TW202342508A - POLYPEPTIDES COMPRISING IMMUNOGLOBULIN SINGLE VARIABLE DOMAINS TARGETING TCRαβ, CD33 and CD123 - Google Patents

Abstract

The present technology aims at providing a novel type of drug for treating a subject suffering from acute myeloid leukemia (AML). Specifically, the present technology provides polypeptides comprising at least three immunoglobulin single variable domains (ISVDs), characterized in that at least one ISVDs binds to T cell receptor [alpha][beta] (TCR[alpha][beta]), at least ISVD binds to CD33, and at least one ISVD binds to CD123. The present technology also provides nucleic acids, vectors and compositions.

Description

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

The present technology relates to polypeptides targeting TCRαβ, CD33 and CD123. It also relates to nucleic acid molecules encoding these polypeptides and vectors containing these nucleic acids. The present technology further relates to compositions comprising such polypeptides, nucleic acids or vectors. The present technology also relates to such compositions for use in methods of treating subjects suffering from acute myelogenous leukemia (AML). Additionally, the present technology relates to methods of producing these compositions.

To this day, treatment of acute myelogenous leukemia (AML) remains challenging. Because of all immune cells, cytotoxic T cells appear to have the highest potential to treat malignant disease, AML therapeutic approaches aim to direct cytotoxic T cells to AML cells. The CD33 and CD123 antigens were found to be overrepresented on blasts and leukemic stem cells in most AML cases and were therefore used as suitable tumor-associated target antigens for antibody-based therapies. Mylotarg (ogemtuzumab; an anti-CD33 antibody drug conjugate) is the first targeted compound registered for the treatment of AML. Recent therapies are based on dual-specific antibody constructs targeting the tumor antigens CD33 or CD123 on AML cells and CD3 on cytotoxic T cells.

Therefore, bispecific antibodies can be anchored on CD33+ or CD123+ AML cells and simultaneously bind to CD3 on T cells. In this way, T cells are brought into close proximity with tumor cells. Multiple binding of bispecific antibodies to tumor antigens (CD33 or CD123) on tumor cells and simultaneously to TCR-associated CD3 molecules on T cells results in TCR clustering. This ultimately leads to efficient T cell activation independent of TCR specificity. Cytotoxic T cells near the AML cells are then primed, leading to tumor cell killing. Dual-specific antibody constructs currently being tested in clinical trials are, for example, Flotetuzumab (MGD006; CD3/CD123 DART), AMG330 or AMG673 (both CD3/CD33 BiTEs).

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

The inventors of the present invention discovered that a polypeptide (or ISVD construct) that dually targets CD33 and CD123 on acute myeloid leukemia (AML) cells in combination with targeting T cell receptor αβ (TCR αβ) on T cells leads to AML Effective killing of cells. Killing activity against CD33/CD123 dual-expressing cells is comparable to single-target benchmarks such as CD33/CD3 AMG 330 BiTE or CD123/CD3 MGD006 DART. However, the polypeptides of the invention showed potent killing activity against CD33 and CD123 single-expressing cells, whereas the single-targeting benchmark only showed 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, polypeptides of the present technology are efficiently produced (eg, in microbial hosts) and exhibit low viscosity at high concentrations, which is advantageous and convenient for subcutaneous administration. Furthermore, such polypeptides have limited reactivity with pre-existing antibodies in the subject to be treated (i.e., antibodies present in the subject prior to first treatment with the antibody construct). In preferred embodiments, such polypeptides exhibit a sufficiently long half-life in the subject to be treated such that successive treatments can be conveniently spaced apart.

Polypeptides of the present technology comprise or consist of at least three immunoglobulin single variable domains (ISVDs), wherein at least one ISVD specifically binds to TCRαβ, at least one ISVD specifically binds to CD33 and at least one ISVD CD123 binds specifically (exemplary polypeptides are shown in Figure 1 ). Preferably, said at least one ISVD binding to TCRαβ specifically binds to human TCRαβ, said at least one ISVD binding to CD33 specifically binds to human CD33 and said at least one ISVD binding to CD123 is specific to human CD123 Sexually combined.

The polypeptide preferably further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, which are identical to the absence of said one or more other groups, residues or binding units. The one or more other groups, residues, moieties or binding units increases the half-life of the polypeptide as compared to the corresponding polypeptide. For example, the binding unit may be an ISVD that binds to a serum protein, preferably to a human serum protein such as human serum albumin.

Also provided are a nucleic acid molecule capable of expressing the polypeptide of the present technology, a nucleic acid or vector comprising the nucleic acid, and a composition comprising the polypeptide, the nucleic acid or the vector. The composition is preferably a pharmaceutical composition.

Also provided is a host or host cell comprising a nucleic acid or vector encoding a polypeptide according to the present technology.

Also provided is a method for producing a polypeptide according to the technology of the present invention, the method at least comprising the following steps: a. Optionally express the nucleic acid sequence encoding the polypeptide according to the technology of the invention in a suitable host cell or host organism or in another suitable expression system, optionally followed by: b. Isolate and/or purify polypeptides according to the technology of the present invention.

Furthermore, the present technology provides the polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid or vector containing a nucleotide sequence encoding the polypeptide for use as a medicament. Preferably, the polypeptide or composition is used to treat acute myeloid leukemia (AML), wherein preferably the AML is relapsed and/or refractory AML.

Additionally, a method of treating AML is provided, wherein the method includes 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 preferred embodiments, the method further comprises administering one or more additional therapeutic agents.

Further provided is the use of polypeptides or compositions of the present technology in the preparation of pharmaceutical compositions for the treatment of AML, wherein preferably AML is relapsed and/or refractory AML.

Specifically, the technology of the present invention provides the following embodiments:

Example 1. A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding the polypeptide, the polypeptide or composition being used as a medicine, wherein the polypeptide comprises at least three or consisting of an immunoglobulin single variable domain (ISVD), wherein each said ISVD comprises three complementarity determining regions (CDR1 to CDR3 respectively) optionally connected via one or more peptide linkers; And among them: a) The first ISVD specifically binds to T cell receptor αβ (TCRαβ) and contains i. CDR1, which contains SEQ ID NO: 6 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 6; ii. CDR2 comprising SEQ ID NO: 10 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 10; and iii. CDR3, which includes SEQ ID NO: 14 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 14; b) The second ISVD specifically binds to CD33 and contains iv. CDR1, which contains SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acid; v. CDR2 comprising an amino acid sequence having SEQ ID NO: 11 or having a 2 or 1 amino acid difference from SEQ ID NO: 11; and vi. A CDR3 comprising SEQ ID NO: 15 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 15; and c) The third ISVD specifically binds to CD123 and contains vii. CDR1, which contains SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acid; viii. CDR2, which contains SEQ ID NO: 12 or an amino acid sequence that differs from SEQ ID NO: 12 by 2 or 1 amino acid; and ix. CDR3 comprising SEQ ID NO: 16 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 16, The order of the ISVD starts from the N-terminus.

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

Embodiment 3. The polypeptide or composition for the use according to Embodiment 1 or 2, wherein: a) The first ISVD includes CDR1 with the amino acid sequence of SEQ ID NO: 6, CDR2 with the amino acid sequence of SEQ ID NO: 10 and CDR3 with the amino acid sequence of SEQ ID NO: 14; b) the second ISVD comprises 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; and c) The third ISVD includes 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 the use according to any one of embodiments 1 to 3, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 2; b) the amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 3; and c) The amino acid sequence of the third ISVD has a sequence identity greater than 90% with SEQ ID NO: 4.

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

Embodiment 6. The polypeptide or composition for the use according to any one of embodiments 1 to 5, wherein the polypeptide further comprises one or more peptides optionally connected via one or more peptide linkers. other groups, residues, moieties or binding units, wherein said one or more other groups, residues, moieties or binding units are less significant than a corresponding polypeptide without said one or more other groups, residues, moieties or binding units. The base, moiety or binding unit increases the half-life of the polypeptide.

Embodiment 7. The polypeptide or composition for the use according to embodiment 6, wherein the one or more other groups, residues, moieties or binding units that increase the half-life of the polypeptide are selected from A group consisting of polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind to serum proteins.

Embodiment 8. The polypeptide or composition for the use according to any one of embodiments 6 to 7, wherein the one or more other groups, residues, which increase the half-life of the polypeptide, The moiety or binding unit is selected from the group of binding units that can bind to serum albumin (eg, human serum albumin) or serum immunoglobulin (eg, IgG).

Embodiment 9. The polypeptide or composition for the use according to embodiment 8, wherein the binding unit that increases the half-life of the polypeptide is an ISVD that can bind to human serum albumin.

Embodiment 10. The polypeptide or composition for the use according to embodiment 9, wherein the ISVD that binds to human serum albumin comprises i. CDR1, which contains SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 9; ii. CDR2 comprising SEQ ID NO: 13 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3, which comprises SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acid.

Embodiment 11. The polypeptide or composition for the use according to any one of embodiments 9 to 10, wherein the ISVD that binds to human serum albumin comprises the amino acid having SEQ ID NO: 9 CDR1 of the sequence, 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 the use according to any one of embodiments 9 to 11, wherein the amino acid sequence of the ISVD that binds to human serum albumin has the same amino acid sequence as SEQ ID NO: 5 Greater than 90% sequence identity.

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

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

Embodiment 15. The polypeptide or composition for the 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 the use according to any one of claims 1 to 15, which is used to treat AML.

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

Example 18. A polypeptide comprising or consisting of at least three immunoglobulin single variable domains (ISVDs), wherein each of said ISVDs comprises, optionally linked via one or more peptide linkers Three complementarity determining regions (CDR1 to CDR3 respectively); and among them: a) The first ISVD specifically binds to T cell receptor αβ (TCRαβ) and contains i. CDR1, which contains SEQ ID NO: 6 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 6; ii. CDR2 comprising SEQ ID NO: 10 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 10; and iii. CDR3, which includes SEQ ID NO: 14 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 14; b) The second ISVD specifically binds to CD33 and contains iv. CDR1, which contains SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acid; v. CDR2 comprising SEQ ID NO: 11 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 11; and vi. A CDR3 comprising SEQ ID NO: 15 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 15; and c) The third ISVD specifically binds to CD123 and contains vii. CDR1, which contains SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acid; viii. CDR2 comprising SEQ ID NO: 12 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 12; and ix. CDR3 comprising SEQ ID NO: 16 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 16, The order of the ISVD starts from the N-terminus.

Embodiment 19. The polypeptide according to embodiment 18, wherein: a) The first ISVD includes CDR1 with the amino acid sequence of SEQ ID NO: 6, CDR2 with the amino acid sequence of SEQ ID NO: 10 and CDR3 with the amino acid sequence of SEQ ID NO: 14; b) the second ISVD comprises 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; and c) The third ISVD includes 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 20. The polypeptide according to any one of embodiments 18 or 19, wherein: a) The amino acid sequence of the first ISVD has greater than 90% sequence identity with SEQ ID NO: 2; b) the amino acid sequence of the second ISVD has greater than 90% sequence identity with SEQ ID NO: 3; and c) The amino acid sequence of the third ISVD has a sequence identity greater than 90% with SEQ ID NO: 4.

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

Embodiment 22. The polypeptide of any one of embodiments 18 to 21, wherein the polypeptide further comprises one or more other groups, residues, moieties, optionally connected via one or more peptide linkers or a binding unit, wherein said one or more other groups, residues, moieties or binding units renders said The half-life of the peptide is increased.

Embodiment 23. The polypeptide of embodiment 22, wherein the one or more other groups, residues, moieties or binding units that increase the half-life of the polypeptide are selected from the group consisting of polyethylene glycol molecules, serum proteins or A group consisting of its fragments, binding units that can bind to serum proteins, Fc portions, and small proteins or peptides that can bind 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 that increase the half-life of the polypeptide are selected from the group consisting of compounds that are compatible with serum A group of binding units that bind to albumin (such as human serum albumin) or serum immunoglobulin (such as IgG).

Embodiment 25. The polypeptide of embodiment 24, wherein the binding unit that increases the half-life of the polypeptide is an ISVD that can bind to human serum albumin.

Embodiment 26. The polypeptide of embodiment 25, wherein the ISVD that binds to human serum albumin comprises i. CDR1, which contains SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 9; ii. CDR2 comprising SEQ ID NO: 13 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3, which comprises SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acid.

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

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

Embodiment 29. The polypeptide according to 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 greater than 90% sequence identity with SEQ ID NO: 1.

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

Embodiment 32. A nucleic acid comprising a nucleotide sequence encoding a 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 the polypeptide according to any one of embodiments 18 to 31, the method at least comprising the following steps: a) expressing the nucleic acid according to Example 32 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 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 according to Embodiment 35, which is a pharmaceutical composition, and the pharmaceutical composition further includes at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant. agent, and optionally includes 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 a pharmaceutically active amount of a polypeptide according to any one of claims 18 to 31 or a polypeptide according to claims 35 to 36 The composition of any one.

Embodiment 38. The method of 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 in 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.

The technology of the present invention aims to provide new drugs for the treatment of acute myeloid leukemia (AML).

The inventors of the present invention discovered that a polypeptide (or ISVD construct) that dually targets CD33 and CD123 on acute myeloid leukemia (AML) cells in combination with targeting T cell receptor αβ (TCR αβ) on T cells leads to AML Effective killing of cells. Killing activity is comparable or even higher than single-target benchmarks such as CD33/CD3 AMG 330 BiTE or CD123/CD3 MGD006 DART. Due to the high heterogeneity of CD33 and CD123 expression on AML cells within and between patient samples, the polypeptides of the invention provide broader patient coverage compared to single-target benchmarks.

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

The polypeptide is at least bispecific, but may also be trispecific, tetraspecific or pentaspecific, for example. Furthermore, the polypeptide is at least tetravalent, but may also be, for example, pentavalent or hexavalent, among others.

The terms " bispecific ,"" trispecific ,"" tetraspecific, " or " pentaspecific " all fall within the term " multispecific " and refer to two, three, four, or five species, respectively. combination of different target molecules. The terms " bivalent ", " trivalent ", " tetravalent ", " pentavalent " or " hexavalent " fall within the scope of the term " polyvalent " and mean two, three, four or five, respectively. The existence of binding units (such as ISVD). For example, the polypeptide may be tetraspecific-tetravalent, such as a polypeptide comprising or consisting of four ISVDs, one ISVD binding to human TCRαβ, one ISVD binding to human CD33, one ISVD binding to CD123 and one ISVD Binds to human serum albumin (ISVD construct as shown in SEQ ID NO: 1). For example, if two ISVDs bind to two different epitopes on the same target, for example, if two ISVDs bind to TCRαβ, the polypeptide may be biparatopic at the same time. The term " biparatope " refers to binding to two different parts (eg, epitopes) of the same target molecule.

As used herein, the terms "first ISVD", "second ISVD", "third ISVD" and the like simply refer to the relative position of the ISVDs to each other, where numbering begins with the N-terminus of the polypeptide of the invention. Therefore, the "first ISVD" is closer to the N-terminus than the "second ISVD", and the "second ISVD" is closer to the N-terminus than the "third ISVD", and so on. Therefore, the ISVD arrangement is reversed when considered from the C terminus. Since the numbering is not absolute and only represents the relative position of the at least three ISVDs, it is not excluded that there may be other binding units/building blocks in the polypeptide, such as additional ISVDs that bind to TCRαβ, CD33 or CD123, or to another Target-binding ISVD. For example, as described further below (see in particular the section "(In Vivo) Half-life Extension"), the polypeptide may further comprise another ISVD that binds to human serum albumin, which may be the C-terminus of at least three ISVDs. Four ISVDs. Furthermore, the possibility that other binding units/building blocks (such as ISVD) could be placed in between is not excluded. For example, the polypeptide may further comprise another ISVD which may even be located between, for example, a "second ISVD" and a "third ISVD".

In view of the above, the present invention provides polypeptides comprising or consisting of at least three ISVDs, wherein at least one ISVD specifically binds to TCRαβ, at least one ISVD specifically binds to CD33 and at least one ISVD specifically binds to CD123.

The components of the polypeptide (preferably ISVD) can be linked to each other via one or more suitable linkers (eg 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 type of commonly used peptide linker is called "Gly-Ser" or "GS" linker. These are linkers consisting essentially of glycine (G) and serine (S) residues and typically contain one or more repeats of a peptide motif such as GGGGS (SEQ ID NO: 77) A motif (eg, having the formula (Gly-Gly-Gly-Gly-Ser)n, where n can 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, for example, Chen et al., Adv. Drug Deliv. Rev. 2013 Oct 15;65(10):1357–1369; and Klein et al., Protein Eng. Des. Sel. (2014) 27(10):325 -330. In the polypeptides of the invention, it is preferred to use a 9GS linker to link the components of the polypeptide to each other.

In preferred embodiments, the ISVD that specifically binds TCRαβ is located at the N-terminus of the polypeptide. The inventors 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 C terminus of ISVD.

Therefore, preferably, the polypeptide comprises or consists of the following in order starting from the N-terminus of the polypeptide: a first ISVD that specifically binds to TCRαβ, a second ISVD that specifically binds to CD33, and a second ISVD that specifically binds to CD123 A third ISVD that binds selectively and an optional binding unit that provides the polypeptide with an increased half-life as defined herein. The binding unit that increases the half-life of the polypeptide is preferably an ISVD.

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

Such configurations of the polypeptides may provide increased production yields, good CMC characteristics as well as optimized functionality and greater potency with respect to the 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 in at least one ISVD, but preferably in each ISVD, methamine (V) at amino acid position 11 and leucine at amino acid position 89 Acid (L) (according to Kabat number). In another embodiment, the polypeptide comprises a stretch of 1 to 5 (preferably naturally occurring) amino acids at the C-terminus of the C-terminal ISVD, such as a single alanine (A) stretch. The C-terminus of ISVD is usually VTVSS (SEQ ID NO: 93). In another embodiment, the polypeptide comprises lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVDs comprise lysine (K) or glutamic acid (Q) at position 112 (according to Kabat numbering) in at least one ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 94), VQVSS (SEQ ID NO: 95), VTVKS (SEQ ID NO: 96), VTVQS (SEQ ID NO: 97), VKVKS (SEQ ID NO: 98), VKVQS (SEQ ID NO: 99), VQVKS (SEQ ID NO: 100) or VQVQS (SEQ ID NO: 101), such that upon addition of a single alanine, the C-terminus of the polypeptide is e.g. Contains the sequences 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, in each ISVD, a jutamine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering), either Optionally comprise lysine (K) or glutamic acid (Q) at position 110 (according to Kabat numbering) in at least one ISVD and at the C-terminus the C-terminus of the ISVD from 1 to 5 (preferably naturally occurring ) amino acid extension, such as a single alanine (A) extension (such that the C-terminus of the polypeptide includes, 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 greater than 90% (eg greater than 95% or greater than 99%) sequence identity with SEQ ID NO: 1, wherein four ISVD The CDRs are as defined by items A to D (or A' to D', if Kabat definitions are used) shown in the following sections "Immunoglobulin single variable domains" and "(In vivo) half-life extension" respectively, where in particular land: • The ISVD that specifically binds to TCRαβ has CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 10, and CDR2 containing the amino acid sequence of SEQ ID NO: 14 CDR3; • The ISVD that specifically binds to CD33 has a CDR1 containing the amino acid sequence of SEQ ID NO: 7, a CDR2 containing the amino acid sequence of SEQ ID NO: 11, and a CDR2 containing the amino acid sequence of SEQ ID NO: 15. CDR3; • The ISVD that specifically binds to CD123 has a CDR1 containing the amino acid sequence of SEQ ID NO: 8, a CDR2 containing the amino acid sequence of SEQ ID NO: 12, and a CDR2 containing the amino acid sequence of SEQ ID NO: 16 CDR3; and • The ISVD that binds to human serum albumin has a CDR1 containing the amino acid sequence of SEQ ID NO: 9, a CDR2 containing the amino acid sequence of SEQ ID NO: 13, and a CDR2 containing the amino acid sequence of SEQ ID NO: 17 CDR3, Or alternatively, if using Kabat definition: • The ISVD that specifically binds to TCRαβ has CDR1 containing the amino acid sequence of SEQ ID NO: 34, CDR2 containing the amino acid sequence of SEQ ID NO: 38, and CDR1 containing the amino acid sequence of SEQ ID NO: 42 CDR3; • The ISVD that specifically binds to CD33 has CDR1 containing the amino acid sequence of SEQ ID NO: 35, CDR2 containing the amino acid sequence of SEQ ID NO: 39, and CDR1 containing the amino acid sequence of SEQ ID NO: 43. CDR3; • The ISVD that specifically binds to CD123 has CDR1 containing the amino acid sequence of SEQ ID NO: 36, CDR2 containing the amino acid sequence of SEQ ID NO: 40, and CDR1 containing the amino acid sequence of SEQ ID NO: 44. CDR3; and • The ISVD that binds to human serum albumin has a CDR1 containing the amino acid sequence of SEQ ID NO: 37, a CDR2 containing the amino acid sequence of SEQ ID NO: 41, and a CDR2 containing the amino acid sequence of SEQ ID NO: 45. CDR3.

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

Compared with the polypeptide consisting of the amino acids of SEQ ID NO: 1, the polypeptide of the present invention preferably has at least half the binding affinity to human TCRαβ and to human CD33 and to human CD123, and more preferably at least the same binding affinity, Where the binding affinity is measured using the same method (eg SPR).

5.1 Immunoglobulin single variable domain

The term "immunoglobulin single variable domain" (ISVD) is used interchangeably with "single variable domain" and defines an immunoglobulin in which the antigen-binding site is present on and formed from a single immunoglobulin domain. molecular. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (e.g. monoclonal antibodies) or fragments thereof (e.g. Fab, Fab', F(ab') ₂ , scFv, di-scFv), where Two immunoglobulin domains, particularly two variable domains, interact to form an antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain ( _VH ) and the light chain variable domain (VL ₎ interact to form the antigen-binding site. In this case, the complementarity determining regions (CDRs) of both _VH and _VL will contribute to the antigen-binding site, that is, a total of 6 CDRs will participate in the formation of the antigen-binding site.

In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or a Fab fragment, F(ab') derived from such a conventional 4-chain antibody ₂ fragments, Fv fragments (such as disulfide-linked Fv or scFv fragments) or diabodies (all known in the art) will generally not be considered immunoglobulin single variable domains because in these cases, It is not a (single) immunoglobulin domain that binds to the corresponding antigenic epitope, but a pair (associated) immunoglobulin domains (such as light chain and heavy chain variable domains), that is, the immunoglobulin structure Binding to the corresponding antigenic epitope occurs with the _VH - _VL pairs of the domains, which collectively bind to the corresponding antigenic epitope.

In contrast, immunoglobulin single variable domains are capable of specifically binding to antigenic epitopes without pairing with additional immunoglobulin variable domains. The binding site of an immunoglobulin single variable domain is formed by a single _VH , a single _VHH , or a single _VL domain.

Thus, the single variable domain may be a light chain variable domain sequence (e.g., a _VL sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a _VH sequence or a _VHH sequence) or a suitable fragment thereof; as 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 the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit).

An immunoglobulin single variable domain (ISVD) may be, for example, a heavy chain ISVD, such as _VH , _VHH , including camelid _VH or humanized _VHH . Preferably, it is a _VHH , including camelid _VH or humanized _VHH . Heavy chain ISVD can be derived from conventional quadribodies 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), a "dAb" or a dAb (or an amino acid sequence suitable for use as a dAb) ( As defined herein, and including, but not limited to, NANOBODY® ISVD); other single variable domains, or any suitable fragment of any of them.

In particular, the immunoglobulin single variable domain may be a NANOBODY® ISVD (including humanized _VHH or camelidized _VH ) or a suitable fragment thereof. [Note: NANOBODY®, NANOBODIES® and NANOCLONE® are registered trademarks of Ablynx NV]

" _VHH domain", also known as _VHH , _VHH antibody fragment and _VHH antibody, has originally been described as "heavy chain antibody" (i.e., "light chain antibody"; Hamers-Casterman et al., Nature 363 : 446-448, 1993) Antigen-binding immunoglobulin variable domains. The term " _V domain" has been chosen to distinguish these variable domains from the heavy chain variable domains found in conventional 4-chain antibodies (which are referred to herein as " _V domains") and from conventional 4-chain antibodies. The light chain variable domain (which is referred to herein as the " _VL domain") is distinguished from the light chain variable domain present in the VL domain. For further description of _VHH , reference is made to Muyldermans' review article (Reviews in Molecular Biotechnology 74: 277-302, 2001), and to the following patent application mentioned as general background: WO 94 of Vrije Universiteit Brussel /04678, WO 95/04079 and WO 96/34103; Unilever’s 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; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and WO 03/055527 from Vlaams Instituut voor Biotechnologie (VIB), Belgium; WO 03/050531 to Algonomics NV and Ablynx NV; WO 01/90190 to the National Research Council of Canada; WO 03/025020 (= EP 1433793) to the Institute of Antibodies; and Ablynx NV 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.

Typically, immunoglobulin production involves immunization of experimental animals, fusion of immunoglobulin-producing cells to produce hybridomas, and screening for desired specificity. Alternatively, immunoglobulins can be generated by screening natural or synthetic libraries (eg, by phage display).

The generation of immunoglobulin sequences such as NANOBODY® ISVD has been extensively described in various publications, among them WO 94/04678, Hamers-Casterman et al., 1993, and Muyldermans et al., 2001 (reviewed in Molecular Biotechnology 74: 277-302 , 2001) can serve as an illustration. In these methods, a camelid is immunized with a target antigen to induce an immune response against the target antigen. The library of NANOBODY® ISVDs obtained from the immunizations is further screened for ISVDs that bind the target antigen.

In these cases, antibody generation requires purified antigen for immunization and/or screening. Antigens can be purified from natural sources or during recombinant production.

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

Immunoglobulin sequences from various sources may be used in the present invention, including mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The invention also encompasses fully human, humanized or chimeric sequences. For example, the invention includes camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelidized domain antibodies, such as camelidized 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). In addition, the present invention also uses fused immunoglobulin sequences, for example to form multivalent and/or multispecific constructs (for multivalent and multispecific polypeptides containing one or more _VHH domains and their preparation, also See Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, and see, e.g., WO 96/34103 and WO 99/23221); and derivable from the immunoglobulin sequences of the invention Immunoglobulin sequences that contain tags or other functional moieties (e.g., toxins, labels, radioactive chemicals, etc.).

"Humanized _VHH " includes an amino acid sequence corresponding to the amino acid sequence of a naturally occurring _VHH domain but that has been "humanized", i.e., by converting said naturally occurring _VHH sequence (and in particular the structure sequence), one or more amino acid residues in the amino acid sequence of a conventional 4-chain antibody from human (e.g., shown above) are present at one or more corresponding positions in the _VH domain Humanized by substitution of one or more amino acid residues. This can be done in a manner known per se, as will be clear to a person of ordinary skill in the art, for example on the basis of the further description herein and the prior art (eg WO 2008/020079). Furthermore, it should be noted that such humanized _VHHs may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring VHH domains as starting material.

"Camelized _VH " includes an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring _VH domain but has been "camelized", i.e., by combining naturally occurring _VH from a conventional 4-chain antibody One or more amino acid residues in the amino acid sequence of the domain are replaced with one or more amino acid residues present at one or more corresponding positions in the _VHH domain of the heavy chain antibody. Generalization. This can be done in a manner known per se, as will be clear to a person of ordinary skill in the art, for example on the basis of the further description herein and the prior art (eg WO 2008/020079). Such "camelized" substitutions, as defined herein, are preferably inserted at the amino acid positions formed and/or present at the _VH - _VL junction and/or at so-called camelid signature residues, As defined herein (see eg WO 94/04678 and Davies and Riechmann (1994 and 1996), supra). Preferably, the _VH sequence used as starting material or starting point for generating or designing camelidized VHs is preferably a _VH sequence from a mammal, more preferably _a human _VH sequence, such as a _VH3 sequence. It should be noted, however, that such camelidized _VHs may be obtained in any suitable manner known per se and are therefore not strictly limited to polypeptides that have been obtained using polypeptides comprising naturally occurring _VH domains as starting material.

It should be noted that one or more immunoglobulin sequences can 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). See, for example, the review by Holliger and Hudson, Nat Biotechnol. 2005 Sep;23(9):1126-36. Generally, where a polypeptide is intended for administration to a subject (eg for prophylactic, therapeutic and/or diagnostic purposes), it preferably comprises an immunoglobulin that is not naturally occurring in the subject. sequence.

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

As further described in paragraph q) on pages 58 and 59 of WO 08/020079 (incorporated by reference), the amino acid residues of the immunoglobulin single variable domain can be determined according to the method described by Kabat et al. (" Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91) are numbered according to the universal numbering used for _VH domains, as in Riechmann and Muyldermans, 2000 (J. Immunol. Methods 240 (1-2): 185-195; see, for example, Figure 2 of this publication as applied to V _HH domains from camelids. It should be noted that, as is well known in the art for _VH domains and _VHH domains, the total number of amino acid residues in each CDR can vary and may not correspond to the amino acid residues represented by Kabat numbering of the total number (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 number allowed by Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in the _VH domain and _VHH domain typically ranges from 110 to 120, often ranging between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.

In this application, unless otherwise stated, CDR sequences are as described in Kontermann and Dübel (eds. 2010, Antibody Engineering, Vol. 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51) The AbM number is determined. According to this method, FR1 contains the amino acid residues at positions 1-25, CDR1 contains the amino acid residues at positions 26-35, FR2 contains the amino acid residues at positions 36-49, and CDR2 contains the amino acid residues at positions 50-58 FR3 contains the amino acid residues at positions 59-94, CDR3 contains the amino acid residues at positions 95-102, and FR4 contains the amino acid residues at positions 103-113 .

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

In such immunoglobulin sequences, the architecture sequence may be any suitable architecture sequence, and examples of suitable architecture sequences are of common knowledge, for example based on standard manuals and additional disclosures and prior art mentioned herein. will be clear.

The architecture sequence is preferably an immunoglobulin architecture sequence or (a suitable combination of) architecture sequences derived from an immunoglobulin architecture sequence (eg, by humanization or camelidization). For example, the framework sequence may be a framework sequence derived from a light chain variable domain (eg, a _VL sequence) and/or a heavy chain variable domain (eg, a _VH sequence or a _VHH sequence). In a particularly preferred aspect, the architectural sequence is an architectural sequence derived from a _VHH sequence (wherein the architectural sequence may optionally have been partially or fully humanized) or a conventional _VH sequence that has been camelized (as herein defined).

In particular, the architectural sequences present in ISVD sequences used in the present invention may contain one or more marker residues (as defined herein) such that the ISVD sequence is a NANOBODY® immunoglobulin variable domain, including humanized _VHH Or camelized V _H . Some preferred but non-limiting examples of (suitable combinations of) such architectural sequences will become clear 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 containing suitably flanked by one or more architectural sequences and/or via one or more A fragment of one or more CDR sequences to which the architecture sequence is linked (eg, in the same order in which these CDR and architecture sequences would appear in the full-size immunoglobulin sequence from which the fragment was derived).

It should be noted, however, that with respect to the origin of the ISVD sequence (or the nucleotide sequence used to represent said ISVD sequence), and with respect to the manner in which said ISVD sequence or nucleotide sequence is generated or obtained (or has been generated or obtained), The invention is not limited. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In specific but non-limiting aspects, the ISVD sequences are naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as 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 modified by technology ( Such as affinity maturation (e.g. starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, surface dressing, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and general knowledge or any suitable combination of any of the foregoing.

Similarly, the nucleotide sequence may be a naturally occurring nucleotide sequence or a synthetic or semi-synthetic sequence, and may be, for example, a sequence isolated by PCR from a suitable naturally occurring template (eg, DNA or RNA isolated from a cell) , nucleotide sequences that have been isolated from libraries (and in particular expression libraries), nuclei that have been prepared by introducing mutations into naturally occurring nucleotide sequences (using any suitable technique known per se, such as mismatch PCR) A nucleotide sequence, 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 mentioned above, the ISVD may be NANOBODY® V _HH or a suitable fragment thereof. For a general description of NANOBODY® ISVD, reference is made to the further description below and to the prior art cited herein. In this regard, however, it should be noted that this description and the prior art primarily describe so-called " _VH class 3" NANOBODY® ISVDs (i.e., human germline sequences with _VH class 3 such as DP-47, DP-51 or DP-29 has high sequence homology to NANOBODY® ISVD). However, it should be noted that the present invention in its broadest sense can generally use any type of NANOBODY® ISVD and for example also use NANOBODY® ISVD belonging to the so-called "V _H 4 category" (i.e. human race with the V _H 4 category). NANOBODY® ISVDs with high sequence homology to sequences such as DP-78, as described for example in WO 2007/118670.

In general, NANOBODY® ISVDs (particularly _VHH sequences, including (parts of) humanized _VHH sequences and camelidized _VH sequences) may be characterized by the presence in one or more architectural sequences (also as further described herein) One or more "Hallmark residues" (as described herein). Therefore, in general, a NANOBODY® ISVD can be defined as an immunoglobulin sequence with the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where FR1 to FR4 refer to architectural 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 marker residues are as further defined herein.

In particular, NANOBODY® ISVD can be an immunoglobulin sequence with the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 wherein FR1 to FR4 refer to architectural regions 1 to 4, respectively, and wherein CDR1 to CDR3 refer to complementarity determining regions 1 to 3, respectively, and wherein the architectural sequence is as further defined herein.

More specifically, a NANOBODY® ISVD may be an immunoglobulin sequence with the following (general) structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 where FR1 to FR4 refer to architectural regions 1 to 4 respectively, and where CDR1 to CDR3 refers respectively to complementarity determining regions 1 to 3, and where: one of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to Kabat numbering or more selected from the marker residues mentioned in Table 1 below.

Table 1 : Hallmark residues in NANOBODY® ISVD Location People V _H 3 Flag residue 11 L, V; mainly L L, S, V, M, W, F, T, Q, E, A, R, G, K, Y, N, P, I; preferably L 37 V, I, F; usually V F ⁽¹⁾ , Y, V, L, A, H, S, I, W, C, N, G, D, T, P; preferably F ⁽¹⁾ or Y 44 ⁽⁸⁾ G E ⁽³⁾ , Q ⁽³⁾ , G ⁽²⁾ , D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G ⁽²⁾ , E ⁽³⁾ or Q ⁽³⁾ ; more preferably G ⁽²⁾ or Q ⁽³⁾ . 45 ⁽⁸⁾ L L ⁽²⁾ , R ⁽³⁾ , P, H, F, G, Q, S, E, T, Y, C, I, D, V; preferably L ⁽²⁾ or R ⁽³⁾ 47 ⁽⁸⁾ W.Y F ⁽¹⁾ , L ⁽¹⁾ or W ⁽²⁾ , G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferred W ⁽²⁾ , L ⁽¹⁾ or F ⁽¹⁾ 83 R or K; usually R R, K ⁽⁵⁾ , T, E ⁽⁵⁾ , Q, N, S, I, V, G, M, L, A, D, Y, H; preferably K or R; most preferably K 84 A, T, D; mainly A P ⁽⁵⁾ , S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E; preferably P 103 W W ⁽⁴⁾ , R ⁽⁶⁾ , G, S, K, A, M, Y, L, F, T, N, V, Q, P ⁽⁶⁾ , E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108 L, M or T; mainly L Q, L ⁽⁷⁾ , R, P, E, K, S, T, M, A, H; preferably Q or L ⁽⁷⁾ Notes: (1) Especially, but not exclusively, in combination with KERE or KQRE at positions 43-46. (2) Typically GLEW at positions 44-47. (3) Typically KERE or KQRE at positions 43-46, such as KEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47. Alternatively, sequences such as TERE (for example TEREL), TQRE (for example TQREL), KECE (for example KECEL or KECER), KQCE (for example KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREF or RQREW), QERE (such as QEREG), QQRE (such as QQREW, QQREL or QQREF), KGRE (such as KGREG), KDRE (such as KDREV). Some other possible but less preferred sequences include, for example, DECKL and NVCEL. (4) With two GLEWs at positions 44-47, and KERE or KQRE at positions 43-46. (5) Often KP or EP at positions 83-84 of the naturally occurring V _HH domain. (6) Especially, but not exclusively, in combination with GLEW at positions 44-47. (7) Provided that when positions 44-47 are GLEW, position 108 in the (non-humanized) V _HH sequence, which also contains a W at 103, is always Q. (8) The GLEW group also contains GLEW-like sequences at positions 44-47, such as, for example, GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER, and ELEW.

The present technology specifically utilizes ISVDs that specifically bind to TCRαβ, CD33 or CD123. In the context of the present technology, "binding" to a specific target molecule has the usual meaning in the art as understood in the context of antibodies and their corresponding antigens.

Polypeptides of the present technology may comprise one or more ISVDs that specifically bind to TCRαβ, one or more ISVDs that specifically bind to CD33, and one or more ISVDs that specifically bind to CD123.

The ISVDs used in the present technology form part of a polypeptide of the present technology that contains or consists of at least three ISVDs such that the polypeptide can specifically bind TCRαβ, CD33, and CD123. Therefore, the polypeptide can be anchored on CD33+CD123+ leukemia stem cells (LSC) and acute myeloid leukemia (AML) blast cells, and can simultaneously bind to TCRαβ on cytotoxic T cells. In this way, the peptide brings T cells into close proximity with LSC and AML blasts. Multiple binding of polypeptides to tumor antigens (CD33 and CD123) on tumor cells and simultaneous binding to TCR molecules on individual T cells results in TCR clustering and ultimately T cell priming. T cell priming in the vicinity of LSCs and AML blasts leads to efficient tumor cell killing, resulting in therapeutic efficacy in AML patients.

Accordingly, the target molecules of the at least three ISVDs as used in the polypeptides of the present technology are TCRαβ, 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 mouse, rat, rabbit, cat, dog, goat, sheep, The TCRαβ, CD33, and CD123 of horses, pigs, nonhuman primates (such as cynomolgus monkeys) (also referred to herein as “ cynos ”), or camelids (such as llamas or alpacas) are also suitable for technology of the present invention.

Specific examples of ISVDs that specifically bind to TCRαβ, CD123, and CD123 that may be used in the present technology are as described in Items A to C below:

A. The following ISVD, which specifically binds to human TCRαβ and contains i. CDR1, which contains SEQ ID NO: 6 or an amino acid sequence that differs from SEQ ID NO: 6 by 2 or 1 amino acid; ii. CDR2 comprising SEQ ID NO: 10 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 10; and iii. CDR3 comprising SEQ ID NO: 14 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 14, Preferably, it is 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 ISVDs that specifically bind to human TCRαβ have one or more (and preferably all) architectural regions as represented in Table A-2 for TCRαβ-V _HH (and as defined in the preceding item A CDR), and most preferably an ISVD having the complete amino acid sequence of TCRαβ- _VHH (SEQ ID NO: 2; see Table A-1 and Table A-2).

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

When such an ISVD that specifically binds to TCRαβ has a 2 or 1 amino acid difference relative to the corresponding reference CDR sequence in at least one CDR (item A above), it is compatible with TCRαβ-V _HH (SEQ ID NO: 2) The ISVD preferably has at least half the binding affinity to human TCRαβ, more preferably at least the same binding affinity, where the binding affinity is measured using the same method (such as SPR).

B. The following ISVD, which specifically binds to human CD33 and contains i. CDR1, which contains SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acid; ii. CDR2 comprising SEQ ID NO: 11 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 11; and iii. CDR3 comprising SEQ ID NO: 15 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 15, Preferably, it is 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 ISVDs that specifically bind to human CD33 have one or more (and preferably all) architectural regions as represented in Table A-2 for CD33-V _HH (and as previously defined in Item B CDR), and most preferably an ISVD having the complete amino acid sequence of CD33-V _HH (SEQ ID NO: 3, see Tables A-1 and A-2).

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

When such an ISVD that specifically binds to CD33 has a 2 or 1 amino acid difference relative to the corresponding reference CDR sequence in at least one CDR (item B above), it is consistent with construct CD33-V _HH (SEQ ID NO: 3) The ISVD preferably has at least half the binding affinity to human CD33, more preferably at least the same binding affinity, wherein the binding affinity is measured using the same method (such as SPR).

C. The following ISVD, which specifically binds to human CD123 and contains i. CDR1, which contains SEQ ID NO: 8 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 8; ii. CDR2 comprising SEQ ID NO: 12 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 12; and iii. CDR3 comprising SEQ ID NO: 16 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 16, Preferably, it is 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 ISVDs that specifically bind to human CD123 have one or more (and preferably all) architectural regions as represented in Table A-2 for CD123-V _HH (and as previously defined in Item C CDR), and most preferably an ISVD having the complete amino acid sequence of CD123-V _HH (SEQ ID NO: 4, see Tables A-1 and A-2).

Furthermore, in a preferred embodiment, the amino acid sequence of the ISVD that specifically binds to human CD123 may have greater than 90% (such as greater than 95% or greater than 99%) sequence identity with SEQ ID NO: 4, wherein Optionally the CDRs are as defined in item C above. In particular, the ISVD that binds to CD123 preferably has the amino acid sequence of SEQ ID NO: 4.

When such an ISVD that specifically binds to CD123 has 2 or 1 amino acid difference relative to the corresponding reference CDR sequence in at least one CDR (item C above), it is consistent with CD123-V _HH (SEQ ID NO: 4) The ISVD preferably has at least half the binding affinity for human CD123, more preferably at least the same binding affinity, where the binding affinity is measured using the same method (such as SPR).

Preferably, each of the ISVDs as defined under items A to C above is comprised in the polypeptide of the invention. Compared to a polypeptide consisting of the amino acid of SEQ ID NO: 1, such a polypeptide of the present invention comprising each of the ISVDs as defined under items A to C above is effective for human TCRαβ, for human CD33 and to human CD123 preferably have at least half the binding affinity, more preferably at least the same binding affinity, where the binding affinity is measured using the same method (eg SPR).

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

Therefore, specific ISVDs that specifically bind to TCRαβ, CD33 or CD123 for use in the present invention can be described using the AbM definition as described above or using the Kabat definition as described in items A' to C' below:

A’. The following ISVD, which specifically binds to human TCRαβ and contains i. CDR1, which contains SEQ ID NO: 34 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 34; ii. CDR2 comprising SEQ ID NO: 38 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 38; and iii. CDR3 comprising SEQ ID NO: 42 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 42, Preferably, it is 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 ISVDs that specifically bind to human TCRαβ have one or more (and preferably all) architectural regions as represented in Table A-2.1 for TCRαβ-V _HH (and as described in the preceding item A' defined CDR), and most preferably an ISVD having the complete amino acid sequence of TCRαβ- _VHH (SEQ ID NO: 2; see Table A-1 and Table A-2.1).

B’. The following ISVD, which specifically binds to human CD33 and contains i. CDR1, which contains SEQ ID NO: 35 or an amino acid sequence that differs from SEQ ID NO: 35 by 2 or 1 amino acid; ii. CDR2 comprising SEQ ID NO: 39 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 39; and iii. CDR3 comprising SEQ ID NO: 43 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 43, Preferably, it is 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 ISVDs that specifically bind to human CD33 have one or more (and preferably all) architectural regions as represented in Table A-2.1 for CD33-V _HH (and as previously represented in item B' defined CDR), and most preferably an ISVD having the complete amino acid sequence of CD33-V _HH (SEQ ID NO: 3, see Tables A-1 and A-2.1).

C’. The following ISVD, which specifically binds to human CD123 and contains i. CDR1, which contains SEQ ID NO: 36 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 36; ii. CDR2 comprising SEQ ID NO: 40 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 40; and iii. CDR3 comprising SEQ ID NO: 44 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 44, Preferably, it is 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 ISVDs that specifically bind to human CD123 have one or more (and preferably all) architectural regions as represented in Table A-2.1 for CD123-V _HH (and as previously represented in item C' defined CDR), and most preferably an ISVD with 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: ([ The amino acid residues in the first amino acid sequence and the amino acid residues in the second amino acid sequence The same number of amino acid residues at the corresponding positions ]/[ the total number of amino acid residues in the first amino acid sequence ]) × 100% , where compared with the first amino acid sequence, in the second Each deletion, insertion, substitution, or addition of an amino acid residue in an amino acid sequence is considered a difference at a single amino acid residue (ie, at a single position).

Typically, 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 greatest number of amino acid residues is considered the "first" amino acid sequence, and another amino acid sequence as the "second" amino acid sequence.

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

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

Particularly preferred conservative substitutions are as follows: Ala is replaced by Gly or replaced by Ser; Arg is replaced by Lys; Asn is replaced by Gln or replaced by His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or replaced by Pro; His is replaced by Asn or replaced by Gln; Ile is replaced by Leu or replaced by Val; Leu is replaced by Ile or replaced by Val; Lys is replaced by Arg, replaced by Gln or replaced by Glu; Met Substituted for Leu, substituted for Tyr or substituted for Ile; Phe substituted for Met, substituted for Leu or substituted for Tyr; Ser substituted for Thr; Thr substituted for Ser; Trp substituted for Tyr; Tyr substituted for Trp; and/or Phe substituted is Val, replaced by Ile or replaced by Leu.

5.2 specificity

The term " specificity ", " specifically binds " or " specifically binds " means that a specific binding unit (e.g. ISVD) can bind with sufficiently high affinity (see below) to different target molecules (e.g. antigens) from the same organism. ) quantity. " Specificity ,"" specifically binds " or " specifically binds " are used interchangeably herein with " selective ,"" selectively binds " or " selectively binds ." A binding unit such as ISVD preferably specifically binds to its designated target.

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

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

Typically, the binding units (such as ISVD) used in the present technology will be from 10 ^-5 to 10 ^-12 mol/L or less, and preferably from 10 ^-7 to 10 ^-12 mol/L or less, and more preferably 10 A dissociation constant (KD) of ^-8 to 10 ^-12 mol/L (i.e., 10 ⁵ to 10 ¹² L/mol or higher, and preferably 10 ⁷ to 10 ¹² L/mol or higher, and more preferably 10 ⁸ to Association constant (KA) of 10 ¹² L/mol) binds to its target.

It is generally accepted that any KD value greater than 10 ⁻⁴ mol/L (or any KA value less than 10 ⁴ L/mol) indicates nonspecific binding.

The KD for a biological interaction considered specific (such as the binding of an immunoglobulin sequence to an antigen) is typically between 10 ^-5 mol/L (10000 nM or 10 µM) and 10 ^-12 mol/L (0.001 nM or 1 pM) or less.

Thus ^, specific/selective binding may mean that the binding unit (or polypeptide containing the same) binds to ^TCRαβ , TCRαβ, CD33 and/or CD123 bind and bind to the relevant target with a KD value greater than 10 ^-4 mol/liter.

Therefore, compared with the polypeptide composed of the amino acids of SEQ ID NO: 1, the polypeptide of the present technology preferably has at least half the binding affinity to human TCRαβ, human CD33 and human CD123, and more preferably at least the same Binding affinity, where the binding affinity is measured using the same method (eg, 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 the binding unit (or a polypeptide comprising the binding unit) may also specifically bind to TCRαβ from cynomolgus monkey. Likewise, 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 a binding unit to its designated target may be achieved by any suitable means known per se, including, for example, Scatchard analysis and/or competitive binding assays such as radioimmunoassays (RIA), enzyme immunoassays ( EIA) and sandwich competition assays) and different variants thereof known per se in the art); as well as other techniques mentioned herein.

The dissociation constant may be an actual or apparent dissociation constant, as will be apparent to one of ordinary skill. Methods of determining dissociation constants will be clear to those of ordinary skill and include, for example, the techniques mentioned below. In this regard, it will also be clear that it may not be possible to measure dissociation constants greater than 10 ⁻⁴ mol/L or 10 ⁻³ mol/L (eg 10 ⁻² mol/L). Optionally, as will also be clear to a person of ordinary skill, the (actual or apparent) dissociation constant can be calculated based on the (actual or apparent) association constant (KA) by the relationship [KD = 1/KA].

The distance between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technology (see, eg, Ober et al. 2001, Intern. Immunology 13: 1551-1559) affinity of molecular interactions. As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that allows the analysis of real-time biospecific interactions by detecting changes in protein concentration in a biosensor matrix in which a molecule is immobilized on a biosensor chip. on, and another molecule passes through the immobilized molecule under flow conditions, producing _kon , _koff measurements, and therefore the _KD (or _KA ) value. This can be done, for example, using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further description, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991 Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8) : 125-131) and Johnson et al. (1991, Anal. Biochem. 198: 268-277).

Another well-known biosensor technique for determining the affinity of biomolecular interactions is biolayer interferometry (BLI) (see, e.g., Abdiche et al. 2008, Anal. Biochem. 377: 209-217). As used herein, the term "biolayer 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 biosensor tip. Immobilized protein layer (signal beam). Changes in the number of molecules bound to the biosensor tip cause a shift in the interference pattern, reported as wavelength shift (nm), the size of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since interactions can be measured instantaneously, association and dissociation rates as well as affinity can be determined. For example, BLI can be performed using the well-known Octet® system (ForteBio, a division of Pall Life Sciences, Menlo Pike, USA).

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

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

5.3 ( inside the body ) half-life extension

The polypeptide may also comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptide linkers, wherein the polypeptide is identical to the absence of the one or more other groups, residues The one or more other groups, residues, moieties or binding units provide the polypeptide with an increased (in vivo) half-life compared to the corresponding polypeptide. Prolonged half-life in vivo means, for example, an increase in the half-life of the polypeptide in a mammal, such as a human subject, following administration. The half-life can be expressed, for example, as t1/2β.

The type of group, residue, moiety or binding unit is generally not limited and may, for example, be selected from polyethylene glycol molecules, serum proteins or fragments thereof, binding units that bind to serum proteins, Fc portions and those that bind to serum proteins. A group of small proteins or peptides.

More specifically, the one or more other groups, residues, moieties or binding units that increase the half-life of the polypeptide may be selected from the group consisting of those that are compatible with serum albumin (e.g., human serum albumin) or serum immunoglobulin ( A group consisting of binding units that bind, such as IgG), and preferably are binding units that can bind to human serum albumin. The binding unit is preferably ISVD.

For example, WO 04/041865 describes NANOBODY® ISVDs that bind to serum albumin (and specifically to human serum albumin), which may be combined with other proteins (such as one or more other NANOBODY® ISVDs that bind to the desired target) Linked to increase the half-life of the protein.

International application WO 06/122787 describes various NANOBODY® ISVDs against (human) serum albumin. These NANOBODY® ISVDs include the NANOBODY® ISVD known as Alb-1 (SEQ ID NO: 52 in WO 06/122787) and its humanized variants, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787 ). Additionally, these can be used to extend the half-life of therapeutic proteins and peptides as well as other therapeutic entities or moieties.

Furthermore, WO 2012/175400 describes an additional improved form of Alb-1, called Alb-23.

In a preferred embodiment, the polypeptide comprises a serum albumin binding portion selected from: Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb- 9. Alb-10 and Alb-23, preferably Alb-8 or Alb-23 or variants thereof, as shown on pages 7 to 9 of WO2012/175400; and WO2012/175741, WO2015/173325, WO2017/080850, WO2017 /085172, WO2018/104444, WO2018/134235, and albumin binding agents described in WO2018/134234. Table A-4 also shows some preferred serum albumin binding agents. Particularly preferred additional components of the polypeptides of the present technology are as described in item C:

C. The following ISVD, which binds to human serum albumin and contains i. CDR1, which contains SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 9; ii. CDR2 comprising SEQ ID NO: 13 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3, which includes SEQ ID NO: 17 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 17; Preferably, CDR1 contains the amino acid sequence of SEQ ID NO: 9, CDR2 contains the amino acid sequence of SEQ ID NO: 13 and CDR3 contains the amino acid sequence of SEQ ID NO: 17.

Preferred examples of such ISVDs that bind human serum albumin have one or more (and preferably all) architectural regions as represented in Table A-2 for construct ALB23002 (and CDRs as previously defined in Item C ), and most preferably an ISVD containing 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 using the Kabat definition as:

C’. The following ISVD, which binds to human serum albumin and contains i. CDR1, which contains SEQ ID NO: 37 or an amino acid sequence that differs from SEQ ID NO: 37 by 2 or 1 amino acid; ii. CDR2 comprising SEQ ID NO: 41 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 41; and iii. CDR3, which includes SEQ ID NO: 45 or an amino acid sequence that differs from SEQ ID NO: 45 by 2 or 1 amino acid; Preferably, CDR1 contains the amino acid sequence of SEQ ID NO: 37, CDR2 contains the amino acid sequence of SEQ ID NO: 41 and CDR3 contains the amino acid sequence of SEQ ID NO: 45.

Preferred examples of such ISVDs that bind human serum albumin have one or more (and preferably all) architectural regions as represented in Table A-2.1 for construct ALB23002 (and as previously defined in item C' CDR), and most preferably an ISVD containing the complete amino acid sequence of construct ALB23002 (SEQ ID NO: 5, see Tables A-1 and A-2.1).

Also in preferred embodiments, the amino acid sequence of the ISVD that binds to human serum albumin may have greater than 90% (such as greater than 95% or greater than 99%) sequence identity with SEQ ID NO: 5, wherein optionally The CDR is as defined in item C above. In particular, the ISVD that binds to human serum albumin preferably contains the amino acid sequence of SEQ ID NO: 5.

When such an ISVD that binds to human serum albumin has 2 or 1 amino acid difference relative to the corresponding reference CDR sequence in at least one CDR (item C above), it is identical to that shown in SEQ ID NO: 5 The ISVD has at least half the binding affinity for human serum albumin compared to construct ALB23002, preferably at least the same binding affinity, where the binding affinity is measured using the same method (such as SPR).

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

5.4 nucleic acid molecules

Nucleic acid molecules encoding 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 each other via a phosphate backbone to form a nucleotide sequence. Nucleic acids can be used to transform/transfect host cells or host organisms, for example, to express and/or produce polypeptides. Suitable hosts or host cells for production purposes will be clear to those of ordinary skill in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell strain or any suitable fungal, prokaryotic or eukaryotic organism. Hosts or host cells containing 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 contain (eg, 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 or cDNA.

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 can, for example, be subjected to site-directed mutagenesis to provide nucleic acid molecules encoding polypeptides with sequence variations. In addition, it will be clear to one of ordinary skill in the art that, in order to prepare a nucleic acid, it is also possible to combine several nucleotide sequences (eg, at least one nucleotide sequence encoding a targeting moiety) and, for example, encoding one or more linkers. The nucleic acids are linked together in a suitable manner.

Techniques for generating nucleic acids will be clear to those of ordinary skill 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 parts) are combined to introduce mutations that result in the expression of a truncated expression product; to introduce one or more restriction sites (e.g., to create a cassette that can be easily digested and/or ligated using appropriate restriction enzymes and/or region), and/or introduce mutations via PCR reactions using one or more "mismatch" primers.

5.5 carrier

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 (such as plasmids or mRNA) or nucleic acids embedded in larger structures (such as liposomes or viral vectors).

Vectors typically comprise at least one nucleic acid optionally linked to one or more regulatory elements (eg, like one or more suitable promoters, enhancers, terminators, etc.). The vector is preferably an expression vector, ie a vector suitable for expressing the encoded polypeptide or construct under suitable conditions, eg when the vector is introduced into (eg human) cells. For DNA-based vectors, this typically includes the presence of elements for transcription (eg promoters and polyA signals) and translation (eg Kozak sequences).

Preferably, in said vector, said at least one nucleic acid and said 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 said to be "operably linked" to a coding sequence if it is capable of initiating or otherwise controlling/regulating the transcription and/or expression of the coding sequence (wherein the coding sequence is understood to be "under the control of" the promoter mentioned above). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They are also usually essentially continuous, although this may not be required.

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, for example, that the promoter should be able to initiate or otherwise control/regulate operably linked to it Transcription and/or expression of nucleotide sequences (e.g., coding sequences).

5.6 Composition

The technology of the present invention also provides a composition comprising at least one polypeptide of the present technology, at least one nucleic acid molecule encoding the polypeptide of the present technology, or at least one vector containing 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 organism

The present technology also relates to a host cell or host organism comprising a polypeptide of the present technology, a nucleic acid encoding a polypeptide of the present technology, and/or a vector containing a nucleic acid molecule encoding a polypeptide of the present technology.

Suitable host cells or host organisms will be clear to one of ordinary skill in the art and are, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell strain or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

5.8 Methods and uses of peptides

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) Express the nucleic acid sequence encoding the polypeptide in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b) Isolate and/or purify the polypeptide.

Suitable host cells or host organisms for production purposes will be clear to those of ordinary skill in the art and may be, for example, any suitable fungal, prokaryotic or eukaryotic cell or cell strain or any suitable fungal, prokaryotic or eukaryotic biology. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

The polypeptide of the technology of the present invention, the nucleic acid molecule or vector as described, or the composition containing the polypeptide, nucleic acid molecule or vector of the technology of the present invention, preferably the polypeptide or the composition containing the polypeptide, can be used as a medicine.

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

Also provided are polypeptides of the technology of the present invention, nucleic acid molecules or vectors as described, or compositions comprising polypeptides of the technology of the present invention, nucleic acid molecules or vectors, for (preventive or therapeutic) treatment of acute myeloid leukemia ( AML) (preferably relapsed and/or refractory AML).

Further provided is a (preventive and/or therapeutic) method of treating AML, wherein the method comprises administering to a subject in need a pharmaceutically active amount of a polypeptide of the present technology, a nucleic acid molecule or a vector as described , or a composition comprising a polypeptide, nucleic acid molecule or vector of the technology of the present invention.

Further provided are the use of polypeptides of the technology of the present invention, nucleic acid molecules or vectors as described, or compositions comprising polypeptides of the technology of the present invention, nucleic acid molecules or vectors, in the preparation of pharmaceutical compositions preferably used to treat AML.

The AML may be relapsed and/or refractory AML.

A "subject" as mentioned in the context of the present technology may be any animal, preferably a mammal. Among mammals, humans and non-human mammals can be distinguished. Non-human animals may be, for example, companion animals (e.g., dogs, cats), domestic animals (e.g., bovine, equine, ovine, goat, or porcine animals) or animals commonly used for research purposes and/or for the production of antibodies (e.g., mice, rats, etc. Rat, rabbit, cat, dog, goat, sheep, horse, pig, non-human primate (such as cynomolgus monkey) or camelid (such as llama or alpaca)).

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.

Substances (including polypeptides, nucleic acid molecules and vectors) or compositions may be administered to a subject by any suitable route of administration, such as enterally (e.g., orally or rectally) or parenterally (e.g., Epidermal, sublingual, buccal, nasal, intraarticular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration. Parenteral administration, such as intramuscular, subcutaneous or intradermal administration is preferred. Most preferred is subcutaneous administration.

An effective amount of a polypeptide, 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 the desired therapeutic result.

One or more doses can 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.

surface A-1 : In tetravalent polypeptide A025001562 ( TCR-CD33-CD123 multispecific ISVD construct ) Different unit prices for internal appraisals _VHH Amino Acid Sequences of Building Blocks ( 「 ID " means as used in this article SEQ ID NO ) Name ID amino acid sequence TCR-V _HH (Building Block 1) 2 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSS CD33-V _HH (Building Block 2) 3 EVQLVESGGGVVQPGGSLRLSCAASGGVFRLNSMGWFRQAPGKQRELVAIITSDGDTNYADSVKGRFTISRDQAKNTVYLQMNSLRPEDTALYYCQITYSTSSYSFPINTWGQGTLVTVSS CD123-V _HH (Building Block 3) 4 EVQLVESGGGVVQPGGSLRLSCAASGVIFSGNVMGWYRRQAPGKEREWVAAIAEGGSILYRDSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCNSHPPVLPYWGQGTLVTVSS ALB23002 (Building Block 4) 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS

surface A-2 :according to ikB numbered CDR and sequence of architectures ( 「 ID " refers to the given SEQ ID NO ) ID _VHH ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 TCR 18 DVQLVESGGGVVQPGGSLRLSCVAS 6 GYVHKINFYG twenty two WYRQAPGKEREKVA 10 HISIGDDQTD 26 YADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRA 14 LSRIWPYDY 30 WGQGTLVTVSS 3 CD33 19 EVQLVESGGGVVQPGGSLRLSCAAS 7 GGVFRLNSMG twenty three WFRQAPGKQRELVA 11 IITSDGDTN 27 YADSVKGRFTISRDQAKNTVYLQMNSLRPEDTALYYCQI 15 TYSTSSYSFPINT 31 WGQGTLVTVSS 4 CD123 20 EVQLVESGGGVVQPGGSLRLSCAAS 8 GVIFSGNVMG twenty four WYRRQAPGKEREWVA 12 AIAEGGSIL 28 YRDSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCNS 16 HPPVLPY 32 WGQGTLVTVSS 5 ALB3002 twenty one EVQLVESGGGVVQPGGSLRLSCAAS 9 GTFTFRSFGMS 25 WVRQAPGKGPEWVS 13 SISGSGSDTL 29 YADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 17 GGSLSR 33 SSQGTLVTVSS

surface A-2.1 :according to Kabat numbered CDR and sequence of architectures ( 「 ID " refers to the given SEQ ID NO ) ID _VHH ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 TCR 46 DVQLVESGGGVVQPGGSLRLSCVASGYVHK 34 INFYG 50 WYRQAPGKEREKVA 38 HISIGDQTDYADSAKG 54 RFTISRDESKNTVYLQMNSLRPEDTAAYYCRA 42 LSRIWPYDY 58 WGQGTLVTVSS 3 CD33 47 EVQLVESGGGVVQPGGSLRLSCAASGGVFR 35 LNSMG 51 WFRQAPGKQRELVA 39 IITSDGDTNYADSVKG 55 RFTISRDQAKNTVYLQMNSLRPEDTALYYCQI 43 TYSTSSYSFPINT 59 WGQGTLVTVSS 4 CD123 48 EVQLVESGGGVVQPGGSLRLSCAASGVIFS 36 GNVMG 52 WYRRQAPGKEREWVA 40 AIAEGGSILYRDSVKG 56 RFTISRDNAKNTVYLQMNSLRPEDTALYYCNS 44 HPPVLPY 60 WGQGTLVTVSS 5 ALB3002 49 EVQLVESGGGVVQPGGSLRLSCAASGFTFR 37 SFGMS 53 WVRQAPGKGPEWVS 41 SISGSGSDTLYADSVKG 57 RFTISRDNSKNTLYLQMNSLRPEDTALYYCTI 45 GGSLSR 61 SSQGTLVTVSS

surface A-3 : Amino acid sequence of the selected multivalent polypeptide ( 「 ID " refers to the given SEQ ID NO ) Name ID amino acid sequence A025001562 1 DVQLVESGGGVVQPGGSLRLSCVASGYVHKINFYGWYRQAPGKEREKVAHISIGDQTDYADSAKGRFTISRDESKNTVYLQMNSLRPEDTAAYYCRALSRIWPYDYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGGVFRLNSMGWFRQAPGKQRELVAIITSDGDTNYADSVKGRFTISRDQAKNTVYL QMNSLRPEDTALYYCQITYSTSSYSFPINTWGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGVIFSGNVMGWYRRQAPGKEREWVAAIAEGGSILYRDSVKGRFTISRDNAKNTVYLQMNSLRPEDTALYYCNSHPPVLPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSW VRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSA

surface A-4 : Bound to serum albumin ISVD sequence ( 「 ID " means as used in this article SEQ ID NO ) Name ID amino acid sequence Alb8 62 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVTVSS Alb23 63 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVTVSS Alb129 64 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSSLRSSQGTLVTVSSA Alb132 65 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSSLRSSQGTLVTVSSA Alb11 66 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVTVSS Alb11 (S112K)-A 67 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSSLRSSQGTLVKVSSA Alb82 68 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS Alb82-A 69 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSA Alb82-AA 70 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSAA Alb82-AAA 71 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSAAA Alb82-G 72 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSG Alb82-GG 73 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISSRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGG Alb82-GGG 74 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSGGG Alb23002 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSS Alb223 75 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSSLRSSQGTLVTVSSA

surface A-5 : Connector sequence ( 「 ID " means as used in this article SEQ ID NO ) Name ID amino acid sequence 3A connector 76 AAA 5GS connector 77 GGGGS 7GS connector 78 SGGSGGS 8GS connector 79 GGGGSGGS 9GS connector 80 GGGGSGGGS 10GS connector 81 GGGGSGGGGS 15GS connector 82 GGGGSGGGGSGGGGS 18GS connector 83 GGGGSGGGGSGGGGSGGS 20GS connector 84 GGGGSGGGGSGGGGSGGGGS 25GS connector 85 GGGGSGGGGSGGGGSGGGGSGGGGS 30GS connector 86 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 35GS connector 87 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 40GS connector 88 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS G1 hinge 89 EPKSCDKTHTCPPCP 9GS-G1 hinge 90 GGGGSGGGSEPKSCDKTHTCPPCP Llama upper long hinge area 91 EPKTPKPQPAAA G3 hinge 92 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

6 Example

6.1 Example 1 :Multiple specificity ISVD construct generation

Multispecific NANOBODY® ISVD protein expressed in P. pastoris Yeast expression vector contains AOX1 promoter and terminator, giomycin resistance gene, and Saccharomyces cerevisiae alpha-mating factor Coding information for signal peptides. NANOBODY® ISVD monovalent building blocks (BB) were combined with the GS linker and transformed into expression vectors via Golden Gate cloning (Engler C, Marillonnet S. Golden Gate cloning. Methods Mol Biol. 2014;1116: 119-31). Expression vectors contain two BpiI restriction sites for transduction of PCR-amplified monovalent NANOBODY® ISVD building blocks and GS linkers contained in one or more vectors. All these elements are flanked by BpiI sites. The use of unique nucleotide overhangs at each position of the cloning cassette allows for seamless ligation in a predetermined sequence. After Sanger sequence confirmation, plasmid DNA derived from E. coli TOP10 was linearized and transformed by electroporation into the in-house prepared hypercompetent Pichia pastoris strain NRRL Y-11430 (ATCC 76273).

E. coli TG1 cells (Lucigen, Cat. No. 60502) containing the NANOBODY ^® ISVD protein expression vector were grown at 37ºC for 2 hours and then in baffled shake flasks containing "5052" auto-induction medium at 30ºC (250 rpm). Grow for 29 hours. Pellet the cells by centrifugation (20 min, 4500 rpm, 4ºC), discard the supernatant, and freeze the pellet at -20ºC overnight. The frozen cell pellet was then dissolved in DPBS at 1/12.5 of the original culture volume and incubated for 1 hour at 4ºC with gentle rotation to disrupt the outer membrane of the cells. Cells were pelleted again (20 minutes, 8500 rpm, 4ºC), and the supernatant containing NANOBODY ^® ISVD protein was collected and filtered for immediate purification.

FLAG3His ₆ -tagged NANOBODY ^® ISVD proteins are purified by immobilized metal affinity chromatography (IMAC) or on NiIDA/NTA (Genscript) resin with imidazole elution (for the former) or acidic elution (for the latter), This was 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, robotic stations or ÄKTA purification systems are used.

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

6.2 Example 2 :Multiple specificity ISVD pair of constructs TCRαβ , CD33 , CD123 binding affinity to serum albumin

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

6.2.1 Humans and cynomolgus monkeys CD33 and CD123 Determination of protein affinity

Measurement of TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) versus huCD33L-Fc, cyCD33L by surface plasmon resonance (SPR)-based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.) at 37ºC - Affinity of the Fc (via capture setup) or huCD123 (via capture setup) protein, expressed as association rate constant ( _ka ), dissociation rate constant ( _kd ) and equilibrium dissociation constant ( _KD ).

Setup for measuring binding affinity to CD33

Anti-huIgG(Fc) was immobilized via amine coupling using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS (N-hydroxysuccinimide ester) chemistry) GLH (long matrix, high capacity) sensor wafer. Next, capture huCD33L-Fc and cyCD33L-Fc (association: 120 s, 25 µL/min). Inject as SEQ at different concentrations (0.4 to 625 nM) Purified TCRαβ-CD33-CD123 multispecific ISVD construct shown in ID NO: 1 for 120 s followed by dissociation for 900 s.

Setup for measuring binding affinity to CD123

Anti-huIgG(Fc) was immobilized via amine coupling using EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and NHS (N-hydroxysuccinimide ester) chemistry) GLH (long matrix, high capacity) sensor wafer. Next, capture huCD123-Fc and cyCD123-Fc (association: 120 s, 25 µL/min). Inject as SEQ at different concentrations (1.2 to 300 nM) Purified TCRαβ-CD33-CD123 multispecific ISVD construct shown in ID NO: 1 for 120 s followed by dissociation for 900 s at 45 µl/min.

Data were double referenced by subtracting the reference analyte lane and blank buffer injection. ProteOn Manager 3.1.0 (version 3.1.0.6) software was used to calculate the affinity constants ( _ka , k _d , K _D ) by applying the Langmuir 1:1 interaction model.

Measurements of the affinity of the TCRαβ-CD33-CD123 multispecific ISVD construct as shown in SEQ ID NO: 1 for human and cynomolgus CD33 and human and cynomolgus CD123 are summarized in Tables 2 and 3 below.

surface 2 : Based on SPR for TCRαβ-CD33-CD123 multispecific ISVD Constructs for humans and cynomolgus monkeys CD33 Determination of protein affinity Human CD33L-Fc Cynomolgus CD33L-Fc ISVD k _a (1/Ms) k _d (1/s) K _D (M) k _a (1/Ms) k _d (1/s) K _D (M) TCRαβ-CD33-CD123 (SEQ ID NO: 1) 3.5E+05 2.7E-03 7.6E-09 2.8E+05 1.9E-03 6.9E-09

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).

surface 3 : Based on SPR for TCRαβ-CD33-CD123 multispecific ISVD Constructs for humans and cynomolgus monkeys CD123 Determination of protein kinetics Human CD123-Fc Cynomolgus CD123-Fc ISVD k _a (1/Ms) k _d (1/s) K _D (M) k _a (1/Ms) k _d (1/s) K _D (M) TCRαβ-CD33-CD123 (SEQ ID NO: 1) 1.3E+06 7.4E-04 5.9E-10 1.2E+06 2.4E-03 2.0E-09

The TCRαβ-CD33-CD123 multispecific ISVD construct was 3.3-fold 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 construct binds human/cynomolgus CD33 and human /cynomolgus CD123 with high affinity.

6.2.2 Humans and cynomolgus monkeys TCRαβ Determination of protein affinity

The response of the TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) to recombinant huTCR(2XN9)-zipperin, cyTCR( AEA41865)-zipper protein (set via coating), expressed as association rate constant ( _Ka ), dissociation rate constant ( _kd ) and equilibrium dissociation constant ( _KD ).

Setup for measuring binding affinity to TCRαβ

huTCR(2XN9)-zipper, cyTCR(AEA41865)-zipper proteins are coated on GLC (short matrix, normal capacity) sensor wafers. 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 120 s, followed by dissociation for 900 s.

Data were double referenced by subtracting the reference analyte lane and blank buffer injection. ProteOn Manager 3.1.0 (version 3.1.0.6) software was used to calculate the affinity constants ( _ka , k _d , K _D ) by applying the Langmuir 1:1 interaction model.

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

surface 4 : Based on SPR for TCRαβ-CD33-CD123 multispecific ISVD Constructs and References TCR-ISVD Constructs for humans and cynomolgus monkeys TCRαβ determination of kinetics Human TCRαβ-zipper Cynomolgus TCRαβ-zipper ISVD n k _a (1/Ms) k _d (1/s) K _D (M) n k _a (1/Ms) k _d (1/s) K _D (M) TCRαβ-CD33-CD123 (SEQ ID NO: 1) 1 8.0E+04 1.7E-03 2.1E-08 1 4.9E+05 3.3E-03 6.7E-09 TCR (refer to) Experiment 1 1.4E+05 1.7E-03 1.2E-08 Experiment 1 4.2E+05 4.4E-03 1.1E-08 Experiment 2 2.6E+05 1.8E-03 7.0E-09 Experiment 2 8.2E+05 3.5E-03 4.3E-09

The TCRαβ-CD33-CD123 multispecific ISVD construct was 3-fold more reactive to cynomolgus monkey TCRαβ (K _D = 6.7 nM) than to human TCRαβ (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 monkey serum albumin

Evaluation of the TCRαβ-CD33-CD123 multispecific ISVD construct (SEQ ID NO: 1) via the C-terminal ALB23002 half-life extender (via coating setup) by SPR-based assay on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.) at 37ºC ) to recombinant human, cynomolgus monkey and mouse serum albumin, expressed as association rate constant ( _ka ), dissociation rate constant ( _kd ) and equilibrium dissociation constant ( _KD ).

Setup for measuring binding affinity to serum albumin

Human, cynomolgus monkey and mouse serum albumin were immobilized on the ProteOn GLC sensor chip using amine coupling using EDC and NHS chemistry (running buffer used: HBS-EP+, pH 7.4). Albumin was immobilized at 2.5 µg/mL (HSA and MSA) and 5 µg/mL (CSA) in pH 4.5 acetate buffer, increasing immobilization levels up to 220 RU for CSA and 150 RU for MSA. , and HSA is 110 RU. Purified polyvalent _VHH was injected at different concentrations (between 4.3 nM and 416 nM) for 2 min (flow rate 45 µL/min), followed by dissociation for 900 s. Regeneration between cycles consisted of injection of 10 mM glycine-HCL (pH 1.5) at 100 µL/min for 47 s.

Data were double referenced by subtracting reference ligand lanes and buffer injections. The processed curves were evaluated by fitting with the Langmuir 1:1 interaction model using the ProteOn Manager 3.1.0 (version 3.1.0.6) software model, and the affinity constants ( _ka , _kd , _KD ) were calculated.

Measurements of the affinity of the TCRαβ-CD33-CD123 multispecific ISVD construct as shown in SEQ ID NO: 1 for human, mouse and cynomolgus monkey serum albumin are summarized in Table 5 below.

surface 5 : Based on SPR for TCRαβ-CD33-CD123 multispecific ISVD Determination of the affinity of the construct for serum albumin MSA CSA HSA ISVD k _a (1/Ms) k _d (1/s) K _D (M) k _a (1/Ms) k _d (1/s) KD(M) k _a (1/Ms) k _d (1/s) K _D (M) TCRαβ-CD33-CD123 (SEQ ID NO: 1) KD＜5E-07 2.5E+05 2.0E-03 7.9E-09 2.2E+05 2.1E-03 9.7E-09

Cross-reactivity to CSA was confirmed. Furthermore, although no kinetic parameters were reported, the affinity for MSA was good enough to obtain half-life extension and use that of serum albumin.

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

6.3 Example 3 :Multiple specificity ISVD Structure and membrane binding CD33 and / or CD123 The binding affinity of

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

Flow cytometry was used to evaluate the binding affinity of the TCRαβ-CD33-CD123 multispecific ISVD construct to target cell lines expressing CD33 and/or CD123. Target cell lines expressing CD123 are described in detail in WO2018/091606A1.

Transfected CD33 cells were generated as follows. Stable CHO Flp recombinantly expressed CD33 was generated using Flp-In™ site-directed recombination technology (Flp-In™ system for generation of stable mammalian expressing cell lines through Flp recombinase-mediated integration (Invitrogen, K601001, K601002)) -In (Invitrogen, R758-07) cell line. Hereby, DNA integration occurs at a specific gene body position at the FRT (Flp recombination target) site by Flp recombinase (pOG44) derived from Saccharomyces cerevisiae. Both the Flp-In™ host cell line and the expression plasmid (pcDNA5) contain this FRT site, 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 genbank number XP_005590138.

Briefly, cells were harvested and transferred to a V-bottom 96-well plate (5x10 ⁴ cells/well) and incubated in FACS buffer ( Serial dilutions of the TCRαβ-CD123-CD33 multispecific ISVD construct in D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)) with A final volume of 100 µL was incubated for 30 min at 4ºC. Next, cells were washed three times with FACS buffer and incubated with 1 µg/mL mouse monoclonal anti-FLAG® M2 antibody (Sigma-Aldrich, F1804) for 30 min at 4ºC to detect FLAG3His _6- tagged CD123-CD33 -TCR multispecific ISVD construct, or incubated with 3 µg/mL mAb anti-V _HH antibody (ABH0077) (APS + in-house, A-0006-00_ABH0077_SF_AB1891) to detect NANOBODY® ISVD with ALB BB. Next, 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) in a final volume of 100 µL and incubated at 4ºC for 30 minutes. Subsequently, cells were resuspended in 50 µL cold FACS buffer supplemented with 1 µg/mL propidium iodide (PI, Sigma P4170) to distinguish live and dead cells. After staining, cells were collected using a MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, the P1 population accounting for more than 80% of the total events was selected based on FSC-SSC distribution to distinguish cells and debris. PI positive (dead) cells were excluded from this population (P1) and the median APC fluorescence intensity of PI negative cells was evaluated.

Setup for measuring binding affinity to primary T cells

The binding affinity of TCRαβ-CD33-CD123 multispecific ISVD constructs to primary T cells was evaluated using flow cytometry in a competition setting using FLAG3His _6- tagged monovalent TCR ISVDs as ligands.

Briefly, human or cynomolgus primary T cells were thawed and transferred to V-bottom 96-well plates ( ^7.5x10 cells/well in 100 µL) and incubated in 30 µM clinical grade HSA (CSL Behring, 2160-679) with TCRαβ-CD33 in FACS buffer (D-PBS (Gibco, 14190) with 10% FBS (Sigma, F7524) and 0.05% sodium azide (Acros organics, 19038)) -Serial dilutions of CD123 multispecific ISVD constructs and fixed concentrations of ligand were incubated in a final volume of 100 µL for 30 min at 4ºC. The ligand concentration used in the assay was below its binding _EC50 . After an incubation period of 90 minutes at 4ºC, ligand binding levels were determined by flow cytometry. Subsequently, the cells were washed three times and incubated with 1 μg/ml mouse monoclonal anti-FLAG® M2 antibody (Sigma-Aldrich, F1804) for 30 min at 4ºC, washed again, and incubated with 5 μg/ml Allophycocyanin Protein (APC) AffiniPure Goat Anti-Mouse IgG (Subclass 1+2a+2b+3) (FCγ Fragment Specific) (Jackson Immunoresearch, 115-136-071) was incubated in a final volume of 100 µL for 30 min at 4ºC. Subsequently, 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 a MACSQuant X flow cytometer (Miltenyi Biotec) and analyzed using FlowLogic (Miltenyi Biotec). First, the P1 population accounting for more than 80% of the total events was selected based on FSC-SSC distribution to distinguish cells and debris. PI positive (dead) cells were excluded from this population (P1) and the median APC fluorescence intensity of PI negative cells was evaluated.

Human-cynomolgus cross-reactivity was evaluated by testing the monovalent CD33 building block (SEQ ID NO: 3) and the monovalent CD123 building block (SEQ ID NO: 4) with human or food using flow cytometry as described above. Cynomolgus CD33-transfected cell lines or combinations with human or cynomolgus CD123-transfected cell lines. The results are graphically represented in Figure 2 . _EC50 values for different experiments are summarized in Table 6 (CD33-transfected target cells) and Table 7 (CD123-transfected target cells).

surface 6 : CD33 building blocks ( SEQ ID NO: 3 ) and performance huCD33 or cyCD33 combination of cells EC ₅₀ value. Sample ID cell lines EC50(M) 95%LCI(M) 95%UCI(M) CD33 binding ISVD CHOcyCD33 5.7E-09 3.2E-09 5.1E-08 CD33 binding ISVD CHOcyCD33 1.2E-08 8.2E-09 2.2E-08 CD33 binding ISVD CHOhuCD33 6.2E-09 4.6E-09 8.5E-09 CD33 binding ISVD CHOhuCD33 5.5E-09 4.1E-09 7.7E-09 CD33 binding ISVD CHOhuCD33 7.7E-09 6.8E-09 8.8E-09

surface 7 : CD123 building blocks ( SEQ ID NO: 4 ) and performance huCD123 or cyCD123 combination of cells EC ₅₀ value. Sample ID cell lines EC50(M) 95%LCI(M) 95%UCI(M) CD123 binds ISVD CHO human CD123 3.1E-10 2.4E-10 4.1E-10 CD123 binds ISVD CHO human CD123 2.0E-10 2.9E-11 Undetermined CD123 binds ISVD HEK293 cynomolgus CD123 8.0E-10 7.2E-10 9.1E-10

Binding of monovalent CD33 and CD123 building blocks to human and cynomolgus monkey membrane targets was confirmed. The difference in _EC50 between humans and cynomolgus monkeys was less than a 2-fold decrease for binding to CD33 and a 3-fold decrease for binding to CD123 cells (Table 7).

In conclusion, in addition to binding affinities for recombinant human and cynomolgus CD33 and CD123 proteins, dose-dependent binding of the TCRαβ-CD33-CD123 multispecific ISVD construct to CD33 and CD123 exhibited by human and cynomolgus cells was confirmed (n =1).

In a competition setting, the CD123-CD33-TCR multispecific ISVD construct and the reference TCR-ISVD construct (consisting only of the TCRαβ building block linked to ALB23002 (SEQ ID NO: 5)) were evaluated using flow cytometry as described above. SEQ ID NO: 2) Binding to human and cynomolgus monkey T cells ( Figure 3 ).

An illustrative example of DRC is depicted in Figure 4 . ISVD constructs were tested on a variety of healthy donor T cells. Overall _IC50 is shown in Table 8 .

Table 8 : Overall _IC50 ( M ) for the TCRαβ-CD33-CD123 multispecific ISVD construct and the reference TCR-ISVD construct in human and cynomolgus monkey T cell competition assays as determined by flow cytometry ISVD Primary human T cell overall IC50* Primary cynomolgus monkey T cell overall IC50* The difference between cynomolgus monkeys and humans TCRαβ-CD33-CD123 (SEQ ID NO: 1) 218nM [190nM – 249nM] n=4 729nM [545nM – 976nM] n=7 3.3 ReferenceTCR-ISVD 187nM [134nM – 261nM] n=4 493nM [314nM – 772nM] n=7 2.6 *Overall IC ₅₀ (weighted)

In conclusion, the cross-reactivity of the TCRαβ-CD33-CD123 multispecific ISVD construct on cynomolgus monkey primary T cells was confirmed. The EC ₅₀ (= 729 nM) on primary cynomolgus monkey T cells is approximately 3 times higher than the EC ₅₀ (= 218 M) on primary human T cells

6.4 Example 4 :Multiple specificity ISVD construct-induced T cell-mediated target cell killing

6.4.1 Impedance-based cytotoxicity assay

Characterization of ISVD constructs for redirected T cell-mediated killing using human or cynomolgus monkey primary effector T cells and adherent target cells in an impedance-based cytotoxicity assay (e.g., as described in WO 2018091606A1) . An xCELLigence instrument (Roche) was used to measure impedance changes caused by adhesion of target cells to the electrode surface. T cells are non-adhesive and therefore do not affect impedance measurements. The xCELLigence® RTCA MP instrument quantifies changes in electrical impedance, displaying it as a dimensionless parameter called the cell index, which is proportional to the total area of the tissue culture well covered by the cells. To each well of a 96 E-plate (ACEA Biosciences; 05 232 368 001), add Assay Medium (Target Cell Growth Medium (without selective antibiotics) + 1% Penicillin/Streptomycin (Life technologies Cat. No. 15140 )) in 50 µL of 4X concentrated HSA solution (in some assays, use 200 µM to have a final concentration of 50 µM, in other assays use 120 µM to have a final concentration of 30 µM). The outer wells are not used and filled with 200 µL medium or D-PBS. Place the 96 E-Plate in the xCELLigence® Station (in a 37ºC, 5% CO2 incubator) and take a single measurement in the absence of cells to measure the background impedance of the assay medium. Subsequently, 50 µL of target cells (2 x ¹⁰ cells/well) in assay medium were plated onto a 96 E-plate and 50 µL of serially diluted ISVD construct solution in assay medium (4X concentration) was added. (Final volume = 200 μL). After 30 min at room temperature, add 50 µL of primary T cells (3 x 105 cells/well) per well in assay medium to achieve an effector to target ratio of 15:1. Place the plate in the xCELLigence® station and measure impedance every 15 min for 4 days. Data were analyzed at fixed time points indicated in the results.

6.4.2 Flow cytometry-based cytotoxicity assay

ISVD constructs were characterized for redirected T cell-mediated killing in flow cytometry-based cytotoxicity assays using human or cynomolgus monkey primary T cells as effector cells and non-adherent target cells. Target cells were labeled with 4 µM PKH26 membrane dye using the PKH26 red fluorescent cell linker kit (Sigma, PKH26GL-1KT) according to the manufacturer's instructions. Effector cells (2.5 x ¹⁰ cells/well) and PKH-labeled target cells (2.5 x ¹⁰ cells/well) were plated in a 96-well V-bottom plate (Greiner Bio-one, #651 180) on target cells. Cell lines were cocultured in assay medium (targeted growth medium with 1% penicillin/streptomycin (Life Technologies, 15140) and 50 µM Alburex HSA (CSL Behring, 2160-679)) (effector to target ratio of 10 :1). To analyze concentration-dependent cell lysis, serial dilutions of ISVD constructs in target assay medium were added to cells and incubated for 18 h at 37ºC in a 5% CO2 atmosphere. 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, cells were resuspended in 100 µL FACS buffer supplemented with 5 nM TO-PRO®-3 Iodide (642⁄661) (ThermoFisher Scientific, T3605) to differentiate between live and dead cells. Cells were analyzed using a MACSQuant X flow cytometer (Miltenyi Biotec). The total sample volume collected for each sample was 70 µL. Set a gate for PKH26-positive cells and measure TO-PRO®-3-positive cells within this population. Percent specific lysis = ((% TO-PRO-3+no construct – % TO-PRO-3+with construct)/ % TO-PRO-3+no construct)) x100.

Functional cross-reactivity analysis of TCRαβ-CD33-CD123 multispecific ISVD constructs toward CD33, CD123, and TCR was performed in an impedance-based cytotoxicity assay (xCELLigence) using human or cynomolgus monkey primary T cells and adherent human or Cynomolgus monkeys were determined by transfection of CD33 or CD123 cells.

As described above in 6.4.1 and 6.4.2, all assays are performed in the presence of excess HSA such that NANOBODY® ISVD is fully saturated with HSA as described above. Reference TCR-ISVD was used as a negative control. The results are shown in Figures 5 and 6 and Table 9 .

6.4.3 result

The results of assays comparing human and cynomolgus monkey primary T cells are graphically presented in Figures 5 and 7 . Human T cell-mediated cell killing of human CD33- and human CD123-transfected cells was evaluated using T cells from different human donors, allowing calculation of overall _IC50 values ( Table 9 ) . Cynomolgus T cell-mediated killing of human CD33- and human CD123-transfected cells was evaluated using T cells from one cynomolgus monkey. _IC50 values are summarized in Table 10 .

surface 9 : based on impedance ( xCELLigence ) people T cell mediated CD33+ or CD123+ For cell killing assays, use 15 Compare 1 The effector to target ratio, in 50 µM HSA in the presence of, TCRαβ-CD33-CD123 overall IC ₅₀ ( M ) . Impedance-based human T cell-mediated killing of CHO hu CD33 cells Impedance-based human T cell-mediated killing of CHO hu CD123 cells ISVD n Overall IC50 (M) IC50 (M) 95% LCI IC50 (M) 95% UCI n Overall IC50 (M) IC50 (M) 95% LCI IC50 (M) 95% UCI TCRαβ-CD33-CD123 (SEQ ID NO: 1) 4 1.1E-10 5.4E-11 2.4E-10 7 4.4E-11 2.5E-11 8.0E-11

Regarding CD33 and CD123, the overall _IC50 against human target expressing cells was 5,4.10 ^-11 M and 2,5.10 ^-11 M respectively.

surface 10 : based on impedance ( xCELLigence ) cynomolgus monkey T cell mediated CD33+ or CD123+ For cell killing assays, use 15 Compare 1 The effector to target ratio, in 50 µM HSA in the presence of, TCRαβ-CD33-CD123 of IC ₅₀ ( M ) . Impedance-based T cell-mediated killing of CHO Flp-In cynoCD33 cells in cynomolgus monkeys Impedance-based T cell-mediated killing of CHO Flp-In cynoCD123 cells in cynomolgus monkeys ISVD IC50(M) IC50 (M) 95% LCI IC50 (M) 95% UCI IC50(M) IC50 (M) 95% LCI IC50 (M) 95% UCI TCRαβ-CD33-CD123 (SEQ ID NO: 1) 6.2E-12 5.1E-12 7.4E-12 1.4E-12 1.2E-12 1.7E-12

To confirm the human cynomolgus monkey cross-reactivity of the TCRαβ-CD33-CD123 ISVD construct to TCR, human or primary T cells were used in flow cytometry-based T cell-mediated killing of MOLM-13 cells as described above. ISVD constructs were evaluated in the presence of 50 µM HSA in combination with a human MOLM-13 target cell line dually expressing CD33 and CD123. A graphical representation of these results is shown in Figure 6 . Additional data are available for TCRαβ-CD33-CD123 ISVD constructs tested in human T cell-mediated killing assays using different donors to calculate overall _EC50 values ( Table 11 ). _EC50 values for the cynomolgus monkey T cell-mediated killing assay are shown in Table 12 .

surface 11 : People who are using flow cytometry T cell mediated MOLM-13 For cell killing assays, use 10 Compare 1 The effector to target ratio, in 50 µM HSA in the presence of, A025001562 ( TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1 ) overall EC ₅₀ ( M ) . Impedance-based killing of human T cell-mediated MOLM-13 cells ISVD n Overall EC ₅₀ (M) EC ₅₀ (M) 95% LCI EC ₅₀ (M) 95% UCI TCRαβ-CD33-CD123 (SEQ ID NO: 1) 13 1.80E-11 1.16E-11 2.78E-11

surface 12 : In cynomolgus monkeys T cell mediated MOLM-13 For cell killing assays, use 10 Compare 1 The effector-to-target ratio of TCRαβ-CD33-CD123 ISVD Architectural EC50 ( M ) . T cell-mediated killing of MOLM-13 cells in cynomolgus monkeys ISVD EC ₅₀ (M) 95%LCI 95%UCI TCRαβ-CD33-CD123 (SEQ ID NO: 1) 1.36E-11 1.15E-11 1.62E-11

In summary, the TCRαβ-CD33-CD123 ISVD construct was functional in both human and cynomolgus monkey human target cell-mediated killing assays. The overall killing effect of the ISVD on CD33/CD123 double-positive AML cell lines was 1.8.10 ^-11 M.

6.5 Example 5 : during implantation T Cell humanization NSG in mice CD123 ⁺ Molm-13-luc disseminated AML in model TCRαβ-CD33-CD123 multispecific ISVD Preclinical in vivo efficacy of constructs

6.5.1 Materials and methods

Cell lines and human materials

A 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 (37ºC, 5% _CO2 , 95% humidity) in RPMI1640 Glutamax medium (supplemented with 20% fetal calf serum). Cells were infected with a non-replicating lentivirus-borne luciferase vector (SV40-PGL4-Puro); polyclonal Molm-13-luc was selected using 2 µg/ml puromycin.

For human administration T Cell purification and expansion

EFS (Etablissement Français du Sang, Île-de-France, France) provides fresh human peripheral blood from healthy donors.

Fresh human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation (without braking) at 200 g for 40 min at room temperature. The pellet was washed and resuspended in 50 ml of phosphate buffered saline (PBS). in the final volume. Total viable PBMC numbers were defined by Vi-CELL counter (Beckman Coulter Life Sciences, Brea, CA, USA). The pellet was recovered in autoMACS running buffer (Miltenyi Biotec). According to the manufacturer's Instructions. Isolation of T cells from PBMC using Pan-T Cell Isolation Kit (Miltenyi Biotec) and autoMACS. Priming over 14 days in vitro using CD3 and CD28 costimulation-based T Cell TRANSACT Matrix Priming/Expansion Kit (Miltenyi Biotec) and expansion of purified T cells. According to the Miltenyi protocol, the priming protocol involves priming in TexMACS medium (Miltenyi Biotec) supplemented with 20 000 IU soluble IL-2 and 1% penicillin-streptomycin (Gibco) in the presence of TRANSACT matrix T cells were cultured for 2 weeks. On day 14 of expansion, T cells were harvested and resuspended in PBS at a final concentration of 5 x ¹⁰ cells/ml and administered to each animal via intraperitoneal (IP) injection 10 ⁷ cells. After testing, the viability of T cells in animals before injection was higher than 85%.

In vivo characterization

All in vivo experiments were approved by the Sanofi Ethics Committee and performed in AAALAC-accredited facilities in compliance with local and institutional laws, ethics, and guidelines.

Evaluation of TCRαβ-CD33-CD123 multispecificity after Molm13-luc AML implantation in non-irradiated NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (Charles River Laboratories, Saint-Germain-Nuelles, France) Antitumor activity of ISVD construct (SEQ ID NO: 1). On day 0, 6- to 8-week-old female animals were implanted intravenously (IV) with 10 ⁶ /0.2 ml Molm-13-luc cells per mouse. On day 1, the same animals were implanted intraperitoneally with 10 ⁷ /0.2 ml human T cells per mouse.

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

Table 13 : Compound Evaluation Study Design treatment group Dosage(nmol/Kg) Volume/Injection Route schedule Number of animals control - - - 10 TCR-CD33-CD123 (SEQ ID NO: 1) 12 0.2ml IV Q2dx5 (4,6,8,10,12) ^7a 1.2 0.2ml IV Q2dx5 (4,6,8,10,12) 8 0.12 0.2ml IV Q2dx5 (4,6,8,10,12) 8 0.012 0.2ml IV Q2dx5 (4,6,8,10,12) 8 TCR ISVD construct 1.2 0.2ml IV Qdx10 (4-13) 8 positive control 1.3 0.2ml IV Qdx10 (4-13) 8 a): One unresponsive animal was not included in the analysis due to lack of T cell engraftment

Data collection and efficacy standards

Animal body weights were monitored from day 3 to the end of the assay to track the effects of therapy. A dose that produces 20% body weight loss or 15% body weight loss or 10% or more drug-induced death for 3 consecutive days is considered an excessively toxic dose. Animal body weight includes tumor weight.

Tumor growth was assessed by in vivo BLI on days 3, 7, 10, and 14 after tumor injection using an IVIS Lumina XRMS imager (PerkinElmer, Waltham, CA) with Living Image 4.5.2 acquisition software (PerkinElmer). Massachusetts, USA) by in vivo luciferase activity measurement using beetle luciferin potassium salt 160 mg/kg Ip injected 15 minutes later and kétamine®/Xylazine® (120 mg/ kg; 6 mg/kg IM, 5 ml/kg) anesthetized animals for imaging processing. Tumor growth is based on bioluminescence signal curves (expressed in photons/second).

Tumor growth was tracked throughout the body and in the long bones of the hind legs via BLI signal measurements on days 7, 10, and 14 after tumor implantation. The primary efficacy endpoint was the ratio of change from baseline in tumor signal between treatment and control (T/C), partial regression (PR), and complete regression (CR).

Tumor growth based on bioluminescence signal curves (expressed in photons/second) was plotted over time for each animal in each treatment group and expressed as the median of whole-body (linear scale) and bone segmental signal (logarithmic scale) Curve ± MAD. The tumor signal on the day of the first treatment (staging day) was calculated for each treatment (T{ Bioluminescence signal}) group and control (C { Calculate the median T for the treatment group, and calculate the median C for the control group. The ratio T/C is then calculated and expressed as a percentage:

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

A dose is considered therapeutically active when the dT/dC is below 42%, and a dose is considered very active when the dT/dC is below 10%. If dT/dC is below 0, the dose is considered highly active and the percentage of resolution is recorded with a date:

Percent tumor regression was defined as the % reduction in tumor signal in the treatment group on a specific observation day compared to the signal on the first day of treatment.

For each animal, % extinction was calculated at specific time points. To account for the risk of signal variability due to variability in luciferin kinetics due to missed i.p. injections, true extinction was considered when extinction was observed for at least two consecutive time points per animal.

fade%(at t)=

Partial regression (PR): Regression is defined as 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 start of treatment Partially subsided. Complete regression (CR): Complete regression is defined if the tumor signal decreases to less than 80% of the initial signal.

Biometric analysis

Tumor growth based on the bioluminescence signal curve (over time) was measured for each animal in each treatment group. For the longitudinal in vivo BLI data, two nonparametric two-way analyzes of variance (ANOVA) were performed with repeated measures by day, followed by two contrastive analyzes with Bonferroni-Holm adjustment for multiplicity: p > 0.05: NS, 0.05 < p ＞ 0.01: * , p ＜ 0.01: **. For the terminal ex vivo BLI data, a one-way ANOVA was performed on the rank-transformed bioluminescence signals with factor groups. Descriptive statistics using median ± median absolute deviation are provided by group and day of measurement: p > 0.05: NS, 0.05 < p > 0.01: *, p < 0.01: **.

6.5.2 result

In the Molm-13-luc AML xenograft model, the TCRαβ-CD33-CD123 multispecific ISVD construct induced antileukemic effects in vivo ( Fig. 8A ).

In the Molm-13-luc xenograft model, TCRαβ-CD33-CD123 multispecific ISVD constructs administered intravenously every 2 days in the presence of human effector T cells (T cell/tumor ratio R = 10) It was well tolerated at all doses. No adverse events or evidence of weight changes were observed under treatment. The TCRαβ-CD33-CD123 multispecific ISVD construct performed with the same activity at all doses tested (dT/dC of 2% (p < 0.0001) vs. 2% (p) at 0.012; 0.12; 12 and 12 nmol/kg, respectively. ＜ 0.0001), 2% (p ＜ 0.0001) and 3% (p ＜ 0.0001)) inhibited systemic tumor growth ( Figure 8A , Figure 8B ).

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

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

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

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

6.6 Example 6 : T Cell-mediated killing of human target cells

6.6.1 Materials and methods

ISVD constructs were characterized for redirecting T cell-mediated killing in a flow cytometry-based cytotoxicity assay using human primary T cells as effector cells and non-adherent target cells. CD123- and/or CD33-positive target cells (MOLM-13, DSMZ ACC 554, U-937, ATCC® CRL1593.2 and KG-1a, ATCC® CCL246.1™). Effector cells (2.5 x ¹⁰ cells/well) and PKH-labeled target cells (2.5 x ¹⁰ cells/well) were plated in a 96-well V-bottom plate (Greiner Bio-one, #651 180) on target cells. Cell lines were cocultured in assay medium (targeted growth medium without antibiotics) (effector to target ratio 10:1). To analyze concentration-dependent cell lysis, serial dilutions of compounds in target assay medium were added to cells and incubated for 18 h at 37ºC in 5% CO2. 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, cells were resuspended in FACS buffer supplemented with 5 nM TO-PRO®-3 Iodide (642⁄661) (ThermoFisher Scientific, T3605) to differentiate between live and dead cells. Cells were analyzed using a FACS array flow cytometer (BD Biosciences). The total sample volume collected per sample was 80 µL. Set a gate for PKH26-positive cells and measure TO-PRO®-3-positive cells within this population. Percent specific lysis = ((% TO-PRO®-3 + no ISVD – % TO-PRO®-3 + with ISVD)/ (% TO-PRO®-3 + no ISVD))x100.

Multispecific TCRαβ-CD33-CD123 ISVD constructs according to the invention are compared with corresponding constructs in which CD33-binding ISVD or CD123-binding ISVD are replaced by irrelevant ISVD IRR (does not bind to CD33 and does not bind to CD123 binding; Table 23 ) and CD123/CD3 positive control and CD33/CD3 positive control substitutions. The results are shown in Figure 10 , Figure 11 and Figure 12 .

6.6.2 result

As can be observed in Figure 10 , in MOLM-13 cells, all compounds tested triggered tumor cell killing (which was expected) since MOLM-13 cells are positive for both CD123 and CD33. Among ISVD constructs, the dual-targeting form (CD33/CD123 TCE) has the most potent tumor cell killing effect.

Killing potency and lysis percentage in U-937 and KG-1a cells are depicted in Table 24 and Figures 11 and 12 .

surface twenty three : sample ID as well as NANOBODY® ISVD and the composition of the connector Sample ID Target ISVD1 Connector Target ISVD2 Connector Target ISVD3 A TCR 9GS CD33 9GS CD123 B TCR 9GS CD33 9GS IRR C TCR 9GS IRR 9GS CD123

surface 2 4 : in person T cell mediated U-937 or KG-1a Cell killing assay in progress , use 10 Compare 1 effector-to-target ratio , trivalent NANOBODY® ISVD of EC ₅₀ ( M ) and lysis % . Flow cytometry-based human T cell-mediated killing of U-937 cells Flow cytometry-based human T cell-mediated killing of KG-1a cells Sample ID EC ₅₀ (M) EC ₅₀ (M) 95% LCI EC ₅₀ (M) 95% UCI lysis% EC ₅₀ (M) EC ₅₀ (M) 95% LCI EC ₅₀ (M) 95% UCI lysis% CD33/CD3 1.1E-11 9.8E-12 1.3E-11 55 8.7E-12 6.9E-12 1.1E-11 15 CD123/CD3 8.9E-13 7.0E-13 1.1E-12 13 3.0E-12 2.4E-12 4.0E-12 58 A 5.9E-11 4.6E-11 7.9E-11 57 3.6E-11 2.8E-11 4.9E-11 54 B 4.7E-11 4.2E-11 5.2E-11 55 3.0E-10 2.3E-10 4.2E-10 30 C ~4.4E-12 / / 17 3.8E-11 2.9E-11 5.1E-11 64

TCR-CD123 single-targeting ISVD induced almost no killing of CD123-U-937 cells, which was also observed for the CD123/CD3 positive control. Similarly, CD33 single-targeting ISVD induced only low levels of CD33-/+ KG-1a cell killing, which was also observed for the CD33/CD3 positive control. On the other hand, the dual-targeting TCRαβ-CD33-CD123 ISVD (Construct A) demonstrated effective tumor cell killing against both CD33- and CD123- cell lines, thus exemplifying 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 the T cell engager, cytokines according to the TCRαβ-CD33-CD123 multispecific ISVD construct of the present invention were determined in an autologous healthy donor PBMC assay. Release features. In parallel, autologous depletion of monocytes within human PBMCs was evaluated. The functionality of the ISVD according to the invention was compared with the following 2 tool molecules: CD123/CD3 positive control and CD33/CD3 positive control. Non-targeting T cell engager (TCE) ISVD was used as a negative control.

6.7.1 Materials and methods

A total of 200 000 PBMCs (100 µL, 2 x ¹⁰ cells/mL in culture medium) isolated from whole blood using Leucosep ^TM tubes containing Lymphoprep ^TM solution were transferred to a 96-well V-bottom clear well plate (Greiner CELLSTAR ® 96-well plate; 651 180).

Next, add 50 µL of serial dilutions of the compound to be tested or 50 µL of medium for empty wells to the wells.

Culture plates containing treated PBMC were incubated at 37ºC, 5% CO2 for 20 h. After overnight (20 h) incubation, PBMC were centrifuged at 300 g for 2 min. Supernatants were collected and transferred to new 96-well storage plates and frozen at -20ºC for cytokine measurements. The cell pellet was suspended in 100 μL of cold FACS buffer and washed once with 100 μL of FACS buffer.

Afterwards, centrifuge the PBMC at 300 g for 2 min at 4ºC. Discard the supernatant and resuspend cells in 30 µL of diluted Fc block (1/200 in FACS buffer) (BD, 564220) and incubate for 10 minutes at room temperature.

Next, add 30 µL (2X) of the antibody staining mix (CD123, HLA-DR, and CD14 antibodies) to the PBMC suspension and incubate in the dark at 4ºC for 30 minutes.

The cell suspension was then washed 2 times and resuspended in 50 µL of diluted TO-PRO™-3 iodide. Read the plate on MACSQuantX. The readout is the number of viable cells in each subset detected by flow cytometry. Quantification of monocytes by SSC %CD14+ ( Figure 13 ).

Frozen supernatants from overnight cultured human PBMC were used to measure a panel of cytokines including IL-2, IL-6, IFNγ, TNFα using a multiplex bead assay from Bio-rad. Assays were performed according to the manufacturer's guidelines. Use the Luminex FlexMAP 3D system to acquire data from the reaction ( Figure 14 ).

6.7.2 result

The TCRαβ-CD33-CD123 multispecific ISVD construct induced depletion of monocytes associated with cytokine production in all donors. Non-targeting TCE did not show any killing or cytokine release (see Figure 13 ). The data shown in Figure 13 are from 1 donor and represent data from all 6 donors who provided PBMC.

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

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

6.8 Example 8 : Isolated primary AML Cell killing assay

6.8.1 Materials and methods

Lysis of AML blasts mediated by CD33/CD3 positive controls was tested as follows: AML samples from patients with primary diagnosis or relapse were used in an ex vivo co-culture system without the addition of any other cells. Therefore, the E:T ratio is determined by the number of remaining T cells within the primary AML sample. Whole blood samples from AML patients were provided by public hospitals or central laboratories in Marseille (La Conception, AP-HM) or Montpellier.

First, dispense 1 mL of whole blood sample per 50 mL tube and add 40 mL (1x) red blood cell lysis buffer and incubate at room temperature for 10 minutes. After incubation, the pellet was washed with PBS (Eurobio CS1PBS01-01) and resuspended in 1 mL of medium (RPMI (Eurobio Catalog No. CM1RPM00-01), FCS 10% (Sigma Catalog No. F2442-500ml 17L484), Optional Amine Amino acid 1% (Eurobio catalog number CSTAAN00), Glutamic acid 1% (Eurobio catalog number CSTGLU00), Sodium pyruvate 1% (Eurobio catalog number CSTVAT00), Penicillin/Streptomycin (Eurobio catalog number CABPES01)).

Next, in the presence of 1 mL (2x) saturating concentration of TCRαβ-CD33-CD123 multispecific ISVD construct according to the invention (CD33/CD123 TCE), positive control (CD33/CD3 or CD123/CD3) or negative control ( In the case of non-targeting TCE), add 300 µL of AML blasts to 700 µL of culture medium per well in a 6-well plate and incubate at 37ºC, 5% _CO2 . After 4 days of incubation, cells were harvested and washed once with PBS.

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

The cells were then washed and stained with a cocktail of antibodies (CD45, CD33, CD34, CD38, CD14, CD123, CD11 antibodies) and incubated on ice for 10 minutes.

Next, cells were washed and fixed with 1.5 mL PBS + 2% paraformaldehyde for 30 min at 4ºC. After 30 minutes of incubation, dilute the paraformaldehyde with 9 mL of diluent. Data were analyzed on a Cytoflex cytometer. The results are shown in Figures 15 to 17 .

6.8.2 result

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 wide range of disease subtypes. Therefore, AML samples are suitable for this assay.

In Figure 15 , AML blast cell killing in all AML patient samples is shown. Each point represents the count of viable blasts normalized to the negative control (non-targeted TCE) after treatment with one of the ISVD according to the invention or the positive control. Results from several individual patients are highlighted in Figure 17 .

As can be observed, blasts treated with ISVD according to the invention had on average the lowest cell viability compared to the positive control. Cells from all patients showed a clear response, although responses varied from patient to patient. This indicates that the 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 primates ( NHP ) Research

The pharmacokinetic (PK), pharmacodynamic (PD) and non-clinical safety profile of the TCRαβ-CD33-CD123 multispecific ISVD construct according to the invention was evaluated in a study with cynomolgus monkeys.

6.9.1 Materials and methods

A total of 4 male cynomolgus monkeys were administered an ISVD construct according to the invention as a single dose as a 1 hour continuous intravenous (IV) infusion, followed by an observation period of 21 days. Systemic exposure and potential toxicity of ISVD were determined, and PD endpoints (immune cell assessment) were evaluated. The study design is shown in Table 25 .

surface 25 : Study design: Exploratory single dose in male cynomolgus monkeys IV Research group Dosage(µg/kg) Number of animals Gender and animal identification number 1 0.04 2 M1, M2 2 0.4 2 M3, M4 Abbreviation: M :male

6.9.2 safety

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

Cytokine evaluation showed only a very minor increase in IL-6 associated with the ISVD construct administered at a single dose of 0.04 μg/kg (peaking at 2 and/or 6 hours after the start of infusion and returning to baseline within 24 hours ). No changes in IFN-γ, IL-1β, IL-2, IL-8, and TNF-α levels were observed at 0.04 μg/kg. Very small to minimal increases in IL-2, IL-6, IFN-γ, and TNF-α (peaking at 2 hours and/or 6 hours after the start of the infusion and returning to baseline within 24 hours) versus 0.4 μg/kg Correlates of ISVD constructs administered at a single dose. At 0.4 μg/kg, no changes related to the TCR-CD33-CD123 multispecific ISVD construct according to the invention were observed in monkey IL-1β and IL-8 levels.

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

6.9.3 Pharmacokinetics

Following 1 hour infusion of the ISVD construct, serum levels were quantifiable in monkeys at 0.04 μg/kg up to 3 days and at 0.4 μg/kg up to 7 days. Overall, clearance of approximately 0.07 L/day/kg was observed following IV infusion at the 0.04 μg/kg and 0.4 μg/kg dose levels. The terminal elimination half-life is estimated to be approximately 1 day. The increase in ISVD construct exposure (AUC) from 0.04 to 0.4 μg/kg was slightly smaller than expected on a dose-proportional basis, with a 7.7-fold increase in exposure for a 10-fold increase in dose, possibly due to the increase in exposure in one monkey (M3) Lower exposure was observed in . PK parameters are reported in Table 26 .

surface 26.TCRαβ-CD33-CD123 multispecific ISVD Construct toxicokinetic parameters. dose (µg/kg) C _eoi (pg/mL) T _max (day) AUC _last (day*pg/mL) T _last (day) AUC (day*pg/mL) CL (L/day/kg) V _ss (L/kg) T _1/2z (day) 0.04 830 0.06 [0.04-0.08] 523 2.5 [2-3] 621 0.0664 0.0776 1.12 0.4 7980 0.04 [0.04-0.04] 4590 5 [3-7] 4790 0.100 0.0867 1.37 Abbreviation: AUC :from 0 to infinite area under the curve, AUClast :from 0 The area under the plasma concentration versus time curve from the time to the last measurable concentration; CL :clearance rate; Ceoi : Plasma concentration at the end of infusion; t1/2 : Terminal half-life; tlast :The last measurable concentration; tmax :reached for the first time Cmax time; Vss : Steady state distribution volume

6.9.4 pharmacodynamics

IV infusion of ISVD constructs into cynomolgus monkeys at single doses of 0.04 and 0.4 μg/kg induced changes in CD123+ cell populations and CD33+ monocytes at both dose levels. These changes consist of: - An overall decrease in total CD123+ cell numbers was observed in all animals starting 6 hours after the start of infusion. This decrease was maintained in all animals throughout the study. - An overall decrease in monocyte CD33+ cell numbers was observed in all animals starting 6 hours after the start of infusion (and for animal M1 receiving the ISVD construct at 0.04 μg/kg until day 1). Complete recovery of animal M3 was observed on day 1.

Furthermore, an overall decrease in CD4+ and CD8+ cell numbers was observed in all animals starting 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 on day 3 was observed for animal M4.

Cell counts have been visualized in Figure 18 .

7 Industrial applicability

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

without

Figure 1 : Schematic representation of a multispecific ISVD construct according to the invention, showing from N-terminus to C-terminus the monovalent building blocks/ISVD TCRαβ, CD33, CD123 and Alb connected via linkers.

Figure 2 : Binding of monovalent CD33 (left) and CD123-bound BB (right) to human (top) or cynomolgus monkey (bottom) transfected CD33 and CD123 cells, respectively.

Figure 3 : Binding of A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO: 1) to human CD33 and/or human CD123 expressing cells.

Figure 4 : A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO: 1) (black square) and reference in the absence (dashed curve) or presence (solid curve) of clinical grade HSA Dose-dependent inhibition of primary T cells by TCR (gray dots) in a competition assay.

Figure 5 : Dose-dependent human (top) or cynomolgus monkey (bottom) T cells in an impedance-based assay (xCELLigence) in the presence of 50 µM HSA using an effector to target ratio of 15 to 1 Mediated killing of cells transfected with CD33 (left) or CD123 (right) of the corresponding species.

Figure 6 : Dose-dependent human (left) or cynomolgus monkey (right) T cell-mediated killing of MOLM-13 cells in a flow cytometry-based assay using an effector to target ratio of 10:1. Plot % TO-PRO®-3 positive target cells versus ISVD concentration.

Figure 7 : Dose-dependent human T cell-mediated cell killing in an impedance-based assay (xCELLigence) using an effector to target ratio of 15 to 1. The cell index (CI) after 32 to 35 hours of incubation was plotted against the concentration of ISVD.

Figure 8 : Inhibition of Molm13-luc AML tumor growth by A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO.: 1) by in vivo bioluminescence imaging.

Figure 9 : Inhibition of Molm13-luc AML tumor growth by A025001562 (TCR-CD33-CD123 multispecific ISVD construct, SEQ ID NO: 1) by ex vivo bioluminescence imaging.

Figure 10 : Dose-dependent human T cell-mediated killing of ISVD according to the invention in MOLM-13 cells compared to CD123 and CD33 positive controls.

Figure 11 : Dose-dependent human T cell-mediated killing of ISVD according to the invention in KG-1a cells compared to CD123 and CD33 positive controls.

Figure 12 : Dose-dependent human T cell-mediated killing of ISVD according to the invention in U-937 cells compared to CD123 and CD33 positive controls.

Figure 13 : Dose-dependent monocyte depletion of ISVD according to the invention in human peripheral blood mononuclear cells (PBMC) compared to CD123 and CD33 positive controls and a negative control group (non-targeted TCE).

Figure 14 : Dose-dependent cellularity of ISVD according to the invention compared to CD123 and CD33 positive controls and negative control (non-targeted TCE) in human PBMC from healthy donors using a panel of different cytokines Hormone release. A.IL -6. B. IFNγ. C. TNFα. D. IL-2.

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

Figure 16 : Scatter plot depicting the percentage of CD33 or CD123 positive cells for each AML sample.

Figure 17 : Cell viability of primary blasts from AML patients with broad disease subtypes relative to ISVD according to the invention (compared to CD123 and CD33 positive controls and negative controls). A. Patient number 3. B. Patient number 4. C. Patient number 5.

Figure 18 : Total CD123+ T cells ( A ), monocyte CD33+ cells ( B ), CD4+ T cells ( C ) and CD8+ T cells as measured in the peripheral blood of cynomolgus monkeys treated with ISVD according to the invention ( D ) Changes in individual absolute cell counts over time. Animals M1 and M2 received 0.04 μg/kg, while M3 and M4 received a single 1 hour continuous intravenous infusion of 0.4 μg/kg of a solution of the ISVD according to the invention.

TW202342508A_111148430_SEQL.xml

without.

Claims (26)

A polypeptide, a composition comprising the polypeptide, or a composition comprising a nucleic acid containing a nucleotide sequence encoding the polypeptide, wherein the polypeptide comprises or consists of at least three immunoglobulin single variable domains (ISVDs), wherein the ISVDs each comprise three complementarity determining regions (CDR1 to CDR3, respectively) optionally linked via one or more peptide linkers; and wherein: a. A first ISVD that specifically binds to T cell receptor αβ (TCRαβ) and contains i. CDR1, which contains SEQ ID NO: 6 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 6; ii. CDR2 comprising SEQ ID NO: 10 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 10; and iii. CDR3, which includes SEQ ID NO: 14 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 14; b. A second ISVD that specifically binds to CD33 and contains iv. CDR1, which contains SEQ ID NO: 7 or an amino acid sequence that differs from SEQ ID NO: 7 by 2 or 1 amino acid; v. CDR2, which contains SEQ ID NO: 11 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 11; and vi. CDR3 comprising SEQ ID NO: 15 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 15; and c. A third ISVD that specifically binds to CD123 and contains vii. CDR1, which contains SEQ ID NO: 8 or an amino acid sequence that differs from SEQ ID NO: 8 by 2 or 1 amino acid; viii. CDR2, which contains SEQ ID NO: 12 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 12; and ix. CDR3 comprising SEQ ID NO: 16 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 16, The sequence of the ISVDs represents the relative positions of the ISVDs to each other from the N-terminus to the C-terminus of the polypeptide. The polypeptide or composition of claim 1, wherein: a. The first ISVD includes CDR1 containing the amino acid sequence of SEQ ID NO: 6, CDR2 containing the amino acid sequence of SEQ ID NO: 10, and CDR3 containing the amino acid sequence of SEQ ID NO: 14; b. The second ISVD includes CDR1 containing the amino acid sequence of SEQ ID NO: 7, CDR2 containing the amino acid sequence of SEQ ID NO: 11, and CDR3 containing the amino acid sequence of SEQ ID NO: 15; and c. The third ISVD includes CDR1 containing the amino acid sequence of SEQ ID NO: 8, CDR2 containing the amino acid sequence of SEQ ID NO: 12, and CDR3 containing the amino acid sequence of SEQ ID NO: 16. The polypeptide or composition according to any one of claims 1 or 2, wherein: a. The amino acid sequence of the first ISVD contains greater than 90% sequence identity with SEQ ID NO: 2; b. The amino acid sequence of the second ISVD contains greater than 90% sequence identity with SEQ ID NO: 3; and c. The amino acid sequence of the third ISVD contains greater than 90% sequence identity with SEQ ID NO: 4. The polypeptide or composition according to any one of claims 1 to 3, wherein: a. The first ISVD contains the amino acid sequence of SEQ ID NO: 2; b. The second ISVD contains the amino acid sequence of SEQ ID NO: 3; and c. The third ISVD contains the amino acid sequence of SEQ ID NO: 4. The polypeptide or composition of any one of claims 1 to 4, wherein the polypeptide further comprises one or more other groups, residues, moieties or combinations optionally connected via one or more peptide linkers Units wherein the one or more other groups, residues, moieties or binding units increase the half-life of the polypeptide compared to the corresponding polypeptide without the one or more other groups, residues, moieties or binding units. The polypeptide or composition for the use as described in claim 5, wherein the one or more other groups, residues, moieties or binding units that increase the half-life of the polypeptide are selected from the group consisting of: : Polyethylene glycol molecules, serum proteins or fragments thereof, binding units that can bind to serum proteins, Fc portions and small proteins or peptides that can bind to serum proteins. The polypeptide or composition of any one of claims 5 to 6, wherein the one or more other groups, residues, moieties or binding units that increase the half-life of the polypeptide are selected from the group consisting of: : A binding unit that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin (such as IgG). The polypeptide or composition of claim 7, wherein the binding unit that increases the half-life of the polypeptide is a fourth ISVD that can bind to human serum albumin. The polypeptide or composition of claim 8, wherein the fourth ISVD binding to the human serum albumin comprises i. CDR1, which contains SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid difference from SEQ ID NO: 9; ii. CDR2 comprising SEQ ID NO: 13 or an amino acid sequence that differs by 2 or 1 amino acid from SEQ ID NO: 13; and iii. CDR3, which comprises SEQ ID NO: 17 or an amino acid sequence that differs from SEQ ID NO: 17 by 2 or 1 amino acid. The polypeptide or composition of any one of claims 8 to 9, wherein the ISVD binding to the human serum albumin includes a CDR1 containing the amino acid sequence of SEQ ID NO: 9, a CDR1 containing the amino acid sequence of SEQ ID NO: 13 The CDR2 of the amino acid sequence and the CDR3 of the amino acid sequence of SEQ ID NO: 17. The polypeptide or composition of any one of claims 8 to 10, wherein the amino acid sequence of the ISVD that binds to the human serum albumin comprises greater than 90% sequence identity with SEQ ID NO: 5. The polypeptide or composition of any one of claims 8 to 11, wherein the ISVD binding to the human serum albumin comprises the amino acid sequence of SEQ ID NO: 5. The polypeptide or composition of any one of claims 1 to 12, wherein the amino acid sequence of the polypeptide contains greater than 90% sequence identity with SEQ ID NO: 1. 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. A nucleic acid comprising a nucleotide sequence encoding the polypeptide of any one of claims 1 to 14. A host or host cell comprising the nucleic acid of claim 15. A method for producing the polypeptide described in any one of claims 1 to 14, the method at least includes the following steps: a. Express the nucleic acid of claim 15 in a suitable host cell or host organism or in another suitable expression system; optionally followed by: b. Isolate and/or purify the polypeptide as described in any one of claims 1 to 14. A composition comprising at least one polypeptide according to any one of claims 1 to 14 or a nucleic acid according to claim 15. The composition according to claim 18, which is a pharmaceutical composition, which further contains at least one pharmaceutically acceptable carrier, diluent or excipient and/or adjuvant, and optionally Contains one or more additional pharmaceutically active polypeptides and/or compounds. The polypeptide according to any one of claims 1 to 14 or the composition according to any one of claims 18 or 19, which is used as a medicine. The polypeptide according to any one of claims 1 to 14 or the composition according to any one of claims 18 or 19, which is used to treat acute myelogenous leukemia (AML). The polypeptide or composition for the use as described in claim 21, wherein the AML is relapsed and/or refractory AML. A method of treating acute myeloid leukemia (AML), wherein the method comprises administering to a subject in need a pharmaceutically active amount of the polypeptide of any one of claims 1 to 14 or claim 18 or 19 The composition described in any one of them. The method of claim 23, wherein the AML is relapsed and/or refractory AML. Use of the polypeptide as described in any one of claims 1 to 14 or the composition as described in any one of claims 18 or 19 in the preparation of a pharmaceutical composition for the treatment of acute myelogenous leukemia (AML). The use of a polypeptide or composition as described in claim 25, wherein the AML is relapsed and/or refractory AML.