the medical treatment comprises administering a therapeutically effective amount of the cell or cell population expressing the first isoform of the surface protein in combination with a therapeutically effective amount of a depleting agent (e.g., CAR cells or antibodies) that specifically binds the first isoform of the surface protein (e.g., a first isoform of CD 123).

Cells or cell populations, preferably immune cells expressing a first isotype of the disclosure, are particularly useful in adoptive transfer cell transfer therapy into a patient. The transplanted cells expressing the first isoform of the surface protein may be further depleted in a patient by administering a therapeutically effective amount of an depleting agent comprising a second antigen binding region that specifically binds to the first isoform and does not bind to a second isoform of the surface protein expressed by patient cells to avoid eventual serious side effects such as graft versus host disease due to transplantation. In this case, the agent comprising a second antigen binding region that specifically binds the first isoform of the surface protein (expressed by the transplanted cells) is administered to deplete specific transplanted cells rather than patient cells. By providing a "safety switch," selective depletion of transplanted cells constitutes an important safety feature.

Graft versus host disease (GvHD) is associated with medical complications after receiving transplanted tissue from genetically diverse people. Immune cells in the donor tissue (graft) recognize the recipient (host) as foreign. In certain embodiments, the medical condition is graft versus host disease caused by hematopoietic stem cell transplantation or adoptive cell transfer therapy in which immune cells are transferred into a patient.

Such side effects may also occur when transplanted cells, particularly immune cells carrying CARs, have serious side effects, such as cytokine release syndrome and/or neurotoxicity. In this case, the transplanted cells expressing the first isoform may be eliminated as a safety switch when the cells become malignant or cause any type of undesired on-target or off-target damage.

The present disclosure relates to methods for adoptive cell transfer therapy in a patient having cells that express a second isoform of a surface protein (e.g., CD 123), the methods comprising:

(i) Administering an effective amount of cells expressing a first isoform of the surface protein (e.g., CD 123), wherein the cells expressing the first isoform (e.g., CD 123) comprise genomic DNA having at least one polymorphic allele, preferably a Single Nucleotide Polymorphism (SNP) allele, or a genetically engineered allele in a nucleic acid encoding the first isoform, and wherein the polymorphism is not present in the genome of a patient having cells expressing the second isoform of the surface protein (e.g., a second isoform of CD 123) or a pharmaceutical composition thereof; and

(ii) Administering a therapeutically effective amount of an agent comprising at least a second antigen binding region that specifically binds the first isoform of the surface protein (e.g., a first isoform of CD 123) and does not bind the second isoform of the surface protein (e.g., a second isoform of CD 123) to specifically deplete cells expressing the first isoform.

The cells expressing the first isoform or a pharmaceutical composition thereof are administered to a subject in combination (e.g., prior to, concurrently with, or subsequent to) an agent comprising a second antigen-binding region as described above.

In a preferred embodiment, the depleting agent (e.g., CAR cells or antibodies that target the second isotype of CD 123) is administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells that express the first isotype (e.g., the first isotype of CD 123) or is administered 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, 8 weeks, 12 weeks, or 16 weeks) a dose of hematopoietic stem cells that express the first isotype (e.g., the first isotype of CD 123).

Thus, in a particular embodiment, the disclosure relates to a depleting agent (e.g., CAR cells or antibodies) for preventing or reducing the risk of serious side effects in a patient who has received cells expressing a first isoform of a cell surface protein as described above, wherein the patient has primary cells expressing a second isoform of the cell surface protein, and wherein the depleting agent comprises at least a second antigen binding region that specifically binds the first isoform of the cell surface protein but not the second isoform of the cell surface protein.

Kit for detecting a substance in a sample

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

The invention is disclosed in the following claims:

1. a mammalian cell or population of cells expressing a first isoform of a surface protein for use in medical treatment of a patient in need thereof, said patient having cells expressing a second isoform of said surface protein,

Wherein the cell expressing the first isoform comprises genomic DNA having at least one polymorphism or genetically engineered allele in a nucleic acid encoding the first isoform, preferably wherein the polymorphism is a Single Nucleotide Polymorphism (SNP) allele, wherein the polymorphism allele is not present in the genome of a patient having cells expressing the second isoform of the surface protein.

2. The mammalian cell or cell population for use according to claim 1, wherein the first and second isoforms of surface proteins are functional.

3. The mammalian cell or cell population for use according to claim 1 or 2, wherein the surface protein is CD123.

4. A mammalian cell or cell population for use according to claim 3, wherein the polymorphism or genetically engineered allele is characterized by at least one amino acid substitution at E51, S59 and/or R84 relative to position SEQ ID No. 1.

5. Mammalian cell or cell population for use according to claim 4, wherein the residue E51 is substituted with an amino acid selected from the group consisting of K, N, T, R, M, G and a, preferably with an amino acid selected from K or T.

6. The mammalian cell or cell population for use according to claim 4 or 5, wherein the residue S59 is substituted with an amino acid selected from the group consisting of I, P, E, L, K, F, R and Y; preferably by amino acid substitutions selected from the group consisting of P, E.

7. The mammalian cell or cell population for use according to any one of claims 4 to 6, wherein the residue R84 is substituted with an amino acid selected from the group consisting of T, S, Q, N, H and a.

8. The mammalian cell or population of cells for use according to any one of claims 1 to 7, wherein the cell expressing the first isoform is selected from a subject comprising native genomic DNA having at least one native polymorphic allele in a nucleic acid encoding the first isoform.

9. The mammalian cell or cell population for use according to any one of claims 1 to 7, wherein the first isoform is obtained by ex vivo modification of a nucleic acid sequence encoding the surface protein via genetic editing.

10. The mammalian cell or cell population for use according to claim 9, wherein the nucleic acid sequence encoding a surface protein is modified by: introducing into the cell a gene-editing enzyme capable of inducing a site-specific mutation within a target sequence encoding a surface protein region involved in the binding of an agent comprising at least a first antigen binding region.

11. Mammalian cell or cell population for use according to claim 10, wherein the gene-editing enzyme is a site-specific nuclease, base editor or primer editor, preferably a CRISPR/Cas gene-editing enzyme comprising a guide RNA comprising a sequence complementary to the target sequence.

12. The mammalian cell or cell population for use according to claim 11, wherein the surface protein is CD123, and wherein the

The guide RNA sequence is SEQ ID NO. 16 or 17 and targets the sequence encoding the substitution E51K relative to SEQ ID NO. 1,

the guide RNA sequence is selected from the group consisting of SEQ ID NOS.18 to 20 and targets the sequence encoding the substitution S59P relative to SEQ ID NO. 1 and/or

The guide RNA sequence is selected from the group consisting of SEQ ID NOS.21 to 23 and targets the sequence encoding the substitution R84E relative to SEQ ID NO. 1.

13. The mammalian cell of any one of claims 9 to 12, wherein the nucleic acid sequence encoding the surface protein is modified by further introducing an HDR template.

14. The mammalian cell or cell population of any one of claims 1-13, wherein the medical treatment comprises:

Administering to the patient in need thereof a therapeutically effective amount of the cell or cell population expressing the first isoform in combination with a therapeutically effective amount of a depleting agent comprising at least a first antigen binding region that specifically binds the second isoform to specifically deplete patient cells expressing the second isoform.

15. Mammalian cell or cell population for use according to claim 14, wherein the cell expressing the first isoform is a hematopoietic cell, preferably a hematopoietic stem cell.

16. Mammalian cell or cell population for use according to claim 14 or 15 for restoring normal hematopoiesis after immunotherapy in the treatment of hematopoietic diseases, preferably malignant hematopoietic diseases, such as Acute Myeloid Leukemia (AML), blast plasmacytoid dendritic cell tumor (BPDCN) or B-acute lymphoblastic leukemia (B-ALL).

17. The mammalian cell or cell population for use according to any one of claims 14 to 16, wherein the depleting agent is an antibody or antibody-drug conjugate comprising a first antigen binding region that specifically binds the second isoform and does not bind the first isoform.

18. Mammalian cell or cell population for use according to claims 14 to 16, wherein the depleting agent is an immune cell, preferably a T cell, carrying a Chimeric Antigen Receptor (CAR) comprising a first antigen binding region that specifically binds the second isoform but not the first isoform.

19. The mammalian cell or cell population for use according to claim 17 or 18, wherein the surface protein is CD123, and wherein the first antigen binding region of the depleting agent specifically binds an epitope comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1.

20. The mammalian cell or population of cells for use according to claim 19, wherein the first antigen binding region comprises:

c) An antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO 2, VHCD2 is SEQ ID NO 3 and VHCDR3 is SEQ ID NO 4; and

d) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7 and VLCDR3 is SEQ ID NO 8.

21. The mammalian cell or cell population for use according to claim 20, wherein the first antigen binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 10, 12 and 14.

22. The mammalian cell or cell population for use according to any one of claims 1 to 13, wherein the medical treatment comprises:

administering to said patient in need thereof a therapeutically effective amount of said cell or cell population expressing said first isoform in combination with a therapeutically effective amount of a depleting agent comprising at least a second antigen binding region that specifically binds said first isoform to specifically deplete expressing first isoform-transferred cells.

23. Mammalian cells or cell populations for use according to claim 22, for adoptive cell transfer therapy, preferably for the treatment of malignant hematopoietic diseases such as Acute Myeloid Leukemia (AML), blast plasmacytoid dendritic cell tumor (BPDCN) or B-acute lymphoblastic leukemia (B-ALL).

24. Mammalian cells or cell population for use according to claim 22 or 23, wherein the depleting agent is administered after the cells or cell population expressing the first isoform surface protein, in order to avoid eventual serious side effects such as graft versus host disease due to transplantation.

25. Mammalian cell or cell population for use according to any one of claims 22 to 24, wherein the cell expressing the first isoform is a hematopoietic cell, preferably an immune cell, more preferably a T cell.

26. Mammalian cell or cell population for use according to claim 25, wherein the immune cell expressing the first isotype is an immune cell carrying a Chimeric Antigen Receptor (CAR), preferably a T cell.

27. The mammalian cell or population of cells for use according to claim 26, wherein the Chimeric Antigen Receptor (CAR) targets the second isoform expressed by cells of the patient.

28. The mammalian cell or cell population for use according to claim 27, wherein the CAR comprises an antigen binding region that specifically binds an epitope of CD123 located within the third extracellular loop or within the polypeptide comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1.

29. The mammalian cell or population of cells for use according to claim 28, wherein the CAR comprises an antigen binding region comprising:

-an antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO 2 and VHCD2 is SEQ ID NO:3, VHCDR3 is SEQ ID NO. 4; and

-an antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7 and VLCDR3 is SEQ ID NO 8.

30. Mammalian cell or cell population for use according to claim 29, wherein the CAR comprises an antigen binding region comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 10, 12 and 14, preferably wherein the CAR comprises or consists of the amino acid sequence SEQ ID NO 15.

31. The mammalian cell or cell population for use according to any one of claims 22 to 30, wherein the depleting agent is an antibody or antibody-drug conjugate comprising a second antigen binding region that specifically binds the first isotype and does not bind the second isotype.

32. Mammalian cell or cell population for use according to claims 22 to 30, wherein the depleting agent is an immune cell, preferably a T cell, carrying a Chimeric Antigen Receptor (CAR) comprising a second antigen binding region that specifically binds the first isoform and not the second isoform.

33. A pharmaceutical composition comprising a mammalian cell, preferably a hematopoietic stem cell or an immune cell such as a T cell, as defined in any one of claims 1 to 13, and a pharmaceutically acceptable carrier.

34. The pharmaceutical composition according to claim 33, wherein the immune cells expressing the first isoform are immune cells carrying a Chimeric Antigen Receptor (CAR), preferably T cells, targeting the CAR of the second isoform of CD123 expressed by cells of the patient.

35. The pharmaceutical composition of claim 34, wherein the CAR comprises an antigen binding region that specifically binds an epitope of CD123 that is located within a third extracellular loop or within a polypeptide comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1.

36. The pharmaceutical composition of claim 35, wherein the CAR comprises an antigen binding region comprising:

-an antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO 2, VHCD2 is SEQ ID NO 3 and VHCDR3 is SEQ ID NO 4; and

37. The pharmaceutical composition according to claim 36, wherein the CAR comprises an antigen binding region comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from SEQ ID NOs 10, 12 and 14, preferably wherein the CAR comprises or consists of the amino acid sequence SEQ ID NO 15.

38. The pharmaceutical composition according to any one of claims 33 to 37, further comprising a consumable as defined in claims 27 to 27, 31 and 32.

39. A depleting agent for preventing or reducing the risk of serious side effects in a patient who has received cells expressing a first isoform of a surface protein, wherein primordial cells of the patient express a second isoform of a surface protein, and wherein the depleting agent comprises at least a second antigen binding region that specifically binds the first isoform and does not bind the second isoform.

40. The depleting agent according to claim 39, wherein said depleting agent is an antibody or antibody-drug conjugate comprising a second antigen binding region that specifically binds a first isoform of a surface protein and does not bind a second isoform of said surface protein.

41. The depleting agent according to claim 40, wherein said depleting agent is an immune cell, preferably a T cell, carrying a Chimeric Antigen Receptor (CAR) comprising a second antigen binding region that specifically binds said first isoform and does not bind said second isoform.

42. The consumable of any one of claims 39 to 41, wherein said surface protein is CD123.

43. A depleting agent for selectively depleting host cells in a patient in need thereof, wherein the patient's primordial cells express a second isoform of a surface protein, and wherein the depleting agent comprises at least a first antigen binding region that specifically binds to the second isoform.

44. The consumable of claim 44, wherein the consumable is an antibody or antibody-drug conjugate comprising a first antigen binding region that specifically binds a second isoform of the surface protein.

45. The depleting agent according to claim 44, wherein said depleting agent is an immune cell, preferably a T cell, carrying a Chimeric Antigen Receptor (CAR) comprising a first antigen binding region that specifically binds to said second isoform.

46. The consumable of any one of claims 44 to 46, wherein said surface protein is CD123.

47. The depleting agent according to claim 47, wherein said first antigen binding region of said depleting agent specifically binds to an epitope that is within a third extracellular loop or within a polypeptide comprising amino acids T48, D49, E51, a56, D57, Y58, S59, M60, P61, a62, V63, N64, T82, R84, V85, a86, N87, P89, F90, S91 of SEQ ID No. 1.

48. The consumable of claim 48, wherein the first antigen binding region comprises:

e) An antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO 2, VHCD2 is SEQ ID NO 3 and VHCDR3 is SEQ ID NO 4; and

f) An antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO 5 or 6, VLCDR2 is SEQ ID NO 7 and VLCDR3 is SEQ ID NO 8.

49. The consumable of claim 49, wherein the first antigen binding region comprises a heavy chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 9, 11 and 13 and/or a light chain variable domain comprising or consisting of any one of the amino acid sequences selected from the group consisting of SEQ ID NOs 10, 12 and 14, preferably wherein the consumable is a CAR comprising or consisting of SEQ ID NO 15.

The invention will now be illustrated by the following examples, which are not limiting.

Examples

Computational analysis of CD123

The identity and position of epitopes on cell surface proteins can be extracted from the 3D structure of the complex or predicted based on sequence-based and/or structure-based epitope prediction tools. Methods for predicting linear or conformational B cell epitopes include, but are not limited to, bepippred PMID 28472356,DiscoTope PMID:26424260,ElliPro PMID:19055730 and SVMTrip PMID 32162263.

In order to estimate the effect of variants on the structural and functional status of the protein and possible pathogenic effects, information including biophysical characteristics, evolutionary conservation patterns, proximity to biologically relevant sites (i.e., interaction sites, functionally and structurally related sites), protein stability is considered.

The position of the variant on the protein is based on: i) A useful or predicted 3D structure of the protein; ii) sequence comparison with homologous proteins of known structure. Solvent accessibility or Accessible Surface Area (ASA) of the amino acid sites comprising the variant and the entire protein region is calculated based on 3D structural information (when available) or predicted from sequence-based features.

Methods for predicting ASA from amino acid sequences include sequence spectra, structural similarity, and machine learning methods such as neural networks, support Vector Machines (SVMs), and bayesian statistics PMID:2217139, PMID:7892171, PMID:15281128, PMID:15814555, PMID:8727318. Methods for predicting the effect of a variant include, but are not limited to SIFT, polyPhen, mutationTaster, condel, fashmm.

Structure-based methods for predicting the effect of amino acid variations on protein stability are based on statistical potential, physical or empirical energy functions, including but not limited to: foldX, rosetta, CC/PBSA (PMID: 12079393, PMID: 1843248, PMID:19116609, PMID: 15063647).

Global and local probability models calculated from multiple sequence alignments can be used to quantify the effect of single or higher order substitutions based solely on sequence information and include mutual information, direct coupling analysis and covariance estimation PMID 28092658, PMID 22101153, PMID 23458856, PMID 24573474.

Analysis of the CD123-CSL362 Complex

CSL362 epitope: open and closed state

The three-dimensional structure of the CD123-CSL362 interaction complex is used to identify key interaction sites as preferred sites for the generation of protein variants. Protein sites and variants were selected based on the following: i) Relative solvent accessibility of each residue observed in the CSL 362-bound state and CSL 362-free state, ii) evolutionary conservation and amino acid usage from each site of multiple sequence alignment; iii) Predicted stability changes after computer mutagenesis based on sequence (EVmutation) and structure-based simulation (FoldX) (fig. 1).

SNP analysis of CD123

In combination with the nature of the amino acid changes, each variant will be analyzed and prioritized based on the distribution or frequency of alleles in a given population, homozygous state, disease-related data, phenotype-related data, and the effect of variation on protein, cellular and organism levels.

Allele frequency data can be obtained from a series of programs and libraries, including 1000 genome programs; genome aggregation database (gnomAD); dbSNP; ensembl; search in Uniprot (table 1);

phenotype data and disease-related variants can be obtained from a range of programs and libraries, including ClinVar; COSMIC phenotype variants; HGMD-publish variants; a NHGRI-EBI phenotype variant catalog; an OMIM phenotype variant; phenCode; GWAS; genotype tissue expression (GTEx).

Chromosome location

rsID

Reference alleles

Alternative alleles

Protein results

Total number of genotypes

Allele frequency

X:1464295

rs189735318

G

A

p.Glu51Lys

250792

3.59e-5

X:1464295

rs189735318

G

C

p.Glu51Gln

282020

3.19e-5

X:1464297

rs144300259

G

C

p.Glu51Asp

281874

1.06e-5

X:1464320

rs1318699562

C

G

p.Ser59Cys

247760

4.04e-6

X:1467391

rs138837148

G

C

p.Arg84Pro

251178

3.98e-6

X:1467391

rs138837148

G

A

p.Arg84Gln

282560

1.06e-4

Table 1 CD123 SNP. The data relates to SNPs that produce non-synonymous variants at amino acid residues 51, 59 and 84. The source is as follows: gnomAD v2.1.1.

4. FACS scanning of living cells

Expression and purification of CSL362/Okt3

The plasmid encoding CSL362/Okt3-BiTE is a friendly gift from Dario Neri (Hutmacher et al, leuk Res.2019Sep; 84:106178).

CHO-S had grown to a density of 2000 ten thousand/ml in Power CHO2 medium (Lonza: BELN12-771Q supplemented with 1XHT, glutamax, antibiotic antifungal).

Then 2.10 ⁹ The individual cells were centrifuged and resuspended in 500ml ProCHO4 medium (Lonza: BEBP12-029Q supplemented with 1XHT, glutamax, antibiotic-antifungal). 1.7mg CSL362/Okt3 BiTE DNA was added to the cells together with 5ml PEI (at 1 mg/ml). The cells were then distributed in 4X 500ml roller bottles and kept in CO ₂ The cells were grown in an incubator at 31℃and 140rpm for 6 days.

CHO-S cells were then centrifuged at 3000rpm for 20mns. The supernatant was filtered through a 0.22mM filter and applied to a 5ml Ni-NTA column (ThermoFisher) pre-washed with 100ml of washing solution (PBS containing 150mM NaCl and 5mM imidazole, pH 7.4).

The column was washed in 100ml of washing medium. Elution was performed in PBS,150mM NaCl,250mM imidazole, ph=8.0. Fractions of 0.5ml were collected and OD was measured at 280 mM. The high concentration fractions were pooled and subjected to two O/N dialysis against PBS. The Bite was sterile filtered through 0.22 μm, aliquoted at 1mg/ml, and stored at-80 ℃.

CSL362 and MIRG123

CSL362 is a chimeric antibody carrying the S239D and I332E mutations in the Fc region (Leukemia (2014) 28:2213-21). To generate the monoclonal IgG1 anti-CD 123 antibody MIRG123, the heavy and kappa light chain variable regions (VH and VKL) were derived from CSL362/OKT3 BiTE. Under the control of the hCMV promoter, the VH and VKL sequences were cloned into AbVec2.0-IGHG1 (Addgene plasmid # 80795) and AbVec1.1-IGKC (Addgene plasmid # 80796), respectively (Tiller et al J Immunol methods.20088 Jan 1;329 (1-2): 112-24.Epub 2007Oct 31).

VH sequence (SEQ ID NO: 9):

EVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSNGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSS

VKL sequence (SEQ ID NO: 10)

DIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK

Both plasmids were then co-transfected in CHO-S cells for expression. CHO-S has been grown to a density of 2000 tens of thousands/ml in Power CHO2 medium (Lonza: BELN12-771Q, supplemented with glutamax, HT supplement, antibiotic-antifungal).

Then 2X 10 ⁹ The individual cells were centrifuged and resuspended in 500ml ProCHO4 medium (Lonza: BEBP12-029Q supplemented with 1XHT, glutamax, antibiotic-antifungal). 0.6mg of VH and 0.6mg of VKL DNA were added to the cells together with 5ml PEI (at 1 mg/ml). The cells were then distributed in 4X 500ml roller bottles and kept in CO ₂ The cells were grown in an incubator at 31℃and 140rpm for 6 days.

CHO-S cells were then centrifuged at 3000rpm for 20mns. The supernatant was filtered through a 0.22 μm filter and applied to a protein a column pre-washed with PBS. The column was then washed with 100ml PBS. The antibody was eluted with 0.1M glycine (ph=2.2). Fractions of 0.5ml were collected and OD was measured at 280 mM. The high concentration fractions were pooled and subjected to two O/N dialysis against PBS.

5. Binding assays by FACS

5.1 materials and methods

Eukaryotic cell lines

The human embryonic kidney 293 cell line (HEK-293) is a friendly gift of M.Zavalan (Biozendrum Basel). All cell lines were thawed freshly and passaged 3-6 times before use in the assay. At 37℃at 5% CO ₂ HEK-293 was cultured in Dulbecco's modified Eagle's Medium-high glucose (Sigma-Aldrich) supplemented with 10% heat-inactivated fetal calf serum (FCS; gibco Life Technologies) and 2mM Glutamax and split three times per week. Geneticin G418 (50 mg/ml; bioConcept) was added at a concentration of 350. Mu.g/ml to the medium of all stable cell lines expressing the neomycin resistance cassette.

Wild-type HEK-293, which was negatively stained for CD123, was designated HEK, HEK-293 expressing wild-type CD123 was labeled HEK-CD123, and CD123 variants were described by the position of residues and altered amino acids.

Full-length cDNA (NM-002183.2) of the human interleukin 3 receptor (CD 123) was purchased from Sino Biological in pCMV3 vector (catalog number: HG 10518-NF). For stable cell line production, hygromycin was replaced with neomycin.

E51 has been mutated to: k, N, T, S, Q, R, M, G, A.

S59 has been mutated to: i, G, P, E, L, T, K, F, R, Y.

R84 has been mutated to: t, K, S, Q, N, E, H, A, L.

All point mutations have then been introduced by PCR, resulting in single amino acid changes.

Using a Neon electroporation apparatus (Invitrogen; 1100V-20ms-2 pulse), a pair of 2X 10 DNA (pCMV plasmid-huCD 123 wild type or variant) was used with 6. Mu.g DNA ⁶ The HEK cells were electroporated. After 48 hours, cells were used for binding analysis by FACS (as transient transfection). They were then stored for 2 weeks under G418 selection (350. Mu.g/ml medium) and then tested for binding as stable cell lines.

FACS：

2X 10 pairs with anti-HuCD 123-Buv clone 6H6 (Biolegend catalog: 30020-1/100) together with MIRG 123-biotin (1/50) +streptavidin-FITC (1/200) ⁵ Individual HEK cells were stained and analyzed by FACS.

Binding assays based on flow cytometry:

to test binding of CD123 variants to MIRG123, high antibody concentrations were used: stable cell lines were stained with anti-HuCD 123-Buv clone 6H6 (1/12:4.2 mg/ml) and biotinylated MIRG123-Bio (1/7.5:50 mg/ml) +streptavidin PE (1/200).

5.2 results

FIG. 2 shows a flow cytometry plot showing the binding of anti-human CD123 antibody clone 6H6 (x-axis) and MIRG123 (y-axis) to wild-type CD123 and variants thereof stably expressed in HEK-293 cells in vitro. Strong staining of 6H6 showed stable expression of CD123 protein, while different CD123 variant isoforms showed elimination of binding to clone MIRG123<1 MIRG123 ⁺ 6H6 ⁺ Cells (upper right quadrant)), weak (1-20% MIRG123 ⁺ 6H6 ⁺ Cells) or strong%>20% MIRG123 ⁺ 6H6 ⁺ Cells). Control conditions (grey) were HEK-293 cells stably expressing human wild-type CD123 (HEK-CD 123) and untransduced HEK-293-cells (HEK). Representative flow cytometry data from 3 independent experiments.

The target cells are stable HEK-293 cell lines, or non-transduced (HEK), expressing wild-type CD123 (HEK-CD 123) or variant isoforms thereof (indicated with altered amino acid residue positions). CD123 variants are encoded by their binding to the anti-CD 123 antibody clone MIRG123, either eliminated (underlined), weak (circles) or strong (asterisks). The assembled data of 3 independent flow cytometry experiments is shown in figure 3.

Adcc assay: in vitro assay for quantifying MIRG 123-mediated ADCC activity

6.1ADCC assay method

ADCC activity was predicted from fcyriiia activation assays with ADCC reporter bioassays, V variants (Promega, reference G7015). HEK293 cell lines stably expressing hCD123 variants were seeded in white 96-well plate clear bottoms (Corning costar # 3610). On day 0, 4400 cells were inoculated in 100ul of medium, whereby 6250 cells were obtained after 24h (E: T: 12:1). On day 1, the medium was removed from HEK293 cell culture and added: (i) 25 μl ADCC buffer (Promega), (ii) 25 μl MIRG123 antibody, diluted in ADCC buffer at 3 Xfinal concentration (final concentration 1 ug/ml), (iii) Mu.l effector cells (Jurkat/FcgammaRIIIa/NFAT-Luc cells) were diluted in ADGC buffer at a concentration of 3M cells/ml. The mixture was subjected to 5% CO at 37 ℃C ₂ Incubate for 5h. Luciferase activity was measured using Bio-GloTM luciferase assay reagent (Promega). Mu.l of reagent was added and the cells were incubated for 10 minutes at Room Temperature (RT) with stirring. The bottom of the plate was covered with an opaque label (Elmer 6005199) and luminescence was read with the pheasatart FSX (BMG LABTECH) program Luc-Glo (LUM), gaina=3600, optical module=lumplus.

In this assay Raji cells incubated with 1 μg/ml rituximab were used as positive control.

6.2 results

Relative luminescence signal measured after co-culturing HEK, HEK-CD123 (wild type) and HEK CD123 variant isoforms with Jurkat/fcyriiia/NFAT-Luc reporter cells. RLU was normalized to the signal measured with HEK-CD123 (wild type) (fig. 4).

7.T cell adaptor assay: quantification of T cell activation and target attenuation by CSL362 derived T cell adaptors In vitro assay of cell killing.

7.1 materials and methods

Isolated culture of primary human T cells

White blood cell Buffy coat from anonymous healthy human donors was purchased from Basel blood donation center (Blutspendezentrum SRK beider Basel, BSZ). Peripheral Blood Mononuclear Cells (PBMCs) were isolated by density centrifugation using a sepmatettm tube (Stemcell technologies) according to the manufacturer's protocol in the case of density gradient media Ficoll paque (GE Healthcare). Human T cells were then purified (> 96% purity) by magnetic negative selection using easy sep human T cell isolation kit (Stemcell Technologies) according to manufacturer's instructions. If frozen PBMC are used, T cells are isolated after thawing and cultured overnight in supplemented medium without stimulation. T cells were cultured in RPMI-1640 medium (Sigma-Aldrich) supplemented with: 10% heat-inactivated human serum (AB+, male; from BSZ Basel), 2mM Glutamax,10mM HEPES,1mM sodium pyruvate, 0.05mM 2-mercaptoethanol and 1% MEM nonessential amino acids (100X) (all from Gibco Life Technologies).

In vitro BiTE-mediated killing assay

For the BiTE killing assay, HEK-293 target cells were co-cultured with primary human effector T cells and 300ng/mL CD3/CSL365 BiTE at an effector to target ratio (E: T ratio) of 10:1 for 72h.

HEK-CD123 and HEK stably expressing CD123 variants were stained with CellTraceViolet (CTV) and at 37 ℃,5% co, according to the manufacturer's protocol before starting co-cultivation of HEK ₂ The culture was performed overnight in 96-well plates in complete human medium. The next day, thawed T cells (kept overnight in supplemented medium) or freshly isolated T cells were added to HEK-293 cells at a concentration of 300ng/ml together with BiTE and kept at 37℃for 72h. T cell activation was assessed by quantifying the proportion of CD69% positive T cells via flow cytometry. Cytotoxicity and activation of T cells were analyzed by flow cytometry. The specific killing was calculated as follows: (1-number of live target cells in the presence of BiTE/number of live target cells in the absence of BiTE) ×100. Cell morphology was assessed with an optical microscope Axio ver A1 (Zeiss) at 20 x magnification.

7.2 results:

t cell adaptor mediated T cell activation

Stable target cell lines HEK, HEK-CD123 and CD123 variants were co-cultured with human primary pan T cells in the presence of 300ng/mL CD3/CSL362 BiTE at an E:T ratio of 10:1. FIG. 5 shows the% CD69 measured after co-culture with HEK, HEK-CD123 or all CD123 variants ⁺ Summary of T cells. Data were normalized to% CD69 in the presence of HEK target cells ⁺ And (3) cells. Error bars show mean ± SD. Data represent 5 independent blood donors and experiments with 2 technical replicates per group.

T cell adaptor mediated cell killing

Stable target cell lines HEK, HEK-CD123 and CD123 variants were co-cultured with human primary pan T cells in the presence of 300ng/mL CD3/CSL362 BiTE at an E:T ratio of 10:1. FIG. 6 shows specific BiTE mediated killing (in%) of HEK, HEK-CD123 and all CD123 variants after 72h co-culture. Error bars show mean ± SD. Data represent 5 independent blood donors and experiments with 2 technical replicates per group.

CAR T killing

8.1 materials and methods

Flow cytometry and cell sorting

Flow cytometry was performed on BD LSRFortessa using BD FACSDiva software and data was analyzed using FlowJo software (FlowJo version 10.7.1).

Primary T cells were washed in ice-cold PBS and then stained with fixable feasibility dye for 20min in the dark. Thereafter, in the dark at room temperature, 50. Mu.l FACS buffer (PBS+2% FCS+0.1% NaN) with fluorescently labeled antibody ₃ ) The cell surface staining was performed for 20min. In the case of biotin-labeled antibodies, the second antibody with streptavidin is then stained with the same protocol. The stained cells were washed once in FACS buffer and then immediately obtained.

For cell sorting, the cell pellet was resuspended in FACS buffer (pbs+2% fcs) supplemented with 1mM EDTA and kept on ice until analysis. FACS was performed on a BD FACSAria or BD FACSMelody cell sorter. Control cells were also subjected to the sorting process.

Design and production of CD123-CAR HDRT

CAR T cells are generated by co-electroporation of CRISPR-Cas9 Ribonucleoprotein (RNP) specific for the TRAC locus (SEQ ID NO: 24) and double stranded DNA HDR template (HDRT). Clone CSL362 (a friendly gift from D.neri and single chain variable fragment (scFv) previously disclosed (HUTMACHER et al 2019), CD8 alpha hinge (SEQ ID NO: 25) and transmembrane domain (SEQ ID NO: 26) (Gen CD8AENSG 00000153563), intracellular signaling moiety 4-1BB (SEQ ID NO: 27) (Gen TNFRSF9 ENSG 00000049249) and CD3 zeta (SEQ)ID NO:28 (Gen CD247ENSG 00000198821) and a fluorescent reporter GFP to encode a second generation CD123 specific CAR (SEQ ID NO: 15). It is flanked by symmetrical homology arms (300 bp) complementary to exon 1 of the TRAC locus (FIG. 7). The construct is constructed byThe HDRT was synthesized and cloned into a cloning vector (pUC 57 backbone) and PCR amplified from the plasmid using kappa high fidelity polymerase (Kapa Hifi Hotstart Ready Mix, roche). The PCR amplicons were purified using a Nucleospin gel and a PCR clean-up kit (Macherey-Nagel) according to the manufacturer's instructions and the correct size was verified by gel electrophoresis on a 1% agarose gel. HDRT was then concentrated to a final concentration of 1ug/μl using vacuum concentration and stored at-20 ℃ until use.

Genomic DNA extraction and sequencing from human T cells

By re-suspending in 100. Mu.l Tail Lysis buffer (100mM Tris[pH 8.5) containing proteinase K (0.1. Mu.g; sigma-Aldrich)]5mM Na-EDTA,0.2% SDS,200mM NaCl) and incubated at 56℃on a Eppendorf Thermomix Comfort shaker at 1000 rpm. After 1h, the enzyme was heat inactivated at 95℃for 15min. After centrifugation (14000 rpm,10 min), the DNA was precipitated by mixing the sample with isopropanol in a 1:1 volume ratio. The DNA was pelleted, washed in 70% ethanol, air dried and resuspended in Milli-Q water. Finally, using NanoDrop ^TM The device measures DNA concentration (Thermo Fisher). To verify correct insertion of the CAR transgene into the TRAC locus, primers were designed outside the 5 'and 3' arms with homology. After PCR with Phusion high fidelity DNA polymerase (Thermo Scientific), the PCR products were size separated by gel electrophoresis on a 0.8% agarose gel and the correct amplicon length was purified using a Nucleospin PCR and gel clean-up kit (Macherey-Nagel; according to manufacturer's protocol). 100ng of eluted DNA was used for additional PCR amplification with the same primers, thereby increasing the amount of DNA. PCR and gel cleaning kit (Macherey-N were used Gel) and ligating it into pJET 1.2/count cloning vector at 22℃for 2h using ConeJET PCR cloning kit (Thermo Scientific). After transformation of the bacteria into competent bacteria (JM 109) and incubation overnight at 37℃32 colonies were picked and screened by PCR. Colonies with the correctly integrated transgene were inoculated in 5ml LB medium supplemented with 50ug/ml ampicillin and grown overnight at 37 ℃. Plasmid DNA was isolated from the cultured bacteria using the GenElute plasmid Miniprep kit (Sigma-Aldrich) according to the manufacturer's protocol. Sanger sequencing was performed at Microsynth AG Switzerland. The sequence was analyzed using MegAlign Pro (DNASTAR, version 17.0.1.183).

Generation of human CD123-CAR T cells by non-viral CRISPR/Cas 9-based editing

Cas9 Ribonucleoprotein (RNP) was freshly generated prior to each electroporation. Thawed crrnas (specific for the TRAC locus and previously disclosed, ROTH et al 2018) and tracrRNA (both from IDT Technologies, resuspended at 200 μm) were mixed at a 1:1 molar ratio (120 pmol each), denatured at 95 ℃ for 5min, and annealed at room temperature for 10 to 20min, thus complexing 80 μm single guide RNA (sgRNA) solutions. Polyglutamic acid (PGA; 15-50 kDa; 100mg/ml; sigma-Aldrich) was added to the sgRNA at a volume ratio of 0.8:1. Finally, 60pmol of recombinant Cas9 (University of California Berkeley, at 40 μm) was mixed with freshly prepared sgrnas (molar ratio Cas9: sgrnas=1:2) into a complex Ribonucleoprotein (RNP), incubated in the dark for 20min at room temperature.

Before electroporation, 1.5-2X 10 ⁶ Cell density of individual cells/ml at 37℃at 5% CO ₂ With CD3/CD28 Dynabeads (Thermofisher) (at a cell to bead ratio of 1:1) and recombinant human cytokine IL-2 (150U/ml; proleukin from University Hospital Basel), IL-7 (5 ng/ml; R)&D Systems) and IL-15 (5 ng/ml; r is R&D Systems) the isolated human T cells are activated.

With the program EH-115 using a 4D Nucleplay ^TM Electroporation was performed by the system (Lonza). After activation, the cells were isolated by placing resuspended T cells in EasyStep ^TM Removal from magnetsCD3/CD28-Dynabeads, for 2min. For each electroporation, 1X 10 was used ⁶ The activated T cells were washed once in PBS and then resuspended in 20 μl Lonza-supplemented P3 electroporation buffer. HDRT (3-4 ug) and RNP (60 pmol) were well mixed and incubated for 5min in separate 96 well plates. Cells were then added, mixed, and the entire volume was transferred to 16-well Nucleocuvette ^TM On the strip. Immediately after electroporation 80. Mu.l of pre-warmed supplemented medium was added to each cuvette and incubated at 37 ℃. After 20min, the cells were incubated at 1X 10 ⁶ Individual cells/ml were transferred to 48-well plates and supplemented with IL-2 500U/ml. Media and IL-2 were supplemented every 2 days and cells were maintained at 1X 10 ⁶ Cell density of individual cells/ml. After flow sorting on days 3-5 after electroporation, the supplemented medium was supplemented with 1% penicillin-streptomycin (10' 000U/ml) and IL-2 (50U/ml) and the cells were allowed to expand for 5-6 days until used in the subsequent experiments. Control T cells were electroporated with incomplete RNP (deletion of specific crrnas), and treated exactly the same as CAR T cells.

In vitro human CD123-CAR killing assay

On the day before co-cultivation, HEK-293 target cells (HEK, HEK-CD123, CD-123 variants) were stained with CTV according to manufacturer's instructions and incubated at 37℃with 5% CO ₂ The cells were kept overnight in the supplemented human medium. Flow-sorted, expanded gfp+ CAR T cells and control cells were added to target cells at an effector to target ratio of 10:1, and at 37 ℃,5% co ₂ Co-cultivation was performed for 24 hours. Specific killing and T cell activation were measured by flow cytometry. Specific killing was calculated according to the formula indicated below: (1-number of live target cells co-cultured with CAR T cells/number of live target cells co-cultured with control cells) ×100. Cell morphology was recorded using a microscope Axio ver A1 (Zeiss).

Human cytokine measurement (ELISA)

Supernatants from co-culture experiments (BiTE and CAR) were harvested and stored at-20 ℃ for human cytokine measurement. Ifnγ was measured using a colorimetric ELISA MAX Standard Set Human IFN γ kit (BioLegend) according to the manufacturer's instructions. Briefly, human ifnγ -specific capture antibodies were coated on 96-well plates and incubated overnight at 4 ℃. The next day, the sample (dilution 1:10) and standard were added and incubated with biotinylated anti-human ifnγ detection antibody. Subsequently, avidin-horseradish peroxidase solution was added and color development was induced using colorimetric substrate TMB terminated with a stop solution. The optical density was read at 450nm using a microplate reader. For each experiment, the standard curve calculated from the standard diluent was run in duplicate. Data are expressed in pg/ml.

Statistical analysis

Statistical analysis was performed on Prism 9.1.2 software (GraphPad). There are N values in each legend.

8.2 results

CAR T cell mediated T cell activation

Fig. 8 shows FACS plots presenting effector T cell activation (CD 69) after co-culturing target cells HEK, HEK-CD123, E51K with control cells or CD123 specific CAR T cells for 1 day. Cd69+ CAR T cells alone (effector T cells) or in the presence of HEK, HEK-CD123 or all CD123 variants after 24h co-culture. Data were normalized to% CD69 in the presence of HEK target cells ⁺ And (3) cells.

CART cell mediated cell killing

Figure 9 shows quantification of HEK, HEK-CD123 and variants thereof by specific killing of CD123 specific CAR T cells measured by flow cytometry on day 1 of co-culture. Error bars show mean ± SD. Data were from 3 independent blood donors and experiments with 2 technical replicates per group.

9. Affinity measurement of selected variants:

9.1 materials and methods

All BLI experiments were performed on Octet RED96e or Octet R8 at 25℃with shaking at 1000rpm using 1 Xkinetic buffer (Sartorius, PN: 18-1105).

Binding of CSL362hIgG1 to CD123ECD wild type and variants thereof

Binding of antibody CSL362 hgg 1 to CD123ECD wild type and variants (analyte) was performed at low (50 nM) and high (300 nM) concentrations of analyte.

The antibody CSL362hIgG1 was captured by an anti-human Fc capture biosensor (AHC) (Sartorius, PN: 18-5060) at 0.5 μg/mL for 300s. The analyte CD123ECD wild-type and variants (CD 123ECD variants only were assayed at high analyte concentrations) were titrated at 7 concentrations of 50nM to 0.78nM (300 nM to 4.7 nM). Association with the analyte was monitored for 300s and dissociation was monitored for 600s (900 s). Double reference subtraction was performed on the complete buffer only and the biosensor loaded with negative hIgG1 control. Regeneration was performed in 10mM Gly-HCl (ph 1.7). The data were analyzed using the Octet data analysis software HT 12.0. The data is fit (where possible) to a 1:1 binding model. The kinetic rates ka and kd were globally fitted. In the qualitative illustrations of fig. 10a,10b,10e and 10F, binding levels were achieved at 280s association for all concentrations.

Binding of 6H6mIgG1 to CD123ECD wild type and variants

Binding of antibody 6H6mIgG1 (bioleged, PN: 30682) to CD123ECD wild-type and variants (analytes) was performed using a streptavidin capture biosensor (SA) (Sartorius, PN: 18-5019). Captureselect was captured at 1 μg/mL on SA tip ^TM Biotin is anti-LC-kappa (murine) (Thermo Fischer, PN: 7103152100) for 600s. The biosensor was then used to capture the antibody 6H6mIgG1 at 2.5. Mu.g/mL for 300s. The analyte CD123ECD wild type and variants were titrated at 7 concentrations of 50 to 0.78 nM. Association with the analyte was monitored for 300s and dissociation was monitored for 600s. Only buffer wells were used entirely as references. Regeneration was performed in 10mM Gly-HCl (pH 1.7). The data were analyzed using the Octet data analysis software HT 12.0. The data is fit (where possible) to a 1:1 binding model. The kinetic rates ka and kd were globally fitted. In the qualitative illustrations in FIGS. 10C and 10D, the values at 250s association were taken for all concentrationsBinding level.

Binding of Fu Tuozhu monoclonal antibody hIgG1 to CD123ECD wild type and variants thereof

Binding of antibody trastuzumab hIgG1 to CD123ECD wild-type was performed at low concentration (50 nM) analyte, and binding of antibody trastuzumab hIgG1 to CD123 variant (analyte) was performed at high concentration (300 nM) analyte.

Antibody Fu Tuozhu monoclonal antibody hIgG1 was captured by an anti-human Fc capture biosensor (AHC) (Sartorius, PN: 18-5060) at 0.5 μg/mL for 300s. The analyte CD123 ECD wild-type and variants (CD 123 ECD variants only were assayed at high analyte concentrations) were titrated at 7 concentrations of 50nM to 0.78nM or 300nM to 4.7nM, respectively. Association with the analyte was monitored for 300s and dissociation was monitored for 600s (CD 123 wild-type) and 900s (CD 123 variant). Double reference subtraction was performed on the complete buffer only and the biosensor loaded with negative hIgG1 control. Regeneration was performed in 10mM Gly-HCl (pH 1.7). The data were analyzed using the Octet data analysis software HT 12.0. The data is fit (where possible) to a 1:1 binding model. Global fits are made to the kinetic rates ka and kd. In the qualitative illustration of fig. 10G, binding levels were obtained at 250s association for all concentrations.

9.2 results

CSL362 antibody was immobilized and binding to recombinant CD123 wild type or indicated variants was measured at various concentrations as a function of time. Although wild-type CD123 bound in a dose-dependent manner (fig. 10a, 10B), variants E51T, E51K, E51Q, S59P, S E and R84E did not bind CSL362 up to 50nM (fig. 10B). Because there is no binding, the inventors were unable to determine affinity. In contrast, wild-type CD123 and all tested variants bound to control antibody 6H6, which bound an epitope that did not overlap with any of the variants. The only variant with reduced binding was R84E, which may indicate that R84E leads to reduced protein stability (fig. 10D). Since the inventors were unable to determine affinity for CSL362 at a maximum analyte concentration of 50nM, experiments were repeated with analytes up to 300 nM. At these concentrations, wild-type CD123 was no longer measured. Even at this very high protein concentration, the test variants characterized as "non-binders" (e.g., fig. 10E, 10F) did not produce detectable binding (e.g., fig. 51T, E51K, E51A, S59P, S59E, S59R; S59F; R84E). Variants that gave very weak binding at 300nM (E51Q, S59Y, R84T and R84Q) were characterized as "weak binders". Thus, for all non-conjugates tested, no interaction was detected between the variant and CSL362, while binding to 6H6 was maintained, except for variant R84E.

CD123 isoforms retain Interleukin-3 binding

10.1 materials and methods

Binding of hIL3 to CD123 ECD wild type and variants thereof

Binding of hIL3 (SinoBiological, PN: 11858-H08H) (analyte) to CD123 ECD wild-type and variants (ligands) was performed using a streptavidin capture biosensor (SA) (Sartorius, PN: 18-5019). CD123 ECD wild type and variants were biotinylated using a type B biotinylation kit (Abcam, PN: ab 201796) according to the manufacturer's instructions. Biotinylated CD123 ECD wild-type and variants (ligands) were captured on the SA tip for 1000s to achieve a nm shift of 1.5 to 2 nm. The analyte hIL3 was titrated at 7 concentrations of 500 to 7.8 nM. Association with the analyte was monitored for 300s and dissociation was detected for 120s. Only buffer was used entirely as reference. No regeneration was performed and a new set of tips was used for each biotinylated capture ligand. The data were analyzed using the Octet data analysis software HT 12.0. Due to the rapid on/off nature of the interaction, the data was analyzed using steady state analysis. In the qualitative illustration in fig. 11, the binding level was obtained at 250s association for all concentrations.

10.2 results

Recombinant wild-type CD123 or indicated variants were biotinylated and captured. Interleukin-3 (IL-3) binding was determined as a function of time.

Recombinant wild-type CD123 and the indicated variants bind IL-3 in a dose-dependent manner. Thus, the non-binding variants did not bind to CSL362, but the protein was normally bound by the control antibodies 6H6 and IL-3 (fig. 11).

Variants S59P and R84E showed slightly reduced binding to IL3 compared to the wild type.

11. Thermally induced refolding

11.1 materials and methods

DSF analysis was performed on a Bio-Rad CFX96 Touch Deep Well RT PCR detection system. Sypro Orange 5000X (Sigma, PN: S5692) in DMSO was used at a final concentration of 5X. In a reaction volume of 20uL, the temperature gradient was from 25 ℃ to 95 ℃ in 1.5 ℃ increments. The "FRET" scan mode is used to monitor fluorescence. All samples were analyzed in triplicate at a final concentration of 0.25 mg/mL. The temperature Tm of the protein unfolding transition is calculated using the first derivative method.

11.2 results

Heat-induced unfolding measurements showed that the thermostability of most variants was comparable to wild-type CD123 (fig. 12). The thermal stability of R84E decreases. This result may explain why R84E exhibits reduced binding to control antibody 6H6, which control antibody 6H6 binds to an epitope that is not affected by the R84E mutation itself. In addition, variant R84Q has a lower Tm.

12. Genetically engineered TF-1 cells expressing CD123 variants

12.1 materials and methods

A red and white blood patient cell line TF-1 (DSMZ No. ACC 334) was used as a model for Hematopoietic Stem Cells (HSC). TF-1 cells were electroporated with CRISPR-Cas9 and HDR templates to knock the E51K and E51T variants into the endogenous DNA of CD 123.

Cas9 Ribonucleoprotein (RNP) was freshly generated prior to each electroporation. Thawed crRNA and tracrRNA (both from IDT Technologies, resuspended at 200. Mu.M) were mixed at a 1:1 molar ratio (120 pmol each), denatured at 95℃for 5min, and annealed at room temperature for 10 to 20min, thereby complexing 80. Mu.M single guide RNA (sgRNA) solutions. Polyglutamic acid (PGA; 15-50 kDa; 100mg/ml; sigma-Aldrich) was added to the sgRNA at a volume ratio of 0.8:1. Finally, 60pmol of recombinant Cas9 (University of California Berkeley, at 40 μm) was mixed with freshly prepared sgrnas (molar ratio Cas9: sgrnas=1:2) into a complex Ribonucleoprotein (RNP), incubated in the dark for 20min at room temperature. 5min before electroporation, 50pmol of HDR template was added to the complex RNP (60 pmol).

Using Neon ^TM The transfection system (Thermo Fischer) was used for electroporation. For each electroporation, 0.2X10 was used ⁶ TF-1 cells were washed twice in PBS and then resuspended in 10. Mu. l R buffer. Cells in R buffer were mixed with the complex RNP/HDR template and electroporated at 1200v,40ms,1 pulse. After electroporation, the cells were incubated in fresh medium with GM-CSF (RPMI-1640, 10% FCS,1% Glutamax,2ng/ml GM-CSF) at 0.4X10 × ⁶ Individual cells/ml were transferred to 48-well plates and split every 2 days.

Engineered cells were batch sorted by flow cytometry based on their CD123 variant expression 12 days after electroporation. Cells were stained with MIRG123 biotin/Strep-PE and 6H6-Bv 650. E51K and E51T knockins (MIRG 123-, 6H2+), CD123 knockouts (MIRG 123-, 6H2-) and wild-type cells (MIRG123+, 6H2+) were sorted and cultured for 14 days, and then the variants were tested for function.

sgRNA for CD123 KO:

GTCTTTAACACACTCGATAT(SEQ ID NO：29)

E51K HDR template:

TTTTAGATCCAAACCCACCAATCACGAACCTAAGGATGAAAGCAAAGGCTCAGCAGTTGACCTGGGACCTTAACAGAAATGTGACaGAcATtaagTGTGTTAAAGACGCCGACTATTCTATGCCGGTAAATCATACTCTCTATTGTTTTTTTATTTTTATTTTATTTATTTATGTATTTA(SEQ ID NO:30)

E51T HDR template:

TTTTAGATCCAAACCCACCAATCACGAACCTAAGGATGAAAGCAAAGGCTCAGCAGTTGACCTGGGACCTTAACAGAAATGTGACaGAcATtaccTGTGTTAAAGACGCCGACTATTCTATGCCGGTAAATCATACTCTCTATTGTTTTTTTATTTTTATTTTATTTATTTATGTATTTA(SEQ ID NO:31)

12.2 results

TF-1 cells with E51K and E51T knockins can be successfully generated. Sorting of the cells confirmed that the E51K and E51T variants showed loss of binding to antibody MIRG123 (data not shown). The cells were classified and analyzed for subsequent experiments.

13. Unbound variants can be enriched by incubation with IL3

13.1 materials and methods

Cells were washed twice in PBS 12 days after electroporation, and then resuspended in 1ml medium containing 2ng/ml GM-CSF (RPMI 1640, 10% FCS,1% Glutamax) or 10ng/ml hIL3 (RPMI 1640, 10% FCS,1% Glatamax) at a concentration of 30 ten thousand cells/ml. Cells were cultured for 6 days and analyzed by flow cytometry on days 0,2,4 and 6. On the day of analysis, 200 μl of cells were removed from the culture and washed in PBS. They were then resuspended in 200. Mu.l of medium containing 2ng/ml GM-CSF in 96-well plates and incubated at 37℃and 5% CO ₂ Incubation was performed for 7H, followed by flow cytometry staining with MIRG 123-boot/Strep-PE and 6H6-Bv 650. Genomic DNA was extracted from 200. Mu.l of culture on days 0,2,4 and 6. On days 2,4 and 6, 400. Mu.l of medium was added to the culture.

13.2 results

Bulk TF-1 cells were cultured as controls (no crRNA), KO (crRNA but no HDRT) or KI (crRNA+KI HDRT, E51K or E51T). Control cells cultured with IL-3 or GM-CSF maintained MIRG123+, 6H2+. KO cells cultured with GM-CSF largely retain MIRG123-, 6H2+. In contrast, MIRG123+, 6H2+ cell populations were detectable in cultures containing KO cells gradually cultured with IL-3. On day 6, the mirg123+,6h6+ population predominated, indicating that cells expressing CD123 receptor have a competitive advantage in the presence of IL 3. In KI cells (E51K or E51T), the population of MIRG 123-6H26+ cells and the population of MIRG123+6H26+ cells gradually increased with IL 3. This is less pronounced in cells cultured with GM-CSF. Thus, KI cells (MIRG 123-6H2+) have functional receptors. See fig. 13.

14. Gene-edited cells retain response to stimulation by IL3

14.1 materials and methods

TF-1 cells are known to respond to stimulation by GM-CSF, IL3, and SCF. TF-1 cells (wild-type, knocked-out, and E51K and E51T knockin cells) were tested for their ability to be stimulated by IL 3.

TF-1 (ACC 334, DSMZ) cells were maintained in the presence of 2mM Glutamax supplemented with 10% heat-inactivated FCS ^TM (Gibco) and 2ng/ml hGM-CSF (215-GM, boot-techne) in RPMI-1640 medium. For hybridization of Cas9 with the tracRNA/crRNA mixture (SEQ ID Nos. 29-31), PGA was added (see example 8.1)"by non-disease based Editing of toxic CRISPR/Cas9 produces human CD123-CAR T cells'The method described in (a). Immediately prior to electroporation, 50pmol of 180bp long ssDNA HDR template (Ultramer DNA oligonucleotide, IDT) was added to the RNP. For each electroporation, 200,000 TF-1 cells were washed twice with PBS and resuspended in 10. Mu. l R buffer (Neon ^TM Transfection system 10 uL). The RNP/HDR template mixture was slowly added and cells were electroporated using a Neon transfection system/Thermo Fisher with 1200V,40ms,1 pulse setup. The electroporated cells were expanded every 2 to 3 days for 11 days for enrichment assays and for 12 days, then flow cytometry sorted for functional assays.

The sorted cells were washed once in PBS and then they were distributed in 96-well white microplate clear flat bottom (Greiner bio-one). In each well, 10,800 cells were cultured in 150. Mu.l of medium without GM-CSF (RPMI-1640, 10% FCS,1% Glutamax) at increasing concentrations of hIL3 (0.2 ng/ml,0.8ng/ml,3.13ng/ml,12.5n g/ml,50 ng/ml). Using 15. Mu.l (Promega) 5% CO at 37 ℃C ₂ Cell proliferation was measured after 72 hours down. Luminescence was read using a Synergy H1 (BioTek), with an integration time of 1s.

14.2 results

The results are shown in fig. 14. TF-1 knock-in cells expressing E51K and E51T variants of CD123 can proliferate to a similar extent as TF-1 cells expressing wild-type CD123 upon addition of hIL 3. The knockdown cells showed only minimal response to hll 3.

15. The genetically edited cells were protected from blockage by MIRG123

15.1 materials and methods

Antibody MIRG123 was tested for its ability to bind to TF-1 cells (wild type, knocked-out, and E51K and E51T knockin cells).

The sorted cells were washed once in PBS and then they were distributed in a 96-well white microplate transparent flat bottom (Greiner bio-one). In each well, 10,800 cells were cultured with fixed concentrations of hIL3 (2.5 ng/ml) but different concentrations of MIRG123 (0.0013 nM, 0.04 nM,0.012nM,0.036nM,0.11nM,0.33nM,1 nM) in 150. Mu.l medium without GM-CSF (RPMI-1640, 10% FCS,1% Glutamax). Using 15. Mu.l(Promega) 5% CO at 37 ℃C ₂ Cell proliferation was measured after 72 hours down. Luminescence was read using a Synergy H1 (BioTek), with an integration time of 1s.

15.2 results

The results are shown in fig. 15. MIRG123 resulted in dose-dependent proliferation blocking and apoptosis of IL3 stimulated wild-type TF-1 cells. In contrast, TF-1 knockdown cells expressing E51K and E51T variants of CD123 were effectively protected from the blocking effect of MIRG 123.

Editing of HSPC

16.1 materials and methods

HSPC was supplemented with HSC Brew GMP supplement, 2% human serum albumin, 100ng/mL SCF,100ng/mL TPO,100ng/mL Flt3L and 60ng/mL IL3 (Miltenyi) at 0.5X10 in HSC Brew-GMP basal medium (Miltenyi) ⁶ cell/mL density thawing. Cells were electroporated after 2 days. Thawed crRNA and tracrRNA (both from IDT Technologies, resuspended at 200. Mu.M) were mixed at a 1:1 molar ratio (120 pmol each), denatured at 95℃for 5min, and annealed at Room Temperature (RT) for 5min to complex 80. Mu.M guide RNA (gRNA) solutions. Finally, 1. Mu.M Spyfi Cas9 (Alvetron, at 61.889. Mu.M) was combined with freshly prepared gRNA (molar ratio Cas9: gRN) into a complex Ribonucleoprotein (RNP)A=1:2) and incubated at room temperature for 20min. During RNP complexing, HSPC cells were collected, washed twice with electroporation buffer (Miltenyi) and incubated at 1X 10 ⁶ Each cell was resuspended in electroporation buffer solution at 50. Mu.l. Cells were then mixed with RNP (5 μl) and HDRT (5 μl, corresponding to 500 pmol) and the whole volume was transferred to electroporation nucleocuvette. Electroporation was performed with Miltenyi CliniMACS Prodigy using the following setup: 600V 100 μs shock wave (burst)/400V 750 μs square wave (square). Immediately after electroporation, cells were transferred to 6-well plates and allowed to stand at room temperature for 20min. After 20min, 2mL of pre-warmed HSPC medium supplemented with 100ng/mL SCF,100ng/mL TPO and 100ng/mL Flt3L was added to each well and the plates were incubated at 37 ℃. Cells were collected at different time points and stained with antibody 6H6-BV650 and MIRG 123-boot/Strep-PE for flow cytometry. Genomic DNA (gDNA) was extracted for sequencing analysis using rapid DNA extraction (Lucigen). The sgRNA for CD123 KO and the HDR templates for E51K and E51T are shown in example 12 as SEQ ID NOs 29-31, respectively.

16.2 results

Mobilized peripheral blood cd34+hspcs carrying E51K and E51T knockins could be successfully generated (fig. 16). Cells were analyzed by FACS and successful knockins were verified by Sanger sequencing. HSPCs electroporated with RNP (KO) alone showed an increase in the fraction of CD123 negative cells. In contrast, the E51K and E51T variants showed a loss of binding to antibody MIRG123, but maintained expression of CD123 as assessed by control antibody 6H6 (fig. 17). Using CD90 and CD45RA we demonstrate that LT HSCs (long term repopulating hematopoietic stem cells) as well as MPP1 cells (cd34+cd38-CD 90-CD45 RA-) and MPP2 cells (cd34+cd38-CD 90-CD45 ra+) were also edited (fig. 18).

17. In vitro HSPC killing assay with BiTE

17.1 materials and methods

For the BiTE killing assay, HSPCs were compiled as described above. Human T cells of the same HSPC donor were isolated from PBMCs by magnetic negative selection using EasySep human T cell isolation kit (Stemcell Technologies) according to the manufacturer's instructions. Isolated T cells were cultured overnight in non-stimulated supplemented medium. 2 days after electroporation, edited HSPC were co-cultured with human effector T cells (at 3:1 effector to target ratio) and CD3/CSL362BiTE (at 100 ng/ml) in 96U-shaped bottom plates for 72h at 37 ℃. Cytotoxic activity (specific killing and elimination of HSPCs) was analyzed by flow cytometry. The specific killing was calculated as follows: (number of viable target cells in the presence of BiTE/number of viable target cells in the absence of BiTE) ×100.

17.2 results

Co-culture of human effector T cells with control or edited HSPC cells (E: T=3:1) in the presence of CD3/CSL362 BiTE at a concentration of 100ng/ml resulted in a reduction of wild type HSPC when treated with BiTE, as measured by quantification of flow cytometry patterns. In contrast, HSCs expressing CD 123E 51K or E51T variants were protected and enriched as demonstrated by the increased MIRG123-6H6+ cell population. See fig. 19 and 20. In addition, after 72 hours of co-culture, HSC cells were sorted into CD123 antibody clones 6h6+ and 6H 6-cells using flow cytometry.

Enrichment of knockin cells was confirmed by Sanger sequencing of the corresponding cell population.

18. In vivo transplantation of edited HSPC

18.1 materials and methods

HSC cells were edited as described above. After 1 day of electroporation, cells were collected, washed once with PBS, and washed at 10X 10 ⁶ The concentration of viable cells/ml was resuspended in PBS. Cells were injected intravenously into NSG-SGM3 female mice (3 weeks old) irradiated with 200cGy the previous day. Transplantation was monitored by FACS by analyzing peripheral blood after 6 and 10 weeks of staining for mouse and human CD 45. Mice were euthanized 13 weeks after humanization to analyze blood, spleen and bone marrow.

18.2 results

HSPCs were quantified by measuring the percentage of human CD45 (human chimerism) in the spleen of mice 13 weeks after HSPCs injection. Mice that received E51K or E51T knockin HSPCs were successfully transplanted and showed evidence of human cd45+ immune cell development. Furthermore, the presence of B cells (measured by CD 19), T cells (measured by CD 3) and cd33+ myeloid cells among human CD45 cells in spleen and bone marrow demonstrated the successful multilineage differentiation potential of engineered E51K or E51T knockins into HSPCs. Human HSPCs were detected in bone marrow.

Sequences for practicing the invention

Human CD123 (SEQ ID NO: 1)

MVLLWLTLLLIALPCLLQTKEDPNPPITNLRMKAKAQQLTWDLNRNVTDIECVKDADYSMPAVNNSYCQFGAISLCEVTNYTVRVANPPFSTWILFPENSGKPWAGAENLTCWIHDVDFLSCSWAVGPGAPADVQYDLYLNVANRRQQYECLHYKTDAQGTRIGCRFDDISRLSSGSQSSHILVRGRSAAFGIPCTDKFVVFSQIEILTPPNMTAKCNKTHSFMHWKMRSHFNRKFRYELQIQKRMQPVITEQVRDRTSFQLLNPGTYTVQIRARERVYEFLSAWSTPQRFECDQEEGANTRAWRTSLLIALGTLLALVCVFVICRRYLVMQRLFPRIPHMKDPIGDSFQNDKLVVWEAGKAGLEECLVTEVQVVQKT

anti-CD 123 antibody (Fu Tuozhu mab) VH (SEQ ID NO: 11)

EVQLVQSGAELKKPGASVKVSCKASGYTFTDYYMKWVRQAPGQGLEWIGDIIPSNGATFYNQKFKGRVTITVDKSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSS

anti-CD 123 antibody (Fu Tuozhu mab) VL (SEQ ID NO: 12)

DFVMTQSPDSLAVSLGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIK

anti-CD 123 antibody (Vibecostab) VH CDR1 (SEQ ID NO: 13)

QVQLQQSGAEVKKPGASVKVSCKASGYTFTDYYMKWVKQSHGKSLEWMGDIIPSNGATFYNQKFKGKATLTVDRSTSTAYMELSSLRSEDTAVYYCARSHLLRASWFAYWGQGTLVTVSS

anti-CD 123 antibody (Vibecostab) VL CDR1 (SEQ ID NO: 14)

DFVMTQSPDSLAVSLGERATINCKSSQSLLNTGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGGGTKLEIK

anti-CD 123 CAR (SEQ ID NO: 15)

MALPVTALLLPLALLLHAARPDIVMTQSPDSLAVSLGERATINCESSQSLLNSGNQKNYLTWYQQKPGQPPKPLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPYTFGQGTKLEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTDYYMKWARQMPGKGLEWMGDIIPSNGATFYNQKFKGQVTISADKSISTTYLQWSSLKASDTAMYYCARSHLLRASWFAYWGQGTMVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

gRNATRAC(SEQ ID NO:24)

AGAGTCTCTCAGCTGGTACA

CD8 hinge Domain (SEQ ID NO: 25)

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

CD8 transmembrane domain (SEQ ID NO: 26)

IYIWAPLAGTCGVLLLSLVIT

4-1BB cytoplasmic domain (SEQ ID NO: 27)

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

CD 3-zeta domain (SEQ ID NO: 28)

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. A mammalian cell or cell population expressing a first isoform of CD123 for use in medical treatment in a patient in need thereof having a second isoform expressing said surface protein Cell,

wherein said cell expressing said first isoform comprises genomic DNA having at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is absent In the genome of said patient having cells expressing said second isoform of said surface protein, and wherein said first isoform and said second isoform are functional.

2. The mammalian cell or cell population for use according to claim 1, wherein the first isoform and the second isoform of CD123 are involved in IL-3 binding, IL-3-dependent proliferation. , are functional in terms of cell surface expression or intracellular signaling capabilities.

3. A mammalian cell or cell population for use according to claim 1 or 2, wherein the polymorphic or genetically engineered allele is characterized by at least one substitution of an amino acid at position E51 or S59 of SEQ ID NO: 1.

4. The mammalian cell or cell population for use according to claim 3, wherein the residue E51 is substituted with an amino acid selected from the group consisting of K, N, T, R, M, G and A, preferably with an amino acid selected from the group consisting of K, A or T, and/or the residue S59 is substituted with an amino acid selected from the group consisting of I, P, E, L, K, F, R and Y; preferably with an amino acid selected from the group consisting of P, E, R or F.

5. A mammalian cell or cell population for use according to any one of claims 1 to 4, wherein said cells expressing said first isoform are selected from a subject comprising native genomic DNA, The native genomic DNA has at least one native polymorphic allele in the nucleic acid encoding the first isoform.

6. A mammalian cell or cell population for use according to any one of claims 1 to 4, wherein the first isoform is obtained by ex vivo modification of the nucleic acid sequence encoding the surface protein via gene editing, preferably by introducing into the cell a gene editing enzyme capable of inducing site-specific mutations within a target sequence encoding a surface protein region involved in the binding of an agent comprising at least a first antigen binding region.

7. The mammalian cell or cell population according to any one of claims 1 to 6, preferably hematopoietic stem cells, wherein the medical treatment includes:

A therapeutically effective amount of said cell or cell population expressing said first isoform is administered to said patient in need thereof, preferably in the treatment of a hematopoietic disease, preferably in the treatment of a malignancy, in combination with a therapeutically effective amount of a depleting agent. To restore normal hematopoiesis after immunotherapy for hematopoietic diseases, the depleting agent includes at least a first antigen-binding region, and the at least first antigen-binding region specifically binds to the second isotype to specifically deplete expression of the second isotype. Types of patient cells with hematopoietic malignancies such as acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN), or B-acute lymphoblastic leukemia (B-ALL).

8. Mammalian cell or cell population for use according to claim 7, wherein the depleting agent is an antibody, an antibody-drug conjugate or an immune cell, preferably bearing a chimeric antigen receptor (CAR) T cells, the chimeric antigen receptor (CAR) comprising a first antigen-binding region that specifically binds the second isotype without binding the first isotype.

9. A mammalian cell or cell population for use according to claim 8, wherein said surface protein is CD123, and wherein said first antigen-binding region of said depleting agent specifically binds to an epitope, said Said epitope includes amino acids T48, D49, E51, A56, D57, Y58, S59, M60, P61, A62, V63, N64, T82, R84, V85, A86, N87, P89, F90, S91 of SEQ ID NO: 1, Preferably wherein said first antigen binding region comprises an antigen binding region having the same epitope specificity as an antigen binding region comprising:

a) An antibody heavy chain variable domain (VH) comprising three CDRs: VHCDR1, VHCDR2 and VHCDR3, wherein VHCD1 is SEQ ID NO:2, VHCD2 is SEQ ID NO:3 and VHCDR3 is SEQ ID NO:4; and

b) an antibody light chain variable domain (VL) comprising three CDRs: VLCDR1, VLCDR2 and VLCDR3, wherein VLCDR1 is SEQ ID NO: 5 or 6, VLCDR2 is SEQ ID NO: 7, and VLCDR3 is SEQ ID NO: 8, more preferably wherein the first antigen-binding region comprises a heavy chain variable domain and/or a light chain variable domain, the heavy chain variable domain comprising any one of the amino acid sequences selected from SEQ ID NOs: 9, 11 and 13 or consisting of any one of the amino acid sequences of SEQ ID NOs: 9, 11 and 13, and the light chain variable domain comprising any one of the amino acid sequences selected from SEQ ID NOs: 10, 12 and 14 or consisting of any one of the amino acid sequences of SEQ ID NOs: 10, 12 and 14.

10. A mammalian cell or cell population for use according to any one of claims 1 to 6, wherein the medical treatment comprises:

A therapeutically effective amount of said cell or cell population expressing said first isoform is administered to said patient in need thereof, preferably for adoptive cell transfer therapy, more preferably with a therapeutically effective amount of a depleting agent. For treating malignant hematopoietic diseases, the depleting agent includes at least a second antigen-binding region, and the at least second antigen-binding region specifically binds to the first isotype to specifically deplete transferred cells expressing the first isotype. , the malignant hematopoietic disease such as acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm (BPDCN) or B-acute lymphoblastic leukemia (B-ALL), again more preferably, wherein the The depleting agent is subsequently administered to the cell or cell population expressing the first isoform of the surface protein to avoid eventual serious side effects, such as graft versus host disease due to transplantation.

11. A mammalian cell or cell population for use according to claim 10, wherein the cell or cell population expressing the first isotype is an immune cell, preferably a T cell carrying a chimeric antigen receptor (CAR).

12. A mammalian cell or cell population for use according to claim 11, wherein the CAR comprises an antigen binding region that specifically binds to an epitope of CD123 located within the third extracellular loop or within a polypeptide, the polypeptide comprising amino acids T48, D49, E51, A56, D57, Y58, S59, M60, P61, A62, V63, N64, T82, R84, V85, A86, N87, P89, F90, S91 of SEQ ID NO: 1, preferably wherein the first antigen binding region comprises an antigen binding region having the same epitope specificity as an antigen binding region comprising:

b) Antibody light chain variable domain (VL), which contains three CDRs: VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 5 or 6, VLCDR2 is SEQ ID NO: 7, and VLCDR3 is SEQ ID NO: 8. wherein the antigen-binding region comprises a heavy chain variable domain and/or a light chain variable domain, the heavy chain variable domain comprising any one of the amino acid sequences selected from SEQ ID NO: 9, 11 and 13 One or consisting of any one of the amino acid sequences of SEQ ID NO: 9, 11 and 13, the light chain variable domain comprising any one of the amino acid sequences of SEQ ID NO: 10, 12 and 14 or consisting of Consisting of any one of the amino acid sequences of SEQ ID NO: 10, 12 and 14, again more preferably, wherein the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 15.

13. A pharmaceutical composition comprising mammalian cells, preferably hematopoietic stem cells or immune cells, such as hematopoietic stem cells or immune cells, and preferably a consuming agent as defined in claim 8 or 9 and a pharmaceutically acceptable carrier. A T cell as defined in any one of claims 1 to 12.

14. A depleting agent for preventing or reducing the risk of serious side effects in a patient who has received cells expressing a first isoform of a surface protein, wherein the patient's native cells express a second isoform of the surface protein type, and wherein said depleting agent comprises at least a second antigen-binding region that specifically binds said first isotype without binding said second isotype, preferably wherein said surface The protein is CD123.

15. A depleting agent for selectively depleting host cells of a patient in need thereof, wherein the patient's native cells express a second isoform of a surface protein, and wherein the depleting agent comprises at least one that specifically binds a first antigen binding region of said second isotype, preferably wherein said surface protein is CD123, and wherein said first antigen binding region of said depleting agent specifically binds to an epitope comprising SEQ Amino acids T48, D49, E51, A56, D57, Y58, S59, M60, P61, A62, V63, N64, T82, R84, V85, A86, N87, P89, F90, S91 of ID NO: 1, more preferably among which The first antigen-binding region includes an antigen-binding region having the same epitope specificity as an antigen-binding region including:

b) Antibody light chain variable domain (VL), which contains three CDRs: VLCDR1, VLCDR2 and VLCDR3, where VLCDR1 is SEQ ID NO: 5 or 6, VLCDR2 is SEQ ID NO: 7, and VLCDR3 is SEQ ID NO: 8. Again more preferably, wherein the first antigen binding region comprises a heavy chain variable domain and/or a light chain variable domain, the heavy chain variable domain comprising one selected from the group consisting of SEQ ID NO: 9, 11 and 13 Any one of the amino acid sequences or consisting of any one of the amino acid sequences of SEQ ID NO: 9, 11 and 13, the light chain variable domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 12 and 14 or consisting of any one of the amino acid sequences of SEQ ID NO: 10, 12 and 14.