A bispecific antibody that binds CD3 and a fluorophore

Abstract

The present invention relates to a bispecific antibody comprising at least a first binding domain and a second binding domain, wherein said first binding domain is bound to an organic fluorophore and said second binding domain is bound to CD3, as well as to a combination thereof with a labeled binding agent that specifically binds to a target antigen, wherein the labeled binding agent is labeled with an organic fluorophore.

Description

[Technical field]

[0001] The present invention relates to bispecific antibodies. [Background technology]

[0002] Bispecific T cell-inducing activating antibodies and chimeric antigen receptor (CAR) T cells (CAR T) cells are promising antibody-based approaches for treating various types of cancer (or other diseases such as autoimmune, inflammatory, or infectious diseases). Both therapies rely on (re)directing the killing activity of T cells against target cells.

[0003] Bispecific antibodies are biologics that can simultaneously bind two different antigens with the aim of linking effector cells (e.g. T cells) to target antigen-expressing cells, leading to effector cell-mediated killing of the target antigen-carrying cells. In particular, bispecific antibodies can bind to CD3 and tumor-associated antigens on T cells, cross-linking tumor cells and effector T cells. Cross-linking leads to T cell activation and induces potent and selective (re)targeting of their cytolytic activity to target-carrying cells, independent of MHC. Clinical-stage bispecific antibodies range from small proteins consisting of two linked antigen-binding fragments to large immunoglobulin G (IgG)-like molecules with additional domains added.

[0004] CAR-T cells are genetically modified T cells that overexpress an artificial CAR, which usually consists of a single-chain variable fragment (scFv) binding domain linked to a T cell via a flexible hinge and a transmembrane domain. Additional CAR components, the intracellular costimulatory domain and the stimulatory domain, mediate the activation signal. The surface scFv redirects the CAR-T cell cytotoxic activity against target cells expressing the alloantigen, independent of MHC.

[0005] One bispecific T cell conjugate (BiTE™), consisting of two scFvs against CD3 and CD19, has received regulatory approval (blinatumomab; Blycinto®), as well as two CAR-T cell products (Kymriah® and Yescarta®) as salvage therapies for certain subtypes of B cell leukemia or lymphoma.

[0006] However, despite high rates of initial response, relapse is observed in a notable proportion of patients. Bispecific antibodies and CAR-T cells, which can target single tumor cell antigens, cannot address antigen escape or overcome tumor heterogeneity. Improved outcomes using combinatorial strategies require the development of new GMP products for each new antigen that may be desired to be targeted.

[0007] Compared to recombinant protein development, GMP production of CAR-T cells is the cost driver of personalized medicine, as each batch is manufactured from the patient's cells, thus limiting its clinical applicability. Furthermore, while the activity of antibodies can be controlled by injection schedules, this on / off safety switch is not currently possible when using CAR-T cells. Their activation lacks downstream control mechanisms after injection and can result in severe side effects resulting from systemic toxicity or on-target off-tumor reactions due to strong levels of CAR T cell activation.

[0008] However, so far only a very small selection of bispecific T cell-inducing binding agents is available on the market, which severely limits the clinical potential of T cell-inducing therapy for diseases that represent targets for which only a few bispecific T cell-inducing binding agents are already available, leaving physicians with no suitable second-line therapy if the respective disease develops resistance to said therapy. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides a bispecific antibody comprising at least a first binding domain and a second binding domain, wherein the first binding domain binds to an organic fluorophore and the second binding domain binds to CD3, as well as a combination thereof with a labeled binding agent that specifically binds to a target antigen, wherein the labeled binding agent is labeled with an organic fluorophore.

[0010] Such new constructs are termed "Universal T Cell Inducing Activating Bispecific Antibodies" and are abbreviated herein as UniTEA. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a schematic diagram and characterization of UniTEA. (A) Universal T cell inducible activating bispecific antibody (UniTEA) has two specificities for human CD3 (e.g., CD3ε) and fluorescein expressed on T cells. T cell cytotoxicity is elicited only in the presence of a fluorophore-tagged target binding adapter. The fluorophore is shown as a star (☆). Different types of binders that can be used as adapters are shown, i.e., antibodies (left, represented by IgG-like shapes, including other fragments or derivatives such as scFv or diabodies, and small molecules (middle) or peptides (right). (B) Size-exclusion chromatography profiles and SDS PAGE gels showing the purity and molecular weight of three different UniTEA formats, called 4m5.3 / BlinCD3, E2 / BlinCD3, and FluA / BlinCD3. [Diagram 2]FIG. 2 shows T cell-mediated depletion of CD19, CAIX, and CD117 expressing cell lines in vitro. (A) NALM-6 (CD19+) B-ALL cell line was used to compare T cell-mediated killing induced by Blinatumomab™ (anti-CD19 / BlinCD3 T cell-inducing activating bispecific antibody, TEA) with 4m5.3 / BlinCD3 and FluA / BlinCD3 UniTEA formats. Target and T cells were co-incubated for 24 and 48 hours at an E:T ratio of 10:1. (B) Background killing of UniTEA or commercial anti-human CD19 IgG-FITC adapter molecule alone at the concentrations shown. (C) Equimolar concentrations of 4m5.3 / BlinCD3 and FluA / BlinCD3 UniTEA induced T cell-mediated target cell lysis similar to Blinatumomab™ when 10 nM adapter was added. (D) Renal cell carcinoma cell line SK-RC-52 (CAIX+) was used to determine T cell-mediated killing induced by 4m5.3 / BlinCD3, FluA / BlinCD3, and E2 / BlinCD3 UniTEA in the presence of 10 nM site-specifically fluoresceinated anti-CAIX IgG. (E) All three UniTEA formats induced T cell-mediated tumor cell lysis in a concentration-dependent manner after 24 h at an E:T ratio of 10:1. Background killing after co-culture of T cells and tumor cells alone or by adding 10 nM adapter molecule alone is also reported. (F) Comparison between CAR T cells and TEA and UNiTEA was performed by targeting CD117 on MOLM14 (CD117+) AML cells. 4m5.3 / BlinCD3 UNiTEA (BiTE format) was used in combination with a site-specifically fluoresceinated anti-CD117 diabody (Db). Target and effector cells were co-incubated at a 1:1 E:T ratio for 24 and 48 h. (G) Addition of 4m5.3 / BlinCD3 UniTEA in the presence of 10 nM fluoresceinated anti-CD117 Db resulted in T cell-mediated target cell lysis similar to direct anti-CD117 CAR-T cells and anti-CD117 / BlinCD TEA at 24 and 48 h. [Diagram 3] Figure 3 shows the persistence of FITC-antibodies in vivo. (A) Commercially available anti-mouse CD19 (mCD19) IgG-FITC was injected iv (50ug / mouse) into two C57BL / 6J mice. Peripheral blood was analyzed by FACS at various time points over 8 days. (B) Mean fluorescence intensity (MFI) at each time point of mCD19+ cells stained with and without additional mCD19 IgG-FITC is shown by filled and open circles, respectively. (C) Live mCD19+ / mCD20+ B cells expressed as percentage of mCD19+ / mCD20+ cells at each time point compared to baseline counts after 4 hours stained with and without additional mCD19 IgG-FITC, shown by filled and open circles, respectively. (D) Representative FACS plots showing peripheral blood CD19+CD20+ mouse B cell populations at the indicated time points with and without additional CD19-FITC labeling. [Figure 4] FIG. 4 shows the structures of FITC and fluorescein-5-maleimide, the latter used for site-specific conjugation of FITC to antibodies labeled with a terminal cysteine ​​residue. [Diagram 5] Figure 5 is a diagram showing an overview of various bispecific antibody formats. This diagram shows various formats of bispecific antibodies that can be used in the present invention. It is taken from Spiess et al. 2015, the contents of which are incorporated herein by reference, especially to take responsibility for the possible loss of color information. [Figure 6]Figure 6 shows T cell-mediated elimination of PSMA+ LNCaP human prostate adenocarcinoma cells by fluoresceinated PSMA targeting ligand and E2 / OKT3 UniTEA in vitro. (A) UniTEA has dual specificity for human CD3ε expressed on T cells (OKT3 binder) and for fluorescein (E2 binder). T cell cytotoxic activity against PSMA+ cells is elicited only in the presence of a fluorescein-tagged PSMA-specific adaptor, the small molecule DUPA-FITC (DF). (B) Representative FACS plots showing PSMA+ target cells co-cultured with T cells, 1 nM UniTEA, and the indicated concentrations of DUPA-FITC (DF) for 24 hours. (C) Quantification of the percentage of specific lysis of PSMA+ CD3- cells achieved after 24 hours of co-culture with T cells (E:T 10:1), 1 nM UniTEA, and the indicated concentrations of DUPA-FITC (DF). Specific lysis was calculated as follows: (1-number of viable target cells / number of viable target cells without antibody)*100. [Figure 7]Figure 7 shows T cell-mediated elimination of double positive CD20+ / CD38+ human B cell lymphoma cells by fluoresceinated clinically approved antibodies and E2 / OKT3 UniTEA in vitro. (A) UniTEA has dual specificity for human CD3ε expressed on T cells (OKT3 binder) and for fluorescein (E2 binder). T cell cytotoxicity against CD20+ / CD38+ cells is elicited only in the presence of fluorescein-tagged clinically approved anti-CD20 or anti-CD38 antibodies. (B) Representative FACS plots showing cell surface expression of human CD3, CD19, CD20, and CD38 on DOHH-2 NHL cell line. (C) Flow cytometry histograms showing DOHH-2 cells labeled with clinically available anti-CD20 (Rituximab) and anti-CD38 human (Daratumomab) IgG1 at the indicated concentrations, labeled with FITC (fluorescein:protein ratio=2). (D) Representative FACS plots showing the percentage of CD19+CD3- cells remaining over 24 hours after co-culture with T cells (E:T 1:1) and the indicated antibodies. We used CD19 to identify target cells, since DOHH2 cells are positive for CD19, CD20, and CD38, thus avoiding epitope blocking of targeting and detection antibodies (FIG. 5B). (E) Quantification of the percentage of specific lysis of CD3-CD19+CD20+CD38+DOHH-2 cells achieved after 24 hours of co-culture with T cells and the indicated antibodies. [Figure 8]FIG. 8 shows that T cell-mediated elimination of human AML cell lines is improved by combinatorial targeting of tumor-associated antigens using a combination of fluoresceinated tumor-specific binders and UniTEA in vitro. (A) The diagram shows a target cell population expressing antigen A or antigen B or both antigens A and B. Fluoresceinated adapters for antigen A or B bind to the respective cells via the expressed antigen. This leads to a higher degree of labeling and therefore more fluorescein expression of cells expressing both antigens A and B. Thus, the addition of UniTEA leads to improved T cell activation and killing of cells expressing both antigens A and B, but killing of cells expressing only antigen A or B is less efficient. Thus, this system allows for improved killing of populations positive for multiple selected target antigens by combinatorial use of linkers, while at the same time cells expressing a single target antigen are killed less efficiently. (B) Representative FACS plots showing cell surface expression of CD117 and CD371 on a mixture of MOLM13 human AML cell lines transduced to express CD117 (lower right corner), CD371 (upper left corner), or both CD117 and CD371 (upper right corner). In addition, CD117- and CD371-negative T cells in the cell mixture are shown (lower left corner). (C) Quantification of the percentage of specific lysis of CD33+CD3-MOLM13 cells after 24 hours of co-culture with T cells with the indicated concentrations of E2 / OKT3 UniTEA and fluoresceinated tumor antigen-specific adaptors used in combination and as single agents (E:T 1:10). (D) Representative FACS plots showing the percentage of CD33+CD3- cells remaining over 24 hours after co-culture with T cells, the indicated fluoresceinated tumor antigen-specific adaptors, and UniTEA. [Figure 9]FIG. 9 shows T cell-mediated elimination of transplanted human AML cell lines in immunodeficient NSG mice treated with fluoresceinated tumor antigen-specific adaptors and UniTEA. (A) Experimental design of prophylactic in vivo AML treatment using AML cell lines, human primary T cells, and UniTEA and fluoresceinated diabody. (B) Quantification of raw bioluminescence imaging of immunodeficient mice 7, 14, and 19 days after CD117+ human AML cell line injection. Treatment groups are indicated. (C) Quantification of flow cytometry data showing the percentage of CD117+GFP+ human AML MOLM14 cells in the bone marrow of mice at the end of the experiment (day 19). (D) Representative FACS plots showing MOLM14 AML cells (identified by human CD45+GFP+ cells) in the bone marrow, peripheral blood, and spleen of mice at the end of the experiment (day 19). Cells are shown as a percentage of total live nucleated cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Before describing the invention in detail, it should be understood that the invention is not limited to the specific components of the described devices or process steps of the described methods, as such devices and methods may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include single and / or plural referents unless clearly indicated by the context. When a range is given for a parameter bounded by numerical values, the range is considered to include the upper values.

[0013] It should be further understood that the embodiments disclosed herein are not to be understood as individual embodiments that may be unrelated to each other. Features described according to one embodiment are also to be disclosed in relation to other embodiments shown herein. In some cases, if a particular feature is not disclosed according to one embodiment but is disclosed according to another embodiment, a person skilled in the art will understand that this does not necessarily mean that said feature is not disclosed according to said other embodiment. A person skilled in the art will understand that it is the essence of the present application to disclose said features also for other embodiments, but this has not been done merely for the sake of clarity and to keep the specification manageable.

[0014] Furthermore, the contents of the prior art documents referred to in this specification are incorporated by reference, especially those prior art documents that disclose standard or routine methods, in which case incorporation by reference has the primary purpose of providing a full and enabling disclosure and avoiding redundant repetition.

[0015] According to a first aspect of the invention, a bispecific antibody comprises at least a first binding domain and a second binding domain, said first binding domain binding to an organic fluorophore and said second binding domain binding to CD3.

[0016] Such new constructs are termed "Universal T Cell Inducing Activating Bispecific Antibodies" and are abbreviated herein as UniTEA.

[0017] As used herein, the term "organic fluorophore" refers to a small organic molecule that has the ability to re-emit light upon photoexcitation. The organic fluorophores disclosed herein have a maximum molecular weight not exceeding 2500 Daltons. The term "organic fluorophore" excludes fluorescent proteins such as green fluorescent protein (GFP), which has a molecular weight of 27 kDa, and some phycobiliproteins, which have a molecular weight of about 240 kDa.

[0018] In some embodiments, the organic phosphor is Xanthene derivatives such as fluorescein, rhodamine, Oregon green, eosin, and Texas red Cyanine derivatives such as cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine Squaraine derivatives and ring-substituted squaraines, including Seta and Square dyes Squaraine rotaxane derivatives, e.g. See Tau dye Naphthalene derivatives (dansyl derivatives and prodan derivatives) Coumarin derivatives Oxadiazole derivatives, such as pyridyloxazoles, nitrobenzoxadiazoles, and benzoxadiazoles Anthracene derivatives such as anthraquinones, including DRAQ5, DRAQ7, and CyTRAK Orange Pyrene derivatives such as Cascade Blue Oxazine derivatives such as Nile red, Nile blue, cresyl violet, oxazine 170, etc. Acridine derivatives such as proflavine, acridine orange, acridine yellow, etc. Arylmethine derivatives such as auramine, crystal violet, and malachite green Tetrapyrrole derivatives, such as, for example, porphines, phthalocyanines, bilirubins, and / or Dipyrromethene derivatives such as BODIPY and aza-BODIPY The compound belongs to at least one of the groups selected from the list consisting of:

[0019] The inventors surprisingly discovered that such constructs can be used together with a second binding agent (also referred to herein as an "adapter") labeled with a corresponding fluorophore to deliver T cell directed therapy to almost any conceivable target within a patient's body.

[0020] Thus, a modular approach is provided that facilitates the application of T cell-directed therapies to targets not previously accessible to this approach, and may further facilitate and accelerate regulatory approval of T cell-directed therapies against new targets.

[0021] The resulting UniTEA and fluorophore-tagged adaptor complex facilitates the formation of cytolytic junctions between CD3-expressing T cells and target cells, leading to efficient target cell killing.

[0022] UniTEA can be provided as a commercially available GMP manufactured biologic and can be used in combination with clinically approved therapeutic antibodies or other target binders (referred to herein as "adapters") that are minimally modified (fluorophore tagged) to render them suitable for redirecting T cell cytotoxicity to the respective target of interest.

[0023] Organic fluorophores can be linked to a wide range of adaptors so that T cells can be universally redirected to any target cell of choice, provided there is a clinical grade bridge available. Furthermore, antibodies (or peptides, small molecules, or any other antigen binders) that would serve as suitable adaptors would have their functionality enhanced by UniTE to recruit and redirect T cells via the adaptor.

[0024] The UniTEA modular approach allows for on-demand T cell activation, which would only be induced if UniTEA was co-present with the fluorophore-tagged target-binding adaptor, whereas T cell cytotoxic activity cannot be induced in the absence of UniTEA, even though the fluorophore-tagged bridge may remain in the body. Thus, the short half-life of UniTEA (~30 min) allows for tight control over endogenous T cell activity and fine tuning of dosing effects.

[0025] Antigen-negative recurrence or tumor heterogeneity will be addressed by utilizing combinatorial strategies that can be further individualized for each patient or disease. This approach therefore allows existing clinically used target binding molecules to be utilized in combination or sequentially to target broader and more heterogeneous tumor cell antigens (blood and non-blood tumors), overcoming the limitations of monospecific tumor antigen targeting approaches.

[0026] The investment to develop the protein is much lower than the cost of manufacturing new monovalent or multivalent CAR-T cells and modular CAR-T cells, all of which require dedicated GMP facilities to engineer the patient's T cells. The advantage of generating a single generic bispecific protein makes the production of new bispecific antibodies obsolete. The development of UniTE molecules will be an alternative to the highly labor-intensive multivalent antibodies and CAR T cells, while retaining the benefits of personalization of tailored therapeutics and the GMP production costs of a single recombinant protein.

[0027] According to one embodiment, said second binding domain binds to the CD3 epsilon chain and / or the CD3 gamma chain.

[0028] The T cell receptor (TCR) complex is composed of ligand-binding subunits (TCRα and TCRβ) and signaling subunits (CD3γ, CD3δ, CD3ε, and CD3ζ [CD247]). The ligand for the TCR consists of peptide antigens bound to major histocompatibility complex (MHC) class I or class II molecules. Assembly studies, transfection and reconstitution experiments, and detergent dissociation studies suggest that the components of the TCR complex are organized as dimers [reviewed in ]. CD3ε forms non-covalently distinct dimers with CD3γ and CD3δ, TCRα forms a disulfide-linked dimer with TCRβ, and CD3ζ is expressed in the form of a disulfide-linked homodimer.

[0029] T cells can be induced by binding molecules capable of binding to CD3, particularly CD3ε, the invariant signaling component of the T cell receptor complex. This exploits the ability to utilize a polyclonal population of cytotoxic T cells [including CD8+, CD4+, and regulatory T cells (Treg)] and does not rely on MHC class I for antigen presentation. Monovalent binding of CD3 does not activate the TCR unless target cell binding is induced by combining a bispecific antibody and a labeled binding agent, as described below. In this manner, T cell induction leads to T cell activation and resulting proliferation, which contributes to improved efficacy. Furthermore, T cell induction contributes to continuous lysis, which further improves efficacy.

[0030] According to one embodiment of the invention, the bispecific antibodies are formatted as full-length antibodies or antibody fragments, the latter retaining target binding properties.

[0031] In general, many formats for bispecific antibodies have been evaluated. For a review, see, for example, Spiess et al. (2015), the contents of which are incorporated herein by reference. Bispecific antibodies are classified into five different structural groups: (i) Bispecific IgG (BsIgG) (ii) IgG with additional antigen-binding moieties (iii) Bispecific antibody fragment (iv) a bispecific fusion protein, and (v) Bispecific antibody conjugate

[0032] Moreover, it is usually (i) Asymmetric bispecific antibodies (heteromultimeric antibodies having different chains with binding moieties for different antigens) (ii) Symmetric bispecific antibodies (heteromultimeric or monomeric / single-chain antibodies, where different binding moieties for different antigens are on the same chain) It is distinguished by:

[0033] Generally, small formats, often in monomeric / single-chain conformation, have short serum half-lives, which necessitate frequent or even constant dosing, but offer the option to discontinue treatment immediately upon the onset of side effects such as cytokine storm.

[0034] Asymmetric bispecific antibodies often suffer from problems when it comes to post-translational pairing of the different chains. In the co-expression of four different antibody chains (heavy and light chains with each specificity), improper pairing can occur, leading to 10 different products, of which only one is the correct variant. However, these mispairing challenges can be addressed by various technical approaches (see Wang et al. 2018).

[0035] For an overview of the various formats, see Figure 5. Each of these formats offers different properties and advantages and therefore, inter alia: - valency of binding to each antigen, The geometrical arrangement of the antigen binding site c, -Accuracy of strand pairing, Pharmacokinetic half-life and possibly effector functions can be selected as the preferred format for

[0036] According to an embodiment of the invention, the bispecific antibody comprises: DVD-Ig Knobs in Holes Crossmab ·BiTE(trademark) Nanobody Diamond Body ·DART(trademark) TandAb™, and / or scDb-scFv The image has at least one format selected from the group consisting of:

[0037] These formats are familiar to those skilled in the art (see Spiess et al. (2015) for definitions). One skilled in the art can use the sets of variable domains and / or CDRs disclosed herein to generate the above-mentioned formats by routine methods.

[0038] According to one embodiment of the invention, the bispecific antibody is in a BiTE format.

[0039] The BiTE format essentially consists of two scFv format antibodies linked together by a peptide linker, usually a linker containing the amino acid sequence GGGG ("G4S"). The resulting single chain has a weight of about 55 kD. The BiTE can have the following format (the dash between the variable domains indicates an optional linker): VH1-VL1-VL2-VH2 VL1-VH1-VH2-VL2 VL1-VH1-VL2-VH2 VH1-VL1-VH2-VL2

[0040] Due to its small size, the BiTE format is associated with a very rapid clearance profile in patients. Blinatumomab is the only clinically approved BiTE on the market and is administered as a continuous infusion for 28 days thanks to the use of an intravenous pump with a constant flow rate. In this way, there are circumstances to immediately discontinue antibody administration if a serious adverse event occurs.

[0041] The scDb-scFv is similar to the BiTE format, the only difference being that the variable domains directed to a first or second target are overlapped, e.g., as shown below: VH1-VL1-VH1-VL1-VL2-VH2 VL1-VH1-VL1-VH1-VH2-VL2 VL1-VH1-VL1-VH1-VL2-VH2 VH1-VL1-VH1-VL1-VH2-VL2

[0042] TandAb (tandem diabody) formats are, for example, as follows: JPEG2024504354000002.jpg33170

[0043] The DART format is a dimeric format in which the two chains are connected to each other by a cysteine ​​linker: ·Chain 1: VL1-VH2 ·Chain 2: VH1-VL2

[0044] Anticalin proteins are artificial proteins that can bind to antigens, proteins, or small molecules. Anticalin proteins are not structurally related to antibodies, making them a type of antibody mimetic. Instead, they are derived from human lipocalins, a family of natural binding proteins. Anticalin proteins are used in place of monoclonal antibodies, but are about one-eighth the size of monoclonal antibodies, with a size of about 180 amino acids and a mass of about 20 kDa.

[0045] According to one embodiment, said first binding domain of the bispecific antibody binds to fluorescein, preferably fluorescein when bound to a protein (conjugated fluorescein).

[0046] As used herein, the terms "fluorescein", "fluorescein isothiocyanate" and "FITC" are used interchangeably. Binding agents, including antibodies that bind fluorescein, preferably when bound to a protein (conjugated fluorescein), are widely available. Several embodiments are described below, but reference is also made to Biocomepare, an internet source that lists >78,000 anti-FITC antibody products, including ab19224 (Abcam, goat polyclonal IgG) and LS C34608 (LSBio, monoclonal mouse FITC antibody (clone F4 / 1, IHC), to name just a few.

[0047] According to one embodiment, the first binding domain of the bispecific antibody that binds fluorescein or conjugated fluorescein comprises anticalin FluA (SEQ ID NO: 47), which is disclosed in Vopel et al. (2005), the contents of which are incorporated herein by reference.

[0048] As described elsewhere herein, Anticalin proteins are artificial proteins capable of binding to antigens, proteins or small molecules. Anticalin proteins are derived from human lipocalins, a family of natural binding proteins.

[0049] Preferably, the first binding domain of a bispecific antibody that binds fluorescein or conjugated fluorescein comprises anticalin FluA having >80%, >81%, >82%, >83%, >84%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, and most preferably >99% sequence identity to SEQ ID NO:47.

[0050] In the following, embodiments are described in which the sequence of a specific antibody is referred to. In some embodiments, alternative CDR (complementarity determining region) sequences are disclosed. These embodiments relate to the same antibody, but differ only in the nomenclature by which the CDRs are defined.

[0051] As used herein, the term "CDR" or "complementarity determining region" is intended to mean the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. These particular regions are described by Kabat et al. (1977), Kabat et al. (1991), Chothia et al. (1987), and MacCallum et al., (1996), whose definitions, when compared with each other, include overlapping or subsets of amino acid residues. Nevertheless, application of either definition to refer to the CDR of an antibody or grafted antibody or variant thereof is intended to be within the scope of the term as defined and used herein. The amino acid residues that comprise the CDRs defined by each of the above cited references are set forth below in Table 1 as a comparison. Please note that this numbering may actually differ from the CDRs disclosed in the enclosed sequence listing, as the definition of CDRs may vary depending on the context.

[0052] [Table 1]

[0053] As used herein, the term "framework" when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions in the variable region of an antibody. Thus, the variable region framework is between about 100-120 amino acids in length, but is intended to refer only to amino acids excluding the CDRs.

[0054] In one embodiment, the term "capable of binding to target X" should be understood to mean binding with sufficient binding affinity. In such an embodiment, each binding domain is 10 -4 The following K D Binds to the target at K D is the equilibrium dissociation constant between an antibody and its antigen, k off / k on It is the ratio of K D and affinity are inversely proportional. D The value relates to the antibody concentration (the amount of antibody needed for a particular experiment), K D The lower the value (lower concentration), the higher the affinity of the binding domain. The table below shows typical K values ​​for monoclonal antibodies. D Indicates the range.

[0055] [Table 2]

[0056] According to one embodiment, the first binding domain that binds fluorescein or conjugated fluorescein comprises: a) comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in one of the heavy chain / light variable region sequence pairs of anti-fluorescein antibody E2 or 4m5.3, as set forth in SEQ ID NOs: 27 and 31 or SEQ ID NOs: 36 and 40, respectively; b) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 28) HC CDR2 (SEQ ID NO:29) HC CDR3 (SEQ ID NO: 30) LC CDR1 (SEQ ID NO: 32) LC CDR2 (SEQ ID NO: 33), and LC CDR3 (SEQ ID NO:34) c) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 37) HC CDR2 (SEQ ID NO: 38) HC CDR3 (SEQ ID NO: 39) LC CDR1 (SEQ ID NO: 41) LC CDR2 (SEQ ID NO: 42), and LC CDR3 (SEQ ID NO: 43) d) comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), with the proviso that at least one of the CDRs has no more than three amino acid substitutions compared to SEQ ID NOs: 28-30, 32-34, 37-39, or 41-43, respectively; and / or e) comprising the heavy / light chain complementarity determining regions (CDRs) of b), with the proviso that at least one of the CDRs has a sequence identity of ≧66% to each of SEQ ID NOs: 28-30, 32-34, 37-39, or 41-43, The CDRs are embedded in a suitable protein framework such that they can bind to fluorescein or conjugated fluorescein.

[0057] Anti-fluorescein antibody E2 is disclosed in Vaughan et al. (1996), the contents of which are incorporated herein by reference. Anti-fluorescein antibody 4m5.3 is disclosed in Boder et al. (2000), the contents of which are incorporated herein by reference.

[0058] Preferably, this applies to all CDR sets disclosed herein, where at least one CDR has no more than two amino acid substitutions, more preferably no more than one amino acid substitution.

[0059] Preferably, this applies to all CDR sets disclosed herein, where at least one of the CDRs has >67%, >68%, >69%, >70%, >71%, >72%, >73%, >74%, >75%, >76%, >77%, >78%, >79%, >80%, >81%, >82%, >83%, >84%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, and most preferably >99% sequence identity to the respective SEQ ID NO.

[0060] As used herein, the term "sequence identity %" should be understood as follows: the two sequences to be compared are aligned to maximize the correlation between the sequences. This may include inserting "gaps" in one or both sequences to improve the degree of alignment. The identity % may then be determined over the entire length of each of the sequences being compared (so-called global alignment), which is particularly suitable for sequences of the same or similar length, or over a shorter, defined length (so-called local alignment), which is more suitable for sequences of unequal length. In the above context, an amino acid sequence having at least, for example, 95% "sequence identity" to a query amino acid sequence is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence, except that the subject amino acid sequence may contain no more than 5 amino acid modifications per each 100 amino acids of the query amino acid sequence. In other words, to obtain an amino acid sequence having at least 95% identity to the query amino acid sequence, no more than 5% (5 out of 100) of the amino acid residues in the subject sequence may be inserted or substituted by another amino acid or deleted. Methods for comparing the identity and homology of two or more sequences are well known in the art. The percentage of identity of two sequences can be determined, for example, by using a mathematical algorithm. Preferred but non-limiting examples of mathematical algorithms are summarized in the BLAST family of programs, such as BLAST or NBLAST programs, and FASTA. Sequences that are identical to other sequences to a certain extent can be identified by these programs. In addition, programs available in the Wisconsin Sequence Analysis Package, version 9.1, such as the programs BESTFIT and GAP, may be used to determine the percent identity between two polypeptide sequences. When reference is made herein to an amino acid sequence that shares a significant degree of sequence identity with a reference sequence, the differences in the sequence are preferably due to conservative amino acid substitutions.Preferably, such sequences retain the activity of the reference sequence, although perhaps at a slower rate.

[0061] Preferably, this applies to all sets of CDRs disclosed herein, wherein at least one of the CDRs comprises: ·Affinity maturation Reduced immunogenicity The CDR sequences have been modified to include:

[0062] Affinity maturation is a process that increases the affinity of a particular antibody in vitro. Like natural affinity maturation, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been used successfully to optimize antibodies, antibody fragments, or other peptide molecules such as antibody mimetics. Random mutations within the CDRs are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range. For the principles, see Eylenstein et al. (2016), the contents of which are incorporated herein by reference.

[0063] Humanized antibodies contain CDR regions derived from mouse sequences grafted into V regions derived from human sequences with any necessary framework back mutations. Therefore, the CDRs themselves may cause immunogenic reactions when the humanized antibody is administered to a patient. Methods for reducing the immunogenicity caused by CDRs are disclosed in Harding et al. (2010), the contents of which are incorporated herein by reference. According to one embodiment of the present invention, the framework is a human VH / VL framework. VH represents the heavy chain variable domain of an IgG-like antibody, whereas VL represents the light chain variable domain (kappa or lambda).

[0064] According to one embodiment of the invention, the first binding domain that binds fluorescein or conjugated fluorescein comprises: a) the variable domain of one antibody selected from the group consisting of E2 or 4m5.3 b) Heavy / Light Chain Variable Domains (VD) HC VD (SEQ ID NO: 27 or 36) and LC VD (SEQ ID NO: 31 or 40) c) the heavy / light chain variable domains (VD) of b), except the HCVD has ≥80% sequence identity to SEQ ID NO: 27 or 36, respectively; and / or LCDVD has a sequence identity of ≥ 80% to SEQ ID NO: 31 or 40, respectively d) the heavy / light chain variable domains (VD) of b), provided that at least one of the HCVD or LCVD has no more than 10 amino acid substitutions compared to the respective SEQ ID NO: Including, The bispecific antibody is still capable of binding to fluorescein or conjugated fluorescein.

[0065] "Variable domain", when used in reference to an antibody or its heavy or light chain, is intended to mean the portion of the antibody that confers antigen-binding properties to the molecule and is not the constant region. The term is intended to include functional fragments thereof that maintain some of all of the binding functions of the entire variable region. Variable region binding fragments include functional fragments such as, for example, Fab, F(ab)2, Fv, single chain Fv (scfv), and the like. Such functional fragments are well known to those skilled in the art. Thus, the use of these terms in describing functional fragments of heteromeric variable regions is intended to be consistent with the definitions well known to those skilled in the art. Such terms are described, for example, in Huston et al., (1993) or Plueckthun and Skerra (1990).

[0066] Preferably, this applies to all variable domains disclosed herein, where at least one of the variable domains has < 9, < 8, < 7, < 6, < 5, < 4, < 3, < 2, < 1 amino acid substitutions with respect to the respective SEQ ID NO.

[0067] Preferably, this applies to all variable domains disclosed herein, where at least one of the variable domains has a sequence identity of >81%, >82%, >83%, >84%, >85%, >86%, >87%, >88%, >89%, >90%, >91%, >92%, >93%, >94%, >95%, >96%, >97%, >98%, >99 to the respective SEQ ID NO.

[0068] Preferably, this applies to all variable domains disclosed herein, with at least one amino acid substitution in any of the variable domains disclosed herein being a conservative amino acid substitution.

[0069] "Conservative amino acid substitutions" have less impact on antibody function than non-conservative substitutions. Although there are many ways to classify amino acids, they are often divided into six main groups based on the general chemical characteristics of the structure and R groups.

[0070] In one embodiment, a "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include: Basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), Uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), Non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), Beta-branched side chains (e.g., threonine, valine, isoleucine), and Aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) The amino acid sequence includes:

[0071] Other conservative amino acid substitutions may also be made across amino acid side chain families, such as when substituting asparagine for aspartic acid to modify the charge of the peptide. Thus, a predicted non-essential amino acid residue in an HR domain polypeptide, for example, is preferably replaced with another amino acid residue from the same side chain family or a homolog across families (e.g., asparagine for aspartic acid, glutamine for glutamic acid). Conservative changes can further include the substitution of a chemically homologous non-natural amino acid (i.e., a synthetic non-natural hydrophobic amino acid for leucine, a synthetic non-natural aromatic amino acid for tryptophan).

[0072] According to an embodiment of the invention, the second binding domain that binds to CD3 comprises the CDRs and / or variable domains of an antibody selected from the group consisting of OKT3 or BlinCD3 and UCHT-1 (Ceuppens et al, 1986), BMA031 (Borst et al, 1990), and 12F6 (Li et al, 2005).

[0073] The anti-CD3 antibody OKT3 is disclosed in Horn et al. (2017), the contents of which are incorporated herein by reference. The anti-CD3 antibody BlinCD3 is a modified variant of OKT3.

[0074] According to one embodiment of the invention, the second binding domain that binds to CD3 comprises: a) comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in one of the heavy / light variable region sequence pairs of the anti-CD3 antibodies BlinCD3 and OKT3, as set forth in SEQ ID NOs: 1 and 8 or SEQ ID NOs: 16 and 20, respectively; b) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 2 or 5) HC CDR2 (SEQ ID NO: 3 or 6) HC CDR3 (SEQ ID NO: 4 or 7) LC CDR1 (SEQ ID NO: 9 or 12) LC CDR2 (SEQ ID NO: 10 or 13), and LC CDR3 (SEQ ID NO: 11 or 14) c) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 17) HC CDR2 (SEQ ID NO: 18) HC CDR3 (SEQ ID NO: 19) LC CDR1 (SEQ ID NO: 21) LC CDR2 (SEQ ID NO: 22), and LC CDR3 (SEQ ID NO: 23) d) comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), with the proviso that at least one of the CDRs has no more than 3 amino acid substitutions compared to SEQ ID NOs: 2-4, 9-11, 17-19, or 21-23, respectively; and / or e) comprising the heavy / light chain complementarity determining regions (CDRs) of b), with the proviso that at least one of the CDRs has 66% sequence identity to SEQ ID NOs: 2-4, 9-11, 17-19, or 21-23, respectively; The CDRs are embedded in a suitable protein framework such that they can bind to CD3.

[0075] Regarding item b), two different sets of CDRs are disclosed because the counts of CDRs may vary from one counting method to the other (eg, Kabat, Chothia, or MacCallum).

[0076] According to one embodiment of the invention, the second binding domain that binds to CD3 comprises: a) the variable domain of an antibody selected from the group consisting of OKT3 and BlinCD3 b) Heavy / Light Chain Variable Domains (VD) HC VD (SEQ ID NO: 1 or 16) and LC VD (SEQ ID NO: 8 or 20) c) the heavy / light chain variable domains (VD) of b), except the HCVD has ≥80% sequence identity to SEQ ID NO: 1 or 16, respectively; and / or LCDVD has a sequence identity of ≥ 80% to SEQ ID NO: 8 or 20, respectively d) the heavy / light chain variable domains (VD) of b), provided that at least one of the HCVD or LCVD has no more than 10 amino acid substitutions compared to the respective SEQ ID NO: Including, The bispecific antibody is still capable of binding to CD3.

[0077] According to one embodiment, the framework is a human VH / VL framework. According to one embodiment, at least one amino acid substitution is a conservative amino acid substitution.

[0078] According to one embodiment of the bispecific antibody, the second binding domain that binds to CD3 comprises: a) comprising an scFv-like sequence as set out in any one of SEQ ID NOs: 15 or 24; or b) comprises an amino acid sequence having ≧80% sequence identity to any one of SEQ ID NOs: 15 or 24.

[0079] According to one embodiment of the bispecific antibody, the first binding domain that binds fluorescein or conjugated fluorescein comprises: a) comprising an scFv-like sequence as set out in any one of SEQ ID NOs: 35 or 44; or b) comprises an amino acid sequence having ≧80% sequence identity to any one of SEQ ID NOs: 35 or 44.

[0080] According to one embodiment of the bispecific antibody, the first binding domain that binds fluorescein or conjugated fluorescein comprises: a) comprising at least one sequence selected from SEQ ID NOs: 48 to 50; or b) containing an amino acid sequence having ≥ 80% sequence identity to SEQ ID NOs: 48 to 50; Optionally, the C-terminal cysteine ​​residue and / or the His tag are removed or left intact.

[0081] According to one embodiment of the bispecific antibody, the agent has, compared to the bispecific antibody according to the above description: ≥ 50% target binding affinity as measured by SPR to CD3 and / or ≥ 50% target binding affinity as measured by SPR to fluorescein or conjugated fluorescein It has at least one of the following.

[0082] According to another aspect of the invention there is provided a bispecific antibody which competes with the bispecific antibody described above for binding to fluorescein or conjugated fluorescein and / or CD3, respectively.

[0083] According to another aspect of the present invention, a nucleic acid is provided that codes for the binding agent according to the above description. Such a nucleic acid can be an mRNA, a cDNA, a DNA, or a genomic DNA, which may contain non-coding stretches (introns). Due to the degeneracy of the genetic code, a large number of different nucleic acid sequences can be included for the same binding agent. However, based on the sequence information of the binding agent, a person skilled in the art can determine or identify one or more such nucleic acid molecules.

[0084] According to another aspect of the present invention, a) a bispecific antibody according to the above description and b) a labeled binding agent that specifically binds to the target antigen A combination comprising: A labelled binding agent is provided in combination, in which the first binding domain of the bispecific antibody according to the above description is labelled with an organic fluorophore which specifically detects and / or binds to it.

[0085] According to another aspect of the present invention, a) a bispecific antibody according to the above description and b) a labeled binding agent that specifically binds to the target antigen A dosing regimen comprising administering to a patient sequentially or simultaneously: A regimen is provided in which the labelled binding agent is labelled with an organic fluorophore which is specifically detected and / or bound by the first binding domain of the bispecific antibody according to the above description.

[0086] The labeled binding agent is also referred to herein as an "adapter molecule" or "adapter," and the labeled binding agent provides target specificity for the combination. Technically, the labeled binding agent can be selected from a large library of binding agents, including antibodies, that are specific for every possible target antigen.

[0087] In one embodiment, the target antigen is an antigen presented by tumor cells of a hematological tumor.In one embodiment, the target antigen is an antigen presented by tumor cells of a solid tumor.

[0088] The advantages of using organic fluorophores in adapter molecules are manifold: a) For many organic fluorophores there are commercially available antibodies with high affinity that can be used in bispecific antibodies. b) organic fluorophores are relatively small (fluorescein, for example, has a molecular weight of about 390 Da); c) Organic fluorescers are non-immunogenic and non-toxic (fluorescein is used, for example, in eye drops). d) Binding agents labeled with organic fluorophores can be monitored, for example, in preclinical trials. e) There is a proven chemistry to stably conjugate organic fluorophores to binding agents such as antibodies, avoiding dissociation of the organic fluorophores from the binding agent in the bloodstream (which can occur with peptide labels).

[0089] With regard to the bispecific antibodies in said combination, the same embodiments apply as disclosed above for the bispecific antibodies alone.

[0090] According to one embodiment of the combination or administration regimen according to the above description, the bispecific antibody and the labeled binding agent are administered individually or first mixed together and then administered.

[0091] According to one embodiment of the combination or dosing regimen described above, the organic fluorophore in the labeled binding agent is fluorescein.

[0092] As used herein, the terms "fluorescein," "fluorescein isothiocyanate," and "FITC" are used interchangeably.

[0093] According to one embodiment of the combination or dosing regimen described above, the labeled binding agent is site-specifically labeled with an organic fluorophore.

[0094] In this way it is possible to ensure that: a) A constant stoichiometric ratio between binding agent and organic fluorophore can be achieved, which is crucial for reproducible dosing of the bispecific antibody and labeled binding agent combination. b) the label does not interfere with the binding of the labeled bispecific agent to its target.

[0095] In one embodiment, site-specific labeling with fluorescein ("FITCylation") can be achieved by the use of (i) fluorescein-5-maleimide (5-MF) and (ii) a proteinaceous binder, such as an antibody, fragment, or derivative thereof, having a C-terminal cysteine.

[0096] Such an approach is disclosed in the Experimental Section herein for enablement purposes and has been described, for example, in Pellegrino et al. 2020, the contents of which are incorporated herein by reference.

[0097] According to one embodiment of the combination or dosing regimen according to the above description, the labeled binding agent comprises an antibody or a target-binding fragment or derivative thereof.

[0098] In a preferred embodiment, the tagged binding agent is at least one selected from the group consisting of: ·antibody, Antibody-based binding proteins Modified antibody formats that retain target binding ability; antibody derivatives or fragments that retain target binding ability, and / or Antibody mimetics.

[0099] As used herein, the term "binding agent" is meant to define an entity, agent, or molecule that has affinity for a particular target, such as a receptor, cell surface protein, cytokine, or the like. Such binding agents may optionally block or attenuate agonist-mediated responses or inhibit receptor-agonist interactions. Most importantly, however, binding agents may act as shuttles to deliver payloads to specific sites defined by the target recognized by said binding agent. Thus, for example, but not limited to, a binding agent that targets a receptor will deliver its payload to a site characterized by an abundance of said receptor.

[0100] Binding agents include, but are not limited to, antibodies, antibody fragments, antibody-based binding proteins, antibody mimetics, receptors, soluble decoy receptors, scaffold proteins with affinity for a particular target, and ligands for receptors.

[0101] An "antibody", also referred to interchangeably as "immunoglobulin" (Ig), generally comprises four polypeptide chains, two heavy (H) chains and two light (L) chains, and is thus a multimeric protein, or includes its equivalent Ig homologues (e.g., camelid antibodies, which comprise only heavy chains, single domain antibodies (dAbs) which can be derived from either the heavy or light chains), and includes full-length functional mutants, variants, or derivatives thereof (including but not limited to murine, chimeric, humanized, and fully human antibodies which retain the essential epitope binding properties of Ig molecules, and includes bispecific, bispecific, multispecific, and dual variable domain immunoglobulins. Immunoglobulin molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2) and allotype.

[0102] An "antibody-based binding protein," as used herein, refers to a protein that contains at least one antibody-derived V in association with other non-immunoglobulin or non-antibody derived components. H , V L , or C H This may refer to any protein that contains an immunoglobulin domain. Such antibody-based proteins include (i) Immunoglobulin C H F of a binding protein containing all or part of a domain and a receptor or receptor component c fusion protein, (ii) V H and / or V L (iii) a binding protein in which the domain is linked to an alternative molecular scaffold; or (iv) immunoglobulin V. H and / or V L and / or C H These include, but are not limited to, molecules in which domains are combined and / or assembled in a manner not normally found in naturally occurring antibodies or antibody fragments.

[0103] "Antibody derivative or fragment," as used herein, refers to a molecule that comprises at least one polypeptide chain derived from an antibody that is not full-length, and that has (i) a variable light (V L ), variable weight (V H ), Stationary Light (C L ), and steady state weight 1 (C H (I) a Fab fragment, which is a monovalent fragment consisting of a domain; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V H and C H F consisting of I domain ab (Fa) the heavy chain portion of the fragment; (iv) the V of a single arm of an antibody L and V H A variable fragment consisting of the domain (F v (v) domain antibody (dAb) fragments consisting of a single variable domain; (vi) isolated complementarity determining regions (CDRs); (vii) single chain F v Fragment (scFv); (viii)V H and V L (ix) diabodies, which are bivalent, bispecific antibodies in which the domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen-binding sites; (ix) a pair of tandem Fv segments (V H -C H 1-V H -C H (xI) other non-full-length portions of immunoglobulin heavy and / or light chains or mutants, variants, or derivatives thereof, alone or in any combination.

[0104] The term "modified antibody formats", as used herein, includes antibody-drug conjugates, polyalkylene oxide modified scFvs, monobodies, diabodies, camelid antibodies, domain antibodies, bi- or tri-specific antibodies, IgA, i.e. two IgG constructs linked by a J chain and secretory component, shark antibodies, New World primate framework + non-New World primate CDRs, an IgG4 antibody with the hinge region removed, an IgG with two additional binding sites engineered into the CH3 domain, antibodies with modified Fc regions to improve affinity for Fc gamma receptors, dimerization constructs containing CH3+VL+VH, and the like.

[0105] The term "antibody mimetics" as used herein refers to proteins that do not belong to the immunoglobulin family and also non-proteins such as aptamers or synthetic polymers. Some types have an antibody-like beta-sheet structure. Potential advantages of "antibody mimetics" or "alternative scaffolds" over antibodies are better solubility, higher tissue penetration, higher thermal and enzymatic stability, and relatively low production costs.

[0106] Several antibody mimetics can be provided in large libraries that offer specific binding candidates for every possible target. As with antibodies, target-specific antibody mimetics can be developed by the use of high-throughput screening (HTS) techniques and by established display technologies such as phage display, bacterial display, yeast or mammalian display. Antibody mimetics that have been developed so far include, for example, ankyrin repeat proteins (called DARPins), C-type lectins, A-domain proteins of S. aureus, transferrin, lipocalins, the 10th fibronectin type III domain, Kunitz domain protease inhibitors, ubiquitin-derived binders (called affilins), gamma crystallin-derived binders, cysteine ​​knots or knottins, thioredoxin A scaffold-based binders, nucleic acid aptamers, artificial antibodies produced by molecular imprinting of polymers, peptide libraries derived from bacterial genomes, SH-3 domains, stradobodies, "A domains" of membrane receptors stabilized by disulfide bonds and Ca2+, CTLA4-based compounds, and Fyn SH3.

[0107] In one embodiment, the labeled binding agent is an scfV fragment, e.g., having one of the following structures: VL1-VH1 VH1-VL1

[0108] In one embodiment, the labeled binding agent is a diabody, e.g., having one of the following structures: VL1-VH1-VL1-VH1 VH1-VL1-VH1-VL1

[0109] According to an embodiment of the combination or dosing regimen described above, the labeled binding agent specifically binds to a target antigen selected from the group consisting of CD117 and CAIX.

[0110] Preferably, CD117 / c-KIT is human CD117 / c-KIT, for example as disclosed under UniProt identifier P10721. The obesity / stem cell growth factor receptor (SCFR), also known as the proto-oncogene c-KIT or tyrosine-protein kinase KIT or CD117, is a receptor tyrosine kinase protein encoded by the KIT gene in humans. Multiple transcript variants encoding different isoforms have been found for this gene. KIT was first described in 1987 by the German biochemist Axel Ullrich as the cellular homologue of the feline sarcoma virus oncogene v-kit.

[0111] CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells and other cell types. Modified forms of this receptor may be associated with some types of cancer. CD117 is a receptor tyrosine kinase type III that binds to stem cell factor, also known as "hematopoietic stem cell factor" or "c-kit ligand," a substance that triggers the growth of certain types of cells. When this receptor binds to stem cell factor (SCF), a dimer is formed that activates the activity of its intrinsic tyrosine kinase, which then phosphorylates and activates signaling molecules that transmit signals in the cell. After activation, the receptor is ubiquitinated to mark the receptor for delivery to lysosomes and eventual destruction. Signaling through CD117 plays a role in cell survival, proliferation, and differentiation. For example, CD117 signaling is required for melanocyte survival, which is also involved in hematopoiesis and gametogenesis.

[0112] Like other members of the receptor tyrosine kinase III family, CD117 consists of an extracellular domain, a transmembrane domain, a juxtamembrane domain, and an intracellular tyrosine kinase domain. The extracellular domain is composed of five immunoglobulin-like domains, and the protein kinase domain is interrupted by a hydrophilic insert of about 80 amino acids. The ligand stem cell factor binds via the second and third immunoglobulin domains.

[0113] Cluster of differentiation (CD) molecules, as recognized by a specific set of antibodies, are markers on the cell surface and are used to identify cell type, stage of differentiation and activity of cells. CD117 is an important cell surface marker used to identify certain types of hematopoietic (blood) progenitor cells in bone marrow. In particular, hematopoietic stem cells (HSCs), multipotent progenitors (MPPs) and common myeloid progenitors (CMPs) express high levels of CD117. Common lymphoid progenitors (CLPs) express low surface levels of CD117. CD117 also identifies the earliest thymocyte progenitors in thymus-early T lineage progenitors (ETPs / DN1), and DN2 thymocytes express high levels of c-Kit. CD117 is also a marker for mouse prostate stem cells. In addition, mast cells, melanocytes in the skin, and interstitial cells of Cajal in the gastrointestinal tract express CD117. In humans, expression of c-kit on helper-like innate lymphoid cells (ILCs), which lack expression of CRTH2 (CD294), is used to mark the ILC3 population.

[0114] In humans, CD117 / c-Kit is present primarily in hematopoietic cells and plays a critical role in the early stages of hematopoiesis, but is also expressed in AML blasts.

[0115] Preferably, CAIX (also called carbonic anhydrase IX, CA09) is human CAIX (UniProt identifier: A0A087WYS4. Carbonic anhydrase IX (CAIX) is a suitable target for various anti-cancer strategies. CAIX is a cell surface protein present in human tumors but not in corresponding normal tissues. CAIX expression is induced by hypoxia and correlates with cancer prognosis in many tumor types. Furthermore, CAIX has been functionally implicated in cancer progression as a pro-survival factor that protects cancer cells from hypoxia and acidosis via its ability to regulate pH and cell adhesion.

[0116] The cancer-associated distribution of CAIX allows targeting of cancer cells by antibodies binding to its extracellular domain, whereas the functional engagement of CAIX opens the possibility of attacking cancer cells by blocking their adaptation to physiological stresses via inhibition of CAIX enzymatic activity. The latter strategy has received considerable attention recently, and extensive efforts have been made to generate CAIX-selective inhibitor derivatives with anticancer effects.

[0117] Targeting CAIX-expressing cells by immunotherapy has reached clinical trials and is approaching application in the treatment of renal cell carcinoma patients. Nevertheless, the development and characterization of new CAIX-specific antibodies is still ongoing.

[0118] According to one embodiment of the combination or dosing regimen described above, the labeled binding agent that binds to CD117 comprises a) comprising a set of heavy chain / light chain complementarity determining regions (CDRs) contained in the heavy chain / light variable region sequence pair of anti-CD117 antibody 79D, as set forth in SEQ ID NOs: 52 and 59; b) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 53 or 56) HC CDR2 (SEQ ID NO: 54 or 57) HC CDR3 (SEQ ID NO: 55 or 58) LC CDR1 (SEQ ID NO: 60 or 63) LC CDR2 (SEQ ID NO: 61 or 64), and LC CDR3 (SEQ ID NO: 62 or 65) c) comprising the heavy / light chain complementarity determining regions (CDRs) of b) or c), with the proviso that at least one of the CDRs has no more than 3 amino acid substitutions compared to SEQ ID NOs: 53-55, 60-62, 56-57, or 63-65, respectively; and / or d) comprising the heavy / light chain complementarity determining regions (CDRs) of b), with the proviso that at least one of the CDRs has a sequence identity of ≧66% to SEQ ID NOs: 53-55, 60-62, 56-57, or 63-65, respectively; The CDRs are embedded in a suitable protein framework such that they are capable of binding to CD117.

[0119] The anti-CD117 antibody 79D is disclosed in X, the contents of which are incorporated herein by reference.

[0120] Regarding item b), two different sets of CDRs are disclosed because the counts of CDRs may vary from one counting method to the other (eg, Kabat, Chothia, or MacCallum).

[0121] According to one embodiment of the combination or dosing regimen described above, the labeled binding agent that binds to CD117 comprises a) Variable domains of antibody 79D b) Heavy / Light Chain Variable Domains (VD) HC VD (SEQ ID NO: 52) and LC VD (SEQ ID NO:59) c) the heavy / light chain variable domains (VD) of b), except HCVD has ≥80% sequence identity to each SEQ ID NO:52; and / or LCDVD has ≥80% sequence identity to each of SEQ ID NO:59 Subject to the condition d) the heavy / light chain variable domains (VD) of b), provided that at least one of the HCVD or LCVD has no more than 10 amino acid substitutions compared to the respective SEQ ID NO: Including, The labeled binding agent is still capable of binding to CD117.

[0122] With regard to the various alternatives disclosed, the same embodiments apply as for the bispecific antibodies disclosed elsewhere herein, particularly with regard to the range of homology and conservative amino acid substitutions.

[0123] With regard to the various alternatives disclosed, the same embodiments apply as for the bispecific antibodies disclosed elsewhere herein, particularly with regard to the range of homology and conservative amino acid substitutions.

[0124] According to another aspect of the present invention there is provided a pharmaceutical composition comprising a combination according to the above description, the pharmaceutical composition optionally comprising one or more pharma- ceutically acceptable excipients.

[0125] According to one aspect of the present invention, the combination or dosing regimen or pharmaceutical composition as described above is administered to treat or prevent neoplastic disease. Have been diagnosed with - Are suffering from what they are doing, or There is a risk of Provided for use (in the manufacture of a medicament) in the treatment of a human or animal subject or for the prevention of such conditions.

[0126] The above description with the term "in the manufacture of a medicament" in brackets is meant to provide disclosure both in so-called Swiss style claim writing (the brackets would be considered deleted) and in claim writing allowable under, for example, EPC 2000 (the content between the brackets would be considered deleted).

[0127] In some embodiments thereof, the bispecific antibody (a) and the labeled binding agent (b) are a) before b) or b) before a) or ·at the same time is administered.

[0128] In preferred embodiments, such neoplastic diseases are solid tumors or hematological tumors, and the examples provided herein have provided effective support for both applications.

[0129] According to one aspect of the present invention there is provided a method for treating or preventing a neoplastic disease comprising administering to a subject in need thereof an effective amount of a combination or pharmaceutical composition as described above or subjecting said patient to a dosing regimen as described above.

[0130] In some embodiments thereof, the bispecific antibody (a) and the labeled binding agent (b) are a) before b) or b) before a) or ·at the same time is administered.

[0131] According to one aspect of the present invention, a therapeutic kit of parts is provided comprising: a) Combinations or pharmaceutical compositions according to the above description b) a device for administering the combination or composition, and c) Optionally, instructions for use. EXAMPLES

[0132] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustrations and descriptions are to be considered as illustrative or exemplary, and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprises" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be interpreted as limiting the scope.

[0133] All amino acid sequences disclosed herein are presented from N-terminus to C-terminus, and all nucleic acid sequences disclosed herein are presented from 5' to 3'.

[0134] Materials and Methods cell line NALM-6 (ACC128, DSMZ) and MOLM-14 (ACC777, DSMZ) cells were cultured in RPMI-1640 supplemented with 20% FBS and 1% penicillin / streptomycin. Cell lines were expanded and stored as frozen aliquots in liquid nitrogen. Cells were grown according to the supplier's protocol and kept in culture for up to 2 weeks. Authentication of cell lines further included checking viability after freezing, growth characteristics, morphology, testing for mycoplasma contamination, isoenzyme assays, and sterility testing, and was performed by the cell bank before dispatch. MOLM-14 cells were lentivirally transduced to uniformly express CD117 on their surface and then selected (Myburgh et al. 2020).

[0135] T cell isolation and purification Buffy coats from healthy donors were obtained from the Zurich blood donation service (Blutspende Zuerich, Zuerich, Switzerland). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Ficoll Paque Plus (GE Healthcare) according to the manufacturer's instructions. T cells were then negatively isolated with EasySep™ beads (Human T cell isolation kit, STEMCELL Technologies). Purified T cell samples were resuspended in 90% FCS supplemented with 10% DMSO and stored frozen at -80°C and then in liquid nitrogen.

[0136] Cloning of UniTE(4m5.3 / BlinCD3), UniTE(E2 / BlinCD3), and UniTE(FluA / BlinCD3) The sequences of the humanized version of the anti-human CD3 antibody OKT3, called Blin CD3 (SEQ ID NOs: 1-14), and the sequences of the anti-fluorescein mouse 4m5.3 (SEQ ID NOs: 36-34) or human E2 antibody (27.35) (disclosed in Boder et al. (2000) and Vaughan et al. (1996)) or anticalin™ FluA (disclosed in Vopel et al. (2005) (SEQ ID NO: 47)) were genetically assembled by PCR in the following format to obtain UniTE 4m5.3 / BlinCD3, FluA / BlinCD3, and E2 / BlinCD3 (the bottom row shows the SEQ ID NOs):

[0137] [Table 3]

[0138] It was then cloned into the mammalian expression vector pcDNA3.1(+) (Invitrogen).

[0139] Cloning of the antibody that will be used as the adapter Two different labeled binding agents ("adapters") were generated, both antibodies. One adapter binds to CD117, which, as described elsewhere herein, is a target expressed on blood and hematopoietic cells. CD117 is therefore representative of hematological cancers.

[0140] The other adaptor binds to CAIX (also called carbonic anhydrase IX, CA09), which, as described elsewhere herein, is a cell surface protein that is present in human tumors but not in corresponding normal tissues, and is therefore representative of solid tumors.

[0141] The anti-CD117 antibody was described in Reshetnyak et al. (2013) and has the clone name antibody 79D. The anti-CD117 antibody comprises the heavy / light chain variable domains HC VD (SEQ ID NO: 52) and LC VD (SEQ ID NO: 59).

[0142] The anti-CAIX antibody was described in Cazzamalli et al (2018) and has the clone name antibody XE114. The anti-CAIX antibody comprises the heavy / light chain variable domains HC VD (SEQ ID NO: 68) and LC VD (SEQ ID NO: 72).

[0143] Anti-CD117 antibody The sequences of the variable regions of the light and heavy chains of the anti-CD117 antibody were cloned into pcDNA3.1(+) in diabody (Db) format (VL-linker 5aa-VH) with a C-terminal polyhistidine tag. An additional C-terminal cysteine ​​was added for site-selective conjugation.

[0144] Anti-CAIX antibody Anti-CAIX antibodies were cloned and prepared as IgG as described in Cazzamalli et al. (2018), the contents of which are incorporated herein by reference.

[0145] Antibody expression Antibodies were produced by transient gene expression using polyethylenimine in CHO-S cells (Invitrogen) according to standard protocols. 8 Proteins were purified by Ni-NTA agarose resin (ThermoFisher) and analyzed using SDS-PAGE and size-exclusion chromatography (Superdex 200 Increase, 10 / 300, GE Healthcare).

[0146] Conjugation of anti-CD117 and anti-CAIX to fluorescein Antibody solutions (1 mg / ml) in PBS were reduced with 50 eq. DTT (1,4-dithio-DL-threitol) in PBS. After reduction, the protein solution was washed three times with PBS using Vivaspin centrifugal columns (Sartorius) to dilute the DTT. Fluorescein-5-maleimide (Thermo Scientific) was dissolved in anhydrous DMSO to obtain a final concentration of 10% (v / v) when added to the reduced protein. The antibody was treated with 50 eq. fluorescein per cysteine, and homogenous conjugates with a single modification in the light chain were observed by LC-MS after 1 h. The conjugated protein was purified by PD-10 desalting columns (GE Healthcare) and eluted in acetate buffer, pH 5. Aliquots were flash frozen in liquid nitrogen and stored at -80°C until further use.

[0147] CD19 + UniTE-mediated T cell killing of tumor cells in vitro Healthy donor T cells were thawed one day before the assay and cultured overnight in modified RPMI (T cell medium) supplemented with 1x Glutamax (Gibco), 10% FBS, and 1% penicillin / streptomycin. T cells and CD19+ B acute lymphoblastic leukemia (B-ALL) NALM-6 cells were co-incubated at a 10:1 ratio (E:T=10:1) in the presence of 10 nM of commercially available FITC-anti-human CD19 mouse IgG1 (Biolegend; clone HIB19). UniTE(4m5.3 / BlinCD3) and UniTE(FluA / BlinCD3) were compared with the clinically approved (anti-CD19 / BlinCD3) bispecific T cell conjugate (BiTE™) Blinatumomab (Blycinto™) at the same concentrations (100 nM and 10 nM). Specific lysis of target cells was analyzed by flow cytometry using an LSR II Fortessa cell analyzer (BD Biosciences) and quantified using the following formula: (1-number of live target cells / number of live target cells without antibody)*100.

[0148] CD117+ UniTE-mediated T cell killing of tumor cells in vitro T cells were thawed 1 day prior to the assay and cultured overnight in T cell medium. T cells and acute myeloid leukemia (AML) MOLM-14 cells (transduced to express CD117 on the surface) were co-cultured in T cell medium (E:T=1:1) in the presence of 10 nM of anti-CD117 diabody site-specifically conjugated to fluorescein. Killing by UniTE (4m5.3 / BlinCD3) was compared to the anti-CD117 / BlinCD3 bispecific T cell conjugate at equimolar concentrations (100 nM and 1 nM) and to direct anti-CD117 CAR-T cells. 1 , both of which carry scFv from the same clone of the cross-linked Db. Specific lysis of target cells was analyzed as previously described.

[0149] CAIX + UniTEA-Mediated T Cell Killing of Tumor Cells In Vitro T cells were thawed one day before the assay and cultured overnight in T cell medium. + Renal cell carcinoma SK-RC-52 cells were harvested one day before the assay and the membranes were stained using the PKH26 Red Fluorescent Cell Linker Kit for General Membrane (Sigma-Aldrich) according to the manufacturer's instructions. Stained SK-RC-52 cells were seeded in 96-well plates and incubated overnight. The next day, SK-RC-52 cells and T cells were co-cultured in T cell medium (E:T=10:1) in the presence of 10 nM site-specifically fluoresceinated anti-CAIX IgG and various concentrations of 4m5.3 / BlinCD3, FluA / BlinCD3, and E2 / BlinCD3 UniTEAs (1, 10, and 100 nM). After 24 hours, specific lysis of target cells was analyzed as described previously. Background killing after co-culture of T cells and tumor cells alone or by adding only 10 nM adapter molecule was also analyzed.

[0150] In vivo stability of fluorescein molecules on antibodies Eight-week-old C57BL / 6J mice were injected iv with 50ug of anti-mouse CD19-FITC antibody (Biolegend; clone 1D3). Mouse B cells were analyzed by flow cytometry from peripheral blood at 4 hours, 1 day, 4 days, and 8 days after injection. Peripheral blood was lysed with RBC lysis reagent (Biolegend) for 10 minutes and washed 1x with PBS (Invitrogen). Each sample was split into two and stained with anti-mouse CD20 (Biolegend; clone SA271G2) alone or with anti-mouse CD20 (Biolegend; clone SA271G2) and anti-mouse CD19-FITC (Biolegend; clone 1D3). Samples were stained in FACS buffer (PBS with 2% FBS and 2mM EDTA) and washed twice after incubation before FACS analysis.

[0151] statistical analysis Data are reported as mean ± standard deviation (SD) unless otherwise stated. Statistical analysis was performed with GraphPad Prism 8.0 software. FlowJo software (v10.0.7, FlowJo) was used for data analysis and presentation.

[0152] result Production of various UniTEA formats Aiming to redirect, activate and trigger T cell-mediated killing of potentially any target cell via fluorescein-tagged target-binding molecules [Figure 1A], we generated three versions of UniTE that all bind human CD3ε and fluorescein [Figure 1B]. The three versions are named as follows; 4m5.3 / BlinCD3, E2 / BlinCD3, and FluA / BlinCD3. Each UniTEA version differs in its fluorescein-binding moiety. 4m5.3 and E2 are scFv-formatted antibody fragments against fluorescein of mouse and human origin, respectively. FluA is a lipocalin fluorescein-binding molecule derived from Pieris brassicae.

[0153] UniTE redirects T cells to eliminate CD19- and CD117-expressing leukemia cell lines in vitro. + and CD117 + They were functionally tested in vitro by determining their potential to redirect T cell killing against human cell lines [Figure 2]. +The NALM6 cell line was used to compare T cell-mediated killing induced by 4m5.3 / BlinCD3 and FluA / BlinCD3 UniTEA formats compared to clinical grade BiTE™ Blinatumomab (anti-CD19 / BlinCD3) [Figure 2A]. We confirmed that T cell activation and killing was induced only in the presence of both UniTEA and the adaptor molecule, as target cells and T cells co-incubated with UniTEA or the bridge molecule alone led to minimal (~5%) background lysis of target cells [Figure 2B]. Using effector-to-target ratios as high as 10:1, it is expected that some spontaneous activation of T cells against target cells will be seen in the absence of any T cell activator. After 24 hours, Blinatumomab led to >50% to 77% T cell-mediated target cell lysis [Figure 2C]. Addition of 4m5.3 / BlinCD3 or FluA / BlinCD3 UniTEA was able to achieve similar cell lysis as equimolar concentrations of blinatumomab when 10 nM of commercially available anti-CD19 IgG-FITC (Biolegend; clone HIB1) was used as a bridging molecule [Figure 2C]. + The MOLM14 cell line was used to compare T cell-mediated killing induced by 4m5.3 / BlinCD3 and FluA / BlinCD3 UniTEA formats compared to conventional CAR-T cells and BiTEs™ directed against CD117 [Figure 2D]. CD117-targeted CAR T cells and BiTEs™ were able to achieve >95% target cell lysis after 24 hours [Figure 2E]. Similar target cell lysis occurred at the same time point when 100 nM 4m5.3 / BlinCD3 UniTEA was added in combination with 10 nM fluorescein-labeled anti-CD117 Db. Addition of 1 nM 4m5.3 / BlinCD3 UniTEA resulted in >95% target cell lysis after 48 hours [Figure 2E].

[0154] Persistence of fluoresceinated antibodies in vivo Stability of fluorescein in blood over time is necessary for efficient UniTE-fluorescein bridge complex formation and T cell redirection. To determine the effectiveness of structurally intact fluorescein on the bridge molecule, we administered 50ug of commercially available anti-mouse CD19 (mCD19) IgG-FITC and analyzed peripheral blood at various time points over an 8-day period [Figure 3A]. At each time point analyzed, mCD19 IgG belonging to the same mouse was detected. + Cells showed no difference in mean fluorescence intensity (MFI) when stained with or without additional mCD19-FITC [Figure 3B]. + The MFI of the population decreased after 24 hours but remained stable until day 8 [Figure 3B]. mCD19 stained with and without additional CD19-FITC labeling + / mCD20 + There was no difference in the proportion of B cells (mCD19 compared to baseline counts after 4 hours + / mCD20 + (expressed as a percentage of cells) [Figure 3C]. Additional CD19-FITC labeling for flow cytometry was performed to determine CD19 + All available CD19 epitopes are still occluded by the CD19-FITC antibody injected on day 0, as this did not increase the percentage of cells or MFI, and FITC remains stable in vivo over the course of 5 days. Only on day 8 did additional mCD19-FITC labeling increase the CD19 + This resulted in an increase in the MFI of the population, indicating that some of the injected mCD19-FITC antibody had been cleared from the body by day 8. Peripheral blood mCD19 at the indicated time points with and without additional mCD19-FITC labeling. + / mCD20 + Representative FACS plots showing B cell populations are reported in Figure 3D. These populations were pre-gated on live single cells.

[0155] References Myburgh,R.et al.Anti-human CD117 CAR T-cells efficiently eliminate healthy and malignant CD117-expressing hematopoietic cells.Leukemia (2020). Horn,L.A.et al.CD3xPDL1 bi-specific T cell engager(BiTE)simultaneously activates T cells and NKT cells,kills PDL1+ tumor cells,and extends the survival of tumor-bearing humanized mice.Oncotarget 8,57964(2017). Boder,E.T.,Midelfort,K.S.& Wittrup,K.D.Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.Proc.Natl.Acad.Sci.97,10701-10705(2000). Vaughan,T.J.et al.Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library.Nat.Biotechnol.14,309-314(1996). Vopel,S.,Muehlbach,H.& Skerra,A.Rational engineering of a fluorescein-binding anticalin for improved ligand affinity.Biol.Chem.386,1097-1104(2005). Reshetnyak,A.V.et al.Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region.Proc.Natl.Acad.Sci.U.S.A.110,17832-17837(2013). Casi,G.,Huguenin-Dezot,N.,Zuberbuehler,K.,Scheuermann,J.& Neri,D.Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery.J.Am.Chem.Soc.134,5887-5892(2012). Rajendra,Y.,Kiseljak,D.,Baldi,L.,Hacker,D.L.& Wurm,F.M.A simple high-yielding process for transient gene expression in CHO cells.J.Biotechnol.153,22-26(2011). Plueckthun and Skerra,Meth.Enzymol.,178:497-515(1989)and in Day,E.D.,Advanced Immunochemistry,Second Ed.,Wiley-Liss,Inc.,New York,N.Y.(1990) Huston et al.,Cell Biophysics,22:189-224(1993) Pellegrino et al,Bioconjugate Chem.2020,31,1775-1783 Eylenstein R,Weinfurtner D,Haertle S,Strohner R,Boettcher J,Augustin M,Ostendorp R,Steidl S.Molecular basis of in vitro affinity maturation and functional evolution of a neutralizing anti-human GM-CSF antibody.MAbs.2016;8(1):176-86 Harding FA,Stickler MM,Razo J,DuBridge RB.The immunogenicity of humanized and fully human antibodies:residual immunogenicity resides in the CDR regions.MAbs.2010 May-Jun;2(3):256-65. Schanzer,J.M.,et al.,A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor(EGFR)and insulin-like growth factor receptor type I(IGF-1R)demonstrating unique molecular properties.J Biol Chem,2014.289(27): p.18693-706. Goebeler,M.E.and R.Bargou,Blinatumomab: a CD19 / CD3 bispecific T cell engager(BiTE)with unique anti-tumor efficacy.Leuk Lymphoma,2016.57(5): p.1021-32. Neri,D.,et al.,High-affinity antigen binding by chelating recombinant antibodies(CRAbs).J Mol Biol,1995.246(3): p.367-73. Nagorsen,D.,et al.,Blinatumomab:a historical perspective.Pharmacol Ther,2012.136(3):p.334-42 Cazzamalli S.et al,Chemically Defined Antibody- and Small Molecule-Drug Conjugates for in vivo Tumor Targeting Applications: A Comparative Analysis.J Am Chem Soc.2018 Feb 7;140(5):1617-1621 Spiess C et al.,Alternative molecular formats and therapeutic applications for bispecific antibodies.Mol Immunol.2015 Oct;67(2 Pt A):95-106. Wang,A systematic approach for analysis and characterization of mispairing in bispecific antibodies with asymmetric architecture.MAbs.2018 Nov-Dec;10(8):1226-1235 Ceuppens JL et al,CD3 modulation inhibits pokeweed mitogen-induced T-cell help for immunoglobulin production.Cell Immunol.1986 Oct 1;102(1):144-51. Borst J et al.,BMA031,a monoclonal antibody suited to identify the T-cell receptor alpha beta / CD3 complex on viable human T lymphocytes in normal and disease states. Hum Immunol.1990 Nov;29(3):175-88. Li B et al.,Construction and characterization of a humanized anti-human CD3 monoclonal antibody 12F6 with effective immunoregulation functions.Immunology.2005 Dec;116(4):487-98.

[0156] array The following sequences form part of the disclosure of this application. An electronic sequence listing conforming to WIPO ST25 is also submitted with this application. For the avoidance of doubt, in case of discrepancies between the sequences in the following table and the electronic sequence listing, the sequences in the table shall be considered accurate.

[0157] [Table 4] JPEG2024504354000007.jpg243170JPEG2024504354000008.jpg241170JPEG2024504354000009.jpg244170JPEG2024504354000010.jpg104170

Claims

1. A bispecific antibody comprising at least a first binding domain and a second binding domain, wherein the first binding domain binds to an organic fluorophore and the second binding domain binds to CD3.

2. The bispecific antibody of claim 1 , wherein the first binding domain binds to fluorescein or conjugated fluorescein.

3. The first binding domain that binds fluorescein or conjugated fluorescein is a) comprising a set of heavy / light chain complementarity determining regions (CDRs) contained in the heavy / light variable region sequence pair of one of anti-fluorescein antibodies E2 or 4m5.3, as set forth in SEQ ID NOs: 27 and 31, or SEQ ID NOs: 36 and 40, respectively; b) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO:28) HC CDR2 (SEQ ID NO:29) HC CDR3 (SEQ ID NO: 30) LC CDR1 (SEQ ID NO: 32) LC CDR2 (SEQ ID NO: 33), and LC CDR3 (SEQ ID NO: 34), or c) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 37) HC CDR2 (SEQ ID NO: 38) HC CDR3 (SEQ ID NO: 39) LC CDR1 (SEQ ID NO: 41) LC CDR2 (SEQ ID NO: 42), and LC CDR3 (SEQ ID NO: 43) 3. The bispecific antibody of claim 1 or 2, wherein the CDRs are embedded in a human VH / VL framework such that they can bind to fluorescein or conjugated fluorescein.

4. The first binding domain that binds fluorescein or conjugated fluorescein is a) the variable domain of an antibody selected from the group consisting of E2 or 4m5.3 b) Heavy / Light Chain Variable Domains (VD) -HC VD (SEQ ID NO: 27 or 36) and LC VD (SEQ ID NO: 31 or 40) c) the heavy / light chain variable domains (VDs) of b), provided that - the HCVD has ≧80% sequence identity to SEQ ID NO: 27 or 36, respectively; and / or - with the proviso that said LCVD has ≧80% sequence identity to SEQ ID NO: 31 or 40, respectively; or d) the heavy / light chain variable domains (VD) of b), with the proviso that at least one of the HCVD or LCVD has no more than 10 amino acid substitutions compared to the respective SEQ ID NO. Including, The bispecific antibody of any one of claims 1 to 3, wherein the bispecific antibody is still capable of binding to fluorescein or conjugated fluorescein.

5. The second binding domain that binds to CD3 comprises: a) a set of heavy / light chain complementarity determining regions (CDRs) contained in the heavy / light variable region sequence pair of one of the anti-CD3 antibodies BlinCD3 and OKT3, as set forth in SEQ ID NOs: 1 and 8 or SEQ ID NOs: 16 and 20, respectively; b) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 2 or 5) HC CDR2 (SEQ ID NO: 3 or 6) HC CDR3 (SEQ ID NO: 4 or 7) LC CDR1 (SEQ ID NO: 9 or 12) LC CDR2 (SEQ ID NO: 10 or 13), and LC CDR3 (SEQ ID NO: 11 or 14), or c) a set of heavy / light chain complementarity determining regions (CDRs) comprising the following sequences: HC CDR1 (SEQ ID NO: 17) HC CDR2 (SEQ ID NO: 18) HC CDR3 (SEQ ID NO: 19) LC CDR1 (SEQ ID NO:21) LC CDR2 (SEQ ID NO: 22), and LC CDR3 (SEQ ID NO: 23) The bispecific antibody according to any one of claims 1 to 4, wherein the CDRs are embedded in a human VH / VL framework such that they are capable of binding to CD3.

6. The second binding domain that binds to CD3 comprises: a) the variable domain of an antibody selected from the group consisting of OKT3 and BlinCD3 b) Heavy / Light Chain Variable Domains (VD) HC VD (SEQ ID NO: 1 or 16) and LC VD (SEQ ID NO: 8 or 20) c) the heavy / light chain variable domains (VDs) of b), provided that - the HCVD has ≥ 80% sequence identity to SEQ ID NO: 1 or 16, respectively; and / or - with the proviso that said LCVD has ≧80% sequence identity to SEQ ID NO: 8 or 20, respectively; or d) the heavy / light chain variable domains (VD) of b), with the proviso that at least one of the HCVD or LCVD has no more than 10 amino acid substitutions compared to the respective SEQ ID NO. Including, The bispecific antibody of any one of claims 1 to 5, wherein the bispecific antibody is still capable of binding to CD3.

7. The second binding domain that binds to CD3 comprises: a) comprising an scFv-like sequence as set forth in any one of SEQ ID NOs: 15 or 24, or b) comprises an amino acid sequence having ≥ 80% sequence identity to any one of SEQ ID NOs: 15 or 24 A bispecific antibody according to any one of claims 1 to 6.

8. The first binding domain that binds fluorescein or conjugated fluorescein is c) comprising an scFv-like sequence as set forth in any one of SEQ ID NOs: 35 or 44; or d) comprises an amino acid sequence having ≧80% sequence identity to any one of SEQ ID NOs: 35 or 44 A bispecific antibody according to any one of claims 1 to 7.

9. a) comprising at least one of the sequences selected from SEQ ID NOs: 48 to 50; or b) comprises an amino acid sequence having ≧80% sequence identity to SEQ ID NOs: 48-50; 9. The bispecific antibody of claim 1, wherein optionally the C-terminal cysteine ​​residue and / or the His tag are removed or left intact.

10. a) a bispecific antibody according to any one of claims 1 to 9, and b) a labeled binding agent that specifically binds to the target antigen Including, A combination, wherein the labelled binding agent is labelled with an organic fluorophore to which the first binding domain of the bispecific antibody according to any one of claims 1 to 9 specifically binds.

11. The combination of claim 10, wherein the organic fluorophore in the tag binder is fluorescein.

12. 12. The combination of claim 10 or 11, wherein the labeled binding agent comprises an antibody or a target-binding fragment or derivative thereof.

13. A pharmaceutical composition comprising a combination according to any one of claims 10 to 12 and optionally one or more pharma- ceutically acceptable excipients.

14. Developing a neoplastic disease - Have been diagnosed with - Are suffering from what they are doing, or There is a risk of A combination or pharmaceutical composition according to any one of claims 10 to 13 for use in the treatment of a human or animal subject or for the prevention of such a condition.

15. a) and b) are - a) before b) or b) before a) or ·at the same time The combination for use according to claim 14, wherein the

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