Polypeptides containing immunoglobulin single variable domains that target TCRαβ, CD33 and CD123

Abstract

The present technology aims to provide a new type of drug for treating subjects suffering from acute myeloid leukemia (AML). Specifically, the present technology provides a polypeptide comprising at least three immunoglobulin single variable domains (ISVDs), characterized in that at least one ISVD binds to T cell receptor αβ (TCRαβ), at least one ISVD binds to CD33, and at least one ISVD binds to CD123. The present technology also provides nucleic acids, vectors, and compositions.

Description

[Technical field]

[0001] 1 Field of this technology The present technology relates to polypeptides that target TCRαβ, CD33, and CD123. The present invention also relates to nucleic acid molecules encoding these polypeptides, as well as vectors that contain these nucleic acids. The present technology further relates to compositions that contain such polypeptides, nucleic acids, or vectors. The present technology also relates to such compositions for use in methods of treating subjects suffering from acute myeloid leukemia (AML). The present technology also relates to methods of producing these compositions. [Background technology]

[0002] 2 Technical background To date, the treatment of acute myeloid leukemia (AML) remains challenging. Cytotoxic T cells appear to have the greatest therapeutic potential for malignant disease among all immune cells, and thus AML therapeutic approaches aim to direct cytotoxic T cells to AML cells. CD33 and CD123 antigens were found to be overexpressed on blasts and leukemic stem cells in most cases of AML and were therefore used as suitable tumor-associated target antigens for antibody-based therapy. Mylotarg (Gemtuzumab Ozogamicin; an anti-CD33 antibody drug conjugate) was the first targeted compound registered for the treatment of AML. Recent therapeutic approaches are based on bispecific antibody constructs that target one of the tumor antigens CD33 or CD123 on AML cells and CD3 on cytotoxic T cells.

[0003] Thereby, the bispecific antibody can fix to CD33+ or CD123+ AML cells and simultaneously bind to CD3 on T cells. In this way, the T cells are in close proximity to the tumor cells. The multiple binding of the bispecific antibody to tumor antigens (CD33 or CD123) on the tumor cells and the simultaneous binding to the TCR-associated CD3 molecules on the T cells leads to TCR clustering. This ultimately leads to efficient T cell activation regardless of TCR specificity. Cytotoxic T cell activation in the vicinity of the AML cells can then lead to tumor cell killing. Bispecific antibody constructs currently being tested in clinical trials are, for example, flotetuzumab (MGD006; CD3 / CD123 DART), AMG330 or AMG673 (both CD3 / CD33 BiTEs).

[0004] Effective treatment of AML is complicated, given the heterogeneity of CD33 and CD123 expression found in both AML patient populations (interpatient) and in the AML blast population of individual patients (intrapatient). Moreover, targeting a single tumor antigen carries the risk of losing expression of this antigen on tumor cells due to selective pressure induced by therapeutic intervention (Gardner et al., Blood, 127(20), 2406-2410(2016); Blood. 2017 Jan 5; 129(1): 100-104). Therefore, there is a strong need for new therapeutic approaches that overcome the limitations of single targeted therapy and have a broader patient range. Summary of the Invention [Means for solving the problem]

[0005] 3. Overview of this technology The present inventors have found that polypeptides (or ISVD constructs) that dually target CD33 and CD123 on acute myeloid leukemia (AML) cells, combined with targeting T cell receptor αβ (TCRαβ) on T cells, result in effective killing of AML cells. The killing activity against CD33 / CD123 dual expressing cells was comparable to single targeting benchmarks such as CD33 / CD3 AMG 330 BiTE or CD123 / CD3 MGD006 DART. However, the polypeptides of the present invention showed strong killing activity against CD33 and CD123 single expressing cells, whereas the single targeting benchmarks only showed activity against cells expressing their specific targets. Furthermore, the polypeptides of the present invention induce similar or even lower levels of inflammatory cytokines compared to the benchmarks.

[0006] In some embodiments, the polypeptide of the present technology is efficiently produced (e.g., in a microbial host) and exhibits low viscosity at high concentration, which is advantageous and convenient for subcutaneous administration.Furthermore, such polypeptide has limited reactivity to the existing antibodies in the treated subject (i.e., the antibodies that exist in the subject before the first treatment with the antibody construct).In a preferred embodiment, such polypeptide exhibits a sufficiently long half-life in the treated subject so that successive treatments can be conveniently spaced apart.

[0007] The polypeptide of the present technology comprises or consists of at least three immunoglobulin single variable domains (ISVDs), where at least one ISVD specifically binds to TCRαβ, at least one ISVD specifically binds to CD33, and at least one ISVD specifically binds to CD123 (exemplary polypeptides are shown in FIG. 1). Preferably, at least one ISVD that binds to TCRαβ specifically binds to human TCRαβ, at least one ISVD that binds to CD33 specifically binds to human CD33, and at least one ISVD that binds to CD123 specifically binds to human CD123.

[0008] The polypeptide preferably further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, which provide the polypeptide with an increased half-life compared to a corresponding polypeptide that does not comprise said one or more other groups, residues, moieties or binding units. For example, the binding unit may be an ISVD that binds to a serum protein, preferably a human serum protein such as human serum albumin.

[0009] Also provided are nucleic acid molecules capable of expressing the polypeptides of the present technology, vectors comprising the nucleic acids or nucleic acids, and compositions comprising the polypeptides, nucleic acids or vectors. The compositions are preferably pharmaceutical compositions.

[0010] Also provided are hosts or host cells comprising a nucleic acid or vector encoding the polypeptides of the present technology.

[0011] A method for producing a polypeptide in accordance with the present technology comprising: a. expressing a nucleic acid sequence encoding a polypeptide according to the present technology, optionally in a suitable host cell or host organism, or in another suitable expression system, and then, optionally b. Isolating and / or purifying the polypeptide according to the present technology There is further provided a method comprising at least

[0012] Furthermore, the present technology provides a polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid or vector comprising a nucleotide sequence encoding the polypeptide, for use as a medicament.Preferably, the polypeptide or composition is for use in the treatment of acute myeloid leukemia (AML), preferably the AML is relapsed and / or refractory AML.

[0013] Further provided is a method for treating AML, comprising administering to a subject in need thereof a pharma- ceutically active amount of a polypeptide or composition according to the present technology. AML is preferably relapsed and / or refractory AML. In a preferred embodiment, the method further comprises administering one or more additional therapeutic agents.

[0014] There is further provided a use of the polypeptide or composition of the present technology in the preparation of a pharmaceutical composition for treating AML, preferably relapsed and / or refractory AML.

[0015] In particular, the present technology provides the following embodiments:

[0016] Embodiment 1. A polypeptide, a composition comprising a polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding a polypeptide, for use as a medicament, wherein the polypeptide comprises or consists of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptide linkers: a) a first ISVD specifically binds to T cell receptor αβ (TCRαβ); i. a CDR1 having the amino acid sequence of SEQ ID NO:6 or having 2 or 1 amino acid difference from SEQ ID NO:6; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 10 or having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 14 or having 2 or 1 amino acid difference from SEQ ID NO: 14; b) the second ISVD specifically binds to CD33; iv. CDR1 having the amino acid sequence of SEQ ID NO:7 or having 2 or 1 amino acid difference from SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11 or having 2 or 1 amino acid difference from SEQ ID NO:11; and vi. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 15 or having 2 or 1 amino acid difference from SEQ ID NO: 15; and c) the third ISVD specifically binds to CD123; vii. CDR1 having the amino acid sequence of SEQ ID NO:8 or having 2 or 1 amino acid difference from SEQ ID NO:8; viii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or having 2 or 1 amino acid difference from SEQ ID NO: 12; and ix. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 16 or having 2 or 1 amino acid difference from SEQ ID NO: 16; A polypeptide or composition in which the ISVDs are ordered starting from the N-terminus.

[0017] Embodiment 2. A composition for use according to embodiment 1, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more further pharma- ceutically active polypeptides and / or compounds.

[0018] Embodiment 3. A polypeptide or composition for use according to embodiment 1 or 2, a) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO:10, and a CDR3 having the amino acid sequence of SEQ ID NO:14; b) the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:11, and a CDR3 having the amino acid sequence of SEQ ID NO:15; and c) A polypeptide or composition, wherein the third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 8, a CDR2 having the amino acid sequence of SEQ ID NO: 12, and a CDR3 having the amino acid sequence of SEQ ID NO: 16.

[0019] Embodiment 4. A polypeptide or composition for use according to any one of embodiments 1 to 3, a) the amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO:3; and c) A polypeptide or composition, wherein the amino acid sequence of the third ISVD has a sequence identity of greater than 90% identity to SEQ ID NO:4.

[0020] Embodiment 5. A polypeptide or composition for use according to any one of embodiments 1 to 4, a) the first ISVD has the amino acid sequence of SEQ ID NO:2; b) the second ISVD has the amino acid sequence of SEQ ID NO:3; and c) A polypeptide or composition, wherein the third ISVD has the amino acid sequence of SEQ ID NO:4.

[0021] Embodiment 6. A polypeptide or composition for use according to any of embodiments 1 to 5, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, and wherein said one or more other groups, residues, moieties or binding units provide a polypeptide with an increased half-life compared to a corresponding polypeptide not comprising said one or more other groups, residues, moieties or binding units.

[0022] Embodiment 7. A polypeptide or composition for use according to embodiment 6, wherein the one or more other groups, residues, moieties or binding units that confer an increased half-life to the polypeptide are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties and small proteins or peptides capable of binding to serum proteins.

[0023] Embodiment 8. A polypeptide or composition for use according to any one of embodiments 6 to 7, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with an increased half-life are selected from the group consisting of binding units capable of binding to serum albumin (e.g. human serum albumin) or serum immunoglobulin (e.g. IgG).

[0024] Embodiment 9. A polypeptide or composition for use according to embodiment 8, wherein the binding unit providing the polypeptide with increased half-life is an ISVD capable of binding to human serum albumin.

[0025] Embodiment 10. The ISVD that binds to human serum albumin is i. a CDR1 having the amino acid sequence of SEQ ID NO: 9 or having 2 or 1 amino acid difference from SEQ ID NO: 9; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 13 or having 2 or 1 amino acid difference from SEQ ID NO: 13; and iii. A polypeptide or composition for use according to embodiment 9, comprising a CDR3 having the amino acid sequence of SEQ ID NO: 17 or having 2 or 1 amino acid difference from SEQ ID NO: 17.

[0026] Embodiment 11. A polypeptide or composition for use according to any one of embodiments 9 to 10, wherein the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0027] Embodiment 12. A polypeptide or composition for use according to any one of embodiments 9 to 11, wherein the amino acid sequence of the ISVD that binds to human serum albumin has more than 90% sequence identity with SEQ ID NO:5.

[0028] Embodiment 13. A polypeptide or composition for use according to any one of embodiments 9 to 12, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID NO:5.

[0029] Embodiment 14. A polypeptide or composition for use according to any one of embodiments 1 to 13, wherein the amino acid sequence of the polypeptide has more than 90% sequence identity with SEQ ID NO:1.

[0030] Embodiment 15. A polypeptide or composition for use according to any one of embodiments 1 to 14, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:1.

[0031] Embodiment 16. A polypeptide or composition for use according to any one of claims 1 to 15 for use in the treatment of AML.

[0032] Embodiment 17. A polypeptide or composition for use according to claim 16, wherein the AML is relapsed and / or refractory AML.

[0033] Embodiment 18. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers: a) a first ISVD specifically binds to T cell receptor αβ (TCRαβ); i. a CDR1 having the amino acid sequence of SEQ ID NO:6 or having 2 or 1 amino acid difference from SEQ ID NO:6; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 10 or having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 14 or having 2 or 1 amino acid difference from SEQ ID NO: 14; b) the second ISVD specifically binds to CD33; iv. CDR1 having the amino acid sequence of SEQ ID NO:7 or having 2 or 1 amino acid difference from SEQ ID NO:7; v. a CDR2 having the amino acid sequence of SEQ ID NO:11 or having 2 or 1 amino acid difference from SEQ ID NO:11; and vi. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 15 or having 2 or 1 amino acid difference from SEQ ID NO: 15; and c) the third ISVD specifically binds to CD123; vii. CDR1 having the amino acid sequence of SEQ ID NO:8 or having 2 or 1 amino acid difference from SEQ ID NO:8; viii. CDR2 having the amino acid sequence of SEQ ID NO: 12 or having 2 or 1 amino acid difference from SEQ ID NO: 12; and ix. comprises a CDR3 having the amino acid sequence of SEQ ID NO: 16 or having 2 or 1 amino acid difference from SEQ ID NO: 16; A polypeptide in which the ISVDs are ordered starting from the N-terminus.

[0034] Embodiment 19. A polypeptide according to embodiment 18, comprising: a) the first ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO:10, and a CDR3 having the amino acid sequence of SEQ ID NO:14; b) the second ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:11, and a CDR3 having the amino acid sequence of SEQ ID NO:15; and c) A polypeptide, wherein the third ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:16.

[0035] Embodiment 20. A polypeptide according to any one of embodiments 18 or 19, comprising: a) the amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO:2; b) the amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO:3; and c) A polypeptide, wherein the amino acid sequence of said third ISVD has a sequence identity of greater than 90% identity to SEQ ID NO:4.

[0036] Embodiment 21. A polypeptide according to any one of embodiments 18 to 20, comprising: a) the first ISVD has the amino acid sequence of SEQ ID NO:2; b) the second ISVD has the amino acid sequence of SEQ ID NO:3; and c) A polypeptide, wherein the third ISVD has the amino acid sequence of SEQ ID NO:4.

[0037] Embodiment 22. A polypeptide according to any one of embodiments 18 to 21, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, and wherein the one or more other groups, residues, moieties or binding units provide a polypeptide having an increased half-life compared to a corresponding polypeptide not comprising the one or more other groups, residues, moieties or binding units.

[0038] Embodiment 23. The polypeptide of embodiment 22, wherein the one or more other groups, residues, moieties or binding units that confer an increased half-life to the polypeptide are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties and small proteins or peptides capable of binding to serum proteins.

[0039] Embodiment 24. A polypeptide according to any one of embodiments 22 to 23, wherein the one or more other groups, residues, moieties or binding units which confer an increased half-life to the polypeptide are selected from the group consisting of binding units capable of binding to serum albumin (e.g. human serum albumin) or serum immunoglobulin (e.g. IgG).

[0040] Embodiment 25. The polypeptide of embodiment 24, wherein the binding unit that provides the polypeptide with increased half-life is an ISVD capable of binding to human serum albumin.

[0041] Embodiment 26. An ISVD that binds to human serum albumin i. a CDR1 having the amino acid sequence of SEQ ID NO: 9 or having 2 or 1 amino acid difference from SEQ ID NO: 9; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 13 or having 2 or 1 amino acid difference from SEQ ID NO: 13; and iii. The polypeptide of embodiment 25, comprising a CDR3 having the amino acid sequence of SEQ ID NO: 17 or having 2 or 1 amino acid difference from SEQ ID NO: 17.

[0042] Embodiment 27. A polypeptide described in any one of embodiments 25 to 26, wherein the ISVD that binds to human serum albumin comprises a CDR1 having the amino acid sequence of SEQ ID NO: 9, a CDR2 having the amino acid sequence of SEQ ID NO: 13, and a CDR3 having the amino acid sequence of SEQ ID NO: 17.

[0043] Embodiment 28. A polypeptide described in any one of embodiments 25 to 27, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO:5.

[0044] Embodiment 29. A polypeptide described in any one of embodiments 25 to 28, wherein the ISVD that binds to human serum albumin has the amino acid sequence of SEQ ID NO:5.

[0045] Embodiment 30. A polypeptide according to any one of embodiments 18 to 29, wherein the amino acid sequence of the polypeptide has greater than 90% sequence identity with SEQ ID NO:1.

[0046] Embodiment 31. A polypeptide according to any one of embodiments 18 to 29, comprising or consisting of the amino acid sequence of SEQ ID NO:1.

[0047] Embodiment 32. A nucleic acid comprising a nucleotide sequence encoding a polypeptide according to any one of embodiments 18 to 31.

[0048] Embodiment 33. A host or host cell comprising a nucleic acid according to embodiment 32.

[0049] Embodiment 34. A method for producing a polypeptide according to any one of embodiments 18 to 31, comprising: a) expressing in a suitable host cell or host organism or in another suitable expression system a nucleic acid according to embodiment 32; optionally followed by: b) A method comprising at least the step of isolating and / or purifying a polypeptide according to any one of embodiments 18 to 31.

[0050] Embodiment 35. A composition comprising at least one polypeptide according to any one of embodiments 18 to 31 or a nucleic acid according to embodiment 32.

[0051] Embodiment 36. The composition according to embodiment 35, which is a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharma- ceutically active polypeptides and / or compounds.

[0052] Embodiment 37. A method for treating AML, comprising administering to a subject in need thereof a pharma- ceutical active amount of a polypeptide described in any one of claims 18-31 or a composition described in any one of claims 35-36.

[0053] Embodiment 38. The method of claim 37, wherein the AML is relapsed and / or refractory AML.

[0054] Embodiment 39. Use of a polypeptide according to any one of claims 18 to 31 or a composition according to any one of claims 35 to 36 in the preparation of a pharmaceutical composition for treating AML.

[0055] Embodiment 40. Use of a polypeptide or composition according to claim 39, wherein the AML is relapsed and / or refractory AML.

[0056] 4. Brief description of the drawings [Brief description of the drawings]

[0057] [Figure 1] FIG. 1 : Schematic diagram of a multispecific ISVD construct according to the invention showing monovalent components / ISVD TCRαβ, CD33, CD123 and Alb linked via linkers from N-terminus to C-terminus. [Diagram 2] Binding of monovalent CD33 (left) and CD123 bound to BB (right) to human (top) or cynomolgus monkey (bottom) transfected CD33 and CD123 cells, respectively. [Diagram 3] Binding of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO:1) to human CD33 and / or human CD123 expressing cells. [Figure 4] Dose-dependent inhibition of A025001562, a TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO:1 (black boxes) and a reference TCR (grey dots) in a competition assay against primary T cells in the absence (dotted curve) or presence (full curve) of clinical grade HSA. [Diagram 5] Dose-dependent human (top) or cynomolgus monkey (bottom) T cell-mediated killing of CD33 (left) or CD123 (right) transfected cells of the corresponding species using an effector-to-target ratio of 15:1 in an impedance-based assay (xCELLigence) in the presence of 50 μM HSA. [Figure 6] Dose-dependent human (left) or cynomolgus monkey (right) T cell-mediated MOLM-13 cell killing in a flow cytometry-based assay using an effector-to-target ratio of 10:1. % TO-PRO®-3 positive target cells are plotted against the concentration of ISVD. [Figure 7] Dose-dependent human T cell-mediated cell killing using an effector-to-target ratio of 15 to 1 in an impedance-based assay (xCELLigence). The cell index (CI) after 32-35 hours of incubation is plotted against the concentration of ISVD. [Figure 8] Inhibition of Molm13-luc AML tumor growth by in vivo bioluminescence imaging of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO:1). [Figure 9] Inhibition of Molm13-luc AML tumor growth by ex vivo bioluminescence imaging of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO:1). [Figure 10] Dose-dependent human T cell-mediated killing of ISVD according to the invention compared to CD123 and CD33 positive controls in MOLM-13 cells. [Figure 11] Dose-dependent human T cell-mediated killing of ISVD according to the invention compared to CD123 and CD33 positive controls in KG-1a cells. [Figure 12] Dose-dependent human T cell-mediated killing of ISVD according to the invention compared to CD123 and CD33 positive controls in U-937 cells. [Figure 13] Dose-dependent monocyte depletion of ISVD according to the invention compared to CD123 and CD33 positive and negative controls (non-targeted TCE) in human peripheral blood mononuclear cells (PBMC). [Figure 14] Dose-dependent cytokine release of ISVD according to the invention compared to CD123 and CD33 positive controls and a negative control (non-targeted TCE) in human PBMCs from healthy donors using a panel of different cytokines. A. IL-6. B. IFNγ. C. TNFα. D. IL-2. [Figure 15] AML blast killing of ISVD according to the invention versus CD33 and CD123 positive controls in all study patients. [Figure 16] Scatter plots showing the percentage of CD33 or CD123 positive cells per AML sample. [Figure 17] Cell viability of primary blasts from AML patients with a wide range of disease subtypes for ISVD according to the present invention compared to CD123 and CD33 positive and negative controls. A. Patient #3. B. Patient #4. C. Patient #5. [Figure 18] Individual absolute cell counts of total CD123+ T cells (A), monocytic CD33+ cells (B), CD4+ T cells (C) and CD8+ T cells (D) measured over time in peripheral blood of cynomolgus monkeys treated with an ISVD according to the invention. Animals M1 and M2 received 0.04 μg / kg and M3 and M4 received a single 1 hour continuous intravenous infusion of a solution of an ISVD according to the invention at 0.4 μg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] 5 Detailed description of this technology The present technology aims to provide a new type of drug for treating acute myeloid leukemia (AML).

[0059] The present inventors have found that a polypeptide (or ISVD construct) that dually targets CD33 and CD123 on acute myeloid leukemia (AML) cells, combined with targeting T cell receptor αβ (TCRαβ) on T cells, results in effective killing of AML cells. Killing activity was comparable to or even higher than single targeting benchmarks such as CD33 / CD3 AMG 330 BiTE or CD123 / CD3 MGD006 DART. Due to the high heterogeneity of CD33 and CD123 expression on AML cells in intra- and inter-patient samples, the polypeptides of the present invention provide broader patient coverage compared to single targeting benchmarks.

[0060] In some embodiments, the polypeptide of the present technology is efficiently produced (e.g., in a microbial host) and exhibits low viscosity at high concentration, which is advantageous and convenient for subcutaneous administration.Furthermore, such polypeptide has limited reactivity to the existing antibodies in the treated subject (i.e., the antibodies that exist in the subject before the first treatment with the antibody construct).In a preferred embodiment, such polypeptide exhibits a sufficiently long half-life in the treated subject so that successive treatments can be conveniently spaced apart.

[0061] The polypeptides are at least bispecific, but may be, for example, trispecific, tetraspecific, or pentaspecific. Further, the polypeptides are at least tetravalent, but may be, for example, pentavalent, hexavalent, etc.

[0062] The terms "bispecific", "trispecific", "tetraspecific" or "pentaspecific" all fall under the term "multispecific" and refer to binding to two, three, four or five different target molecules, respectively. The terms "bivalent", "trivalent", "tetravalent", "pentavalent" or "hexavalent" all fall under the term "multivalent" and refer to the presence of two, three, four or five binding units (such as ISVDs), respectively. For example, a polypeptide can be tetraspecific tetravalent, such as a polypeptide comprising or consisting of four ISVDs, one ISVD binding to human TCRαβ, one ISVD binding to human CD33, one ISVD binding to CD123 and one ISVD binding to human serum albumin (e.g., the ISVD construct shown in SEQ ID NO:1). For example, a polypeptide can be bispecific at the same time when two ISVDs bind to two different epitopes on the same target, e.g., when two ISVDs bind to TCRαβ. The term "biparatopic" refers to binding to two different portions (eg, epitopes) of the same target molecule.

[0063] The terms "first ISVD", "second ISVD", "third ISVD" etc. as used herein merely indicate the relative positions of the ISVDs with respect to each other, where the numbering starts from the N-terminus of the polypeptide of the invention. Thus, the "first ISVD" is closer to the N-terminus than the "second ISVD", while the "second ISVD" is closer to the N-terminus than the "third ISVD". The ISVD arrangement is therefore reversed when considered from the C-terminus. As the numbering is not absolute but only indicates the relative positions of at least three ISVDs, it is not excluded that other binding units / components such as further ISVDs that bind to TCRαβ, CD33 or CD123, or ISVDs that bind to another target, may be present in the polypeptide. For example, as described further below (see in particular the "(in vivo) half-life extension" section), the polypeptide may further comprise another ISVD that binds to human serum albumin, which may be a fourth ISVD located at the C-terminus of the at least three ISVDs. Furthermore, it is not excluded that other binding units / components such as ISVDs may be located therebetween. For example, the polypeptide may further comprise another ISVD, which may even be located between, for example, the "second ISVD" and the "third ISVD".

[0064] In light of the above, the present invention provides a polypeptide comprising or consisting of at least three ISVDs, wherein at least one ISVD specifically binds to TCRαβ, at least one ISVD specifically binds to CD33, and at least one ISVD specifically binds to CD123.

[0065] The components of the polypeptide, preferably the ISVDs, may be linked to each other by one or more suitable linkers, eg peptide linkers.

[0066] The use of linkers to join two or more (poly)peptides is well known in the art. Exemplary peptide linkers are shown in Table A-5. A commonly used class of peptide linkers is known as the "Gly-Ser" or "GS" linkers. These are linkers that consist essentially of glycine (G) and serine (S) residues, and usually have a GGGGS (SEQ ID NO: 77) motif (e.g., the formula (Gly-Gly-Gly-Gly-Ser) n (wherein n can be 1, 2, 3, 4, 5, 6, 7 or more). Some frequently used examples of such GS linkers are the 9GS linker (GGGGSGGGS, SEQ ID NO:80), the 15GS linker (n=3) and the 35GS linker (n=7). See, for example, Chen et al. Adv. Drug Deliv. Rev. 2013 Oct 15; 65(10); 1357-1369 and Klein et al., Protein Eng. Des. Sel. (2014) 27(10); 325-330. In the polypeptides of the present invention, the use of 9GS linkers to link the components of the polypeptide to each other is preferred.

[0067] In a preferred embodiment, the ISVD that specifically binds to TCRαβ is located at the N-terminus of the polypeptide. The present inventors have surprisingly found that such a configuration can increase the production yield of the polypeptide.

[0068] Furthermore, in a preferred embodiment, the ISVD that specifically binds to CD33 is located C-terminal to the ISVD that specifically binds to TCRαβ.

[0069] In an even more preferred embodiment, the ISVD that specifically binds CD123 is positioned C-terminal to the ISVD that specifically binds CD33, which is itself positioned C-terminal to the ISVD that specifically binds TCRαβ.

[0070] Thus, the polypeptide preferably comprises or consists of, in order starting from the N-terminus of the polypeptide: a first ISVD that specifically binds to TCRαβ, a second ISVD that specifically binds to CD33, and a third ISVD that specifically binds to CD123, and any binding unit that provides a polypeptide with increased half-life as defined herein. The binding unit that provides a polypeptide with increased half-life is preferably an ISVD.

[0071] It is even more preferred that the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: an ISVD that specifically binds to TCRαβ, a linker, an ISVD that specifically binds to CD33, a linker, an ISVD that specifically binds to CD123, a linker and an ISVD that binds to human serum albumin. More particularly, the polypeptide comprises or consists of, in order starting from the N-terminus of the polypeptide: an ISVD that specifically binds to TCRαβ, a 9GS linker, an ISVD that specifically binds to CD33, a 9GS linker, an ISVD that specifically binds to CD123, a 20GS linker and an ISVD that binds to human serum albumin.

[0072] Such configuration of the polypeptides can provide increased manufacturing yields, good CMC properties, as well as optimized functionality and greater potency with respect to modulating the immune response.

[0073] Preferably, the polypeptide of the present technology exhibits reduced binding by pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least one ISVD, preferably in each ISVD. In another embodiment, the polypeptide comprises an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD. The C-terminus of the ISVD is typically VTVSS (SEQ ID NO: 93). In another embodiment, the polypeptide comprises a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD comprises a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO:94), VQVSS (SEQ ID NO:95), VTVKS (SEQ ID NO:96), VTVQS (SEQ ID NO:97), VKVKS (SEQ ID NO:98), VKVQS (SEQ ID NO:99), VQVKS (SEQ ID NO:100) or VQVQS (SEQ ID NO:101) and, after the addition of a single alanine, the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO:102), VKVSSA (SEQ ID NO:103), VQVSSA (SEQ ID NO:104), VTVKSA (SEQ ID NO:105), VTVQSA (SEQ ID NO:106), VKVKSA (SEQ ID NO:107), VKVQSA (SEQ ID NO:108), VQVKSA (SEQ ID NO:109) or VQVQSA (SEQ ID NO:110), preferably VTVSSA (SEQ ID NO:102).In another embodiment, the polypeptide comprises a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) of each ISVD, and optionally a lysine (K) or glutamine (Q) at position 110 of at least one ISVD, and comprises an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension at the C-terminus of the C-terminal ISVD (such that the C-terminus of the polypeptide comprises, for example, the sequence VTVSSA (SEQ ID NO: 102), VKVSSA (SEQ ID NO: 103) or VQVSSA (SEQ ID NO: 104), preferably VTVSSA (SEQ ID NO: 102). For further information in this regard, see, for example, WO 2012 / 175741 and WO 2015 / 173325.

[0074] In a preferred embodiment, the polypeptide of the invention comprises or consists of an amino acid sequence having more than 90%, such as more than 95% or more than 99% sequence identity to SEQ ID NO: 1, and the CDRs of the four ISVDs are as defined in the sections "Immunoglobulin Single Variable Domains" and "(In Vivo) Half-Life Extension" below, respectively, items A to D (or A' to D' if the Kabat definitions are used), in particular: an ISVD that specifically binds to TCRαβ has a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO:10, and a CDR3 having the amino acid sequence of SEQ ID NO:14; an ISVD that specifically binds to CD33 has a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:11, and a CDR3 having the amino acid sequence of SEQ ID NO:15; an ISVD that specifically binds to CD123, having a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:16; and the ISVD that binds to human serum albumin has a CDR1 having the amino acid sequence of SEQ ID NO:9, a CDR2 having the amino acid sequence of SEQ ID NO:13, and a CDR3 having the amino acid sequence of SEQ ID NO:17; Or, using the Kabat definition: an ISVD that specifically binds to TCRαβ has a CDR1 having the amino acid sequence of SEQ ID NO: 34, a CDR2 having the amino acid sequence of SEQ ID NO: 38, and a CDR3 having the amino acid sequence of SEQ ID NO: 42; an ISVD that specifically binds to CD33 has a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 43; an ISVD that specifically binds to CD123, having a CDR1 having the amino acid sequence of SEQ ID NO: 36, a CDR2 having the amino acid sequence of SEQ ID NO: 40, and a CDR3 having the amino acid sequence of SEQ ID NO: 44; and The ISVD that binds to human serum albumin has a CDR1 having the amino acid sequence of SEQ ID NO:37, a CDR2 having the amino acid sequence of SEQ ID NO:41, and a CDR3 having the amino acid sequence of SEQ ID NO:45.

[0075] In particular, the polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 1. In a most preferred embodiment, the polypeptide consists of the amino acid sequence of SEQ ID NO:1.

[0076] The polypeptides of the present invention preferably have at least half the binding affinity, and more preferably at least the same binding affinity, for human TCRαβ, human CD33 and human CD123 compared to a polypeptide consisting of the amino acid sequence of SEQ ID NO:1, and the binding affinity is measured using the same method, such as SPR.

[0077] 5.1 Immunoglobulin Single Variable Domains The term "immunoglobulin single variable domain" (ISVD), used interchangeably with "single variable domain", defines an immunoglobulin molecule in which an antigen-binding site is present on and formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g., monoclonal antibodies) or fragments thereof (e.g., Fab, Fab', F(ab')2, scFv, di-scFv), in which two immunoglobulin domains, in particular two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (V H ) and the light chain variable domain (V L ) interact to form the antigen-binding site. In this case, V H and V L Both complementarity determining regions (CDRs) of each gene contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site.

[0078] In view of the above definition, fragments of conventional four chain antibodies (e.g. IgG, IgM, IgA, IgD or IgE molecules known in the art), or Fab fragments, F(ab')2 fragments, Fv fragments such as disulfide-linked Fv or scFv fragments, or bispecific antibodies derived from such conventional four chain antibodies (all known in the art) are not usually considered as immunoglobulin single variable domains, because in these cases binding to the respective epitope of an antigen is usually not by one (single) immunoglobulin domain, but by a pair of (related) immunoglobulin domains such as light and heavy chain variable domains, i.e. the V of immunoglobulin domains which jointly bind to the respective epitope of the antigen. H -V L Because it happens in pairs.

[0079] In contrast, an immunoglobulin single variable domain can specifically bind to an epitope of an antigen without pairing with other immunoglobulin variable domains. The binding site of an immunoglobulin single variable domain is a single V H , single V HH or single VL It is formed by domains.

[0080] Thus, a single variable domain may be any combination of a light chain variable domain sequence (e.g., V), as long as it is capable of forming a single antigen-binding unit (i.e., a functional antigen-binding unit that consists essentially of a single variable domain and that does not require interaction with other variable domains to form a functional antigen-binding unit). L -sequence) or a suitable fragment thereof, or a heavy chain variable domain sequence (e.g., H -sequence or V HH sequence) or a suitable fragment thereof.

[0081] Immunoglobulin single variable domains (ISVDs) can be, for example, heavy chain ISVDs, e.g., camelized V H or humanized V HH Contains V H ,V HH Preferably, this is a camelized V H or humanized V HH Contains V HH The heavy chain ISVD can be derived from a traditional four chain antibody or from a heavy chain antibody.

[0082] For example, the immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) (as defined herein, including but not limited to, NANOBODY® ISVD); other single variable domains, or any suitable fragment of any one of them.

[0083] In particular, the immunoglobulin single variable domain is a humanized V HH Or camelization V H or a NANOBODY® ISVD containing a suitable fragment thereof. [Note: NANOBODY®, NANOBODIES® and NANOCLONE® are registered trademarks of Ablynx NV]

[0084] "V HH Domain" is V HH , V HH Antigen fragments, and V HH Also known as antibodies, they were originally described as the antigen-binding immunoglobulin variable domains of "heavy chain antibodies" (i.e., "antibodies without light chains"; Hamers-Casterman et al. Nature 363:446-448 (1993)). HH The term "variable domain" refers to the heavy chain variable domains present in a conventional four-chain antibody (heavy chain variable domains, herein referred to as "V H domains”) present in conventional four-chain antibodies (herein referred to as “V L The domain was chosen to distinguish it from the domains that are called "domains." HHFor further description, see the review article by Muyldermans (review in Molecular Biotechnology 74:277-302, 2001), as well as the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 to Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193 to Unilever; Instituut voor Biotechnologie (VIB) in WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; Algonomics NV and Ablynx NV in WO 03 / 050531.WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 (=EP 1433793) by the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825 by Ablynx NV.

[0085] Typically, production of immunoglobulins involves immunization of laboratory animals, fusing immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins can be produced by screening naive or synthetic libraries, for example by phage display.

[0086] The generation of immunoglobulin sequences such as NANOBODY® ISVDs has been extensively described in WO 94 / 04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 (Reviews in Molecular Biotechnology 74:277-302, 2001). In these methods camelids are immunized with a target antigen to elicit an immune response against the target antigen. The repertoire of NANOBODY® ISVDs resulting from the immunization is further screened for ISVDs that bind to the target antigen.

[0087] In these instances, the generation of antibodies requires purified antigen for immunization and / or screening. The antigen may be purified from natural sources or during recombinant production.

[0088] Immunization and / or screening for immunoglobulin sequences can be carried out using peptide fragments of such sources.

[0089] The invention may use immunoglobulin sequences of different origins, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The invention also includes fully human, humanized or chimeric sequences. For example, the invention includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, such as camelized dAbs described by Ward et al (e.g. WO 94 / 04678 and Riechmann, Febs Lett., 339:285-290, 1994 and Prot. 9:531-537, 1996). Furthermore, the invention also uses fused immunoglobulin sequences, for example to form multivalent and / or multispecific constructs (one or more V HH For multivalent and multispecific polypeptides containing domains and their preparation, see Conrath et al., J. Biol. Chem., Vol. 276.10.7346-7350, 2001, and for example WO 96 / 34103 and WO 99 / 23221), as well as immunoglobulin sequences containing tags or other functional moieties derivable from the immunoglobulin sequences of the invention, such as toxins, labels, radiochemicals, etc.

[0090] "Humanized V HH " is a naturally occurring V HH domain, but "humanized," i.e., the naturally occurring V HH One or more amino acid residues in the amino acid sequence (and particularly in the framework sequences) of the sequence are replaced with a V HThe humanized V domain comprises an amino acid sequence substituted with one or more of the amino acid residues (e.g., as described above) occurring at the corresponding positions in the V domain. This can be done in a manner known per se, which will be clear to the skilled person, for example, based on the further description herein and the prior art (e.g., WO 2008 / 020079). Again, such a humanized V domain may be used as a substitute for one or more of the amino acid residues (e.g., as described above) occurring at the corresponding positions in the V domain. HH It should be noted that the VHH domain-containing polypeptides can be obtained by any conventional method known per se and are therefore not strictly limited to polypeptides obtained using naturally occurring VHH domain-containing polypeptides as starting material.

[0091] "Camelization V H " is a naturally occurring V H The amino acid sequence of the V domain corresponds to that of the naturally occurring V from a conventional four-chain antibody, but has been "camelized", i.e. H One or more amino acid residues in the amino acid sequence of the domain are HH The amino acid sequence includes an amino acid sequence in which one or more of the amino acid residues occurring at the corresponding positions in the V domain are substituted. This can be done in a manner known per se, which will be clear to the skilled person, for example, on the basis of the further explanations herein and the prior art (for example, WO 2008 / 020079). Such "camelization" substitutions are preferably made in the V domain, as defined herein, of one or more of the amino acid residues occurring at the corresponding positions in the V domain. H -V L They are inserted at the positions of amino acids that form and / or are present at interfaces and / or so-called Camelidae hallmark residues (see, for example, WO 94 / 04678 and Davies and Riechmann, supra (1994 and 1996)). H V, which is used as a starting material or starting point for generating or designing H The sequence is a mammalian V H Sequences, e.g., V H Array, e.g. V H 3 sequence. However, such camelized V Hcan be obtained by any suitable method known per se, and therefore can be obtained without using the naturally occurring V as starting material. H It should be noted that the polypeptide obtained using the polypeptide containing the domain is not strictly limited.

[0092] It should be noted that one or more immunoglobulin sequences may be linked to each other and / or to other amino acid sequences (e.g., via disulfide bridges) to provide peptide constructs (e.g., Fab' fragments, F(ab')2 fragments, scFv constructs "diabodies" and other multispecific constructs) that may also be useful in the present invention. See, for example, the review by Holliger and Hudson, Nat Biotechnol 2005 Sep;23(9):1126-36). In general, when a polypeptide is intended for administration to a subject (e.g., for prophylactic, therapeutic and / or diagnostic purposes), the polypeptide preferably comprises an immunoglobulin sequence that does not naturally occur in said subject.

[0093] The preferred structure of an immunoglobulin single variable domain sequence can be considered to consist of four framework regions ("FRs"), which are referred to in the art and herein as "framework region 1" ("FR1"), "framework region 2" ("FR2"), "framework region 3" ("FR3"), and "framework region 4" ("FR4"), respectively, interrupted by three "complementarity determining regions" ("CDRs"), which are referred to in the art and herein as "complementarity determining region 1" ("CDR1"), "complementarity determining region 2" ("CDR2"), and "complementarity determining region 3" ("CDR3"), respectively.

[0094] As further described in paragraph q) of pages 58 and 59 of WO 08 / 020079 (hereby incorporated by reference), the amino acid residues of an immunoglobulin single variable domain are HThe domain of V from Camelids in the article by Riechmann and Muyldermans, 2000, given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91). HH The numbers can be numbered according to the common numbering scheme applied to the domains (J. Immunol. Methods 240(1-2):185-195; see, for example, Fig. 2 of this document). H Domain and V HH It should be noted that, as is well known in the art for domains, the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This generally means that the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. H Domain and V HH The total number of amino acid residues in a domain will usually be in the range of 110 to 120, often 112 to 115. It should be noted, however, that shorter and longer sequences may also be suitable for the purposes described herein.

[0095] In this application, unless otherwise indicated, the CDR sequences were determined by the AbM numbering as described by Kontermann and Duebel (Eds. 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According to this method, FR1 contains amino acid residues at positions 1-25, CDR1 contains amino acid residues at positions 26-35, FR2 contains amino acids at positions 36-49, CDR2 contains amino acid residues at positions 50-58, FR3 contains amino acid residues at positions 59-94, CDR3 contains amino acid residues at positions 95-102, and FR4 contains amino acid residues at positions 103-113.

[0096] The determination of the CDR regions can also be performed according to different methods. In the CDR determination according to Kabat, FR1 of an immunoglobulin single variable domain comprises amino acid residues at positions 1 to 30, CDR1 of an immunoglobulin single variable domain comprises amino acid residues at positions 31 to 35, FR2 of an immunoglobulin single variable domain comprises amino acid residues at positions 36 to 49, CDR2 of an immunoglobulin single variable domain comprises amino acid residues at positions 50 to 65, FR3 of an immunoglobulin single variable domain comprises amino acid residues at positions 66 to 94, CDR3 of an immunoglobulin single variable domain comprises amino acid residues at positions 95 to 102, and FR4 of an immunoglobulin single variable domain comprises amino acid residues at positions 103 to 113.

[0097] In such immunoglobulin sequences, the framework regions may be any suitable framework region sequence, and examples of suitable framework sequences will be clear to the skilled person, e.g. from standard handbooks and on the basis of the further disclosure and prior art set out herein.

[0098] The framework sequences are preferably immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization) (a suitable combination of such sequences). For example, the framework sequences may be those of a light chain variable domain (e.g., L -sequence) and / or heavy chain variable domain (e.g., V H -sequence or V HH In one particularly preferred embodiment, the framework sequence is derived from the V HH -framework sequences derived from conventional V sequences (wherein said framework sequences are optionally partially or fully humanized) or camelized (as defined herein) H It can be one of the arrays.

[0099] In particular, the framework sequences present in the ISVD sequences used in the present invention are HH or Camelization V H A NANOBODY® immunoglobulin variable domain may include one or more Hallmark residues (as defined herein), such that the NANOBODY® immunoglobulin variable domain includes: Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

[0100] Again, as generally described herein for immunoglobulin sequences, it is also possible to use any suitable fragment (or combination of fragments) of the foregoing, for example a fragment comprising one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences (e.g. in the same order as those CDR and framework sequences may be present in the full-sized immunoglobulin sequence from which the fragment is derived).

[0101] However, it should be noted that the present invention is not limited with respect to the origin of the ISVD sequence (or the nucleotide sequence used to express it), nor with respect to the manner in which the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequence may be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In specific, but non-limiting embodiments, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHV sequences). HH Immunoglobulin sequences include, but are not limited to, "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences obtained by techniques such as affinity maturation (e.g., starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, joining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar immunoglobulin sequence engineering techniques well known to those skilled in the art, or any suitable combination of any of the foregoing.

[0102] Likewise, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, for example a sequence isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), a nucleotide sequence isolated from a library (and in particular an expression library), a nucleotide sequence prepared by mutagenesis (using any suitable technique known per se, such as mismatch PCR) into a naturally occurring nucleotide sequence, a nucleotide sequence prepared by PCR using overlapping primers, or a nucleotide sequence prepared using DNA synthesis techniques known per se.

[0103] As mentioned above, ISVD is a HHor a suitable fragment thereof. For a general description of NANOBODY® ISVDs, see the further description below and the prior art cited therein. However, in this regard, the description and prior art are not intended to be limiting. H 3 classes" (i.e., V H It should be noted that the present invention primarily describes NANOBODY® ISVDs of the three classes of NANOBODY® ISVDs with a high degree of sequence homology to human germline sequences (such as DP-47, DP-51 or DP-29). However, the present invention, in its broadest sense, can generally use any type of NANOBODY® ISVD, including, for example, the so-called "V- H 4 class" (i.e., V such as DP-78) H It should be noted that we also use NANOBODY® ISVDs that belong to four classes of NANOBODY® ISVDs that have a high degree of sequence homology to human germline sequences.

[0104] Generally, NANOBODY® ISVDs (especially (partially) humanized VSVDs) HH Sequence and camelized V H V containing arrays HH A NANOBODY® ISVD may be characterized by the presence of one or more "hallmark residues" (also as further described herein) in one or more framework sequences (as described herein). Thus, in general, a NANOBODY® ISVD may be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1-FR4 refer to framework regions 1-4, respectively, CDR1-CDR3 refer to complementarity determining regions 1-3, respectively, and one or more of the hallmark residues are as further defined herein.

[0105] Specifically, a NANOBODY® ISVD can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and framework sequences are as further defined herein.

[0106] More specifically, the NANOBODY® ISVD can be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 Here, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively. One or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, 108 according to the Kabat numbering are selected from the hallmark residues set out in Table 1 below.

[0107] [Table 1]

[0108] [Table 2]

[0109] The present technology employs ISVDs that can specifically bind to TCRαβ, CD33 or CD123, among others. In the context of the present technology, "binding" a particular target molecule has its normal meaning in the art as understood in the context of antibodies and their respective antigens.

[0110] The polypeptides of the present technology may comprise one or more ISVDs that specifically bind to TCRαβ, one or more ISVDs that specifically bind to CD33, and one or more ISVDs that specifically bind to CD123.

[0111] The ISVD used in the present technology forms part of the polypeptide of the present technology, which comprises or consists of at least three ISVDs, so that the polypeptide can specifically bind to TCRαβ, CD33 and CD123. Thereby, the polypeptide can be fixed on CD33+CD123+ leukemia stem cells (LSC) and acute myeloid leukemia (AML) blasts and can simultaneously bind to TCRαβ on cytotoxic T cells. In this way, the polypeptide brings T cells into close proximity to LSC and AML blasts. Multiple binding of the polypeptide to tumor antigens (CD33 and CD123) on tumor cells and simultaneous binding to TCR molecules on a single T cell leads to TCR cluster formation and ultimately T cell activation. Activation of T cells in close proximity to LSC and AML blasts can result in efficient tumor cell killing in AML patients, resulting in a therapeutic effect.

[0112] Thus, the target molecules of at least three ISVDs used in the polypeptide of the present technology are TCRαβ, CD33 and CD123. Examples are mammalian CD33, CD123 and TCRαβ. Human TCRαβ (Uniprot accession), human CD33 (Uniprot accession) and human CD123 (Uniprot accession) are preferred, but versions from other species, such as mouse, rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primates such as cynomolgus monkeys (also referred to herein as "cyno"), or camelids such as llamas or alpacas, are also suitable for the present technology.

[0113] Specific examples of TCRαβ, CD123, and ISVDs that specifically bind to CD123 that can be used in the present technology are shown in A to C below: A. Binds specifically to human TCRαβ i. a CDR1 having the amino acid sequence of SEQ ID NO:6 or having 2 or 1 amino acid difference from SEQ ID NO:6; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 10 or having 2 or 1 amino acid difference from SEQ ID NO: 10; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 14 or having 2 or 1 amino acid difference from SEQ ID NO: 14; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:6, a CDR2 having the amino acid sequence of SEQ ID NO:10, and a CDR3 having the amino acid sequence of SEQ ID NO:14.

[0114] A preferred example of such an ISVD that specifically binds to human TCRαβ is (in addition to the CDRs defined in the previous section A) the TCRαβ-V HH and most preferably has one or more, and preferably all, of the framework regions shown in HH( It is an ISVD having the complete amino acid sequence of SEQ ID NO:2 (see Tables A-1 and A-2).

[0115] Also, in a preferred embodiment, the amino acid sequence of the ISVD that specifically binds to human TCRαβ may have greater than 90%, such as greater than 95% or greater than 99% sequence identity to SEQ ID NO:2, and optionally the CDRs are as defined in the preceding section A. In particular, the ISVD that specifically binds to TCRαβ preferably has the amino acid sequence of SEQ ID NO:2.

[0116] If such an ISVD that specifically binds to TCRαβ has two or one amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item A above), the ISVD is preferably a TCRαβ-V HH (SEQ ID NO: 2), has at least half the binding affinity to human TCRαβ, more preferably at least the same binding affinity, as measured using the same methods, e.g., SPR.

[0117] B. Specific binding to human CD33; i. a CDR1 having the amino acid sequence of SEQ ID NO:7 or having 2 or 1 amino acid difference from SEQ ID NO:7; ii. a CDR2 having the amino acid sequence of SEQ ID NO:11 or having 2 or 1 amino acid difference from SEQ ID NO:11; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 15 or having 2 or 1 amino acid difference from SEQ ID NO: 15; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:7, a CDR2 having the amino acid sequence of SEQ ID NO:11, and a CDR3 having the amino acid sequence of SEQ ID NO:15.

[0118] Preferred examples of such ISVDs that specifically bind to human CD33 include (in addition to the CDRs defined in section B above) the CD33-V HH and most preferably has one or more, and preferably all, of the framework regions as shown for CD33-V. HH (SEQ ID NO: 3, see Tables A-1 and A-2).

[0119] Also, in a preferred embodiment, the amino acid sequence of the ISVD that specifically binds to human CD33 may have more than 90%, such as more than 95% or more than 99% sequence identity with SEQ ID NO:3, and optionally the CDRs are as defined in the previous section B. In particular, the ISVD that binds to CD33 preferably has the amino acid sequence of SEQ ID NO:3.

[0120] If such an ISVD that specifically binds to CD33 has two or one amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item B above), the ISVD is preferably HH (SEQ ID NO: 3), has at least half the binding affinity to human CD33, more preferably at least the same binding affinity, as measured using the same methods, e.g., SPR.

[0121] C. Specific binding to human CD123; i. a CDR1 having the amino acid sequence of SEQ ID NO:8 or having 2 or 1 amino acid difference from SEQ ID NO:8; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 12 or having 2 or 1 amino acid difference from SEQ ID NO: 12; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 16 or having 2 or 1 amino acid difference from SEQ ID NO: 16; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:8, a CDR2 having the amino acid sequence of SEQ ID NO:12, and a CDR3 having the amino acid sequence of SEQ ID NO:16.

[0122] Preferred examples of such ISVDs that specifically bind to human CD123 include (in addition to the CDRs defined in section C above) the CD123-V HH and most preferably has one or more, and preferably all, of the framework regions as shown for CD123-V. HH (SEQ ID NO: 4, see Tables A-1 and A-2).

[0123] Also, in a preferred embodiment, the amino acid sequence of the ISVD that specifically binds to human CD123 may have more than 90%, such as more than 95% or more than 99% sequence identity with SEQ ID NO:4, and optionally the CDRs are as defined in the previous section C. In particular, the ISVD that binds to CD123 preferably has the amino acid sequence of SEQ ID NO:4.

[0124] If such an ISVD that specifically binds to CD123 has two or one amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item C above), the ISVD is preferably HH (SEQ ID NO: 4), has at least half the binding affinity to human CD123, more preferably at least the same binding affinity, as measured using the same methods, e.g., SPR.

[0125] Preferably, each of the ISVDs defined under items A to C above is comprised in a polypeptide of the invention. Such a polypeptide of the invention comprising each of the ISVDs defined under items A to C above preferably has at least half, more preferably at least the same binding affinity to human TCRαβ, human CD33 and human CD123 as the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, the binding affinity being measured using the same method, such as SPR.

[0126] The sequence numbers referred to in the above items A-C are based on the CDR definition according to the AbM definition (see Table A-2). It should be noted that sequence numbers defining the same CDRs according to the Kabat definition (see Table A-2.1) can be used in the above items A-C as well.

[0127] Thus, specific ISVDs that specifically bind to TCRαβ, CD33 or CD123 that can be used in the present invention as described above using the AbM definition can also be described using the Kabat definition as shown in sections A'-C' below: A'. Binds specifically to human TCRαβ i. a CDR1 having the amino acid sequence of SEQ ID NO: 34 or having 2 or 1 amino acid difference from SEQ ID NO: 34; ii. CDR2 having the amino acid sequence of SEQ ID NO: 38 or having 2 or 1 amino acid difference from SEQ ID NO: 38; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 42 or having 2 or 1 amino acid difference from SEQ ID NO: 42; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 34, a CDR2 having the amino acid sequence of SEQ ID NO: 38, and a CDR3 having the amino acid sequence of SEQ ID NO: 42.

[0128] Preferred examples of such ISVDs that specifically bind to human TCRαβ include (in addition to the CDRs defined in the previous section A') the TCRαβ-V HHand most preferably has one or more, and preferably all, of the framework regions shown in HH (SEQ ID NO: 2, see Tables A-1 and A-2.1).

[0129] B'. Binds specifically to human CD33 i. a CDR1 having the amino acid sequence of SEQ ID NO: 35 or having 2 or 1 amino acid difference from SEQ ID NO: 35; ii. a CDR2 having the amino acid sequence of SEQ ID NO: 39 or having 2 or 1 amino acid difference from SEQ ID NO: 39; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 43 or having 2 or 1 amino acid difference from SEQ ID NO: 43; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO: 35, a CDR2 having the amino acid sequence of SEQ ID NO: 39, and a CDR3 having the amino acid sequence of SEQ ID NO: 43.

[0130] Preferred examples of such ISVDs that specifically bind to human CD33 include (in addition to the CDRs defined in the previous section B) the CD33-V HH and most preferably has one or more, and preferably all, of the framework regions shown for CD33-V. HH (SEQ ID NO: 3, see Tables A-1 and A-2.1).

[0131] C'. Binds specifically to human CD123 i. a CDR1 having the amino acid sequence of SEQ ID NO: 36 or having 2 or 1 amino acid difference from SEQ ID NO: 36; ii. CDR2 having the amino acid sequence of SEQ ID NO: 40 or having 2 or 1 amino acid difference from SEQ ID NO: 40; and iii. a CDR3 having the amino acid sequence of SEQ ID NO: 44 or having 2 or 1 amino acid difference from SEQ ID NO: 44; Preferably, the ISVD comprises a CDR1 having the amino acid sequence of SEQ ID NO:36, a CDR2 having the amino acid sequence of SEQ ID NO:40, and a CDR3 having the amino acid sequence of SEQ ID NO:44.

[0132] Preferred examples of such ISVDs that specifically bind to human CD123 include (in addition to the CDRs defined in the previous section C) the CD123-V HH and most preferably has one or more, and preferably all, of the framework regions shown for CD123-V. HH (SEQ ID NO: 4, see Tables A-1 and A-2.1).

[0133] The percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated by dividing the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at corresponding positions in the second amino acid sequence by the total number of amino acid residues in the first amino acid sequence, and multiplying by 100%, where each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence is considered to be a difference in a single amino acid residue (i.e., at a single position) compared to the first amino acid sequence.

[0134] Typically, for purposes of determining the percentage of "sequence identity" between two amino acid sequences according to the calculation methods outlined above, the amino acid sequence having the greatest number of amino acid residues is considered to be the "first" amino acid sequence, and the other amino acid sequence is considered to be the "second" amino acid sequence.

[0135] As used herein, an "amino acid difference" refers to a deletion, insertion or substitution, preferably a substitution, of a single amino acid residue relative to the reference sequence.

[0136] The amino acid substitutions are preferably conservative substitutions. Such conservative substitutions are preferably those in which one amino acid residue in the following groups (a) to (e) is replaced by another amino acid residue in the same group: (a) small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic non-polar residues: Met, Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0137] Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val; Lys to Arg, Gln or Glu; Met to Leu, Tyr, or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, Ile, or Leu.

[0138] 5.2 Specificity The terms "specificity", "specifically binds" or "specific binding" refer to the number of different target molecules, such as antigens from the same organism, that a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). "Specificity", "specifically binds" or "specific binding" are used interchangeably herein with "selectivity", "selectively binds" or "selective binding". Binding units, such as ISVDs, preferably bind specifically to their designated targets.

[0139] The specificity / selectivity of a binding unit can be determined based on affinity. Affinity indicates the strength or stability of a molecular interaction. Affinity is generally given by KD or dissociation constant with units of mol / l (or M). Affinity can also be expressed as an association constant KA, where KA is equal to 1 / KD and is expressed as (mol / l) -1 (or M -1 )

[0140] Affinity is a measure of the binding strength between a moiety on a target molecule and a binding site. The smaller the value of KD, the stronger the binding strength between the target molecule and the targeting moiety.

[0141] Typically, the binding units used in this technology (such as ISVD) are 10 -5 ~10 -12 mol / l or less, preferably 10 -7 ~10 -12 mol / l or less, more preferably 10 -8 ~10 -12 mol / l (i.e., association constant (KA) is 10 5 ~10 12 l / mol or more, preferably 10 7 ~10 12 l / mol or more, more preferably 10 8 ~10 12 They bind to their targets with a dissociation constant (KD) of 100 μg / mol.

[0142] 10 -4 Any KD value greater than mol / l (or 10 4 Any K A value less than 1 l / mol) is generally considered to indicate nonspecific binding.

[0143] The KD of a biological interaction, such as the binding of an immunoglobulin sequence to an antigen, that is considered to be specific is typically around 10 -5 mol / l (10000nM or 10μM) to 10 -12 The range is below mol / l (0.001 nM or 1 pM).

[0144] Thus, specific / selective binding can be achieved by determining - using the same measurement method, e.g., SPR - whether the binding unit (or a polypeptide comprising it) is more than 10 -5 ~10 -12 Binds to TCRαβ, CD33 and / or CD123 with a KD value of 10 -4 It may mean binding to a relevant target with a K value of greater than mol / l.

[0145] Thus, the polypeptides of the present technology preferably have at least half the binding affinity, and more preferably at least the same binding affinity, for human TCRαβ, human CD33, and human CD123 compared to a polypeptide consisting of the amino acids of SEQ ID NO:1, where the binding affinity is measured using the same method, such as SPR.

[0146] Specific binding to a particular target from a particular species does not exclude that the binding unit can also specifically bind to a similar target from a different species. For example, specific binding to human TCRαβ does not exclude that the binding unit (or a polypeptide comprising it) can also specifically bind to TCRαβ from cynomolgus monkeys. Similarly, specific binding to, for example, human CD33 or CD123 does not exclude that the binding unit (or a polypeptide comprising it) can also specifically bind to CD33 or CD123 from cynomolgus monkeys ("cyno").

[0147] Specific binding of a binding unit to a designated target can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and their various variants known per se in the art; as well as other techniques mentioned herein.

[0148] The dissociation constant may be an actual dissociation constant or an apparent dissociation constant, as will be appreciated by those skilled in the art. Methods for determining dissociation constants will be apparent to those skilled in the art and include, for example, the techniques described below. In this regard, -4mol / l or 10 -3 mol / l (e.g., 10 -2 It will also be clear that it may not be possible to measure dissociation constants greater than 100 kD (mol / l). In some cases, as will be clear to the skilled artisan, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) according to the relationship [KD=1 / KA].

[0149] The affinity of the molecular interaction between two molecules can be determined using the well-known surface plasmon resonance (SPR) biosensor technology (see, e.g., Ober et al. 2001, Intern. Immunology 13:1551-1559). As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detection of changes in protein concentration within a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes over the immobilized molecule under flow conditions to obtain k on , k off measurements, and therefore K D (or K A ) values. This can be performed, for example, using the well-known BIAcore® system (BIAcore International AB, GE Healthcare, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51:19-26), Jonsson et al. (1991 Biotechniques 11:620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al. (1991, Anal. Biochem. 198:268-277).

[0150] Another well-known biosensor technique for determining the affinity of biomolecular interactions is Biolayer Interferometry (BLI) (see, for example, Abdiche et al. 2008, Anal. Biochem. 377:209-217). The term "Biolayer Interferometry" or "BLI" as used herein refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized proteins on the biosensor chip (signal beam). Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Interactions can be measured in real time, so that association and dissociation rates as well as affinity can be determined. BLI can be performed, for example, using well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0151] Alternatively, affinity can be measured in a kinetic exclusion assay (KinExA) (see, e.g., Drake et al. 2004, Anal. Biochem., 328:35-43) using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of unmodified molecules. An equilibrium solution of antibody / antigen complex is passed through a column containing beads precoated with antigen (or antibody), allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is achieved using a fluorescently labeled protein that binds to the antibody (or antigen).

[0152] The GYROLAB® Immunoassay System provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5:1765-74).

[0153] 5.3 (In vivo) Half-life extension The polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, which provide the polypeptide with an increased (in vivo) half-life compared to a corresponding polypeptide that does not comprise the one or more other groups, residues, moieties or binding units. In vivo half-life extension means, for example, that the polypeptide has an increased half-life in a mammal, such as a human subject, after administration. Half-life can be expressed, for example, as t1 / 2 beta.

[0154] The type of group, residue, moiety or binding unit is generally not limited and may be selected, for example, from the group consisting of a polyethylene glycol molecule, a serum protein or fragment thereof, a binding unit capable of binding to a serum protein, an Fc portion, and a small protein or peptide capable of binding to a serum protein.

[0155] More specifically, said one or more other groups, residues, moieties or binding units conferring an increased half-life to the polypeptide may be selected from the group consisting of binding units capable of binding to serum albumin, such as human serum albumin, or serum immunoglobulin, such as IgG, preferably a binding unit capable of binding to human serum albumin. The binding unit is preferably an ISVD.

[0156] For example, WO 04 / 041865 describes NANOBODY® ISVDs that bind serum albumin (in particular, that bind human serum albumin), which can be linked to other proteins (such as one or more other NANOBODY® ISVDs that bind to a desired target) to increase the half-life of the protein.

[0157] The international application WO 06 / 122787 describes several NANOBODY® ISVDs against (human) serum albumin. These NANOBODY® ISVDs include the NANOBODY® ISVD called Alb-1 (SEQ ID NO: 52 in WO 06 / 122787) and its humanized variants such as Alb-8 (SEQ ID NO: 62 in WO 06 / 122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptides as well as other therapeutic entities or moieties.

[0158] Furthermore, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0159] In a preferred embodiment, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23, preferably Alb-8 or Alb-23 or variants thereof, as set out on pages 7 to 9 of WO 2012 / 175400, and the albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, WO 2018 / 134234. Some preferred serum albumin binders are also shown in Table A-4. Particularly preferred additional components of the polypeptides of the present technology are as described in Section C.

[0160] C. binds to human serum albumin i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR1 having 2 or 1 amino acid difference from SEQ ID NO: 9; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a CDR2 having 2 or 1 amino acid difference from SEQ ID NO: 13; and iii. a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a CDR3 having 2 or 1 amino acid difference from SEQ ID NO: 17; Preferably, the ISVD comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:9, a CDR2 comprising the amino acid sequence of SEQ ID NO:13, and a CDR3 comprising the amino acid sequence of SEQ ID NO:17.

[0161] A preferred example of such an ISVD that binds to human serum albumin is an ISVD that has (in addition to the CDRs defined in section C above) one or more (preferably all) framework regions as shown for construct ALB23002 in Table A-2, and most preferably includes the complete amino acid sequence of construct ALB23002 (see SEQ ID NO:5, Tables A-1 and A-2).

[0162] Item C can also be explained using the Kabat definition as follows:

[0163] C'. Binds to human serum albumin i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 37, or a CDR1 having 2 or 1 amino acid difference from SEQ ID NO: 37; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 41, or a CDR2 having 2 or 1 amino acid difference from SEQ ID NO: 41; and iii. a CDR3 comprising the amino acid sequence of SEQ ID NO: 45, or a CDR3 having 2 or 1 amino acid difference from SEQ ID NO: 45; Preferably, the ISVD comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:37, a CDR2 comprising the amino acid sequence of SEQ ID NO:41, and a CDR3 comprising the amino acid sequence of SEQ ID NO:45.

[0164] A preferred example of such an ISVD that binds to human serum albumin is an ISVD that has (in addition to the CDRs defined in the previous section C') one or more, preferably all, of the framework regions shown for construct ALB23002 in Table A-2.1, and most preferably includes the complete amino acid sequence of construct ALB23002 (see SEQ ID NO:5, Tables A-1 and A-2.1).

[0165] Also in a preferred embodiment, the amino acid sequence of the ISVD that binds human serum albumin may have more than 90%, such as more than 95% or more than 99% sequence identity with SEQ ID NO:5, and optionally the CDRs are as defined in the previous section C. In particular, the ISVD that binds human serum albumin preferably comprises the amino acid sequence of SEQ ID NO:5.

[0166] If such an ISVD binding to human serum albumin has two or one amino acid difference in at least one CDR compared to the corresponding reference CDR sequence (item C above), the ISVD has at least half the binding affinity, preferably at least the same binding affinity, to human serum albumin as construct ALB23002 shown in SEQ ID NO:5, the binding affinity being measured using the same methods, such as SPR.

[0167] In a preferred embodiment, when such an ISVD binding to human serum albumin has a C-terminal position, it exhibits a C-terminal alanine (A) or glycine (G) extension, and is preferably selected from SEQ ID NO: 64, 65, 67, 69, 70, 71, 72, 73, 74 and 75 (see Table A-4 below). When the ISVD binding to human serum albumin includes a position other than the C-terminal position (i.e., not the C-terminal ISVD of the polypeptide of the present technology), it is selected from SEQ ID NO: 5, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 66 and SEQ ID NO: 68 (see Table A-4 below).

[0168] 5.4 Nucleic acid molecules Nucleic acid molecules encoding the polypeptides of the present technology are also provided.

[0169] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked together via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform / transfect a host cell or host organism, for example for expression and / or production of a polypeptide. Suitable hosts or host cells for production purposes will be apparent to the skilled artisan and can be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Hosts or host cells comprising a nucleic acid encoding a polypeptide of the present technology are also encompassed by the present technology.

[0170] The nucleic acid may be, for example, DNA, RNA, or a hybrid thereof, and may also include (for example, chemically) modified nucleotides such as PNA. It may be single-stranded or double-stranded, and is preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the present technology may be genomic DNA, cDNA.

[0171] The nucleic acid of the present technology can be prepared or obtained in a manner known per se and / or isolated from a suitable natural source. A nucleotide sequence encoding a naturally occurring (poly)peptide can be subjected to site-directed mutagenesis, for example, to provide a nucleic acid molecule encoding a polypeptide having sequence variations. Also, as will be clear to those skilled in the art, several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and, for example, a nucleic acid encoding one or more linkers, can also be linked together in a suitable manner to prepare a nucleic acid.

[0172] Techniques for generating nucleic acids will be apparent to those of skill in the art and may include, for example and without limitation, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more portions thereof), introducing mutations that result in expression of truncated expression products; introducing one or more restriction sites (e.g., to create cassettes and / or regions that can be easily digested and / or ligated using appropriate restriction enzymes), and / or introducing mutations by PCR reactions using one or more "mismatched" primers.

[0173] 5.5 Vectors Also provided is a vector that comprises the nucleic acid molecule that codes for the polypeptide of the present technology.A vector as used herein is a vehicle that is suitable for carrying genetic material into cells.Vector includes naked nucleic acid such as plasmid or mRNA, or nucleic acid that is incorporated into larger structure such as liposome or viral vector.

[0174] A vector generally comprises at least one nucleic acid, optionally linked to one or more regulatory elements (e.g., one or more suitable promoters, enhancers, terminators, etc.). The vector is preferably an expression vector, i.e., a vector suitable for expressing the encoded polypeptide or construct under appropriate conditions, e.g., when the vector is introduced into a (e.g., human) cell. In the case of a DNA-based vector, this usually includes the presence of elements for transcription (e.g., promoters and polyA signals) and translation (e.g., Kozak sequences).

[0175] Preferably, in a vector, the at least one nucleic acid and the regulatory element are "operably linked" to each other, which generally means that they are in a functional relationship with each other. For example, a promoter is considered to be "operably linked" to a coding sequence if the promoter is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence (wherein the coding sequence should be understood to be "under the control" of the promoter). Generally, when two nucleotide sequences are operably linked, they are in the same orientation and usually also in the same reading frame. They are usually essentially contiguous, although this may not be necessary either.

[0176] Preferably, any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.

[0177] For example, a promoter, enhancer or terminator must be "operable" in the intended host cell or host organism, meaning, for example, that the promoter must be capable of initiating or otherwise controlling / regulating the transcription and / or expression of a nucleotide sequence, e.g., a coding sequence, to which it is operably linked.

[0178] 5.6 Composition The present technology also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding the polypeptide of the present technology, or at least one vector comprising such a nucleic acid molecule.The composition can be a pharmaceutical composition.The composition can further comprise at least one pharma- ceutically acceptable carrier, diluent or excipient and / or adjuvant, and can comprise one or more additional pharma- ceutically active polypeptides and / or compounds.

[0179] 5.7 Host organisms The present technology also relates to host cells or host organisms comprising a polypeptide of the present technology, a nucleic acid encoding a polypeptide of the present technology, and / or a vector comprising a nucleic acid molecule encoding a polypeptide of the present technology.

[0180] Suitable host cells or host organisms are clear to those skilled in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism, such as HEK293 cells, CHO cells, E. coli, Pichia pastoris, etc. The most preferred host is Pichia pastoris.

[0181] 5.8 Methods and Uses of Polypeptides The present technology also provides a method for producing the polypeptide of the present technology. The method may include transforming / transfecting a host cell or host organism with a nucleic acid encoding the polypeptide, expressing the polypeptide in the host, optionally followed by one or more isolation and / or purification steps. Specifically, the method includes: a) expressing a nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism, or in another suitable expression system; optionally followed by: b) isolating and / or purifying the polypeptide.

[0182] Suitable host cells or host organisms for production purposes will be clear to the skilled artisan and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell line, or any suitable fungal, prokaryotic or eukaryotic organism, such as HEK293 cells, CHO cells, E. coli, Pichia pastoris, etc. The most preferred host is Pichia pastoris.

[0183] The polypeptides of the present technology, the described nucleic acid molecules or vectors, or compositions comprising the polypeptides, nucleic acid molecules or vectors of the present technology, preferably the polypeptides or compositions comprising them, are useful as pharmaceuticals.

[0184] Thus, the present technology provides a polypeptide of the present technology, a nucleic acid molecule or vector described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology, for use as a medicament.

[0185] Also provided is a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology, for use in the treatment (prophylactic or therapeutic) of acute myeloid leukemia (AML), preferably relapsed and / or refractory AML.

[0186] Further provided is a method of treating (prophylactic and / or therapeutic) AML comprising administering to a subject in need thereof a pharma- ceutically active amount of a polypeptide, nucleic acid molecule, or vector of the present technology, or a composition comprising the polypeptide, nucleic acid molecule, or vector of the present technology.

[0187] Further provided is the use of a polypeptide of the present technology, a nucleic acid molecule or vector described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology, preferably in the preparation of a pharmaceutical composition for treating AML.

[0188] The AML can be relapsed and / or refractory AML.

[0189] The "subject" referred to in the context of the present technology can be any animal, preferably a mammal. Among mammals, humans and non-human mammals can be distinguished. Non-human animals can be, for example, companion animals (e.g., dogs, cats), livestock animals (e.g., bovine, equine, ovine, caprine, or porcine animals), or animals generally used for research purposes and / or to produce antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, ovine, equine, porcine, non-human primates, such as cynomolgus monkeys, or camelids, such as llamas or alpacas).

[0190] In the context of prophylactic and / or therapeutic purposes, the subject may be any animal, more particularly any mammal, but is preferably a human subject.

[0191] Substances (including polypeptides, nucleic acid molecules and vectors) or compositions may be administered to a subject by any suitable route of administration, for example enteral (such as oral or rectal) or parenteral (e.g. epicutaneous, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration. Parenteral administration, such as intramuscular, subcutaneous, or intradermal, is preferred. Subcutaneous administration is most preferred.

[0192] To provide the intended therapeutic result, an effective amount of the polypeptide, the described nucleic acid molecule or vector, or a composition comprising the polypeptide, nucleic acid molecule or vector, can be administered to a subject.

[0193] One or more doses may be administered. When more than one dose is administered, the doses may be administered at appropriate intervals to maximize the effect of the polypeptide, composition, nucleic acid molecule or vector.

[0194] [Table 3]

[0195] [Table 4]

[0196] [Table 5]

[0197] [Table 6]

[0198] [Table 7]

[0199] [Table 8]

[0200] [Table 9] EXAMPLES

[0201] 6. Working Example 6.1 Example 1: Generation of multispecific ISVD constructs Multispecific NANOBODY® ISVD proteins were expressed in P. pastoris. The yeast expression vector contains the AOX1 promoter and terminator, a resistance gene for Zeocin and coding information for the Saccharomyces cerevisiae alpha mating factor signal peptide. The NANOBODY® ISVD monovalent components (BB) were combined with GS linkers and cloned into the expression vector by Golden Gate cloning (Engler C, Marillonnet S. Golden Gate Cloning Methods Mol Biol. 2014;1116:119-31). The expression vector contains two BpiI restriction sites for cloning PCR amplified monovalent NANOBODY® ISVD components with GS linkers included in one or more vectors. All these elements are flanked by BpiI sites. The use of unique nucleotide overhangs at each position of the cloning cassette allows seamless ligation in a defined order. After Sanger sequence confirmation, plasmid DNA from E. coli TOP10 was linearized and transformed into an in-house prepared highly competent P. pastoris strain NRRL Y-11430 (ATCC 76273) by electroporation.

[0202] E. coli TG1 cells (Lucigen, Cat. No. 60502) (containing NANOBODY® ISVD protein expression vector) were grown in baffled shake flasks containing "5052" autoinduction medium at 37°C for 2 hours followed by 29 hours at 30°C (250 rpm). The cells were pelleted by centrifugation (20 min, 4500 rpm, 4°C), the supernatant was discarded and the pellet was frozen overnight at -20°C. The frozen cell pellet was then dissolved in DPBS at 1 / 12.5 of the original culture volume and incubated at 4°C for 1 hour with gentle rotation to disrupt the outer membrane of the cells. The cells were pelleted again (20 min, 8500 rpm, 4°C) and the supernatant containing the NANOBODY® ISVD protein was collected and filtered to proceed immediately with purification.

[0203] FLAG3His6-tagged NANOBODY® ISVD proteins were purified in D-PBS by immobilized metal affinity chromatography (IMAC) on NiIDA / NTA (Genscript) resin with imidazole (former) or acidic elution (latter), followed by a desalting step (PD column with Sephadex G25 resin, GE Healthcare) and, if necessary, preparative size-exclusion chromatography (SEC) (Superdex 75 column, GE Healthcare). For this purpose, a robot station or an AKTA purification system was used.

[0204] Constructs containing tagless NANOBODY® ISVD proteins or ALB components were purified on Amsphere A3 (JSR) or MabCaptureA (Poros) resins followed by a desalting step (PD column with Sephadex G25 resin, GE Healthcare) and, if necessary, preparative SEC (Superdex 75 column, GE Healthcare) in D-PBS. N-octyl-β-d-glucopyranoside (OGP; Alpha Aesar, Cat. No. J67390) treatment was performed during purification / gel filtration chromatography whenever low LPS levels were required. Concentrations were determined by OD280 / OD340 measurements. Quality control was performed by SDS-PAGE and mass spectrometry.

[0205] 6.2 Example 2: Multispecific ISVD Construct Binding Affinities to TCRαβ, CD33, CD123 and Serum Albumin A TCRαβ-CD33-CD123 multispecific ISVD construct was generated as shown in SEQ ID NO:1.

[0206] 6.2.1 Affinity Determination for Human and Cynomolgus Monkey CD33 and CD123 Proteins Association rate constants (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D Affinities, expressed as affinity per unit time (A / A), were measured at 37° C. by a surface plasmon resonance (SPR)-based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.).

[0207] Setup for measuring binding affinity to CD33 Anti-huIgG(Fc) was immobilized on a GLH (long matrix, high capacity) sensor chip via amine coupling using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide ester) chemistry. Then, huCD33L-Fc and cyCD33L-Fc were captured (association: 120 s, 25 μL / min). Purified TCRαβ-CD33-CD123 multispecific ISVD constructs shown in SEQ ID NO:1 were injected at different concentrations (0.4 nM to 625 nM) for 120 s and dissociation was followed for 900 s.

[0208] Setup for measuring binding affinity to CD123 Anti-huIgG(Fc) was immobilized on a GLH (long matrix, high capacity) sensor chip via amine coupling using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide ester) chemistry. Then, huCD123-Fc and cyCD123-Fc were captured (association: 120 s, 25 μL / min). Purified TCRαβ-CD33-CD123 multispecific ISVD constructs shown in SEQ ID NO:1 were injected at different concentrations (1.2 nM to 300 nM) for 120 s and dissociation was followed for 900 s at 45 μl / min.

[0209] Data were double-referenced by subtracting the reference analyte lane and blank buffer injections. The affinity constant (k a , k d , K D ) was calculated applying the Langmuir 1:1 interaction model using ProteOn Manager 3.1.0 (version 3.1.0.6) software.

[0210] The results of affinity measurements of the TCRαβ-CD33-CD123 multispecific ISVD construct set forth in SEQ ID NO: 1 for human and cynomolgus CD33 and human and cynomolgus CD123 are summarized in Tables 2 and 3 below.

[0211] [Table 10]

[0212] Cynomolgus monkey CD33(K D = 6.9 nM) was observed in the reactivity of the TCRαβ-CD33-CD123 multispecific ISVD construct against human CD33 (K D =7.6nM).

[0213] [Table 11]

[0214] Cynomolgus monkey CD123(K D = 2 nM) was observed in the reactivity of the TCRαβ-CD33-CD123 multispecific ISVD construct against human CD123 (K D = 0.59 nM), which was 3.3 times lower.

[0215] The results (Tables 2 and 3) demonstrate that the multispecific ISVD constructs bind with high affinity to human / cynomolgus CD33 and human / cynomolgus CD123.

[0216] 6.2.2 Affinity Determination for Human and Cynomolgus TCR αβ Proteins The association rate constant (k) of the TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) to recombinant huTCR(2XN9)-zipper, cyTCR(AEA41865)-zipper (with coating configuration) a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D The affinity, expressed as affinity per unit area (A), was evaluated at 37° C. by an SPR-based assay based on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.).

[0217] Setup for measuring binding affinity to TCRαβ huTCR(2XN9)-zipper, cyTCR(AEA41865)-zipper proteins were coated onto a GLC (short matrix, normal volume) sensor chip. Purified TCRαβ-CD33-CD123 multispecific ISVD construct shown in SEQ ID NO:1 was injected at different concentrations (0.4 nM to 625 nM) for 120 s and dissociation was followed for 900 s.

[0218] Data were double-referenced by subtracting the reference analyte lane and blank buffer injections. The affinity constant (k a , k d , K D ) was calculated applying the Langmuir 1:1 interaction model using ProteOn Manager 3.1.0 (version 3.1.0.6) software.

[0219] The results of affinity measurements of the TCRαβ-CD33-CD123 multispecific ISVD construct shown in SEQ ID NO: 1 for human and cynomolgus TCRαβ are summarized below in Table 4. As a reference, an ISVD consisting only of the TCRαβ component (SEQ ID NO: 2) linked to ALB23002 (SEQ ID NO: 5) was taken.

[0220] [Table 12]

[0221] Cynomolgus monkey TCRαβ(K D = 6.7 nM) of the TCRαβ-CD33-CD123 multispecific ISVD construct against human TCRαβ (K D = 21 nM).

[0222] The results (Table 4) demonstrate that the multispecific ISVD constructs bind with high affinity to human / cynomolgus TCR αβ.

[0223] 6.2.3 Affinity determination for human, mouse and cynomolgus monkey serum albumin Binding rate constants (k a ), dissociation rate constant (k d ) and the equilibrium dissociation constant (K D Binding affinities (according to the coating setup), expressed as affinity (%), were assessed at 37° C. by an SPR-based assay based on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.).

[0224] Setup for measuring serum albumin binding affinity Human, cynomolgus and mouse serum albumin were immobilized on a ProteOn GLC sensor chip using amine coupling with EDC and NHS chemistry (running buffer used: HBS-EP+, pH 7.4). Albumin was immobilized at two concentrations, 2.5 μg / mL (HSA and MSA) and 5 μg / mL (CSA) in pH 4.5 acetate buffer, resulting in immobilization levels of up to 220 RU for CSA, 150 RU for MSA and 110 RU for HSA. Purified multivalent V HH was injected at different concentrations (4.3 nM to 416 nM) for 2 min (flow rate 45 μL / min) and dissociation was followed for 900 s. Regeneration between cycles consisted of injecting 10 mM glycine-HCL, pH 1.5 at 100 μL / min for 47 s.

[0225] Data were double-referenced by subtracting the reference ligand lane and buffer injections. The ProteOn Manager 3.1.0 (version 3.1.0.6) software model was used to evaluate the treatment curves by fitting with a Langmuir 1:1 interaction model, and affinity constants (k a , k d , K D ) was calculated.

[0226] The results of affinity measurements of the TCRαβ-CD33-CD123 multispecific ISVD construct shown in SEQ ID NO:1 against human, mouse and cynomolgus serum albumin are summarized in Table 5 below.

[0227] [Table 13]

[0228] Cross-reactivity to CSA was confirmed. Furthermore, although kinetic parameters were not reported, the affinity for MSA was good enough to obtain half-life extension and adopt the half-life of serum albumin.

[0229] The results (Table 5) demonstrate that the multispecific ISVD constructs bind with high affinity to human / mouse / cynomolgus TCR αβ.

[0230] 6.3 Example 3: Multispecific ISVD Construct Binding Affinity to Membrane-bound CD33 and / or CD123 Setup for measuring binding affinity to CD33 / CD123 on target cell lines The binding affinity of the TCRαβ-CD33-CD123 multispecific ISVD constructs to target cell lines expressing CD33 and / or CD123 was assessed using flow cytometry. The target cell lines expressing CD123 are described in detail in WO 2018 / 091606 A1.

[0231] Transfected CD33 cells were generated as follows: Stable CHO Flp-In (Invitrogen, R758-07) cell lines with recombinant overexpression of CD33 were generated using Flp-In (Flp-In™ System for generating stable mammalian expression cell lines by Flp recombinase-mediated integration (Invitrogen, K601001, K601002)) site-specific recombination technology, which allows DNA integration at the specific genomic location of the FRT (Flp Recombination Target) site by Flp recombinase (pOG44) from Saccharomyces cerevisiae. Both the Flp-In™ host cell line and the expression plasmid (pcDNA5) contain this FRT site, thereby allowing single homologous DNA recombination. The sequence of human CD33 was derived from NCBI RefSeq NP_001763, and the sequence of cynomolgus CD123 was derived from NCBI genbank no. XP_005590138.

[0232] Briefly, cells were harvested and transferred to V-bottom 96-well plates (5 × 10 4 Cells were then incubated with serial dilutions of TCRαβ-CD123-CD33 multispecific ISVD constructs for 30 min at 4° C. in FACS buffer (D-PBS (Gibco, 14190) containing 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros Organics, 19038)) in a final volume of 100 μL in the presence of 30 μM clinical grade HSA (CSL Behring, 2160-679). Cells were then washed three times with FACS buffer and incubated for 30 min at 4° C. with 1 μg / mL mouse monoclonal anti-FLAG® M2 antibody (Sigma-Aldrich, F1804) for detection of FLAG3His6-tagged CD123-CD33-TCR multispecific ISVD constructs or with 3 μg / mL mAb anti-V for detection of NANOBODY® ISVD by ALB BB. HHThe cells were then incubated with antibody (ABH0077) (APS+ in-house, A-0006-00_ABH0077_SF_AB1891). Cells were then washed three times with FACS buffer and incubated with 5 μg / mL Allophycocyanin (APC) AffiniPure Goat Anti-Mouse IgG (Subclasses 1+2a+2b+3), Fcγ Fragment Specific (Jackson Immunoresearch, 115-136-071) in a final volume of 100 μL for 30 min at 4 °C. Cells were then resuspended in 50 μL of cold FACS buffer supplemented with 1 μg / mL propidium iodide (PI, Sigma P4170) to distinguish between live and dead cells. After staining, cells were acquired using a MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, to distinguish cells from debris, we selected the P1 population, which represents >80% of all events, based on FSC-SSC distribution. From this population (P1), we excluded PI-positive (dead) cells and assessed the median APC fluorescence intensity of PI-negative cells.

[0233] Setup for measuring binding affinity to primary T cells The binding affinity of the TCRαβ-CD33-CD123 multispecific ISVD constructs to primary T cells was assessed using flow cytometry in a competitive setting using FLAG3His6-tagged monovalent TCR ISVD as the ligand.

[0234] Briefly, human primary T cells or cynomolgus primary T cells were thawed and plated in a V-bottom 96-well plate (7.5 x 10 cells in 100 µL). 4Cells were transferred to the wells (cells / well) and incubated with serial dilutions of TCRαβ-CD33-CD123 multispecific ISVD constructs and a fixed concentration of ligand in FACS buffer (D-PBS (Gibco, 14190) containing 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros Organics, 19038)) in the presence of 30 μM clinical grade HSA CSL Behring, 2160-679 for 30 min at 4 °C in a final volume of 100 μL. The concentration of ligand used in the assay was determined based on its binding EC 50 After a 90 minute incubation period at 4°C, the level of ligand binding was determined by flow cytometry. There, cells were washed three times, incubated with 1 μg / ml mouse monoclonal ANTI-FLAG® M2 antibody (Sigma-Aldrich, F1804) for 30 minutes at 4°C, washed again, and incubated with 5 μg / ml allophycocyanin (APC) AffiniPure goat anti-mouse IgG (subclasses 1+2a+2b+3), Fcγ fragment specific (Jackson Immunoresearch, 115-136-071) in a final volume of 100 μL for 30 minutes at 4°C. Cells were then resuspended in FACS buffer supplemented with 1 μg / ml propidium iodide (PI, Sigma, P4170) to distinguish between live and dead cells. After staining, cells were acquired using a MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, to distinguish cells from debris, the P1 population was selected, representing >80% of all events, based on FSC-SSC distribution. From this population (P1), PI-positive (dead) cells were excluded and the median APC fluorescence intensity of PI-negative cells was evaluated.

[0235] Human-cynomolgus cross-reactivity was assessed by testing the binding of monovalent CD33 components (SEQ ID NO:3) and monovalent CD123 components (SEQ ID NO:4) to human or cynomolgus CD33 or human or cynomolgus CD123 transfected cell lines using flow cytometry as described above. The results are graphically represented in Figure 2. EC of different experiments 50 The values ​​are summarized in Table 6 (CD33 transfected target cells) and Table 7 (CD123 transfected target cells).

[0236] [Table 14]

[0237] [Table 15]

[0238] Binding of monovalent CD33 and CD123 building blocks to human and cynomolgus membrane targets was confirmed. 50 The difference was less than a two-fold decrease in binding to CD33 and a three-fold decrease in binding to CD123 cells (Table 7).

[0239] In conclusion, in addition to the binding affinity to recombinant human and cynomolgus CD33 and CD123 proteins, dose-dependent binding of the TCRαβ-CD33-CD123 multispecific ISVD construct to human and cynomolgus cell-expressed CD33 and CD123 was also confirmed (n=1).

[0240] Binding of the CD123-CD33-TCR multispecific ISVD construct and the reference TCR-ISVD construct (consisting only of the TCR αβ component (SEQ ID NO: 2) linked to ALB23002 (SEQ ID NO: 5)) to human and cynomolgus T cells was assessed in a competitive setting using flow cytometry as described above (Figure 3).

[0241] An illustrative example of a DRC is shown in Figure 4. ISVD constructs were tested in multiple healthy donor T cells. Global IC 50 is shown in Table 8.

[0242] [Table 16]

[0243] In summary, cross-reactivity of the TCRαβ-CD33-CD123 multispecific ISVD construct to primary cynomolgus T cells was confirmed. 50 (=729 nM) is the EC 50 (=218M), which was about three times higher.

[0244] 6.4 Example 4: Multispecific ISVD construct-induced T cell-mediated target cell killing 6.4.1 Impedance-Based Cytotoxicity Assay ISVD constructs were characterized for redirected T cell-mediated killing in an impedance-based cytotoxicity assay using primary human or cynomolgus monkey effector T cells and adherent target cells (e.g., as described in WO2018091606A1). An xCELLigence device (Roche) was used to measure the change in impedance induced by the adhesion of target cells to the electrode surface. T cells are non-adherent and therefore do not affect the impedance measurements. The xCELLigence® RTCA MP instrument quantifies the change in electrical impedance and displays it as a dimensionless parameter called the cell index, which is directly proportional to the total area of ​​the tissue culture well covered by the cells. To each well of a 96 E-plate (ACEA Biosciences; 05 232 368 001) in assay medium (target cell growth medium (without selective antibiotics) + 1% penicillin / streptomycin (Life technologies catalogue no. 15140) was added 50 μL of 4x concentrated HSA solution (in some assays 200 μM was used to have a final concentration of 50 μM, in others 120 μM was used to have a final concentration of 30 μM). The outer wells were not used and were filled with 200 μL of medium or D-PBS. The 96 E-plate was placed in a xCELLigence® station (in a 37°C incubator, 5% CO2) and a single measurement was taken to measure the background impedance of the assay medium in the absence of cells. Subsequently, 50 μL of target cells (2 × 10 4 10 cells / well) were seeded into a 96 E-plate and 50 μL of serially diluted ISVD construct solution (4X concentration) in assay medium was added. (Final volume=200 μL). After 30 minutes at room temperature, 50 μL of primary T cells (3×105 cells / well) in assay medium were added per well to achieve an effector to target ratio of 15:1. The plate was placed in the xCELLigence® station and impedance was measured every 15 minutes for 4 days. Data was analyzed at fixed time points indicated in Results.

[0245] 6.4.2 Flow cytometry-based cytotoxicity assays ISVD constructs were characterized for redirected T cell-mediated killing in a flow cytometry-based cytotoxicity assay using human or cynomolgus primary T cells as effector cells and non-adherent target cells. Target cells were labeled with 4 μM PKH26 membrane dye using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions. Effector cells (2.5 × 10 5 cells / well) and PKH-labeled target cells (2.5 × 10 4 Cells / well) were co-incubated in 96-well V-bottom plates (Greiner Bio-one, #651 180) (effector to target ratio 10:1) in assay medium of the target cell line (target growth medium containing 1% penicillin / streptomycin (Life Technologies, 15140) and 50 μM Al-Brex HSA (CSL Behring, 2160-679)). For analysis of concentration-dependent cell lysis, serial dilutions of ISVD constructs in target assay medium were added to the cells and incubated for 18 h at 37 °C in a 5% CO2 atmosphere. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (D-PBS (Gibco, 14190) containing 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros Organics, 19038)). Cells were then resuspended in 100 μL of FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642-661) (ThermoFisher Scientific, T3605) to distinguish between live and dead cells. Cells were analyzed using a MACSQuant X flow cytometer (Miltenyi Biotec). A total sample volume of 70 μL was acquired per sample. Gating was set on PKH26 positive cells, and TO-PRO®-3 positive cells were determined within this population. Percent specific lysis = ((% TO-PRO-3+ no construct - % TO-PRO-3+ with construct) / % TO-PRO-3+ no construct) × 100.

[0246] Functional cross-reactivity analysis of the TCRαβ-CD33-CD123 multispecific ISVD constructs against CD33, CD123 and TCR was determined in an impedance-based cytotoxicity assay (xCELLigence) using primary human or cynomolgus monkey T cells and adherent human or cynomolgus monkey transfected CD33 or CD123 cells.

[0247] To fully saturate the NANOBODY® ISVD with HSA as described above, all assays were performed in the presence of excess HSA, as described above in 6.4.1 and 6.4.2. A reference TCR-ISVD was used as a negative control. The results are shown in Figures 5 and 6 and Table 9.

[0248] 6.4.3 Results The results of the assay comparing human primary T cells with primary cynomolgus monkey T cells are shown graphically in Figures 5 and 7. Evaluation of human T cell-mediated cell killing of human CD33 and human CD123 transfected cells was performed using T cells from different human donors, demonstrating the global IC 50 Values ​​could be calculated (Table 9). Evaluation of cynomolgus T cell-mediated killing of human CD33 and human CD123 transfected cells was performed using T cells from one cynomolgus monkey. IC 50 The values ​​are summarized in Table 10.

[0249] [Table 17]

[0250] Global IC for human target expressing cells 50 were 5 and 4.10 for CD33 and CD123, respectively. -11 M and 2, 5.10 -11 It was M.

[0251] [Table 18]

[0252] To confirm human-cynomolgus cross-reactivity of the TCRαβ-CD33-CD123 ISVD constructs for TCR, the ISVD constructs were evaluated in flow cytometry-based T cell-mediated MOLM-13 cell killing using either human or primary T cells in combination with the CD33, CD123 dual-expressing human MOLM-13 target cell line in the presence of 50 μM HSA as described above. A graphical representation of these results is shown in Figure 6. Further data are available for the TCRαβ-CD33-CD123 ISVD constructs tested in human T cell-mediated killing assays using different donors, which will allow for the identification of global EC 50 Values ​​could be calculated (Table 11). EC 50 The values ​​are shown in Table 12.

[0253] [Table 19]

[0254] [Table 20]

[0255] In conclusion, the TCRαβ-CD33-CD123 ISVD construct was functional in both human and cynomolgus human target cell-mediated killing assays. The global killing potency of the described ISVD against CD33 / CD123 double positive AML cell lines was 1, 8, 10 -11 It was M.

[0256] 6.5 Example 5: CD123 Engrafted into T Cell-Humanized NSG Mice + Preclinical in vivo efficacy of TCRαβ-CD33-CD123 multispecific ISVD constructs in the Molm-13-luc disseminated AML model 6.5.1 Materials and Methods Cell lines and human materials Human AML-derived cell lines expressing CD123 Molm-13 were obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany). Molm-13 cells were grown in culture in RPMI1640 Glutamax medium (complete with 20% fetal bovine serum) at 37°C, 5% CO2, 95% humidity. Cells were infected with a luciferase vector carried by a non-replicating lentivirus (SV40-PGL4-Puro). Polyclonal Molm-13-luc was selected using 2 μg / ml puromycin.

[0257] Purification and expansion of human T cells for in vivo administration Fresh human peripheral blood from healthy donors was provided by the EFS (Etablissement Francais du Sang, Ile-de-France, France).

[0258] Fresh human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation at 200 g for 40 min at room temperature without brake. The pellet was washed and resuspended in a final volume of 50 ml completed with phosphate-buffered saline (PBS). Total viable PBMC numbers were determined by Vi-CELL counter (Beckman Coulter Life Sciences, Brea, CA, USA). The pellet was collected in autoMACS Running Buffer (Miltenyi Biotec). T cells were isolated from PBMCs using the Pan T Cell Isolation Kit (Miltenyi Biotec) and autoMACS according to the manufacturer's instructions. Purified T cells were activated and expanded in vitro for 14 days using the T Cell TRANSACT matrix activation / expansion kit (Miltenyi Biotec) based on CD3 and CD28 co-stimulation. According to the Miltenyi procedure, the activation protocol involved culturing T cells for 2 weeks in the presence of TRANSACT matrix in TexMACS medium (Miltenyi Biotec) supplemented with 20 000 IU soluble IL-2 and 1% penicillin-streptomycin (Gibco). On day 14 of expansion, T cells were harvested and cultured at 5 × 10 7 Resuspend in PBS at a final concentration of 10 cells / ml. 7 Cells were administered to each animal via intraperitoneal (IP) injection. T cell viability prior to animal injection was tested to be greater than 85%.

[0259] In vivo characterization All in vivo experiments were approved by the Sanofi Ethics Committee and were performed in accordance with local and institutional laws, ethics and guidance in an AAALAC accredited facility.

[0260] Antitumor activity of the TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) was assessed in non-irradiated NOD.Cg-Prkdc scid Il2rg tm1WjlThe engraftment of Molm13-luc AML in 6- to 8-week-old female animals was evaluated after immunization with 10 6 Molm-13-luc cells were implanted intravenously (IV) at 0.2 ml / mouse. 7 Human T cells were intraperitoneally transplanted at 0.2 ml per day.

[0261] Animals were distributed among groups based on tumor bone marrow engraftment assessed by whole body bioluminescence imaging (BLI) signal uniformity and long bone signal segmentation on day 3. Mice were treated IV with TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) at 12, 1.2, 0.12 and 0.012 nmol / kg Q2D from days 4 to 12, or with a reference without CD33 or CD123 binding ISVD (TCRαβ-ISVD (SEQ ID NO: 2) linked to Alb-ISVD (SEQ ID NO: 5)) at 1.2 nmol / kg QD (days 4-13) or CD123 / CD3 positive control at 1.3 nmol / kg QD (days 4-13), see Table 13. Longitudinal in vivo bioluminescence imaging (BLI) was performed to monitor disseminated tumor growth. Mice were sacrificed on day 14 and necropsied under BLI for impact assessment in deep soft tissues such as liver, spleen, ovaries and abdominal fat.

[0262] [Table 21]

[0263] Data Collection and Validity Criteria Animal body weights were monitored from day 3 until the end of the assay to follow the effects of treatment. Doses resulting in 20% or 15% body weight loss or 10% or more drug deaths for three consecutive days were considered excessively toxic doses. Animal body weights included tumor weights.

[0264] Tumor growth was assessed by in vivo BLI using an IVIS Lumina XRMS imager (PerkinElmer, Waltham, MA, USA) with Living Image 4.5.2 acquisition software (PerkinElmer) via luciferase activity measurements in vivo at days 3, 7, 10, and 14 after tumor injection using beetle luciferin potassium salt 160 mg / kg Ip injection 15 min prior to imaging in animals anesthetized with Ketamine® / Xylazine® (120 mg / kg; 6 mg / kg IM, 5 ml / kg) 5 min prior to imaging. Tumor growth was based on bioluminescence signal curves (expressed in photons / second).

[0265] Tumor growth was followed in the whole body and in the long bones of the hind limbs by BLI signal measurements on days 7, 10 and 14 after tumor implantation. The primary efficacy endpoints were the ratio of change in tumor signal change from baseline between the treatment and control groups (T / C), partial regression (PR) and complete regression (CR).

[0266] Tumor growth based on bioluminescence signal curves (expressed in Phot / sec) was plotted over time for each animal in each treatment group and expressed as median curve ± MAD for both whole body (linear scale) and bone segmentation signals (Log scale). Changes in tumor bioluminescence signal for each treatment (T) and control (C) are calculated for each animal and each day by subtracting the tumor signal on the first treatment day (staging day) from the tumor signal on the designated observation day. Median T is calculated for the treatment group and median C is calculated for the control group. The ratio T / C is then calculated and expressed as a percentage. dT / dC = [(median T end value - median T day3) / (median C end value - median C day3)] x 100

[0267] A dose is considered therapeutically active if dT / dC is less than 42% and highly active if dT / dC is less than 10%. If dT / dC is less than 0, the dose is considered highly active and the percentage of regression is dated.

[0268] Tumor regression rate is defined as the % of tumor signal reduction in a treatment group on a particular observation day compared to the signal on day 1 of treatment.

[0269] Calculate the % regression for each animal at specific time points. Considering the risk of signal variation due to luciferin kinetic fluctuations due to ip injection errors, consider true regression if observed at least two consecutive time points for each animal.

number

[0270] Partial Regression (PR): Regression is defined as partial if the tumor signal decreases below the tumor signal at the start of treatment at two consecutive time points, one of which is less than 50% of the starting signal. Complete Regression (CR): Regression is defined as complete if the tumor signal decreases below 80% of the starting signal.

[0271] Biostatistical analysis Tumor growth based on bioluminescence signal curves over time was measured for each animal in each treatment group. For longitudinal in vivo BLI data, two-way nonparametric analysis of variance (ANOVA type) followed by two-contrast analysis with Bonferroni-Holm adjustment for multiplicity was performed for daily repeated measures: p>0.05: NS, 0.05 0.01: *, p<0.01: **. For final ex vivo BLI data, one-way ANOVA with factor group on rank-transformed bioluminescence signals was performed. Descriptive statistics with median ± median absolute deviation are provided by group and measurement date: p>0.05: NS, 0.05 0.01:*,p<0.01:**.

[0272] 6.5.2 Results The TCRαβ-CD33-CD123 multispecific ISVD construct induces anti-leukemic effects in a Molm-13-luc AML xenograft model in vivo (Figure 8A).

[0273] In the presence of human effector T cells (T cell / tumor ratio R=10), TCRαβ-CD33-CD123 multispecific ISVD constructs administered intravenously every 2 days in a Molm-13-luc xenograft model were well tolerated at all doses. No evidence of adverse events or changes in body weight were observed under treatment. TCRαβ-CD33-CD123 multispecific ISVD constructs inhibited systemic tumor growth with the same activity at all doses tested (dT / dC of 2% (p<0.0001) vs. 2% (p<0.0001), 2% (p<0.0001) and 3% (p<0.0001) at 0,012; 0,12; 12 and 12 nmol / kg, respectively) (Figures 8A, 8B).

[0274] The sum of the longest diameter (LD) of all target lesions was the baseline sum LD, which was used as a reference to characterize objective tumor response.

[0275] In long bones, 3 / 8 complete responses (CR; disappearance of all lesions) and 1 / 8 partial response (PR; at least a 30% reduction in the sum of the LD of target lesions, taking the baseline sum LD as reference) were observed at 0.012 nmol / kg.

[0276] 3 / 8 CR and 1 / 8 PR were observed at 0.12 nmol / kg, 4 / 8 CR and 3 / 8 PR at 1.2 nmol / kg, and 3 / 7 CR and 3 / 7 PR at 12 nmol / kg (Figure 8C). The reference TCR-ISVD construct was totally inactive against Molm13-luc tumor growth at 1.2 nmol / kg (80% dT / dC). The CD123 / CD3 positive control 1, 3 nmol / kg inhibited tumor growth at dT / dC 8%, with 4 / 8 CR in long bones (NS treated with A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO: 1)) (Figures 8A, 8B).

[0277] Based on final ex vivo bioluminescence imaging, the TCRαβ-CD33-CD123 multispecific ISVD construct significantly inhibited tumor growth in the liver (p<0.0001), spleen (p<0.0001) and ovaries (p<0.0001), but not in abdominal fat at all doses tested, whereas the reference ISVD TCR-HLE was inactive in all tissues and the CD123 / CD3 positive control significantly inhibited tumor burden in the liver (p<0.0001) and spleen (p<0.0001), but not in the ovaries (NS) or abdominal adipose tissue (Figure 9).

[0278] 6.6 Example 6: T Cell-Mediated Human Target Cell Killing 6.6.1 Materials and Methods ISVD constructs were characterized for redirected T cell-mediated killing in a flow cytometry-based cytotoxicity assay using human primary T cells as effector cells and non-adherent target cells. CD123 and / or CD33 positive target cells (MOLM-13, DSMZ ACC 554, U-937, ATCC® CRL1593.2, and KG-1a, ATCC® CCL246.1™) were labeled with 4 μM PKH-26 membrane dye using the PKH26 Red Fluorescent Cell Linker Kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions. Effector cells (2.5 × 10 5 cells / well) and PKH-labeled target cells (2.5 × 10 4 Cells / well) were co-incubated in 96-well V-bottom plates (Greiner Bio-one, #651 180) (effector to target ratio 10:1) in the assay medium of the target cell line (target growth medium without antibiotics). For analysis of concentration-dependent cell lysis, serial dilutions of compounds in target assay medium were added to the cells and incubated for 18 hours at 37°C under a 5% CO2 atmosphere. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (Gibco's D-PBS with 10% FBS from Sigma and 0.05% sodium azide from Merck). Cells were then resuspended in FACS buffer supplemented with 5 nM TO-PRO®-3 iodide (642-661) (ThermoFisher Scientific, T3605) to distinguish between live and dead cells. Cells were analyzed using a FACS Array flow cytometer (BD Biosciences). A total sample volume of 80 μL was acquired per sample. Gating was set on PKH26 positive cells and TO-PRO®-3 positive cells were determined within this population. Percent specific lysis = ((% TO-PRO®-3+ ISVDなし -%TO-PRO(R)-3+ ISVDあり ) / (%TO-PRO(R)-3+ ISVDなし )) x 100.

[0279] The multispecific TCRαβ-CD33-CD123 ISVD constructs according to the invention were compared with the corresponding constructs in which either the CD33-binding ISVD or the CD123-binding ISVD was replaced by an irrelevant ISVD IRR (does not bind to CD33, does not bind to CD123; Table 23) as well as a CD123 / CD3 positive control and a CD33 / CD3 positive control. The results are shown in Figures 10, 11 and 12.

[0280] 6.6.2 Results As seen in Figure 10, in MOLM-13 cells, all compounds tested induced tumor cell killing, which is expected since MOLM-13 cells are positive for both CD123 and CD33. Among the ISVD constructs, the dual targeting format (CD33 / CD123 TCE) had the most potent tumor cell killing.

[0281] The killing efficacy and percent lysis in U-937 and KG-1a cells are shown in Table 24 and in Figures 11 and 12.

[0282] [Table 22]

[0283] [Table 23]

[0284] The TCR-CD123 single-targeting ISVD induced little killing of CD123-U-937 cells, as was also observed for the CD123 / CD3 positive control. Similarly, the CD33 single-targeting ISVD induced only low levels of killing of CD33- / +KG-1a cells, as was also observed for the CD33 / CD3 positive control. On the other hand, the dual-targeting TCRαβ-CD33-CD123 ISVD (construct A) showed potent tumor cell killing in both CD33- and CD123- cell lines, thus illustrating the advantage of the dual-targeting approach.

[0285] 6.7 Example 7: Cytokine Release Assay Since cytokine release syndrome (CRS) is a known side effect of T cell engagers, the cytokine release profile of the TCRαβ-CD33-CD123 multispecific ISVD construct according to the invention was determined in an autologous healthy donor PBMC assay. In parallel, autologous depletion of monocytes in human PBMCs was evaluated. The functionality of the ISVD according to the invention was compared with two tool molecules: a CD123 / CD3 positive control and a CD33 / CD3 positive control. A non-targeting T cell engager (TCE) ISVD was used as a negative control.

[0286] 6.7.1 Materials and Methods A total of 200,000 PBMCs (100 μL at 2×106 cells / mL in culture medium) isolated from whole blood using Leucosep™ tubes containing Lymphoprep™ solution were transferred to a 96-well V-bottom clear well plate (Greiner CELLSTAR® 96-well plate; 651:180).

[0287] Then, 50 μL of serial dilutions of the compounds to be tested or 50 μL of culture medium for blank wells were added to the wells.

[0288] Culture plates containing treated PBMCs were incubated at 37°C, 5% CO2 for 20 hours. After overnight (20 hours) incubation, PBMCs were centrifuged at 300g for 2 minutes. Supernatants were collected, transferred to new 96-well storage plates, and frozen at -20°C for cytokine measurements. Cell pellets were suspended in 100 μL cold FACS buffer and washed once with 100 μL FACS buffer.

[0289] The PBMCs were then centrifuged at 300 g for 2 minutes at 4° C. The supernatant was discarded and the cells were resuspended in 30 μL of diluted Fc block (1 / 200 in FACS buffer) (BD, 564220) and incubated for 10 minutes at room temperature.

[0290] Next, 30 μL (2X) antibody staining mix (CD123, HLA-DR and CD14 antibodies) was added to the PBMC suspension and incubated for 30 minutes at 4° C. in the dark.

[0291] The cell suspension was then washed twice and resuspended in 50 μL of diluted TO-PRO™-3 iodide. The plates were read on a MACSQuantX. Readings included detection of viable cell numbers of each subset by flow cytometry. Monocytes were quantified by SSC%CD14+ (Figure 13).

[0292] Frozen supernatants from overnight incubations of human PBMCs were used to measure a range of cytokines including IL-2, IL-6, IFNγ, and TNFα using multiplex bead assays from Bio-Rad. Assays were performed according to the manufacturer's guidelines. Data from the reactions were acquired using a Luminex FlexMAP 3D system (Figure 14).

[0293] 6.7.2 Results The TCRαβ-CD33-CD123 multispecific ISVD construct induced monocyte depletion associated with cytokine production in all donors. Non-targeted TCE showed no killing or cytokine release (see FIG. 13). The data shown in FIG. 13 is from one donor and represents data from all six donors who provided PBMC.

[0294] Furthermore, both the potency and maximum levels of cytokines induced by the ISVD constructs according to the invention were substantially lower compared to the CD123 / CD3 control compound. IL-6 and TNFα production induced by the ISVD constructs according to the invention was comparable compared to the CD33 / CD3 control. Although IL-2 and IFNγ levels were higher than the CD33 / CD3 control, the levels were still acceptable.

[0295] On this basis it can be concluded that the ISVD constructs according to the invention do not pose any greater risk of inducing CRS than other CD123 or CD33 targeted compounds and should be safe for use in humans.

[0296] 6.8 Example 8: Ex vivo primary AML cell killing assay 6.8.1 Materials and Methods Lysis of AML blasts mediated by CD33 / CD3 positive controls was tested as follows: AML samples from patients with primary diagnosis or relapse were used in an ex vivo co-culture system without the addition of other cells. The E:T ratio was therefore determined by the number of remaining T cells within the primary AML sample. Whole blood samples from AML patients were provided by the public hospitals or central laboratories of Marseille (La Conception, AP-HM) or Montpellie.

[0297] First, 1 mL of whole blood sample was distributed per 50 mL tube, 40 mL (1x) red blood cell lysis buffer was added and incubated for 10 min at room temperature. After incubation, the pellet was washed with PBS (Eurobio CS1PBS01-01) and resuspended in 1 mL of culture medium (RPMI (Eurobio Cat. No. CM1RPM00-01) FCS 10% (Sigma Cat. No. F2442-500ml 17L484), non-essential amino acids 1% (Eurobio Cat. No. CSTAAN00), glutamine 1% (Eurobio Cat. No. CSTGLU00), sodium pyruvate 1% (Eurobio Cat. No. CSTVAT00), penicillin / streptomycin, (Eurobio Cat. No. CABPES01)).

[0298] Then, 300 μL of AML blast cells were added to 700 μL of culture medium per well in a 6-well plate in the presence of 1 mL (2×) of a saturating concentration of TCRαβ-CD33-CD123 multispecific ISVD construct according to the invention (CD33 / CD123 TCE), positive control (CD33 / CD3 or CD123 / CD3) or negative control (non-targeted TCE) and incubated at 37° C. with 5% CO2. After 4 days of incubation, the cells were harvested and washed once with PBS.

[0299] Zombie Violet viability marker (1 / 100 dilution in PBS) was then added to the cell pellet and incubated for 5 minutes at room temperature in the dark.

[0300] Cells were then washed and stained with an antibody cocktail (CD45, CD33, CD34, CD38, CD14, CD123, CD11 antibodies) and incubated on ice for 10 min.

[0301] The cells were then washed and fixed with 1.5 mL of PBS + 2% paraformaldehyde for 30 minutes at 4°C. After 30 minutes of incubation, the paraformaldehyde was diluted with 9 mL of diluent. The data was analyzed on a Cytoflex cytometer. The results are shown in Figures 15-17.

[0302] 6.8.2 Results The percentage of CD33 or CD123 positive cells per AML sample is shown in Figure 16. As can be seen, all patients had high numbers of both CD33 and CD123 positive cells. Furthermore, the AML patients had a wide range of disease subtypes. Therefore, the AML samples were suitable for use in this assay.

[0303] AML blast cell killing in all AML patient samples is shown in Figure 15. Each dot represents the number of surviving blast cells after treatment with one of the ISVDs according to the invention or the positive control normalized to the negative control (non-targeted TCE). Results from several separate patients are highlighted in Figure 17.

[0304] As can be seen, the cell viability of blast cells treated with the ISVD according to the invention was, on average, the lowest compared to the positive control. Cells from all patients showed a clear response, but the response varied from patient to patient. This indicates that the ISVD according to the invention can target cell killing of CD123 and CD33 positive tumor cells.

[0305] 6.9 Example 9: Non-Human Primate (NHP) Studies The pharmacokinetics (PK), pharmacodynamics (PD) and non-clinical safety profile of the TCRαβ-CD33-CD123 multispecific ISVD construct according to the invention were evaluated in a study in cynomolgus monkeys.

[0306] 6.9.1 Materials and Methods The ISVD construct according to the invention was administered to a total of four male cynomolgus monkeys as a single dose of 1-hour continuous intravenous (IV) infusion followed by a 21-day observation period. Systemic exposure and potential toxicity of the ISVD were determined and PD endpoints (immune cell assessment) were evaluated. The study design is shown in Table 25.

[0307] [Table 24]

[0308] 6.9.2 Safety A single 1-hour continuous IV infusion of the ISVD construct to cynomolgus monkeys was well tolerated. No deaths or treatment-related clinical signs occurred during the course of the study. No treatment-related weight or temperature changes were observed.

[0309] Cytokine evaluation showed only a very minimal increase in IL-6 (peak occurring 2 and / or 6 hours after initiation of infusion, returning to baseline within 24 hours) associated with the ISVD construct administered as a single dose at 0.04 μg / kg. No changes in IFN-γ, IL-1β, IL-2, IL-8 and TNF-α levels were observed at 0.04 μg / kg. Very minimal to minimal increases in IL-2, IL-6, IFN-γ and TNF-α (peak occurring 2 and / or 6 hours after initiation of infusion, returning to baseline within 24 hours) were associated with the ISVD construct administered as a single dose at 0.4 μg / kg. No changes associated with the TCR-CD33-CD123 multispecific ISVD construct according to the invention were observed in IL-1β and IL-8 levels in monkeys at 0.4 μg / kg.

[0310] It can therefore be concluded that the ISVD construct according to the invention was well tolerated in this NHP model.

[0311] 6.9.3 Pharmacokinetics After 1-h infusion of the ISVD construct, serum levels could be quantified in monkeys for up to 3 days at 0.04 μg / kg and up to 7 days at 0.4 μg / kg. Overall, a clearance of approximately 0.07 L / day / kg was observed after IV infusion at dose levels of 0.04 μg / kg and 0.4 μg / kg. The terminal elimination half-life is estimated to be close to 1 day. From 0.04 μg / kg to 0.4 μg / kg, ISVD construct exposure (AUC) showed a slightly less pronounced increase than would be expected by dose proportionality, with a 7.7-fold increase in exposure for a 10-fold increase in dose, likely due to the low exposure observed in one monkey (M3). PK parameters are reported in Table 26.

[0312] [Table 25]

[0313] 6.9.4 Pharmacodynamics Single dose IV injection of the ISVD construct into cynomolgus monkeys at 0.04 and 0.4 μg / kg induced changes in CD123+ cell populations and CD33+ monocytes at both dose levels. These changes consisted of the following: - A total decrease in total CD123+ cell counts was observed in all animals starting 6 hours after the start of the infusion. This decrease was maintained in all animals throughout the study. - A total decrease in the number of monocyte CD33+ cells was observed for all animals, starting 6 hours after the start of the infusion (and until day 1 for animal M1 receiving 0.04 μg / kg of the ISVD construct). For animal M3, a complete recovery was observed on day 1.

[0314] Also, a total decrease in CD4+ and CD8+ cell counts was observed in all animals starting 6 hours after the start of the infusion. For CD8+ cells, a complete recovery was observed on day 3, with a rebound effect for animal M4. For CD4+ cells, a complete recovery was observed on day 3 for animal M4.

[0315] The cell numbers are visualized in FIG.

[0316] 7 Industrial Applicability The polypeptides described herein, nucleic acid molecules encoding them, vectors containing the nucleic acids, and compositions can be used, for example, to treat subjects suffering from acute myeloid leukemia.

Claims

1. A polypeptide, said polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), each of said ISVDs comprising three complementarity determining regions (CDR1 to CDR3, respectively), optionally linked via one or more peptide linkers; and a) the first ISVD specifically binds to T cell receptor αβ (TCRαβ); i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 6, or a CDR1 having 2 or 1 amino acid difference from SEQ ID NO: 6; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 10, or a CDR2 having two or one amino acid difference from SEQ ID NO: 10; and iii. A CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or a CDR3 having two or one amino acid difference from SEQ ID NO: 14; b) the second ISVD specifically binds to CD33; iv. a CDR1 comprising the amino acid sequence of SEQ ID NO: 7, or a CDR1 having two or one amino acid difference from SEQ ID NO: 7; v. a CDR2 comprising the amino acid sequence of SEQ ID NO: 11, or a CDR2 having two or one amino acid difference from SEQ ID NO: 11; and vi. A CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or a CDR3 having two or one amino acid difference from SEQ ID NO: 15; and c) the third ISVD specifically binds to CD123; vii. CDR1 comprising the amino acid sequence of SEQ ID NO: 8, or a CDR1 having two or one amino acid difference from SEQ ID NO: 8; viii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 12, or a CDR2 having two or one amino acid difference from SEQ ID NO: 12; and ix. a CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or a CDR3 having 2 or 1 amino acid difference from SEQ ID NO: 16; The order of the ISVDs relative to each other is considered from the N-terminus to the C-terminus of the polypeptide. Relative positions of polypeptides are shown.

2. 2. The polypeptide of claim 1, a. the first ISVD comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:6, a CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a CDR3 comprising the amino acid sequence of SEQ ID NO:14; b. the second ISVD comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:7, a CDR2 comprising the amino acid sequence of SEQ ID NO:11, and a CDR3 comprising the amino acid sequence of SEQ ID NO:15; and c. A polypeptide wherein the third ISVD comprises a CDR1 comprising the amino acid sequence of SEQ ID NO:8, a CDR2 comprising the amino acid sequence of SEQ ID NO:12, and a CDR3 comprising the amino acid sequence of SEQ ID NO:

16.

3. 2. The polypeptide of claim 1, a. the amino acid sequence of the first ISVD comprises greater than 90% sequence identity with SEQ ID NO:2; b. the amino acid sequence of the second ISVD comprises greater than 90% sequence identity with SEQ ID NO:3; and c. A polypeptide wherein the amino acid sequence of said third ISVD comprises a sequence identity of greater than 90% identity to SEQ ID NO:

4.

4. 2. The polypeptide of claim 1, a. the first ISVD comprises the amino acid sequence of SEQ ID NO:2; b. the second ISVD comprises the amino acid sequence of SEQ ID NO:3; and c. A polypeptide wherein said third ISVD comprises the amino acid sequence of SEQ ID NO:

4.

5. 2. The polypeptide of claim 1, wherein the polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, and wherein the one or more other groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide that does not comprise the one or more other groups, residues, moieties or binding units.

6. 6. The polypeptide of claim 5, wherein the one or more other groups, residues, moieties or binding units that confer an increased half-life to the polypeptide are selected from the group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc moieties and small proteins or peptides capable of binding to serum proteins.

7. 6. The polypeptide of claim 5, wherein the one or more other groups, residues, moieties or binding units that confer an increased half-life to the polypeptide are selected from the group consisting of binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

8. 8. The polypeptide of claim 7, wherein the binding unit that provides the polypeptide with an increased half-life is a fourth ISVD capable of binding to human serum albumin.

9. 9. The polypeptide of claim 8, wherein the fourth ISVD that binds human serum albumin is: i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 9, or a CDR1 having 2 or 1 amino acid difference from SEQ ID NO: 9; ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 13, or a CDR2 having two or one amino acid difference from SEQ ID NO: 13; and iii. A polypeptide comprising a CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or a CDR3 having two or one amino acid difference from SEQ ID NO:

17.

10. The polypeptide of claim 8, wherein the ISVD that binds to human serum albumin comprises CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO:

17.

11. The polypeptide of claim 8, wherein the amino acid sequence of the ISVD that binds to human serum albumin has greater than 90% sequence identity with SEQ ID NO:

5.

12. The polypeptide of claim 8, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO:

5.

13. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide has greater than 90% sequence identity with SEQ ID NO:

1.

14. 2. The polypeptide of claim 1, wherein the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

1.

15. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of claim 1.

16. A host or host cell comprising the nucleic acid of claim 15.

17. A method for producing the polypeptide of claim 1, comprising: a. expressing the nucleic acid of claim 15 in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b. Isolating and / or purifying the polypeptide of claim 1. The method includes at least

18. A composition comprising at least one polypeptide according to claim 1 or a nucleic acid according to claim 15.

19. 19. The composition of claim 18, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and / or compounds.

20. 20. A composition according to claim 18 or 19 for use as a medicament.

21. 20. The composition of claim 18 or 19 for use in the treatment of acute myeloid leukemia (AML).

22. 22. The composition for use according to claim 21, wherein the AML is relapsed and / or refractory AML.

23. 20. Use of the composition of claim 18 or 19 in the preparation of a pharmaceutical composition for treating acute myeloid leukemia (AML).

24. 24. The use of the composition of claim 23, wherein the AML is relapsed and / or refractory AML.

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