Siglec-6 binding polypeptides

Abstract

The problem to be solved by the present invention is to overcome the unmet clinical need by providing improved compositions for the therapeutic treatment of patients.SOLUTION: The present invention relates to a Siglec-6 binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to Siglec-6 or comprising or consisting of a Siglec-6 binding chimeric antigen receptor (CAR), a polynucleotide encoding the Siglec-6 binding polypeptide, an expression vector comprising the polynucleotide, an immune cell comprising the polypeptide, polynucleotide or expression vector, a method for producing the immune cell and a pharmaceutical composition comprising the immune cell. The immune cells and pharmaceutical compositions of the invention may be used in methods for treating a disease, such as cancer, in a patient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to Siglec-6-binding polypeptides comprising or consisting of an antibody or fragment thereof that binds to Siglec-6, or a Siglec-6-binding chimeric antigen receptor (CAR), polynucleotides encoding the Siglec-6-binding polypeptides, expression vectors comprising the polynucleotides, immune cells comprising the polypeptides, polynucleotides or expression vectors, and methods for producing such immune cells and pharmaceutical compositions comprising such immune cells. The immune cells and pharmaceutical compositions of the present invention may be used in methods for treating disease in patients. [Background technology]

[0002] T cells harboring synthetic chimeric antigen receptors (CARs) have demonstrated long-term remission in B-cell and plasma cell malignancies [1-4], and targeting of other hematologic and solid tumors with CAR T cells is being explored. Acute myeloid leukemia (AML) represents a disease with unmet medical need, necessitating novel curative therapies. Although several preclinical studies have demonstrated the feasibility of targeting AML with CAR T cells, identifying CAR target antigens with acceptable safety profiles remains challenging [5]. This is primarily due to the expression in AML of previously identified candidate CAR target antigens on normal hematopoietic stem and progenitor cells (HSC / Ps) [6, 7]. Therefore, novel CAR targets that are not expressed by HSC / Ps and are not or minimally expressed on other healthy cells and tissues are highly desirable.

[0003] CAR T cells targeting B-cell and plasma cell malignancies have shown unprecedented clinical responses in patients with multiple first-line treatments and advanced hematologic malignancies [1-4], spurring interest in the use of CAR T-cell therapy in AML. Because CAR T cells target cell surface molecules, it is desirable that the target antigen be uniformly expressed on tumor cells and minimally or even absent on healthy cells and tissues. This requirement poses a challenging challenge for CAR T therapy in AML, as most of the candidate antigens proposed to date are expressed by healthy HSCs / Ps and innate immune cells

[20] . Therefore, directing CAR T cells against such antigens is predicted to cause unwanted toxicities, including the reduction or elimination of healthy hematopoiesis. Indeed, CAR T cells directed against myeloid antigens, such as CD123 and CD33, have been shown to be myeloablative and require allogeneic hematopoietic stem cell transplantation (allo-HSCT) to reconstitute normal hematopoiesis [19, 22]. Of note, the clinical use of CD123-specific CAR T cells has resulted in significant and unexpected toxicity and fatal adverse events in a first-in-human clinical trial utilizing an allogeneic CD123-CAR T cell product, resulting in the trial being put on hold

[21] . To prevent the elimination of normal HSC / Ps, Kim et al. suggested that gene editing using CRISPR / Cas9 to knock down CD33 in donor HSC / P cells could prevent the elimination of HSC / Ps by CD33-CAR T cells

[22] . However, clinical implementation of this strategy, which entails the administration of gene-edited HSCs (in addition to the genetically modified CD33-CAR T cells), is labor-intensive, complex, and expensive. Additionally, the use of gene-edited HSCs entails a substantially greater risk of unwanted genotoxicity, including the risk of malignant transformation.

[0004] Additionally, other potential CAR targets of interest in AML include FLT3, CLL-1, CD44v6, CD7, folate receptor β, and Lewis-y antigen. +We have previously shown that targeting FLT3 with CAR T cells in AML can induce complete remission in mouse xenografts, and the anti-leukemia efficacy of FLT3 CAR T cells can be enhanced with a FLT3 inhibitor

[23] . However, FLT3 is also expressed on HSC / P, and FLT3-CAR T cell treatment is predicted to induce myeloablation

[23] . C-type lectin-like molecule 1 (CLL-1) is expressed by AML blasts and is also present on lung and gastrointestinal epithelial cells

[24] . We observed CLL-1 expression on HSC / P, suggesting that this may result in severe toxicity when targeted by CLL-1-specific CAR T cells. Although CD44v6 is not present on HSC / P, its expression in critical cells and tissues, such as keratinocytes, oral mucosa, and monocytes, can cause fatal toxicity when targeted by CD44v6-CAR T cells

[25] . Because CD7 is expressed at high levels on T cells by only approximately 30% of AML patients

[26] , it is necessary to knock out CD7 from T cells to generate anti-CD7-CAT T cells, which further complicates clinical application.

[0005] Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a member of the immunoglobulin superfamily of cell surface receptors primarily expressed by leukocytes and associated with inhibitory signaling in human immune cells [8]. Notably, Siglec-2 (CD22) and Siglec-3 (CD33), members of the Siglec superfamily, are of interest as CAR target antigens in hematological malignancies, such as B-cell acute lymphoblastic leukemia (B-ALL) and AML, respectively. Encouragingly, CD22-targeted CAR T cells have been shown to induce complete remission in patients with relapsed / refractory (R / R) B-ALL [9], indicating that targeting Siglecs with favorable expression profiles can induce remission of leukemia and potentially cure patients.

[0006] Baskar et al. generated a monoclonal antibody (mAb) from a repertoire of antibodies following allogeneic hematopoietic stem cell transplantation (allo-HSCT), which potentially contributed to the graft-versus-leukemia (GVL) response in patients with chronic lymphocytic leukemia (CLL)

[10] . Subsequent target discovery analysis revealed the mAb "JML-1" as a candidate that binds to and recognizes the human Siglec-6 protein

[11] . Siglec-6 belongs to the CD33-related Siglec subfamily and is structurally closely related to Siglec-3 (CD33). Siglec-6 consists of three extracellular immunoglobulin (Ig) domains and two intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motifs [12-14]. Due to these ITIM motifs, Siglec-6, like other CD33-related Siglecs, is thought to act as a regulator of pathway activation

[13] . Siglec-6 expression has been reported in primary B cells [10, 12], and aberrant expression in CLL [10, 11] and MALT lymphoma

[15] . Siglec-6 has also been found to be expressed on the placenta [12, 16] and human mast cells [17, 18]. However, unlike other Siglec proteins, it is not present on NK cells, T cells, neutrophils, macrophages, or monocytes

[13] .

[0007] In view of the above, there remains a significant need for new therapies that provide safe and effective treatment for leukemias and lymphomas, particularly AML, CLL, MALT lymphomas and clonal mast cell diseases. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2008 / 119567 [Non-patent literature]

[0009] [Non-Patent Document 1] Kim MY, Yu KR, Kenderian SS, et al. Genetic inactivation of CD33 in hematopoietic stem cells to enable CAR T cell immunotherapy for acute myeloid leukemia. Cell. 2018; 173(6): 1439-1453. e1419. [Non-patent document 2] BaskarS, Suschak JM, Samija I, et al. A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display. Blood, The o\Journal of the American Society of Hematology. 2009; 114(20): 4494-4502. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention aims to overcome unmet clinical needs by providing improved compositions for the therapeutic treatment of patients.

[0011] We demonstrate Siglec-6 expression on primary AML blasts from newly diagnosed and relapsed / refractory AML patients. Interestingly, we also demonstrate Siglec-6 expression on AML leukemia stem cells (LSCs). Human CD4 + and CD8 +T cells were equipped to express a Siglec-6-specific CAR (JML-1-CAR) with a targeting domain derived from the fully human JML-1 IgG1 mAb. The anti-leukemic reactivity of JML-1-CAR T cells derived from AML patients and HD patients against primary "bulk" AML blasts, AML leukemia stem cells, and AML cell lines was evaluated and demonstrated. Furthermore, we assessed Siglec-6 expression on normal hematopoietic stem / progenitor cells (HSC / P) and mature peripheral blood cells to evaluate potential hematologic toxicity in on-target and off-tumor JML-1-CAR T cell-mediated tumor responses. We demonstrated that Siglec-6 is not expressed on normal HSC / P, demonstrating that normal HSC / P are not recognized by JML-1-CAR T cells. High levels of Siglec-6 were confirmed on malignant B-CLL cells and healthy B cells from untreated CLL patients, demonstrating the anti-leukemic activity of JML-1 CAR-T cells against CLL.

[0012] This application provides the first valid demonstration that a treatment using immune cells that bind to Siglec 6, such as immune cells that contain a CAR that binds to Siglec 6, is effective. Such a treatment involves the removal of Siglec 6-expressing cells.

[0013] Furthermore, the present application confirms that Siglec-6 is not expressed on non-cancerous hematopoietic stem / progenitor cells (HSC / P), suggesting that targeting Siglec-6 with immune cells, e.g., CAR-T cells, may be a safe approach for treating cancer, e.g., AML, and may not require subsequent allo-HSCT. Thus, the present application presents for the first time a treatment using immune cells that bind to Siglec-6, which does not involve the removal of non-cancerous HSC / P, thereby eliminating the need for allo-HSCT after such immunotherapy. Furthermore, the use of immune cells that bind to Siglec-6 may obviate the need for post-treatment depletion of the immune cells. [Means for solving the problem]

[0014] Therefore, the present invention provides the following preferred embodiments. [1] A Siglec-6-binding polypeptide comprising or consisting of an antibody or a fragment thereof that binds to Siglec-6, or comprising or consisting of a chimeric antigen receptor (CAR). [2] The Siglec-6-binding polypeptide according to [1], which comprises or consists of an antibody that binds to Siglec-6 or a fragment thereof. [3] The Siglec-6-binding polypeptide according to [1] or [2], which is at least bispecific. [4] A Siglec-6-binding polypeptide according to [2] or [3], comprising or consisting of a first antibody or fragment thereof that binds to Siglec-6 and a second antibody or fragment thereof that binds to a target other than Siglec-6, optionally linked to each other via a linker. [5] A Siglec-6-binding polypeptide described in any one of [2] to [4], wherein the antibody or fragment thereof that binds to Siglec-6 is represented by the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 25. [6] The Siglec-6-binding polypeptide according to [4] or [5], which is capable of binding to immune cells, such as T cells or NK cells, preferably T cells. [7] The Siglec-6-binding polypeptide according to any one of [4] to [6], which further binds to CD3, for example, CD3 zeta or CD3 epsilon, preferably CD3 zeta. [8] A Siglec-6-binding polypeptide according to any one of [4] to [7], which is capable of recruiting immune cells, such as T cells or NK cells, preferably T cells, to target cells expressing Siglec-6 on their surface. [9] The Siglec-6-binding polypeptide according to any one of [2] to [5], which is conjugated to a drug.

[10] The Siglec-6-binding polypeptide according to [9], wherein the drug is a toxin.

[11] The Siglec-6-binding polypeptide according to [1] or [3], which comprises or consists of a Siglec-6-binding CAR.

[12] The Siglec-6-binding polypeptide according to

[11] , wherein the CAR comprises at least one extracellular ligand-binding domain, a transmembrane domain, and at least one intracellular signaling domain.

[13] The Siglec-6-binding polypeptide according to

[12] , wherein the extracellular ligand-binding domain comprises a Siglec-6-binding element.

[14] The Siglec-6-binding polypeptide according to

[13] , wherein the Siglec-6-binding element is represented by the amino acid sequence shown in SEQ ID NO: 25 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 25.

[15] The Siglec-6-binding polypeptide according to any one of

[12] to

[14] , wherein the extracellular ligand-binding domain comprises a spacer domain, for example, a spacer domain derived from CD8α, IgG3, or IgG4.

[16] The Siglec-6-binding polypeptide according to any one of

[12] to

[15] , wherein the transmembrane domain preferably comprises a CD28 transmembrane domain represented by the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13.

[17] The Siglec-6-binding polypeptide according to any one of

[12] to

[16] , wherein the intracellular signaling domain comprises a costimulatory domain and a CD3 zeta domain, and the costimulatory domain is preferably a CD28 cytoplasmic domain or a 4-1BB costimulatory domain.

[18] The Siglec-6-binding polypeptide according to

[17] , wherein the costimulatory domain is a CD28 cytoplasmic domain.

[19] The Siglec-6-binding polypeptide according to

[17] or

[18] , wherein the CD28 cytoplasmic domain is represented by the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 15.

[20] The Siglec-6-binding polypeptide according to

[17] , wherein the 4-1BB costimulatory domain is represented by the amino acid sequence set forth in SEQ ID NO: 17 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 17.

[21] The Siglec-6-binding polypeptide according to any one of

[17] to

[20] , wherein the CD3 zeta domain is represented by the amino acid sequence set forth in SEQ ID NO: 19 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 19.

[22] A Siglec-6-binding polypeptide according to any one of

[12] to

[19] and

[21] , comprising an amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33.

[23] A polynucleotide or a set of polynucleotides encoding the Siglec-6-binding polypeptide according to any one of [1] to

[22] .

[24] A polynucleotide or set of polynucleotides according to

[23] , comprising a nucleotide sequence represented by SEQ ID NO: 26 or a nucleotide sequence having at least 80% identity to the nucleotide sequence shown in SEQ ID NO: 26.

[25] A polynucleotide or set of polynucleotides according to

[23] or

[24] , comprising a nucleotide sequence represented by any one of SEQ ID NOs: 28, 30, 32 or 34, or a nucleotide sequence having at least 80% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 28, 30, 32 or 34.

[26] The polynucleotide according to any one of

[23] to

[25] , further comprising adjacent segments in the 5' and 3' directions of the polynucleotide encoding the polypeptide.

[27] The polynucleotide according to

[26] , wherein the adjacent segment in the 5' direction is a left inverted repeat / direct repeat (IR / DR) segment and the adjacent segment in the 3' direction is a right inverted repeat / direct repeat (IR / DR) segment.

[28] The polynucleotide according to

[27] , wherein the left IR / DR segment is represented by SEQ ID NO: 43 and the right IR / DR segment is represented by SEQ ID NO: 44.

[29] The polynucleotide according to any one of

[23] to

[28] , comprising a nucleotide sequence of a left IR / DR, a polynucleotide sequence encoding a Siglec-6-binding polypeptide, and a nucleotide sequence of a right IR / DR.

[30] An expression vector comprising the polynucleotide or set of polynucleotides described in any one of

[23] to

[29] .

[31] The expression vector according to

[30] , which is a non-viral vector or a viral vector.

[32] The expression vector according to

[31] , which is a non-viral vector.

[33] The expression vector according to

[32] , which is a minimal DNA expression cassette.

[34] The expression vector according to

[32] or

[33] , which is a transposon donor DNA molecule.

[35] The expression vector according to

[34] , wherein the transposon donor DNA molecule is a Sleeping Beauty or PiggyBac transposon donor DNA molecule.

[36] The expression vector according to any one of

[32] to

[35] , which is a minicircle DNA.

[37] The expression vector according to

[31] , which is a viral vector.

[38] The expression vector according to

[37] , which is a lentiviral or gamma-retroviral vector.

[39] An immune cell comprising an expression vector comprising a Siglec-6-binding polypeptide described in any one of

[11] to

[22] , and / or a polynucleotide or set of polynucleotides encoding a Siglec-6-binding polypeptide described in any one of

[11] to

[22] , and / or a polynucleotide or set of polynucleotides encoding a Siglec-6-binding polypeptide described in any one of claims

[11] to

[22] .

[40] The immune cell according to

[39] , which expresses a polynucleotide or a set of polynucleotides and / or a vector.

[41] The immune cell according to any one of

[39] to

[40] , which is a lymphocyte.

[42] The immune cell according to

[41] , wherein the lymphocyte is a T cell or an NK cell.

[43] The above T cells are CD4 + cells or CD8 +

[42] The immune cell described in

[42] .

[44] The immune cell according to any one of

[39] to

[43] , which further expresses a detectable marker.

[45] The immune cell according to any one of

[39] to

[44] , which is a human cell.

[46] (a) isolating immune cells from a blood sample of a subject; (b) transforming or transducing immune cells with the polynucleotide according to any one of

[23] to

[29] or the expression vector according to any one of

[30] to

[38] ; (c) optionally purifying the transformed or transduced immune cells; 1. A method for generating (recombinant) immune cells, comprising:

[47] The method according to

[46] , wherein in step (b), immune cells are transformed using 1) a transposable element comprising the polynucleotide of any one of

[23] to

[29] and 2) a transposase (a polynucleotide encoding the transposase).

[48] ​​The method according to

[47] , wherein the transposase is Sleeping Beauty transposase or PiggyBac transposase.

[49] The method according to

[48] , wherein the Sleeping Beauty transposase is represented by the amino acid sequence shown in SEQ ID NO: 45.

[50] The method according to any one of

[47] to

[49] , wherein the transposable element is integrated into the genome of the immune cell by the action of the transposase.

[51] The method according to any one of

[46] to

[50] , wherein the immune cells are lymphocytes.

[52] The method according to

[51] , wherein the lymphocytes are T cells or NK cells.

[53] The above T cells are CD4 + cells or CD8 + The method according to

[52] , wherein the cell is a cell.

[54] The method according to any one of

[46] to

[53] , wherein the subject is a human. An immune cell obtainable by the method according to any one of

[55]

[46] to

[54] .

[56] A pharmaceutical composition comprising a plurality of immune cells according to any one of

[39] to

[45] or

[55] , wherein the plurality of immune cells optionally comprises CD4 + Cells and CD8 + A pharmaceutical composition that is a mixture of cells.

[57] The immune cell according to any one of

[39] to

[45] or

[55] or the pharmaceutical composition according to

[56] for use as a pharmaceutical.

[58] An immune cell according to any one of

[39] to

[45] or

[55] or a pharmaceutical composition according to

[56] for use in a method for treating cancer, to be administered to a subject.

[59] The immune cell or pharmaceutical composition for use as defined in

[57] or

[58] , wherein the pharmaceutical composition is administered intravenously.

[60] The immune cell or pharmaceutical composition for use as defined in any one of

[57] to

[59] , wherein the immune cell is a lymphocyte.

[61] The immune cell or pharmaceutical composition for use as defined in

[60] , wherein the lymphocyte is a T cell or an NK cell.

[62] The above T cells are CD4 + T cells and / or CD8 + The immune cell or pharmaceutical composition for use as defined in

[61] , wherein the immune cell or pharmaceutical composition is a T cell.

[63] The immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[62] , wherein the subject is a human.

[64] The immune cell or pharmaceutical composition for use according to any one of

[58] to

[63] , wherein the cancer is a cancer that expresses Siglec-6.

[65] The immune cell or pharmaceutical composition for use according to any one of

[58] to

[64] , wherein the cancer is leukemia.

[66] The immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[65] , wherein the cancer is primary acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), MALT lymphoma, clonal mast cell disease or thymoma.

[67] The immune cell or pharmaceutical composition for use according to any one of

[58] to

[66] , wherein the cancer is AML.

[68] An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[67] , wherein the method for treating cancer comprises the elimination of cancer stem cells of the cancer by the immune cells.

[69] The cancer stem cells express CD45 dim cells, preferably CD45 dim CD34 + cells, most preferably CD45 dim CD34 + CD38 -

[68] The immune cell or pharmaceutical composition for use as defined in

[68] .

[70] An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[69] , wherein the method for treating cancer does not involve the removal of non-cancerous hematopoietic stem or progenitor cells by the immune cells.

[71] An immune cell or pharmaceutical composition for use as defined in any one of

[68] to

[70] , further comprising a step of monitoring the elimination of said cancer stem cells and / or said non-cancerous hematopoietic stem or progenitor cells.

[72] An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[71] , wherein the method for treating cancer does not include subsequent allogeneic hematopoietic stem cell transplantation, or the subject has experienced a recurrence of the cancer after allogeneic hematopoietic stem cell transplantation.

[73] An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[72] , wherein the method does not include further chemotherapy after administration of the immune cell or pharmaceutical composition and / or after termination of treatment with the immune cell or pharmaceutical composition.

[74] The immune cells or pharmaceutical composition for use as defined in any one of

[58] to

[73] , wherein the method for treating cancer does not include depletion of the immune cells after treatment.

[75] The method, 1) determining the expression level of Siglec-6 on cancer cells obtained from the subject; and then 2) administering the immune cells or the pharmaceutical composition to the subject; An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[74] , comprising:

[76] The immune cell or pharmaceutical composition for use as defined in

[75] , wherein the immune cell or pharmaceutical composition is administered in step 2) only if Siglec-6 is expressed on the cancer cells.

[77] The method, (i) a CD70-binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to CD70, or comprising or consisting of a chimeric antigen receptor (CAR); or (ii) an immune cell comprising a CD70-binding polypeptide according to (i), and / or a polynucleotide or set of polynucleotides encoding the CD70-binding polypeptide according to (i), and / or an expression vector comprising a polynucleotide or set of polynucleotides encoding the CD70-binding polypeptide according to (i). further treatment with The immune cells are preferably T cells, e.g., CD4 + T cells or CD8 + An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[76] , wherein the immune cell or pharmaceutical composition is a T cell or an NK cell.

[78] The immune cell or pharmaceutical composition for use as defined in

[77] , wherein said CD70-binding polypeptide comprises or consists of a chimeric antigen receptor (CAR).

[79] The method, (i) a TIM-3-binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to TIM-3, or comprising or consisting of a chimeric antigen receptor (CAR); or (ii) an immune cell comprising the TIM-3-binding polypeptide described in (i), and / or a polynucleotide or set of polynucleotides encoding the TIM-3-binding polypeptide described in (i), and / or an expression vector comprising the polynucleotide or set of polynucleotides encoding the TIM-3-binding polypeptide described in (i). further treatment with The immune cells are preferably T cells, e.g., CD4 + T cells or CD8 + An immune cell or pharmaceutical composition for use as defined in any one of

[58] to

[78] , wherein the immune cell or pharmaceutical composition is a T cell or an NK cell.

[80] The immune cell or pharmaceutical composition for use as defined in

[79] , wherein the TIM-3-binding polypeptide comprises or consists of a chimeric antigen receptor (CAR). [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1 shows the recognition and elimination of Siglec-6+ AML cell lines by JML-1-CAR T cells in vitro. (A) Flow cytometry analysis of Siglec-6 expression on AML cell lines (U937, MV4;11, MOLM13, and K562) is shown. Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inserted numbers indicate normalized mean fluorescence intensity (NMFI). (B) Specific cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, and untransduced (UTD) T cells against AML cell lines in a luminescence-based assay (4 hours). Assays were performed in triplicate wells with 5,000 target cells / well. Values ​​are presented as mean ± SD. (C) ELISA was performed to detect IFN-γ and IL-2 in supernatants obtained after 24 hours of coculture of CD4+ or CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, or UTD T cells with target cells. T cells and target cells were seeded in triplicate wells in an effector:target (2:1) ratio. Values ​​are presented as mean ± SD. (D) Proliferation of CD4+ and CD8+ JML-1_28z CAR and JML-1_BBz CAR T cells after 72 hours of coculture with target cells is shown, as assessed by CFSE dye dilution. Assays were performed in triplicate wells in an effector:target (2:1) ratio. Histograms show proliferation of viable (7-AAD-) T cells. No exogenous cytokines were added. Data shown in B–D are representative of results obtained with CAR and control T cell lines prepared from n>5 healthy donors (HD). [Figure 2]Figure 1 shows the recognition and elimination of primary AML cells in vitro by JML-1-CAR T cells. (A) Flow cytometry analysis of Siglec-6 expression on bulk AML and AML LSCs (CD45dim CD34+ CD38-) in n=5 representative AML patient samples (see Table 1). Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inset numbers show normalized mean fluorescence intensity (NMFI) obtained by staining with anti-Siglec-6 mAb and isotype control. Plots show the cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, and untransduced (UTD) T cells against LSCs and bulk AML blasts in a flow cytometry-based assay (24-hour co-culture). Experiments were performed in triplicate wells with 10,000 target cells / well. Counting beads were used to quantify the number of viable target cells remaining at the end of co-culture. (B) Correlation between tumor-specific cell killing by CD8+ JML-1_BBz CAR T cells (flow cytometry-based assay, 24-hour co-culture; E:T ratio 2.5:1) and Siglec-6 NMFI expression on primary AML cells. (C) Siglec-6 expression on bulk AML and AML LSCs from n=10 AML patients. Patients are ranked by ascending NMFI (see Table 1). [Figure 3]Figure 1 shows that JML-1-CAR T cells confer potent anti-leukemic activity in vivo in a xenograft model of AML. Female NSG mice were inoculated with 2 x 10 U937 AML cells (ffluc+GFP+) and treated with 5 x 10 CAR-modified or untransduced (UTD) T cells on day 6. T cells were administered at a 1:1 ratio of CD4+:CD8+. (A) Serial bioluminescence (BL) imaging is shown to assess leukemic progression and / or regression. Note that the scale indicates the upper and lower limits of BL at each analysis time point (right). (B) Flow cytometry analysis of PB on days 10, 14, and 45 to detect T cells and leukemic cells is shown. Human T cells in mouse PB were defined as 7-AAD-CD45+CD3+ cells. Leukemic cells were defined as 7-AAD-CD45+GFP+ cells. ****p<.0001 (Student's t-test). (C) Waterfall plot showing the change in absolute BL values ​​between days 6 and 10 after tumor inoculation. BL values ​​were obtained as photons / second / cm2 / sr in a region of interest encompassing the entire body of each mouse. (D) The percentage of leukemic cells detected in the BM, spleen, and PB by flow cytometry at the end of the experiment is shown. ****p<0.0001 **p<0.05 *p<0.5 (Student's t-test). (E-F) Kaplan-Meier survival analysis is shown. (E) Overall survival of the various treatment groups is shown. (F) Progression-free survival of the various treatment groups is shown. Data shown are representative of results obtained in independent experiments with JML-1-CAR T cells from n=2 donors. ****p<0.0001, log-rank (Mantel-Cox) test. (G-H) Female NSG mice were inoculated with 1 x 106 MOLM-13 AML cells (ffluc+ GFP+) and treated with 5 x 106 CAR-modified or untransduced (UTD) T cells on days 4 and 7. T cells were administered at a 1:1 ratio of CD4+:CD8+. (G) Waterfall plot showing the change in absolute BL scores between days 7 and 10 after tumor inoculation. (H) Kaplan-Meier survival analysis for each treatment group. ****p<0.0001, log-rank (Mantel-Cox) test. [Figure 4]Figure 1 shows that human HSC / P do not express Siglec-6 and are preserved after in vitro coculture with JML-1-CAR T cells. (A) Flow cytometry analysis of Siglec-6 expression on G-CSF-mobilized CD34+CD38- HSCs and CD34+CD38+ progenitor cells from the PB of n=5 HD patients. The inset values ​​indicate NMFI. NMFI is calculated by dividing the MFI of anti-Siglec-6 mAb (gray) by the MFI of the isotype control (white histogram). (B) Right panel: The percentage of viable (7-AAD-) HSCs after 24 hours of co-incubation with CD8+ JML-1_BBz CAR, CD123 CAR, or untransduced T cells is shown. Assays were performed in triplicate wells with 5,000 target cells / well. Counting beads were used to quantitate the number of remaining viable HSCs at the end of co-culture. Data from n=3 independent experiments are shown. Left panel: Colony formation assay performed with remaining viable HSCs after 24 hours of co-incubation with CD8+ JML-1_BBz CAR, CD123 CAR, or untransduced T cells. The figure shows the absolute number of colonies (mean ± SD) per 55 mm plate as determined by microscopy on day 14 from n = 3 independent experiments. GEMM (granulocyte / erythrocyte / macrophage / megakaryocyte); GM (granulocyte / macrophage); CFU-E (colony-forming unit-erythrocyte); CFU-M (colony-forming unit-macrophage); CFU-G (colony-forming unit-granulocyte). (C) Flow cytometry analysis of cell surface expression of various CAR target antigens on CD34+ and CD34+CD38+ cells from HD (n = 5). Values ​​indicate normalized mean fluorescence intensity. (D) ****p<0.0001 **p<0.05 *p<0.5 (Student's t-test). [Figure 5]Figure 1 shows that Siglec-6 is expressed on malignant B lymphocytes and healthy memory B cells in B-CLL. (A) Flow cytometry analysis of Siglec-6 expression on CLL cells from n=10 patients. Patient characteristics are summarized in Table 2. Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inserted numbers indicate NMFI. (B) Specific cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, CD19_BBz CAR, and untransduced (UTD) T cells against CLL cells in a flow cytometry-based assay. Target cells were seeded in triplicate wells (10,000 cells / well) and cocultured with effector cells at an E:T ratio of 5:1. Counting beads were used to quantify the number of remaining viable target cells after 4 hours of coculture. (C) Correlation between CLL-specific cell killing by CD8+ JML-1_BBz or JML-1_28z CAR T cells (after 4 hours of co-culture, E:T ratio 5:1) and normalized Siglec-6 expression on primary B cells. Simple linear correlations were calculated (R-squared = 0.54; p = 0.01 and R-squared 0.29; p = 0.1 for JML-1_BBz CAR and JML-1_28z CAR, respectively). (D) Flow cytometry analysis of Siglec-6 expression on healthy B cells (CD45+CD19+CD5-CD20high) from B-ALL patients. Left: Pooled data of Siglec-6 expression on B-CLL cells from n = 10 patients and healthy B cell subsets from 5 of 10 B-CLL patients are shown. The remaining patients (n = 5) did not have enough healthy B cells in their PB for subset analysis. Right: Representative histogram from patient 3 showing Siglec-6 expression on healthy immature (CD45+CD19+CD5-CD20highCD10+), naive (CD45+CD19+CD20highCD5-CD10-CD27-), and memory (CD45+CD19+CD5-CD20highCD10-CD27+) B cells compared with B-CLL cells. (E) Flow cytometry analysis of Siglec-6 expression on healthy PBMCs from HD n=7.Siglec-6 expression by B cells (CD45+CD19+), myeloid cells (CD45+CD33+), T cells (CD45+CD3+CD56-), NK cells (CD45+CD56+CD3-), and NKT cells (CD45+CD3+CD56+) from n=7 HD patients is shown. Siglec-6 expression by Siglec-6-positive (U937, TF-1, MV4;11, and MOLM-13) and -negative (K562, JeKo-1) cell lines is plotted for reference. The graph and histogram on the right show Siglec-6 expression on memory, naive, and immature cells from n=5 HD patients (left histogram: memory B cells, right histogram: naive / immature B cells). (F) Siglec-6 expression on healthy B cells from CLL patients and HD patients. *p<0.5, **p<0.05 (Student's t-test). [Figure 6] Figure 1 shows the design of CAR constructs, CAR expression, and phenotypes of CD4+ and CD8+ T cells. (A) The design of the CARs used in the study is shown. Single-chain variable fragments (scFv; VH-linker-VL) were derived from mAbs JML-1 (Siglec-6-specific CAR), 4G8 (FLT3-specific CAR), FMC63 (CD19-specific CAR), and 32716 (CD123-specific CAR). The scFvs were fused to an IgG4 hinge spacer, and the CD28 transmembrane domain was fused to an intracellular signaling module. CD28 or 4-1BB and CD3z were incorporated as costimulatory and signaling domains, respectively. A truncated epidermal growth factor receptor (EGFRt) (separated from the CAR transgene by a T2A ribosomal skip sequence) was incorporated for detection and enrichment of CAR-positive T cells. (B) Dot plots show the expression of the EGFRt marker on CD4+ and CD8+ T cells after transduction. (C) Purity of CAR-positive T cells after enrichment of EGFRt+ CD8+ and CD4+ T cells before functionality testing is shown. Non-transduced T cells are included in comparisons B-C. (D) Summary data of the percentage of HD-derived CAR-positive T cells after enrichment is shown. [Figure 7]Figure 1 shows the specificity and selectivity of JML-1-CAR T cells for Siglec-6-expressing target cells. (A) Flow cytometry analysis of Siglec-6 expression by naive K562 and K562 / Siglec-6 cells. Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inserted numbers indicate NMFI. (B) Left panel: Specific cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, and untransduced (UTD) T cells against K562 / Siglec-6 cells analyzed in a bioluminescence-based assay after 4 hours of co-culture. Right panel: Summarized data from a cytotoxicity assay of CAR T cells from HD n=3 (24 hours of co-culture, E:T ratio 10:1). Values ​​are shown as mean ± sd. (C) ELISA detecting IFN-γ and IL-2 in supernatants after 24 hours of co-culture. T cells and target cells were seeded in triplicate wells at an E:T ratio of 2:1. Values ​​are shown as mean ± SD. (D) Summary data for cytokine production (IFN-γ and IL-2) by CD4+ T cells from various donors (n=3) are shown. (E) T cell proliferation after 72 hours of co-culture analyzed by CFSE dye dilution. Assays were performed in triplicate wells at an E:T ratio of 2:1. Histograms show proliferation of live (7-AAD-) T cells. No exogenous cytokines were added to the assay medium. ***p<0.001, ****p<0.0001 (Student's t-test). [Figure 8]Figure 1 shows recognition of TF-1 and Kasumi-1 tumor cell lines by JML-1-CAR T cells. (A) Flow cytometry analysis of Siglec-6 expression on TF-1 (erythroleukemia) and Kasumi-1 (AML with t(8;21)). Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inserted numbers indicate NMFI. (B) Specific cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, and untransduced (UTD) T cells against TF-1 and Kasumi-1 after 4 hours of co-culture, analyzed in a luminescence-based assay. The TF-1 cell line does not express FLT3, while Kasumi-1 expresses low levels of FLT3. (C) ELISA detecting IFN-γ and IL-2 in the supernatant after 24 hours of co-culture. T cells and target cells were seeded in triplicate wells at an E:T ratio of 2:1. Values ​​are presented as mean ± SD. (D) Proliferation of CD4+ T cells after 72 hours of co-culture analyzed by CFSE dye dilution. T cells and target cells were seeded in triplicate wells at an E:T ratio of 2:1. Proliferation of live (7-AAD) T cells is shown in the histogram. No exogenous cytokines were added to the assay medium. [Figure 9] Figure 1 shows leukemia stem cells (LSCs) in primary AML samples. (A) Flow cytometry gating strategy for primary AML blasts. Siglec-6 expression is analyzed on live (7-AAD-) bulk AML cells (CD45dim) and AML LSCs (CD45dimCD34+CD38-). (B) Siglec-6 expression on AML blasts with phenotypic heterogeneity. Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inset numbers indicate NMFI. [Figure 10]Figure 1 shows the generation and functional analysis of JML-1-CAR T cells obtained from AML patients. (A) CAR transduction efficiency in CD4+ and CD8+ T cells, shown as EGFRt expression, before and after CAR-positive T cell enrichment. (B) CD4+ and CD8+ T cell proliferation after CAR transduction. (C) Cytolytic activity of CD8+ JML-1_BBz CAR, FLT3_28z CAR, CD123_28z CAR, and untransduced (UTD) T cells against AML cell lines. (D) IFN-γ and IL-2 production (ELISA) after 24 hours of co-culture of CD4+ T cells with AML cell lines. T cells and target cells were seeded in triplicate wells at an E:T ratio of 2:1. Values ​​are shown as mean ± SD. (E) CD4+ T cell proliferation after 72 hours of co-culture, analyzed by CFSE dye dilution. T cells and target cells were seeded in triplicate wells at an E:T ratio of 2:1. Live T cell proliferation is shown in histograms. No exogenous cytokines were added to the assay medium. Data shown in B-E correspond to CAR T cells from one representative patient out of at least n=3 AML patients. [Figure 11] Figure 1 shows the anti-leukemic activity of patient-derived JML-1-CAR T cells against autologous AML blasts. (A) Specific cytolysis by CD8+ JML-1_28z CAR, JML-1_BBz CAR, FLT3_28z CAR, and untransduced (UTD) T cells against autologous "bulk" AML blasts, AML LSCs, and U937 cells is shown. (B) IFN-γ production (ELISA) of CD4+ T cells after co-culture with autologous AML blasts and U937 cells within 24 hours. T cells and target cells were plated in triplicate wells at an E:T ratio of 2:1. Values ​​are shown as mean ± SD. (C) Proliferation of CD4+ T cells after 72 hours of co-culture analyzed by CFSE dilution is shown. T cells and target cells were plated in triplicate wells at an E:T ratio of 2:1. Proliferation of live (7-AAD-) T cells is shown in the histogram. No exogenous cytokines were added to the assay medium. Data shown correspond to CAR T cells from one representative patient out of at least n=3 patients. [Figure 12]Figure 1 shows leukemia burden in mouse BM and CAR T cell persistence in an AML xenograft model. (A) Dot plots show the frequencies of T cells (CD45+CD3+) and leukemia cells (CD45+GFP+) in the BM as a percentage of live (7-AAD-) cells in one representative mouse per group. [Figure 13] Figure 1 shows the expression of candidate target antigens for CAR T cells in AML on normal HSC / P. (A) Gating strategy for identifying HSC (CD34+) and HPC (CD34+CD38-) cells derived from G-CSF-mobilized PB cells of HD is shown. (B) Expression of various potential CAR antigens on HSC (upper panel) and HPC (lower panel). Histograms show expression of antigens (gray) relative to staining with isotype control mAb (white histogram). Inserted numbers indicate NMFI. Data are representative of HD n=5. [Figure 14] Figure 1 shows malignant and normal B cells in patients with B-CLL and HD. (A) Flow cytometry gating strategy for primary CLL cells. Siglec-6 expression is analyzed on live (7-AAD-) healthy B cells (CD45+CD19+CD20highCD5-) and B-CLL cells (CD45+CD19+CD20mid / lowCD5+). (B and C) Immature cells are CD45highCD19+CD10+CD5-, naive B cells are CD45highCD19+CD5-CD27-CD38-, and memory B cells are CD45highCD19+CD5-CD27+. Histograms show staining with anti-Siglec-6 mAb (gray) and isotype control antibody (white histogram). Inset numbers indicate NMFI. [Figure 15] Figure 1 shows recognition of normal B cells from B-CLL patients by JML-1-CAR T cells. (A) Specific cytolytic activity of CD8+ JML-1_28z CAR, JML-1_BBz CAR, CD19_BBz CAR, and untransduced (UTD) T cells against healthy CD19+ B cells in a flow cytometry-based assay. Counting beads were used to quantitate the number of remaining viable target cells after 4 hours of co-culture. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the fields of cancer immunotherapy, gene therapy, immunology, biochemistry, genetics, and molecular biology.

[0017] Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials described herein.

[0018] The term "about" as used in the context of the present invention means that the value following the term "about" can vary within a range of + / -20%, preferably within a range of + / -15%, more preferably within a range of + / -10%.

[0019] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes. References mentioned herein are designated by a reference number in square brackets (e.g., "

[31] " or "See

[31] "), which refers to the respective reference in the reference list at the end of the specification. In case of conflict, the present specification, including definitions, will take precedence over the cited reference. Furthermore, the materials, methods, and examples are illustrative only and, unless otherwise specified, are not intended to be limiting.

[0020] As used herein, each occurrence of terms such as "comprising" or "comprises" may optionally be substituted with "consisting of."

[0021] Siglec-6 binding polypeptide The present invention relates to a Siglec-6-binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to Siglec-6, or a chimeric antigen receptor (CAR), preferably a CAR.

[0022] In particular, the present invention provides a Siglec-6 binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to Siglec-6.

[0023] The term "antibody or fragment thereof" includes, for example, monoclonal, chimeric, single-chain, humanized, and human antibodies. It also includes, for example, Fab fragments, F(ab'), Fv, scFv fragments, or single-domain antibodies, e.g., domain antibodies or nanobodies comprising only one variable domain, such as a VHH, VH, or VL, single variable domain antibodies, or immunoglobulin single variable domains, which specifically bind to an antigen or epitope independently of other variable regions or domains. The term also includes diabodies or dual affinity retargeting (DART) antibodies. (Bispecific) single-chain diabodies, tandem diabodies, bispecific T cell-inducing (BiTE) antibodies, and trispecific T cell-inducing antibodies, e.g., hemibodies, are further contemplated. Any such antibodies and fragments thereof, and their generation, are generally known in the art.

[0024] Preferably, a polypeptide comprising or consisting of an antibody or fragment thereof that binds to Siglec-6 is at least bispecific. However, it may also be multispecific, e.g., trispecific or tetraspecific. Bispecific, trispecific, etc. means that the polypeptide is capable of binding to two, three, etc. different target antigens simultaneously or sequentially.

[0025] Thus, a Siglec-6 binding polypeptide may comprise or consist of a first antibody or fragment thereof that binds to Siglec-6 and a second antibody or fragment thereof that binds to a target other than Siglec-6, which may optionally be linked to each other via a linker.

[0026] The antibody or fragment thereof that binds to Siglec 6 can be represented, for example, by the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 25, and has the ability to bind to Siglec 6. Preferably, the antibody or fragment thereof that binds to Siglec 6 is represented by the amino acid sequence shown in SEQ ID NO: 25.

[0027] The at least bispecific Siglec-6-binding polypeptide is preferably capable of binding to an immune cell, such as a T cell or an NK cell, preferably a T cell, but also encompasses binding to other immune cells, such as macrophages.

[0028] Thus, at least the bispecific Siglec-6-binding polypeptide preferably further binds to (human) CD3, e.g., CD3 epsilon or CD3 zeta, preferably CD3 zeta. CD3 is expressed on T cells and forms part of the T cell receptor. Thus, the bispecific polypeptide can simultaneously bind to Siglec-6 and, e.g., CD3, thereby recruiting effector cells, e.g., T cells or NK cells, to target cells expressing Siglec-6 on their surface. Antibodies against human CD3 are well known in the art; see, for example, the antibodies against the N-terminal amino acids 1-27 of CD3 epsilon in WO 2008 / 119567, which is incorporated herein by reference.

[0029] Thus, at least bispecific Siglec-6 binding polypeptides are capable of recruiting preferably immune cells, such as T cells, NK cells, preferably T cells, to target cells expressing Siglec-6 on their surface.

[0030] In another embodiment, the Siglec-6-binding polypeptide is conjugated to another compound, such as a detectable marker or a drug. In this embodiment, the polypeptide is preferably conjugated to a drug. The drug can be, for example, a toxin. The toxin is preferably capable of killing target cells that express Siglec-6 on their surface. Examples of such toxins include maytansine, auristatin, taxoids, and PNU anthracyclines.

[0031] In a preferred embodiment, the Siglec-6 binding polypeptide is a chimeric antigen receptor (CAR). A CAR is a receptor that can be expressed on the surface of a cell, for example, that can bind to a ligand expressed on the surface of another cell. This allows the receptor to mobilize the cell that expresses the receptor to a target cell that expresses the ligand on its surface. Furthermore, optionally, the CAR can transmit an intracellular signal in the cell in which it is expressed upon binding to the ligand. Thus, for example, a CAR can be expressed on a T cell, and upon binding to its ligand, activate the T cell.

[0032] Thus, in more specific embodiments, the CAR comprises at least one extracellular ligand-binding domain, a transmembrane domain, and at least one intracellular signaling domain, wherein the extracellular ligand-binding domain preferably comprises a Siglec 6 binding element. The extracellular domain may further comprise a spacer domain, e.g., a spacer domain derived from CD8α, IgG3, or IgG4. The transmembrane domain may comprise a CD28 transmembrane domain. The intracellular signaling domain may comprise a costimulatory domain and a CD3 zeta (CD3ζ) domain.

[0033] In one embodiment of the present invention, the Siglec-6 binding element is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 25, and has Siglec-6 binding ability. Preferably, the Siglec-6 binding element is represented by the amino acid sequence set forth in SEQ ID NO: 25.

[0034] In one embodiment of the invention, the spacer domain is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 9 or 11. Preferably, the spacer domain is represented by the amino acid sequence set forth in SEQ ID NO: 9 or 11. The spacer binds to the extracellular target and the transmembrane domain. This affects the flexibility of the Siglec-6 binding element, reducing the spatial constraint from the CAR to the ligand and thus affecting epitope binding. Binding to membrane-distal epitopes often requires a CAR with a short spacer domain, while binding to cell surface-proximal epitopes often requires a CAR with a long spacer.

[0035] In one embodiment of the invention, the transmembrane domain is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity with the amino acid sequence set forth in SEQ ID NO: 13. Preferably, the transmembrane domain is represented by the amino acid sequence set forth in SEQ ID NO: 13. The CD28 transmembrane domain consists of a hydrophobic alpha helix, which crosses the membrane of the cell and anchors the CAR to the cell surface, which affects the expression of the CAR on the cell surface.

[0036] In one embodiment of the invention, the costimulatory domain of the Siglec-6-CAR polypeptide is the CD28 cytoplasmic domain or the 4-1BB costimulatory domain.

[0037] In one embodiment of the invention, the intracellular signaling domain comprises a CD28 cytoplasmic domain and a CD3 zeta domain. In another embodiment of the invention, the intracellular signaling domain comprises a 4-1BB costimulatory domain and a CD3 zeta domain.

[0038] In one embodiment of the invention, the CD28 cytoplasmic domain is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. Preferably, the CD28 cytoplasmic domain is represented by the amino acid sequence set forth in SEQ ID NO: 15. The CD28 cytoplasmic domain is a costimulatory domain and is derived from the intracellular signaling domain of a costimulatory molecule.

[0039] In one embodiment of the invention, the 4-1BB costimulatory domain is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. Preferably, the 4-1BB costimulatory domain is represented by the amino acid sequence set forth in SEQ ID NO: 17.

[0040] In one embodiment of the invention, the CD3 zeta domain is represented by an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 19. Preferably, the CD3 zeta domain is represented by the amino acid sequence set forth in SEQ ID NO: 19. The CD3 zeta domain mediates downstream signaling in T cell activation. It is derived from the intracellular signaling domain of the T cell receptor and contains an ITAM (immunoreceptor tyrosine-based activation motif).

[0041] In one embodiment of the invention, the extracellular domain comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 35 or 37. Preferably, the extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 35 or 37. More preferably, the extracellular domain consists of the amino acid sequence set forth in SEQ ID NO: 35 or 37.

[0042] In certain embodiments of the invention, the intracellular signaling domain comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 39 or 41. Preferably, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 39 or 41. More preferably, the intracellular signaling domain consists of the amino acid sequence set forth in SEQ ID NO: 39 or 41.

[0043] Thus, the extracellular domain may comprise the amino acid sequence set forth in SEQ ID NO: 35 or 37, or an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 35 or 37; the transmembrane domain may comprise the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 13; and the intracellular signaling domain may comprise the amino acid sequence set forth in SEQ ID NO: 39 or 41, or an amino acid sequence with at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 39 or 41.

[0044] In one preferred embodiment of the invention, the Siglec 6-CAR polypeptide comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33. Preferably, the Siglec 6-CAR polypeptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33. More preferably, the Siglec 6-CAR polypeptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33.

[0045] In embodiments relating to Siglec-6-binding CARs that include variant elements defined by percent sequence identity to a particular SEQ ID NO: 11, the CAR ideally retains the ability to function as a Siglec-6-binding CAR (e.g., including ligand binding and / or lymphocyte activation), and the ability to function as a Siglec-6-binding CAR is ideally at least the same as for a CAR of the same sequence, where the element in question is represented by the SEQ ID NO: 11, without variation. For example, if a CAR includes a spacer domain that has at least 90% sequence identity to SEQ ID NO: 11, the CAR ideally has the same ability to function as a Siglec-6-binding CAR (i.e., without variation) as a CAR of the same sequence except for the spacer represented by SEQ ID NO: 11.

[0046] A CAR polypeptide can also be specific for more than one target. Accordingly, the present invention also provides Siglec-6 binding polypeptides comprising or consisting of a CAR, which comprises at least two binding elements, at least one of which binds to Siglec-6, and / or comprises at least one binding element that is a switchable / programmable binding domain that can be switched / programmed to bind to Siglec-6.

[0047] Polynucleotides encoding Siglec-6 binding polypeptides The present invention relates to a polynucleotide or a set of polynucleotides that encodes a Siglec-6-binding polypeptide of the invention as defined above.

[0048] In one embodiment of the invention, a polynucleotide encoding a polypeptide of the invention is further flanked by left and right inverted repeat / direct repeat (IR / DR) segments, where the flanking segment on the 5' direction is represented by a left inverted repeat / direct repeat (IR / DR) segment and the flanking segment on the 3' direction is represented by a right inverted repeat / direct repeat (IR / DR) segment.

[0049] The nucleotide sequence of the left IR / DR segment and the nucleotide sequence of the right IR / DR segment can be recognized by a transposase protein. The transposase is not particularly limited and can be, for example, Sleeping Beauty transposase or PiggyBac transposase.

[0050] Preferably, the left IR / DR segment comprises a nucleotide sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99%, or even 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 43. Similarly, the right IR / DR segment comprises a nucleotide sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99%, or even 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 44.

[0051] The term "flanking with" indicates that additional nucleotides are present in the 5' and 3' regions of the polynucleotide sequence encoding the polypeptide, all located on the same polynucleotide. Thus, the polynucleotide sequence encoding the polypeptide is flanked by the IR / DR sequences, i.e., the flanking segments, such that the presence of a transposase allows for the integration of the polynucleotide encoding the polypeptide and the nucleotide sequence corresponding to the flanking segments into the genome of the transfected cell. In one embodiment, the polynucleotide integrated into the genome comprises the polynucleotide encoding the polypeptide and an optional detectable marker gene, e.g., the EGFRt marker, and is flanked by the flanking segments. In this embodiment, the nucleotide sequences corresponding to the coding region of the polypeptide and the region of the EGFRt marker are considered to represent the reference segment.

[0052] As used herein, the term "adjacent to" also means that the distance between the adjacent segment and the reference segment is less than 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400, 300 bp, 200 bp, 100 bp, 50 bp, 20 bp, or less than 10 bp.

[0053] In this regard, the reference segment is the region corresponding to the coding region of the polynucleotide to be integrated into the genome. The overall structure of the polynucleotide to be integrated into the genome of the transfected cell can be as follows (5' to 3' direction): [left IR / DR sequence] - [reference segment] - [right IR / DR sequence].

[0054] The distance between an adjacent segment and a reference segment can be determined by counting the nucleotides between the 3' end of the left IR / DR sequence and the 5' end of the reference segment. Similarly, the distance between an adjacent segment and a reference segment can be determined by counting the distance between the 3' end of the reference segment and the 5' end of the right IR / DR sequence. Both distances can be the same, so as to center the reference segment between the adjacent segments, or the distances can be different.

[0055] The distance between the 3' end of the left IR / DR sequence and the 5' end of the reference segment can be less than 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, or 100 bp.

[0056] The distance between the 3' end of the reference segment and the 5' end of the right IR / DR sequence can be 200 bp, 100 bp, 50 bp, less than 20 bp, or less than 10 bp.

[0057] In an exemplary embodiment of the invention, the distance between the 3' end of the left IR / DR sequence and the 5' end of the reference segment may be less than 700 bp, and the distance between the 3' end of the reference segment and the 5' end of the right IR / DR sequence may be less than 10 bp.

[0058] In an exemplary embodiment of the present invention, the distance between the 3' end of the left IR / DR sequence and the 5' end of the reference segment may be more than 600 bp and less than 700 bp, and the distance between the 3' end of the reference segment and the 5' end of the right IR / DR sequence may be more than 5 bp and less than 10 bp.

[0059] In one embodiment, the polynucleotide to be integrated into the genome comprises a polynucleotide encoding a Siglec-6-binding polypeptide and a detectable marker gene, such as an EGFRt marker, and is flanked by flanking segments. In this embodiment, the nucleotide sequences corresponding to the coding region of the Siglec-6-binding polypeptide and the region of the EGFRt marker are considered to represent the reference segment.

[0060] In one embodiment, the polynucleotide sequence of the invention comprises the sequence represented by SEQ ID NO: 26, or a nucleotide sequence having at least 80% sequence identity, such as at least 90%, preferably at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 26. The polynucleotide sequence of the invention preferably comprises the nucleotide sequence represented by SEQ ID NO: 26.

[0061] Thus, in a related embodiment of the invention, a polynucleotide of the invention relates to a polynucleotide sequence comprising a sequence having at least 90%, preferably 95%, more preferably 97%, or most preferably 99%, or even 100% sequence identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 28, 30, 32 or 34. Preferably, a polynucleotide of the invention comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 28, 30, 32 or 34. Alternatively, a polynucleotide of the invention may consist of a nucleotide sequence set forth in any one of SEQ ID NOs: 28, 30, 32 or 34.

[0062] Expression vector The present invention relates to an expression vector comprising a polynucleotide or set of polynucleotides of the invention as defined herein.

[0063] A wide variety of expression vectors for polypeptides are known in the art and are further described herein. For example, in some embodiments of the invention, the expression vector is a non-viral or viral vector, and in the context of medical purposes, preferably a non-viral vector.

[0064] The expression vector may be a minimal DNA expression cassette. Moreover, the expression vector may be a DNA expression vector, such as a plasmid, a linear expression vector, or an episome. In certain embodiments, the vector comprises additional sequences, such as sequences that facilitate expression of a polypeptide, such as a promoter, an enhancer, a polyA signal, and / or one or more introns. In certain embodiments, the expression vector may be a transposon donor DNA molecule, preferably a minicircle DNA.

[0065] The present invention also relates to minicircle DNAs comprising the polynucleotides of the present invention as defined herein. As used herein, the term "minicircle DNA" refers to vectors that are supercoiled DNA molecules lacking a bacterial replication origin and antibiotic resistance genes. Thus, they primarily consist of eukaryotic expression cassettes.

[0066] In one useful embodiment, the minicircle DNA of the present invention, in combination with a transposase protein or a nucleic acid (e.g., DNA or mRNA) encoding a transposase protein (e.g., Sleeping Beauty or PiggyBac), is introduced into cells by electrophoretic transfer, e.g., electroporation, nucleofection; chemical transfer using substances such as lipofectamine, fugene, calcium phosphate; nanoparticles, or any other conceivable method suitable for introducing substances into cells.

[0067] The viral vector may be, for example, a gammaretroviral vector or a lentiviral vector. Such vectors and their construction and production are generally known in the art.

[0068] Polynucleotides or expression vectors can be introduced into immune cells by any suitable means, for example, transfection or transduction. Transfection refers to chemical or physical delivery into cells, for example, by electrophoretic transfer, e.g., electroporation, nucleofection; chemical transfer using substances such as lipofectamine, fugene, calcium phosphate, or PEI. Transduction refers to other means of (targeted) delivery into cells, including delivery by viral vectors or nanoparticles. However, the present invention is not limited to any particular method of delivery of genetic material into immune cells, and therefore, any other conceivable method suitable for introducing genetic material into cells can also be used in the context of the present invention.

[0069] immune cells The present invention also relates to an immune cell (preferably a lymphocyte, more preferably a T cell) comprising a polypeptide and / or a polynucleotide or set of polynucleotides and / or an expression vector of the invention as defined herein.

[0070] The present invention also relates to immune cells (preferably lymphocytes, more preferably T cells) comprising a polypeptide of the invention as defined herein.

[0071] The present invention also relates to an immune cell (preferably a lymphocyte, more preferably a T cell) comprising a polynucleotide or set of polynucleotides of the invention as defined herein.

[0072] The present invention also relates to an immune cell (preferably a lymphocyte, more preferably a T cell) comprising an expression vector of the invention as defined herein.

[0073] The immune cell may be a recombinant immune cell. A "recombinant immune cell" refers to an immune cell, e.g., a naturally occurring immune cell obtained from a (human) subject, that has been modified to contain a molecule, e.g., a polypeptide or polynucleotide, particularly a polynucleotide, that is not contained in the same cell without such modification.

[0074] Immune cells (preferably lymphocytes, more preferably T cells) are also preferably capable of expressing the polynucleotide of the present invention, such that the Siglec-6-binding polypeptide encoded by the polynucleotide of the present invention is translated and incorporated into the cell membrane of the immune cell.

[0075] Expression of the Siglec-6-binding polypeptide enables immune cells (preferably lymphocytes, more preferably T cells) of the present invention to acquire specific reactivity against target cells expressing the Siglec-6 antigen, including leukemia cells. Such immune cells, for example, Siglec-6 CAR-T cells, can recognize and (antigen-specifically) eradicate leukemia cells, more particularly, AML, CLL, MALT lymphoma, clonal mast cell disease cells, or thymoma. Such cells can proliferate and induce immune responses after encountering the Siglec-6 antigen.

[0076] In some useful applications, immune cells can also be modified to bind at least one additional target other than Siglec 6. Accordingly, the present invention also provides immune cells comprising one, two, or more CAR constructs, each targeting a distinct target antigen, at least one of which is Siglec 6, and / or a polynucleotide or set of polynucleotides encoding such CARs, and / or an expression vector comprising a polynucleotide or set of polynucleotides encoding such CARs.

[0077] The present invention also provides immune cells comprising a single CAR construct comprising one, two or more binding elements, at least one of which binds to Siglec-6, and / or a polynucleotide or set of polynucleotides encoding such a CAR, and / or an expression vector comprising a polynucleotide or set of polynucleotides encoding such a CAR.

[0078] The present invention also provides an immune cell comprising a CAR, wherein at least one binding domain is a switchable / programmable binding domain that can be switched / programmed to bind to Siglec-6, and / or a polynucleotide or set of polynucleotides encoding such a CAR, and / or an expression vector comprising a polynucleotide or set of polynucleotides encoding such a CAR.

[0079] The immune cells are preferably lymphocytes, such as T cells or NK cells, but also include other immune cells, such as macrophages. T cells are particularly preferred.

[0080] In one embodiment of the invention, the immune cells (preferably lymphocytes, more preferably T cells) are CD4 + T cells or CD8 + T cells.

[0081] In one embodiment of the invention, the immune cells (preferably lymphocytes, more preferably T cells) are CD4 + T cells.

[0082] In an embodiment of the invention, the immune cells (preferably lymphocytes, more preferably T cells) are CD8 + T cells.

[0083] In one embodiment of the present invention, the immune cells of the present invention may further express a marker gene, such as an EGFRt marker, on the cell surface. The EGFRt marker can be used to detect, track, select, and deplete the immune cells of the present invention. This allows for analysis of the persistence of the formulation after immune cell administration. Furthermore, the EGFRt marker makes the immune cells of the present invention susceptible to ADCC / CDC by the antibody cetuximab, allowing this antibody to be used as a safety switch.

[0084] The amino acid sequence of EGFRt that can be used in the present invention is represented by SEQ ID NO:23.

[0085] In one embodiment of the invention, the immune cells are obtained (or have been obtained) from immune cells (preferably lymphocytes, more preferably T cells) derived from a mammal, preferably a human. Preferably, the immune cells are obtained (or have been obtained) from a subject to be treated with the immune cells after modification to contain a Siglec-6-binding polypeptide, e.g., by the methods described herein. Alternatively, the immune cells modified to contain a Siglec-6-binding polypeptide are obtained (or have been obtained) from a healthy (allogeneic) donor, an umbilical cord blood unit (autologous or allogeneic) or an induced pluripotent stem cell.

[0086] Methods for generating immune cells The present invention also relates to a method for generating an immune cell (preferably a lymphocyte, more preferably a T cell) of the invention as defined herein.

[0087] In certain embodiments of the invention, a method for generating immune cells comprises the steps of (a) isolating immune cells from a (peripheral) blood sample of a subject, (b) transforming or transducing the immune cells with the polynucleotide or expression vector described above, and then optionally (c) purifying the transfected or transduced immune cells. The method may further comprise formulating the immune cells into a formulation suitable for administration to a human subject.

[0088] Preferably, in step (b), the immune cells are transformed using a transposable element comprising a polynucleotide or set of polynucleotides described herein and a transposase.

[0089] The transposase is not limited to, for example, Sleeping Beauty transposase or PiggyBac transposase. Sleeping Beauty transposase can be represented by, for example, the amino acid sequence shown in SEQ ID NO: 45.

[0090] The transposable element preferably integrates into the genome of the immune cell by the action of a transposase.

[0091] In one embodiment, the immune cell is a lymphocyte, more preferably a T cell or an NK cell. However, other immune cells, such as macrophages, are also encompassed. Most preferably, the immune cell is a T cell.

[0092] In a further embodiment, the T cells are CD4 + T cells and / or CD8 + T cells.

[0093] In a further embodiment of the invention, the blood sample is obtained (or has been obtained) from a human subject, preferably a human subject diagnosed with cancer, preferably a human subject diagnosed with leukemia, such as AML.

[0094] In another embodiment, the present invention provides a method for generating immune cells, comprising administering to a subject (in vivo transfer) an expression vector encoding a Siglec-6-binding polypeptide described herein. See, e.g., references

[38] -

[40] , all of which are incorporated by reference. Preferably, the expression vector for in vivo gene transfer is a lentiviral vector pseudotyped to transduce human immune cells (preferably T cells), or a nanoparticle containing a non-viral vector suitable for delivery of the non-viral vector to human immune cells (preferably T cells).

[0095] The present invention also relates to immune cells (preferably lymphocytes, more preferably T cells) obtainable by the above-mentioned methods or formulations of immune cells (preferably lymphocytes, more preferably T cells).

[0096] Pharmaceutical Composition The present invention also relates to pharmaceutical compositions comprising a plurality of immune cells (preferably lymphocytes, more preferably T cells) as described herein. The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutical composition may optionally contain a mixture of different cells, e.g., CD4 + and CD8 + It may comprise a mixture of T cells.

[0097] In one embodiment of the present invention, the pharmaceutical composition may be formulated as an infusion solution containing NaCl, glucose and human serum albumin in amounts of 0.45%, 2.5% and 1%, respectively.

[0098] medical use The present invention also relates to an immune cell or pharmaceutical composition described herein for use as a medicament.

[0099] In one embodiment of the invention, the immune cells or pharmaceutical compositions are for use in a method of treating cancer, wherein the immune cells or pharmaceutical compositions of the invention are administered to a subject (in need thereof), preferably a human subject.

[0100] The present invention also relates to a method for treating cancer, comprising the step of administering the immune cells or pharmaceutical compositions of the present invention to a subject, preferably a human subject.

[0101] In certain embodiments of the invention, the immune cells or pharmaceutical compositions are administered intravenously.

[0102] In one embodiment of the present invention, the cancer is a Siglec-6-expressing cancer, i.e., a cancer arising from abnormal cells that express and display Siglec-6 protein on their cell surface. Preferably, the cancer is selected from the group consisting of leukemia, such as AML or CLL, MALT lymphoma or clonal mast cell disease, and solid tumors, such as thymoma. Preferably, the cancer is a leukemia, such as AML or CLL, most preferably AML.

[0103] The (use in) method for treating cancer preferably comprises the elimination of cancer stem cells of said cancer by said immune cells. Such cancer stem cells are preferably leukemia stem cells, e.g., AML stem cells. Cancer stem cells are defined as a subset of cancer cells that have enhanced tumorigenic potential and / or the capacity for self-renewal and differentiation, and / or determinable phenotypic, functional and / or genetic characteristics that distinguish them from non-cancer stem cells.

[0104] Also, the (use in) method of treating cancer preferably does not include the removal of non-cancerous hematopoietic stem or progenitor cells by said immune cells. + Hematopoietic stem cells can be identified as cells that typically express CD38 - and hematopoietic progenitor cells are typically CD38 + Non-cancerous hematopoietic stem or progenitor cells typically express CD45 + is.

[0105] With this in mind, the (use in) method of treating cancer may further comprise the step of monitoring the elimination of said cancer stem cells and / or said non-cancerous hematopoietic stem or progenitor cells.

[0106] Thus, (use in) a method of treating cancer may include a step of monitoring the elimination of said cancer stem cells.

[0107] The (use in) method of treating cancer may comprise the step of monitoring the removal of said non-cancerous hematopoietic stem or progenitor cells.

[0108] The (use in) method of treating cancer may further comprise the step of monitoring the elimination of said cancer stem cells and said non-cancerous hematopoietic stem or progenitor cells.

[0109] Depletion of cancer stem cells and / or non-cancerous hematopoietic stem or progenitor cells can be monitored by bone marrow analysis, including flow cytometry analysis and other phenotyping methods, including, for example, high-resolution flow cytometry for minimal residual disease (MRD) analysis, as well as next-generation sequencing and other genotyping methods. Depletion of cancer stem cells and / or non-cancerous hematopoietic stem or progenitor cells can also be monitored by peripheral blood analysis, including blood counts and differential counts, as well as liquid biopsies and other genotyping methods.

[0110] Phenotypic markers typically used to identify AML LSCs by flow cytometry are CD45, CD34, and CD38 (AML LSC phenotype: CD45 dimCD34 + CD38 - ). Further optional markers (and corresponding phenotypes) that have been used to identify and / or characterize AML LSCs include HLA-DR(+), CD25(+), CD26(+), CD32(+), CD33(+), CD36(+), CD44(+), CD45RA(+), CD47(+), CD71(+), CD90(+), CD96(+), CD99(+), CD117(+), CD123(+), CD133(+), CD135(+), IL-1RAP(+), CD184(+), CD305(+), CD366(+), CD371(+) [35, 36, 37]. CD45 dim Cells that become CD45 have detectable surface expression of CD45 + For example, (CD45 dim ) Cancer stem cells are expressed in healthy (CD45 + ) may have lower (average) CD45 surface expression than hematopoietic stem or progenitor cells.

[0111] Moreover, not removing non-cancerous hematopoietic stem or progenitor cells can avoid the need to perform a bone marrow transplant after treatment with the modified immune cells. Thus, in one embodiment, the (use in) method of treating cancer does not include allogeneic hematopoietic stem cell transplantation.

[0112] Furthermore, treatment with immune cells comprising a Siglec-6-binding polypeptide does not require post-treatment depletion of said immune cells, and therefore the present invention also provides (use in) the methods of treating cancer described herein that do not involve post-treatment depletion of immune cells.

[0113] Moreover, in one embodiment, the (use in) method of treating cancer does not include further conventional chemotherapy. Preferably, the (use in) method of treating cancer does not include further chemotherapy after administration of the immune cells or pharmaceutical composition and / or after termination of treatment with the immune cells or pharmaceutical composition.

[0114] As used herein, conventional chemotherapy refers to the therapeutic use of chemotherapeutic agents that do not specifically target the given abnormal cells (e.g., cancer cells) being treated. Examples of such chemotherapeutic agents include cytarabine and daunorubicin. As used herein, conventional chemotherapy does not refer to targeted therapies used to more specifically target the given abnormal cells (e.g., cancer cells). Examples of such targeted therapies include Bruton's tyrosine kinase (BTK) inhibitors, such as ibrutinib, or fms-like tyrosine kinase 3 (FLT3) inhibitors, such as midostaurin, or epigenetic therapies, such as the administration of hypomethylating agents (DNA methyltransferase inhibitors), such as 5-azacytidine. Such targeted and / or epigenetic therapies can be used either in combination with or as maintenance therapy with immune cells containing Siglec-6-binding polypeptides.

[0115] Thus, the (use in) method of treating cancer may be, for example, a combination therapy further comprising administering a targeted therapy, such as a Bruton's tyrosine kinase (BTK) inhibitor (e.g., ibrutinib), or an fms-like tyrosine kinase 3 (FLT3) inhibitor (e.g., midostaurin), or an epigenetic therapy, such as a hypomethylating agent (DNA methyltransferase inhibitor), such as 5-azacytidine, either in combination with or as a maintenance therapy following treatment with immune cells comprising a Siglec-6-binding polypeptide.

[0116] Therapy using immune cells that bind to Siglec-6 is most effective against target cells that express Siglec-6. Therefore, the present invention also provides a method for determining the expression level of Siglec-6 on the surface of cancer cells from a (human) subject. This method is preferably an in vitro method. It is preferably performed on a sample obtained from a subject suspected of having or diagnosed with cancer. For example, the sample can be a (peripheral) blood sample or a biopsy of the cancer to be treated.

[0117] The present invention also provides a method for diagnosing cancer, preferably AML, comprising determining the expression level of Siglec-6 on the surface of cancer cells derived from a (human) subject. The method is preferably an in vitro method. It is preferably performed on a sample obtained from a subject suspected of having cancer. For example, the sample can be a (peripheral) blood sample or a biopsy of the cancer to be treated.

[0118] Moreover, in one embodiment, the (use in) the described methods of treatment further comprises, prior to treatment, a step of determining the expression level of Siglec-6 on the surface of designated target cells, e.g., cancer cells of the cancer to be treated, e.g., AML.

[0119] The step of determining the expression level of Siglec-6 is preferably carried out in vitro. It is preferably carried out on a sample obtained from the subject to be treated. For example, the sample may be a (peripheral) blood sample or a biopsy of the cancer to be treated.

[0120] The cancer (eg, AML) biopsy can be, for example, a bone marrow biopsy or a tissue biopsy (eg, of extracellular AML expression).

[0121] Consequently, in certain embodiments, the described methods of treatment (for use in) further comprise administering immune cells only if the designated target cells, e.g., cancer (e.g., AML) cells, express Siglec-6.

[0122] Therefore, in the present invention, 1) determining the expression level of Siglec-6 on cancer cells obtained from a subject; 2) administering the immune cells or pharmaceutical composition of the present invention to the subject; Also provided is a method of treating cancer, comprising: optionally administering immune cells or pharmaceutical compositions only if Siglec-6 is expressed on said cancer cells.

[0123] In addition, in the present invention, 1) determining the expression level of Siglec-6 on cancer cells obtained from a subject; 2) administering the immune cells or pharmaceutical composition of the present invention to the subject; and optionally, administering the immune cells or pharmaceutical composition only if Siglec-6 is expressed on said cancer cells.

[0124] Preferably, the cancer is selected from the group consisting of leukemia, such as AML or CLL, MALT lymphoma or clonal mast cell disease, preferably AML and CLL, most preferably AML.

[0125] Those skilled in the art can establish suitable criteria for determining whether Siglec-6 is expressed on a given cell. For example, surface expression can be determined by flow cytometry as described herein. Briefly, cells can be (surface) stained in two separate samples by conjugating a monoclonal antibody (mAb) against Siglec-6 to a detectable (e.g., fluorescent) dye in the first sample and conjugating an isotype control (i.e., a control monoclonal antibody that does not target Siglec-6 and is of the same isotype as the Siglec-6 mAb used) to the same detectable dye in the second sample. If the detectable (average) intensity (e.g., mean fluorescence intensity, MFI) of the detectable dye in the first sample is higher than that in the second sample, the cells can be classified as expressing Siglec-6. If the detectable (e.g., fluorescent) (average) signal of the detectable dye in the first sample is the same as or lower than that in the second sample, the cells can be classified as not expressing Siglec-6.

[0126] For example, cells can be classified as expressing Siglec-6 if the detectable (mean) intensity (e.g., MFI) of the detectable dye in a first sample divided by the detectable (mean) intensity (e.g., MFI) of the detectable dye in a second sample (e.g., normalized mean fluorescence intensity (NMFI) calculated by dividing the MFI obtained after staining with an anti-Siglec-6 mAb by the MFI of an isotype control) is greater than 1.0, at least 1.1, at least 1.2, at least 1.3, at least 1.4, at least 1.5, or at least 2, preferably at least 1.2.

[0127] Additionally, cells can be classified as expressing Siglec-6 if the value (e.g., NMFI) obtained by dividing the detectable (mean) intensity (e.g., MFI) of the detectable dye in a first sample by the detectable (mean) intensity (e.g., MFI) of the detectable dye in a second sample is at least the same as the value (e.g., NMFI) obtained by dividing the detectable (mean) intensity (e.g., MFI) of the detectable dye in a first sample using U937 cells (stained with Siglec-6 mAb) by the detectable (mean) intensity (e.g., MFI) of the detectable dye in a second sample using MOLM-13 cells (stained with an isotype control).

[0128] The above pharmaceutical compositions containing modified T cells are stored at 2-8° C. The pharmaceutical compositions are stable for (at least) 48 hours after formulation and should be administered to patients within this period. [Example]

[0129] The Siglec-6 CAR-T cells generated in the experimental section of this application relate to a non-limiting exemplary embodiment of the present invention.

[0130] Materials and Methods human subjects Human peripheral blood (PB) T cells were obtained from healthy donors (HD) and buffy coats of AML patients. G-CSF-mobilized PB CD34 +Cells were isolated from HD PB. Primary AML bone marrow (BM) and PB, and CLL PB samples were obtained after written informed consent.

[0131] Structure of the Siglec-6-binding CAR polypeptide A schematic diagram of the gene cassettes expected to be contained in Siglec-6 CAR T cells is shown in Figure 6.

[0132] An exemplary gene cassette comprising a nucleotide sequence encoding a Siglec-6 CAR polypeptide also includes an optional truncated epidermal growth factor receptor (EGFRt) sequence separated from the CAR sequence by a T2A ribosomal skip element to ensure translation of CAR and EGFRt into two separate proteins and stoichiometric expression of both proteins on the surface of T cells.

[0133] The EGFRt protein allows for the detection and selection of CAR-positive cells using the anti-EGFR monoclonal antibody cetuximab (trade name: Erbitux®). Additionally, if unmanageable toxicity occurs, EGFRt allows for selective depletion of EGFRt-expressing cells with cetuximab. Preclinical models have demonstrated that administration of cetuximab results in the depletion of EGFRt-expressing CAR-T cells in vivo within days.

[0134] Exemplary CAR structures and amino acid sequences are shown in Table A.

[0135] [Table 1A]

[0136] [Table 1B]

[0137] CAR construction JML-1mAb V H and VL A codon-optimized target domain containing the segment (GeneArt ThermoFisher, Regensburg, Germany, SEQ ID NO: 25; codon-optimized DNA sequence SEQ ID NO: 26) was synthesized and fused in-frame to a CAR scaffold containing a short IgF4-Fc hinge spacer, a CD28 transmembrane domain, a CD28 or 4-1BB costimulatory moiety, and CD3z (DNA sequence SEQ ID NO: 27 or 29; SEQ ID NO: 28 or 30) along with a T2A element and an EGFRt transduction marker (Figure 6A) [14-16]. The entire transgene was encoded within the lentiviral vector epHIV7 and expressed under the control of an EF1 / HTLV hybrid promoter

[16] . CARs specific for FLT3 (clone 4G8), CD19 (clone FMC63), and CD123 (clone 32716) proteins with CD28 or 4-1BB costimulatory moieties [14, 15, 17-19] were used as controls in this study.

[0138] Primary AML and CLL cells BM and PB from AML patients were processed for mononuclear cell isolation using density gradient centrifugation (Biocoll®, Merck Millipore). Samples with a volume of less than 1 mL were processed directly for flow cytometry analysis after red blood cell lysis. When possible, cytotoxicity analysis was performed immediately after this step; otherwise, cells were frozen and kept at -80°C until experiments were performed. Thawed primary AML cells were maintained in RPMI-1640 supplemented with 10% human serum, 2 mM glutamine, 100 U / mL penicillin / streptomycin, and a cytokine cocktail containing IL-4 (1000 IU / mL), granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 ng / mL), stem cell factor (5 ng / mL), and tumor necrosis factor (TNF)-α (10 ng / mL) (Miltenyi Biotec, Germany). Fresh primary CLL samples were analyzed by flow cytometry, after which cells were frozen and subjected to cytotoxicity assays.

[0139] Tumor cell lines Human tumor cell lines U937 (ATCC CRL-1593.2), MOLM13 (ACC 554), MV4;11 (ACC 102), K562 (ACC 10), TF-1 (ACC 334), and Kasumi-1 (ACC 220) were purchased from the American Type Culture Collection (ATCC, USA) or DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany). Cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, and 100 U / mL penicillin / streptomycin. To enable cell detection by flow cytometry and bioluminescence imaging, we transduced all cell lines with a lentiviral vector encoding a firefly luciferase (ffluc)-green fluorescent protein (GFP) transgene and isolated GFP-positive cells by FACS sorting. + The cells were concentrated and then used for in vitro or in vivo studies.

[0140] K562 / Siglec-6 was generated by electroporation of the full-length human SIGLEC-6 gene in K562 cells. To this end, full-length Siglec-6 DNA (SEQ ID NO: 46) was cloned into the pT2HB vector backbone and nucleofected with SB100x minicircle DNA vector using a 4D nucleofector (Lonza, Switzerland). Nucleofected cells were stained with APC-conjugated anti-Siglec-6 mAb, and Siglec-6-positive cells were enriched by FACS-based cell sorting.

[0141] Generation and in vitro analysis of CAR-modified T cells Preparation of CAR-modified T cells, analysis of CAR-T function in vitro, and colony formation assays were performed as previously described [14, 15, 17, 19, 20]. + and CD8 + T cells were isolated from PB by positive selection, transduced with CAR-encoding lentivirus, and expressed using biotinylated anti-EGFR mAb (see above) and anti-biotin beads (Miltenyi). +T cells were enriched and then expanded using a rapid expansion protocol [ 15 , 20 ].

[0142] Colony formation assay PB-derived G-CSF-mobilized human CD34 + CD38 - and CD34 + CD38 + 5 × 10 peripheral blood HSC / P cells 3 Cells / well were seeded in duplicate wells and incubated with JML-1-CAR or CD123-CAR T cells or untransduced T cells at an E:T ratio of 5:1. After 24 hours, one-fifth of the cell suspension was seeded onto methylcellulose-based medium (Methocult opti H4034, Stem Cell Technologies, Cambridge, MA) in 6-well plates (Smartdish™ plates, StemCell Technologies). Colonies were assessed using criteria established according to the manufacturer's instructions and counted under a light microscope after 14 days.

[0143] Flow cytometry analysis Mononuclear cells from healthy donors or AML patients were stained with one or more of the following conjugated mAbs: CD3, CD4, CD8, CD19, CD33, CD34, CD38, CD45, CD56, CD123, CD135 (FLT3), CD327 (Siglec-6), and CD371 (CLL-1), along with matched isotype controls, and 7-AAD for live / dead cell discrimination (Miltenyi biotec, Bergisch-Gladbach, Germany; BD, Heidelberg, Germany; Biolegend, London, UK). PB mononuclear cells (PBMCs) from CLL patients were stained with one or more of the following conjugated mAbs: CD5, CD10, CD19, CD20, CD27, CD38, and CD327 (Miltenyi biotec, Bergisch-Gladbach, Germany; BD, Heidelberg, Germany; Biolegend, London, UK). Non-transduced or CAR-transduced T cells were stained with one or more of the following conjugated mAbs: CD3, CD4, CD8, and 7-AAD (Miltenyi biotec / BD / Biolegend). CAR T cells were detected using an anti-EGFR antibody (ImClone Systems Inc.) conjugated in-house to AF647 (EZ-Link™ Sulfo-NHS-SSBiotin, ThermoFisher Scientific, IL; according to the manufacturer's instructions). Cells were acquired and analyzed by flow cytometry on a FACSCanto (BD), and data analysis was performed using FlowJo software v9.0.2 (Treestar, Ashland, OR). Primary AML blasts were stained with mAbs against CD45, CD34, CD38, CD123, CD33, FLT3, Siglec-6, CLL-1, and CD117. LSCs were stained with mAbs against CD45. dim CD34 + CD38 - Normalized mean fluorescence intensity (NMFI) was calculated by dividing the MFI obtained by flow cytometry after staining with a specific (e.g., anti-Siglec-6) mAb by the MFI obtained by flow cytometry after staining with an isotype control.

[0144] Siglec-6 expression analysis by flow cytometry Siglec-6 (CD327) expression was assessed using APC-conjugated mouse anti-human Siglec-6 mAb (clone 767329, R&D Systems, USA) or REAfinity™ anti-human Siglec-6 (clone REA852, Milteny biotec, Germany) and mouse IgG1 isotype control (R&D Systems, USA) or REA Control Antibody (S), human IgG1 (Milteny biotec, Germany). Briefly, 1 × 10 6 The cells were washed, resuspended in 100 μL of PBS / 0.5% fetal bovine serum, blocked with human IgG for 20 min at 4°C when using R&D Systems mAbs (Jackson ImmunoResearch, USA), and stained with anti-human Siglec-6 mAb or isotype for 30 min at 4°C.

[0145] In vivo experiments using the U937 xenograft model All in vivo experiments were evaluated and approved by the local animal experiment committee. NOD.Cg-PrkdcscidIl2rgtm1Wj / SzJ (NSG) mice (female, 6-8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Mice were transfected with ffluc_GFP. + U937 cells 2 x 10 6 Mice were randomly assigned to different treatment groups and injected with doses administered via the tail vein on days 6 and 21 using a split-dose strategy of CAR T cells. Each dose contained 5 x 10 T cells. 6 cells (i.e., CD4 in 200 μL of PBS / 0.5% FCS) + 2.5 x 10 6 Cells and CD8 + 2.5 x 10 6The PBs were obtained at regular intervals and analyzed for tumor cell and transduced T cell frequencies. Bioluminescence imaging (BLI) was performed weekly using an IVIS Lumina imaging system (PerkinElmer, Waltham, Massachusetts) after intraperitoneal administration of D luciferin substrate (0.3 mg / g body weight) (Biosynth, Staad, Switzerland). Bioluminescence images were analyzed using Living Image software (PerkinElmer).

[0146] In vivo experiments using the MOLM-13 xenograft model All in vivo experiments were evaluated and approved by the local animal experiment committee. NOD.Cg-PrkdcscidIl2rgtm1Wj / SzJ (NSG) mice (female, 6-8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Mice were transfected with ffluc_GFP. + MOLM-13 cells 1 x 10 6 Mice were randomly assigned to different treatment groups and injected with CAR T cells using a split-dose strategy, with doses administered via the tail vein on days 4 and 7. Each dose contained 5 x 10 T cells. 6 cells (i.e., CD4 in 200 μL of PBS / 0.5% FCS) + 2.5 x 10 6 Cells and CD8 + 2.5 x 10 6 The PBs were obtained at regular intervals and analyzed for tumor cell and transduced T cell frequencies. Bioluminescence imaging (BLI) was performed weekly using an IVIS Lumina imaging system (PerkinElmer, Waltham, Massachusetts) after intraperitoneal administration of D luciferin substrate (0.3 mg / g body weight) (Biosynth, Staad, Switzerland). Bioluminescence images were analyzed using Living Image software (PerkinElmer).

[0147] Flow cytometry-based cytotoxicity assay The cytolytic activity of JML-1-CAR, FLT3-CAR, CD19-CAR, or untransduced T cells against primary AML blasts and CLL cells was analyzed in a FACS-based cytotoxicity assay. T cells and primary cells were transduced at 10 target cells per well. 4 Cells were seeded into 96-well plates at effector:target (E:T) ratios ranging from 10:1 to 2.5:1. After 4 or 24 hours of coculture, the cocultured cells were stained and subjected to flow analysis using the following mAbs: anti-CD3 / anti-CD33 / anti-CD34 / anti-CD45 / anti-EGFRt mAbs for AML samples, and anti-CD3 / anti-CD5 / anti-CD20 / anti-CD19 / anti-CD45 for CLL samples. 7-AAD was used to distinguish live and dead cells. To quantify the number of remaining live AML cells, 123 counting beads (e-bioscience, San Diego, CA) were used according to the manufacturer's instructions. Flow analysis was performed on a FACS Canto II (BD), and data were analyzed using FlowJo software (Treestar).

[0148] statistical analysis Statistical analysis was performed using Prism software v6.07 (GraphPad, San Diego, California). Data obtained in in vitro and in vivo experiments were analyzed using Student's t-test (unpaired). Differences in survival observed in in vivo experiments were analyzed using the log-rank (Mantel-Cox) test. P values ​​of differences <0.05 were considered statistically significant.

[0149] result JML-1-CAR T cells induce Siglec-6 + AML cell lines are recognized and eliminated The inventors isolated CD4 + and CD8 +JML-1-CAR T cells were generated. For this purpose, we used a single-chain variable fragment (scFv) derived from the fully human JML-1-mAb10, linking it to the CD3ζ signaling domain and the CD28 or 4-1BB costimulatory domain (Figure 6A). We transduced T cells with lentivirus (multiplicity of infection = 3) and generated CD4 + JML-1-CAR T cells were 38.9–82.4%, and CD8 + We observed transduction efficiencies of 31.7-66.2% for JML-1-CAR T cells (Figures 6B-6D). Prior to expansion and functional testing of JML-1-CAR T cells, we performed an enrichment step using the EGFRt selection marker, which resulted in a yield of >85% CAR+ T cells (Figures 6C-6D). + and CD8 + JML-1-CAR T cells proliferated similarly to FLT3-CAR T cells after bead stimulation and lentiviral transduction (Figure 6E).

[0150] Next, we modified K562 cells to stably express Siglec-6 (K562 / Siglec-6, Figure 7A) and confirmed the specific recognition of cell surface Siglec-6 by JML-1-CAR T cells on native K562 (Siglec-6 negative) and K562 / Siglec-6 cells (Figures 7B-7E). We then evaluated Siglec-6 expression on various AML cell lines and observed variability in Siglec-6 expression levels (very high to low, normalized MFI = 8.22 to 1.12) (Figures 1A and 8A). We then used the AML cell lines U937 and TF-1 (high expression), MV4;11 (moderate expression), MOLM-13 (weak expression), and K562 and Kasumi-1 (non-expressing) to express CD8 + We assessed Siglec-6 recognition by JML-1-CAR T cells and confirmed high levels of specific cytolytic activity by both JML-1_28z and JML-1_BBz CAR T cells against Siglec-6-positive cell lines (Figure 1B, Figure 8B). Notably, specific lysis and lysis kinetics correlated with antigen density on target cells (R 2= 0.57, p = 0.01) (Figures 1A-1B, 2C, 8A-8B). Of note, based on previous studies, the inventors selected a similarly designed and functionally optimal FLT3 CAR (CD28z) as a control for the assay. + and CD8 + Both JML-1-CAR T cells produced high levels of effector cytokines (i.e., IFN-γ and IL-2) and underwent productive proliferation after co-culture with Siglec-6-positive AML cell lines, whereas in control T cells and after exposure to the antigen-negative cell lines K562 and Kasumi-1, we observed only background reactivity (Figures 1C-D, 8C-D).

[0151] Taken together, the data demonstrate that T cells expressing JML-1-CAR with CD28 or 4-1BB costimulatory domains exhibit antigen-specific and potent anti-leukemia reactivity against AML cell lines in vitro.

[0152] Siglec-6 is highly and uniformly expressed on primary AML blasts, including AML leukemia stem cells; JML-1-CAR T cells recognize and eliminate primary AML cells in vitro We evaluated Siglec-6 expression on primary AML blasts from n = 10 adult AML patients. This patient cohort included patients with newly diagnosed AML, relapsed / refractory AML, and secondary AML. Furthermore, the patient cohort included patients with AML and AML with various molecular and cytogenetic abnormalities (Table 1). We found that Siglec-6 was uniformly expressed on AML blasts in each patient (10 / 10) and ranked the patients according to expression levels based on normalized MFI (Figure 2A, Table 1).

[0153] Interestingly, we also found uniform Siglec-6 expression on subpopulations of AML leukemia stem cells (LSCs) in each patient. Even more interestingly, Siglec-6 expression levels were similar or even higher in AML LSC subpopulations compared to the "bulk" population of AML blasts (Figure 2A, Table 1, Figure 9A). We also detected Siglec-6 expression across diverse AML blast populations in the same patient. This indicates that targeting Siglec-6 leads to complete and definitive elimination of AML blasts, resulting in an effective and even potentially curative treatment (Figure 9B).

[0154] To assess the recognition of primary "bulk" AML blasts and AML leukemia stem cells, the inventors used CD8 + Cytolytic experiments were performed with JML-1-CAR T cells. We observed high levels of cytolytic activity by JML-1-CAR T cells against primary "bulk" AML blasts and AML leukemic stem cells (Figure 2A, Figure 2B, and Table 1). Importantly, even though leukemic stem cells are known to have a stronger intrinsic resistance to conventional anti-AML treatments, we observed that cytolysis of "bulk" AML blasts and AML LSCs by JML-1-CAR T cells was similar, and AML leukemic stem cells were rapidly eliminated. JML-1-CAR T cells carrying the CD28 versus 4-1BB costimulatory domain (JML-1_28z and JML-1_BBz CAR-T cells) had similarly potent cytolytic activity against AML LSCs (Figure 2A).

[0155] Taken together, the data demonstrate that Siglec-6 is highly and uniformly expressed in primary AML blasts from patients with various AML disease subtypes. The data also demonstrate that Siglec-6 is highly expressed in AML LSCs, with expression levels similar or even higher compared to the "bulk" AML blast population. The data further demonstrate that targeting Siglec-6 confers specific and potent anti-AML activity, leading to specific and potent elimination of bulk AML blasts and AML leukemia stem cells in the example of JML-1-CAR T cells.

[0156] [Table 2]

[0157] Patient-derived JML-1 CAR-T cells potently eliminate autologous AML blasts We then generated JML-1-CAR T cells from AML patients with newly diagnosed AML (n=2) and previously treated AML (MRD+, n=1) and evaluated their anti-leukemic activity against autologous AML blasts. + T cells accounted for 24.9-50.0% and CD8 + We observed transduction efficiencies of 20.4-43.0% for T cells, and for enriched cells, CAR+ T cells were >95% (Figure 10A). + and CD8 +JML-1-CAR T cells expanded 40- to 60-fold within 12 days of culture (Figure 10B) and demonstrated potent anti-leukemic reactivity against Siglec-6-positive cell lines in vitro (Figures 10C-10E). When cocultured with autologous leukemic blasts, JML-1-CAR T cells demonstrated near-complete elimination of AML blasts within 24 hours (Figure 11A) and delivered significant levels of IFN-γ, indicating extensive proliferation (Figures 11B-11C). Again, JML-1-CAR T cells conferred similar potent cytolytic and cytotoxic activity against "bulk" AML blasts and AML leukemic stem cells (Figure 11A). Control non-CAR-modified T cells from the same respective patients did not demonstrate any discernible reactivity in these functional assays. In conclusion, patient-derived JML-1-CAR T cells are highly responsive to Siglec-6-positive autologous AML blasts and AML cell lines.

[0158] JML-1-CAR T cells eradicate aggressive systemic acute myeloid leukemia in vivo The inventors evaluated the anti-leukemic efficacy of JML-1-CAR T cells in an AML xenograft model using immunodeficient NSG mice. + GFP + U937 leukemia cells were inoculated into the mice, and systemic tumor cell engraftment was observed in the BM and spleen of the mice 6 days after tumor inoculation by BLI analysis (Figure 3A). Next, the inventors administered 5x10 JML-1_28z or JML-1_BBz-CAR T cells to the mice. 6 cells, or non-transduced T cells (CD4 + :CD8 +Mice were treated with JML-1-CAR T cells at a ratio of 1:1 (JML-1_BBz CAR-T cells vs. JML-1_28z CAR T cells). We observed engraftment, stable proliferation, and persistence of JML-1-CAR T cells (Figure 3B, left). Notably, in PB, JML-1_BBz CAR-T cells expanded and persisted significantly more than JML-1_28z CAR T cells (Figure 3B, left), and we observed rapid clearance of leukemic cells from PB within 7 days after JML-1-CAR T cell treatment (Figure 3B, right). Furthermore, all mice treated with JML-1-CAR T cells showed rapid regression of leukemia, whereas all mice treated with non-transduced T cells showed an increase in leukemic burden (Figure 3A, Figure 3C).

[0159] At the end of the observation period, the inventors observed leukemia-free BM, spleen, and PB by flow cytometry and confirmed sustained complete remission of AML in mice treated with JML-1-CAR T cells, whereas progressive, aggressive leukemia was observed in all mice treated with control T cells (Figure 3D, Figure 11).

[0160] The inventors observed a significantly higher overall survival rate (Figure 3E) in the group of mice treated with JML-1-CAR T cells compared to control T cells, and a superior progression-free survival rate in mice treated with JML-1-CAR T cells compared to control T cells (Figure 3F).

[0161] We also evaluated the anti-leukemic efficacy of JML-1-CAR T cells in immunodeficient NSG mice inoculated with MOLM-13 cells (low Siglec-6 expression). Treatment with Siglec-6-CAR T cells conferred a significant anti-leukemic effect (Figure 3G) and caused a significant survival benefit (Figure 3H), but this was less effective compared to the NSG / U937 model (high Siglec-6 expression).

[0162] In conclusion, these data demonstrate that targeting Siglec-6 in AML confers potent anti-leukemic activity in vivo. The data also show that JML-1-CAR T cells confer potent anti-leukemic activity and induce long-term complete remission of AML in vivo.

[0163] Human hematopoietic stem and progenitor cells do not express Siglec-6 and this is preserved after exposure to JML-1-CAR T cells in vitro We sought to evaluate the on-target and off-tumor effects of JML-1-CAR T cells on normal hematopoietic stem and progenitor cells (HSC / P). First, we used G-CSF-mobilized PB-derived CD34 T cells from HD (n=5). + CD38 - HSCs and CD34 + CD38 + Siglec-6 expression on HSPCs was evaluated. We observed a lack of Siglec-6 expression on HSCs and HSPCs in all n=5 HDs analyzed by flow cytometry (NMFI<1.0, Figure 4A). We then analyzed CD8 +JML-1-CAR T cells were cocultured with HSC / Ps to assess in vitro target cell recognition. We used CD123-CAR T cells as a positive control for the assay because of their reported myeloablative activity

[19] . We observed that normal HSC / Ps were not lysed by JML-1-CAR T cells, but the majority of HSC / Ps were rapidly eliminated by CD123-CAR T cells after 24 h (Figure 4B, left). To assess the colony-forming ability of the remaining HSC / Ps in vitro, we performed a colony formation assay using the remaining HSC / Ps after 24 h of coculture with JML-1-CAR or CD123-CAR T cells. HSC / Ps treated with JML-1-CAR T cells exhibited colony formation comparable to HSC / Ps exposed to untransduced T cells (Figure 4B, right). As expected, we observed a small number of erythroid colonies when exposed to CD123-CAR T cells, but myeloid colony formation was completely abolished (Figure 4B, right). We then compared Siglec-6 expression on HSC / P with other candidate CAR target antigens for AML (FLT3, CLL1, CD33, and CD123). We observed the absence of Siglec-6 expression on healthy HSC / P in all five HD patients. In contrast, there was strong expression of FLT3, CLL1, CD33, and CD123 on healthy HSC / P in all five HD patients (Figure 4C, Figures 12A-B).

[0164] Collectively, these data indicate that Siglec-6 is a unique AML target antigen in that it is not expressed on normal HSC / P. The data also indicate that normal HSC / P are not recognized by JML-1-CAR T cells. These data suggest that targeting Siglec-6 does not induce myeloablation in humans.

[0165] Malignant B cells in B-CLL express Siglec-6 and are eliminated by JML-1-CAR T cells We evaluated Siglec-6 expression on primary B-CLL cells derived from the PB of untreated CLL patients (n=10, Table 2). We show that Siglec-6 is uniformly and highly expressed on primary B-CLL cells from 9 out of 10 such patients (Figure 5A, Table 2, and Figure 14A). To assess recognition of primary B-CLL cells, we cloned PBMCs from patients with CD8 + The JML-1_BBz CAR T cells were co-cultured with JML-1-CAR T cells. We observed high levels of cytolytic activity against Siglec-6-positive B-CLL cells by JML-1_28z and JML-1_BBz CAR T cells within 4 hours of co-culture (Figure 5B and Table 2). CLL patients with very high Siglec-6 expression (patients #2, 6, and 8) showed almost complete elimination of B-CLL cells, which was comparable to the lysis observed by CD19-CAR T cells within the 4-hour assay period (Figure 5B). Moreover, the cytolytic activity of JML-1_BBz-CAR T cells showed a linear correlation with the Siglec-6 expression level of B-CLL cells (Figure 5C). We also observed Siglec-6 expression on non-CLL B cells, particularly memory B cells (Figure 5D, Figure 14B). Healthy B cells (CD19) from B-CLL patients + CD5 - CD20 high non-B-CLL cells) were recognized by JML-1-CAR T cells at a similar level to CD19-CAR T cells (Figure 15).

[0166] Collectively, these data demonstrate that Siglec-6 is expressed on malignant B cells in B-CLL and demonstrate that JML-1-CAR T cells rapidly and potently eliminate malignant B-CLL cells.

[0167] Siglec-6 is expressed on a subset of normal B cells and confers recognition by JML-1-CAR T cells Next, we attempted to analyze Siglec-6 expression on HD-derived PBMCs (n=7). We detected high levels of Siglec-6 on the B cell fraction in flow analysis, whereas other healthy PB cells, i.e., NK cells, T cells, and NKT cells, did not express Siglec-6 (Figure 5E). We found that CD33 cells, which express low levels of Siglec-6, + A small fraction of myeloid cells was detected (Figure 5E). We observed significantly higher Siglec-6 expression on memory B cells compared with naive / immature B cells in HD (Figure 5E, Figure 14C). Notably, each HD had memory B cells with low to very high Siglec-6 expression (histogram, Figure 5E). When we compared Siglec-6 expression levels on normal B cells in CLL patients and HD, we observed significantly lower Siglec-6 levels in healthy donors compared with CLL patients (Figure 5F). Therefore, we predict that when patients with B-CLL are treated with JML-1-CAR T cells, Siglec-6+ memory and naive B cells will be sensitive to CAR-mediated recognition and elimination.

[0168] Collectively, these data demonstrate that Siglec-6 is expressed on a subset of normal B cells in healthy donors and patients, and suggest that the predicted on-target, off-tumor effect of targeting Siglec-6 is the selective partial elimination of normal B cells.

[0169] [Table 3]

[0170] Consideration The inventors demonstrate that Siglec-6 is a target for antibody-based cellular immunotherapy in AML. In particular, the inventors demonstrate that Siglec-6 is a target for antibody-based cellular immunotherapy that targets and destroys AML leukemic stem cells. The inventors demonstrate that JML-1-CAR T cells confer potent anti-leukemic efficacy against primary AML blasts in vitro and induce complete remission of leukemia in mice transplanted with AML cell lines.

[0171] Importantly, Siglec-6 was found not to be present on normal HSC / P. Thus, JML-1-CAR T cells did not recognize normal HSC / P and did not cause a reduction in hematopoietic development in colony formation experiments. The potential to eradicate AML blasts and AML leukemia stem cells while sparing normal HSC / P allows for non-myeloablative immunotherapy to effectively treat AML while eliminating the need for allo-HSCT.

[0172] Siglec-6 expression on other healthy tissues is limited to the placenta

[16] , mast cells

[17] , and a subset of normal B cells

[10] , suggesting a favorable safety profile with negligible on-target and off-tumor reactivity. We found that a majority of memory B cells, but a small proportion of naive and immature B cells, express Siglec-6, predicting selective partial depletion of normal B cells after anti-Siglec-6 immunotherapy, such as JML-1 CAR-T cell therapy. Encouragingly, potential hypogammaglobulinemia can be overcome, if needed, by intravenous immunoglobulin (IVIG) replacement therapy, already a common treatment after CD19-CAR T-cell therapy

[27] . Siglec-6 has been reported to be present on mast cells

[18] , which may lead to mast cell reduction or depletion after anti-Siglec-6 immunotherapy.

[0173] Furthermore, we found high expression of Siglec-6 on primary B-CLL cells obtained from untreated CLL patients, consistent with previous findings

[11] . Indeed, we observed potent anti-CLL activity of both JML-1_28z and JML-1_BBz CAR T cells against primary B-CLL cells in vitro. T cells from CLL patients exhibit characteristics of T cell exhaustion and proliferation abnormalities, and therefore, patient-derived JML-1-CAR T cells may not function as well as those from HD patients [28, 23, 24]. Encouragingly, the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib has been shown to improve the anti-leukemia efficacy of CD19-CAR T cells in mouse models and CLL patients [28, 29]. Therefore, combination with ibrutinib may improve the efficacy of JML-1-CAR T-cell therapy in patients with CLL, and preclinical evaluation of the synergistic effects of JML-1-CAR T cells with ibrutinib may be warranted.

[0174] CAR T cell rejection due to transgenes containing murine scFv (e.g., FMC63) is a mechanism contributing to resistance to CAR T cell therapy

[30] . JML-1-CAR is derived from a fully human scFv, making it unlikely to be immunogenic. This allows multiple sequential infusions of JML-1-CAR T cells to be administered to further enhance and sustain the anti-leukemia response, avoiding allo-HSCT if necessary.

[0175] Furthermore, leukemia relapse due to loss or downregulation of target antigens has been observed in 30–60% of all relapses in B-ALL patients treated with CD19-CAR or CD22-CAR T cells [9, 31, 32]. Although there is uniform Siglec-6 expression on AML blasts, there is a potential risk that Siglec-6 expression may change under the therapeutic pressure of anti-Siglec-6 immunotherapy due to clonal diversity. However, no clinical experience with Siglec-6 as an immune target has been reported to date.

[0176] Furthermore, the possibility of proteolytic cleavage resulting in Siglec-6 protein lacking the transmembrane domain and thus resulting in the loss of cell surface Siglec-6 cannot be excluded, as reported for CD19 protein after CD19-CAR T-cell therapy in B-ALL patients

[33] . Preclinical mouse models that mimic antigen loss escape may be useful in investigating strategies to prevent antigen loss escape and resistance to CAR-T-cell therapy after CAR T-cell treatment. Alternatively, the use of tandem or compound CARs that target two or more antigens simultaneously or sequentially may prevent antigen loss escape in AML.

[0177] Our findings regarding Siglec-6 expression and anti-leukemic activity by JML-1-CAR T cells suggest that treatment using Siglec-6-binding immune cells, e.g., JML-1-CAR T cells, may be applicable to patients with AML and CLL and warrants clinical investigation in humans. Furthermore, Siglec-6 expression has also been reported on MALT lymphoma

[15] , clonal mast cell disease

[34] , and thymoma, expanding the application of treatment using Siglec-6-binding immune cells, e.g., JML-1-CAR T cells, to other hematological and oncological conditions as well as other pharmaceutical indications. [Industrial Applicability]

[0178] The Siglec-6-binding polypeptides, nucleotide sequences encoding the Siglec-6-binding polypeptides, expression vectors containing the Siglec-6-binding polypeptides, and immune cells according to the present invention can be industrially produced and sold as products for the described methods and uses (e.g., for treating cancer, as defined herein) in accordance with known standards for the manufacture of pharmaceutical products. Thus, the present invention is industrially applicable.

[0179] array SEQ ID NO: 1 (GMCSF signal peptide)

[0180] [ka]

[0181] SEQ ID NO: 2 (DNA sequence encoding SEQ ID NO: 1)

[0182] [ka]

[0183] SEQ ID NO: 3 (JML-1 heavy chain variable domain (VH))

[0184] [ka]

[0185] SEQ ID NO: 4 (DNA sequence encoding SEQ ID NO: 3)

[0186] [ka]

[0187] SEQ ID NO: 5 (4(GS) x 3 linker)

[0188] [ka]

[0189] SEQ ID NO: 6 (DNA sequence encoding SEQ ID NO: 5)

[0190] [ka]

[0191] SEQ ID NO: 7 (JML-1 light chain variable domain (VL))

[0192] [ka]

[0193] SEQ ID NO: 8 (DNA sequence encoding SEQ ID NO: 7)

[0194] [ka]

[0195] SEQ ID NO: 9 (IgG4 hinge domain)

[0196] [ka]

[0197] SEQ ID NO: 10 (DNA sequence encoding SEQ ID NO: 9)

[0198] [ka]

[0199] SEQ ID NO: 11 (IgG3 hinge)

[0200] [ka]

[0201] SEQ ID NO: 12 (DNA sequence encoding SEQ ID NO: 11)

[0202] [ka]

[0203] SEQ ID NO: 13 (CD28 transmembrane domain)

[0204] [ka]

[0205] SEQ ID NO: 14 (DNA sequence encoding SEQ ID NO: 13)

[0206] [ka]

[0207] SEQ ID NO: 15 (CD28 costimulatory domain)

[0208] [ka]

[0209] SEQ ID NO: 16 (DNA sequence encoding SEQ ID NO: 15)

[0210] [ka]

[0211] SEQ ID NO: 17 (4-1BB costimulatory domain)

[0212] [ka]

[0213] SEQ ID NO: 18 (DNA sequence encoding SEQ ID NO: 17)

[0214] [ka]

[0215] SEQ ID NO: 19 (CD3 zeta signaling domain)

[0216] [ka]

[0217] SEQ ID NO: 20 (DNA sequence encoding SEQ ID NO: 19)

[0218] [ka]

[0219] SEQ ID NO: 21 (T2A ribosomal skip sequence)

[0220] [ka]

[0221] SEQ ID NO: 22 (DNA sequence encoding SEQ ID NO: 21)

[0222] [ka]

[0223] SEQ ID NO: 23 (EGFRt)

[0224] [ka]

[0225] SEQ ID NO: 24 (DNA sequence encoding SEQ ID NO: 23)

[0226] [ka]

[0227] SEQ ID NO: 25 (JML-1scFv)

[0228] [ka]

[0229] SEQ ID NO: 26 (DNA sequence encoding SEQ ID NO: 25)

[0230] [ka]

[0231] SEQ ID NO: 27 (full length CAR with IgG4 hinge and CD28 costimulatory domain)

[0232] [ka]

[0233] SEQ ID NO: 28 (DNA sequence encoding SEQ ID NO: 27)

[0234] [ka]

[0235] SEQ ID NO: 29 (full length CAR with IgG4 hinge and 4-1BB costimulatory domain)

[0236] [ka]

[0237] SEQ ID NO: 30 (DNA sequence encoding SEQ ID NO: 29)

[0238] [ka]

[0239] SEQ ID NO: 31 (full length CAR with IgG3 hinge and CD28 costimulatory domain)

[0240] [ka]

[0241] SEQ ID NO: 32 (DNA sequence encoding SEQ ID NO: 31)

[0242] [ka]

[0243] SEQ ID NO: 33 (full length CAR with IgG3 hinge and 4-1BB costimulatory domain)

[0244] [ka]

[0245] SEQ ID NO: 34 (DNA sequence encoding SEQ ID NO: 33)

[0246] [ka]

[0247] SEQ ID NO: 35 (Extracellular domain with IgG4 hinge)

[0248] [ka]

[0249] SEQ ID NO: 36 (DNA sequence encoding SEQ ID NO: 35)

[0250] [ka]

[0251] SEQ ID NO: 37 (Extracellular domain with IgG3 hinge)

[0252] [ka]

[0253] SEQ ID NO: 38 (DNA sequence encoding SEQ ID NO: 37)

[0254] [ka]

[0255] SEQ ID NO: 39 (intracellular domain with CD28 costimulatory domain)

[0256] [ka]

[0257] SEQ ID NO: 40 (DNA sequence encoding SEQ ID NO: 39)

[0258] [ka]

[0259] SEQ ID NO: 41 (intracellular domain with 4-1BB costimulatory domain)

[0260] [ka]

[0261] SEQ ID NO: 42 (DNA sequence encoding SEQ ID NO: 41)

[0262] [ka]

[0263] SEQ ID NO: 43 (left IR / DR segment)

[0264] [ka]

[0265] SEQ ID NO: 44 (right IR / DR segment)

[0266] [ka]

[0267] SEQ ID NO: 45 (Sleeping Beauty amino acid sequence)

[0268] [ka]

[0269] SEQ ID NO: 46 (DNA sequence encoding full-length Siglec-6)

[0270] [ka] [References] TIFF2026034446000051.tif230169TIFF2026034446000052.tif254169TIFF2026034446000053.tif35169

Claims

1. A Siglec-6-binding polypeptide comprising or consisting of a chimeric antigen receptor (CAR), the CAR comprising at least one extracellular ligand-binding domain, a transmembrane domain and at least one intracellular signaling domain, the extracellular ligand-binding domain comprising a Siglec-6-binding element; the Siglec-6 binding element comprises or consists of a first antibody fragment that binds to Siglec-6 and a second antibody fragment that binds to Siglec-6, which are linked to each other via a linker; A Siglec-6-binding polypeptide, wherein the first antibody fragment comprises a VH region having the amino acid sequence shown in SEQ ID NO: 3, and the second antibody fragment comprises a VL region having the amino acid sequence shown in SEQ ID NO:

7.

2. (i) the Siglec-6 binding element is represented by the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 25; and / or (ii) the polypeptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33, or an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 27, 29, 31, or 33; The Siglec-6 binding polypeptide according to claim 1.

3. (a) the extracellular ligand-binding domain comprises a spacer domain, e.g., a spacer domain derived from CD8α, IgG3, or IgG4; (b) the transmembrane domain comprises a CD28 transmembrane domain; (c) the intracellular signaling domain comprises a costimulatory domain and a CD3 zeta domain; The Siglec-6-binding polypeptide according to claim 1 or 2.

4. the polypeptide comprises a nucleotide sequence represented by SEQ ID NO: 26 or a nucleotide sequence having at least 90% identity to the nucleotide sequence shown in SEQ ID NO: 26; and / or The polypeptide comprises a nucleotide sequence represented by any one of SEQ ID NOs: 28, 30, 32, or 34, or a nucleotide sequence having at least 90% identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 28, 30, 32, or 34. A polynucleotide or set of polynucleotides encoding the Siglec-6-binding polypeptide of any one of claims 1 to 3.

5. The polynucleotide of claim 4, further comprising flanking segments 5' and 3' to the polynucleotide encoding the polypeptide.

6. 6. The polynucleotide of claim 4, comprising a nucleotide sequence of a left IR / DR, a polynucleotide sequence encoding a Siglec-6-binding polypeptide, and a nucleotide sequence of a right IR / DR.

7. 7. An expression vector comprising the polynucleotide or set of polynucleotides according to any one of claims 4 to 6, wherein the expression vector is a non-viral vector or a viral vector.

8. 8. The expression vector of claim 7, which is a minimal DNA expression cassette and is a non-viral vector.

9. 8. The expression vector of claim 7, which is a viral vector that is a lentiviral or gamma-retroviral vector.

10. An immune cell comprising an expression vector comprising a Siglec-6 binding polypeptide described in any one of claims 1 to 3, and / or a polynucleotide or set of polynucleotides encoding a Siglec-6 binding polypeptide described in any one of claims 1 to 3, and / or a polynucleotide or set of polynucleotides encoding a Siglec-6 binding polypeptide described in any one of claims 1 to 3.

11. The immune cell of claim 10, wherein the polynucleotide or set of polynucleotides and / or vector is expressed.

12. The immune cell of claim 10 or 11, which is a lymphocyte.

13. (a) isolating immune cells from a blood sample of a subject; (b) transforming or transducing immune cells with the polynucleotide of any one of claims 4 to 6 or the expression vector of any one of claims 7 to 9; 1. A method for generating (recombinant) immune cells, comprising:

14. 14. The method of claim 13, wherein in step (b) immune cells are transformed using 1) a transposable element comprising the polynucleotide of any one of claims 4 to 6 and 2) a transposase (a polynucleotide encoding the same), or the transposase is PiggyBac transposase.

15. 15. The method of claim 13 or 14, wherein the immune cells are lymphocytes.

16. 16. Immune cells obtainable by the method according to any one of claims 13 to 15.

17. 17. A pharmaceutical composition comprising a plurality of immune cells according to any one of claims 10 to 12 or claim 16.

18. 18. An immune cell according to any one of claims 10 to 12 or claim 16 or a pharmaceutical composition according to claim 17 for use as a medicament.

19. 18. An immune cell according to any one of claims 10 to 12 or claim 16 or a pharmaceutical composition according to claim 17 for use in a method for treating cancer.

20. 20. The immune cell or pharmaceutical composition for use according to claim 18 or 19, wherein the immune cell is a lymphocyte.

21. The cancer is (a) Cancer expressing Siglec-6; (b) the cancer is leukemia, and / or (c) the cancer is primary acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), MALT lymphoma, or clonal mast cell disease; 21. Immune cells or pharmaceutical compositions for use according to claim 19 or 20.

22. (i) the method of treating cancer includes elimination of cancer stem cells of the cancer by the immune cells; (ii) the method of treating cancer does not include depletion of non-cancerous hematopoietic stem or progenitor cells by the immune cells; (iii) the method of treating cancer does not include allogeneic hematopoietic stem cell transplantation, or the subject has experienced a recurrence of the cancer after allogeneic hematopoietic stem cell transplantation; (iv) the method does not include further chemotherapy after administration of the immune cells or the pharmaceutical composition and / or after termination of treatment with the immune cells or the pharmaceutical composition; (v) the method of treating cancer does not include depletion of the immune cells after treatment; (vi) the method comprising: 1) determining the expression level of Siglec-6 on cancer cells obtained from the subject; and then 2) administering the immune cells or the pharmaceutical composition to the subject; Including, (vii) the process comprises: (i) a CD70-binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to CD70, or comprising or consisting of a chimeric antigen receptor (CAR); or (ii) an immune cell comprising a CD70-binding polypeptide according to (i), and / or a polynucleotide or set of polynucleotides encoding the CD70-binding polypeptide according to (i), and / or an expression vector comprising a polynucleotide or set of polynucleotides encoding the CD70-binding polypeptide according to (i). further treatment with (viii) the process comprises: (i) a TIM-3-binding polypeptide comprising or consisting of an antibody or fragment thereof that binds to TIM-3, or comprising or consisting of a chimeric antigen receptor (CAR); or (ii) an immune cell comprising the TIM-3-binding polypeptide described in (i), and / or a polynucleotide or set of polynucleotides encoding the TIM-3-binding polypeptide described in (i), and / or an expression vector comprising the polynucleotide or set of polynucleotides encoding the TIM-3-binding polypeptide described in (i). further treatment with 22. Immune cells or pharmaceutical compositions for use according to any one of claims 19 to 21.

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