CN117986371A

CN117986371A - Antibodies that bind CD123 and gamma-delta T cell receptors

Info

Publication number: CN117986371A
Application number: CN202410080627.6A
Authority: CN
Inventors: R·C·罗弗斯; J·J·范德弗利特; D·卢杰·胡尔西克; P·W·H·I·帕伦; J·M·鲁本; C·M·穆塞特
Original assignee: Lava Therapy Co
Current assignee: Lava Therapy Co
Priority date: 2021-02-26
Filing date: 2022-02-28
Publication date: 2024-05-07
Also published as: CN117545772A

Abstract

The present invention relates to antibodies capable of binding to human CD123 and capable of binding to the vδ2 chain of the human vγ9vδ2T cell receptor. The invention also relates to pharmaceutical compositions comprising the antibodies of the invention and to the use of the antibodies of the invention for medical treatment.

Description

Antibodies that bind CD123 and gamma-delta T cell receptors

The application is a divisional application of China patent application (application day: 2022, 2, 28, title of application: antibodies binding to CD123 and gamma-delta T cell receptors) with application number 202280026880.2.

Technical Field

The present invention relates to novel multispecific antibodies capable of binding to human CD123 and capable of binding to the vδ2 chain of human vγ9vδ2T cell receptor. The invention also relates to pharmaceutical compositions comprising the antibodies of the invention and to the use of the antibodies of the invention for medical treatment.

Background

The CD123 or interleukin-3 (IL 3) receptor alpha chain is a membrane protein that transmits signals through IL3, a cytokine involved in blood cell production. CD123 forms heterodimers with the common beta chain CD 131. CD123 is normally expressed on some types of blood cells (such as plasmacytoid dendritic cells or monocytes) and is expressed by a subset of normal myeloid progenitor cells. However, CD123 is strongly overexpressed on leukemic stem cells in patients with acute myeloid leukemia. CD123 is thus a potential therapeutic target in several hematological malignancies, including acute myeloid leukemia.

Several bispecific CD123-CD 3T cell engagement antibodies have been described (Kuo et al (2012) Protein ENG DES SET 10:561; al-Hussaini et al (2016) Blood 127:122). Bispecific T cell engager antibodies have tumor target binding specificity and T cell binding specificity and thus boost efficacy by redirecting T cell cytotoxicity to malignant cells, see, e.g., huehls et al (2015) Immunol Cell Biol 93:290; ellerman (2019) Methods,154:102; de Bruin et al (2017) Oncoimmunology (1): e1375641 and WO2015156673. However, the results are very different. For example, in one study combining a CD3 binding moiety with binding moieties directed against 8 different B cell targets (CD 20, CD22, CD24, CD37, CD70, CD79B, CD138 and HLA-DR), bispecific antibodies targeting different tumor targets were found to exhibit a large difference in their ability to induce cytotoxicity of the target cells, and cytotoxicity was not correlated with antigen expression levels. For example, CD 3-based bispecific antibodies targeting HLA-DR or CD138 are incapable of inducing cytotoxicity despite moderate to high levels of expression of HLA-DR and CD138 (Engelberts et al (2020) Ebiomedicine 52:102625). Few T cell redirection therapies have reached the later stage of clinical development, possibly due to significant toxicity, manufacturing problems, immunogenicity, narrow therapeutic window, and low response. In particular, when the T cell adapter (T-CELL ENGAGER) includes a CD3 binding arm, toxicity may occur and result in uncontrolled, excessive immune activation and cytokine release.

Thus, despite significant advances, there remains a need for novel CD 123-targeting antibodies that are therapeutically effective and have acceptable toxicity as well as stability and manufacturability.

Disclosure of Invention

The present invention provides novel antibodies for CD 123-based therapies. Bispecific antibodies were constructed in which a single domain CD123 binding region was combined with a binding region capable of binding to the vδ2 chain of a human vγ9vδ2T cell receptor and thereby engaging γδ T cells. Surprisingly, bispecific antibodies were exceptionally effective in mediating activation of vγ9vδ2t cells and inducing killing of CD123 expressing cell lines as well as patient-derived tumor cells in the presence of vγ9vδ2t cells.

Thus, in a first aspect, the invention provides a multispecific antibody comprising a first antigen-binding region capable of binding to human CD123 and a second antigen-binding region capable of binding to the vδ2 chain of a human vγ9vδ2T cell receptor.

In another broad aspect, the invention provides an antibody comprising a first antigen binding region capable of binding to human CD123, wherein the first antigen binding region is a single domain antibody comprising:

(i) A VH CDR1 sequence shown in SEQ ID No. 2, a VH CDR2 sequence shown in SEQ ID No. 3 and a VH CDR3 sequence shown in SEQ ID No. 4, wherein preferably the first antigen binding region comprises or consists of: a sequence selected from the group of sequences set forth in SEQ ID NO. 1, 25 to 34, or a sequence having at least 90%, such as at least 92%, e.g. at least 94%, such as at least 96%, e.g. at least 98% sequence identity to a sequence selected from the group of sequences set forth in SEQ ID NO. 1, 25 to 34, or

(Ii) A VH CDR1 sequence shown in SEQ ID No. 10, a VH CDR2 sequence shown in SEQ ID No. 11 and a VH CDR3 sequence shown in SEQ ID No. 12, wherein preferably the first antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 9, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 9.

In summary, the present invention includes, but is not limited to, the following:

1. A multispecific antibody comprising a first antigen-binding region capable of binding to human CD123 and a second antigen-binding region capable of binding to the vδ2 chain of a human vγ9vδ2T cell receptor.

2. The multispecific antibody of item 1, wherein the multispecific antibody is a bispecific antibody.

3. The multispecific antibody of any one of the preceding claims, wherein the first antigen-binding region is a single domain antibody and/or the second antigen-binding region is a single domain antibody.

4. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody competes for binding to human CD123 with an antibody having the sequence set forth in SEQ ID No. 1, preferably wherein the multispecific antibody binds to the same epitope on human CD123 as an antibody having the sequence set forth in SEQ ID No. 1.

5. A multispecific antibody according to any one of the preceding claims, wherein the first antigen-binding region comprises or consists of a VH CDR1 sequence as set out in SEQ ID No. 2, a VH CDR2 sequence as set out in SEQ ID No. 3 and a VH CDR3 sequence as set out in SEQ ID No. 4, wherein preferably the first antigen-binding region comprises or consists of: a sequence selected from the group of sequences set forth in SEQ ID nos. 1, 25 to 34, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity with a sequence selected from the group of sequences set forth in SEQ ID nos. 1, 25 to 34.

6. A multispecific antibody according to any one of claims 1 to 3, wherein the multispecific antibody competes for binding to human CD123 with an antibody having the sequence set forth in SEQ ID No. 9, preferably wherein the multispecific antibody binds to the same epitope on human CD123 as an antibody having the sequence set forth in SEQ ID No. 9.

7. The multispecific antibody of item 6, wherein the first antigen-binding region comprises or consists of a VH CDR1 sequence set forth in SEQ ID No. 10, a VH CDR2 sequence set forth in SEQ ID No. 11, and a VH CDR3 sequence set forth in SEQ ID No. 12, wherein preferably the first antigen-binding region comprises or consists of: the sequence shown in SEQ ID NO. 9, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 9.

8. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody is capable of activating human vγ9vδ2t cells.

9. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody competes for binding to human vδ2 with an antibody having the sequence shown in SEQ ID No. 17, wherein X ₄ is Y, preferably wherein the multispecific antibody binds to the same epitope on human vδ2 as an antibody having the sequence shown in SEQ ID No. 17, wherein X ₄ is Y,

Or (b)

Wherein the multispecific antibody competes for binding to human V.delta.2 with an antibody having the sequence set forth in SEQ ID NO. 36, preferably wherein the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence set forth in SEQ ID NO. 36,

Or (b)

Wherein the multispecific antibody competes for binding to human V.delta.2 with an antibody having the sequence shown in SEQ ID NO. 37, preferably wherein the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence shown in SEQ ID NO. 37,

Or (b)

Wherein the multispecific antibody competes for binding to human vδ2 with an antibody having a sequence shown in SEQ ID No. 38, preferably wherein the multispecific antibody binds to the same epitope on human vδ2 as an antibody having a sequence shown in SEQ ID No. 38.

10. A multispecific antibody according to any one of the preceding claims, wherein the second antigen-binding region comprises or consists of a VH CDR1 sequence as set out in SEQ ID No. 18, a VH CDR2 sequence as set out in SEQ ID No. 19 and a VH CDR3 sequence as set out in SEQ ID No. 20, wherein preferably the second antigen-binding region comprises or consists of: the sequence shown in SEQ ID NO. 17, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 17,

Or (b)

Wherein the second antigen binding region comprises or consists of a VH CDR1 sequence as set forth in SEQ ID No. 39, a VH CDR2 sequence as set forth in SEQ ID No. 40 and a VH CDR3 sequence as set forth in SEQ ID No. 41, wherein preferably the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 36, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 36,

Or (b)

Wherein the second antigen binding region comprises or consists of a VH CDR1 sequence as set forth in SEQ ID No. 42, a VH CDR2 sequence as set forth in SEQ ID No. 43 and a VH CDR3 sequence as set forth in SEQ ID No. 44, wherein preferably the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 37, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 37,

Or (b)

Wherein the second antigen binding region comprises or consists of a VH CDR1 sequence as set forth in SEQ ID No. 45, a VH CDR2 sequence as set forth in SEQ ID No. 46 and a VH CDR3 sequence as set forth in SEQ ID No. 47, wherein preferably the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 38, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 38.

11. The multispecific antibody of any one of the preceding claims, wherein

(I) The first antigen binding region comprises a VH CDR1 sequence shown in SEQ ID NO. 2, a VH CDR2 sequence shown in SEQ ID NO. 3 and a VH CDR3 sequence shown in SEQ ID NO.4, and the second antigen binding region comprises a VH CDR1 sequence shown in SEQ ID NO. 18, a VH CDR2 sequence shown in SEQ ID NO. 19 and a VH CDR3 sequence shown in SEQ ID NO. 20, or

(Ii) The first antigen binding region comprises a VH CDR1 sequence shown in SEQ ID NO. 10, a VH CDR2 sequence shown in SEQ ID NO. 11 and a VH CDR3 sequence shown in SEQ ID NO. 12, and the second antigen binding region comprises a VH CDR1 sequence shown in SEQ ID NO. 18, a VH CDR2 sequence shown in SEQ ID NO. 19 and a VH CDR3 sequence shown in SEQ ID NO. 20.

12. The multispecific antibody of any one of the preceding claims, wherein the first antigen-binding region capable of binding to human CD123 is located N-terminal to the second antigen-binding region capable of binding to the human vδ2 chain.

13. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody further comprises a half-life extending domain, such as an Fc region, preferably a human Fc region.

14. The multispecific antibody of claim 13, wherein the Fc region is a heterodimer comprising two Fc polypeptides, wherein the first antigen-binding region is fused to the first Fc polypeptide and the second antigen-binding region is fused to the second Fc polypeptide, and wherein the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor heterodimer formation relative to homodimer formation, wherein preferably the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L a and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

15. The multispecific antibody of any one of claims 13 or 14, wherein the cysteine residue at position 220 in the first and second Fc polypeptides has been deleted or substituted, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

16. The multispecific antibody of any one of claims 13 to 15, wherein the first and second Fc polypeptides further comprise mutations at positions 234 and/or 235, preferably wherein the first and second Fc polypeptides comprise L234F and L235E substitutions, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

17. The multispecific antibody of any one of claims 13 to 16, wherein the first Fc polypeptide comprises the sequence set forth in SEQ ID No. 21 and the second Fc polypeptide comprises the sequence set forth in SEQ ID No. 22, or vice versa.

18. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody is capable of mediating killing of CD 123-expressing cells such as C1r-neo cells or THP-1 cells by vγ9vδ2t cells.

19. An antibody comprising a first antigen binding region capable of binding human CD123, wherein the first antigen binding region is a single domain antibody comprising:

20. A pharmaceutical composition comprising the multispecific antibody according to any one of the preceding claims or the antibody according to claim 19 and a pharmaceutically acceptable excipient.

21. The multispecific antibody according to any one of claims 1to 18 or the antibody according to claim 19 for use as a medicament, preferably for use in the treatment of cancer, more preferably for the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, hodgkin lymphoma, blast plasmacytoid dendritic cell tumor, chronic granulocytic leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder or myelodysplastic syndrome.

22. A nucleic acid construct comprising a nucleotide sequence encoding the antibody of any one of claims 1 to 19, or a host cell comprising one or more nucleic acid constructs encoding the antibody of any one of claims 1 to 19.

Additional aspects and embodiments of the invention are described below.

Drawings

Fig. 1: ELISA showing binding of all different bispecific VHHs to CD123, vγ9vδ2 (GDT) TCR and BSA (as negative controls). OD values are depicted; the value is the average of repeated measurements; error bars represent standard error of the mean. Monovalent anti-vδ2vhh was used as a control for TCR staining ("TCR control") and commercially available anti-CD 123 antibodies ("anti-CD 123") were used as controls for CD123 antigen coating. "no AB" means a negative control without primary antibody.

Fig. 2: binding specificity of bispecific VHH as determined using flow cytometry. The geometric mean of the fluorescent signal is plotted as a function of the antibody and cell type used. VHH recognizing EGFR expressed endogenously by 293F cells were included as positive control. Monovalent anti-vδ2vhh was used as negative control VHH.

Fig. 3: the apparent affinity of 1D2-5C8var1 binding to CD123 was determined using flow cytometry. Serial dilutions of purified 1D2-5C8var1 were tested for binding to 293F cells transiently expressing CD123 or CD131 or both CD123 and CD 131. The geometric mean of the fluorescence intensities was plotted as a function of the concentration of antibody used. EC50 values were determined by curve fitting.

Fig. 4: representative BLI analysis of 1D2-5C8var1 binding to CD 123. The light reflectance shift (measured in nm) representing the mass of the bound protein is plotted as a function of time. 0-300 seconds: an association phase; 300-900 seconds: and a dissociation stage.

Fig. 5: C1R-neo target cell-dependent, 1D2-5C8var1 mediated activation of Vγ9Vδ2T cells. The percentage of cd3+ -vγ 9+T cells exhibiting CD107A expression (degranulation) was plotted as a function of the concentration of antibody used. Experiments using two different donors are depicted.

Fig. 6:1D2-5C8var 1-induced, V gamma 9V delta 2T cell-mediated cytotoxicity of C1R-neo target cells. The percentage of viable C1R-neo target cells was plotted as a function of the concentration of bispecific VHH used. Data obtained using two different donors of vγ9vδ2t cells are depicted.

Fig. 7: bispecific VHH-induced vγ9vδ2T cell mediated cytotoxicity of THP-1 target cells. The percentage of viable THP-1 target cells was plotted as a function of the concentration of bispecific VHH used.

Fig. 8: bispecific VHH mediated vγ9vδ2T cell activation and bispecific VHH mediated T cell induced lysis of patient-derived primary AML samples. Upper graph: t cell activation as measured by CD107A expression. The following figures: t cells along with bispecific VHH lyse AML blasts.

Fig. 9: HP-SEC profile of purified anti-CD 123 x V γ9Vδ2TCR bispecific antibody 1D2-5C8var1 (Y105F) -Fc.

Fig. 10:1D2-5C8var1 (Y105F) -Fc induces Vγ9Vδ2T cell activation. A typical experiment is shown. The percentage of CD107a- (lysosomal associated protein-1, or LAMP-1) positive V.gamma.9V.delta.2 cells is plotted as a function of the concentration of compound used. EC50 values (in pM, determined by curve fitting) are depicted in the lower graph. Data points are averages of three replicates; error bars represent standard deviation.

Fig. 11:1D2-5C8var1 (Y105F) -Fc induces T cell mediated lysis of target cells. A typical experiment is shown. The graph shows the percentage of target cells killed after 24 hours of co-culture as a function of the concentration of compound used. EC50 values (in pM, determined by curve fitting) are depicted in the lower graph. Data points are averages of three replicates; error bars represent standard deviation.

Fig. 12: CD123 expression levels on plasmacytoid dendritic cells (upper panel) and THP-1 cell lines (lower panel). Typical staining is shown. Histograms showing unstained cells, isotype control staining (left overlapping histogram) and staining for CD123 (right peak) are depicted. The event number (Y-axis) is shown as a function of fluorescence intensity (X-axis).

Fig. 13: 1D2-5C8var1 (Y105F) -Fc induced preferential killing of THP-1 cells compared to pDC. Representative results are shown. The percentage of target cells killed is plotted as a function of the concentration of compound used per target cell population (i.e., THP-1 or pDC). EC50 values (in pM, determined by curve fitting) are depicted in the lower graph. Data points are averages of three replicates; error bars represent standard deviation.

Fig. 14: the primary amino acid sequence of full-length human CD123 (GenBank accession No. NM-002183.4) (SEQ ID NO: 23). Residues found to cross-link with the 1D2 antibody are indicated in bold and underlined. Residues in italics (flanked by found reactive residues) may also be part of the recognized epitope.

Fig. 15: c-alpha trace model of CD123 (IL-3 receptor alpha chain: broughton et al 2018 2018Nat Commun.9:386); residues found to crosslink with antibodies are indicated. The transmembrane helix will be located on the left side of the figure.

Fig. 16: the stress-induced changes determined by using (a) size exclusion chromatography by ultraviolet absorption (SEC-UV) and (B) capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and performed after reduction were measured for aggregates and fragments.

Fig. 17: (A) Degranulation was analyzed after 4 hours by measuring the percentage of CD107a (lysosomal associated protein-1, or LAMP-1) positive cells using flow cytometry. (B) T cell activation was analyzed by measuring the percentage of CD25 positive cells. (C) Cytotoxicity was analyzed by determining the percentage of viable target cells after 24 hours using flow cytometry.

Detailed Description

Definition of the definition

As used herein, the term "human CD123" refers to the human CD123 protein, also known as the interleukin-3 receptor alpha chain (GenBank accession NM-002183.4, NCBI reference sequence: NP-002174.1). The sequence of human CD123 is shown in SEQ ID NO. 23. IL3 receptor is a heterodimer of CD123 and the common beta chain CD131 (NCBI reference sequence: NP-000386.1). CD131 is shown as SEQ ID NO. 24.

As used herein, the term "human V.delta.2" refers to the rearranged.delta.2 chain of the V.gamma.9V.delta.2-T Cell Receptor (TCR) (SEQ ID NO: 48). UniProtKB-A0JD36 (A0JD36_HUMAN) gives an example of a variable TRDV sequence.

As used herein, the term "human vγ9" refers to the rearranged y9 chain of the vγ9vδ2-T Cell Receptor (TCR). UniProtKB-Q99603_HUMAN gives an example of a variable TRGV sequence.

The term "antibody" is intended to refer to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of any of these, which has the ability to specifically bind to an antigen under typical physiological conditions, and which has a half-life of a substantial period of time, such as at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., or any other relevant functionally defined period of time (such as a period of time sufficient to induce, promote, enhance, and/or modulate a physiological reaction associated with an antibody binding antigen and/or a period of time sufficient for an antibody to recruit effector activity). The antigen binding region that interacts with an antigen may comprise or consist of the variable regions of the heavy and light chains of an immunoglobulin molecule, or may comprise or consist of a single domain antigen binding region, such as a heavy chain only variable region. The constant region of an antibody, if present, may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells and T cells) and components of the complement system, such as C1q, which are the first components of the classical pathway of complement activation. However, in some embodiments, the Fc region of an antibody has been modified to be rendered inert, "inert" means that the Fc region is at least incapable of binding any fcγ receptor, inducing Fc-mediated FcR crosslinking, or inducing FcR-mediated target antigen crosslinking through both Fc regions of a single antibody. In another embodiment, the inert Fc region is not capable of binding C1q in addition. In one embodiment, the antibody comprises mutations at positions 234 and 235 (Canfield and Morrison (1991) J Exp Med 173:1483), e.g., a Leu to Phe mutation at position 234 and a Leu to Glu mutation at position 235 (according to EU numbering, see below). In another embodiment, the antibody comprises a Leu to Ala mutation at position 234, a Leu to Ala mutation at position 235, and a Pro to Gly mutation at position 329. In another embodiment, the antibody comprises a Leu to Phe mutation at position 234, a Leu to Glu mutation at position 235, and an Asp to Ala mutation at position 265.

The Fc region of an immunoglobulin is defined as the antibody fragment that will normally be produced upon digestion of an antibody with papain, and includes the two CH2-CH3 regions of the immunoglobulin and a linking region, such as a hinge region. The constant domains of the antibody heavy chains define antibody isotypes, e.g., igG1, igG2, igG3, igG4, igA1, igA2, igM, igD, or IgE. The Fc region mediates antibody effector functions using cell surface receptors called Fc receptors and proteins of the complement system.

The term "hinge region" as used herein is intended to refer to the hinge region of an immunoglobulin heavy chain. Thus, for example, the hinge region of a human IgG1 antibody corresponds to amino acids 216-230 according to EU numbering.

The term "CH2 region" or "CH2 domain" as used herein is intended to refer to the CH2 region of an immunoglobulin heavy chain. Thus, for example, the CH2 region of a human IgG1 antibody corresponds to amino acids 231-340 according to EU numbering. However, the CH2 region may also be any other subtype as described herein.

The term "CH3 region" or "CH3 domain" as used herein is intended to refer to the CH3 region of an immunoglobulin heavy chain. Thus, for example, the CH3 region of a human IgG1 antibody corresponds to amino acids 341-447 according to EU numbering. However, the CH3 region may also be any other subtype as described herein.

The reference in the present invention to amino acid positions in the Fc region/Fc domain is according to EU numbering (Edelman et al, proc NATL ACAD SCI U S A.1969, month 5; 63 (1): 78-85; kabat et al, sequences of proteins of immunological inter 5 th edition-1991 NIH publication No. 91-3242).

As noted above, the term antibody as used herein includes antibody fragments that retain the ability to specifically bind antigen unless otherwise indicated or clearly contradicted by context. It has been shown that the antigen binding function of an antibody can be performed by fragments of full length antibodies. Examples of binding fragments encompassed within the term "antibody" include (i) Fab' or Fab fragments, i.e. monovalent fragments consisting of VL, VH, CL and CH1 domains, or monovalent antibodies as described in WO 2007059782; (ii) F (ab') 2 fragments, i.e., bivalent fragments comprising two Fab fragments linked at the hinge region by a disulfide bridge; (iii) an Fd fragment consisting essentially of VH and CH1 domains; and (iv) Fv fragments consisting essentially of the VL and VH domains of a single arm of an antibody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker, which enables them to be prepared as a single protein chain, in which the VL and VH regions pair to form monovalent molecules (known as single chain antibodies or single chain Fv (scFv), see, e.g., bird et al, science 242,423-426 (1988) and Huston et al, PNAS USA 85,5879-5883 (1988)). Such single chain antibodies are encompassed within the term antibody unless the context indicates otherwise. Unless otherwise indicated, the term antibody also includes polyclonal antibodies, monoclonal antibodies (mabs), chimeric antibodies, and humanized antibodies, as well as antibody fragments provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

In some embodiments of the antibodies of the invention, the first antigen binding region or the second antigen binding region, or both, are single domain antibodies. Single domain antibodies (sdabs, also known asOr VHH) are well known to the skilled person, see for example Hamers-Casterman et al (1993) Nature 363:446, roovers et al (2007) Curr Opin Mol Ther 9:327 and Krah et al (2016) Immunopharmacol Immunotoxicol 38:21. A single domain antibody comprises a single CDR1, a single CDR2, and a single CDR3. Examples of single domain antibodies are variable fragments of heavy chain-only antibodies, antibodies that do not naturally contain light chains, single domain antibodies derived from conventional antibodies, and engineered antibodies. The single domain antibodies may be derived from any species including mouse, human, camel, llama, shark, goat, rabbit and bovine. For example, naturally occurring VHH molecules may be derived from antibodies produced in camelidae species, such as camels, dromedaries, llamas, alpacas and alpacas. Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may comprise only the variable domains of the immunoglobulin chain, namely CDR1, CDR2 and CDR3, as well as the framework regions.

The term "immunoglobulin" as used herein is intended to refer to a class of structurally related glycoproteins that generally consist of two pairs of polypeptide chains, a pair of light (L) chains and a pair of heavy (H) chains, all of which may be linked to each other by disulfide bonds, although some mammalian species do not produce light chains but only heavy chain antibodies. The term "immunoglobulin heavy chain", "heavy chain of an immunoglobulin" or "heavy chain" as used herein is intended to refer to one of the chains of an immunoglobulin. Heavy chains are typically composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH) that define the isotype of immunoglobulins. The heavy chain constant region is typically composed of three domains, CH1, CH2 and CH 3. The heavy chain constant region also comprises a hinge region. Within the structure of an immunoglobulin (e.g., igG), two heavy chains are connected to each other by disulfide bonds in the hinge region. As with heavy chains, each light chain is typically composed of several regions; namely a light chain variable region (VL) and a light chain constant region (CL). Furthermore, VH and VL regions can be subdivided into regions of hypervariability (or regions of hypervariability in sequence and/or forming structurally defined loops), also known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FR). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.CDR sequences can be determined using various methods, such as those provided in the following references: choitia and Lesk (1987) J.mol.biol.196:901 or Kabat et al (1991) Sequence of protein of immunological interest, fifth edition. Various methods for CDR determination and amino acid numbering can be compared at www.abysis.org (UCL).

The term "isotype" as used herein refers to immunoglobulin (subclass) (e.g., igG1, igG2, igG3, igG4, igD, igA, igE, or IgM) or any isotype thereof, such as IgG1m (za) and IgG1m (f) encoded by heavy chain constant region genes. Each heavy chain isoform may be combined with a kappa (kappa) or lambda (lambda) light chain. The antibodies of the invention may be of any isotype.

The term "parent antibody" is understood to mean an antibody identical to an antibody according to the invention, but wherein the parent antibody does not have one or more of the specified mutations. A "variant" or "antibody variant" or "variant of a parent antibody" of the invention is an antibody molecule comprising one or more mutations compared to the "parent antibody". Amino acid substitutions may exchange a natural amino acid for another naturally occurring amino acid, or for a non-naturally occurring amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the class of amino acids reflected in one or more of the following three tables:

amino acid residue class for conservative substitutions

Acidic residues	Asp (D) and Glu (E)
		Basic residues	Lys (K), arg (R) and His (H)
Hydrophilic uncharged residues	Ser (S), thr (T), asn (N) and Gln (Q)
		Aliphatic uncharged residues	Gly (G), ala (A), val (V), leu (L) and Ile (I)
Nonpolar uncharged residues	Cys (C), met (M) and Pro (P)
		Aromatic residues	Phe (F), tyr (Y) and Trp (W)

Substitution class of alternative conservative amino acid residues

1	A	S	T
				2	D	E
3	N	Q
				4	R	K
5	I	L	M
				6	F	Y	W

Alternative physical and functional classifications of amino acid residues

In the context of the present invention, the substitutions in the variants are expressed as:

Original amino acid-position-substituted amino acid;

Three-letter codes or single-letter codes are used, including codes Xaa and X to indicate amino acid residues. Thus, the notation "T366W" means that the variant comprises a tryptophan to threonine substitution in the variant amino acid position corresponding to amino acid position 366 of the parent antibody.

Furthermore, the term "substitution" includes substitution to any of the other nineteen natural amino acids, or to other amino acids, such as unnatural amino acids. For example, the substitution of amino acid T in position 366 includes each of the following substitutions: 366A, 366C, 366D, 366G, 366H, 366F, 366I, 366K, 366L, 366M, 366N, 366P, 366Q, 366R, 366S, 366E, 366V, 366W, and 366Y.

As used herein, the term "full length antibody" refers to an antibody comprising all the heavy and light chain constant and variable domains corresponding to those typically found in wild-type antibodies of the isotype.

The term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., from a rodent) and the constant region is derived from a different species, such as a human. Chimeric antibodies can be produced by genetic engineering. Chimeric monoclonal antibodies were developed for therapeutic applications to reduce antibody immunogenicity.

The term "humanized antibody" refers to a genetically engineered non-human antibody that comprises a human antibody constant domain and a non-human variable domain modified to comprise a high level of sequence homology to a human variable domain. This can be accomplished by grafting six non-human antibody Complementarity Determining Regions (CDRs) that together form an antigen binding site onto a cognate human acceptor Framework Region (FR). In order to fully reconstruct the binding affinity and specificity of a parent antibody, it may be necessary to replace the framework residues from the parent antibody (i.e., the non-human antibody) with human framework regions (back mutations). Structural homology modeling can help identify amino acid residues in the framework regions that are important for the binding properties of antibodies. Thus, a humanized antibody may comprise non-human CDR sequences, predominantly human framework regions, which optionally comprise one or more amino acid back mutations of a non-human amino acid sequence; and optionally, a fully human constant region. Optionally, additional amino acid modifications (not necessarily back mutations) may be introduced to obtain humanized antibodies with preferred characteristics such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to minimize their immunogenicity in humans, while such humanized antibodies retain the specificity and binding affinity of non-human derived antibodies.

The term "multispecific antibody" refers to an antibody that is specific for at least two different, such as at least three, typically non-overlapping epitopes due to the presence of two or more antigen-binding regions. Such epitopes may be located on the same or different target antigens. If the epitopes are located on different targets, such targets may be located on the same cell or on different cells or cell types.

The term "bispecific antibody" refers to an antibody that has specificity for two different, typically non-overlapping epitopes due to the presence of two antigen binding regions. Such epitopes may be located on the same or different targets. If the epitopes are located on different targets, such targets may be located on the same cell or on different cells or cell types.

Examples of different classes of bispecific antibodies include, but are not limited to (i) IgG-like molecules having complementary CH3 domains to force heterodimerization; (ii) A recombinant IgG-like dual targeting molecule, wherein each side of the molecule comprises Fab fragments or a portion of Fab fragments of at least two different antibodies; (iii) An IgG fusion molecule, wherein a full length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; (iv) An Fc fusion molecule, wherein a single chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, fc region, or portion thereof; (v) Fab fusion molecules, wherein different Fab fragments are fused together, fused to a heavy chain constant domain, fc region, or portion thereof; and (vi) heavy chain antibodies (e.g., domain antibodies) based on scFv and diabodies,) Wherein different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies,) Fused to each other or to another protein or carrier molecule fused to a heavy chain constant domain, fc region or portion thereof.

Examples of IgG-like molecules having complementary CH3 domain molecules include, but are not limited to(Trion Pharma/Fresenius Biotech), knob-wells (Knobs-into-hols) (Genentech), crossMAbs (Roche) and electrostatic matching (electrostatically-mat ched) (amben, chugai, oncomed), LUZ-Y (Genentech, wranik et al j.biol. Chem.2012,287 (52): 43331-9, doi:10.1074/jbc.m112.397869.epub, month 11, 1 day 2012), DIG-bodies and PIG-bodies (Pharmabcine, WO2010134666, WO 2014081202), chain exchange engineering domains (Strand Exchange Engineered Domain body,SEEDbody)(EMD Serono)、Bicl onics(Merus,WO2013157953)、FcΔAdp(Regeneron)、 bispecific IgG1 and IgG2 (Pfizer/Rinat), azymetric scaffolds (Zymeworks/Merck), mAb-Fv (Xencor), bivalent bispecific antibodies (ro, WO 2009080254), and Molecules (Genmab).

Examples of recombinant IgG-like dual targeting molecules include, but are not limited to, dual Targeting (DT) -Ig (GSK/Domanis, WO 2009058383), diabodies (Genntech, bostrom et al 2009.Science 323,1610-1614), cross-linked monoclonal antibodies (Karmanos CANCER CENTER), mAb2 (F-Star), zybodies ^TM (Zyngenia, laFleur et al MAbs.2013 months 3-4 months; 5 (2): 208-18), methods with common light chains kappa lambda Bodies (NovImmune, WO 2012023053) and(CovX/Pfizer, doppalapudi, V.R. et al 2007.Bioorg. Med. Chem. Lett.17, 501-506).

Examples of IgG fusion molecules include, but are not limited to, double Variable Domain (DVD) -Ig (Abbott), double domain double-headed antibodies (Unilever; sanofi Aventis), igG-like bispecific antibodies (IgG-like Bispecific) (ImClone/Eli Lilly, lewis et al Nat Biotechnol.2014, month 2; 32 (2): 191-8), ts2Ab (MedImmune/AZ, dimasi et al J Mol biol.2009, month 30; 393 (3): 672-92) and BsAb (Zymogenics, WO 2010111625), HERCULES (Biogen Idec), scFv fusion (Novartis), scFv fusion (Changzhou Adam Biotech Inc) and TvAb (Roche).

Examples of Fc fusion molecules include, but are not limited to, scFv/Fc fusion (ACADEMIC INSTI tution, pearce et al Biochem Mol Biol int.1997, 9; 42 (6): 1179), SCORPHON (Emergent BioSolutions/Trubion, blancenship JW et al AACR, 100 th annual meeting 2009 (abstract # 5465); zymogenetics/BMS, WO 2010111625), dual affinity re-targeting technology (Fc-DARTTM) (MacroGenics) and bis (ScFv) 2-Fab (National antibody medicine center-China (National RESEARCH CENT ER for Antibody Medicine-China)).

Examples of Fab fusion bispecific antibodies include, but are not limited to, F (ab) 2 (Medarex/AMGEN), dual-Action or Bis-Fab (Genentech),(DN L) (ImmunoMedics), bivalent Bispecific (Biotecnol) and Fab-Fv (UCB-Celltech).

Examples of scFv-based, diabody-based domain antibodies include, but are not limited to, bispecific T cell adaptors (Bispecific T CELL ENGAGER,) (Micromet), tandem diabodies (Tandem Diabody, tandab) (Affimed), dual affinity re-targeting technique (DARTTM) (MacroGenics), single chain diabodies (Academic, lawrence FEBS lett.1998, month 4, 3; 425 (3) 479-84), TCR-like antibodies (AIT, receptorLogics), human serum albumin ScFv fusion (Merrimack, WO 2010059315) and COMBODY molecules (Epigen Biotech, zhu et al Immunol Cell biol.2010 month 8; 88 667-75), double targeting nanobody(Ablynx, hmila et al, FASEB j.2010), dual targeting heavy chain domain only antibody. In some embodiments, the multispecific antibodies of the invention are in VHH-Fc format, i.e., the antibodies comprise two or more single domain antigen-binding regions linked to each other by a human Fc region dimer. In this format, each single domain antigen binding region is fused to an Fc region polypeptide, and the two fusion polypeptides form a dimeric bispecific antibody via a disulfide bridge in the hinge region. Such constructs typically do not comprise the complete or any CH1 or light chain sequences. Fig. 12B of WO06064136 provides an illustration of an example of this embodiment.

In the context of antibody binding to an antigen, the term "binding" or "specific binding" refers to binding of an antibody to a predetermined antigen or target (e.g., human CD123 or vδ2), which typically has an affinity corresponding to about 10 ^-6 M or less, e.g., 10 ^-7 M or less, such as about 10 ^-8 M or less, such as about 10 ^-9 M or less, about 10 ^-10 M or less, or about 10 ^-11 M or even less, K _D, e.g., when determined using flow cytometry as described in the examples herein. Alternatively, the K _D value may be determined using an antigen as ligand and a binding moiety or binding molecule as analyte, using, for example, surface Plasmon Resonance (SPR) techniques in BIAcore T200 or using Biological Layer Interferometry (BLI) in an Octet RED96 instrument. Specific binding means that the binding affinity of the antibody to a predetermined antigen corresponds to a K _D that is at least ten times lower, such as at least 100 times lower, e.g. at least 1,000 times lower, such as at least 10,000 times lower, e.g. at least 100,000 times lower, than its binding affinity to a non-specific antigen other than the predetermined antigen or closely related antigens (e.g. BSA, casein). The degree of affinity reduction depends on the K _D of the binding moiety or binding molecule, so when K _D of the binding moiety or binding molecule is very low (i.e., the binding moiety or binding molecule is highly specific), then the degree of affinity for the antigen may be at least 10,000 times lower than for the non-specific antigen. As used herein, the term "K _D" (M) refers to the dissociation equilibrium constant of a particular interaction between an antigen and a binding moiety or binding molecule.

In the context of the present invention, "competition" or "capable of competing" or "competing (competes)" refers to any detectable significant reduction in the propensity of a particular binding molecule (e.g., a CD123 antibody) to bind to a particular binding partner (e.g., CD 123) in the presence of another molecule (e.g., a different CD123 antibody) that binds to the binding partner. In general, competition refers to a reduction in binding by the presence of another molecule (such as an antibody) of at least about 25%, such as at least about 50%, for example at least about 75%, such as at least 90%, as determined by, for example, ELISA analysis or flow cytometry using a sufficient amount of two or more competing molecules (e.g., antibodies). Additional methods for determining binding specificity by competitive inhibition can be found, for example, in Harlow et al ,Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1988)、Colligan, eds., current Protocols in Immunology, greene Publishing Assoc, AND WILEY INTERSCIENCE N.Y. (1992,1993) and Muller, meth. Zymol.92,589-601 (1983)). In one embodiment, the antibody of the invention binds to the same epitope on CD123 as antibody 1D2 or 1A3 and/or on vδ2 as antibody 5C8, 6H4, 6C1, 5D3 (WO 2015156673) or 5C8var1 (WO 2020060405). It has been determined that the epitope of 5C8 includes residues S33, S43 and K45 (SEQ ID NO: 48). The epitope of 6H4 has been determined to include residues R139, K152, S189 and S191 (SEQ ID NO: 48). There are several methods known in the art that can be used to map antibody epitopes on a target antigen, including but not limited to: cross-linked coupled mass spectrometry, which allows identification of peptides as part of an epitope, and X-ray crystallography, which identifies individual residues on an antigen that form an epitope. Epitope residues can be determined to be less than or equal to at least one atom from an antibodyIs a residue of an amino acid sequence. SelectAs epitope cutoff distance, atoms within the van der waals radius plus possible water-mediated hydrogen bonding are considered. Next, the epitope residues can be determined to have at least one atom less than or equal toIs a residue of an amino acid sequence. Select less than or equal toAs the epitope cutoff distance, the length of the extended arginine amino acid is considered. Cross-linking coupled mass spectrometry first utilizes a mass-labeled chemical cross-linking agent to bind an antibody to an antigen. The presence of complexes is then confirmed using high quality MALDI detection. Because the Ab/Ag complex is extremely stable after crosslinking chemistry, many different enzymes and digestion conditions can be applied to the complex to provide many different overlapping peptides. These peptides were identified using high resolution mass spectrometry and MS/MS techniques. The identification of the cross-linked peptides is determined using a mass tag attached to a cross-linking reagent. Following MS/MS fragmentation and data analysis, the peptides cross-linked and derived from the antigen are part of the epitope, while the peptides derived from the antibody are part of the paratope. All residues found between the N-most and C-terminal crosslinking residues of each crosslinking peptide are considered part of the epitope or paratope.

As used herein, the terms "first" and "second" antigen binding regions do not refer to their orientation/position in an antibody, i.e., they have no meaning with respect to the N-terminus or the C-terminus. The terms "first" and "second" are used only to refer to two different antigen binding regions in the claims and the specification correctly and consistently.

By "capable of binding to human CD123" is meant that the antibody may bind to human CD123 as a separate molecule and/or as part of a CD123/CD131 complex. However, antibodies will not bind CD131 as a separate molecule.

By "a vδ2 chain capable of binding to a vγ9vδ2-TCR" is meant that the antibody may bind to the vδ2 chain as a separate molecule and/or as part of a vγ9vδ2-TCR. However, antibodies will not bind to the vγ9 chain as a separate molecule.

As used herein, "% sequence identity" refers to the number of identical nucleotide or amino acid positions shared by different sequences (i.e., identity% = number of identical positions/total number of positions x 100), taking into account the number of gaps and the length of each gap that need to be introduced to achieve optimal alignment. The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E.Meyers and W.Miller, comput.Appl.Biosci, 4,11-17 (1988) that have been incorporated into the ALIGN program (version 2.0), using the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

Additional aspects and embodiments of the invention

As described above, in a first broad aspect, the present invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human CD123 and a second antigen-binding region capable of binding to the vδ2 chain of the human vγ9vδ2T cell receptor.

In one embodiment, the multispecific antibody is a bispecific antibody. In another embodiment, the multispecific antibody is a trispecific antibody. In another embodiment, the first antigen binding region is a single domain antibody, e.g., a single domain antibody consisting of a heavy chain variable region. In another embodiment, the second antigen binding region is a single domain antibody, e.g., a single domain antibody consisting of a heavy chain variable region. In another embodiment, the first antigen-antigen binding region and the second antigen binding region are both single domain antibodies, e.g., single domain antibodies each consisting of a heavy chain variable region.

In another embodiment, the multispecific antibody is a bispecific antibody, wherein the first antigen-binding region is a single domain antibody and the second antigen-binding region is a single domain antibody. The bispecific antibody may optionally comprise additional sequences, such as a linker and/or an immunoglobulin Fc region.

In one embodiment, the multispecific antibody competes (i.e., is capable of competing) for binding to human CD123 with an antibody having the sequence set forth in SEQ ID No. 1, preferably wherein the multispecific antibody binds to the same epitope on human CD123 as an antibody having the sequence set forth in SEQ ID No. 1. In one embodiment, the multispecific antibody binds to an epitope comprising one or more residues in the S203 to R273 region, such as an epitope fully comprised within the S203 to R273 region, as determined as described in example 11 herein.

In another embodiment, the multispecific antibody binds to an epitope on human CD123 that comprises one or more residues in the S203 to T214 region and one or more residues in the H221 to K227 region and one or more residues in the Y238 to K244 region and one or more residues in the Y268 to R273 region (fig. 14).

In another embodiment, the multispecific antibody binds to an epitope on human CD123 that comprises one, more or all of residues S203, T209, T214, H221, H225, K227, Y238, K244, Y268, T269, and R273 (fig. 14).

In one embodiment, the first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO. 2, the VH CDR2 sequence shown in SEQ ID NO. 3 and the VH CDR3 sequence shown in SEQ ID NO. 4.

In one embodiment, in SEQ ID NO. 2, X ₁ is G. In another embodiment, X ₁ is S.

In one embodiment, in SEQ ID NO. 3, X ₂ is A. In another embodiment, X ₂ is T.

In one embodiment, in SEQ ID NO. 4, X ₃ is Y. In another embodiment, X ₃ is F.

In one embodiment, X ₁ is G, X ₂ is a and X ₃ is Y.

In another embodiment, X ₁ is G, X ₂ is a and X ₃ is F.

In another embodiment, X ₁ is G, X ₂ is T and X ₃ is Y.

In another embodiment, X ₁ is G, X ₂ is T and X ₃ is F.

In one embodiment, X ₁ is S, X ₂ is a and X ₃ is Y.

In another embodiment, X ₁ is S, X ₂ is a and X ₃ is F.

In another embodiment, X ₁ is S, X ₂ is T and X ₃ is Y.

In another embodiment, X ₁ is S, X ₂ is T and X ₃ is F.

In one embodiment, the first antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 1, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 1.

In another embodiment, the first antigen binding region comprises or consists of: a sequence selected from the group of sequences set forth in SEQ ID NOs 25 to 34, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity with a sequence selected from the group of sequences set forth in SEQ ID NOs 25 to 34.

In another embodiment, the multispecific antibody competes for binding to human CD123 with an antibody having the sequence set forth in SEQ ID NO. 9, preferably wherein the multispecific antibody binds to the same epitope on human CD123 as an antibody having the sequence set forth in SEQ ID NO. 9.

In one embodiment, the multispecific antibody binds to an epitope comprising one or more residues in the H225 to T267 region, such as an epitope fully comprising the H225 to T267 region, as determined in example 11 herein.

In another embodiment, the multispecific antibody binds to an epitope on human CD123 that comprises one or more residues in the H225 and R234 regions and one or more residues in the T251 to T267 regions.

In another embodiment, the multispecific antibody binds to an epitope on human CD123 that comprises one, more or all of residues H225, H231, R234, T251, R255 and T267.

In one embodiment, the first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO. 10, the VH CDR2 sequence shown in SEQ ID NO. 11 and the VH CDR3 sequence shown in SEQ ID NO. 12.

In one embodiment, the first antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 9, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 9.

As described above, the multispecific antibodies of the present invention comprise a second antigen-binding region capable of binding to the vδ2 chain of a human vγ9vδ2-T cell receptor. Vδ2 is part of the delta chain of vγ9vδ2-TCR. Antibodies capable of binding to human vδ2 may bind to an epitope located entirely within the vδ2 region or to an epitope that is a combination of residues in the vδ2 region and residues in the constant region of the δ chain. In one embodiment, the multispecific antibody is capable of activating human vγ9vδ2t cells. Activation of vγ9vδ2t cells can be measured by gene expression and/or (surface) marker expression (e.g., activation markers such as CD25, CD69, or CD107 a) and/or secreted protein (e.g., cytokines or chemokines) profiling. In a preferred embodiment, the multispecific antibody is capable of inducing activation (e.g., up-regulation of CD69 and/or CD25 expression) resulting in degranulation, which is marked by increased CD107a expression (see examples herein) and/or cytokine production (e.g., tnfα, ifnγ) of vγ9vδ2T cells. Preferably, the multispecific antibodies of the invention are capable of increasing the number of CD107a positive cells by at least a factor of 2, such as at least a factor of 5, when tested as described in the examples herein. In another preferred embodiment, the multispecific antibodies of the invention have EC50 values that increase the percentage of CD107a positive cells of 50pM or less, such as 25pM or less, for example 20pM or less, such as 15pM or less, for example 10pM or less, when tested using vγ9vδ2t cells and C1r-neo target cells, as described in the examples herein.

Several antibodies that bind vδ2 have been described in WO2015156673 and their antigen binding regions, or at least their CDR sequences, may be incorporated into the multispecific antibodies of the present invention.

In one embodiment, the multispecific antibody competes for binding to human V.delta.2 with an antibody having the sequence set forth in SEQ ID NO. 17, wherein X ₄ is Y.

In another embodiment, the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence set forth in SEQ ID NO. 17.

In one embodiment, the multispecific antibody competes for binding to human V.delta.2 with an antibody having the sequence set forth in SEQ ID NO. 36, preferably the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence set forth in SEQ ID NO. 36.

In one embodiment, the multispecific antibody competes with an antibody having the sequence set forth in SEQ ID NO. 37 for binding to human V.delta.2, preferably the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence set forth in SEQ ID NO. 37.

In one embodiment, the multispecific antibody competes for binding to human V.delta.2 with an antibody having the sequence set forth in SEQ ID NO. 38, preferably the multispecific antibody binds to the same epitope on human V.delta.2 as an antibody having the sequence set forth in SEQ ID NO. 38.

In one embodiment of the multispecific antibody of the present invention, the second antigen-binding region comprises a VH CDR1 sequence shown in SEQ ID No. 18, a VH CDR2 sequence shown in SEQ ID No. 19, and a VH CDR3 sequence shown in SEQ ID No. 20. In one embodiment, in SEQ ID NO. 20, X ₄ is Y. In another embodiment, in SEQ ID NO. 20, X ₄ is F. In another embodiment, in SEQ ID NO. 20, X ₄ is S.

In one embodiment of the multispecific antibody of the present invention, the second antigen-binding region comprises, preferably comprises or consists of the VH CDR1 sequence set forth in SEQ ID No. 40 and the VH CDR3 sequence set forth in SEQ ID No. 41: the sequence shown in SEQ ID NO. 36, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 36.

In one embodiment of the multispecific antibody of the present invention, the second antigen-binding region comprises, preferably comprises or consists of the VH CDR1 sequence shown in SEQ ID No. 42, the VH CDR2 sequence shown in SEQ ID No. 43 and the VH CDR3 sequence shown in SEQ ID No. 44: the sequence shown in SEQ ID NO. 37, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 37.

In one embodiment of the multispecific antibody of the present invention, the second antigen-binding region comprises, preferably comprises or consists of the VH CDR1 sequence shown in SEQ ID No. 45, the VH CDR2 sequence shown in SEQ ID No. 46, and the VH CDR3 sequence shown in SEQ ID No. 47: the sequence shown in SEQ ID NO. 38, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 38.

In one embodiment of the multispecific antibodies of the present invention, the second antigen-binding region is humanized.

In another embodiment, the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 17, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 17. In one embodiment, in SEQ ID NO. 17, X ₄ is Y. In another embodiment, in SEQ ID NO. 17, X ₄ is F. In another embodiment, in SEQ ID NO. 17, X ₄ is S.

In a preferred embodiment of the multispecific antibody of the invention,

(I) The first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO.2, the VH CDR2 sequence shown in SEQ ID NO. 3 and the VH CDR3 sequence shown in SEQ ID NO. 4, and the second antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO. 18, the VH CDR2 sequence shown in SEQ ID NO. 19 and the VH CDR3 sequence shown in SEQ ID NO.20, or

(Ii) The first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO. 10, the VH CDR2 sequence shown in SEQ ID NO. 11 and the VH CDR3 sequence shown in SEQ ID NO. 12, and the second antigen binding region comprises the VH CDR1 sequence shown in SEQ ID NO. 18, the VH CDR2 sequence shown in SEQ ID NO. 19 and the VH CDR3 sequence shown in SEQ ID NO. 20.

In another preferred embodiment of the multispecific antibody of the present invention,

(I) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 1 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Ii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 9 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Iii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 25 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Iv) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 26 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(V) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 27 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Vi) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 28 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Vii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 29 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Viii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 30 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Ix) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 31 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(X) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 32 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Xi) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 33 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y, or

(Xii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 34 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 17, wherein optionally X ₄ is Y.

(I) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 1 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Ii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 9 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Iii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 25 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Iv) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 26 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(V) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 27 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or (vi) the first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 28 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Vii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 29 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Viii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 30 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Ix) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 31 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(X) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 32 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or (xi) the first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 33 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36, or

(Xii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 34 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 36.

(I) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 1 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Ii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 9 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Iii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 25 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Iv) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 26 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(V) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 27 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Vi) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 28 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Vii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 29 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Viii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 30 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Ix) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 31 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(X) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 32 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or (xi) the first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 33 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37, or

(Xii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 34 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 37.

(I) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 1 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Ii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 9 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Iii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 25 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Iv) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 26 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(V) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 27 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or (vi) the first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 28 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Vii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 29 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Viii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 30 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Ix) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 31 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(X) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO.32 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or (xi) the first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 33 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38, or

(Xii) The first antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 34 and the second antigen binding region comprises or consists of the sequence shown in SEQ ID NO. 38.

The first and second antigen binding regions in a multispecific antibody may be arranged in various ways. In one embodiment, the antigen binding regions are linked to each other by a linker, such as a covalent linker. In one embodiment, the first antigen binding region and the second antigen binding region are covalently linked to each other by a peptide linker, e.g. a linker of 1 to 20 amino acids, e.g. 1 to 10 amino acids, such as 2, 3, 4, 5, 6, 7, 8 or 10 amino acids in length. In one embodiment, the peptide linker comprises or consists of a sequence of 4 glycine followed by serine.

In some embodiments, the first antigen binding region capable of binding to human CD123 is located N-terminal to the second antigen binding region capable of binding to human vδ2 chain. In another embodiment, the first antigen binding region capable of binding to human CD123 is located C-terminal to the second antigen binding region capable of binding to human vδ2 chain.

The multispecific antibodies, such as bispecific antibodies, of the present invention may comprise additional molecules, domains, or polypeptide sequences in addition to the first and second antigen-binding regions. In one embodiment, the multispecific antibody further comprises a half-life extending domain, i.e., a domain that extends the half-life of the molecule in the circulation of a human patient. In one embodiment, the multispecific antibody has a terminal half-life of greater than about 168 hours when administered to a human subject. Most preferably, the terminal half-life is 336 hours or more. As used herein, the "terminal half-life" of an antibody refers to the time taken for the serum concentration of a polypeptide to decrease by 50% in vivo during the final elimination stage.

In one embodiment, the multispecific antibody comprises an Fc region, preferably a human Fc region. In one embodiment, the multispecific antibody is in VHH-Fc format, i.e., the antibody comprises two or more single domain antigen-binding regions linked to each other by a human Fc region dimer, wherein each single domain antigen-binding region is fused to an Fc region polypeptide (no CH1 or light chain sequence), and the two fused polypeptides form a dimer bispecific antibody via a disulfide bridge in the hinge region.

Various methods for preparing bispecific antibodies have been described in the art, for example reviewed by Brinkmann and Kontermann (2017) MAbs 9:182 and Labrijn et al (2019) Nature Reviews Drug Discovery 18:18:585. In one embodiment of the invention, the Fc region is a heterodimer comprising two Fc polypeptides, wherein a first antigen binding region is fused to a first Fc polypeptide and a second antigen binding region is fused to a second Fc polypeptide, and wherein the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor heterodimer formation relative to homodimer formation (see, e.g., ridway et al (1996)'Knobs-into-holes'engineering of antibody CH3 domains for heavy chain heterodimerization.Protein Eng 9:617). in another embodiment of this document, the CH3 region of the Fc polypeptide comprises the asymmetric amino acid mutations, preferably the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L a and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human igg1. In another embodiment, the cysteine residues at position 220 in the first and second Fc polypeptides have been deleted or substituted, wherein the amino acid positions correspond to human igg1. In another embodiment, the region comprises the sequence shown in SEQ ID No. 35, according to the EU numbering system.

In some embodiments, the first and/or second Fc polypeptides comprise mutations that render the Fc region inert (i.e., incapable of mediating effector function). In one embodiment, the first and second Fc polypeptides comprise mutations at positions 234 and/or 235, preferably the first and second Fc polypeptides comprise L234F and L235E substitutions, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

In a preferred embodiment of the present invention,

-The first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID No. 2, the VH CDR2 sequence shown in SEQ ID No. 3 and the VH CDR3 sequence shown in SEQ ID No. 4, and the second antigen binding region comprises the VH CDR1 sequence shown in SEQ ID No. 18, the VH CDR2 sequence shown in SEQ ID No. 19 and the VH CDR3 sequence shown in SEQ ID No. 20, and

The first Fc polypeptide comprises the sequence shown in SEQ ID NO. 21 and the second Fc polypeptide comprises the sequence shown in SEQ ID NO. 22 or vice versa.

In a further preferred embodiment of the present invention,

-The first antigen binding region comprises the VH CDR1 sequence shown in SEQ ID No. 10, the VH CDR2 sequence shown in SEQ ID No. 11 and the VH CDR3 sequence shown in SEQ ID No. 12, and the second antigen binding region comprises the VH CDR1 sequence shown in SEQ ID No. 18, the VH CDR2 sequence shown in SEQ ID No. 19 and the VH CDR3 sequence shown in SEQ ID No. 20, and

In another preferred embodiment, the antibody of the invention consists of:

(i) A first polypeptide chain consisting of: a first antigen binding region consisting of a sequence selected from the group consisting of: sequences shown in SEQ ID NOS 1, 9 and 25 to 34, sequence shown in SEQ ID NO 35, sequence shown in SEQ ID NO 21, and

(I) A second polypeptide chain consisting of: a second antigen binding region consisting of: the sequence shown in SEQ ID NO. 17, the sequence shown in SEQ ID NO. 35 and the sequence shown in SEQ ID NO. 22, wherein X ₄ is Y.

In another preferred embodiment, the antibody of the invention consists of:

(i) A first polypeptide chain consisting of: a first antigen binding region consisting of a sequence selected from the group consisting of: sequences shown in SEQ ID NOS 1, 9 and 25 to 34, sequence shown in SEQ ID NO 35, sequence shown in SEQ ID NO 22, and

(I) A second polypeptide chain consisting of: a second antigen binding region consisting of: the sequence shown in SEQ ID NO. 17, the sequence shown in SEQ ID NO. 35 and the sequence shown in SEQ ID NO. 21, wherein X ₄ is Y.

In one embodiment, the multispecific antibodies of the invention are capable of mediating killing of cells expressing CD123, such as C1R-neo cells or THP-1 cells, by Vγ9Vδ2T cells.

Preferably, the antibody is capable of inducing killing of C1R-neo cells by activation of vγ9vδ2t cells, wherein the EC50 value is 50pM or less, such as 25pM or less, for example 20pM or less, such as 15pM or less, for example 10pM or less, or even 5pM or less, such as 2pM or less, when tested as described in example 5 herein.

In another embodiment, the antibody is capable of inducing killing of THP-1 cells by activation of vγ9vδ2t cells, wherein the EC50 value is 100pM or less, such as 50pM or less, such as 25pM or less, e.g. 20pM or less, such as 15pM or less, e.g. 10pM or less, or even 5pM or less, such as 2pM or less, when tested as described in example 5 herein.

In another embodiment, the multispecific antibody is capable of mediating killing of human patient-derived bone marrow-derived AML tumor cells expressing CD 123. Such killing may be determined, for example, as described in example 6 herein. In one embodiment, the multispecific antibodies of the invention are capable of mediating more than 25%, such as more than 50%, of specific cell death at a concentration of 100fM, as determined in the assay described in example 6 herein.

In another embodiment, the multispecific antibody is incapable of mediating killing of CD 123-negative cells, such as CD 123-negative human cells.

In another embodiment, the multispecific antibodies of the invention are capable of binding to 293F cells transiently expressing CD123, wherein the EC50 is 50nM or less, such as 20nM or less, for example 10nM or less, such as 5nM or less, when tested as described in example 3 herein.

In another embodiment, the multispecific antibodies of the invention are capable of binding to a recombinant CD123-Fc fusion protein, wherein the EC50 is 50nM or less, such as 20nM or less, for example 10nM or less, such as 5nM or less, when tested as described in example 4 herein.

In another broad aspect, the invention relates to an antibody comprising a first antigen binding region capable of binding human CD123, wherein the first antigen binding region is a single domain antibody comprising:

In one embodiment, X ₁ is G, X ₂ is a and X ₃ is Y.

In another embodiment, X ₁ is G, X ₂ is a and X ₃ is F.

In another embodiment, X ₁ is G, X ₂ is T and X ₃ is Y.

In another embodiment, X ₁ is G, X ₂ is T and X ₃ is F.

In one embodiment, X ₁ is S, X ₂ is a and X ₃ is Y.

In another embodiment, X ₁ is S, X ₂ is a and X ₃ is F.

In another embodiment, X ₁ is S, X ₂ is T and X ₃ is Y.

In another embodiment, X ₁ is S, X ₂ is T and X ₃ is F.

In another broad aspect, the invention relates to a pharmaceutical composition comprising an antibody according to the invention as described herein (such as a multispecific antibody) and a pharmaceutically acceptable excipient.

Antibodies may be formulated with pharmaceutically acceptable excipients according to conventional techniques, such as those disclosed in (Rowe et al Handbook of Pharmaceutical Excipients, 6 th 2012, ISBN 9780857110275). The pharmaceutically acceptable excipient and any other carrier, diluent or adjuvant should be suitable for the antibody and the mode of administration selected. Suitability of excipients and other components of the pharmaceutical composition is determined based on having no significant negative impact (e.g., less than a substantial impact (relative inhibition of 10% or less, relative inhibition of 5% or less, etc.) upon antigen binding) on the desired biological properties of the selected antibodies or pharmaceutical compositions of the invention.

The pharmaceutical composition may comprise diluents, fillers, salts, buffers, detergents (e.g., nonionic detergents such as Tween-20 or Tween-80), stabilizers (e.g., sugar or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and/or other materials suitable for inclusion in a pharmaceutical composition. Additional pharmaceutically acceptable excipients include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants and absorption delaying agents, and the like, which are physiologically compatible with the antibodies of the present invention.

In another broad aspect, the invention relates to a multispecific antibody according to the invention as described herein for use as a medicament.

The multispecific antibodies according to the invention are capable of creating a microenvironment conducive to killing of tumor cells by vγ9vδ2t cells, particularly CD123 positive tumor cells.

Thus, in another broad aspect, the invention relates to a multispecific antibody according to the invention as described herein for use in the treatment of cancer. In another broad aspect, the invention relates to a multispecific antibody according to the invention as described herein for use in the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, hodgkin's lymphoma, lymphoblastic plasmacytoid dendritic cell tumor, chronic myelogenous leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder, or myelodysplastic syndrome.

Similarly, the present invention relates to a method of treating a disease comprising administering a multispecific antibody according to the present invention as described herein to a human subject in need thereof. In one embodiment, the disease is a cancer, such as acute myeloid leukemia.

In some embodiments, the antibody is administered as a monotherapy. However, the antibodies of the invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents associated with the disease or condition to be treated.

"Treatment" or "Treatment" refers to the administration of an effective amount of an antibody according to the invention, with the aim of alleviating, ameliorating, preventing, eradicating (curing) or preventing a symptom or disease state. An "effective amount" refers to an amount effective to achieve the desired therapeutic result over the necessary dosage and period of time. The effective amount of a polypeptide such as an antibody may vary depending on factors such as the disease stage, age, sex and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the antibody exceed therapeutic benefit. An exemplary, non-limiting range of an effective amount of an antibody of the invention is about 0.1 μg/kg to 100mg/kg, such as about 1 μg/kg to 50mg/kg, e.g., about 0.01 to 20mg/kg, such as about 0.1 to 10mg/kg, e.g., about 0.5, about 0.3, about 1, about 3, about 5, or about 8mg/kg. Administration may be by any suitable route, but will typically be parenteral, such as intravenous, intramuscular or subcutaneous.

The multispecific antibodies of the invention are typically recombinantly produced, i.e., by expressing a nucleic acid construct encoding the antibody in a suitable host cell, and then purifying the resulting recombinant antibody from the cell culture. Nucleic acid constructs can be produced by standard molecular biology techniques well known in the art. The construct is typically introduced into the host cell using an expression vector. Suitable nucleic acid constructs and expression vectors are known in the art. Host cells suitable for recombinant expression of antibodies are well known in the art and include CHO, HEK-293, expi293F, PER-C6, NS/0 and Sp2/0 cells.

Thus, in a further aspect, the invention relates to a nucleic acid construct encoding an antibody of the invention, such as a multispecific antibody according to the invention. In one embodiment, the construct is a DNA construct. In another embodiment, the construct is an RNA construct.

In another aspect, the invention relates to an expression vector comprising a nucleic acid construct encoding a multispecific antibody according to the invention.

In another aspect, the invention relates to a host cell comprising one or more nucleic acid constructs encoding a multispecific antibody according to the invention or an expression vector comprising a nucleic acid construct encoding a multispecific antibody according to the invention.

Table 1: and (5) a sequence table.

All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications herein is not, and should not be taken as, an acknowledgement or any form of suggestion that they form part of the effective prior art or form part of the common general knowledge in any country in the world.

Examples

Example 1: selection and identification of anti-CD 123 VHH from phage display libraries prepared from animals immunized with C1R-CD1d cells

Llama (LLAMA GLAMA) (2 animals) was immunized with CD123 expressing C1R-CD1d cells and then a 'immunized' VHH phage antibody library (Lameris et al, 2016Immunology 149:111) was prepared as described. These libraries were used for phage selection of captured recombinant human CD123 or directly coated CD123 antigen (extracellular domain, sino Biological). Single-round or two-round continuous selection is performed. After one and two rounds of phage selection, the single phage clones were screened for binding to the recombinant capture antigen in ELISA. Those clones that scored positive for binding were sequenced and then all clones with different sequences were tested for binding to the cell line used for immunization in flow cytometry. VHH clones that showed binding in FACS were then selected for further characterization. Eight different clones were identified and named: 1E2, 1B4, 1A3, 2D11, 1D2, 1E4, 1H1 and 1F1.

Example 2: synthesis, production and purification of bispecific VHH' s

The sequence of the CD123 specific VHH domain antibody was then reformatted into a bispecific VHH with a vδ2 specific VHH (5C8var1;SEQ ID NO:17, where X ₄ is Y) in the direction: N-terminal-anti-CD 123 VHH-linker-anti-V delta 2VHH-C tag. The V.delta.2-specific VHH used is shown in SEQ ID NO:17 (wherein X ₄ is Y). The linker between the two VHH domains is a glycine (G) -serine (S) segment with the sequence G4S. The cDNAs encoding these proteins were prepared by synthetic gene synthesis in Genscript and then cloned into eukaryotic expression vector pCDNA3.1+ (Thermofisher Scientific) by directed cloning. Proteins were expressed in Hek293E cells by transient transfection and then (after 5 days of expression) purified from the conditioned cell culture supernatant using Capture Select C tag affinity matrix (Thermo FISHER SCIENTIFIC) according to the protocol of the supplier. Purity of purified bispecific VHH was always >95% and endotoxin levels were very low (< 0.5 EU/mg) as determined by SDS-PAGE analysis using coomassie staining.

Example 3: determination of the binding specificity of bispecific anti-CD 123 x V delta 2VHH in ELISA

Recombinant purified CD123 antigen (extracellular domain; sino Biological) or Fc fusion of CD123 antigen (Bio-technology/R & D Systems) was coated into PBS in wells of ELISA plates (Greiner) at a concentration of 2. Mu.g/ml. As a negative control, wells were coated with 1% (w/v) BSA. The internal design, production and purification of the extracellular domains of human vγ9 and vδ2TCR chains fused to human Fc were also coated as antigens at 2 μg/ml. After coating and blocking the wells with 2% (w/v) BSA, bispecific VHH proteins were tested for binding at 50nM saturation concentration and bound VHH was detected using HRP-labeled anti-VHH antibody (Genscript) and staining with 3,3', 5' -Tetramethylbenzidine (TMB)/H2O 2.

FIG. 1 shows that only 1A3-5C8var1 and 1D2-5C8var1 showed strong and specific binding to CD123 and γδ -TCR (γδ T cell receptor) in ELISA. Other bispecific antibodies bind poorly or not to CD123.

Example 4: determination of binding specificity of bispecific anti-CD 123 x V delta 2VHH Using flow cytometry

Expression constructs for the common beta chain (CD 131) of human CD123 and receptor were purchased from invitogen. The plasmid was transformed into chemically competent DH 5. Alpha. Bacteria and both constructs were amplified using single colonies grown on selective medium inoculated with 50ml of medium. Purified DNA was then transfected into fresh 293F cells using Polyethylenimine (PEI). Either a single plasmid or a mixture of both plasmids was used for transfection. As a negative control, untransfected cells were also used for flow cytometry. One day after transfection, cells were used to test binding of bispecific VHH using staining in FACS. Briefly, binding of 100nM bispecific VHH to transfected cells at saturation concentration was detected with AF 647-labeled anti-VHH (Genscript) antibody and staining was visualized using FACS CELESTA (Becton and Dickinson).

FIG. 2 shows that both 1A3-5C8var1 and 1D2-5C8var1 strongly and specifically recognize CD123 when the antigen is expressed alone or in conjunction with CD 131. 1D2-5C8var1 gave the strongest signal in flow cytometry.

To determine the apparent affinity of 1D2-5C8var1 for CD123 using flow cytometry, a range of concentrations of this bispecific VHH were tested for binding to transiently transfected 293F cells expressing CD123, CD131, or both CD123 and CD 131. The former cell (expressing CD 131) was used as a negative control.

FIG. 3 again shows that 1D2-5C8var1 has very high specificity for CD123, since only cells expressing CD123 but not CD131 are (transiently) recognized. Furthermore, the data show that the apparent affinity of 1D2-5C8var1 binding to CD123, as determined using flow cytometry, was approximately 3nM. This value is comparable to the binding of CD123 alone or to the binding of co-expressed CD123 and CD 131.

Example 5: determination of binding affinity of 1D2-5C8var1 to CD123 using Biological Layer Interferometry (BLI)

To determine the binding kinetics of 1D2-5C8var1 to CD123, recombinant purified CD123-Fc fusion proteins (Bio-technology/R & D Systems) were loaded onto the anti-human IgG Fc capture sensor of the Octet Red96e (Sartorius) instrument at a density of 1nm (using a concentration of 5. Mu.g/ml). Then immersing different sensors into 1D2-5C8var1 with different concentrations; dilutions were prepared in 10x kinetic buffer (10 xKB) supplied by the supplier. From the obtained sensorgrams, the kinetic association and dissociation rate constants were determined by curve fitting.

Fig. 4 shows an actual sensorgram for curve fitting. The latter is depicted as a straight line in the figure. This was used to determine the kinetic association and dissociation rate constants and thus the affinity for anti-CD 123VHH 1D 2. The measurement was performed twice and the affinity of 1D2-5C8var1 for CD123 was measured to be between 3 and 5 nM.

Example 6: CD123 dependent, 1D2-5C8var1 mediated activation of Vγ9Vδ2T cells and T cell mediated cytotoxicity of target cells

Buffy coat (Buffy coat) is obtained from Sanquin (Amsterdam, THE NETHERLANDS). PBMCs were isolated from these buffy coats by Ficoll density gradient centrifugation using the procedure described. High purity vγ9vδ2T cells were obtained by MACS using vδ2 specific antibodies and these cells were expanded using published methods (de Bruin et al 2016Clin Immunol.169:128). CD 123-positive B lymphoblasts, EBV-transformed C1R neo cell line (CRL-2369), and CD 123-positive AML-derived THP-1 cell line (TIB-202) were obtained from the American type culture Collection (AMERICAN TYPE culture collection) and cultured according to the instructions of the supplier. Target cells were labeled with cell microviolet (CTV) at 37 ℃ for 20 minutes. To measure T cell activation, cells were stained for the activation marker CD107a (or LAMP-1, lysosomal associated membrane protein-1), which became exposed to the cell surface once the cells degranulated. A range of bispecific antibodies were incubated with a 1:1 mixture of target cells and expanded vγ9vδ2t cells (50,000 cells each) in the presence of PE-labeled anti-CD 107A antibodies for 4 hours with a final volume of 100 μl. After incubation, cells were washed and stained with a mixture of fluorescently labeled anti-CD 3 and anti-vγ9 antibodies to identify T cells and live/dead stain (7-AAD). Samples were analyzed using FACS CELESTA (Beckton and Dickinson). To assess T cell mediated cytotoxicity of target cells, essentially the same setup was used except that no anti-CD 107A antibody was added and CTV-labeled target cells and vγ9vδ2t cells were incubated for 24 hours in the presence of dual-specific VHH. Cells were again stained for CD3 and vγ9 at the end of the assay and analyzed using flow cytometry in the presence of live/dead stain (7-AAD).

FIG. 5 shows that 1D2-5C8var1 caused potent T cell activation with EC50 in the pM range. In the absence of target cells, high concentrations of 1D2-5C8var1 only caused background activation (data not shown). The efficacy of 1D2-5C8var1 to induce T cell activation is slightly dependent on the T cell donor used; EC50 values range between 3 and 13 pM.

To determine if the observed T cell activation will also result in target cell lysis, cytotoxicity assays were performed using a 1:1 effector to target (E: T) ratio and 24 hour time points. FIG. 6 shows that 1D2-5C8var1 is effective in inducing C1R-neo target cell lysis in the presence of expanded V.gamma.9V.delta.2T cells. The EC50 for cytotoxicity was determined by curve fitting to be between 1 and 2pM, depending on the T cell donor used (data for both donors are depicted).

CD123 positive AML cell line was used: THP-1 was assayed repeatedly for the same cytotoxicity. The efficacy of 1D2-5C8var1 in inducing THP-1 target cell lysis was quite comparable: the EC50 was measured at 1pM. In contrast: the efficacy of 1A3-5C8var1 to induce target cell lysis was measured to be about 50pM: fig. 7.

Example 7: bispecific VHH mediated vγ9vδ2T cell activation and T cell induced primary AML cell lysis

25,000 Bone marrow-derived monocytes from AML patients were co-cultured overnight with expanded vγ9vδ2T cells from healthy donors at a ratio of 1:1. Cells were incubated in the presence of PE-labeled CD107a antibody and in the concentration range of 1D2-5C8var1 (in the range of 10fM to 100 nM). Cells were harvested, washed and labeled 30 "at 4 ℃ with an antibody mixture containing fluorescently labeled anti-CD 45, CD117, CD34, CD33 and CD2 antibodies. After washing, the cells were resuspended in a mixture of live/dead stain (7 AAD) and 123 counting beads and subsequently analyzed using LSRFortessa flow cytometer.

Figure 8 shows that both bispecific VHH compounds were able to induce potent T cell activation dependent on CD123 positive primary AML blasts. Furthermore, the compounds cause high levels of tumor cell lysis and show significant efficacy in this cytotoxicity. EC50 values cannot be determined explicitly from the obtained curves, but are in the fM range (below 1 pM).

Example 8: humanization of anti-CD 123 VHH 1D2 using CDR grafting

The 1d2 VHH antibody fragments are humanised using CDR grafting techniques (see, for example, U.S. Pat. No. 5,225,539 and Williams, d.g. et al 2010,Antibody Engineering, volume 1, chapter 21). First, igBLAST was used to identify human germline sequences (Ye J. Et al, 2013,Nucleic Acids Res.41:W34-40). The V gene IGVH3-23 x 04 was identified as the closest human germline sequence (78.4% identity). Direct grafting of llama CDRs (91.8% identical to human germline IGVH3-23 x 04) using this germline sequence resulted in the following cDNA constructs: SEQ ID NO. 31. Next, the NCBI NR database (downloaded 9.27 days 2020) was queried using BLASTP (version 2.10.0) to identify the human template sequence exhibiting the highest identity to the 1D2 sequence. Two VH sequences were identified which exhibited a similarity score of 70% or higher and exhibited similar CDR lengths, preferably identical to those in 1d2 CDR1, CDR2, CDR3, respectively. The framework encoded by GenBank (Benson, D.A. et al 2013,Nucleic Acids Res.41 (D1): D36-42) accession numbers CAD60357.1 and AKU38567.1 was selected as a template for grafting 1D2 CDRs, resulting in the following cDNA constructs: SEQ ID NOS 28 and 32, respectively. Framework and CDR definitions are those as determined by Kabat et al ("Sequences of Proteins of Immunological Interest", kabat, e. Et al, USDepartment of HEALTH AND Human Services, (1983)). To understand the effect of humanized framework residues on VHH structure BioLuminate 4.2.156 was usedThe 'antibody prediction' tool within (default parameters) generated a homology model for 1d2 VHH. The homology model is built on the basis of PDB ID 6 GKU. CDRs were computer grafted to investigate the effect of human residues on features such as CDR loop conformation, surface hydrophobicity, and structural integrity (e.g., increased rigidity). These features of the resulting construct were examined to design additional constructs: SEQ ID NOS 25, 26, 27, 29, 30, 33 and 34. The sequences of these humanized 1D 2-VHHs were then reformatted into bispecific VHHs with vδ2 specificity VHH (5C8var1;SEQ ID NO:17, where X ₄ is Y) in the direction: n-terminal-humanized anti-1D 2 VHH-linker-anti-V.delta.2 VHH-C tag. The cdnas encoding these molecules were then synthesized and cloned into expression vectors for expression in HEK293E cells. Proteins were prepared by transient transfection of cells and purified from culture supernatants using C-tag affinity chromatography and then preparative size exclusion chromatography.

Example 9: determination of binding affinity of humanized 1D2-5C8var1 (Y105F) variants to CD123 using Biological Layer Interferometry (BLI)

To determine the binding kinetics of the humanized 1D2-5C8var1 variant to CD123, recombinant purified CD123-Fc fusion proteins (Bio-technology/R & D Systems) were loaded onto the anti-human IgG Fc capture sensor of the Octet Red96e (Sartorius) instrument at a density of 1nm (using a concentration of 5. Mu.g/ml). Different sensors were then immersed in different concentrations of the humanized 1D2-5C8var1 variant, starting with 50nM and its double dilution; dilutions were prepared in 10x kinetic buffer (10 xKB) supplied by the supplier. From the obtained sensorgrams, the kinetic association and dissociation rate constants were determined by curve fitting. When fitting was possible, the binding affinity of the humanized 1D2-5C8var1 variant to CD123 was calculated using the association and dissociation rate constants. Two measurements were performed and the affinity of the different humanized 1D2-5C8var1 variants for CD123 ranged from 2.6nM (similar compared to parent 1D 2) to non-binding variants, as shown in table 2. For reference, non-humanized (parent) 1D2 was included in these experiments.

Table 2: affinity of humanized variants of 1D2 to recombinant CD123

* Low binding (> 20 nM), only one measurement, n.b.: no binding was observed

Example 10: bispecific constructs with extended half-life (Fc-containing)

The 1D2-5C8var1 bispecific VHH is reformatted into a therapeutic antibody format that comprises a human Fc domain. Both VHH domains were coupled to human IgG1Fc (i.e. CH2 and CH 3) domains with the following characteristics: VHH is coupled to modified hinge (AAA, heel SDKTHTCPPCP) and human CH2 and CH3 domains. The CH2 domain was silenced by LFLE mutation pair (L234F, L235E) Fc, and the CH3 domain was mutated to have a "knob-hole" mutation (knob: T366W, and hole: T366S, L368A and Y407V) that forced heterodimerization when both chains were co-expressed in the same cell. This mutation pair has been described in the scientific literature (Ridgway et al (1996) Protein Eng 9:617). The C-terminus of the anti-Vγ9Vδ2 heavy chain is equipped with a C-terminal tag (AAAEPEA (SEQ ID NO: 53)) for purification purposes. The sequences of the constructs are shown in SEQ ID NO. 49 and SEQ ID NO. 50. The resulting antibody construct was designated 1D2-5C8var1 (Y105F) -Fc.

Proteins were prepared by co-transfection of the two expression vectors in HEK293E cells and purification from the culture supernatant by C-tag affinity chromatography followed by preparative size exclusion chromatography. This resulted in a high monomer protein preparation of 1D2-5C8var1 (Y105F) -Fc: fig. 9.

Example 11:1D2-5C8var1 (Y105F) -Fc induces target-dependent T cell activation and causes T cell mediated cytotoxicity of target cells with efficacy equivalent to that of bispecific VHH

The ability of 1D2-5C8var1 (Y105F) -Fc to induce target-dependent V.gamma.9V.delta.2T cell activation in co-cultures of V.gamma.9V.delta.2T cells and THP-1 tumor cells (ratio 1:1) was then tested. vγ9vδ2T cells were expanded from the blood of healthy donors using procedures known in the art. The THP-1 cell line (ATCC accession number TIB-202) was cultured according to the supplier's recommendations. Activation of vγ9vδ2t cells was measured by CD107a staining and measuring the percentage of CD107a positive cells by flow cytometry in 4 hour co-cultures of both cell types. FIG. 10 shows that 1D2-5C8var1 (Y105F) -Fc induces target-dependent T cell activation (no activation was observed in the co-culture of V.gamma.9V.delta.2T cells and tumor cells in the absence of compound; data not shown). EC50 is typically in the pM range, ranging from 4 to 16pM (depending on the donor used).

Notably, the potency of Fc-containing molecules to induce vγ9vδ2t cell activation was not measurably different from bispecific VHHs. This was observed in three different independent T cell donors. To determine if this T cell activation also resulted in target cell lysis, viability of THP-1 target cells was measured after 24 hours of co-culture (1:1 ratio) with vγ9vδ2t cells in the presence of antibodies. vγ9vδ2T cells were isolated from the blood of healthy donors and expanded using standardized protocols. The day before the assay, THP-1 target cell lines were labeled with cell microviolet (CTV) to enable differentiation from effector cell populations in flow cytometry. After 24 hours of co-cultivation with increasing concentrations of the compound, the percentage of live target cells was determined: fig. 11.

Figure 11 shows that both bispecific VHH and Fc-containing counterparts induced strong T cell mediated cytotoxicity of target cells, and that the efficacy of both molecules to cause target cell lysis was not measurably different. EC50 values range between 1 and 3pM, depending on the donor used. In the absence of compound, no target cell lysis was observed in the co-culture (data not shown). After 24 hours, all target cells in the assay were killed.

Example 12:1D2-5C8var1 (Y105F) -Fc results in preferential killing of tumor cells relative to target positive normal cells

To determine the extent of antibody-induced CD123 positive normal cell killing, MACS sorting (Miltenyi Biotech, catalog No. 130-097-415) was used to enrich plasma cell-like dendritic cells (pdcs) known to express CD123 (Collin et al, 2013Immunology 140,1:22-30) from Peripheral Blood Mononuclear Cell (PBMC) fractions isolated from blood from two healthy donors. The THP-1 cell line was used as a tumor cell line expressing CD 123. Using staining for CD123 and analysis by flow cytometry, it has been shown that CD123 expression levels on pDC are approximately ten times higher than on THP-1 cell lines: fig. 12.

The cytotoxic effect of the combination of CD123 targeting compound and vγ9vδ2t cells on the target cell mixture was then determined in a co-culture with a ratio of THP-1 cell line, pDC and vγ9vδ2t cells of 1:1:2. vγ9vδ2T cells were isolated from the blood of healthy donors and expanded using standardized protocols. The day before the assay, THP-1 target cell lines were labeled with cell microviolet (CTV) to enable differentiation from effector cell populations and other target cells in flow cytometry. After 24 hours of co-culture in the presence of increasing concentrations of the compounds, the percentage of live target cells was determined by staining for vγ9 (T cells), CD303 (pDC), CTV (THP-1) and CD123 (THP-1 and pDC) and analysis by flow cytometry.

FIG. 13 shows that bispecific antibodies induced lysis of the expected THP-1 target cells (FIG. 11), with an efficacy (EC 50) of about 1pM. However, it is notable that although the expression level of CD123 target molecule on pDC was ten times higher (fig. 12), these cells were much less affected. The maximum lysis observed was lower and the EC50 found in the assay was almost 10 times higher than the EC50 found for THP-1 cell lysis. The results for donor number 2 were similar (EC 50 values for THP-1 and pDC were 1 and 11pM, respectively; data not shown). These data show that compounds and vγ9vδ2t cells preferentially induce lysis of tumor cells relative to target positive normal cells.

Example 13: epitope mapping revealed the binding of 1D2 VHH to membrane proximal epitopes (membrane-proximal epitope)

To determine what epitope on CD123 is recognized by anti-CD 123 VHH 1D2, the epitope is mapped using a mass spectrometry based method (Pimenova et al, 2008J Mass Spectrom.43 (2): 185-95). Many residues in CD123 molecules were found to crosslink with antibodies: fig. 14.

FIG. 14 identifies the epitope of 1D2 as being present in the region of amino acids 203-273 of human CD 123. When these residues are highlighted in the crystal structure of the molecule (PDB ID 5 U.V 8: brougton et al 2018Nat Commun.9:386), this maps to the second domain closest to the membrane and covers itIs a surface area of the substrate.

Figure 15 shows that the epitope recognized by the lead anti-CD 123 antibody is located close to the membrane. Spanning a distance of aboutThis is not uncommon for epitopes. All CDR regions of 1D2VHH were found to be cross-linked to antigen, with a particularly strong signal for CDR 3.

Similar experiments were performed to determine which epitope on CD123 was recognized by anti-CD 123 VHH 1 A3. Residues H225, H231, R234, T251, R255 and T267 of CD123 were found to cross-link with the 1A3 antibody.

Example 14:1D2x5C8var1 (Y105F) -Fc showed an advantageous stability profile

The thermal stability of 1D2x5C8var1 (Y105F) -Fc was analyzed by nano-differential scanning fluorometry (nano-DSF). The protein exhibited high thermal stability with a development temperature >60 ℃ (table 3). In addition, an accelerated stress test was performed on 1D2x5C8var1 (Y105F) -Fc. Samples were incubated at high temperature (40 ℃) and acidic (50 mM acetate buffer, pH 5.0) and basic (100 mM phosphate buffer, pH 8.5) conditions for 1 week and under oxidizing conditions (phosphate buffer, pH 7.4 and 0.05% H ₂O₂) for 6 and 24 hours. Any stress-induced changes were analyzed by measuring aggregates and fragments using (a) size exclusion chromatography by ultraviolet absorption (SEC-UV) and (B) capillary gel electrophoresis under denaturing (SDS) conditions (CE-SDS) and performed after reduction (fig. 16). No detectable degradation of the stressed protein sample was observed compared to the non-stressed reference sample; the protein was found to be very stable.

Table 3:

example 15: the vδ2-CD123 bispecific antibody 1D2x5C8var1 (Y105F) was more potent in inducing T cell activation and T cell mediated cytotoxicity of tumor cells than the 7A5 based vγ9-CD123 bispecific antibody

The potency of the vδ2-CD123 bispecific antibody 1D2x5C8var1 (Y105F) -Fc was compared to a vγ9-CD123-Fc bispecific antibody based on Fab antibody 7A5 binding vγ9. The sequence of 7A5 is provided by Kabelitz professor friendship. Antibodies have been characterized in Oberg et al (2014) CANCER RES (5): 1349. Ganesan et al (2021) Leukemia35 (8): 2274-2284 and WO2020/227457 describe variants of antibody 7A 5. The sequence of Fab 7A5 was fused to human CH2 and CH3 sequences including knob-pore mutations in CH3 and Fc silent mutations L234F and L235E. This vγ9-binding 'half-IgG' (SEQ ID NOs: 51 and 52) was then co-expressed with a CD123 specific VHH-Fc fusion in HEK293E cells to form a bispecific vγ9xcd123 bispecific antibody. The protein was purified by the C-terminal tag of the CH 3C-terminal present in the vγ9 binding arm and then further purified by preparative size exclusion. This results in a protein that is purified and substantially free of endotoxin. The 1D2xFab 7A5-Fc has the same CD123 binding VHH arm as the 1D2x5C8var1 (Y105F) -Fc. For this assay, 50,000 expanded vγ9vδ2T cells were co-cultured with 50,000 Kasumi-3 or THP1 target cells and serial dilutions of both compounds. The results are shown in fig. 17. (A) Degranulation was analyzed after 4 hours by measuring the percentage of CD107a (lysosomal associated protein-1, or LAMP-1) positive cells using flow cytometry. (B) Vγ9vδ2t cell activation was analyzed by measuring the percentage of CD25 positive cells, and (C) cytotoxicity was analyzed by analyzing the percentage of live target cells after 24 hours using flow cytometry. By higher percentage of CD107a positive cells and CD25 positive cells and lower EC50 values than those observed with vγ9 targeting compounds, it can be seen that 1D2x5C8var1 (Y105F) -Fc induces more efficient activation and degranulation of vγ9vδ2t cells. This was demonstrated by the significantly lower EC50 values observed with 1D2x5C8var1 (Y105F) -Fc compared to 1D2xfab 7a 5-Fc.

In summary, the present invention further includes the following:

1. a multispecific antibody comprising

A. A first antigen binding region capable of binding human CD123, said first antigen binding region comprising a Complementarity Determining Region (CDR) 1 sequence shown in SEQ ID NO. 2, a VH CDR2 sequence shown in SEQ ID NO. 3 and a VH CDR3 sequence shown in SEQ ID NO. 4, and

B. a second antigen binding region capable of binding to the vδ2 chain of the human vγ9vδ2T cell receptor.

2. The multispecific antibody of item 1, wherein

X1 in SEQ ID NO. 2 is G;

X2 in SEQ ID NO. 3 is T; and/or

X3 in SEQ ID NO. 4 is Y.

3. The multispecific antibody of item 1 or item 2, wherein the multispecific antibody is a bispecific antibody.

4. The multispecific antibody of any one of the preceding claims, wherein the first antigen-binding region is a single domain antibody and/or the second antigen-binding region is a single domain antibody.

5. The multispecific antibody of any one of the preceding claims, wherein the first antigen-binding region comprises or consists of: the sequence shown in SEQ ID No. 1 or SEQ ID No. 33, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity with the sequence shown in SEQ ID No. 1 or SEQ ID No. 33.

6. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody is capable of activating human vγ9vδ2t cells.

7. The multispecific antibody of any one of the preceding claims, wherein the second antigen-binding region comprises a VH CDR1 sequence shown in SEQ ID No. 18, a VH CDR2 sequence shown in SEQ ID No. 19, and a VH CDR3 sequence shown in SEQ ID No. 20, and wherein X4 in SEQ ID No. 20 is F,

Or (b)

Wherein the second antigen binding region comprises a VH CDR1 sequence shown in SEQ ID NO:39, a VH CDR2 sequence shown in SEQ ID NO:40 and a VH CDR3 sequence shown in SEQ ID NO:41,

Or (b)

Wherein the second antigen binding region comprises a VH CDR1 sequence shown as SEQ ID NO. 42, a VH CDR2 sequence shown as SEQ ID NO. 43 and a VH CDR3 sequence shown as SEQ ID NO. 44,

Or (b)

Wherein the second antigen binding region comprises a VH CDR1 sequence shown as SEQ ID NO. 45, a VH CDR2 sequence shown as SEQ ID NO. 46 and a VH CDR3 sequence shown as SEQ ID NO. 47.

8. The multispecific antibody of any one of the preceding claims, wherein the second antigen-binding region comprises or consists of: the sequence shown in SEQ ID NO. 17, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 17,

Or (b)

Wherein the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 36, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 36,

Or (b)

Wherein the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 37, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 37,

Or (b)

Wherein the second antigen binding region comprises or consists of: the sequence shown in SEQ ID NO. 38, or a sequence having at least 90%, such as at least 92%, for example at least 94%, such as at least 96%, for example at least 98% sequence identity to the sequence shown in SEQ ID NO. 38.

9. The multispecific antibody of any one of the preceding claims, wherein the first antigen-binding region capable of binding to human CD123 is located N-terminal to the second antigen-binding region capable of binding to the human vδ2 chain.

10. The multispecific antibody according to any one of the preceding claims, wherein the multispecific antibody further comprises a half-life extending domain, such as an Fc region, preferably a human Fc region.

11. The multispecific antibody of claim 10, wherein the Fc region is a heterodimer comprising two Fc polypeptides, wherein the first antigen-binding region is fused to the first Fc polypeptide and the second antigen-binding region is fused to the second Fc polypeptide, and wherein the first and second Fc polypeptides comprise asymmetric amino acid mutations that favor heterodimer formation relative to homodimer formation, wherein preferably the first Fc polypeptide comprises a T366W substitution and the second Fc polypeptide comprises T366S, L a and Y407V substitutions, or vice versa, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

12. The multispecific antibody of any one of claims 10 or 11, wherein the cysteine residue at position 220 in the first and second Fc polypeptides has been deleted or substituted, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

13. The multispecific antibody of any one of claims 10 to 12, wherein the first and second Fc polypeptides further comprise mutations at positions 234 and/or 235, preferably wherein the first and second Fc polypeptides comprise L234F and L235E substitutions, wherein the amino acid positions correspond to human IgG1 according to the EU numbering system.

14. The multispecific antibody of any one of claims 10 to 13, wherein the first Fc polypeptide comprises the sequence set forth in SEQ ID No. 21 and the second Fc polypeptide comprises the sequence set forth in SEQ ID No. 22, or vice versa.

15. The multispecific antibody of any one of the preceding claims, wherein the multispecific antibody is capable of mediating killing of CD 123-expressing cells such as C1r-neo cells or THP-1 cells by vγ9vδ2t cells.

16. The multispecific antibody of any one of the preceding claims, wherein

A. The first antigen binding region is a single domain antibody comprising a VH CDR1 domain shown in SEQ ID No. 2, wherein X1 in SEQ ID No. 2 is G; a VH CDR2 sequence shown in SEQ ID No. 3, wherein X2 in SEQ ID No. 3 is T; and the VH CDR3 sequence shown in SEQ ID NO. 4, wherein X3 in SEQ ID NO. 4 is Y.

B. The first antigen binding region is a single domain antibody comprising an amino acid sequence having at least 94% sequence identity to the sequence set forth in SEQ ID NO. 17.

17. The multispecific antibody of item 16, wherein the first antigen-binding region comprises an amino acid sequence that has at least 94% sequence identity to the sequence set forth in SEQ ID No. 1.

18. An antibody comprising a first antigen binding region capable of binding human CD123, wherein the first antigen binding region is a single domain antibody comprising:

19. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-17 or the antibody of claim 18 and a pharmaceutically acceptable excipient.

20. The multispecific antibody according to any one of claims 1to 17 or the antibody according to claim 18 for use as a medicament, preferably for use in the treatment of cancer, more preferably for the treatment of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hairy cell leukemia, hodgkin lymphoma, blast plasmacytoid dendritic cell tumor, chronic granulocytic leukemia, chronic lymphocytic leukemia, B-cell chronic lymphoproliferative disorder or myelodysplastic syndrome.

21. A nucleic acid construct comprising a nucleotide sequence encoding the multispecific antibody according to any one of claims 1 to 17 or the antibody according to claim 18, or a host cell comprising one or more nucleic acid constructs encoding the multispecific antibody according to any one of claims 1 to 17 or the antibody according to claim 18.

Claims

1. A multispecific antibody comprising

2. The multispecific antibody of claim 1, wherein

X ₁ in SEQ ID NO. 2 is G;

X ₂ in SEQ ID NO.3 is T; and/or

X ₃ in SEQ ID NO.4 is Y.

3. The multispecific antibody of claim 1 or claim 2, wherein the multispecific antibody is a bispecific antibody.

7. The multispecific antibody of any one of the preceding claims, wherein the second antigen-binding region comprises a VH CDR1 sequence as set forth in SEQ ID No. 18, a VH CDR2 sequence as set forth in SEQ ID No. 19, and a VH CDR3 sequence as set forth in SEQ ID No. 20, and wherein X ₄ in SEQ ID No. 20 is F,

Or (b)