CN118401560A

CN118401560A - Polypeptides comprising immunoglobulin single variable domains targeting TCRαβ, CD33 and CD123

Info

Publication number: CN118401560A
Application number: CN202280083249.6A
Authority: CN
Inventors: 海伦·博纳沃; 梅丽莎·戴利斯; 安内利斯·罗布鲁克; 斯特凡妮·斯塔埃朗; 迪亚娜·范霍里克; 尤迪特·维尔赫斯特
Original assignee: Ablynx NV; Sanofi Aventis France
Current assignee: Ablynx NV; Sanofi Aventis France
Priority date: 2021-12-17
Filing date: 2022-12-16
Publication date: 2024-07-26

Abstract

The technology of the present invention aims to provide a novel drug for treating subjects with acute myeloid leukemia (AML). Specifically, the technology of the present invention 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 technology of the present invention also provides nucleic acids, vectors and compositions.

Description

Polypeptides comprising immunoglobulin single variable domains targeting TCRαβ, CD33 and CD123

1. Technical field

The present technology relates to polypeptides targeting tcrαβ, CD33 and CD 123. It also relates to nucleic acid molecules encoding these polypeptides and vectors comprising these nucleic acids. The present technology further relates to compositions comprising such polypeptides, nucleic acids or vectors. The present technology also relates to methods of using such compositions in the treatment of subjects with Acute Myeloid Leukemia (AML). Furthermore, the present technology relates to methods of producing these compositions.

2. Background art

Treatment of Acute Myeloid Leukemia (AML) has remained challenging to date. Since cytotoxic T cells show the highest potential for treating malignant diseases among all immune cells, AML treatment methods aim to direct cytotoxic T cells to AML cells. CD33 and CD123 antigens were found to be overexpressed on blast cells and leukemia stem cells in most AML cases and thus were used as suitable tumor-associated target antigens for antibody-based therapies. Mylotarg (Gituzumab Orzomib; an anti-CD 33 antibody drug conjugate) was the first targeting compound registered for the treatment of AML. Recent therapies are based on bispecific antibody constructs targeting the tumor antigen CD33 or CD123 on AML cells and CD3 on cytotoxic T cells.

Thus, bispecific antibodies can anchor to cd33+ or cd123+ AML cells and at the same time can bind to CD3 on T cells. In this way, T cells are brought into close proximity with tumor cells. Multiple binding of the bispecific antibody to a tumor antigen (CD 33 or CD 123) on a tumor cell and simultaneously to a TCR-associated CD3 molecule on a T cell results in TCR clustering. This ultimately results in efficient T cell activation independent of TCR specificity. Cytotoxic T cell activation in the vicinity of AML cells can then lead to tumor cell killing. The bispecific antibody constructs currently tested in clinical trials are, for example, votuzumab (Flotetuzumab) (MGD 006; CD3/CD123 DART), AMG330 or AMG673 (both CD3/CD33 BiTE).

Effective treatment of AML is complicated by the heterogeneity of CD33 and CD123 expression, which is found in both AML patient populations (inter-patient) and AML blast cell populations of individual patients (intra-patient). Furthermore, targeting a single tumor antigen risks losing this antigen expression on tumor cells due to the selection pressure induced by therapeutic intervention (Gardner et al, blood,127 (20), 2406-2410 (2016); blood.2017, month 1, 5; 129 (1): 100-104). Thus, there is a strong need for new therapeutic approaches that overcome the limitations of single targeted therapies and have broader patient coverage.

3. Summary of the invention

The inventors of the present invention found that dual targeting of CD33 and CD123 on Acute Myeloid Leukemia (AML) cells in combination with a polypeptide (or ISVD construct) targeting T cell receptor alpha beta (tcrαβ) on T cells results in efficient killing of AML cells. The killing activity against CD33/CD123 double expressing cells was comparable to a single targeting benchmark (such as CD33/CD3 AMG 330BiTE or CD123/CD3 MGD006 DART). However, the polypeptides of the invention show strong killing activity against CD33 and CD123 single expressing cells, whereas the single targeting benchmark only shows activity against cells expressing their specific targets. Furthermore, the polypeptides of the invention induce similar or even lower levels of inflammatory cytokines compared to baseline.

In some embodiments, the polypeptides of the present technology are produced efficiently (e.g., in a microbial host) and exhibit low viscosity at high concentrations, which is advantageous and convenient for subcutaneous administration. Furthermore, such polypeptides have limited reactivity to pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to the first treatment with the antibody construct). In preferred embodiments, such polypeptides exhibit a sufficiently long half-life in the subject to be treated so that consecutive treatments can be conveniently spaced apart.

The polypeptides of the present technology comprise or consist of at least three Immunoglobulin Single Variable Domains (ISVD), 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 (exemplary polypeptides are shown in fig. 1). Preferably, the at least one ISVD that binds to TCR αβ specifically binds to human TCR αβ, the at least one ISVD that binds to CD33 specifically binds to human CD33 and the at least one ISVD that binds to CD123 specifically binds to human CD 123.

The polypeptide preferably further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units. For example, the binding unit may be an ISVD that binds to serum proteins, preferably to human serum proteins such as human serum albumin.

Also provided are nucleic acid molecules, nucleic acids, or vectors comprising the nucleic acids, that are capable of expressing the polypeptides of the present technology, and compositions comprising the polypeptides, the nucleic acids, or the vectors. The composition is preferably a pharmaceutical composition.

Also provided are hosts or host cells comprising nucleic acids or vectors encoding polypeptides according to the present technology.

Also provided are methods of producing a polypeptide according to the present technology, the method comprising at least the steps of:

a. Optionally expressing a nucleic acid sequence encoding a polypeptide according to the present technology in a suitable host cell or host organism or in another suitable expression system, optionally followed by:

b. isolating and/or purifying the polypeptide according to the present technology.

Furthermore, the present technology provides the polypeptide for use as a medicament, a composition comprising the polypeptide or a composition comprising a nucleic acid or a vector comprising a nucleotide sequence encoding the polypeptide. Preferably, the polypeptide or composition is for use in the treatment of Acute Myeloid Leukemia (AML), wherein preferably the AML is relapsed and/or refractory AML.

In addition, methods of treating AML are provided, wherein the methods comprise administering to a subject in need thereof a pharmaceutically active amount of a polypeptide or composition according to the present technology. Preferably the AML is relapsed and/or refractory AML. In a preferred embodiment, the method further comprises administering one or more additional therapeutic agents.

Further provided is the use of a polypeptide or composition of the present technology in the manufacture of a pharmaceutical composition for the treatment of AML, wherein preferably AML is relapsed and/or refractory AML.

In particular, the present technology provides the following embodiments:

Embodiment 1. A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding the polypeptide, wherein the polypeptide comprises or consists of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of the ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally connected via one or more peptide linkers; and wherein:

a) The first ISVD specifically binds to T cell receptor alpha beta (TCR alpha beta) and comprises

I. CDR1 having the amino acid sequence of SEQ ID NO. 6 or having a2 or 1 amino acid difference from SEQ ID NO. 6;

CDR2 having the amino acid sequence of SEQ ID No. 10 or having 2 or 1 amino acid differences from SEQ ID No. 10; and

CDR3 having the amino acid sequence of SEQ ID No. 14 or having 2 or 1 amino acid differences from SEQ ID No. 14;

b) The second ISVD specifically binds CD33 and comprises

CDR1 having the amino acid sequence of SEQ ID NO. 7 or having 2 or 1 amino acid differences from SEQ ID NO. 7;

v. CDR2 having the amino acid sequence of SEQ ID NO. 11 or having 2 or 1 amino acid differences from SEQ ID NO. 11; and

Amino acid sequence with SEQ ID NO. 15 or CDR3 with 2 or 1 amino acid differences from SEQ ID NO. 15; and

C) The third ISVD specifically binds to CD123 and comprises

CDR1 having the amino acid sequence of SEQ ID NO. 8 or having a2 or 1 amino acid difference from SEQ ID NO. 8;

CDR2 having the amino acid sequence of SEQ ID No. 12 or having 2 or 1 amino acid differences from SEQ ID No. 12; and

Ix. a CDR3 having the amino acid sequence of SEQ ID NO. 16 or having a2 or 1 amino acid difference from SEQ ID NO. 16,

Wherein the sequence of the ISVD starts from the N-terminus.

Embodiment 2. The composition for the use according to embodiment 1, which is a pharmaceutical composition further comprising at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more other pharmaceutically active polypeptides and/or compounds.

Embodiment 3. The polypeptide or composition for use according to embodiment 1 or 2, wherein:

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) The third ISVD comprises CDR1 having the amino acid sequence of SEQ ID NO. 8, CDR2 having the amino acid sequence of SEQ ID NO. 12 and CDR3 having the amino acid sequence of SEQ ID NO. 16.

Embodiment 4. The polypeptide or composition for use according to any one of embodiments 1 to 3, wherein:

a) The amino acid sequence of the first ISVD has more 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) The amino acid sequence of the third ISVD has greater than 90% sequence identity with SEQ ID NO. 4.

Embodiment 5. The polypeptide or composition for use according to any one of embodiments 1 to 4, wherein:

a) The first ISVD has an amino acid sequence of SEQ ID NO. 2;

b) The second ISVD has the amino acid sequence of SEQ ID NO. 3; and

C) The third ISVD has the amino acid sequence of SEQ ID NO. 4.

Embodiment 6. The polypeptide or composition for use according to any one of embodiments 1 to 5, wherein the polypeptide further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units.

Embodiment 7. The polypeptide or composition for use according to embodiment 6, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life 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.

Embodiment 8. The polypeptide or composition for use according to any of embodiments 6 to 7, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

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

Embodiment 10. The polypeptide or composition for use according to embodiment 9, wherein the ISVD that binds human serum albumin comprises

I. CDR1 having the amino acid sequence of SEQ ID NO 9 or having a2 or 1 amino acid difference from SEQ ID NO 9;

CDR2 having the amino acid sequence of SEQ ID No. 13 or having 2 or 1 amino acid differences from SEQ ID No. 13; and

CDR3 having the amino acid sequence of SEQ ID No. 17 or having a 2 or 1 amino acid difference from SEQ ID No. 17.

Embodiment 11. The polypeptide or composition for use according to any of embodiments 9 to 10, wherein the ISVD which 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.

Embodiment 12. The polypeptide or composition for use according to any of embodiments 9 to 11, wherein the amino acid sequence of the ISVD which binds to human serum albumin has more than 90% sequence identity to SEQ ID No. 5.

Embodiment 13. The polypeptide or composition for use according to any of embodiments 9 to 12, wherein the ISVD which binds to human serum albumin has the amino acid sequence of SEQ ID No. 5.

Embodiment 14. The polypeptide or composition for use according to any of embodiments 1 to 13, wherein the amino acid sequence of the polypeptide has more than 90% sequence identity to SEQ ID No. 1.

Embodiment 15. The 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.

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

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

Embodiment 18. A polypeptide comprising or consisting of at least three Immunoglobulin Single Variable Domains (ISVD), wherein each of said ISVD comprises three complementarity determining regions (CDR 1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein:

b) The second ISVD specifically binds CD33 and comprises

C) The third ISVD specifically binds to CD123 and comprises

Wherein the sequence of the ISVD starts from the N-terminus.

Embodiment 19. The polypeptide of embodiment 18, wherein:

Embodiment 20. The polypeptide according to any of embodiments 18 or 19, wherein:

Embodiment 21 the polypeptide according to any one of embodiments 18 to 20, wherein:

a) The first ISVD has an amino acid sequence of SEQ ID NO. 2;

b) The second ISVD has the amino acid sequence of SEQ ID NO. 3; and

C) The third ISVD has the amino acid sequence of SEQ ID NO. 4.

Embodiment 22. The polypeptide according to any one of embodiments 18 to 21, wherein the polypeptide further comprises one or more additional groups, residues, moieties or binding units optionally linked via one or more peptide linkers, wherein the one or more additional groups, residues, moieties or binding units provide the polypeptide with an increased half-life compared to a corresponding polypeptide without the one or more additional groups, residues, moieties or binding units.

Embodiment 23. The polypeptide of embodiment 22, wherein the one or more additional groups, residues, moieties or binding units that provide the polypeptide with increased half-life 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.

Embodiment 24. The polypeptide according to any one of embodiments 22 to 23, wherein the one or more other groups, residues, moieties or binding units providing the polypeptide with increased half-life are selected from binding units capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

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

Embodiment 26. The polypeptide of embodiment 25, wherein the ISVD that binds human serum albumin comprises

Embodiment 27. The polypeptide of any one of embodiments 25 to 26, wherein the ISVD which 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.

Embodiment 28. The polypeptide of 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 to SEQ ID No. 5.

Embodiment 29. The polypeptide of 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.

Embodiment 30. The polypeptide according to any one of embodiments 18 to 29, wherein the amino acid sequence of the polypeptide has more than 90% sequence identity to SEQ ID No. 1.

Embodiment 31 the polypeptide according to any one of embodiments 18 to 29, wherein said polypeptide comprises or consists of the amino acid sequence of SEQ ID No. 1.

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

Embodiment 33. A host or host cell comprising the nucleic acid according to embodiment 32.

Embodiment 34. A method for producing a polypeptide according to any of embodiments 18 to 31, the method comprising at least the steps of:

a) Expressing the nucleic acid according to embodiment 32 in a suitable host cell or host organism or in another suitable expression system; optionally following is:

b) Isolating and/or purifying the polypeptide according to any one of embodiments 18 to 31.

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.

Embodiment 36 the composition of embodiment 35, 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.

Embodiment 37. A method of treating AML, wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide according to any one of claims 18 to 31 or a composition according to any one of claims 35 to 36.

Embodiment 38. The method according to claim 37, wherein the AML is relapsed and/or refractory AML.

Embodiment 39 use of the polypeptide according to any one of claims 18 to 31 or the composition according to any one of claims 35 or 36 for the preparation of a pharmaceutical composition for the treatment of AML.

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

4. Description of the drawings

Fig. 1: schematic representation of a multi-specific ISVD construct according to the invention, showing from N-terminal to C-terminal monovalent building blocks/ISVD tcrαβ, CD33, CD123 and Alb linked via linkers.

Fig. 2: monovalent CD33 (left) and CD123 binding BB (right) binding to CD33, CD123 cells transfected by human (up) or cynomolgus monkey (down), respectively.

Fig. 3: a025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) binding to human CD33 and/or human CD123 expressing cells.

Fig. 4: dose-dependent inhibition of primary T cells in competition assays by A025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) (black squares) and reference TCR (gray dots) in the absence (dashed curve) or presence (solid curve) of clinical grade HSA.

Fig. 5: in an impedance-based assay (xcelligent), a 15 to 1 effector to target ratio was used in the presence of 50 μm HSA, dose dependent human (upper) or cynomolgus monkey (lower) T cell mediated killing of the corresponding species CD33 (left) or CD123 (right) transfected cells.

Fig. 6: in flow cytometry-based assays, dose-dependent human (left) or cynomolgus monkey (right) T cell-mediated MOLM-13 cell killing was used with an effector to target ratio of 10:1. Will be-3% Positive target cells plotted against concentration of ISVD.

Fig. 7: in an impedance-based assay (xcelligent), a 15 to 1 effector to target ratio was used, dose dependent human T cell mediated cell killing. Cell Index (CI) after 32-35h incubation was plotted against ISVD concentration.

Fig. 8: inhibition of Molm-luc AML tumor growth by A025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) derived from in vivo bioluminescence imaging.

Fig. 9: inhibition of Molm-luc AML tumor growth by A025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) derived from ex vivo bioluminescence imaging.

Fig. 10: dose-dependent human T cell-mediated killing by ISVD according to this invention compared to CD123 and CD33 positive controls in MOLM-13 cells.

Fig. 11: dose-dependent human T cell-mediated killing by ISVD according to this invention compared to CD123 and CD33 positive controls in KG-1a cells.

Fig. 12: in U-937 cells, dose-dependent human T cell-mediated killing by ISVD according to the invention compared to CD123 and CD33 positive controls.

Fig. 13: in human Peripheral Blood Mononuclear Cells (PBMC), dose-dependent monocyte depletion in accordance with the ISVD of the present invention compared to CD123 and CD33 positive controls and negative controls (non-targeted TCE).

Fig. 14: dose-dependent cytokine release by ISVD according to the invention compared to CD123 and CD33 positive controls and negative controls (non-targeted TCE) in human PBMC of healthy donors using a different set of cytokines. IL-6. Ifnγ. Tnfα. IL-2.

Fig. 15: AML blast killing by ISVD according to the invention compared to CD33 and CD123 positive controls in all patients tested.

Fig. 16: a scatter plot of the percentage of CD33 or CD123 positive cells for each AML sample is depicted.

Fig. 17: primary parent cells of AML patients with a broad disease subtype were compared to cell viability of ISVD according to the invention (compared to CD123 and CD33 positive and negative controls). A. Patient number 3.B. Patient number 4.C. Patient number 5.

Fig. 18: individual absolute cell counts of total cd123+ T cells (a), monocytes cd33+ cells (B), cd4+ T cells (C) and cd8+ T cells (D) as measured in peripheral blood of cynomolgus monkeys treated with ISVD according to the invention over time. Animals M1 and M2 received 0.04. Mu.g/kg, while M3 and M4 received a single 1 hour continuous intravenous infusion of a solution of ISVD according to the invention.

5. Detailed description of the invention

The present technology aims to provide novel medicaments for the treatment of Acute Myeloid Leukemia (AML).

The inventors of the present invention found that dual targeting of CD33 and CD123 on Acute Myeloid Leukemia (AML) cells in combination with a polypeptide (or ISVD construct) targeting T cell receptor alpha beta (tcrαβ) on T cells results in efficient killing of AML cells. Killing activity was comparable to or even higher than a single targeting benchmark (such as CD33/CD3 AMG 330BiTE or CD123/CD3 MGD006 DART). Because of the high heterogeneity of CD33 and CD123 expression on AML cells in patient and inter-patient samples, the polypeptides of the invention provide a broader range of patient coverage than single targeting references.

The polypeptides are at least bispecific, but may also be, for example, trispecific, tetraspecific or penta-specific. Furthermore, the polypeptide is at least tetravalent, but may also be, for example, pentavalent or hexavalent, and the like.

The terms "bispecific", "trispecific", "tetraspecific" or "penta-specific" are all within the scope of the term "multispecific" and refer to binding to two, three, four or five different target molecules, respectively. The terms "divalent", "trivalent", "tetravalent", "pentavalent" or "hexavalent" all fall within the scope of the term "multivalent" and denote the presence of two, three, four or five binding units (e.g., ISVD), respectively. For example, the polypeptide may be tetra-specific tetravalent, such as a polypeptide comprising or consisting of four ISVD, wherein one ISVD binds to human tcrαβ, one ISVD binds to human CD33, one ISVD binds to CD123 and one ISVD binds to human serum albumin (an ISVD construct as shown in SEQ ID NO: 1). For example, if two ISVD bind to two different epitopes on the same target, e.g., if two ISVD bind to tcrαβ, the polypeptides may be biparatopic at the same time. The term "biparatopic" refers to binding to two different portions (e.g., epitopes) of the same target molecule.

As used herein, the terms "first ISVD", "second ISVD", "third ISVD", etc., merely indicate the relative positions of ISVD to each other, wherein numbering starts from the N-terminus of the polypeptides of the invention. Thus, a "first ISVD" is closer to the N-terminus than a "second ISVD", a "second ISVD" is closer to the N-terminus than a "third ISVD", and so on. Thus, the ISVD arrangement is reversed when considered from the C-terminus. Since numbering is not absolute and only indicates the relative positions of the at least three ISVD, it is not excluded that other binding units/building blocks may be present in the polypeptide, such as additional ISVD that binds to tcrαβ, CD33 or CD123, or ISVD that binds to another target. For example, as described further below (see section "(in vivo) half-life extension) the polypeptide can further comprise another ISVD that binds human serum albumin, which can be a fourth ISVD located at the C-terminus of at least three ISVDs. Furthermore, the possibility that other binding units/building blocks (e.g. ISVD) may be placed in between is not excluded. For example, the polypeptide can further comprise another ISVD that can even be located, for example, between the "second ISVD" and the "third ISVD".

In view of the foregoing, the present invention provides polypeptides comprising or consisting of at least three ISVD, 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 CD 123.

The components of the polypeptide, preferably the ISVD, may be linked to each other by one or more suitable linkers, such as peptide linkers.

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. One class of commonly used peptide linkers is known as "Gly-Ser" or "GS" linkers. These are linkers consisting essentially of glycine (G) and serine (S) residues, and typically comprise one or more repeats of a peptide motif, such as a GGGGS (SEQ ID NO: 77) motif (e.g., having the formula (Gly-Gly-Gly-Gly-Ser) n, where n may be 1, 2, 3,4, 5,6,7 or greater). 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, e.g., chen et al, adv. Drug deliv. Rev.2013, 10 months 15; 65 (10) 1357-1369; and Klein et al, protein Eng. Des. Sel. (2014) 27 (10): 325-330. In the polypeptides of the invention, it is preferred to use a 9GS linker to link the components of the polypeptide to each other.

In a preferred embodiment, the ISVD that specifically binds to tcrαβ is located at the N-terminus of the polypeptide. The inventors have surprisingly found that this configuration can increase the production yield of the polypeptide.

Furthermore, in a preferred embodiment, the ISVD that specifically binds to CD33 is located at the C-terminus of the ISVD that specifically binds to tcrαβ.

In an even more preferred embodiment, the ISVD that specifically binds to CD123 is located at the C-terminus of the ISVD that specifically binds to CD33, which itself is located at the C-terminus of the ISVD that specifically binds to tcrαβ.

Thus, preferably, the polypeptide comprises or consists of, in order 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 an optional binding unit that provides the polypeptide with an increased half-life as defined herein. The binding unit that provides the polypeptide with increased half-life is preferably ISVD.

Even more preferably, the polypeptide comprises or consists of, in order from the N-terminus of the polypeptide: ISVD that specifically binds to tcrαβ, a linker, ISVD that specifically binds to CD33, a linker, ISVD that specifically binds to CD123, a linker, and ISVD that binds to human serum albumin. More specifically, the polypeptide comprises or consists of the following in order from the N-terminus of the polypeptide: ISVD that specifically binds to tcrαβ, a 9GS linker, ISVD that specifically binds to CD33, a 9GS linker, ISVD that specifically binds to CD123, a 20GS linker, and ISVD that binds to human serum albumin.

Such a configuration of the polypeptide may provide increased production yield, good CMC characteristics, and optimized functionality and greater efficacy with respect to modulation of immune responses.

Preferably, the polypeptides of the present technology exhibit reduced binding to pre-existing antibodies in human serum. To this end, in one embodiment, the polypeptide comprises valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (numbering according to Kabat) in at least one ISVD, but preferably in each ISVD. In another embodiment, the polypeptide comprises an extension of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (a) extension. The C-terminus of ISVD is typically VTVSS (SEQ ID NO: 93). In another embodiment, the polypeptide comprises a lysine (K) or a glutamine (Q) at position 110 (numbered according to Kabat) in at least one ISVD. In another embodiment, the ISVD comprises a lysine (K) or a glutamine (Q) at position 112 (numbered according to Kabat) 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) such that upon 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 valine (V) at amino acid position 11 and leucine (L) at amino acid position 89 (according to Kabat numbering) in each ISVD, optionally lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and comprises an extension of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (a) extension (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 more information on this aspect see for example WO 2012/175741 and WO 2015/173325.

In a preferred embodiment, the polypeptide of the invention comprises or consists of an amino acid sequence having more than 90% (e.g. more than 95% or more than 99%) sequence identity to SEQ ID NO:1, wherein the CDRs of the four ISVD are respectively as defined by items a to D (or a 'to D', if defined using Kabat) as shown in the following sections "immunoglobulin single variable domain" and "(in vivo) half-life extension", wherein in particular:

ISVD specifically binding to TCR alpha beta has CDR1 comprising the amino acid sequence of SEQ ID NO:6, CDR2 comprising the amino acid sequence of SEQ ID NO:10 and CDR3 comprising the amino acid sequence of SEQ ID NO: 14;

ISVD specifically binding to CD33 having CDR1 comprising the amino acid sequence of SEQ ID NO. 7, CDR2 comprising the amino acid sequence of SEQ ID NO. 11 and CDR3 comprising the amino acid sequence of SEQ ID NO. 15;

ISVD specifically binding to CD123 having CDR1 comprising the amino acid sequence of SEQ ID NO. 8, CDR2 comprising the amino acid sequence of SEQ ID NO. 12 and CDR3 comprising the amino acid sequence of SEQ ID NO. 16; and

ISVD binding to human serum albumin having 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,

Or alternatively if Kabat definition is used:

ISVD specifically binding to TCR alpha beta has CDR1 comprising the amino acid sequence of SEQ ID NO:34, CDR2 comprising the amino acid sequence of SEQ ID NO:38 and CDR3 comprising the amino acid sequence of SEQ ID NO: 42;

ISVD specifically binding to CD33 having CDR1 comprising the amino acid sequence of SEQ ID NO:35, CDR2 comprising the amino acid sequence of SEQ ID NO:39 and CDR3 comprising the amino acid sequence of SEQ ID NO: 43;

ISVD specifically binding to CD123 having CDR1 comprising the amino acid sequence of SEQ ID NO:36, CDR2 comprising the amino acid sequence of SEQ ID NO:40 and CDR3 comprising the amino acid sequence of SEQ ID NO: 44; and

ISVD binding to human serum albumin has 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.

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.

The polypeptide of the invention preferably has at least half, more preferably at least the same binding affinity, as compared to a polypeptide consisting of the amino acids of SEQ ID NO. 1, for human TCR alpha beta and for human CD33 and for human CD123, wherein said binding affinities are measured using the same method, such as SPR.

5.1 Immunoglobulin Single variable Domains

The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" to define an immunoglobulin molecule in which an antigen binding site is present on and formed from a single immunoglobulin domain. This distinguishes an immunoglobulin single variable domain from a "conventional" immunoglobulin (e.g., monoclonal antibody) or fragment thereof (e.g., fab ', F (ab') ₂, scFv, diav), in which two immunoglobulin domains, particularly two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (V _H) and the light chain variable domain (V _L) interact to form an antigen binding site. In this case, the Complementarity Determining Regions (CDRs) of both V _H and V _L will contribute to the antigen binding site, i.e., a total of 6 CDRs will be involved in the formation of the antigen binding site.

In view of the above definitions, the antigen binding domain of a conventional 4-chain antibody (e.g., igG, igM, igA, igD or IgE molecule; known in the art) or a Fab fragment, F (ab') ₂ fragment, fv fragment (e.g., disulfide linked Fv or scFv fragment) or diabody (all known in the art) derived from such a conventional 4-chain antibody will generally not be considered as an immunoglobulin single variable domain, because in these cases, instead of one (single) immunoglobulin domain binding to the corresponding epitope occurs, a pair of (associated) immunoglobulin domains (e.g., light and heavy chain variable domains) i.e., V _H-V_L pairs of immunoglobulin domains occur binding to the corresponding epitope, which collectively bind to the corresponding epitope.

In contrast, an immunoglobulin single variable domain is capable of specifically binding to an epitope without pairing with another immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single V _H, a single V _HH, or a single V _L domain.

Thus, the single variable domain may be a light chain variable domain sequence (e.g., V _L sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a V _H sequence or a V _HH sequence) or a suitable fragment thereof; so long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit consisting essentially of a single variable domain such that a single antigen binding domain need not interact with another variable domain to form a functional antigen binding unit).

The Immunoglobulin Single Variable Domain (ISVD) may be, for example, a heavy chain ISVD, such as V _H、V_HH, including camelized V _H or humanized V _HH. Preferably, it is V _HH, including camelized V _H or humanized V _HH. The heavy chain ISVD may be derived from conventional four-chain antibodies or heavy chain antibodies.

For example, an immunoglobulin single variable domain may be a single domain antibody (or an amino acid sequence suitable for use as a single domain antibody), "dAb" or dAb (or an amino acid sequence suitable for use as a dAb) (as defined herein, and including but not limited toISVD); other single variable domains, or any suitable fragment of any of these.

In particular, the immunoglobulin single variable domain may beISVD (including humanized V _HH or camelized V _H) or a suitable fragment thereof. Note that: And Is Ablynx N.V. registered trademark ]

The "V _HH domain", also known as the V _HH、V_HH antibody fragment and the V _HH antibody, has been described initially as the antigen-binding immunoglobulin variable domain of a "heavy chain antibody" (i.e., a "light chain-free antibody"; hamers-Casterman et al Nature 363:446-448,1993). the term "V _HH domain" has been selected to distinguish these variable domains from the heavy chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "V ^H domains") and from the light chain variable domains present in conventional 4-chain antibodies (which are referred to herein as "V _L domains"). For further description of V _HH, reference is made to the review article of Muyldermans (REVIEWS IN Molecular Biotechnology 74:277-302,2001), and the following patent applications mentioned as general background: WO 94/04678, WO 95/04079 and WO 96/34103 of the university of Brussell freedom (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 1134231 and WO 02/48193 of Unilever; WO 97/49505, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 of the Belgium Frand Biotechnology institute (Vlaams Instituut voor Biotechnologie, VIB); Algonomics N.V. and Ablynx N.V. WO 03/050531; WO 01/90190 to national institute of Canadian (National Research Council of Canada); WO 03/025020 (=ep 1433793) of the institute of antibodies (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 to Ablynx N.V.

In general, the production of immunoglobulins involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for the desired specificity. Alternatively, immunoglobulins may be produced by screening a naive or synthetic library (e.g., by phage display).

Immunoglobulin sequences (e.gISVD) has been widely described in various publications, with WO 94/04678, hamers-masterman et al 1993 and Muyldermans et al 2001 (REVIEWS IN Molecular Biotechnology 74:277-302,2001) being exemplified. In these methods, a camelid is immunized with a target antigen to induce an immune response against the target antigen. To be obtained from said immunizationThe library of ISVD is used to further screen for ISVD against binding to the target antigen.

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

Immunization and/or screening of immunoglobulin sequences may be performed using peptide fragments of such antigens.

The present 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, e.g., camelized dabs as described by: ward et al (see, e.g., WO 94/04678 and Riechmann, febs Lett.,339:285-290,1994 and prot. Eng.,9:531-537,1996). Furthermore, the present invention also uses fusion immunoglobulin sequences, e.g. to form multivalent and/or multispecific constructs (see also Conrath et al, J.biol.chem., volume 276, 10.7346-7350,2001, and see e.g. WO 96/34103 and WO 99/23221 for multivalent and multispecific polypeptides containing one or more V _HH domains and their preparations); and immunoglobulin sequences comprising tags or other functional moieties (e.g., toxins, labels, radiochemicals, etc.) that may be derived from the immunoglobulin sequences of the invention.

"Humanized V _HH" comprises an amino acid sequence that corresponds to, but has been "humanized" of the amino acid sequence of a naturally occurring V _HH domain, i.e., humanized by substitution of one or more amino acid residues in the amino acid sequence of the naturally occurring V _HH sequence (and in particular the framework sequence) with one or more amino acid residues present at one or more corresponding positions in the V _H domain of a conventional 4 chain antibody from a human (e.g., as set forth above). This can be done in a manner known per se, which is clear to a person skilled in the art, for example based on the further description herein and the prior art (e.g. WO 2008/020079). Furthermore, it should be noted that such humanized V _HH may be obtained in any suitable manner known per se and is thus not strictly limited to polypeptides that have been obtained using polypeptides comprising the naturally occurring V _HH domain as starting material.

"Camelized V _H" comprises an amino acid sequence corresponding to the amino acid sequence of the naturally occurring V _H domain but which has been "camelized", i.e. camelized by substituting one or more amino acid residues in the amino acid sequence of the naturally occurring V _H domain from a conventional 4-chain antibody with one or more amino acid residues present at one or more corresponding positions in the V _HH domain of a heavy chain antibody. This can be done in a manner known per se, which is clear to a person skilled in the art, for example based on the further description herein and the prior art (e.g. WO 2008/020079). Such "camelized" substitutions are preferably inserted at amino acid positions formed and/or present at the V _H-V_L junction and/or at so-called camelid tag residues, as defined herein (see e.g. WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the V _H sequence used as a starting material or point for generating or designing a camelized V _H is preferably a V _H sequence from a mammal, more preferably a human V _H sequence, such as a V _H 3 sequence. It should be noted, however, that such camelized V _H may be obtained in any suitable manner known per se and is thus not strictly limited to polypeptides that have been obtained using a polypeptide comprising the naturally occurring V _H domain as starting material.

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 used in the present invention. For example, refer to reviews by Holliger and Hudson, nat biotechnol.2005, month 9; 23 (9):1126-36. In general, where the polypeptide is intended for administration to a subject (e.g., for prophylactic, therapeutic, and/or diagnostic purposes), it preferably comprises an immunoglobulin sequence that is not naturally occurring in the subject.

The preferred structure of an immunoglobulin single variable domain sequence may be considered to consist of four framework regions ("FR") referred to in the art and herein as "framework region 1" ("FR 1"), "framework region 2" ("FR 2"), "framework region 3" ("FR 3") and "framework region 4" ("FR 4"), respectively, which are interrupted by three complementarity determining regions ("CDRs") referred to in the art and herein as "complementarity determining region 1" ("CDR 1"), "complementarity determining region 2" ("CDR 2") and "complementarity determining region 3" ("CDR 3"), respectively.

Amino acid residues of the immunoglobulin single variable domain may be numbered according to the general numbering for the V _H domain given by Kabat et al ("Sequence of proteins of immunological interest", U.S. Pat. No. 3,35,Bethesda, MD, publication No. 91), as described further in paragraphs q on pages 58 and 59, incorporated by reference, as described in Riechmann and Muyldermans,2000 (J. Immunol. Methods 240 (1-2): 185-195); see, e.g., figure 2 of the publication) for V _HH domains from camelids. It should be noted that the total number of amino acid residues in each CDR may vary, and may not correspond to the total number of amino acid residues indicated by Kabat numbering (that is, one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the Kabat numbering allows), as is well known in the art for V _H domains and V _HH domains. This means that in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. The total number of amino acid residues in the V _H domain and in the V _HH domain is typically in the range of 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.

In the present application, unless otherwise indicated, the CDR sequences are determined according to the AbM numbers as described in Kontermann and Dubel (eds 2010,Antibody Engineering, vol.2, SPRINGER VERLAG Heidelberg Berlin, martin, chapter 3, pages 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acid residues at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

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

In such immunoglobulin sequences, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be apparent to the skilled artisan, e.g., based on standard manuals and the additional disclosure and prior art mentioned herein.

The framework sequences are preferably immunoglobulin framework sequences or framework sequences derived from immunoglobulin framework sequences (e.g., by humanization or camelization) (suitable combinations). For example, the framework sequences can be framework sequences derived from a light chain variable domain (e.g., a V _L sequence) and/or a heavy chain variable domain (e.g., a V _H sequence or a V _HH sequence). In a particularly preferred aspect, the framework sequence is a framework sequence derived from a V _HH sequence (wherein the framework sequence may optionally be partially or fully humanized) or a camelized conventional V _H sequence (as defined herein).

In particular, the framework sequences present in the ISVD sequences used in the present invention may contain one or more tag residues (as defined herein) such that the ISVD sequence isImmunoglobulin variable domains, including humanized V _HH or camelized V _H. Some preferred but non-limiting examples of (suitable combinations of) such framework sequences will become apparent from the further disclosure herein.

Furthermore, as generally described herein for immunoglobulin sequences, suitable fragments (or combinations of fragments) of any of the foregoing may also be used, such as fragments containing one or more framework sequences suitably flanked by and/or linked via one or more CDR sequences (e.g., in the same order as those CDRs and framework sequences might occur in a full-size immunoglobulin sequence from which the fragment was derived).

However, it should be noted that the invention is not limited with respect to the source of the ISVD sequence (or the nucleotide sequence used to express the ISVD sequence), and with respect to the manner in which the ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained). Thus, the ISVD sequence can be a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence. In particular but non-limiting aspects, the ISVD sequences are naturally occurring sequences (from any suitable species) or synthetic or semisynthetic sequences, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (e.g., partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized V _HH sequences), camelized (as defined herein) immunoglobulin sequences, and immunoglobulin sequences that have been obtained by techniques such as affinity maturation (e.g., starting from synthetic, random, or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and the like of engineered immunoglobulin sequences well known to the skilled artisan; or any suitable combination of any of the foregoing.

Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semisynthetic sequence, and may be, 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 that has been isolated from a library (and in particular an expression library), a nucleotide sequence that has been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), a nucleotide sequence that has been prepared by PCR using overlapping primers, or a nucleotide sequence that has been prepared using DNA synthesis techniques known per se.

As described above, the ISVD may beV _HH or a suitable fragment thereof. For the followingFor a general description of ISVD, reference is made to the further description below and to the prior art cited herein. In this respect, however, it should be noted that this description and the prior art mainly describe the so-called "V _H class 3"ISVD (i.e., having a high degree of sequence homology with human germline sequences of V _H class 3, such as DP-47, DP-51 or DP-29ISVD). It should be noted, however, that the present invention in its broadest sense can generally be used with any type ofISVD and also uses, for example, a chemical composition belonging to the so-called "V _H class 4ISVD (i.e., high sequence homology with human germline sequences of V _H class 4, such as DP-78)ISVD) as described, for example, in WO 2007/118670.

In general, the number of the devices used in the system,ISVD (particularly V _HH sequences, including (partially) humanized V _HH sequences and camelized V _H sequences) may be characterized by the presence of one or more "tag residues" (as described herein) in one or more framework sequences (also as described further herein). Thus, in general, the number of the cells,ISVD can be defined as an immunoglobulin sequence having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein one or more of the tag residues are as further defined herein.

In particular the number of the elements to be processed,ISVD can be an immunoglobulin sequence having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the framework sequences are as further defined herein.

More particularly, it is possible to provide,ISVD can be an immunoglobulin sequence having the following (general) structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Wherein FR1 to FR4 refer to framework regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein:

according to Kabat numbering, one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 are selected from the marker residues mentioned in table 1 below.

Table 1: Marker residues in ISVD

The present technology uses, inter alia, ISVD which specifically binds to tcrαβ, CD33 or CD 123. In the context of the present technology, "binding" to a specific target molecule has its ordinary meaning in the art as understood in the context of antibodies and their corresponding antigens.

The polypeptides of the present technology may comprise one or more ISVD that specifically binds to tcrαβ, one or more ISVD that specifically binds to CD33, and one or more ISVD that specifically binds to CD 123.

The ISVD used in the present technology forms part of a polypeptide of the present technology, which comprises or consists of at least three ISVD, such that the polypeptide can specifically bind to tcrαβ, CD33 and CD123. Thus, the polypeptide can anchor to cd33+cd123+ Leukemia Stem Cells (LSCs) and Acute Myelogenous Leukemia (AML) blast cells, while binding to tcrαβ on cytotoxic T cells. In this way, the polypeptide brings the T cells into close proximity with LSC and AML blast cells. Multiple binding of the polypeptide to tumor antigens (CD 33 and CD 123) on tumor cells, and simultaneous binding to TCR molecules on individual T cells, results in TCR clustering and ultimately T cell activation. T cell activation in the vicinity of LSC and AML blast cells can lead to efficient tumor cell killing, resulting in therapeutic efficacy in AML patients.

Thus, the target molecules of the at least three ISVD as used in the polypeptides of the present technology are TCR αβ, CD33 and CD123. Examples are mammalian CD33, CD123 and tcrαβ. Although human tcrαβ (Uniprot accession), human CD33 (Uniprot accession), and human CD123 (Uniprot accession) are preferred, forms from other species (e.g., from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys) (also referred to herein as "cyno"), or camelids (e.g., llamas or alpacas) tcrαβ, CD33, and CD 123) are also suitable for use in the present technology.

Specific examples of ISVD that specifically bind to tcrαβ, CD123 and CD123 that can be used in the present technology are described in the following items a to C:

A. ISVD which specifically binds to human tcrαβ and comprises

Amino acid sequence having SEQ ID NO. 14 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 14,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 6, CDR2 having the amino acid sequence of SEQ ID NO. 10 and CDR3 having the amino acid sequence of SEQ ID NO. 14.

Preferred examples of such ISVD that specifically binds to human tcrαβ have one or more (and preferably all) of the framework regions (and CDRs as defined in item a above) as indicated for tcrαβ -V _HH in table a-2, and most preferably are ISVD with the complete amino acid sequence of tcrαβ -V _HH (SEQ ID NO:2; see tables a-1 and a-2).

Furthermore, in preferred embodiments, the amino acid sequence of the ISVD that specifically binds to human TCRαβ can have greater than 90% (e.g., greater than 95% or greater than 99%) sequence identity to SEQ ID NO:2, wherein optionally the CDRs are as defined in item A above. In particular, ISVD that specifically binds to TCRαβ preferably has the amino acid sequence of SEQ ID NO. 2.

When such ISVD that specifically binds to TCR αβ has a2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (item a above), the ISVD preferably has at least half, more preferably at least the same binding affinity as compared to TCR αβ -V _HH (SEQ ID NO: 2), to human TCR αβ, wherein the binding affinities are measured using the same method (e.g., SPR).

B. ISVD which specifically binds to human CD33 and comprises

I. CDR1 having the amino acid sequence of SEQ ID NO. 7 or having 2 or 1 amino acid differences from SEQ ID NO. 7;

CDR2 having the amino acid sequence of SEQ ID No. 11 or having 2 or 1 amino acid differences from SEQ ID No. 11; and

Amino acid sequence having SEQ ID NO. 15 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 15,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 7, CDR2 having the amino acid sequence of SEQ ID NO. 11 and CDR3 having the amino acid sequence of SEQ ID NO. 15.

Preferred examples of such ISVD that specifically binds to human CD33 have one or more (and preferably all) of the framework regions (and CDRs as defined in item B above) as indicated in Table A-2 for CD33-V _HH, and most preferably are ISVD having the complete amino acid sequence of CD33-V _HH (SEQ ID NO:3, see tables A-1 and A-2).

Furthermore, in preferred embodiments, the amino acid sequence of ISVD that specifically binds to human CD33 may have greater than 90% (e.g., greater than 95% or greater than 99%) sequence identity to SEQ ID NO:3, wherein optionally the CDRs are as defined in item B above. In particular, ISVD that specifically binds CD33 preferably has the amino acid sequence of SEQ ID NO. 3.

When such ISVD specifically binding to CD33 has a2 or 1 amino acid difference in at least one CDR relative to the corresponding reference CDR sequence (upper Wen Xiang B), said ISVD preferably has at least half, more preferably at least the same binding affinity as compared to construct CD33-V _HH (SEQ ID NO: 3), to human CD33, wherein said binding affinity is measured using the same method (e.g. SPR).

C. ISVD which specifically binds to human CD123 and comprises

I. CDR1 having the amino acid sequence of SEQ ID NO. 8 or having a2 or 1 amino acid difference from SEQ ID NO. 8;

Amino acid sequence having SEQ ID NO. 16 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 16,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 8, CDR2 having the amino acid sequence of SEQ ID NO. 12 and CDR3 having the amino acid sequence of SEQ ID NO. 16.

Preferred examples of such ISVD that specifically binds to human CD123 have one or more (and preferably all) of the framework regions (and CDRs as defined in previous item C) as indicated in Table A-2 for CD123-V _HH, and most preferably are ISVD with the complete amino acid sequence of CD123-V _HH (SEQ ID NO:4, see tables A-1 and A-2).

Furthermore, in preferred embodiments, the amino acid sequence of ISVD that specifically binds to human CD123 can have greater than 90% (e.g., greater than 95% or greater than 99%) sequence identity to SEQ ID NO:4, wherein optionally the CDRs are as defined in item C above. In particular, ISVD binding to CD123 preferably has the amino acid sequence of SEQ ID NO. 4.

When such ISVD specifically binding to CD123 has a2 or 1 amino acid difference (upper Wen Xiang C) in at least one CDR relative to the corresponding reference CDR sequence, the ISVD preferably has at least half, more preferably at least the same binding affinity as compared to CD123-V _HH (SEQ ID NO: 4), to human CD123, wherein the binding affinities are measured using the same method (e.g. SPR).

Preferably, each of the ISVD as 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 ISVD as defined under items a to C above preferably has at least half, more preferably at least the same binding affinity for human tcrαβ, for human CD33 and for human CD123, compared to a polypeptide consisting of the amino acids of SEQ ID No.1, wherein the binding affinities are measured using the same method (e.g. SPR).

The SEQ ID NOs mentioned in the above items A to C are based on the CDR definitions according to the AbM definition (see Table A-2). Note that SEQ ID NOs defining the same CDRs according to the Kabat definition (see table a-2.1) can be used equally as well for items a to C above.

Thus, the specific ISVD that can be used in the present invention that specifically binds to tcrαβ, CD33 or CD123 using the AbM definition as described above can also be described using the Kabat definition as described in the following items a 'to C':

a'. ISVD which specifically binds to human tcrαβ and comprises

I. CDR1 having the amino acid sequence of SEQ ID No. 34 or having 2 or 1 amino acid differences from SEQ ID No. 34;

CDR2 having the amino acid sequence of SEQ ID No. 38 or having 2 or 1 amino acid differences from SEQ ID No. 38; and

CDR3 having the amino acid sequence of SEQ ID NO. 42 or having a 2 or 1 amino acid difference from SEQ ID NO. 42,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 34, CDR2 having the amino acid sequence of SEQ ID NO. 38 and CDR3 having the amino acid sequence of SEQ ID NO. 42.

Preferred examples of such ISVD that specifically binds to human tcrαβ have one or more (and preferably all) of the framework regions (and CDRs as defined in the foregoing item a') as indicated for tcrαβ -V _HH in table a-2.1, and most preferably are ISVD with the complete amino acid sequence of tcrαβ -V _HH (SEQ ID NO:2; see tables a-1 and a-2.1).

B'. ISVD which specifically binds to human CD33 and comprises

I. CDR1 having the amino acid sequence of SEQ ID NO. 35 or having a2 or 1 amino acid difference from SEQ ID NO. 35;

CDR2 having the amino acid sequence of SEQ ID No. 39 or having 2 or 1 amino acid differences from SEQ ID No. 39; and

Amino acid sequence having SEQ ID NO. 43 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 43,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 35, CDR2 having the amino acid sequence of SEQ ID NO. 39 and CDR3 having the amino acid sequence of SEQ ID NO. 43.

Preferred examples of such ISVD that specifically binds to human CD33 have one or more (and preferably all) of the framework regions (and CDRs as defined in item B' above) as indicated in Table A-2.1 for CD33-V _HH, and most preferably are ISVD having the complete amino acid sequence of CD33-V _HH (SEQ ID NO:3, see tables A-1 and A-2.1).

C'. ISVD that specifically binds to human CD123 and comprises

I. CDR1 having the amino acid sequence of SEQ ID NO. 36 or having a2 or 1 amino acid difference from SEQ ID NO. 36;

CDR2 having the amino acid sequence of SEQ ID No. 40 or having 2 or 1 amino acid differences from SEQ ID No. 40; and

Amino acid sequence having SEQ ID NO. 44 or CDR3 having 2 or 1 amino acid differences from SEQ ID NO. 44,

Preferably, CDR1 having the amino acid sequence of SEQ ID NO. 36, CDR2 having the amino acid sequence of SEQ ID NO. 40 and CDR3 having the amino acid sequence of SEQ ID NO. 44.

Preferred examples of such ISVD that specifically binds to human CD123 have one or more (and preferably all) of the framework regions (and CDRs as defined in the previous item C') as indicated in Table A-2.1 for CD123-V _HH, and most preferably are ISVD having the complete amino acid sequence of CD123-V _HH (SEQ ID NO:4, see tables A-1 and A-2.1).

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

Generally, to determine the percentage of "sequence identity" between two amino acid sequences according to the calculation method outlined above, the amino acid sequence with the largest number of amino acid residues is taken as the "first" amino acid sequence and the other amino acid sequence is taken as the "second" amino acid sequence.

As used herein, "amino acid difference" refers to a deletion, insertion, or substitution of a single amino acid residue relative to a reference sequence, and is preferably a substitution.

Amino acid substitutions are preferably conservative substitutions. Such conservative substitutions preferably refer to substitutions in which one amino acid in the following groups (a) - (e) is substituted by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amino compounds: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) a large aliphatic, non-polar residue: met, leu, ile, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

Particularly preferred conservative substitutions are as follows: substitution of Ala to Gly or Ser; arg is substituted by Lys; asn is substituted with gin or with His; asp is substituted with Glu; cys is substituted by Ser; gln is substituted with Asn; glu is substituted with Asp; substitution of Gly to Ala or substitution of Gly to Pro; his is substituted by Asn or substituted by Gln; ile is substituted by Leu or by Val; leu is substituted by Ile or by Val; lys is substituted for Arg, for gin, or for Glu; met is substituted with Leu, tyr, or Ile; phe is substituted with Met, with Leu, or with Tyr; substitution of Ser for Thr; thr is substituted by Ser; trp is substituted with Tyr; tyr is substituted with Trp; and/or Phe is substituted for Val, for Ile or for Leu.

5.2 Specificity

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

The specificity/selectivity of the binding unit can be determined based on affinity. Affinity refers to the strength or stability of molecular interactions. Affinity is generally given by KD or dissociation constant, including units of mol/L (or M). Affinity can also be expressed as an association constant KA, which is equal to 1/KD and has units of (mol/L) ^-1 (or M ^-1).

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

In general, the binding units used in the present technology, such as ISVD, will bind to their targets with a dissociation constant (KD) of 10 ^-5 to 10 ^-12 mol/L or less, and preferably 10 ^-7 to 10 ^-12 mol/L or less, and more preferably 10 ^-8 to 10 ^-12 mol/L (i.e., an association constant (KA) of 10 ⁵ to 10 ¹² L/mol or more, and preferably 10 ⁷ to 10 ¹² L/mol or more, and more preferably 10 ⁸ to 10 ¹² L/mol).

Any KD value greater than 10 ^-4 mol/L (or any KA value less than 10 ⁴ L/mol) is generally considered to be indicative of non-specific binding.

The KD for a biological interaction believed to be specific (e.g., binding of an immunoglobulin sequence to an antigen) is typically in the range of 10 ^-5 mol/L (10000 nM or 10. Mu.M) to 10 ^-12 mol/L (0.001 nM or 1 pM) or less.

Thus, specific/selective binding may mean that the binding unit (or polypeptide comprising it) binds to TCR αβ, CD33 and/or CD123 with a KD value of 10 ^-5 to 10 ^-12 mol/L or less and to the relevant target with a KD value of greater than 10 ^-4 mol/L using the same measurement method, e.g., SPR.

Thus, the polypeptide of the present technology preferably has at least half, more preferably at least the same binding affinity for human TCR alpha beta, for human CD33 and for human CD123, as compared to a polypeptide consisting of the amino acids of SEQ ID NO. 1, wherein the binding affinities are measured using the same method (e.g. SPR).

Specific binding to a specific target from a specific species does not exclude that the binding unit may also specifically bind to a similar target from a different species. For example, specific binding to human tcrαβ does not exclude that a binding unit (or a polypeptide comprising said binding unit) may also specifically bind to tcrαβ from cynomolgus monkey. Also, for example, specific binding to human CD33 or CD123 does not exclude that a binding unit (or a polypeptide comprising said binding unit) may also specifically bind to CD33 or CD123 from cynomolgus monkey ("cyno").

Specific binding of the binding unit to its designated target may be achieved by any suitable means known per se, including for example, scatchard (Scatchard) assays and/or competitive binding assays (such as Radioimmunoassays (RIA), enzyme Immunoassays (EIA) and sandwich competition assays) and different variants thereof known per se in the art; and other techniques mentioned herein.

The dissociation constant may be an actual or apparent dissociation constant, as will be clear to the skilled person. The method of determining the dissociation constant is clear to the skilled person and includes, for example, the techniques mentioned below. In this regard, it will also be clear that dissociation constants greater than 10 ^-4 mol/L or 10 ^-3 mol/L (e.g., 10 ^-2 mol/L) may not be measured. Optionally, as also clear to the skilled person, the (actual or apparent) dissociation constant may be calculated by the relation [ kd=1/KA ] based on the (actual or apparent) association constant (KA).

The affinity of the molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known Surface Plasmon Resonance (SPR) biosensor technique (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 for analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix, where one molecule is immobilized on a biosensor chip and the other molecule passes through the immobilized molecule under flow conditions, resulting in a K _on、k_off measurement and thus a K _D (or K _A) value. For example, this may be done using well known techniquesSystem (BIAcore International AB, a GE HEALTHCARE company, uppsala, sweden and Piscataway, N.J.). For further description, see Jonsson et al (1993, ann. Biol. Clin. 51:19-26), jonsson et al (1991Biotechniques 11:620-627), johnsson et al (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al (1991, anal. Biochem. 198:268-277).

Another well-known biosensor technique to determine the affinity of biomolecular interactions is Biological Layer Interferometry (BLI) (see, e.g., abdiche et al 2008, anal biochem. 377:209-217). As used herein, the term "biological layer interferometry" or "BLI" 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 protein-immobilized layer (signal beam) on the biosensor tip. The change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern, reported as wavelength shift (nm), whose size is a direct measure of the number of molecules bound to the surface of the biosensor tip. Since interactions can be measured in real time, association and dissociation rates can be determined. For example, BLI may use well knownThe system (ForteBio, pall Life Sciences, division of Menlopak, USA).

Alternatively, it is possible to use in kinetic exclusion assays (KinExA) (see, e.g., drake et al 2004, anal. Biochem., 328:35-43)Platform (Sapidyne Instruments Inc, boiyi, usa) measures affinity. As used herein, the term "KinExA" refers to a solution-based method for measuring the true equilibrium binding affinity and kinetics of an unmodified molecule. The equilibrated solution of the antibody/antigen complex is passed through a column with beads pre-coated with antigen (or antibody) allowing free antibody (or antigen) to bind to the coated molecule. Detection of the thus captured antibody (or antigen) is accomplished with a fluorescently labeled protein that binds to the antibody (or antigen).

The immunoassay system provides a platform for automated biological analysis and rapid sample turnover (Fraley et al 2013,Bioanalysis 5:1765-74).

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, wherein the one or more other groups, residues, moieties or binding units provide the polypeptide with an increased (in vivo) half-life compared to a corresponding polypeptide without the one or more other groups, residues, moieties or binding units. By in vivo half-life extension is meant, for example, that the polypeptide has an increased half-life in a mammalian, e.g., human subject, after administration. Half-life may be expressed, for example, as t1/2 beta.

The type of group, residue, moiety or binding unit is generally not limited and may be selected, for example, from 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.

More specifically, the one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life may be selected from binding units that are capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG), and preferably are binding units that are capable of binding to human serum albumin. The binding unit is preferably ISVD.

For example, WO 04/041665 describes binding to serum albumin (and in particular to human serum albumin)ISVD, which can be used with other proteins (e.g., one or more other proteins that bind to a desired targetISVD) to increase the half-life of the protein.

International application WO 06/122787 describes a number of methods directed against (human) serum albuminISVD. These areISVD includes a process called Alb-1ISVD (SEQ ID NO:52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO:62 in WO 06/122787). In addition, these can be used to extend the half-life of therapeutic proteins and polypeptides, as well as other therapeutic entities or moieties.

Furthermore, WO 2012/175400 describes a further improved form of Alb-1, referred to as Alb-23.

In a preferred embodiment, the polypeptide comprises a serum albumin binding moiety selected from the group consisting of: 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 shown on pages 7-9 of WO 2012/175400; and albumin binders described in WO2012/175741, WO2015/173325, WO2017/080850, WO2017/085172, WO2018/104444, WO2018/134235, WO 2018/134234. Tables a-4 also show some preferred serum albumin binders. Particularly preferred additional components of the polypeptides of the present technology are as described in item C:

C. ISVD which binds to human serum albumin and comprises

I. Comprising the amino acid sequence of SEQ ID NO. 9 or CDR1 having a2 or 1 amino acid difference from SEQ ID NO. 9;

A CDR2 comprising the amino acid sequence of SEQ ID No. 13 or having a 2 or 1 amino acid difference from SEQ ID No. 13; and

CDR3 comprising the amino acid sequence of SEQ ID No. 17 or having 2 or 1 amino acid differences from SEQ ID No. 17;

preferably CDR1 comprising the amino acid sequence of SEQ ID NO. 9, CDR2 comprising the amino acid sequence of SEQ ID NO. 13 and CDR3 comprising the amino acid sequence of SEQ ID NO. 17.

Preferred examples of such ISVD binding to human serum albumin have one or more (and preferably all) framework regions (and CDRs as defined in previous item C) as indicated in table a-2 for construct ALB23002, and most preferably are ISVD comprising the complete amino acid sequence of construct ALB23002 (SEQ ID NO:5, see tables a-1 and a-2).

Item C can also be described as using the Kabat definition:

C'. ISVD which binds to human serum albumin and comprises

I. Comprising the amino acid sequence of SEQ ID NO. 37 or CDR1 having a2 or 1 amino acid difference from SEQ ID NO. 37;

a CDR2 comprising the amino acid sequence of SEQ ID No. 41 or having a 2 or 1 amino acid difference from SEQ ID No. 41; and

CDR3 comprising the amino acid sequence of SEQ ID No. 45 or having 2 or 1 amino acid differences from SEQ ID No. 45;

Preferably CDR1 comprising the amino acid sequence of SEQ ID NO. 37, CDR2 comprising the amino acid sequence of SEQ ID NO. 41 and CDR3 comprising the amino acid sequence of SEQ ID NO. 45.

Preferred examples of such ISVD binding to human serum albumin have one or more (and preferably all) framework regions (and CDRs as defined in the previous item C) as indicated in table a-2.1 for construct ALB23002, and most preferably are ISVD comprising the complete amino acid sequence of construct ALB23002 (SEQ ID NO:5, see tables a-1 and a-2.1).

Also in a preferred embodiment, the amino acid sequence of the ISVD which binds to human serum albumin can have more than 90% (e.g. more than 95% or more than 99%) sequence identity to SEQ ID No. 5, wherein optionally the CDRs are as defined in item C above. In particular, ISVD binding to human serum albumin preferably comprises the amino acid sequence of SEQ ID NO. 5.

Where such ISVD which binds human serum albumin has a2 or 1 amino acid difference (upper Wen Xiang C) in at least one CDR relative to the corresponding reference CDR sequence, said ISVD has at least half, preferably at least the same, binding affinity to human serum albumin as compared to construct ALB23002 shown in SEQ ID No. 5, wherein said binding affinity is measured using the same method (e.g. SPR).

In a preferred embodiment, when such 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). If the ISVD binding to human serum albumin has another position than the C-terminal position (i.e., is not the C-terminal ISVD of the polypeptides of the present technology) and is selected from SEQ ID NOs: 5, 62, 63, 66 and 68 (see Table A-4 below).

5.4 Nucleic acid molecules

Nucleic acid molecules encoding the polypeptides of the present technology are also provided.

A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked to one another via a phosphate backbone to form a nucleotide sequence. The nucleic acids may be used to transform/transfect a host cell or host organism, for example, to express and/or produce a polypeptide. Suitable hosts or host cells for production purposes will be apparent to those skilled in the art and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism. Hosts or host cells comprising nucleic acids encoding polypeptides of the present technology are also encompassed by the present technology.

The nucleic acid may be, for example, DNA, RNA or hybrids thereof, and may also comprise (e.g. chemically) modified nucleotides, like 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.

The nucleic acids of the present technology may be prepared or obtained in a manner known per se and/or may be isolated from suitable natural sources. Nucleotide sequences encoding naturally occurring (poly) peptides may, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides having sequence variations. In addition, it will be clear to the person skilled in the art that several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety, and for example nucleic acids encoding one or more linkers may be joined together in a suitable manner for the preparation of a nucleic acid.

Techniques for generating nucleic acids will be apparent to the skilled artisan and may include, for example, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more portions thereof), introducing a mutation that results in expression of the truncated expression product; the introduction of one or more restriction sites (e.g., to create cassettes and/or regions that are easily digested and/or ligated using appropriate restriction enzymes), and/or the introduction of mutations by a PCR reaction using one or more "mismatched" primers.

5.5 Vectors

Vectors comprising nucleic acid molecules encoding polypeptides of the present technology are also provided. A vector as used herein is a vehicle suitable for carrying genetic material into a cell. Vectors include naked nucleic acids (e.g., plasmids or mRNA) or nucleic acids embedded in larger structures (e.g., liposomes or viral vectors).

Vectors typically comprise 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 suitable conditions, e.g. when said vector is introduced into a (e.g. human) cell. For DNA-based vectors, this typically includes the presence of elements for transcription (e.g., promoters and poly a signals) and translation (e.g., kozak sequences).

Preferably, in the 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 "operably linked" to a coding sequence (where the coding sequence is understood to be "under the control" of the promoter) if the promoter is capable of promoting or otherwise controlling/regulating transcription and/or expression of the coding sequence. Typically, when two nucleotide sequences are operably linked, they will be in the same orientation and typically also in the same reading frame. They are also typically substantially continuous, although this may not be necessary.

Preferably, any regulatory elements of the vectors enable them to provide their intended biological function in the intended host cell or host organism.

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

5.6 Compositions

The present technology also provides compositions comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding a polypeptide of the present technology, or at least one vector comprising such a nucleic acid molecule. The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally one or more additional pharmaceutically active polypeptides and/or compounds.

5.7 Host organisms

The present technology also relates to host cells or host organisms comprising the polypeptides of the present technology, nucleic acids encoding the polypeptides of the present technology and/or vectors containing nucleic acid molecules encoding the polypeptides of the present technology.

Suitable host cells or host organisms will be apparent to those skilled in the art and are, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, E.coli (ESCHERICHIA COLI) or Pichia pastoris. The most preferred host is Pichia pastoris.

5.8 Methods and uses of polypeptides

The present technology also provides methods for producing the polypeptides of the present technology. The method may comprise 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 may include:

a) Expressing a nucleic acid sequence encoding said polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally following is:

b) Isolating and/or purifying the polypeptide.

Suitable host cells or host organisms for production purposes will be apparent to those skilled in the art and may be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line or any suitable fungus, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, e.coli or pichia pastoris. The most preferred host is Pichia pastoris.

The polypeptide of the present technology, the nucleic acid molecule or vector as described, or a composition comprising the polypeptide, nucleic acid molecule or vector of the present technology, preferably the polypeptide or the composition comprising the polypeptide, may be used as a medicament.

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

Also provided are polypeptides of the present technology, nucleic acid molecules or vectors as described, or compositions comprising polypeptides, nucleic acid molecules or vectors of the present technology, for use in (prophylactic or therapeutic) treatment of Acute Myeloid Leukemia (AML), preferably relapsed and/or refractory AML.

Further provided are (prophylactic and/or therapeutic) methods of treating AML, wherein the methods comprise administering to a subject in need thereof a pharmaceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or vector as described, or a composition comprising a polypeptide, nucleic acid molecule or vector of the present technology.

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

The AML may be relapsed and/or refractory AML.

The "subject" as referred to in the context of the present technology may be any animal, preferably a mammal. In mammals, humans can be distinguished from non-human mammals. The non-human animal may be, for example, a companion animal (e.g., a dog, cat), livestock (e.g., a cow, horse, sheep, goat, or pig animal), or an animal commonly used for research purposes and/or for the production of antibodies (e.g., mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates (e.g., cynomolgus monkeys) or camels (e.g., llamas or alpacas)).

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

The substance (including polypeptide, nucleic acid molecule, and vector) or composition may be administered to the subject by any suitable route of administration, for example, enterally (e.g., orally or rectally) or parenterally (e.g., epidermically, sublingually, buccally, nasally, intra-articular, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, transdermally, or transmucosally). Parenteral administration, such as intramuscular, subcutaneous or intradermal administration is preferred. Most preferred is subcutaneous administration.

An effective amount of a polypeptide, a nucleic acid molecule or vector as described herein, or a composition comprising the polypeptide, nucleic acid molecule or vector, can be administered to a subject to provide a desired therapeutic result.

One or more doses may be administered. If 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.

Table a-1: the amino acid sequences of the different monovalent V _HH building blocks identified within tetravalent polypeptide A025001562 (TCR-CD 33-CD123 multispecific ISVD construct) ("ID" refers to SEQ ID NO as used herein)

Table a-2: the sequences of the CDRs and framework according to AbM numbering ("ID" refers to the given SEQ ID NO

Table a-2.1: the sequences of the CDRs and the frameworks according to Kabat numbering ("ID" refers to the given SEQ ID NO)

Table a-3: the amino acid sequence of the multivalent polypeptide of choice ("ID" refers to the given SEQ ID NO)

Table a-4: the serum albumin binding ISVD sequence ("ID" refers to SEQ ID NO as used herein

Table a-5: the linker sequence ("ID" refers to SEQ ID NO as used herein)

6. Examples

6.1 Example 1: multispecific ISVD construct generation

MultispecificityISVD proteins are expressed in pichia pastoris (p.pastoris). The yeast expression vector contains the AOX1 promoter and terminator, the bleomycin resistance gene and the coding information of Saccharomyces cerevisiae (Saccharomyces cerevisiae) alpha-mating factor signal peptide. Will beISVD monovalent Building Blocks (BB) were combined with GS linkers and cloned into expression vectors via gold cloning (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 contained in one or more vectorsISVD building blocks and GS linkers. All of these elements flank the BpiI site. The use of unique nucleotide overhangs at each position of the cloning cassette allows for seamless ligation in a predetermined order. After Sanger sequence confirmation, plasmid DNA derived from e.coli (e.coli) TOP10 was linearized and transformed by electroporation into the internally prepared highly competent pichia pastoris strain NRRL Y-11430 (ATCC 76273).

To make it containColi TG1 cells (Lucigen, catalog No. 60502) of the ISVD protein expression vector were grown at 37 ℃ for 2 hours and then 29 hours at 30 ℃ (250 rpm) in a baffled shake flask containing "5052" auto-induction medium. The cells were pelleted by centrifugation (20 min, 4500rpm,4 ℃), the supernatant was discarded, and the pellet was frozen at-20 ℃ overnight. The frozen cell pellet was then solubilized in DPBS at 1/12.5 of the original culture volume and incubated at 4 ℃ for 1 hour while gently rotating to disrupt the outer membrane of the cells. The cells were reprecipitated (20 min, 8500rpm,4 ℃) and would containThe supernatant of the ISVD protein was collected and filtered for immediate purification.

Labeling FLAG3His ₆ The ISVD proteins were purified by Immobilized Metal Affinity Chromatography (IMAC) or elution with imidazole (for the former) or acid (for the latter) on NiIDA/NTA (Genscript) resin, followed by a desalting step (PD column with Sephadex G25 resin, GE HEALTHCARE) and, if necessary, preparative Size Exclusion Chromatography (SEC) in D-PBS (Superdex 75 column, GE HEALTHCARE). For this purpose, a robot station orA purification system.

Label-free to ALB building blocksThe ISVD protein or construct is purified on AMSPHERE A (JSR) or MabCaptureA (Poros) resin, followed by a desalting step (PD column with Sephadex G25 resin, GE HEALTHCARE) and, if necessary, preparative SEC in D-PBS (Superdex 75 column, GE HEALTHCARE). N-octyl- β -d-glucopyranoside (OGP; ALPHA AESAR, catalog number J67390) treatment was performed during purification/gel filtration chromatography whenever low LPS levels were required. The concentration was determined via OD280/OD340 measurement. Quality control was performed by SDS-PAGE and mass spectrometry.

6.2 Example 2: binding affinity of multispecific ISVD constructs to TCR αβ, CD33, CD123 and serum albumin

A TCRαβ -CD33-CD123 multispecific ISVD construct as shown in SEQ ID NO. 1 was generated.

6.2.1 Determination of affinity for human and cynomolgus monkey CD33 and CD123 proteins

The affinity of the tcrαβ -CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) to huCD33L-Fc, cyCD33L-Fc (via capture set) or huCD123 (via capture set) proteins, expressed as association rate constant (K _a), dissociation rate constant (K _d) and equilibrium dissociation constant (K _D) was measured on a ProteOn XPR36 instrument (BioRad Laboratories, inc.) by a Surface Plasmon Resonance (SPR) based assay at 37 ℃.

Settings for measuring binding affinity to CD33

Anti-huIgG (Fc) was immobilized on GLH (long matrix, high capacity) sensor chips by ammonia coupling using EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and NHS (N-hydroxysuccinimide ester) chemistry next, huCD33L-Fc and cyCD L-Fc (association: 120s, 25. Mu.L/min) were captured and purified TCRαβ -CD33-CD123 multispecific ISVD constructs as shown in SEQ ID NO:1 were injected at different concentrations (0.4 to 625 nM) for 120s followed by dissociation 900s.

Arrangement for measuring binding affinity to CD123

Anti-huIgG (Fc) was immobilized on GLH (long matrix, high capacity) sensor chips by ammonia coupling using EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and NHS (N-hydroxysuccinimide ester) chemistry following capture of huCD123-Fc and cyCD-Fc (association: 120s, 25. Mu.L/min.) purified TCRαβ -CD33-CD123 multispecific ISVD constructs as shown in SEQ ID NO:1 were injected at different concentrations (1.2 to 300 nM) for 120s followed by dissociation for 900s at 45. Mu.l/min.

The data were double-referenced by subtracting the reference analyte lanes and blank buffer injections. Affinity constants (k _a、k_d、K_D) were calculated using Proteon Manager 3.1.0 (version 3.1.0.6) software using Langmuir 1:1 interaction model.

The results of the measurement of the affinity of the tcrαβ -CD33-CD123 multispecific ISVD construct for human and cynomolgus monkey CD33 and human and cynomolgus monkey CD123 as shown in SEQ ID No. 1 are summarized in tables 2 and 3 below.

Table 2: determination of affinity of TCR αβ -CD33-CD123 multispecific ISVD constructs for human and cynomolgus monkey CD33 proteins based on SPR

The reactivity of the tcrαβ -CD33-CD123 multispecific ISVD construct to cynomolgus monkey CD33 (K _D =6.9 nM) was comparable to the reactivity to human CD33 (K _D =7.6 nM).

Table 3: determination of kinetics of human and cynomolgus monkey CD123 proteins based on SPR for tcrαβ -CD33-CD123 multispecific ISVD constructs

The TCR αβ -CD33-CD123 multispecific ISVD construct was 3.3 times more reactive to human CD123 (K _D =0.59 nM) than to cynomolgus monkey CD123 (K _D =2 nM).

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

6.2.2 Determination of affinity for human and cynomolgus monkey TCR alpha beta protein

Affinity of TCR αβ -CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) to recombinant huTCR (2 XN 9) -zipper protein, cyTCR (AEA 41865) -zipper protein (set via the package) was evaluated on a ProteOn XPR36 instrument (BioRad Laboratories, inc.) at 37 ℃ by SPR-based assay, expressed as association rate constant (K _a), dissociation rate constant (K _d) and equilibrium dissociation constant (K _D).

Arrangement for measuring binding affinity to TCRαβ

HuTCR (2 XN 9) -zipper proteins, cyTCR (AEA 41865) -zipper proteins were coated on GLC (short matrix, normal volume) sensor chips. Purified TCRαβ -CD33-CD123 multispecific ISVD constructs as shown in SEQ ID NO:1 were injected at various concentrations (0.4 to 625 nM) for 120s followed by dissociation 900s.

The results of the measurement of the affinity of the tcrαβ -CD33-CD123 multispecific ISVD construct for human and cynomolgus tcrαβ as shown in SEQ ID No. 1 are summarized in table 4 below. ISVD consisting of only the TCRαβ building block (SEQ ID NO: 2) linked to ALB23002 (SEQ ID NO: 5) was used as a reference.

Table 4: determination of kinetics of human and cynomolgus monkey tcrαβ based on SPR for tcrαβ -CD33-CD123 multispecific ISVD constructs and reference TCR-ISVD constructs

The TCR αβ -CD33-CD123 multispecific ISVD construct was 3-fold more reactive to cynomolgus TCR αβ (K _D =6.7 nM) than to human TCR αβ (K _D =21 nM).

The results (table 4) demonstrate that the multispecific ISVD construct binds human/cynomolgus tcrαβ with high affinity.

6.2.3 Determination of affinity for human, mouse and cynomolgus serum albumin

The binding affinity of the tcrαβ -CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) to recombinant human, cynomolgus monkey and mouse serum albumin via the C-terminal ALB23002 half-life extension (via coating setup) was evaluated on a ProteOn XPR36 instrument (BioRad Laboratories, inc.) at 37 ℃ as an association rate constant (K _a), dissociation rate constant (K _d) and equilibrium dissociation constant (K _D).

Arrangement for measuring binding affinity to serum albumin

Human, cynomolgus and mouse serum albumin were immobilized on a Proteon GLC sensor chip by using EDC and NHS chemistry using ammonia coupling (running buffer used: HBS-EP+, pH 7.4). Albumin was immobilized at both 2.5 μg/mL (HSA and MSA) and 5 μg/mL (CSA) concentrations in pH 4.5 acetate buffer, raising the immobilization level of CSA up to 220RU, MSA 150RU, and HSA 110RU. Purified multivalent V _HH was injected at different concentrations (between 4.3nM and 416 nM) for 2 min (flow rate 45. Mu.L/min) followed by dissociation for 900s. Regeneration between cycles consists of: 10mM glycine-HCl (pH 1.5) was injected at 100. Mu.L/min for 47s.

The data were double-referenced by subtracting the reference ligand lanes and buffer injections. The treated curves were evaluated by fitting with langmuir 1:1 interaction model using the Proteon Manager 3.1.0 (version 3.1.0.6) software model and affinity constants (k _a、k_d、K_D) were calculated.

The results of the measurement of the affinity of the tcrαβ -CD33-CD123 multispecific ISVD construct for human, mouse and cynomolgus serum albumin as shown in SEQ ID No. 1 are summarized in table 5 below.

Table 5: determination of affinity of serum albumin based on SPR for TCRαβ -CD33-CD123 multispecific ISVD constructs

Cross-reactivity to CSA was confirmed. Furthermore, although no kinetic parameters are reported, the affinity for MSA is good enough to obtain a half-life extension and a half-life with serum albumin.

The results (table 5) demonstrate that the multispecific ISVD construct binds human/mouse/cynomolgus monkey tcrαβ with high affinity.

6.3 Example 3: binding affinity of multispecific ISVD constructs to membrane-bound CD33 and/or CD123

Arrangement for measuring binding affinity to CD33/CD123 on target cell lines

The binding affinity of tcrαβ -CD33-CD123 multispecific ISVD constructs to target cell lines expressing CD33 and/or CD123 was evaluated using flow cytometry. Target cell lines expressing CD123 are described in detail in WO2018/091606A 1.

Transfected CD33 cells were generated as follows. A stable CHO Flp-In (Invitrogen, R758-07) cell line that has recombinant over-expressed CD33 was generated using Flp-In ^TM site-directed recombination technology (Flp-In ^TM system (Invitrogen, K601001, K601002) for the generation of stable mammalian expression cell lines by Flp recombinase-mediated integration). Thus, DNA integration occurs by the Saccharomyces cerevisiae-derived Flp recombinase (pOG 44) at specific genomic locations of the FRT (Flp recombination target) site. Both the Flp-In ^TM host cell line and the expression plasmid (pcDNA 5) contained 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 monkey CD123 was derived from NCBI genebank number XP-005590138.

Briefly, cells were harvested and transferred to V-bottom 96-well plates (5 x10 ⁴ cells/well) and serial dilutions of tcrαβ -CD123-CD33 multispecific ISVD constructs were incubated at 4 ℃ for 30min in the presence of 30 μΜ clinical grade HSA (CSL Behring, 2160-679) in FACS buffer (D-PBS (Gibco, 14190), with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038). Next, the cells were washed three times with FACS buffer and incubated with 1. Mu.g/mL mouse monoclonal antibodyM2 antibody (Sigma-Aldrich, F1804) was incubated at 4℃for 30min to detect FLAG3His ₆ -tagged CD123-CD33-TCR multispecific ISVD construct, or 3 μg/mL mAb anti-V _HH antibody (ABH 0077) (APS+ internal, A-0006-00_ABH0077_SF_AB1891) to detect ALB BB-bearing antibodiesISVD. Next, the cells were washed 3 times with FACS buffer and incubated with 5 μg/mL Allophycocyanin (APC) AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3) (fcγ fragment specific) (Jackson Immunoresearch, 115-136-071) at a final volume of 100 μl for 30min at 4 ℃. Subsequently, the cells were resuspended in 50. Mu.L of cold FACS buffer supplemented with 1. Mu.g/mL propidium iodide (PI, sigma P4170) to differentiate between live and dead cells. After staining, cells were collected using MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, a P1 population that accounts for more than 80% of the total events is selected based on FSC-SSC distribution to distinguish cells from debris. PI positive (dead) cells were excluded from this population (P1) and the median APC fluorescence intensity of PI negative cells was assessed.

Arrangement for measuring binding affinity to primary T cells

The binding affinity of tcrαβ -CD33-CD123 multispecific ISVD constructs to primary T cells was evaluated in a competition setting using flow cytometry using a monovalent tcrisvd tagged with FLAG3His ₆ as ligand.

Briefly, human or cynomolgus primary T cells were thawed and transferred to V-bottom 96-well plates (in 100 μl, 7.5x10 ⁴ cells/well) and incubated with serial dilutions of tcrαβ -CD33-CD123 multispecific ISVD constructs and fixed concentrations of ligand in 30 μl final volume at 4 ℃ for 30min in FACS buffer (D-PBS (Gibco, 14190), with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)) in the presence of 30 μl clinical grade HSA (CSL Behring, 2160-679). The ligand concentration used in the assay was lower than its binding EC ₅₀. After incubation at 4 ℃ for a period of 90min, the ligand binding level was determined by flow cytometry. Subsequently, the cells were washed 3 times and incubated with 1. Mu.g/ml mouse monoclonal antibodyM2 antibody (Sigma-Aldrich, F1804) was incubated at 4℃for 30min, washed again, and incubated with 5. Mu.g/ml Allophycocyanin (APC) AffiniPure goat anti-mouse IgG (subclass 1+2a+2b+3) (Fcγ fragment specific) (Jackson Immunoresearch, 115-136-071) at 4℃for 30min at a final volume of 100. Mu.L. Subsequently, the cells were resuspended in FACS buffer supplemented with 1 μg/ml propidium iodide (PI, sigma, P4170) to differentiate between live and dead cells. After staining, cells were collected using MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, a P1 population that accounts for more than 80% of the total events is selected based on FSC-SSC distribution to distinguish cells from debris. PI positive (dead) cells were excluded from this population (P1) and the median APC fluorescence intensity of PI negative cells was assessed.

Human-cynomolgus cross-reactivity was assessed by: the monovalent CD33 building block (SEQ ID NO: 3) and monovalent CD123 building block (SEQ ID NO: 4) were tested for binding to human or cynomolgus CD33 transfected cell lines or to human or cynomolgus CD123 transfected cell lines using flow cytometry as described above. The results are graphically represented in fig. 2. EC ₅₀ values for the different experiments are summarized in table 6 (CD 33 transfected target cells) and table 7 (CD 123 transfected target cells).

Table 6: EC ₅₀ values for binding of CD33 building block (SEQ ID NO: 3) to huCD33 or cyCD expressing cells.

Table 7: EC50 values for binding of CD123 building block (SEQ ID NO: 4) to cells expressing huCD123 or cyCD 123.

Monovalent CD33 and CD123 building blocks were confirmed to bind to human and cynomolgus monkey membrane targets. The difference in EC ₅₀ between human and cynomolgus monkey was less than 2-fold reduction with respect to binding to CD33, and 3-fold reduction with respect to binding to CD123 cells (table 7).

In summary, in addition to binding affinity for recombinant human and cynomolgus monkey CD33 and CD123 proteins, dose-dependent binding of tcrαβ -CD33-CD123 multispecific ISVD constructs to CD33 and CD123 expressed by human and cynomolgus monkey cells was also confirmed (n=1).

In the competition setting, binding of the CD123-CD33-TCR multispecific ISVD construct and the reference TCR-ISVD construct (consisting only of the TCR. Alpha. Beta. Building block (SEQ ID NO: 2) linked to ALB23002 (SEQ ID NO: 5)) to human and cynomolgus monkey T cells was evaluated using flow cytometry as described above (FIG. 3).

An illustrative example of DRC is depicted in fig. 4. The ISVD constructs were tested on a variety of healthy donor T cells. The overall IC ₅₀ is shown in table 8.

Table 8: TCRαβ -CD33-CD123 multispecific ISVD construct and Total IC ₅₀ (M) reference TCR-ISVD construct as determined by flow cytometry in human and cynomolgus monkey T cell competition assays

* Overall IC ₅₀ (weight)

In summary, the cross-reactivity of tcrαβ -CD33-CD123 multispecific ISVD constructs to cynomolgus primary T cells was confirmed. EC ₅₀ (=729 nM) on primary cynomolgus T cells is about 3 times that of EC ₅₀ (=218M) on human primary T cells

6.4 Example 4: t cell mediated killing of target cells induced by multi-specific ISVD constructs

6.4.1 Impedance-based cytotoxicity assays

In an impedance-based cytotoxicity assay (e.g., as described in WO 2018091606 A1), the ISVD construct is characterized for redirected T cell-mediated killing using human or cynomolgus primary effector T cells and adherent target cells. The impedance change induced by target cell adhesion to the electrode surface was measured using an xcelligent instrument (Roche). T cells are non-adherent and therefore do not affect impedance measurements.The RTCA MP instrument quantifies the change in electrical impedance, which is displayed as a dimensionless parameter called the cell index, which is proportional to the total area of the cell-covered tissue culture well. To each well of the 96E-plate (ACEA Biosciences;05 232 368 001), 50. Mu.L of 4X concentration HSA solution (200. Mu.M was used to have a final concentration of 50. Mu.M in some assays and 120. Mu.M was used to have a final concentration of 30. Mu.M in other assays) was added in assay medium (target cell growth medium (without selection antibiotic) +1% penicillin/streptomycin (Life technologies catalog number 15140)). The outer wells were not used and filled with 200. Mu.L of medium or D-PBS. Placing a 96E-plate onIn a station (in a 5% CO ₂ incubator at 37 ℃) and in the absence of cells, a single measurement was made to measure the background impedance of the assay medium. Subsequently, 50. Mu.L of target cells (2X 10 ⁴ cells/well) in the assay medium were inoculated onto 96E-plates and 50. Mu.L of serial dilutions of ISVD construct solution (4X concentration) in the assay medium were added. (final volume=200 μl). After 30min at room temperature, 50 μl of primary T cells (3 x 10 ⁵ cells/well) in assay medium were added per well to achieve a 15:1 effector to target ratio. Placing the plate onIn the station, and impedance was measured every 15min for 4 days. The data were analyzed at fixed time points indicated in the results.

6.4.2 Flow cytometry-based cytotoxicity assays

In flow cytometry-based cytotoxicity assays, the ISVD constructs were characterized for redirected T cell mediated killing using human or cynomolgus primary T cells as effector cells as well as non-adherent target cells. Target cells were labeled with 4. Mu.M PKH26 membrane dye using PKH26 red fluorescent cell adapter kit (Sigma, PKH26GL-1 KT) according to the manufacturer's instructions. Effector cells (2.5x10 ⁵ cells/well) and PKH-labeled target cells (2.5x10 ⁴ cells/well) were co-incubated (effector to target ratio 10:1) in 96-well V-bottom plate (Greiner Bio-one, #651 180) in assay medium of target cell lines (target growth medium with 1% penicillin/streptomycin (Life Technologies, 15140) and 50 μ M Alburex HSA (CSL Behring, 2160-679). To analyze the concentration-dependent cell lysis, serial dilutions of ISVD constructs in target assay medium were added to the cells and incubated at 37 ℃ for 18h in a 5% CO ₂ environment. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (D-PBS (Gibco, 14190), with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)). Subsequently, the cells were resuspended in the medium supplemented with 5nM3 Iodide (642/661) (ThermoFisher Scientific, T3605) in 100. Mu.L FACS buffer to distinguish between live and dead cells. Cells were analyzed using MACSQuant X flow cytometer (Miltenyi Biotec). The total sample volume collected for each sample was 70 μl. Gating on PKH 26-positive cells and assaying in this population-3 Positive cells. Percent specific lysis = (% TO-PRO-3+ no construct-% TO-PRO-3+ with construct)/% TO-PRO-3+ no construct)) x100.

Functional cross-reactivity assays of tcrαβ -CD33-CD123 multispecific ISVD constructs on CD33, CD123 and TCRs were determined in an impedance-based cytotoxicity assay (xcelligent) using human or cynomolgus primary T cells and adherent human or cynomolgus transfected CD33 or CD123 cells.

As described above in 6.4.1 and 6.4.2, all assays were performed in the presence of excess HSA, such thatISVD is fully saturated with HSA as described above. The reference TCR-ISVD was used as a negative control. The results are shown in fig. 5 and 6 and in table 9.

6.4.3 Results

The results of the assay comparing human and cynomolgus primary T cells are graphically shown in fig. 5 and 7. The overall IC ₅₀ values can be calculated by evaluating human T cell mediated cell killing of human CD33 and human CD123 transfected cells using T cells from different human donors (table 9). Cynomolgus T cell mediated killing of human CD33 and human CD123 transfected cells was evaluated using T cells from 1 cynomolgus monkey. The IC ₅₀ values are summarized in table 10.

Table 9: in an impedance (xcelligent) -based human T cell mediated cd33+ or cd123+ cell killing assay, a 15 to 1 effector to target ratio was used, overall IC ₅₀ (M) of tcrαβ -CD33-CD123 in the presence of 50 μΜ HSA.

With respect to CD33 and CD123, the overall IC ₅₀ for human target expressing cells was 5,4.10 ^-11 M and 2,5.10 ^-11 M, respectively.

Table 10: in a cynomolgus T cell mediated cd33+ or cd123+ cell killing assay based on impedance (xcelligent), a 15 to 1 effector to target ratio was used, IC ₅₀ (M) of tcrαβ -CD33-CD123 in the presence of 50 μ MHSA.

To confirm the human cynomolgus monkey cross-reactivity of the tcrαβ -CD33-CD123 ISVD construct to TCRs, the ISVD construct was evaluated in the presence of 50 μm HSA as described above using human or primary T cells in combination with CD33, CD123 double expressed human MOLM-13 target cell lines in flow cytometry-based T cell mediated MOLM-13 cell killing. A graphical representation of these results is shown in fig. 6. Additional data for tcrαβ -CD33-CD123 ISVD constructs tested in human T cell mediated killing assays using different donors were available to calculate overall EC ₅₀ values (table 11). EC ₅₀ values for cynomolgus T cell mediated killing assays are shown in table 12.

Table 11: in a flow cytometry human T cell mediated MOLM-13 cell killing assay, a 10 to 1 effector to target ratio was used, overall EC ₅₀ (M) of A025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) in the presence of 50 μM HSA.

Table 12: in the cynomolgus monkey T cell mediated MOLM-13 cell killing assay, EC ₅₀ (M) of the tcrαβ -CD33-CD123 ISVD construct was used with a 10 to 1 effector to target ratio.

In summary, the tcrαβ -CD33-CD123 ISVD construct is functional in both human and cynomolgus monkey target cell mediated killing assays. The overall killing efficacy of the ISVD on CD33/CD123 double positive AML cell line is 1,8.10 ^-11 M.

6.5 Example 5: preclinical in vivo efficacy of tcrαβ -CD33-CD123 multispecific ISVD constructs in CD123 ⁺ Molm-13-luc-disseminated AML model in T cell-engrafted humanized NSG mice

6.5.1 Materials and methods

Cell lines and human material

The human AML derived cell line expressing CD123 Molm-13 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, germany). Molm-13 cells were grown in culture (37 ℃,5% CO ₂, 95% humidity) in RPMI1640 Glutamax medium (supplemented with 20% fetal bovine serum). Infecting the cells with a non-replicating lentiviral-carried luciferase vector (SV 40-PGL 4-Puro); the polyclonal Molm-13-luc was selected using 2. Mu.g/ml puromycin.

Human T cell purification and expansion for in vivo administration

EFS(EtablissementDu Sang, francis island-De-France), france) provides fresh human peripheral blood from healthy donors.

Fresh human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll gradient centrifugation (without braking) at room temperature for 40min at 200 g. The pellet was washed and resuspended in a final volume of 50ml supplemented with Phosphate Buffered Saline (PBS). The total viable PBMC numbers were defined by a Vi-CELL counter (Beckman Coulter LIFE SCIENCES, brea, calif., U.S.A.). The pellet was recovered in autoMACS run buffer (Miltenyi Biotec). T cells were isolated from PBMCs using 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 a CD3 and CD28 co-stimulated based T cell TRANSACT matrix activation/expansion kit (Miltenyi Biotec). According to the Miltenyi procedure, the activation protocol involved culturing T cells in the presence of TRANSACT matrix for 2 weeks in TexMACS medium (Miltenyi Biotec) supplemented with 20,000 iu of soluble IL-2 and 1% penicillin-streptomycin (Gibco). On day 14 of expansion, T cells were harvested and resuspended in PBS at a final concentration of 5x 10 ⁷ cells/ml and 10 ⁷ cells were administered to each animal by Intraperitoneal (IP) injection. The animal was tested for greater than 85% T cell viability prior to injection.

In vivo characterization

All in vivo experiments were approved by the Sanofi ethics committee and conducted in compliance with local and institutional laws, ethics and guidelines at AAALAC approved facilities.

The TCRαβ -CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) was evaluated for anti-tumor activity after Molm-luc AML implantation in non-irradiated NOD.Cg-Prkdc ^scid Il2rg^tm1Wjl/SzJ (NSG) mice (CHARLES RIVER Laboratories, saint-German-Nuelles, france). On day 0, females of 6-8 weeks of age were implanted Intravenously (IV) with 10 ⁶/0.2 ml Molm-13-luc cells per mouse. On day 1, 10 ⁷/0.2 ml human T cells were implanted intraperitoneally for the same animals per mouse.

Animals were distributed in groups based on systemic bioluminescence imaging (BLI) signal uniformity and tumor bone marrow transplantation assessed by day 3 long bone signal segmentation. Mice were treated with TCRαβ -CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) at 12, 1.2, 0.12 and 0.012nmol/kg Q2D from day 4 to day 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 1.2nmol/kg QD (days 4-13), or with CD123/CD3 positive control at 1.3nmol/kg QD (days 4-13) IV, see Table 13. Longitudinal in vivo bioluminescence imaging (BLI) was performed to monitor the growth of disseminated tumors. Mice were sacrificed on day 14 and necropsied at BLI to assess effects in deep soft tissues such as liver, spleen, ovary and abdominal fat.

Table 13: compound evaluation study design

A) : one non-responding animal due to lack of T cell implantation is not included in the assay

Data collection and efficacy criteria

Animal body weight was monitored from day 3 to the end of the assay to track the effect of the therapy. Doses that produced 20% weight loss or 15% weight loss or 10% or more of drug death for 3 consecutive days were considered to be overly toxic doses. Animal body weight includes tumor weight.

Tumor growth was assessed by: on days 3, 7, 10 and 14 after tumor injection, by in vivo BLI using IVIS Lumina XRMS imager (Perkinelmer, walltherm, mass., USA) with LIVING IMAGE.5.2 acquisition software (Perkinelmer) by in vivo luciferase activity measurement, injection with beetle potassium luciferin salt 160mg/kg Ip, 15 minutes later, 5 minutes prior to imaging(120 Mg/kg;6mg/kg IM,5 ml/kg) of the anesthetized animals were subjected to image processing. Tumor growth is based on a bioluminescence signal curve (expressed in photons/second).

Tumor growth in the whole body and long bones of the hind leg was tracked by BLI signal measurements on days 7, 10 and 14 after tumor implantation. The primary efficacy endpoint was the rate of change of tumor signal from baseline change between treatment and control (T/C), partial Regression (PR) and Complete Regression (CR).

Tumor growth based on bioluminescence signal curves (expressed in photons/second) were plotted in time for each animal of each treatment group and expressed as median curves ± MAD of whole body (linear scale) and bone segmentation signals (logarithmic scale). Tumor bioluminescence signal change per animal per day was calculated for each treatment (T) group and control (C) by subtracting the tumor signal on the day of first treatment (day of planning) from the tumor signal on the indicated day of observation. The median T of the treatment group was calculated and the median C of the control group was calculated. The ratio T/C is then calculated and expressed in percent:

dT/dC= [ (median at end T-median day 3T)/(median at end C-median day 3C) ] x 100

When dT/dC is below 42%, the dose is considered to be therapeutically active, and when dT/dC is below 10%, the dose is considered to be very active. If dT/dC is below 0, the dose is considered highly active and the percentage of regression is recorded on the date:

Percent tumor regression is defined as the% decrease in tumor signal in the treatment group at a particular day of observation compared to the signal at the first day of treatment.

For each animal,% regression was calculated at a specific time point. Considering the risk of variability in the signal due to variability in fluorescein kinetics due to missing ip injections, actual regression was only seen when regression was observed for at least two consecutive time points per animal.

Partial Regression (PR): regression is defined as partial regression if the tumor signal at two consecutive time points (where the signal at one time point is less than 50% of the starting signal) decreases below the tumor signal at the beginning of the treatment. Complete Regression (CR): complete regression is defined if the tumor signal decreases below 80% of the initial signal.

Biometric analysis

Tumor growth based on bioluminescence signal curves (over time) was measured for each animal of each treatment group. For longitudinal in vivo BLI data, two non-parametric two-way analysis of variance (ANOVA) types were performed in duplicate measurements on a daily basis, followed by two comparative analyses adjusted with Bonferroni-Holm for multiplicity: p >0.05:NS,0.05< p >0.01, p < 0.01. For end ex vivo BLI data, the rank-converted bioluminescence signal was analyzed by one-factor variance with a factor group. Descriptive statistics using median ± median absolute deviation are provided by group and measurement day: p >0.05:NS,0.05< p >0.01, p < 0.01.

6.5.2 Results

In Molm-13-luc AML xenograft models, TCRαβ -CD33-CD123 multispecific ISVD constructs induced anti-leukemia effects in vivo (FIG. 8A).

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

The sum of the Longest Diameters (LD) of all target lesions is the baseline LD sum. The baseline LD sum was used as a reference to characterize the target tumor response.

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

3/8CR and 1/8PR were observed at 0.12nmol/kg, 4/8CR and 3/8PR were observed at 1.2nmol/kg, and 3/7CR and 3/7PR were observed at 12nmol/kg (FIG. 8C). The reference TCR-ISVD construct was totally inactive (dT/dC 80%) against Molm-luc tumor growth at 1.2 nmol/kg. CD123/CD3 positive control 1,3nmol/kg inhibited tumor growth, with dT/dC of 8% (NS compared to A025001562 (TCR-CD 33-CD123 multispecific ISVD construct, SEQ ID NO: 1) treatment), correlated with 4/8CR in long bones (FIGS. 8A, 8B).

Based on end-point ex vivo bioluminescence imaging, when the reference ISVD TCR-HLE was inactive in all tissues, the tcrαβ -CD33-CD123 multispecific ISVD construct significantly inhibited tumor growth in the liver (p < 0.0001), spleen (p < 0.0001), and ovary (p < 0.0001) at all doses tested, but not in abdominal fat, and the CD123/CD3 positive control significantly inhibited tumor burden in the liver (p < 0.0001) and spleen (p < 0.0001), but not in ovarian (NS) or abdominal adipose tissue. (FIG. 9)

6.6 Example 6: t cell mediated killing of human target cells

6.6.1 Materials and methods

In flow cytometry-based cytotoxicity assays, ISVD constructs were characterized for redirected T cell mediated killing using human primary T cells as effector cells as well as non-adherent target cells. CD123 and/or CD33 positive target cells (MOLM-13, DSMZ ACC 554, U-937, programming with 4. Mu.M PKH-26 membrane dye using PKH26 red fluorescent cell adapter kit (Sigma, PKH26GL-1 KT) according to the manufacturer's instructions,CRL1593.2 and KG-1a,CCL246.1 ^TM). Effector cells (2.5x10 ⁵ cells/well) and PKH-labeled target cells (2.5x10 ⁴ cells/well) were co-incubated in 96-well V-bottom plate (Greiner Bio-one, #651 180) in assay medium of target cell lines (target growth medium without antibiotic) (effector to target ratio 10:1). For analysis of concentration-dependent cell lysis, serial dilutions of compounds in target assay medium were added to cells and incubated at 37 ℃ for 18h in a 5% CO ₂ environment. After incubation, cells were pelleted by centrifugation and washed with FACS buffer (D-PBS from Gibco, with 10% FBS from Sigma and 0.05% sodium azide from Merck). Subsequently, the cells were resuspended in the medium supplemented with 5nM-3 Iodide (642/661) (ThermoFisher Scientific, T3605) in FACS buffer to distinguish between live and dead cells. Cells were analyzed using FACS array flow cytometry (BD Biosciences). The total sample volume collected for each sample was 80 μl. Gating on PKH 26-positive cells and assaying in this population-3 Positive cells. Percent specific lysis= (%-3+ ISVD-%-3+ With ISVD)/(%-3+ No ISVD)) x100.

The multi-specific tcrαβ -CD33-CD123 ISVD constructs according to the invention were compared to the corresponding constructs in which CD33 binding ISVD or CD123 binding ISVD was replaced by unrelated ISVD IRR (not binding to CD33 and not binding to CD 123; table 23) as well as CD123/CD3 positive control and CD33/CD3 positive control. The results are shown in fig. 10, 11 and 12.

6.6.2 Results

As can be observed in fig. 10, in MOLM-13 cells, all tested compounds triggered tumor cell killing (which is expected) because MOLM-13 cells were positive for both CD123 and CD 33. In the ISVD construct, the dual targeting form (CD 33/CD123 TCE) has the most potent tumor cell killing.

The killing efficacy and percent lysis in U-937 and KG-1a cells are depicted in Table 24 and FIGS. 11 and 12.

Table 23: sample IDISVD and linker composition

Sample ID	Target ISVD1	Joint	Target ISVD2	Joint	Target ISVD3
						A	TCR	9GS	CD33	9GS	CD123
B	TCR	9GS	CD33	9GS	IRR
						C	TCR	9GS	IRR	9GS	CD123

Table 24: in a human T cell mediated U-937 or KG-1a cell killing assay, a 10 to 1 effector to target ratio, trivalent, was usedEC ₅₀ (M) and% lysis of ISVD.

TCR-CD123 single targeting ISVD hardly induced killing of CD123-U-937 cells, which was also observed for CD123/CD3 positive controls. Similarly, CD33 single-targeting ISVD induced only low levels of CD33-/+ KG-1a cell killing, which was also observed for CD33/CD3 positive controls. On the other hand, dual targeting tcrαβ -CD33-CD123 ISVD (construct a) exhibited effective tumor cell killing for both CD 33-and CD 123-cell lines, thus exemplifing the advantages of the dual targeting approach.

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 TCR αβ -CD33-CD123 multispecific ISVD constructs according to the invention was determined in an autologous healthy donor PBMC assay. In parallel, autologous depletion of monocytes in human PBMCs was assessed. The functionality of the ISVD according to the invention is compared with the following 2 tool molecules: CD123/CD3 positive control and CD33/CD3 positive control. Non-targeted T Cell Engager (TCE) ISVD was used as a negative control.

6.7.1 Materials and methods

A total of 200 000 PBMC (100. Mu.L, 2X 106 cells/mL in culture) isolated from whole blood using Leucosep ^TM tubes containing Lymphoprep ^TM solution were transferred to a 96-well V-bottom transparent well plate (Greiner)A 96-well plate; 651 180).

Next, 50 μl of serial dilutions of the compounds to be tested or 50 μl of medium for the empty wells are added to the wells.

The culture plates containing treated PBMC were incubated at 37℃for 20h with 5% CO ₂. After overnight (20 h) incubation, PBMCs were centrifuged at 300g for 2min. Supernatants were collected and transferred to fresh 96-well storage plates, frozen at-20 ℃ for cytokine measurement. The cell pellet was suspended in 100 μl cold FACS buffer and washed 1 time with 100 μl FACS buffer.

Thereafter, the PBMC were centrifuged at 300g for 2min at 4 ℃. 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 min at room temperature.

Next, 30 μl of the (2X) antibody staining mixture (CD 123, HLA-DR and CD14 antibodies) was added to the PBMC suspension and incubated in the dark at 4 ℃ for 30 minutes.

The cell suspension was then washed 2 times and resuspended in 50. Mu.L of diluted TO-PRO ^TM -3 iodide. The plate was read on MACSQuantX. The readout is the number of viable cells per subset detected by flow cytometry. Monocytes were quantified by SSC% cd14+ (fig. 13).

Frozen supernatants from overnight incubated human PBMC were used to measure a panel of cytokines including IL-2, IL-6, IFNγ, TNF α using a multiplex bead assay from Bio-rad. The measurements were made according to manufacturer's guidelines. Data from the reaction was acquired using Luminex FlexMAP D system (fig. 14).

6.7.2 Results

TCR αβ -CD33-CD123 multispecific ISVD constructs induce depletion of monocytes associated with cytokine production in all donors. The non-targeted TCE did not show any killing or cytokine release (see fig. 13). The data shown in fig. 13 are data from 1 donor, which represents data from all 6 donors who provided PBMCs.

Furthermore, both the potency and the highest level of cytokines induced by the ISVD construct according to the invention are significantly lower compared to CD123/CD3 control compounds. IL-6 and TNFα production induced by the ISVD constructs according to this invention was comparable to the CD33/CD3 control. Although the IL-2 and IFN gamma levels were higher than the CD33/CD3 control, the levels were still acceptable.

Based on this, it can be concluded that ISVD constructs according to the invention do not have a higher risk of inducing CRS compared to other CD123 or CD33 targeting compounds, and should be safe for humans.

6.8 Example 8: in vitro primary AML cell killing assay

6.8.1 Materials and methods

The lysis of AML blast cells mediated by CD33/CD3 positive control was tested as follows: AML samples from patients with primary diagnosis or recurrence were used in an ex vivo co-culture system without the addition of any other cells. Thus, the E:T ratio is determined by the number of residual T cells in the primary AML sample. Whole blood samples from AML patients were provided by the public hospitals or central laboratories of mosaic (La Conception, AP-HM) or montreal.

First, 1mL of whole blood sample was dispensed per 50mL tube, and 40mL (1×) of erythrocyte lysis buffer was added and incubated at room temperature for 10 minutes. After incubation, the pellet was washed with PBS (Eurobio CS PBS 01-01) and resuspended in 1mL of medium (RPMI (Eurobio catalog CM1RPM 00-01), FCS10% (Sigma catalog F2442-500mL 17 L484), nonessential amino acid 1% (Eurobio catalog CSTAAN 00), glutamine 1% (Eurobio catalog CSTGLU 00), sodium pyruvate 1% (Eurobio catalog CSTVAT 00), penicillin/streptomycin (Eurobio catalog CABPES 01)).

Next, 300 μl of AML blast cells were added to 700 μl of medium per well in 6 well plates in the presence of 1mL (2 x) saturation concentration of tcrαβ -CD33-CD123 multispecific ISVD construct according to the invention (CD 33/CD123 TCE), positive control (CD 33/CD3 or CD123/CD 3) or negative control (non-targeted TCE) and incubated at 37 ℃, 5% co ₂. After 4 days of incubation, cells were harvested and washed 1 time with PBS.

Next, the Zombie violet viability marker (1/100 dilution in PBS) was added to the cell pellet and incubated in the dark for 5 minutes at room temperature.

The cells were then washed and stained with antibody mixtures (CD 45, CD33, CD34, CD38, CD14, CD123, CD11 antibodies) and incubated on ice for 10 minutes.

Next, the cells were washed at 4 ℃ and fixed with 1.5ml pbs+2% paraformaldehyde for 30 min. After 30 minutes incubation, paraformaldehyde is diluted with 9mL of diluent. The data were analyzed on a Cytoflex cytometer. The results are shown in FIGS. 15-17.

6.8.2 Results

The percentage of CD33 or CD123 positive cells in each AML sample is shown in figure 16. As can be observed, all patients had a large number of both CD33 and CD123 positive cells. In addition, AML patients have a broad disease subtype. Accordingly, AML samples are suitable for this assay.

In fig. 15, AML blast killing in all AML patient samples is shown. Each dot represents the count of viable blast cells following treatment with either ISVD according to the invention or one of the positive controls normalized to the negative control (non-targeted TCE). The results for several individual patients are highlighted in fig. 17.

As can be observed, the cell viability of the parent cells treated with the ISVD according to the invention was on average lowest compared to the positive control. All patients showed a clear response in cells, although the response was patient-to-patient. This indicates that ISVD according to the present invention is capable of targeted cell killing of CD123 and CD33 positive tumor cells.

6.9 Example 9: non-human primate (NHP) study

Pharmacokinetic (PK), pharmacodynamic (PD) and non-clinical safety profiles of TCR αβ -CD33-CD123 multispecific ISVD constructs according to the invention were evaluated in cynomolgus monkey studies.

6.9.1 Materials and methods

The ISVD construct according to the present invention was administered as a single dose of 1 hour continuous Intravenous (IV) infusion to a total of 4 male cynomolgus monkeys, followed by a 21 day observation period. Systemic exposure and potential toxicity of ISVD were determined and PD endpoint (immune cell evaluation) was assessed. The study design is shown in table 25.

Table 25: study design: exploratory single dose IV study in male cynomolgus monkeys

Group of	Dosage (μg/kg)	Number of animals	Sex and animal identification number
				1	0.04	2	M1、M2
2	0.4	2	M3、M4

Abbreviations: m: male male

6.9.2 Security

A single 1 hour continuous IV infusion of the ISVD construct was well tolerated by cynomolgus monkeys. There was no death during the study history and no clinical signs associated with treatment. No treatment-related body weight changes and body temperature changes were observed.

Cytokine evaluation showed only a very slight increase in IL-6 (peaks at 2 hours and/or 6 hours after infusion initiation and returns to baseline within 24 hours) associated with the ISVD construct administered at a single dose of 0.04 μg/kg. No changes in IFN-gamma, IL-1β, IL-2, IL-8 and TNF- α levels were observed at 0.04 μg/kg. Very small to slight increases in IL-2, IL-6, IFN-gamma and TNF-alpha (peaking at 2 hours and/or 6 hours after the start of infusion and returning to baseline within 24 hours) were associated with ISVD constructs administered at a single dose of 0.4. Mu.g/kg. At 0.4. Mu.g/kg, no changes were observed for IL-1β and IL-8 levels in the monkeys, which were associated with the TCR-CD33-CD123 multispecific ISVD constructs according to the invention.

Thus, it can be concluded that the ISVD construct according to the present invention is well tolerated in this NHP model.

6.9.3 Pharmacokinetics

Serum levels of up to 0.04 μg/kg for 3 days and up to 0.4 μg/kg for 7 days in monkeys were quantifiable after 1 hour of infusion of the ISVD construct. Overall, clearance of about 0.07L/day/kg was observed following IV infusion at dose levels of 0.04 μg/kg and 0.4 μg/kg. The estimated terminal elimination half-life is approximately 1 day. From 0.04 to 0.4 μg/kg, the increase in ISVD construct exposure (AUC) was slightly less than expected at the dose scale, with a 7.7-fold increase in exposure for a 10-fold increase in dose, probably due to the lower exposure observed in one monkey (M3). PK parameters are reported in table 26.

TABLE 26 toxicity kinetics parameters for TCRαβ -CD33-CD123 multispecific ISVD constructs.

Abbreviations: AUC: area under the curve from 0 to infinity, AUClast: plasma concentration versus time area under the curve from 0 to the time of last measurable concentration; CL: the clearance rate; ceoi: plasma concentration at the end of infusion; t1/2: terminal half-life; tlast: the last measurable concentration; tmax: time to first reach Cmax; vss: steady state distribution volume

6.9.4 Pharmacodynamics

Infusion of the ISVD construct into cynomolgus monkeys at a single dose IV of 0.04 and 0.4 μg/kg resulted in a change in both cd123+ cell populations and cd33+ monocytes at both dose levels. These variations consist of:

Starting from 6 hours after the start of infusion, a total decrease in total cd123+ cell number was observed for all animals. This decline was maintained for all animals throughout the study.

Starting from 6 hours after the start of infusion (and for animal M1 receiving the ISVD construct at 0.04 μg/kg, up to day 1), a total decrease in the number of monocyte cd33+ cells was observed for all animals. Complete recovery of animal M3 was observed on day 1.

Furthermore, a total decrease in the numbers of cd4+ and cd8+ cells was observed for all animals starting from 6 hours after the start of infusion. For cd8+ cells, complete recovery was observed on day 3, with animal M4 having a rebound effect. For cd4+ cells, complete recovery of animal M4 was observed on day 3.

Cell counts have been visualized in figure 18.

7 Industrial applicability

The polypeptides described herein, nucleic acid molecules encoding the polypeptides, vectors and compositions comprising the nucleic acids may be used, for example, in the treatment of subjects suffering from acute myeloid leukemia.

Claims

1. A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid comprising a nucleotide sequence encoding the polypeptide, wherein the polypeptide comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein each of the ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively) optionally connected via one or more peptide linkers; and wherein:

a. The first ISVD specifically binds to the T cell receptor αβ (TCRαβ) and contains

i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 6 or having 2 or 1 amino acid differences with SEQ ID NO: 6;

ii. an amino acid sequence comprising SEQ ID NO: 10 or a CDR2 having 2 or 1 amino acid differences from SEQ ID NO: 10; and

iii. a CDR3 comprising the amino acid sequence of SEQ ID NO: 14 or having 2 or 1 amino acid differences with SEQ ID NO: 14;

b. The second ISVD specifically binds to CD33 and contains

iv. an amino acid sequence comprising SEQ ID NO: 7 or a CDR1 having 2 or 1 amino acid differences from SEQ ID NO: 7;

v. a CDR2 comprising the amino acid sequence of SEQ ID NO: 11 or having 2 or 1 amino acid differences with SEQ ID NO: 11; and

vi. a CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or having 2 or 1 amino acid differences with SEQ ID NO: 15; and

c. The third ISVD specifically binds to CD123 and contains

vii. A CDR1 comprising the amino acid sequence of SEQ ID NO: 8 or having 2 or 1 amino acid differences with SEQ ID NO: 8;

viii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 12 or having 2 or 1 amino acid differences with SEQ ID NO: 12; and

ix. a CDR3 comprising the amino acid sequence of SEQ ID NO: 16 or having 2 or 1 amino acid differences with SEQ ID NO: 16,

The order of the ISVDs indicates the relative positions of the ISVDs to each other from the N-terminus to the C-terminus of the polypeptide.

2. The polypeptide or composition of claim 1, wherein:

a. The first ISVD comprises a CDR1 comprising an amino acid sequence of SEQ ID NO: 6, a CDR2 comprising an amino acid sequence of SEQ ID NO: 10, and a CDR3 comprising an 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. 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. The polypeptide or composition according to any one of claims 1 or 2, wherein:

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 to SEQ ID NO: 3; and

c. The amino acid sequence of the third ISVD comprises a sequence identity greater than 90% identical to SEQ ID NO:4.

4. The polypeptide or composition according to any one of claims 1 to 3, wherein:

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. The third ISVD comprises the amino acid sequence of SEQ ID NO: 4.

5. A polypeptide or composition as described in any one in claims 1 to 4, wherein the polypeptide also comprises one or more other groups, residues, parts or binding units optionally connected via one or more peptide linkers, wherein the one or more other groups, residues, parts or binding units provide an increased half-life for the polypeptide compared to a corresponding polypeptide without the one or more other groups, residues, parts or binding units.

6. The polypeptide or composition for use as claimed in claim 5, wherein the one or more other groups, residues, parts or binding units that provide the polypeptide with an increased half-life are selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units capable of binding to serum proteins, Fc parts and small proteins or peptides capable of binding to serum proteins.

7. The polypeptide or composition of any one of claims 5 to 6, wherein the one or more other groups, residues, parts or binding units that provide the polypeptide with an increased half-life are selected from binding units that are capable of binding to serum albumin (such as human serum albumin) or serum immunoglobulins (such as IgG).

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

9. The polypeptide or composition of claim 8, wherein the fourth ISVD that binds to human serum albumin comprises

i. a CDR1 comprising the amino acid sequence of SEQ ID NO: 9 or having 2 or 1 amino acid differences with SEQ ID NO: 9;

ii. a CDR2 comprising the amino acid sequence of SEQ ID NO: 13 or having 2 or 1 amino acid differences with SEQ ID NO: 13; and

iii. A CDR3 comprising the amino acid sequence of SEQ ID NO: 17 or having 2 or 1 amino acid differences with SEQ ID NO: 17.

10. The polypeptide or composition of any one of claims 8 to 9, wherein the ISVD that binds to human serum albumin 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.

11. The polypeptide or composition of any one of claims 8 to 10, wherein the amino acid sequence of the ISVD that binds to human serum albumin comprises greater than 90% sequence identity to SEQ ID NO:5.

12. The polypeptide or composition of any one of claims 8 to 11, wherein the ISVD that binds to human serum albumin comprises the amino acid sequence of SEQ ID NO: 5.

13. The polypeptide or composition of any one of claims 1 to 12, wherein the amino acid sequence of the polypeptide comprises greater than 90% sequence identity with SEQ ID NO: 1.

14. The polypeptide or composition of any one of claims 1 to 13, 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 any one of claims 1 to 14.

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

17. A method for producing a polypeptide as claimed in any one of claims 1 to 14, said method comprising at least the following steps:

a. expressing the nucleic acid according to 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 according to any one of claims 1 to 14.

18. A composition comprising at least one polypeptide according to any one of claims 1 to 14 or a nucleic acid according to claim 15.

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 comprising one or more additional pharmaceutically active polypeptides and/or compounds.

20. A polypeptide as claimed in any one of claims 1 to 14 or a composition as claimed in any one of claims 18 or 19 for use as a medicament.

21. The polypeptide of any one of claims 1 to 14 or the composition of any one of claims 18 or 19 for use in the treatment of acute myeloid leukemia (AML).

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

23. A method for treating acute myeloid leukemia (AML), wherein the method comprises administering to a subject in need thereof a pharmaceutically active amount of a polypeptide as claimed in any one of claims 1 to 14 or a composition as claimed in any one of claims 18 or 19.

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

25. Use of the polypeptide according to any one of claims 1 to 14 or the composition according to any one of claims 18 or 19 in the preparation of a pharmaceutical composition for treating acute myeloid leukemia (AML).

26. Use of a polypeptide or composition according to claim 25, wherein the AML is relapsed and/or refractory AML.