Patent Application
20230287129
The present invention relates to a siglec-6-binding polypeptide that comprises or consists of an antibody or a fragment thereof binding siglec-6 or 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 such immune cells and a pharmaceutical composition comprising such immune cells. The immune cells and the pharmaceutical composition of the present invention may be used in methods for treating a disease in a patient.
T-cells endowed with synthetic chimeric antigen receptor (CAR) have shown durable remissions in B-cell and plasma cell malignancies [1-4] and there is a quest for targeting other hematological and solid cancers with CAR T-cells. Acute Myeloid Leukemia (AML) is an entity with unmet medical need and requires novel curative therapies. Although several preclinical studies show feasibility of targeting AML with CAR-T cells, the identification of a CAR target antigen with acceptable safety profile remains challenging [5]. This is mainly due to the expression of previously identified candidate CAR target antigens in AML on normal hematopoietic stem and progenitor cells (HSC/P) [6,7]. Therefore, there is a strong desire for a novel CAR target that is not expressed by HSC/P and that has no or minimal expression on other healthy cells and tissues.
CAR-T cells targeting B-cell and plasma cell malignancies have shown unprecedented clinical responses in patients with multiple prior lines of treatments and advanced hematologic malignancies [1-4], raising interest in using CAR T-cell therapy in AML. Because CAR T-cells target cell surface molecules, it is desirable that a target antigen is uniformly expressed on tumor cells and has minimal or even absent expression on healthy cells and tissues. This prerequisite is challenging for CAR-T therapy in AML because most of the candidate antigens that have previously been proposed are expressed by healthy HSC/P and by innate immune cells [20]. Therefore, directing CAR T-cells against these antigens is anticipated to lead to undesired toxicities including reduction or ablation of healthy hematopoiesis. Indeed, CAR T-cells against myeloid-lineage antigens, e.g. CD123, CD33 has been shown to be myeloablative, and to necessitate allogeneic hematopoietic stem cell transplantation (alloHSCT) 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 severe adverse events in a first-in-man clinical trial that employed an allogeneic CD123-CAR T-cell product and as a consequence, this trial had been placed on hold [21]. In order to prevent elimination of normal HSC/P, Kim et al. suggested that gene editing using CRISPR/Cas9 to knockdown CD33 in donor HSC/P cells may prevent elimination of HSC/P by CD33-CAR T-cells [22]. However, clinical implementation of this strategy—that entails the administration of gene-edited HSC (in addition to gene-modified CD33-CAR T-cells)—would be extremely laborious, complex, and expensive. In addition, the use of gene-edited HSCs entails a substantially greater risk for undesired genotoxicity, including a risk for malignant transformation.
Additionally, other potential CAR targets of interest in AML include FLT3, CLL-1, CD44v6, CD7, folate receptor p, Lewis-y antigen. The inventors have previously shown that targeting FLT3 with CAR T-cells in high-risk FLT3-ITD+AML can induce complete remissions in mouse xenografts and that the anti-leukemia efficacy of FLT3 CAR-T cells can be enhanced by FLT3-inhibitors [23]. However, FLT3 is also expressed on HSC/P and FLT3-CAR T-cells treatment is anticipated 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], and the inventors observed CLL-1-expression on HSC/P, suggesting the potential for severe toxicities if targeted by CLL-1-specific CAR T-cells. Although CD44v6 is absent on HSC/P, its expression in vital cells and tissues such as keratinocytes, oral mucosa and monocytes may lead to lethal toxicities when targeted by CD44v6-CAR T-cells [25]. CD7 is expressed only by ˜30% AML patients and at high-levels on T-cells [26], requiring CD7 knockout from T-cells to manufacture anti-CD7-CAR T-cells, further making clinical application complex.
Sialic-acid-binding immunoglobulin-like lectins (siglecs) are a immunoglobulin superfamily of cell surface receptors that are expressed mainly by leukocytes and are associated with inhibitory signaling in human immune cells [8]. Notably, siglec-2 (CD22) and siglec-3 (CD33) are member of siglec superfamily and 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 shown complete remissions in relapsed/refractory (R/R) B-ALL patients [9], indicating that targeting siglecs with favorable expression profile can induce leukemia remissions and potentially cure patients.
Baskar et al. generated monoclonal antibodies (mAbs) from a post-allogeneic hematopoietic stem cell transplantation (alloHSCT) repertoire that potentially contributed to the graft-versus leukemia (GVL) response in a chronic lymphocytic leukemia (CLL) patient [10]. Subsequent target discovery analyses revealed mAb ‘JML-1’ as a candidate which binds and recognizes human siglec-6 protein [11]. Siglec-6 belongs to CD33-related siglec subfamily and its structure closely relates to siglec-3 (CD33). Siglec-6 consists of three extracellular immunoglobulin (Ig) domains and two intracellular immunoreceptor tyrosine-based inhibition motifs (ITIM) motifs [12-14]. Due to these ITIM motifs, siglec-6 is thought to serve as regulator of activating pathways like other CD33-related siglecs [13]. Siglec-6-expression is reported in primary B-cells [10,12] and aberrantly in CLL [10,11] and in MALT lymphoma [15]. Siglec-6 is also known to be expressed on placenta [12,16] and human mast cells [17,18]. However, unlike other siglec proteins, it is absent on NK cells, T cells, neutrophils, macrophages and monocytes [13].
In view of the above, there still remains the critical need for novel therapies that provide a safe and effective treatment for leukemia and lymphoma, and especially AML, CLL, MALT lymphoma and clonal mast cell diseases.
The present invention aims to overcome the unmet clinical needs by providing an improved composition for therapeutic treatment of patients.
The inventors demonstrate siglec-6-expression on primary AML blasts derived from newly diagnosed and relapsed/refractory AML patients. Intriguingly, the inventors also demonstrate siglec-6-expression on AML leukemic stem cells (LSCs). Human CD4+ and CD8+ T-cells were equipped to express a siglec-6-specific CAR with a targeting domain derived from the fully human JML-1 IgG1 mAb (JML-1-CAR). The anti-leukemia reactivity of AML patient and HD derived JML-1-CAR T-cells against primary ‘bulk’ AML blasts, AML leukemic stem cells and AML cell lines was assessed and demonstrated. Further, the inventors evaluated the expression of siglec-6 on normal hematopoietic stem/progenitor cells (HSC/P) and mature peripheral blood cells, in order to assess potential on-target off-tumor hematologic toxicity mediated by JML-1-CAR T-cells. The inventors show that siglec-6 is not expressed on normal HSC/P and demonstrate that normal HSC/P are not recognized by JML-1-CAR T-cells. High levels of siglec-6 on malignant B-CLL cells from treatment-naïve CLL patients and in healthy B-cells were confirmed and anti-leukemia activity of JML-1 CAR-T cells against CLL demonstrated.
The present application plausibly shows for the first time that a therapy using immune cells binding to siglec-6, such as immune cells comprising a CAR binding to siglec-6, is efficacious. Such therapy involves the elimination of siglec-6 expressing cells.
Furthermore, the present application confirms that siglec-6 is not expressed on non-cancerous hematopoietic stem/progenitor cells (HSC/P), suggesting targeting siglec-6 with immune cells, such as CAR-T cells, could be a safe approach in treating cancer, such as AML, and may not require subsequent alloHSCT. The application therefore for the first time presents a treatment using immune cells binding to siglec-6 that does not involve elimination of non-cancerous HSC/P and consequently obviates the need to perform alloHSCT after such immunotherapy. Moreover, the use of immune cells binding siglec-6 can obviate the need to deplete said immune cells after the treatment.
Accordingly, the present invention provides the following preferred embodiments:
[1] A siglec-6-binding polypeptide that comprises or consists of an antibody or a fragment thereof binding siglec-6 or that comprises or consists of a chimeric antigen receptor (CAR).
[2] The siglec-6-binding polypeptide according to [1] that comprises or consists of an antibody or a fragment thereof binding siglec-6.
[3] The siglec-6-binding polypeptide according to [1] or [2] that is at least bispecific.
[4] The siglec-6-binding polypeptide according to [2] or [3] that comprises or consists of a first antibody or a fragment thereof binding siglec-6 and a second antibody or fragment thereof binding to a target other than siglec-6, optionally connected to each other via a linker.
[5] The siglec-6-binding polypeptide according to any one of [2]-[4], wherein the antibody or a fragment thereof binding siglec-6 is represented by an amino acid sequence shown in SEQ ID NO: 25 or by an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 25.
[6] The siglec-6-binding polypeptide according to [4] or [5] that is capable of binding to an immune cell, such as a T cell or an NK cell, preferably to a T cell.
[7] The siglec-6-binding polypeptide according to any one of [4]-[6] that additionally binds to CD3, such as CD3zeta or CD3epsilon, preferably CD3zeta.
[8] The siglec-6-binding polypeptide according to any one of [4]-[7] that is capable of recruiting an immune cell, such as a T cell or an NK cell, preferably a T cell, to a target cell expressing siglec-6 on its surface.
[9] The siglec-6-binding polypeptide according to any one of [2]-[5] that 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] that 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 signalling domain.
[13] The siglec-6-binding polypeptide according to [12], wherein said 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 an amino acid sequence shown in SEQ ID NO: 25 or by an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 25.
[15] The siglec-6-binding polypeptide according to any one of [12]-[14], wherein the extracellular ligand binding domain comprises a spacer domain, such as a spacer domain from CD8a, IgG3 or IgG4.
[16] The siglec-6-binding polypeptide according to any one of [12]-[15], wherein said transmembrane domain comprises a CD28 transmembrane domain, preferably represented by an amino acid sequence shown in SEQ ID NO: 13 or by an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 13.
[17] The siglec-6-binding polypeptide according to any one of [12]-[16], wherein said intracellular signalling domain comprises a costimulatory domain and a CD3 zeta domain, wherein 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 any one of [17] or [18], wherein the CD28 cytoplasmic domain is represented by an amino acid sequence shown in SEQ ID NO: 15 or by an amino acid sequence having at least 90% identity to an 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 an amino acid sequence shown in SEQ ID NO: 17 or by an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 17.
[21] The siglec-6-binding polypeptide according to any one of [17]-[20], wherein the CD3 zeta domain is represented by an amino acid sequence shown in SEQ ID NO: 19 or by an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 19.
[22] The siglec-6-binding polypeptide according to any one of [12]-[19] and [21], wherein the polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 27, 29, 31 or 33 or an amino acid sequence having at least 90% identity to an amino acid sequence shown in any one of SEQ ID NOs: 27, 29, 31 or 33.
[23] A polynucleotide or set of polynucleotides encoding the siglec-6-binding polypeptide according to any one of the preceding items.
[24] The polynucleotide or set of polynucleotides according to [23], wherein the polynucleotide comprises a nucleotide sequence represented by SEQ ID NO: 26 or a nucleotide sequence having at least 80% identity to nucleotide sequence shown in SEQ ID NO: 26.
[25] The polynucleotide or set of polynucleotides according to [23] or [24], wherein the polynucleotide comprises a nucleotide sequence represented by any one of SEQ ID NO: 28, 30, 32 or 34, or a nucleotide sequence having at least 80% identity to nucleotide sequence shown in any one of SEQ ID NO: 28, 30, 32 or 34.
[26] The polynucleotide according to any one of [23]-[25], wherein the polynucleotide further comprises flanking segments in 5′-direction and in 3′-direction of the polynucleotide encoding the polypeptide.
[27] The polynucleotide according to [26], wherein the flanking segment in 5′-direction is a left inverted repeat/direct repeat (IR/DR) segment and the flanking segment in 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 right IR/DR segment is represented by SEQ ID NO: 44.
[29] The polynucleotide according to any one of [23]-[28], wherein the polynucleotide comprises a nucleotide sequence of a left IR/DR, a polynucleotide sequence encoding the siglec-6-binding polypeptide and a nucleotide sequence of a right IR/DR.
[30] An expression vector comprising a polynucleotide or set of polynucleotides according to any one of [23]-[29].
[31] The expression vector according to [30] that is a non-viral vector or a viral vector.
[32] The expression vector according to [31] that is a non-viral vector.
[33] The expression vector according to [32], wherein the expression vector is a minimal DNA expression cassette.
[34] The expression vector according to [32] or [33], wherein the expression vector 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]-[35], wherein the expression vector is a minicircle DNA.
[37] The expression vector according to [31] that is a viral vector.
[38] The expression vector according to [37] that that is a lentiviral or gamma-retroviral vector.
[39] An immune cell comprising a siglec-6-binding polypeptide according to any one of [11]-[22] and/or a polynucleotide or set of polynucleotides encoding a siglec-6-binding polypeptide according to any one of [11]-[22] and/or an expression vector comprising a polynucleotide or set of polynucleotides encoding a siglec-6-binding polypeptide according to any one of claims [11]-[22].
[40] The immune cell according to [39], wherein the polynucleotide or set of polynucleotides and/or the vector is expressed.
[41] The immune cell according to any one of the [39]-[40], wherein said immune cell is a lymphocyte.
[42] The immune cell according to [41], wherein said lymphocyte is a T cell or an NK cell.
[43] The immune cell according to [42], wherein said T cell is a CD4+ cell or a CD8+ cell.
[44] The immune cell according to any one of the [39]-[43], further expressing a detectable marker.
[45] The immune cell according to any one of the claims [39]-[44], wherein said immune cell is a human cell.
[46] Method for producing (recombinant) immune cells, comprising the steps of
[47] The method according to [46], wherein, in step (b), the immune cells are transformed using 1) a transposable element comprising a polynucleotide according to any one of [23] to [29] and 2) a (polynucleotide encoding a) 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 an amino acid sequence shown in SEQ ID NO: 45.
[50] The method according to any one of [47]-[49], wherein the transposable element is integrated into the genome of the immune cells by the action of the transposase.
[51] The method according to any one of [46]-[50], wherein the immune cell is a lymphocyte.
[52] The method according to [51], wherein the lymphocyte is a T cell or an NK cell.
[53] The method according to [52], wherein the T cell is a CD4+ cell or a CD8+ cell.
[54] The method according to any one of [46]-[53], wherein the subject is a human.
[55] An immune cell obtainable by the method of any one of [46]-[54].
[56] A pharmaceutical composition comprising a plurality of immune cells according to any one of [39]-[45] or of [55], wherein the plurality of immune cells is optionally be a mixture of CD4+ and CD8+ cells.
[57] The immune cell according to any one of [39]-[45] or of [55], or the pharmaceutical composition according to [56] for use as a medicament.
[58] The immune cell according to any one of [39]-[45] or of [55], or the pharmaceutical composition according to [56] for use in a method of treating cancer, wherein the immune cell or the pharmaceutical composition is to be administered to a subject.
[59] The immune cell or the pharmaceutical composition for use according to [57] or [58], wherein the pharmaceutical composition is to be administered intravenously.
[60] The immune cell or the pharmaceutical composition for use according to any one of [57]-[59], wherein said immune cell is a lymphocyte.
[61] The immune cell or the pharmaceutical composition for use according to [60], wherein said lymphocyte is a T cell or an NK cell.
[62] The immune cell or the pharmaceutical composition for use according to [61], wherein the T cell is a CD4+ T cell and/or CD8+ T cell.
[63] The immune cell or the pharmaceutical composition for use according to any one of [58]-[62], wherein said subject is a human.
[64] The immune cell or the pharmaceutical composition for use according to any one of [58]-[63], wherein said cancer is a siglec-6 expressing cancer.
[65] The immune cell or the pharmaceutical composition for use according to any one of [58]-[64], wherein said cancer is leukemia.
[66] The immune cell or the pharmaceutical composition for use according to any one of [58]-[65], wherein said cancer is primary acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), MALT lymphoma, clonal mast cell disease or thymoma.
[67] The immune cell or the pharmaceutical composition for use according to any one of [58]-[66], wherein the cancer is AML.
[68]. The immune cell or the pharmaceutical composition for use according to any one of [58]-[67], wherein the method of treating cancer involves the elimination of cancer stem cells of said cancer by said immune cells.
[69] The immune cell or the pharmaceutical composition for use according to [68], wherein the cancer stem cells are CD45dim cells, preferably CD45dimCD34+ cells, and most preferably CD45dimCD34+ CD38-cells.
[70] The immune cell or the pharmaceutical composition for use according to any one of [58]-[69], wherein the method of treating cancer does not involve the elimination of non-cancerous hematopoietic stem or progenitor cells by said immune cells.
[71] The immune cell or the pharmaceutical composition for use according to any one of [68]-[70], further comprising monitoring the elimination of said cancer stem cells and/or of said non-cancerous hematopoietic stem or progenitor cells.
[72] The immune cell or the pharmaceutical composition for use according to any one of [58]-[71], wherein the method of treating cancer does not involve subsequent allogeneic hematopoietic stem cell transplantation, or wherein the subject is a subject having a relapse of the cancer after allogeneic hematopoietic stem cell transplantation.
[73] The immune cell or the pharmaceutical composition for use according to any one of [58]-[72], wherein the method does not involve additional chemotherapy after administration of the immune cells or the pharmaceutical composition and/or after the termination of the therapy with the immune cells or the pharmaceutical composition.
[74] The immune cell or the pharmaceutical composition for use according to any one of [58]-[73], wherein the method of treating cancer does not involve depletion of said immune cells after treatment.
[75] The immune cell or pharmaceutical composition for use according to any one of [58]-[74], wherein the method comprises:
[76] The immune cell or pharmaceutical composition for use according to [75], wherein the immune cell or pharmaceutical composition is administered in step 2) only if siglec-6 is expressed on said cancer cells.
[77] The immune cell or pharmaceutical composition for use according to any one of [58]-[76], wherein the method involves additional therapy with
[78] The immune cell or pharmaceutical composition for use according to [77], wherein the CD70-binding polypeptide comprises or consists of a chimeric antigen receptor (CAR).
[79] The immune cell or pharmaceutical composition for use according to any one of [58]-[78], wherein the method involves additional therapy with
[80] The immune cell or pharmaceutical composition for use according to [79], wherein the TIM-3-binding polypeptide comprises or consists of a chimeric antigen receptor (CAR).
Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of cancer immunotherapy, gene therapy, immunology, biochemistry, genetics, and molecular biology.
All 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 being described herein.
The term “about” used in the context of the present invention means that the value following the term “about” may vary within the range of +/−20%, preferably in the range of +/−15%, more preferably in the range of +/−10%.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. References referred to herein are indicated by a reference number in square brackets (e.g. as “[31]” or as “reference [31]”), which refers to the respective reference in the list of references at the end of the description. In case of conflict, the present specification, including definitions, will prevail over the cited references. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.
As used herein, each occurrence of terms such as “comprising” or “comprises” may optionally be substituted with “consisting of” or “consists of”.
Siglec-6-Binding Polypeptide
The present invention relates to a siglec-6-binding polypeptide that comprises or consists of an antibody or a fragment thereof binding siglec-6 or a chimeric antigen receptor (CAR), preferably a CAR.
Specifically provided is a siglec-6-binding polypeptide that comprises or consists of an antibody or a fragment thereof binding to siglec-6.
The term “antibody or a fragment thereof” includes, for example, monoclonal, chimeric, single chain, humanized and human antibodies. It also includes, for example, Fab fragments, F(ab′)2, Fv, scFv fragments or single domain antibodies such as domain antibodies or nanobodies, single variable domain antibodies or immunoglobulin single variable domain comprising merely one variable domain, which might be VHH, VH or VL, that specifically bind an antigen or epitope independently of other variable regions or domains. Said term also includes diabodies or Dual-Affinity ReTargeting (DART) antibodies. Further envisaged are (bispecific) single chain diabody, tandem diabody (Tandab), bispecific T cell engager (BiTE) antibodies and tri-specific T cell engaging antibodies such as hemibodies. Any such antibodies and fragments thereof, as well as their production is commonly known in the art.
Preferably, the polypeptide comprising or consisting of antibody or the fragment thereof binding siglec-6 is at least bispecific. However, it can also be multispecific, such as trispecific or tetra specific. Bispecific, trispecific, etc. means that that the polypeptide is able to bind to two, three, etc. different target antigens simultaneously or sequentially.
Thus, the siglec-6-binding polypeptide can comprise or consist of a first antibody or a fragment thereof binding to siglec-6 and a second antibody or fragment thereof binding to a target other than siglec-6, that can optionally be connected to each other via a linker.
The antibody or a fragment thereof binding to siglec-6 can for example be represented by an amino acid sequence shown in SEQ ID NO: 25 or 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 shown in SEQ ID NO: 25 and has siglec-6-binding ability. Preferably, the antibody or fragment thereof binding to siglec-6 is represented by an amino acid sequence shown in SEQ ID NO: 25.
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 to a T cell. However, binding to other immune cells, such as macrophages, is also encompassed.
Accordingly, the at least bispecific siglec-6-binding polypeptide preferably additionally binds to (human) CD3, such as CD3 epsilon or CD3zeta, preferably CD3zeta. CD3 is expressed on T cells and forms part of the T cell receptor. Thus, the bispecific polypeptide can recruit effector cells, such as T cells or NK cells, to target cells expressing siglec-6 on their surface, by binding simultaneously to siglec-6 and to e.g. CD3. Antibodies to human CD3 are well known in the art, see for example the antibodies against N-terminal amino acids 1-27 of CD3 epsilon in WO 2008/119567, incorporated herein by reference.
Thus, the at least bispecific siglec-6-binding polypeptide is preferably capable of recruiting an immune cell, such as a T cell, an NK cell, preferably a T cell, to a target cell expressing siglec-6 on its surface.
In another embodiment, the siglec-6-binding polypeptide is conjugated to another compound, such as a detectable marker or a drug. It is preferred in this embodiment that the polypeptide is conjugated to a drug. The drug can, for example, be a toxin. The toxin is preferably capable of killing target cells expressing siglec-6 on their surface. Examples of such toxins include Maytansin, Auristatin, Taxoid and PNU anthracycline.
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 and that can bind to a ligand, e.g. expressed on the surface of another cell. The receptor can thereby lead to recruitment of a cell expressing the receptor to target cells that express the ligand on their surface. Moreover, upon binding to the ligand the CAR can optionally transmit an intracellular signal within the cells on which it is expressed. Thus, for example the CAR can be expressed on a T cell, and upon binding to its ligand activate the T cell.
In a more specific embodiment, the CAR thus comprises at least one extracellular ligand binding domain, a transmembrane domain and at least one intracellular signalling domain, wherein said extracellular ligand binding domain preferably comprises a siglec-6-binding element. The extracellular domain can further comprise a spacer domain, such as a spacer domain from CD8a, IgG3 or IgG4. The transmembrane domain can comprise a CD28 transmembrane domain. The intracellular signalling domain can comprise a costimulatory domain and a CD3 zeta (CD3i) domain.
In an embodiment of the 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 an amino acid sequence shown in SEQ ID NO: 25 and has siglec-6-binding ability. Preferably, the siglec-6-binding element is represented by an amino acid sequence shown in SEQ ID NO: 25.
In an 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 an amino acid sequence shown in SEQ ID NO: 9 or 11. Preferably, the spacer domain is represented by an amino acid sequence shown in SEQ ID NO: 9 or 11. The spacer connects the extracellular targeting and the transmembrane domain. It affects the flexibility of the siglec-6-binding element, reduces the spatial constraints from CAR to ligand and therefore impacts epitope binding. Binding to epitopes with a membrane-distal position often require CARs with shorter spacer domains, binding to epitopes which lie proximal to the cell surface often require CARs with long spacer.
In an 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 an amino acid sequence shown in SEQ ID NO: 13. Preferably, the transmembrane domain is represented by an amino acid sequence shown in SEQ ID NO: 13.
The CD28 transmembrane domain consists of a hydrophobic alpha helix, traverses the membrane of the cell and anchors the CAR to the cell surface. It impacts the expression of the CAR on the cell surface.
In an embodiment of the invention, the costimulatory domain of the siglec-6-CAR polypeptide is a CD28 cytoplasmic domain or a 4-1BB costimulatory domain.
In an embodiment of the invention, the intracellular signalling domain comprises a CD28 cytoplasmic domain and a CD3 zeta domain. In another embodiment of the invention, the intracellular signalling domain comprises a 4-1BB costimulatory domain and a CD3 zeta domain.
In an 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 with an amino acid sequence shown in SEQ ID NO: 15. Preferably, the CD28 cytoplasmic domain is represented by an amino acid sequence shown in SEQ ID NO: 15. The CD28 cytoplasmic domain is a costimulatory domain and is derived from intracellular signaling domains of costimulatory molecules.
In an 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 with an amino acid sequence shown in SEQ ID NO: 17. Preferably, the 4-1BB costimulatory domain is represented by an amino acid sequence shown in SEQ ID NO: 17.
In an 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 with an amino acid sequence shown in SEQ ID NO: 19. Preferably, the CD3 zeta domain is represented by an amino acid sequence shown in SEQ ID NO: 19. The CD3 zeta domain mediates downstream signaling during the T cell activation. It is derived from the intracellular signaling domain of the T cell receptor and contains ITAMs (immunoreceptor tyrosine based activation motifs).
In an 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 an amino acid sequence shown in SEQ ID NO: 35 or 37. Preferably, the extracellular domain comprises an amino acid sequence shown in SEQ ID NO: 35 or 37. More preferably, the extracellular domain consists of an amino acid sequence shown in SEQ ID NO: 35 or 37.
In an embodiment of the invention, the intracellular signalling domain comprises an amino acid sequence having at least 90%, preferably 95%, more preferably 97% or most preferably 99% sequence identity to an amino acid sequence shown in SEQ ID NO: 39 or 41. Preferably, the intracellular signalling domain comprises an amino acid sequence shown in SEQ ID NO: 39 or 41. More preferably, the intracellular signalling domain consists of an amino acid sequence shown in SEQ ID NO: 39 or 41.
Thus, said extracellular domain can comprise an amino acid sequence shown in SEQ ID NO: 35 or 37 or an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 35 or 37, said transmembrane domain can comprise an amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 13 and said intracellular signalling domain can comprise an amino acid sequence shown in SEQ ID NO: 39 or 41 or an amino acid sequence having at least 90% identity to an amino acid sequence shown in SEQ ID NO: 39 or 41.
In a 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 an amino acid sequence shown in any one of SEQ ID NOs: 27, 29, 31 or 33. Preferably, the siglec-6-CAR polypeptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 27, 29, 31 or 33. More preferably, the siglec-6-CAR polypeptide consists of an amino acid sequence shown in any one of SEQ ID NOs: 27, 29, 31 or 33.
In embodiments that relate to a siglec-6-binding CAR comprising a variant element defined by % sequence identity with a specific SEQ ID NO, the CAR ideally retains its ability to function as siglec-6-binding CAR (including e.g. ligand binding and/or lymphocyte activation), and the ability to function as siglec-6-binding CAR is ideally at least the same as for a CAR of the same sequence but in which the element in question is represented by the SEQ ID NO without variation. For example, if a CAR comprises a spacer domain having at least 90% sequence identity with SEQ ID NO: 11, the CAR ideally has the same ability to function as siglec-6-binding CAR as a CAR of the same sequence except for a spacer represented by SEQ ID NO: 11 (i.e. without variation).
A CAR polypeptide can also be specific to more than one target. Thus, the invention also provides a siglec-6-binding polypeptide comprising or consisting of a CAR that comprises at least two binding elements at least one of which binds to siglec-6 and/or that comprises at least one binding element that is a switchable/programmable binding domain, that can be switched/programmed to bind to siglec-6.
Polynucleotide Encoding the Siglec-6-Binding Polypeptide
The present invention relates to a polynucleotide or set of polynucleotides encoding the siglec-6-binding polypeptide of the present invention as defined above.
In an embodiment of the present invention, the polynucleotide encoding the polypeptide of the present invention is further flanked by a left and a right inverted repeat/direct repeat (IR/DR) segments. The flanking segment in 5′-direction is represented by a left inverted repeat/direct repeat (IR/DR) segment and the flanking segment in 3′-direction is represented by a right inverted repeat/direct repeat (IR/DR) segment.
The nucleotide sequences of the left IR/DR segment and the nucleotide sequences of right IR/DR segment may be recognized by a transposase protein. The transposase is not particularly limited and can be, for example, Sleeping Beauty transposase or PiggyBac transposase.
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 shown 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 shown in SEQ ID NO: 44.
The term “is flanked by” indicates that further nucleotides are present in the 5′-region and in the 3′-region of the polynucleotide sequence encoding the polypeptide which are all located on the same polynucleotide. Hence, the polynucleotide sequence encoding the polypeptide is flanked by IR/DR sequences, i.e. flanking segments, such that the presence of a transposase allows the integration of the polynucleotide encoding the polypeptide as well as the nucleotide sequences corresponding to the flanking segments into the genome of the transfected cell. In an aspect, the polynucleotide which is integrated into the genome comprises a polynucleotide encoding the polypeptide and an optional detectable marker gene such as an EGFRt marker and is flanked by flanking segments. In this aspect, the region of the nucleotide sequence corresponding to the coding regions of the polypeptide and the EGFRt marker is considered to represent the reference segment.
When used in the present invention, the term “is flanked by” also means that the distance between a flanking segment and a reference segment to be 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.
In this respect, the reference segment is the region corresponding to the coding region of the polynucleotides which are integrated into the genome. The overall architecture of the polynucleotide which is integrated into the genome of the transfected cell may be as follows (5′ to 3′ direction): [left IR/DR sequence]—[reference segment]—[right IR/DR sequence].
The distance between a flanking segment and a reference segment may 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 a flanking segment and a reference segment may 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 may be in the same such that the reference segment is centred between the flanking segments or the distances may be different.
The distance between the 3′-end of the left IR/DR sequence and the 5′-end of the reference segment may be less than 1000 bp, 900 bp, 800 bp, 700 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp or less than 100 bp.
The distance between the 3′-end of the reference segment and the 5′-end of the right IR/DR sequence may be less than 200 bp, 100 bp, 50 bp, 20 bp or less than 10 bp.
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.
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 more than 600 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 and more than 5 bp.
In an aspect, the polynucleotide which is integrated into the genome comprises a polynucleotide encoding the siglec-6-binding polypeptide and a detectable marker gene such as an EGFRt marker and is flanked by flanking segments. In this aspect, the region of the nucleotide sequence corresponding to the coding regions of the siglec-6-binding polypeptide and the EGFRt marker is considered to represent the reference segment.
In an embodiment, the polynucleotide sequence of the invention comprises a 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 nucleotide sequence shown in SEQ ID NO: 26. The polynucleotide sequence of the invention preferably comprises a nucleotide sequence represented by SEQ ID NO: 26.
Thus, in a related embodiment of the present invention, the 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 a nucleotide sequence shown in any one of SEQ ID NO: 28, 30, 32 or 34. Preferably, the polynucleotide of the invention comprises a nucleotide sequence shown in any one of SEQ ID NO: 28, 30, 32 or 34. The polynucleotide of the invention can also consist of a nucleotide sequence shown in any one of SEQ ID NO: 28, 30, 32 or 34.
Expression Vector
The present invention relates to an expression vector comprising a polynucleotide or set of polynucleotides of the present invention as defined herein.
A wide range of expression vectors for polypeptides are known in the art and are further detailed 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.
The expression vector can be a minimal DNA expression cassette. Moreover, an expression vector may be a DNA expression vector such as a plasmid, linear expression vector or an episome. In certain aspects, the vector comprises additional sequences, such as sequences that facilitate expression of the polypeptide, such as a promoter, enhancer, poly-A signal, and/or one or more introns. In certain aspects, the expression vector may be a transposon donor DNA molecule, preferably a minicircle DNA.
The present invention also relates to minicircle DNA comprising a polynucleotide of the present invention as defined herein. As used herein, the term “minicircle DNA” refers to vectors which are supercoiled DNA molecules that lack a bacterial origin of replication and an antibiotic resistance gene. Therefore, they are primarily composed of a eukaryotic expression cassette.
In a useful embodiment the minicircle DNA of the invention is introduced into the cell in combination with the transposase protein or a nucleic acid (e.g. DNA or mRNA) encoding a transposase protein (such as Sleeping Beauty or PiggyBac) by electrotransfer, such as electroporation, nucleofection; chemotransfer with substances such as lipofectamin, fugene, calcium phosphate; nanoparticles, or any other conceivable method suitable to transfer material into a cell.
A viral vector can be, for example, a gamma retroviral vector or a lentiviral vector. Such vectors and their construction and production are commonly known in the art.
The polynucleotide or expression vector can be introduced into immune cells by any suitable means, such as by transfection or by transduction. Transfection refers to chemical or physical delivery into the cells, e.g. by electrotransfer, such as electroporation, nucleofection; chemotransfer with substances such as lipofectamin, fugene, calcium phosphate, PEI. Transduction refers to other means of (targeted) delivery into the cells including delivery by a viral vector or nanoparticles. However, the present invention is not limited to any particular method of delivery of genetic material into immune cells, such that also any other conceivable method suitable to transfer genetic material into a cell can be used in the context of the invention.
Immune Cell
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 present invention as defined herein.
The present invention also relates to an immune cell (preferably a lymphocyte, more preferably a T cell) comprising a polypeptide of the present invention as defined herein.
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 present invention as defined herein.
The present invention also relates to an immune cell (preferably a lymphocyte, more preferably a T cell) comprising an expression vector of the present invention as defined herein.
The immune cell can be a recombinant immune cell. A “recombinant immune cell” refers to an immune cell that has been modified to comprise a molecule, such as a polypeptide or a polynucleotide, in particular a polynucleotide, that is not comprised in the same cell without said modification, e.g. a native immune cell obtained from a (human) subject.
The immune cell (preferably lymphocyte, more preferably T cell) is preferably also capable of expressing the polynucleotide of the present invention. Thereby, the siglec-6-binding polypeptide which is encoded by the polynucleotide of the invention is translated and integrated into the cell membrane of the immune cell.
Expression of the siglec-6-binding polypeptide allows the immune cell (preferably lymphocyte, more preferably T cell) of the present invention to acquire specific reactivity against target cells expressing the siglec-6 antigen, including leukemia cells. Such immune cells, e.g. siglec-6 CAR-T cells, are able to recognize and (antigen-specifically) eradicate leukemia cells, and more specifically AML, CLL, MALT lymphoma, clonal mast cell disease cells or thymoma. Such cells are able to proliferate and to induce an immune response after encountering the siglec-6 antigen.
In some useful applications, the immune cell can also be modified to bind to at least one additional target apart from siglec-6. Thus, the invention also provides an immune cell that comprises one, two or more CAR constructs that each targets a distinct target antigen, at least one of them being 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.
The invention also provides an immune cell that comprises a single CAR construct that comprises one, two or more binding elements, at least one of them binding 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.
The invention also provides an immune cell that comprises a CAR where at least one binding domain is a switchable/programmable binding domain, that can be switched/programmed to be 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.
The immune cell is preferably a lymphocyte, such as a T cell or an NK cell. However, other immune cells, such as macrophages, are also encompassed. AT cell is especially preferred.
In an embodiment of the present invention, the immune cell (preferably lymphocyte, more preferably T cell) is a CD4+ T cell or a CD8+ T cell.
In an embodiment of the present invention, the immune cell (preferably lymphocyte, more preferably T cell) is a CD4+ T cell.
In an embodiment of the present invention, the immune cell (preferably lymphocyte, more preferably T cell) is a CD8+ T cell.
In an embodiment of the invention, the immune cell of the present invention may further express a marker gene, e.g. the EGFRt marker on the cell surface. The EGFRt marker can be used to detect, track, select and deplete the immune cell of the present invention.
Therefore, analysis of drug product persistence following administration of the immune cell is made available. Furthermore, the EGFRt marker makes immune cells of the invention sensitive to ADCC/CDC through the antibody Cetuximab which can therefore be used as safety switch.
The amino acid sequence of the EGFRt which may be used in the present invention is represented by SEQ ID NO: 23.
In an embodiment of the invention, the immune cell is (or has been) obtained from an immune cell (preferably lymphocyte, more preferably T cell) derived from a mammal, preferably a human. Preferably, the immune cell is (or has been) obtained from a subject that is to be treated with the immune cell after it has been modified to comprise the siglec-6-binding polypeptide, e.g. by a method as described herein. Alternatively, the immune cell that has been modified to comprise the siglec-6-binding polypeptide is (or has been) obtained from a healthy (allogeneic) donor, an (autologous or allogeneic) cord blood unit, or an induced pluripotent stem cell.
Method for Producing Immune Cells
The present invention also relates to a method for producing immune cells (preferably lymphocyte, more preferably T cell) of the present invention as defined herein.
In an embodiment of the present invention, the method for producing 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 a polynucleotide or expression vector as described above, optionally followed by (c) purifying the transfected or transduced immune cells. The method may further comprise formulating the immune cells into a formulation that is suitable for administration to a human subject.
Preferably, in step (b), the immune cells are transformed using a transposable element comprising a polynucleotide or set of polynucleotides as described herein and a transposase.
The transposase is not further limited and can be, for example, Sleeping Beauty transposase or PiggyBac transposase. The Sleeping Beauty transposase can be, e.g., represented by an amino acid sequence shown in SEQ ID NO: 45.
The transposable element is preferably integrated into the genome of the immune cells by the action of the transposase.
In an embodiment, the immune cells are lymphocytes, more preferably T cells or NK cells.
However, other immune cells, such as macrophages are also encompassed. Most preferably, the immune cells are T cells.
In a further embodiment, the T cell is a CD4+ T cell and/or a CD8+ T cell.
In a further embodiment of the invention, the blood sample is (or has been) obtained from a human subject, preferably a human subject diagnosed with cancer, preferably diagnosed with leukemia, such as AML.
In another embodiment, the invention provides a method for producing immune cells, comprising administering an expression vector encoding the siglec-6-binding polypeptide as described herein to a subject (in vivo gene transfer), see e.g. references [38]-[40], all 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 delivering the non-viral vector to human immune cells (preferably T cells).
The invention also relates to an immune cell (preferably lymphocyte, more preferably T cell) or a formulation of immune cells (preferably lymphocytes, more preferably T cells) obtainable by the method as described above.
Pharmaceutical Composition
The present invention also relates to a pharmaceutical composition comprising a plurality of immune cells (preferably lymphocyte, more preferably T cell) as described herein. The pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutical composition can optionally comprise a mixture of different cells, such as a mixture of CD4+ and CD8+ T cells.
In one embodiment of the invention, the pharmaceutical composition may be formulated as infusion solution comprising NaCl, glucose and human serum albumin in an amount of 0.45%, 2,5% and 1%, respectively.
Medical Uses
The present invention also relates to the immune cell or pharmaceutical composition as described herein for use as a medicament.
In an embodiment of the invention, the immune cell or pharmaceutical composition is for use in a method of treating cancer, wherein in said method the immune cell or pharmaceutical composition of the invention is to be administered to a subject (in need thereof), preferably a human subject.
The invention also relates to a method of treating cancer, comprising administering the immune cell or pharmaceutical composition of the invention to a subject, preferably a human subject.
In an embodiment of the invention, the immune cell or pharmaceutical composition is to be administered intravenously.
In an embodiment of the invention, the cancer is a siglec-6 expressing cancer, i.e. a cancer caused by abnormal cells expressing and displaying the siglec-6 protein on the 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 leukemia, such as AML or CLL, and most preferably AML.
The (use in) the method of treating cancer preferably involves the elimination of cancer stem cells of said cancer by said immune cells. Such cancer stem cells are preferably leukemic stem cells, such as AML stem cells. Cancer stem cells are defined as a subset of cancer cells with enhanced tumorigenic potential, and/or capabilities of self-renewal and differentiation, and/or phenotypic, functional and/or genetic features that can be determined in order to distinguish them from non-cancer stem cells.
The (use in) the method of treating cancer also preferably does not involve the elimination of non-cancerous hematopoietic stem or progenitor cells by said immune cells. Hematopoietic stem or progenitor cells can be identified as CD34+ cells. Hematopoietic stem cells are typically CD38− and hematopoietic progenitor cells are typically CD38+. Non-cancerous hematopoietic stem or progenitor cells are typically CD45*.
In light of this, the (use in) the method of treating cancer can further comprise monitoring the elimination of said cancer stem cells and/or of said non-cancerous hematopoietic stem or progenitor cells.
Thus, the (use in) a method of treating cancer can comprise monitoring the elimination of said cancer stem cells.
The (use in) a method of treating cancer can comprise monitoring the elimination of said non-cancerous hematopoietic stem or progenitor cells.
The (use in) a method of treating cancer can further comprise monitoring the elimination of said cancer stem cells and of said non-cancerous hematopoietic stem or progenitor cells. Elimination of cancer stem cells and/or non-cancerous hematopoietic stem or progenitor cells can be monitored, for example by bone marrow analyses that include flow cytometric analyses and other methods of phenotyping, including high-resolution flow cytometry for minimal residual disease (MRD) analyses, as well as next-generation sequencing and other methods of genotyping. Elimination of cancer stem cells and/or non-cancerous hematopoietic stem or progenitor cells can also be monitored by peripheral blood analyses that include blood counts and differential blood counts, as well as liquid biopsies and other methods of genotyping.
The phenotypic markers that are typically being used to identify AML LCS by flow cytometry include CD45, CD34 and CD38 (AML LSC phenotype: CD45dimCD34+CD38−). Additional optional markers (and corresponding phenotype) 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]. A cell being CD45dim means that the detectable surface expression of CD45 is lower than that of cells classified as CD45+. For example, (CD45dim) cancer stem cells may have a lower (mean) CD45 surface expression than healthy (CD45+) hematopoietic stem or progenitor cells.
Moreover, by not eliminating non-cancerous hematopoietic stem or progenitor cells, the need of conducting a bone marrow transplantation after the therapy using the modified immune cells can be avoided. Thus, in one embodiment, the (use in) the method of treating cancer does not involve allogeneic hematopoietic stem cell transplantation.
The treatment with the immune cells comprising the siglec-6-binding polypeptide also does not require depletion of said immune cells after the treatment. Thus, the invention also provides a (use in) the method of treating cancer as described herein that does not involve depletion of the immune cells after treatment.
Furthermore, in one embodiment, the (use in) the method of treating cancer does not involve additional conventional chemotherapy. Preferably, the (use in) the method of treating cancer does not involve additional chemotherapy after administration of the immune cells or the pharmaceutical composition and/or after the termination of the therapy with the immune cells or the pharmaceutical composition.
Conventional chemotherapy as used herein refers to the therapeutic use of chemotherapeutic agents that do not specifically target a given diseased cell (e.g. cancer cell) to be treated. Such chemotherapeutic agents include, for example, cytarabine and daunorubicin. Conventional chemotherapy as used herein does not refer to targeted therapies that are used to target a given diseased cell (e.g. cancer cell) more specifically. Such targeted therapies include, for example, the administration of a Bruton Tyrosine Kinase (BTK) inhibitor, such as ibrutinib; or an fms-like tyrosine kinase 3 (FLT3) inhibitor such as midostaurin; or an epigenetic therapy such as a hypomethylating agent (DNA methyltransferase inhibitor) such as 5-Azacitidin. Such targeted and/or epigenetic therapies can be used either in combination or as maintenance therapy with the immune cells comprising the siglec-6-binding polypeptide.
Thus, the (use in) a method of treating cancer can be a combination therapy, further comprising administration of, e.g., a targeted therapy such as a Bruton 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-Azacitidin either in combination or as maintenance therapy after the treatment with the immune cells comprising the siglec-6-binding polypeptide.
The treatment using immune cells binding siglec-6 is most effective against target cells expressing siglec-6. Therefore, the invention also provides a method of determining the expression level of siglec-6 on the surface of cancer cells from a (human) subject. The method is preferably an in vitro method. It is preferably conducted on a sample that has been obtained from a subject suspected of having cancer or diagnosed with having cancer. For example, the sample may be a (peripheral) blood sample or biopsy of the cancer to be treated.
The invention also provides a method of diagnosing cancer, preferably AML, the method comprising determining the expression level of siglec-6 on the surface of cancer cells from a (human) subject. The method is preferably an in vitro method. It is preferably conducted on a sample that has been obtained from a subject suspected of having cancer. For example, the sample may be a (peripheral) blood sample or biopsy of the cancer to be treated.
Moreover, in one embodiment, the (use in) the method of treatment as described further comprises determining, before treatment, the expression level of siglec-6 on the surface of the designated target cells, such as cancer cells of the cancer to be treated, e.g. AML.
The step of determining the expression level on siglec-6 is preferably conducted in vitro. It is preferably conducted on a sample that has been obtained from the subject to be treated. For example, the sample may be a (peripheral) blood sample or biopsy of the cancer to be treated.
A biopsy of a cancer (such as AML) can be, for example, a bone marrow biopsy or a tissue biopsy (e.g. of extracellular AML manifestation).
Consequently, in an embodiment, the (use in) the method of treatment as described further comprises administering the immune cells only if the designated target cells, such as cancer (e.g. AML) cells, express siglec-6.
Thus, the invention also provides a method of treating cancer, the method comprising:
The invention also provides an immune cell or pharmaceutical composition for use in a method of treating cancer in a subject, the method comprising:
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, and most preferably AML.
The skilled artisan is able to set suitable criteria for determining if siglec-6 is expressed on a given cell. For example, surface expression can be determined by flow cytometry as described herein. Briefly, the cells can be (surface-)stained in two separate samples, with a monoclonal antibody (mAb) against siglec-6 conjugated to a detectable (e.g. fluorescent) dye in the first sample and with an isotype control (i.e. a control monoclonal antibody not targeting siglec-6 that is of the same isotype as the siglec-6 mAb used) conjugated to the same detectable dye in the second sample. If the (mean) detectable intensity of the detectable dye (such as the mean fluorescence intensity, MFI) in the first sample is higher than in the second sample, the cells can be classified as expressing siglec-6. If the (mean) detectable (e.g. fluorescent) signal from the detectable dye in the first sample is the same or lower than in the second sample, the cells can be classified as not expressing siglec-6.
For example, the cells can be classified as expressing siglec-6 if the (mean) detectable intensity of the detectable dye (such as the MFI) in the first sample divided by the (mean) detectable intensity of the detectable dye (such as the MFI) in the second sample (such as the normalized mean fluorescence intensity (NMFI) that is calculated by dividing MFI obtained after staining with anti-siglec-6 mAb with MFI of 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.
The cells can also be classified as expressing siglec-6 if the value obtained by dividing the (mean) detectable intensity of the detectable dye (such as the MFI) in the first sample by the (mean) detectable intensity of the detectable dye (such as the MFI) in the second sample (such as the NMFI) is at least the same as the value obtained by dividing the (mean) detectable intensity of the detectable dye (such as the MFI) in a first sample (stained with siglec-6 mAb) using U937 cells by the (mean) detectable intensity of the detectable dye (such as the MFI) in a second sample (stained with isotype control) using MOLM-13 cells (such as the NMFI).
The pharmaceutical composition as described above comprising the modified T cells are stored at 2-8° C. The pharmaceutical composition is stable for (at least) 48 hours after formulation and ought to be administered to the patient within this period.
Siglec-6 CAR-T cells as generated in the experimental section of the application relate to a non-limiting exemplified embodiment of the present invention.
Materials and Methods
Human Subjects
Human peripheral blood (PB) T-cells were obtained from buffy coat of healthy donors (HDs) and 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.
Structure of a Siglec-6-Binding CAR Polypeptide
A schematic representation of the gene cassette as expected to be contained in a siglec-6 CAR T-cell is shown in
The exemplary gene cassette comprising a nucleotide sequence encoding a siglec-6 CAR polypeptide also contains 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 stochiometric expression of both proteins on the T cell surface.
The EGFRt protein enables detection and selection of CAR-positive cells using the anti-EGFR monoclonal antibody cetuximab (trade name: Erbitux®). In addition, EGFRt opens the option for selective depletion of cells expressing EGFRt with cetuximab in the event of unmanageable toxicity. It was demonstrated in pre-clinical models that administration of cetuximab leads to depletion of CAR-T cells that express EGFRt within few days in vivo.
The structure and amino acid sequence of exemplary CARs are given in Table A.
CAR Construction
A codon-optimized targeting domain containing the VH and the VL segments of the JML-1 mAb (GeneArt ThermoFisher, Regensburg, Germany, SEQ ID NO: 25; codon-optimized DNA sequence SEQ ID NO: 26) was synthesized and fused to a CAR backbone comprising a short IgG4-Fc hinge spacer, a CD28 transmembrane domain, CD28 or 4-1BB costimulatory moiety, and CD3z (SEQ ID NO: 27 or 29; DNA sequence SEQ ID NO: 28 or 30), in-frame with a T2A element and EGFRt transduction marker (
Primary AML and CLL Cells
AML patients' BM and PB were processed for mononuclear cell isolation using density-gradient centrifugation (Biocoll®, Merck Millipore). The samples with less than 1 mL volume were directly processed for flow cytometry analysis after red blood cell lysis. If possible, cytotoxicity analysis was performed directly after this step or else cells were frozen down and kept at −80° C. until the experiment was performed. Thawed primary AML cells were maintained in RPMI-1640 supplemented with 10% human serum, 2 mM glutamine, 100U/mL penicillin/streptomycin, and a cytokine cocktail including IL-4 (10001U/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) (cytokines from Miltenyi Biotec, Germany). Fresh primary CLL samples were analyzed by flow cytometry and subsequently cells were frozen for cytotoxicity assays.
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), Kasumi-1 (ACC 220) were purchased from American Type Culture Collection (ATCC, USA) or DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen, 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 detection of the cells by flow cytometry and by bioluminescence imaging, the inventors transduced all cell lines with a lentiviral vector encoding a firefly luciferase (ffluc)_green fluorescent protein (GFP) transgene, enriched GFP+ cells by FACS sorting before utilizing in in vitro or in vivo studies.
K562/siglec-6 was generated by electroporation of full-length human SIGLEC-6 gene in K562 cells. For this, full-length siglec-6 DNA (SEQ ID NO: 46) was cloned in pT2HB vector backbone and nucleofected with SB100x minicircle DNA vector using 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.
Generation of CAR-Modified T-Cells and In Vitro Analyses
Preparation of CAR-modified T-cells, analyses of CAR-T function in vitro, and colony formation assays were performed as previously described [14, 15, 17, 19, 20]. Patient-derived CD4+ and CD8+ T-cells were isolated from PB by positive selection, transduced with CAR encoding lentiviruses, and CAR+ T-cells were enriched using biotinylated anti-EGFR mAb (see above) and anti-biotin beads (Miltenyi), prior to expansion using a rapid expansion protocol [15, 20].
Colony Formation Assay
G-CSF-mobilized human CD34+CD38− and CD34+CD38+peripheral blood HSC/P cells from PB were seeded at 5×103 cells/well in duplicate wells and incubated with JML-1-CAR or CD123-CAR T-cells or untransduced T-cells at an E:T ratio=5:1. After 24 hours, 1/5th of cell suspension was plated onto methyl cellulose-based medium (Methocult opti H4034, Stem Cell Technologies, Cambridge, MA) in 6-well plates (Smartdish™ plate, StemCell Technologies). Colonies were evaluated using established criteria according to the manufacturer's instructions and counted under an optical microscope 14 days later.
Flow Cytometric Analyses
Mononuclear cells from healthy donors or AML patients were stained with 1 or more of the following conjugated mAbs: CD3, CD4, CD8, CD19, CD33, CD34, CD38, CD45, CD56, CD123, CD135 (FLT3), CD327 (siglec-6), CD371 (CLL-1) 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 the 1 or more of following conjugated mAbs: CD5, CD10, CD19, CD20, CD27, CD38, CD327 (Miltenyi biotec, Bergisch-Gladbach, Germany; BD, Heidelberg, Germany; Biolegend, London, UK). Untransduced 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). An anti-EGFR antibody (ImClone Systems Inc.) that had been conjugated to AF647 in-house (EZ-Link™Sulfo-NHS-SSBiotin, ThermoFisher Scientific, IL; according to the manufacturer's instructions) was used to detect CAR T-cells. Cells were acquired on FACSCanto (BD) for flow cytometry analyses and data analysis was performed using FlowJo software v9.0.2 (Treestar, Ashland, OR). Primary AML blasts were stained with mAb against CD45, CD34, CD38, CD123, CD33, FLT3, Siglec-6, CLL-1, and CD117. LSC were identified as CD45dimCD34*CD38− cells. The normalized mean fluorescence intensity (NMFI) was calculated by dividing the MFI obtained by flow cytometry after staining with a specific mAb (e.g. anti-siglec-6) through the MFI obtained by flow cytometry after staining with an isotype control.
Siglec-6-Expression Analysis by Flow Cytometry
Expression of siglec-6 (CD327) 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, 1×106 cells were washed, re-suspended in 100 μL PBS/0.5% fetal calf serum, blocked with human IgG when R&D systems mAB was used (Jackson ImmunoResearch, USA) at 42C for 20 minutes and stained with anti-human siglec-6 mAb or isotype for 30 minutes at 4° C.
In Vivo Experiments with U937 Xenograft Model
The competent Institutional Animal Care and Use Committees evaluated and approved all in vivo experiments. NOD.Cg-Prkdcscidll2rgtm1Wj/SzJ (NSG) mice (female, 6-8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Mice were inoculated with 2×106 ffluc_GFP+ U937 cells. Mice were randomly allocated to different treatment groups, and injected using a split CAR T-cell dosing strategy, with doses administered on day 6 and on day 21 via tail vein. Each dose contained 5×106 T-cells (i.e. 2.5×106 CD4+ and 2.5×106 CD8+ in 200 μL of PBS/0.5% FCS). PB was obtained at regular intervals to analyze the frequency of tumor cells and transferred T-cells. Bioluminescence imaging (BLI) was performed weekly after intraperitoneal administration of D-luciferin substrate (0.3 mg/g body weight) (Biosynth, Staad, Switzerland) using an IVIS Lumina imaging system (PerkinElmer, Waltham, Massachusetts). Bioluminescence images were analyzed using Living Image software (PerkinElmer).
In Vivo Experiments with MOLM-13 Xenograft Model
The competent Institutional Animal Care and Use Committees evaluated and approved all in vivo experiments. NOD.Cg-Prkdcscidll2rgtmlWj/SzJ (NSG) mice (female, 6-8 weeks old) were purchased from Charles River (Sulzfeld, Germany). Mice were inoculated with 1×106 ffluc_GFP+ MOLM-13 cells. Mice were randomly allocated to different treatment groups, and injected using a split CAR T-cell dosing strategy, with doses administered on day 4 and on day 7 via tail vein. Each dose contained 5×106 T-cells (i.e. 2.5×106 CD4+ and 2.5×106 CD8+ in 200 μL of PBS/0.5% FCS). PB was obtained at regular intervals to analyze the frequency of tumor cells and transferred T-cells. Bioluminescence imaging (BLI) was performed weekly after intraperitoneal administration of D-luciferin substrate (0.3 mg/g body weight) (Biosynth, Staad, Switzerland) using an IVIS Lumina imaging system (PerkinElmer, Waltham, Massachusetts). Bioluminescence images were analyzed using Living Image software (PerkinElmer).
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 seeded into 96-well plates at effector: target (E:T) ratios ranging from 10:1 to 2.5:1 with 104 target cells per well. Co-cultured cells were stained after 4 or 24-hour co-culture for flow analysis with following mAbs: For AML samples, anti-CD3/anti-CD33/anti-CD34/anti-CD45/anti-EGFRt mAbs and for CLL samples, anti-CD3/anti-CD5/anti-CD20/anti-CD19/anti-CD45 were used to distinguish CAR T-cells and target cells. 7-AAD was used to discriminate live and dead cells. To quantitate the number of residual live AML cells, 123-counting beads (e-bioscience, San Diego, CA) were used according to the manufacturer's instructions. Flow analyses were done on a FACS Canto II (BD) and data analyzed using FlowJo software (Treestar).
Statistical Analyses
Statistical analyses were performed using Prism software v6.07 (GraphPad, San Diego, California). Student's t-test (Unpaired) was used to analyze data obtained in in vitro and in vivo experiments. The differences in survival observed in in vivo experiments were analyzed using Logrank (Mantel-Cox) test. P value with difference <0.05 were considered statistically significant.
Results
JML-1-CAR T-Cells Recognize and Eliminate Siglec-6+ AML Cell Lines
The inventors generated CD4+ and CD8+JML-1-CAR T-cells from healthy donors (HD) (n >5).
For this purpose, the inventors used single-chain fragment variable (scFv) derived from fully human JML-1- mAb10 and linked it to the CD3i signaling domain and CD28 or 4-1BB costimulatory domains (
Next, the inventors engineered K562 cells to stably express siglec-6 (K562/siglec-6,
Taken together, the data show that T-cells expressing the JML-1-CAR with CD28 or 4-1BB co-stimulatory domains show antigen-specific potent anti-leukemia reactivity against AML cell lines in vitro.
Siglec-6 is Highly and Uniformly Expressed on Primary AML Blasts, Including AML Leukemic Stem Cells/JML-1-CAR T-Cells Recognize and Eliminate Primary AML Cells In Vitro
The inventors evaluated siglec-6-expression on primary AML blasts from n=10 adult AML patients. This patient cohort comprised patients with newly diagnosed AML, relapsed/refractory AML and secondary AML. Further, the patient cohort comprised patients and AML with various molecular and cytogenetic abnormalities (Table-1). The inventors found siglec-6 to be uniformly expressed on AML blasts in each of the patients (10/10), and ranked the patients according to the expression level based on normalized MFI (
Intriguingly, the inventors also found uniform siglec-6 expression on the subpopulation of AML leukemia stem cells (LSCs) in each of the patients. Even more intriguingly, the expression level of siglec-6 was similar or even higher in the subpopulation of AML LSCs compared to the ‘bulk’ population of AML blasts (
To assess recognition of primary ‘bulk’ AML blasts and AML leukemic stem cells, the inventors performed cytolysis experiments with CD8+JML-1-CAR T-cells that were derived from a healthy donor. The inventors observed high-levels of cytolytic activity by JML-1-CAR T cells against primary ‘bulk’ AML blasts and AML leukemic stem cells (
Taken together, the data show that siglec-6 is highly and uniformly expressed in primary AML blasts in patients with various AML disease subtypes. The data also show that siglec-6 is highly expressed in AML LSCs with an expression level that is similar or even higher compared to the ‘bulk’ AML blast population. The data further show that targeting siglec-6 confers specific and potent anti-AML activity and—on example of JML-1-CAR T-cells—leads to specific and potent elimination of bulk AML blasts and AML leukemic stem cells.
Patient Derived JML-1 CAR-T Cells Potently Eliminate Autologous AML Blasts
Next, the inventors generated JML-1-CAR T-cells from AML patients with newly diagnosed (n=2) and previously treated AML (MRD+, n=1) and assessed their anti-leukemia activity against autologous AML blasts. The inventors observed transduction efficiency of 24.9-50.0% in CD4+ and 20.4-43.0% in CD8+ T-cells and enriched cells were >95% CAR+ T-cells (
JML-1-CAR T-Cells Eradicate Aggressive Systemic Acute Myeloid Leukemia In Vivo
The inventors evaluated the anti-leukemia efficacy of JML-1-CAR T-cells in AML xenograft models using immunodeficient NSG mice. The inventors inoculated female NSG mice with ffLuc+GFP+ U937 leukemic cells and observed systemic engraftment of tumor cells in BM and spleen of mice after 6 days of tumor inoculation by BLI analysis (
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, deleterious leukemia was observed in all mice that had been treated with control T-cells (
The inventors observed significantly higher overall survival among groups of mice treated with JML-1-CAR T-cells compared to control T-cells (
The inventors also evaluated the anti-leukemia efficacy of JML-1-CAR T-cells in immunodeficient NSG mice that had been inoculated with MOLM-13 cells (low Siglec-6-expression). Treatment with Siglec-6-CAR T-cells conferred a significant anti-leukemia effect (
In conclusion, these data demonstrate that targeting siglec-6 in AML confers potent anti-leukemia activity in vivo. The data also show that JML-1-CAR T-cells confer potent anti-leukemia efficacy and induce durable complete remissions of AML in vivo.
Human Hematopoietic Stem and Progenitor Cells do not Express Siglec-6 and are Preserved after Exposure to JML-1-CAR T-Cells In Vitro
The inventors sought to evaluate on-target off-tumor effect of JML-1-CAR T-cells on normal hematopoietic stem and progenitor cells (HSC/P). First, the inventors assessed siglec-6-expression on G-CSF mobilized PB derived CD34+CD38− HSC and CD34+CD38+HSPC from HD (n=5). The inventors observed lack of siglec-6-expression on HSC and HSPC in all n=5 HD analyzed by flow cytometry (NMFI <1.0,
Taken together, these data show that siglec-6 is a unique AML target antigen in that it is not expressed on normal HSC/P. The data also show that normal HSC/P are not recognized by JML-1-CAR T-cells. These data suggest that targeting siglec-6 will not induce myeloablation in humans.
Malignant B-Cells in B-CLL Express Siglec-6 and are Eliminated by JML-1-CAR T-Cells
The inventors evaluated siglec-6-expression on PB derived primary B-CLL cells from treatment-naïve CLL patients (n=10, Table-2). The inventors show that siglec-6 is expressed uniformly and at high levels on primary B-CLL cells in 9 out of these 10 patients (
Taken together, these data show 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.
Siglec-6 is Expressed on a Subset of Normal B-Cells and Confers Recognition by JML-1-CAR T-Cells
Next, the inventors sought to analyze siglec-6-expression on HD derived PBMCs (n=7). The inventors detected high-levels of siglec-6 on fraction of B-cells in flow analysis while other healthy PB cells i.e. NK cells, T-cells, NKT cells do not express siglec-6 (
Taken together, these data show that siglec-6 is expressed on a subset of normal B-cells in healthy donors and patients, suggesting that an anticipated on-target off-tumor effect of targeting siglec-6 will be selective, partial deletion of normal B-cells.
The present inventors demonstrate that siglec-6 is a target for antibody-based and cellular immunotherapy in AML. In particular, the inventors demonstrate that siglec-6 is a target for antibody-based and cellular immunotherapy to target and destroy AML leukemic stem cells. The inventors demonstrate that JML-1-CAR T-cells confer potent anti-leukemia efficacy against primary AML blasts in vitro and induce complete remissions of leukemia in mice engrafted with AML cell lines.
Importantly, siglec-6 was found to be absent on normal HSC/P. Accordingly, JML-1-CAR T-cells did not recognize normal HSC/P and did not lead to a reduction in hematopoietic lineage development in colony formation experiments. The possibility of sparing normal HSC/P while eradicating AML blast and AML leukemic stem cells enables a non-myeloablative immunotherapy to effectively treat AML while eliminating the need for alloHSCT.
Siglec-6-expression on other healthy tissues is restricted to placenta [16], mast cells [17] and a subset of normal B-cells [10], suggesting a favorable safety profile with negligible on-target, off-tumor reactivity. The inventors found that the majority of memory B-cells but only a small proportion of naïve and immature B-cells express siglec-6, and therefore anticipate selective, partial deletion of normal B-cells to occur after anti-siglec-6 immunotherapy as e.g. JML-1 CAR-T cell therapy. Encouragingly, potential hypogammaglobulinemia can be overcome by intravenous immunoglobulin (IVIG) replacement therapy if needed, which is already common practice after CD19-CAR T-cell therapy [27]. Siglec-6 is reported to be present on mast cells [18] and therefore, mast cell reduction or depletion may occur after anti-siglec-6 immunotherapy.
Moreover, the inventors found high siglec-6 expression on primary B-CLL cells obtained from treatment-naïve CLL patients, which is in line with previous observations [11]. Indeed, the inventors 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 features of T-cell exhaustion and proliferative defects and therefore, patient-derived JML-1-CAR T-cells might not function equally well to that of HD-derived CAR T-cells [28, 23,24]. Encouragingly, a Bruton Tyrosine Kinase (BTK) inhibitor ibrutinib has shown to improve the anti-leukemia efficacy of CD19-CAR T-cells in mouse models and in CLL patients [28,29]. Therefore, the combination of ibrutinib could improve effectiveness of JML-1-CAR T-cell therapy in CLL patients and warrants preclinical evaluation for synergy of JML-1-CAR T-cell with ibrutinib.
CAR T-cell rejection due to transgene containing murine scFv (e.g. FMC63) is a mechanism that contributes to the resistance to CAR T-cell therapy [30]. JML-1-CAR is derived from a fully human scFv and therefore it is unlikely to be immunogenic. This enables the administration of multiple, sequential infusions of JML-1-CAR T-cells to further augment and to sustain the anti-leukemia response, and to avoid alloHSCT, if needed.
Moreover, leukemia relapse due to target antigen loss or downregulation 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 a uniform siglec-6-expression on AML blasts, due to clonal heterogeneity, there is a potential risk that siglec-6 expression may change under the therapeutic pressure from anti-siglec-6 immunotherapy. However, there is no prior clinical experience with siglec-6 as an immune target.
Furthermore, the possibility of protein truncation resulting in siglec-6 protein without transmembrane domain and therefore to a loss of cell surface siglec-6 cannot be eliminated, as reported with CD19 protein after CD19-CAR T-cell therapy in B-ALL patients [33]. Preclinical mouse models mimicking antigen loss escape could help to investigate antigen loss escape after CAR T-cell treatment and strategies to prevent resistance to CAR-T cell therapy. Alternatively, the use of tandem or compound CARs targeting two or more antigens simultaneously or sequentially may prevent antigen loss escape in AML.
The inventors' findings on siglec-6-expression and anti-leukemia activity by JML-1-CAR T-cells suggests that a therapy using siglec-6-binding immune cells, such as JML-1 CAR-T cells, would be applicable to AML and CLL patients and warrants clinical investigation in humans. Moreover, siglec-6-expression is also reported on MALT lymphoma [15], in clonal mast cell disease [34], and in thymoma, extending the application of therapy using siglec-6-binding immune cells, such as JML-1-CAR T-cells, to other hematologic and oncologic indications, and to other applications in medicine.
The siglec-6-binding polypeptide, the nucleotide sequence encoding the siglec-6-binding polypeptide, the expression vector as well as the immune cell comprising the siglec-6-binding polypeptide according to the invention, can be industrially manufactured and sold as products for the methods and uses as described (e.g. for treating a cancer as defined herein), in accordance with known standards for the manufacture of pharmaceutical products. Accordingly, the present invention is industrially applicable.
Frontiers in immunology. 2018; 9:2138.
Molecular Therapy. 2017; 25(9):2202-2213.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20190301.0 | Aug 2020 | EP | regional |
| 21183475.9 | Jul 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/072160 | 8/9/2021 | WO |
