Improved Anti-CD3 antibodies

Abstract

The present invention relates to novel anti-CD3 antibodies with improved properties. These antibodies can be used on their own, but also can be "split" for use with a pair of recombinant binding molecules. Such molecules conditionally form active effector domains upon co-localization on tumor cells. Specifically, affinity optimizations between the two counterparts provide advantageous properties, such as increased therapeutic index and reduced target-mediated drug-disposition.

Description

[0001] IMPROVED ANTI-CD3 ANTIBODIES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel anti-CD3 antibodies with improved properties. These antibodies can be used on their own, but also can be "split" for use with a pair of recombinant binding molecules. Such molecules conditionally form active effector domains upon co-localization on tumor cells. Specifically, affinity optimizations between the two counterparts provide advantageous properties, such as increased therapeutic index and reduced target-mediated drug-disposition.

[0004] BACKGROUND OF THE INVENTION

[0005] T cell engaging antibodies are a class of bispecific antibodies designed to redirect polyclonal T cells towards tumor cells. By simultaneous binding of tumor associated antigens (TAA) on the target cell and a receptor, e.g. CD3, on the T cell (effector cell) a crosslinking is achieved that leads to T cell activation, proliferation and cytokine release which results in T cell dependent cytotoxicity towards the target cell.

[0006] The majority of clinical approved T cell engagers show promising efficacy and durable responses in hematologic malignancies such as Blinatumumab (CD19 x CD3) in acute lymphoblastic leukemia (ALL) or Teclistamab (BCMA x CD3) in multiple myeloma. Nevertheless, even in solid cancer promising response rates with manageable safety profiles could be achieved with Tebentafusp (gplOO xCD3) for uveal melanoma or Tarlatamab (DLL3 x CD3) in Small Cell Lung Cancer (SCLC).

[0007] Despite these achievements, many clinical trial and investigational therapies in solid cancer had to be discontinued due to several liabilities that conventional T cell engagers face.

[0008] • The basal expression of TAA in healthy tissue led to on-target off tumor toxicities, that were dose-limiting in many cases.

[0009] • The systemic, TAA-independent, activation of peripheral T cells led to strong cytokine release syndrome despite prophylactic corticosteroid treatments.

[0010] • The binding of T cell engagers to peripheral T cells led to target-mediated drug disposition (TMDD) and poor biodistribution.

[0011] In particular, the lack of tumor-specific targets limits the application of this potent therapeutic class. The high therapeutic potential combined with the safety liabilities of T cell engagers led to the development of T cell engagers with tumor restricted activity. Examples include proteolytic activation of binding entities (aTAA or aCD3) by proteases in the tumor microenvironment, approaches that rely on avidity-mediated selectivity gain, other environmental factors that enhance binding (TAA or CD3) such as acidic milieu or ATP levels, or on-cell assembly approaches that mediate specificity gain by logic AND-gates.

[0012] The latter was pioneered by Stuhler et al.. In this split concept, the active CD3 binder is split into two halves, namely the VH and VL, which alone can neither bind nor activate T cells. The single domains (VH or VL) were fused to two separate targeting moieties (TAA1, TAA2). These precursor molecules are able to bind tumor cells, however, T cell binding remains inactive. Upon co-localization on dual positive (TAA1+TAA2) tumor cells the split CD3 binder gets restored, and the T cell activation induced. By this logic AN D-gate, the T cell killing capacity is meant to be unleashed specifically at the tumor with limited activity in healthy (single TAA positive) tissue. Thereby, the therapeutic window and hence the amount of safely druggable targets, especially in solid cancer, is increased.

[0013] The split VH and VL domains, however, have a certain affinity towards each other. While this is of advantage to induce the CD3 binder restoration at the tumor, it poses the risk of pre-mature assembly in the periphery at higher concentrations, e.g. after co-administration of the two counterparts. This would again limit the therapeutic index of the technology.

[0014] Various anti-CD3 binders with different affinities to CD3 are know in the art (Scientific reports, vol. 11, no. 1, 13 July 2021, DOI: 10.1038 / s41598-021-93842-0; Eur J Immunol, vol. 32, no. 11, 16 October 2002, pages 3102-3107; WO 2017 / 053856 Al; Molecular Cancer Therapeutics, vol. 17, no. 4, 16 January 2018, pages 776-785; JCI Insight, vol. 5, no. 7, 9 April 2020, DOI: 10.1172 / jci. insight.133757; WO 92 / 22653 Al). These binders are however not used or described in a split system, but are used as regular anti-CD3 binding domains).

[0015] Of importance, for use of the anti-CD3 binders of the present disclosure, not only the affinity of the anti-CD3 binding domain to the CD3 target molecule is relevant, but also the affinity between the anti- CD3 variable heavy chain and the anti-CD3 variable light chain. Only this will allow the formation of a functional anti-CD3 binding domain (and hence full binding molecule composed of a pair of molecules, wherein each of said half-molecules of the pair comprises either an anti-CD3 variable heavy chain or an anti-CD3 variable light chain), as required for the formation of a functional molecule under physiological conditions, in particular upon co-localization on cells.

[0016] Thus, there remains a need for optimizing the binding kinetics of the split domains towards each other to reduce target-independent complementation while retaining on-tumor complementation of the active CD3 binder. Additionally, optimized binding kinetics allow the simultaneous administration of the two precursor molecules with reduced complex formation and rapid dissociation upon dilution after administration, e.g. co-vialed.

[0017] The present invention provides novel anti-CD3 antibodies and antibody fragments which can, for example, be utilized as split domains, but also as classical antibodies.

[0018] SUMMARY OF THE INVENTION

[0019] The present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment has a reduced binding affinity to CD3 while retaining its efficacy as T cell engager on tumor cells compared to an antibody or antibody fragment comprising

[0020] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0021] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0022] In certain embodiments, said antibody or antibody fragment according to claim 1, wherein said antibody or antibody fragment comprises

[0023] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0024] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein said antibody or antibody fragment comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q of the heavy chain variable region (VH).

[0025] In certain embodiments, said antibody or antibody fragment comprises

[0026] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0027] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8. In certain embodiments, said antibody or antibody fragment comprises

[0028] (a) a heavy chain variable region ( VH ) comprising the amino acid sequence of SEQ ID NO: 11, and

[0029] (b) a light chain variable region ( VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0030] The present disclosure also relates to a pair of polypeptides comprising

[0031] (a) a first polypeptide (Pl) comprising

[0032] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0033] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0034] (b) a second polypeptide (P2) comprising

[0035] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0036] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0037] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0038] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0039] The present disclosure also relates to a pair of molecules comprising

[0040] (a) a first molecule (Ml) comprising

[0041] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0042] (b) a second molecule (M2) comprising

[0043] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0044] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0045] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0046] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0047] In certain embodiments, said heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

[0048] In certain embodiments, said heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and said light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0049] In certain embodiments, said heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and said light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, said first targeting moiety (Tl) and said second targeting moiety (T2) are an antibody or an antibody fragment.

[0050] In certain embodiments, said first tumor associated antigen (TAA1) and said second tumor associated antigen (TAA2) are selected from HER2, EpCAM, CD19, MSLN, DLL3, FLT3, EGFR, CD33, CD38, MUC17, CLDN18.2, CDH3, CD70, BCMA ,PSMA, CEACAM5, HER3, STEAP-1, TROP-2, NECTIN-4, B7-H3, B7-H4, MUC16, FOLR1, CD200, CLDN6, GPRC5D, EGFRviii, MUC1 and 5T4.

[0051] In certain embodiments, the present disclosure relates to a nucleic acid molecule encoding aforementioned antibody or antibody fragment according or aforementioned pair of polypeptides.

[0052] In certain embodiments, the present disclosure relates to a nucleic acid molecule encoding aforementioned antibody or antibody fragment according or aforementioned pair of molecules.

[0053] In certain embodiments, the present disclosure relates to a vector comprising aforementioned nucleic acid molecules.

[0054] In certain embodiments, the present disclosure relates to a host cell comprising aforementioned nucleic acid molecule or aforementioned vector.

[0055] In certain embodiments, the present disclosure relates to aforementioned antibodies or antibody fragments or aforementioned pair of polypeptides for use in medicine.

[0056] In certain embodiments, the present disclosure relates to aforementioned antibodies or antibody fragments or aforementioned pair of molecules for use in medicine.

[0057] In certain embodiments, said used in medicine is the treatment of a cancer, an inflammatory disease or an autoimmune diseases.

[0058] DESCRIPTION OF THE FIGURES

[0059] Figure 1 shows a schematic illustration of exemplary BiTAC precursor molecules according to the invention. Figure 1A: Two BiTAC precursors, each comprising one half of a split anti CD3 Fv, a targeting moiety for the binding of TAA and an Fc backbone for half-life extension. Circles in the Fc region indicate modifications to promote Fc heterodimerization following the knob-into-hole principle. Figure IB: Upon co-localization on dual TAA positive target cells and high local concentration of the two precursors, a CD3 binder restoration leads to the crosslinking and activation of polyclonal T cells. This artificial immune synapse formation triggers T cell-dependent cytotoxicity towards the target cell. Figure 2 shows the BiTAC mode of action with no activity of single BiTAC precursors and activity gain with the combination thereof in cellular assays. Figure 2A: Tumor cell killing of the breast cancer cell line SK-BR-3 (HER2, EPCAM positive) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2 respectively) either alone or in combination. As a positive control Runimotamab (HER2-TCE) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours. The used CD3 clone is derived from the UCHT-1. Figure 2B: Binding of BiTAC precursor molecules to CD3+ T cells (EpCAM and HER2 negative) was assessed by flow cytometry. Precursor molecules were incubated separately with pan T cells at the indicated concentrations overnight at 37°C under cell culture conditions. As a positive control Runimotamab was included. Bound molecules were stained with a fluorescence- labelled secondary antibody and analyzed by flow cytometry.

[0060] Figure 3 shows a schematic illustration of the target-independent assembly of BiTAC precursors in the periphery. The intrinsic affinity of the split anti CD3Fv parts (VH and VL) can lead to off-target formation of active trispecific antibodies without target cell encountering. In contrast, the desired targetdependent assembly on tumor cells is illustrated underneath. Figure created with Biorender.com.

[0061] Figure 4 shows the effect of target-independent assembly in cellular assays. Figure 4A: Tumor cell killing of the breast cancer cell line SK-BR-3 (HER2, EpCAM positive) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2 or a nonbinding isotype-like control) in combination. As a positive control Runimotamab (HER2-TCE) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours. The used CD3 clone is derived from the UCHT-1. Figure 4B: Binding of BiTAC precursor molecules to CD3+ T cells (EpCAM and HER2 negative) was assessed by flow cytometry. Precursor molecules were incubated in combination with pan T cells at the indicated concentrations overnight at 37°C under cell culture conditions. As a positive control Runimotamab was included. Bound molecules were stained with a fluorescence-labelled secondary antibody and analyzed by flow cytometry. Target-independent complementation is indicated by binding of the complex by the restored CD3 binder to CD3.

[0062] Figure 5 schematically illustrates exemplary BiTAC precursor molecules according to the present invention. Figure 5A: Two BiTAC precursors each comprising one half of a split anti CD3 Fv, a targeting moiety for the binding of TAA and an Fc backbone for half-life extension. Circles in the Fc region indicate modifications to promote Fc heterodimerization following the knob-into-hole principle. Stars indicate point mutations, either in the VH / VL interface region (conserved backbone) or in the CDRs. Thereby, both the affinity towards the split VH and VL and the affinity to CD3 can be modulated. Figure 5B: Upon co-localization on dual TAA positive target cells and high local concentration of the two precursors, a CD3 binder restoration leads to the crosslinking and activation of polyclonal T cells. This artificial immune synapse formation triggers ? cell-dependent cytotoxicity towards the target cell even with altered affinities between the split anti CD3Fv VH and VL or towards the effector target CD3 itself.

[0063] Figure 6 shows a cell-based assessment to measure target-independent complementation. Figure 6A: A schematic illustration of the assay setup. HER2-positive SK-BR-3 cells were incubated with a HER2- targeting BiTAC precursor (CD3-VL) which includes a kappa light chain in the Fab for TAA targeting. A second, non-targeted (isotype) BiTAC precursor (CD3-VH WT or mutant) including a lambda light chain is added to the reaction and incubated for lh at 37°C. Using labelled secondary anti-kappa or anti lambda antibodies, the amount of bound molecules with the respective constant LC on the cell could be measured. Figure 6B: The readout was done in a flow cytometry setting, the signal depicted as histograms.

[0064] Figure 7 shows the effect of target-independent assembly and the impact of CD3 binder engineering in cellular assays. Figure 7A: Tumor cell killing of the breast cancer cell line SK-BR-3 (HER2, EpCAM positive) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2 or a non-binding isotype-like control) in combination. As a positive control Runimotamab (HER2-TCE) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours. The used CD3 clone is derived from the UCHT-1 clone. Figure 7B: Binding of BiTAC precursor molecules to CD3+ T cells (EpCAM and HER2 negative) was assessed by flow cytometry. Precursor molecules were incubated in combination with pan T cells at the indicated concentrations overnight at 37°C under cell culture conditions. As a positive control Runimotamab was included. Bound molecules were stained with a fluorescence-labelled secondary antibody and analyzed by flow cytometry. Target-independent complementation is indicated by binding of the complex by the restored CD3 binder to CD3. The used CD3 clone is derived from the UCHT-1 clone.

[0065] Figure 8 shows the tumor cell killing of the different breast cancer cell lines displaying different expression levels of EpCAM and HER2. Figure 8A: SK-BR-3 (EpCAM medium, HER2high), Figure 8B: JIMT-l(EpCAM high, HER2medium), Figure 8C: BT-474 (EpCAM high, HER2high), Figure 8D: a healthy tissue surrogate cell line MDA-MB-231 (EpCAMIow, HER2low) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2) in combination. As a positive control Runimotamab (HER2-TCE, open symbols) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours. The used CD3 clone is derived from the UCHT-1 clone, either as WT (circle symbols) or variant. (star symbols). Figure 9 shows IL-2 secretion in a co-culture of healthy tissue surrogate cell line MDA-MB-231 (EpCAMIow, HER2low) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2) in combination. As a positive control Runimotamab (HER2-TCE, open symbols) was included. IL-2 quantification from the supernatant after 48 hours. The used CD3 clone is derived from the UCHT-1 clone, either as WT (circle symbols) or variant(star symbols).

[0066] Figure 10: Cytotoxicity on a healthy tissue surrogate cell line MDA-MB-231 (EpCAMIow, HER2low) with pan T cells from a healthy human donor when treated with the BiTAC precursor molecules (targeting EpCAM or HER2) in combination. Complementary constructs were either diluted separately and combined on the assay plate (star symbols) reflecting a separate administration or incubated at 8.5 pM in PBS overnight at 4°C in advance mimicking a co-vialed approach (asterisk symbols). As a positive control Runimotamab (HER2-TCE, open symbols) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours.

[0067] Figure 11 shows the characteristics of T cell engagers directed against HER2 carrying either a scFv consisting of the UCHT-l-WT or UCHT-1 variants. Panel A: Tumor cell killing of the breast cancer cell line SK-BR-3 (HER2 positive) with pan T cells from a healthy human donor when treated with T cell engagers directed against HER2 carrying either a scFv consisting of the UCHT-l-WT (VBP-007) or UCHT- 1 variant G96E (VBP-008). As a positive control Runimotamab (HER2-TCE) was included. Tumor cell killing was measured by quantification of Lactate dehydrogenase (LDH) in the supernatant after 48 hours. PanelB: Binding of T cell engagers directed against HER2 carrying either a scFv consisting of the UCHT-l-WT (VBP-007) or UCHT-1 variant G96E (VBP-008) to CD3+ T cells (HER2 negative) was assessed by flow cytometry. T cell engagers were incubated in combination with pan T cells at the indicated concentrations overnight at 37°C under cell culture conditions. As a positive control Runimotamab was included. Bound molecules were stained with a fluorescence-labelled secondary antibody and analyzed by flow cytometry.

[0068] Figure 12: Binding of BiTAC precursors and T cell engagers according to the present invention to SK- BR-3 cells (EpCAM medium, HER2high, CD38 negative, SLAMF7 negative). Antibody derivatives were incubated in combination with SK-BR-3 cells at the indicated concentrations for 1 hour on ice. Bound molecules were stained with a fluorescence-labelled secondary antibody and analyzed by flow cytometry.

[0069] Figure 13: Surface Plasmon Resonance (SPR) sensograms for the interaction of BiTAC precursor carrying the UCHT-1 VL (VBP-001) with immobilized BiTAC precursor carrying the UCHT-1 VH (WT; VBP- 002) or immobilized BiTAC precursor carrying the UCHT-1 VH (G96E; VBP-003). (a) Binding of serially diluted BiTAC precursor carrying the UCHT-1 VL (VBP-001) to the BiTAC precursor carrying the UCHT-1 VH (WT; VBP-002)( KD=0.89nM). (b) Binding of serially diluted BiTAC precursor carrying the UCHT-1 VL (VBP-001) to BiTAC precursor carrying the UCHT-1 VH (G96E; VBP-003)(KD=8.12 nM).

[0070] Figure 14: Figure 14A shows the results of a TDCC assay obtained with SK-BR3 cells (HER2 high, EpCAM high; expression levels correspond to tumor tissue). Figure 14B shows the results of a TDCC assay obtained with MDA-MB-231 cell line (HER2 low, EpCAM low; expression levels correspond to tumor healthy tissue (Cancer Res (2023) 83 (7_Supplement): 6337)).

[0071] Figure 15: Figure 15A shows the results of a TDCC assay with SK-BR3 cells (HER2 high, EpCAM high; representative for tumor tissue), Figure 15B the results of a TDCC assay with MDA-MB-231 cells (HER2 low, EpCAM low; representative for healthy tissue).

[0072] DEFINITIONS

[0073] The term "targeting moiety" as used herein refers to a polypeptide-based binding molecule that specifically binds to an antigen such as a tumor associated antigen. The term includes antibodies, antibody fragments, antibody-based and non-antibody based binding molecules.

[0074] The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FR's arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., Igd , lgG2, lgG3, lgG4, IgAl and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0075] The term "antibody fragment", as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879- 5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1 -VH-CH1 ) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U.S. Pat. No. 5,641 ,870).

[0076] The structures and locations of immunoglobulin variable domains, e.g., CDRs, may be defined using well known numbering schemes, e.g., the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g. Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991 ), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901 -917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997).

[0077] A "human antibody" or "human antibody fragment", as used herein, is an antibody and antibody fragment having variable regions in which both the framework and CDR regions are from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or from transgenic mice (e.g. Xenomouse, OmniMouse, Harbour Mouse, ATX-Gx Mouse, Trianni Mouse) provided the respective system yield in antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region also is derived from such sequences. Human origin includes, e.g., human germline sequences, or mutated versions of human germline sequences or antibody containing consensus framework sequences derived from human framework sequences analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86).

[0078] A "humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule, which has constant antibody regions derived from sequences of human origin and the variable antibody regions or parts thereof or only the CDRs are derived from another species. For example, a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domain are from a non-human origin, while one or more frameworks of the variable domain are of human origin and the constant domain (if any) is of human origin.

[0079] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule, which has constant antibody regions derived from, or corresponding to, sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from, or corresponding to, sequences found in humans, and the variable antibody regions (e.g. VH, VL, CDR or FR regions) are derived from sequences found in a non-human animal, e.g. a mouse, rat, rabbit or hamster.

[0080] The term "isolated antibody" or "isolated antibody fragment" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in some aspects, antibodies provided are isolated antibodies, which have been separated from antibodies with a different specificity. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. An isolated antibody that specifically binds to an epitope, isoform or variant of a target may, however, have crossreactivity to other related antigens, e.g., from other species (e.g., species homologs). The term "recombinant antibody" or "recombinant antibody fragment", as used herein, includes all antibodies or antibody fragment that are prepared, expressed, created or segregated by means not existing in nature. For example, antibodies isolated from a host cell transformed to express the antibody, antibodies selected and isolated from a recombinant, combinatorial human antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of all or a portion of a human immunoglobulin gene, sequences to other DNA sequences or antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo. A recombinant antibody may be a monoclonal antibody.

[0081] The term "BiTAC", as used herein, refers to a intermolecular complex comprising two pairs of polypeptides or molecules, wherein each of the polypeptides or molecule pairs comprises a targeting moiety that specifically binds to a first tumor associated antigen and a heavy chain variable region or a light chain variable region of a binding domain that specifically binds to a third antigen, such as CD3. If the first polypeptide or molecule pair comprises a heavy chain variable region, then the second polypeptide or molecule pair comprises a light chain variable region of the binding domain that specifically binds to the third antigen. If the first polypeptide or molecule pair comprises a light chain variable region, then the second polypeptide or molecule pair comprises a heavy chain variable region of the binding domain that specifically binds to the third antigen. The heavy chain variable region of the binding domain that specifically binds to the third antigen is capable form a functional binding domain with the light chain variable region of the binding domain that specifically binds to the third antigen.

[0082] The terms "BiTAC precursor", "BiTAC precursor molecule" or "hemibody" as used herein refers to one of said two pairs of polypeptides or molecules that form a BiTAC.

[0083] As used herein, an antibody "binds specifically to", "specifically binds to", is "specific to / for" or "specifically recognizes" an antigen, such as human CD45, if such antibody is able to discriminate between such antigen and one or more reference antigen(s), since binding specificity is not an absolute, but a relative property. For example, a standard ELISA assay or standard flow cytometry assay can be carried out. The scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide) or by binding of a secondary antibody labeled with PE or another dye or marker. The reaction in certain wells is scored by the optical density (OD), for example, at 450 nm or by mean or median fluorescence intensity (MFI) in flow cytometry. Typical background (=negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. Background and positive reaction MFI are highly dependent on instrument settings. The difference positive / negative can be more than 10-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of about three to five unrelated antigens, such as milk powder, BSA, transferrin or the like. For flow cytometry various antigen-negative cells can be used. An antibody that specifically binds to an antigen may however have cross-reactivity to the respective orthologous antigen from other species (e.g., species homologs). In certain embodiments such cross-reactivity to an orthologous antigen is even preferred.

[0084] The term "internalizing" as used herein, refers to an antibody or antigen-binding fragment that is capable of being taken through the cell's lipid bilayer membrane to an internal compartment (i.e., "internalized") upon binding to the cell, such as into a degradative compartment in the cell. Antibodies and antibody fragments may for example be taken into the cell via receptor-mediated endocytosis.

[0085] As used herein, the term "affinity" refers to the strength of interaction between the polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts through weak non-covalent forces with its target at numerous sites; the more interactions, the stronger the affinity.

[0086] The terms "knob into hole", "knob-into-hole" and "KiH" as used herein, refer to engineering the CH3 domain of antibody Fc region to create either a "knob" or a "hole" in each heavy chain to promote heterodimerization. Generally, a "knob" is created by replacing T366 with a bulky residue W on one heavy chain, and the corresponding "hole" is made by triple mutations of T366S, L368A and Y407V on the other heavy chain. The knob-into-hole structure may comprise other substitutions, as familiar in the art.

[0087] The term "antigen" as used herein refers to an protein or polypeptide comprising an epitope which is recognized by an antigen binding domain, such as an antibody or antibody fragment. The present invention relates to a pair of two antibodies or antibody fragments which bind to a first and a second antigen. The first and the second antigen recognized by these antibodies are preferably different antigens. This is important in the context of the avoidance of off-target toxicities. Nevertheless, if the target antigen is highly specific for cancerous tissues or cells, the first and the second antigen recognized by the two antibodies may principally also be the same antigen.

[0088] The terms "tumor associated antigen", "TAA" and "tumor antigen" are used interchangeably herein and refer to antigens that are presented on tumor cells. These antigens can be presented on the cell surface with an extracellular part, which is often combined with a transmembrane and cytoplasmic part of the molecule. These antigens can sometimes be presented only by tumor cells and never by the normal ones. Tumor antigens can be exclusively expressed on tumor cells or might represent a tumor specific mutation compared to normal cells. In this case, they are called tumorspecific antigens. More common are antigens that are presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens can be overexpressed compared to normal cells or are accessible for antibody binding in tumor cells due to the less compact structure of the tumor tissue compared to normal tissue. Non-limiting examples of tumor antigens include HER2, EpCAM, CD19, MSLN, DLL3, FLT3, EGFR, CD33, CD38, MUC17, CLDN18.2, CDH3, CD70, BCMA,PSMA, CEACAM5, HER3, STEAP-1, TROP-2, NECTIN-4, B7-H3, B7-H4, MUC16, FOLR1, CD200, CLDN6, GPRC5D, EGFRviii, MUC1 and 5T4.

[0089] The term "epitope" includes any proteinaceous region which is specifically recognized by an antibody or antibody fragment thereof or otherwise interacts with a molecule. Generally, epitopes are of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by one of skill in the art, practically anything to which an antibody can specifically bind could be an epitope.

[0090] The terms "ADC" and "antibody drug conjugate" as used herein refer to a conjugate that includes (a) an antibody or an antibody fragment and (b) another moiety that is linked to said antibody or antibody fragment. The other moiety is typically referred to as "payload". Said payload is classically a toxic payload, i.e. a compounds that kills or otherwise harms the cell after internalization of the ADC. The payload may however also be of other nature. For example, and as used within the spirit of the present invention, said antibody may also be a prodrug or an activator. In the present invention one antibody or antibody fragment is conjugated to a prodrug and the second antibody or antibody fragment is conjugated to an activator.

[0091] "Compositions" or of the present disclosure may be used for therapeutic or prophylactic applications. The present disclosure, therefore, includes a pharmaceutical composition containing a pair of antibodies or antibody fragments as disclosed herein and a pharmaceutically acceptable carrier or excipient therefore. In a related aspect, the present disclosure provides a method for treating inflammatory diseases, autoimmune diseases, hematologic malignancies and potentially other diseases. Such method contains the steps of administering to a subject in need thereof an effective amount of the pharmaceutical composition that contains the antibodies or antibody fragments as described herein.

[0092] The present disclosure provides therapeutic methods comprising the administration of a therapeutically effective amount of an antibody or antibody fragment as disclosed herein to a subject in need of such treatment. A "therapeutically effective amount" or "effective amount", as used herein, refers to the amount of the antibodies necessary to elicit the desired biological response. In accordance with the subject disclosure, the therapeutic effective amount is the amount of antibodies necessary to treat and / or prevent a disease.

[0093] "Administered" or "administration" includes but is not limited to delivery of a drug by an injectable form, such as, for example, an intravenous, intramuscular, intradermal or subcutaneous route or mucosal route, for example, as a nasal spray or aerosol for inhalation or as an ingestible solution, capsule or tablet. Preferably, the administration is by an injectable form.

[0094] As used herein, "treatment", "treat" or "treating" and the like refers to clinical intervention in an attempt to alter the natural course of a disease in the subject being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments according to the preset disclosure are used to delay development of a disease or to slow the progression of a disease.

[0095] "Preventing" or "prevention" refers to a reduction in risk of acquiring or developing a disease (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to a disease-causing agent, or predisposed to the disease in advance of disease onset). "Prevention" also refers to methods which aim to prevent the onset of a disease or its symptoms or which delay the onset of a disease or its symptoms.

[0096] "Subject" or "species" or as used in this context refers to any mammal, including rodents, such as mouse or rat, and primates, such as cynomolgus monkey (Macaca fascicularis), Marmoset monkey (Callithrix jacchus), rhesus monkey (Macaca mulatta) or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0097] The "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an IgG heavy chain might vary slightly, the human IgG heavy chain Fc region is usually defined to extend from Cys226, or from Pro230, to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Various Fc modifications are commonly used. For a review see for example Antibodies (2020) 9: 64. Silencing functions include (numbering according EU index) the LALA (L234A / L235A), the PA-LALA (L234A / L235A / P329A) and the PG-LALA (L234A / L235A / P329G) mutations, as well as the AEASS mutations (L234A / L235E / G237A / A330S / P331S). A preferred FC modification is PA-LALA. The mutation may also be a mutation which leads to a reduced binding to FcRn, thereby decreasing the in vivo halflife of the antibody. Such mutations include I253A, H310A, H435A and H435Q. Alternatively, the mutation may also a mutation which leads to an increased binding to FcRn, thereby increasing the in vivo half-life of the antibody. Such mutations include T250Q / M428L, M252Y / S254T / T256E (YTE), H433K / N434F and M252Y / S254T / T256E / H433K / N434F.

[0098] The term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Non-limiting examples of antibody effector functions include Cl q binding and complement dependent cytotoxicity (CDC); Fc receptor binding and antibodydependent cell-mediated cytotoxicity (ADCC) and / or antibody- dependent cellular phagocytosis (ADCP); down regulation of cell surface receptors (e.g. B cell receptor); and direct cell activation or direct cell inhibition.

[0099] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g. NK cells, neutrophils, and macrophages) enable these cytotoxic effector cells to bind specifically to an antigenbearing target cell and subsequently kill the target cell with cytotoxins. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes / macrophages express FcyRI, FcyRII, and FcyRIII. "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (Clq) to antibodies (of the appropriate subclass) of the present disclosure, which are bound to their cognate antigen.

[0100] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism of elimination of antibody-coated target cells by internalization by phagocytic cells, such as macrophages or dendritic cells.

[0101] As used herein, the term "antibody-drug conjugate" or "ADC" refers to an antibody or an antibody fragment that is chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent.

[0102] As used herein, the terms "TDCC" and "T cell dependent cytotoxicity" refers to a mechanism of elimination of target cells by cytotoxic T cells. This effect may be induced by the coating of target cells with engineered molecules known as T cell engaging antibodies which enhance the ability of T cells to selectively recognize and kill target cells, such as cancer cells. More precisely, T cell engagers activate T cells by simultaneously binding to receptors e.g. CD3 and tumor associated antigens on tumor cells which elicits the effector function of T cells and result in the lysis of the cross-linked target cells.

[0103] The terms "engineered" or "modified" as used herein includes manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical coupling of peptides or some combination of these techniques). Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety and the like.

[0104] "Variant" as used herein refers to a polypeptide that differs from a reference polypeptide by one or more modifications for example amino acid substitutions, insertions or deletions. Variant polypeptides typically retain most of the properties of the reference polypeptide, e.g. binding to the target antigen, but introduce a novel, additional feature or property, e.g. the variant polypeptide has a higher affinity to the target antigen compared to the reference polypeptide or the variant polypeptide is a humanized version of the reference polypeptide.

[0105] The term "amino acid mutation" as used herein is meant to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitution, deletion, insertion, and modification can be made as long as the final construct possesses the desired characteristics, e.g., reduced binding to an Fc receptor. Amino acid sequence deletions and insertions include N-and / or C- terminal deletions and insertions of amino acid residues. Particular amino acid mutations are amino acid substitutions. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid residue by methods other than genetic engineering, such as chemical modification, may also be useful. Various designations may be used herein to indicate the same amino acid mutation. For example, a substitution of glycine at position 237 of the antibody Fc region to alanine can be indicated as 237A, G237, G237A, or Gly237Ala.

[0106] The terms "UCHT" refers to an anti-CD3 antibody known in the art and having the amino acid sequence shown in the following Table. This antibody is also referred to as, "UCHT wildtype", "UCHT- wt", "UCHT-WT".

[0107] The terms "SP34" refers to an anti-CD3 antibody known in the art and having the amino acid sequence shown in the following Table. This antibody is also referred to as "SP34-wt".

[0108] DETAILED DESCRIPTION OF THE INVENTION Introduction to the molecules used in the present invention

[0109] Various binding domains were exemplary used in the present invention.

[0110] The antigen-binding domain specific for HER2 comprises the following amino acid sequence:

[0111] The antigen-binding domain specific for SLAMF7 comprises the following amino acid sequence:

[0112] The antigen-binding domain specific for CD38 comprises the following amino acid sequence:

[0113] The antigen-binding domain specific for EpCAM comprises the following amino acid sequence: As positive control the HER2-T cell engager Runimotamab was utilized. The amino acid sequence of

[0114] Runimotamab is as follows:

[0115] Amino acid sequences of certain parts of the antibodies generated and utilized in the present invention include:

[0116] The full length sequences of the heavy chains of the BiTAC precursor molecules generated and utilized in the present invention are as follows: The full length sequences of the light chains of the BiTAC precursor molecules generated and utilized in the present invention are as follows:

[0117] Improved variants of UCHT

[0118] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment has a reduced binding affinity to CD3 while retaining its efficacy as T cell engager on tumor cells compared to an antibody or antibody fragment comprising

[0119] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0120] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0121] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0122] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein said antibody or antibody fragment comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q of the heavy chain variable region (VH).

[0123] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0124] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0125] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0126] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0127] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and

[0128] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0129] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0130] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and

[0131] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0132] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0133] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0134] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0135] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 81, and

[0136] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0137] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0138] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0139] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0140] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0141] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 83, and

[0142] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0143] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0144] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and

[0145] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0146] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0147] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 85, and

[0148] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0149] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises (a) a heavy chain variable region ( VH ) comprising the amino acid sequence of SEQ ID NO: 84, and

[0150] (b) a light chain variable region ( VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0151] In certain embodiments, said an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) is an antibody. In certain embodiments, said an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) is an antibody fragment. In certain embodiments, antibody fragment is a scFv. In other embodiments, antibody fragment is a Fab. In certain embodiments, said an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) is a humanized antibody. In certain embodiments, said an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) is an isolated antibody. In certain embodiments, said an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) is a recombinant antibody.

[0152] Improved variants of SP34

[0153] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment has a reduced binding affinity to CD3 while retaining its efficacy as T cell engager on tumor cells compared to an antibody or antibody fragment comprising

[0154] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 92, and

[0155] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0156] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0157] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 92, and

[0158] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95, wherein said antibody or antibody fragment comprises a substitution of an amino acid selected from V37A, L45A, H95A, G96A or G96E of the heavy chain variable region (VH). In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0159] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 92, and

[0160] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0161] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0162] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 98, and

[0163] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0164] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0165] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 99, and

[0166] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0167] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0168] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 101, and

[0169] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0170] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0171] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 100, and

[0172] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89. In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0173] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 103, and

[0174] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0175] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0176] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 102, and

[0177] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0178] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0179] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 105, and

[0180] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0181] In certain embodiments, the present disclosure relates to an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0182] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 102, and

[0183] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0184] Use of the improved variants as BiTACs

[0185] The novel anti-CD3 binding domains also may be used as BiTACs. BiTACs are pairs of binding molecules comprising parts of an effector domain, such binding molecules being capable of restoring a functional effector domain when accumulating on target cells upon binding of distinct tumor associated antigens on the surface of a target cell. One specific effector domain is CD3. Each binding molecule of said pairs of binding molecules may consist of one or more polypeptides.

[0186] It was surprisingly found that altering the binding affinities of the two complementing split domains of the anti-CD3 binding moiety reduces the propensity of target-independent restoration of the effector domain while maintaining the efficacy on target cells in a target-dependent manner.

[0187] The introduction of specific point mutations into the binding domain of the CD3 antibody UCHT-1 led to an altered binding kinetics which leads to reduced complementation and / or increased dissociation at low concentrations (e.g. in the periphery), but retains sufficiently association competent at high concentrations upon binding to the target antigens.

[0188] Therefore, in certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0189] (a) a first polypeptide (Pl) comprising

[0190] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0191] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0192] (b) a second polypeptide (P2) comprising

[0193] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0194] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0195] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO:

[0196] 4 and a HCDR3 of SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0197] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0198] (a) a first molecule (Ml) comprising

[0199] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0200] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0201] (b) a second molecule (M2) comprising

[0202] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0203] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0204] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO:

[0205] 4 and a HCDR3 of SEQ ID NO: 5, and

[0206] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0207] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0208] (a) a first polypeptide (Pl) comprising

[0209] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0210] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0211] (b) a second polypeptide (P2) comprising (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0212] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain comprises

[0213] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0214] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

[0215] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0216] (a) a first molecule (Ml) comprising

[0217] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0218] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0219] (b) a second molecule (M2) comprising

[0220] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0221] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain comprises (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0222] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

[0223] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0224] (a) a first polypeptide (Pl) comprising

[0225] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0226] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0227] (b) a second polypeptide (P2) comprising

[0228] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0229] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said functional anti-CD3 binding domain comprises

[0230] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0231] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

[0232] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0233] (a) a first molecule (Ml) comprising (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0234] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0235] (b) a second molecule (M2) comprising

[0236] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0237] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said functional anti-CD3 binding domain comprises

[0238] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0239] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

[0240] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0241] (a) a first polypeptide (Pl) comprising

[0242] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0243] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0244] (b) a second polypeptide (P2) comprising

[0245] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0246] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0247] In certain embodiments the present disclosure relates to a pair of molecules comprising (a) a first molecule (Ml) comprising

[0248] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0249] (ii) a heavy chain variable region ( VH ) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0250] (b) a second molecule (M2) comprising

[0251] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0252] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0253] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0254] (a) a first polypeptide (Pl) comprising

[0255] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0256] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and

[0257] (b) a second polypeptide (P2) comprising

[0258] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0259] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0260] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0261] (a) a first molecule (Ml) comprising

[0262] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0263] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and

[0264] (b) a second molecule (M2) comprising

[0265] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0266] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0267] In certain embodiments the present disclosure relates to a pair of polypeptides comprising (a) a first polypeptide (Pl) comprising

[0268] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0269] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and

[0270] (b) a second polypeptide (P2) comprising

[0271] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0272] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0273] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0274] (a) a first molecule (Ml) comprising

[0275] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0276] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and

[0277] (b) a second molecule (M2) comprising

[0278] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0279] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0280] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0281] (a) a first polypeptide (Pl) comprising

[0282] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0283] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0284] (b) a second polypeptide (P2) comprising

[0285] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0286] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0287] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0288] (a) a first molecules (Ml) comprising (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0289] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0290] (b) a second molecule (M2) comprising

[0291] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0292] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0293] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0294] (a) a first polypeptide (Pl) comprising

[0295] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0296] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0297] (b) a second polypeptide (P2) comprising

[0298] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0299] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0300] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0301] (a) a first molecule (Ml) comprising

[0302] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0303] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0304] (b) a second molecule (M2) comprising

[0305] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0306] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0307] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0308] (a) a first polypeptide (Pl) comprising

[0309] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and

[0310] (b) a second polypeptide (P2) comprising

[0311] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0312] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0313] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0314] (a) a first molecule (Ml) comprising

[0315] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0316] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and

[0317] (b) a second Molecule (M2) comprising

[0318] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0319] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0320] In certain embodiments the present disclosure relates to a pair of polypeptides comprising

[0321] (a) a first polypeptide (Pl) comprising

[0322] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0323] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 84, and

[0324] (b) a second polypeptide (P2) comprising

[0325] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0326] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0327] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0328] (a) a first molecule (Ml) comprising

[0329] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0330] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 84, and (b) a second molecule (M2) comprising

[0331] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0332] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ I D NO: 2.

[0333] In certain embodiments the pair of polypeptides disclosed herein comprise a Fc region.

[0334] In certain embodiments the pair of molecules disclosed herein comprise a Fc region.

[0335] In certain embodiments, said first tumor associated antigen (TAA1) and said second tumor associated antigen (TAA2) are selected from HER2, EpCAM, CD19, MSLN, DLL3, FLT3, EGFR, CD33, CD38, MUC17, CLDN18.2, CDH3, CD70, BCMA,PSMA, CEACAM5, HER3, STEAP-1, TROP-2, NECTIN-4, B7-H3, B7-H4, MUC16, FOLR1, CD200, CLDN6, GPRC5D, EGFRviii, MUC1 and 5T4. In certain embodiments, said first tumor associated antigen (TAA1) and said second tumor associated antigen (TAA2) are different.

[0336] In certain embodiments, said first tumor associated antigen (TAA1) or said second tumor associated antigen (TAA1) is HER2.

[0337] In certain embodiments, said targeting domain specifically binding to HER2 comprises (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ I D No. 16, a HCDR2 of SEQ ID NO: 17 and a HCDR3 of SEQ ID NO: 18, and (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 19, a LCDR2 of SEQ ID NO: 20 and a LCDR3 of SEQ ID NO: 21.

[0338] In certain embodiments, said targeting domain specifically binding to HER2 comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 14, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 15.

[0339] In certain embodiments, said first tumor associated antigen (TAA1) or said second tumor associated antigen (TAA1) is EpCAM.

[0340] In certain embodiments, said targeting domain specifically binding to EpCAM comprises (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ I D No. 46, a HCDR2 of SEQ ID NO: 47 and a HCDR3 of SEQ ID NO: 48, and (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 49, a LCDR2 of SEQ ID NO: 50 and a LCDR3 of SEQ ID NO: 51.

[0341] In certain embodiments, said targeting domain specifically binding to EpCAM comprises (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 44, and (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 45. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen is an antibody. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen is an antibody fragment. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen is a scFv. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen is a Fab. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen is a humanized antibody or antibody fragment. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen) is an isolated antibody or antibody fragment. In certain embodiments, said targeting domain specifically binding to said first tumor associated antigen) is an recombinant antibody or antibody fragment.

[0342] In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen is an antibody. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen is an antibody fragment. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen is a scFv. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen is a Fab. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen is a humanized antibody or antibody fragment. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen) is an isolated antibody or antibody fragment. In certain embodiments, said targeting domain specifically binding to said second tumor associated antigen) is an recombinant antibody or antibody fragment.

[0343] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0344] (a) a first molecule (Ml) comprising

[0345] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0346] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 92, and

[0347] (b) a second molecule (M2) comprising

[0348] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0349] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0350] In certain embodiments the present disclosure relates to a pair of molecules comprising (a) a first molecule (Ml) comprising

[0351] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0352] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 98, and

[0353] (b) a second molecule (M2) comprising

[0354] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0355] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0356] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0357] (a) a first molecule (Ml) comprising

[0358] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0359] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 99, and

[0360] (b) a second molecule (M2) comprising

[0361] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0362] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0363] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0364] (a) a first molecule (Ml) comprising

[0365] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0366] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 101, and

[0367] (b) a second molecule (M2) comprising

[0368] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0369] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0370] In certain embodiments the present disclosure relates to a pair of molecules comprising (a) a first molecule (Ml) comprising

[0371] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0372] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 100, and

[0373] (b) a second molecule (M2) comprising

[0374] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0375] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0376] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0377] (a) a first molecule (Ml) comprising

[0378] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0379] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 103, and

[0380] (b) a second molecule (M2) comprising

[0381] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0382] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0383] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0384] (a) a first molecule (Ml) comprising

[0385] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0386] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 102, and

[0387] (b) a second molecule (M2) comprising

[0388] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0389] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0390] (a) a first molecule (Ml) comprising

[0391] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0392] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 90, a HCDR2 of SEQ ID NO: 91 and a HCDR3 of SEQ ID NO: 105, and

[0393] (b) a second molecule (M2) comprising

[0394] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0395] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 93, a LCDR2 of SEQ ID NO: 94 and a LCDR3 of SEQ ID NO: 95.

[0396] In certain embodiments the present disclosure relates to a pair of molecules comprising

[0397] (a) a first molecule (Ml) comprising

[0398] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0399] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 104, and

[0400] (b) a second molecule (M2) comprising

[0401] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0402] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 89.

[0403] Nucleic acids, vectors and host cells

[0404] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibodies, the antibody fragments or the pair of polypeptides of the present disclosure. In certain em bodiments, the present disclosure provides a nucleic acid molecule or a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the antibodies, the antibody fragments or the pair of molecules of the present disclosure.

[0405] In certain em bodiments, the present disclosure provides a nucleic acid molecule or a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibodyfragmentspecific forclusterof differentiation 3 (CD3), wherein said antibody or antibody fragment has a reduced binding affinity to CD3 while retaining its efficacy as T cell engager on tumor cells compared to an antibody or antibody fragment comprising

[0406] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0407] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0408] In certain em bodiments, the present disclosure provides a nucleic acid molecule or a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0409] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0410] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein said antibody or antibody fragment comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q of the heavy chain variable region (VH).

[0411] In certain em bodiments, the present disclosure provides a nucleic acid molecule or a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0412] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID

[0413] NO: 4 and a HCDR3 of SEQ ID NO: 12, and (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0414] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0415] (a) a heavy chain variable region ( VH ) comprising the amino acid sequence of SEQ ID NO: 11, and

[0416] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0417] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0418] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and

[0419] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0420] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0421] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0422] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0423] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0424] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0425] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0426] (a) a heavy chain variable region ( VH ) comprising the amino acid sequence of SEQ ID NO: 82, and

[0427] (b) a light chain variable region ( VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0428] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises

[0429] (a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 84, and

[0430] (b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0431] In an embodiment, said nucleic acid composition and / or said nucleic acid sequence and / or said plurality of nucleic acid sequences are isolated.

[0432] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0433] (a) a first polypeptide (Pl) comprising

[0434] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0435] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0436] (b) a second polypeptide (P2) comprising

[0437] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0438] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0439] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0440] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0441] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0442] (a) a first molecule (Ml) comprising

[0443] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0444] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0445] (b) a second molecule (M2) comprising

[0446] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0447] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain has reduced target-independent complementation while retaining on-tumor complementation compared to an antibody or antibody fragment comprising

[0448] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO:

[0449] 4 and a HCDR3 of SEQ ID NO: 5, and (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0450] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0451] (a) a first polypeptide (Pl) comprising

[0452] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0453] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0454] (b) a second polypeptide (P2) comprising

[0455] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0456] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain comprises

[0457] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0458] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q or the light chain variable region (VL) derived from an antibody.

[0459] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising (a) a first molecule (Ml) comprising

[0460] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0461] (ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and

[0462] (b) a second molecule (M2) comprising

[0463] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0464] (ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain, and wherein said functional anti-CD3 binding domain comprises

[0465] (a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and

[0466] (b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96E or G96Q or the light chain variable region (VL) derived from an antibody.

[0467] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0468] (a) a first polypeptide (Pl) comprising

[0469] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0470] (ii) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0471] (b) a second polypeptide (P2) comprising (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0472] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0473] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0474] (a) a first molecule (Ml) comprising

[0475] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0476] (ii) a heavy chain variable region ( VH ) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and

[0477] (b) a second molecule (M2) comprising

[0478] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0479] (ii) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

[0480] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0481] (a) a first polypeptide (Pl) comprising

[0482] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0483] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and

[0484] (b) a second polypeptide (P2) comprising

[0485] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0486] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0487] (a) a first molecule (Ml) comprising

[0488] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0489] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and

[0490] (b) a second molecule (M2) comprising

[0491] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0492] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0493] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0494] (a) a first polypeptide (Pl) comprising

[0495] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0496] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and

[0497] (b) a second polypeptide (P2) comprising

[0498] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0499] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0500] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0501] (a) a first molecule (Ml) comprising

[0502] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0503] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 78, and (b) a second molecule (M2) comprising

[0504] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0505] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0506] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0507] (a) a first polypeptide (Pl) comprising

[0508] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0509] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0510] (b) a second polypeptide (P2) comprising

[0511] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0512] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0513] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0514] (a) a first molecule (Ml) comprising

[0515] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0516] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 79, and

[0517] (b) a second molecule (M2) comprising

[0518] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0519] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0520] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising (a) a first polypeptide (Pl) comprising

[0521] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0522] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0523] (b) a second polypeptide (P2) comprising

[0524] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0525] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0526] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0527] (a) a first molecule (Ml) comprising

[0528] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0529] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 80, and

[0530] (b) a second molecule (M2) comprising

[0531] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0532] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0533] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0534] (a) a first polypeptide (Pl) comprising

[0535] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0536] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and

[0537] (b) a second polypeptide (P2) comprising

[0538] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0539] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0540] (a) a first molecule (Ml) comprising

[0541] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0542] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 82, and

[0543] (b) a second molecule (M2) comprising

[0544] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0545] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0546] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of polypeptides comprising

[0547] (a) a first polypeptide (Pl) comprising

[0548] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and

[0549] (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 84, and

[0550] (b) a second polypeptide (P2) comprising

[0551] (i) a second targeting moiety (T2) specifically binding to a second tumor associated antigen (TAA2), and

[0552] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0553] In certain embodiments, the present disclosure provides a nucleicacid molecule ora nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a pair of molecules comprising

[0554] (a) a first molecule (Ml) comprising

[0555] (i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and (ii) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 84, and

[0556] (b) a second molecule (M2) comprising

[0557] (i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and

[0558] (ii) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

[0559] In certain embodiments, the present disclosure provides a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid molecule or a nucleic acid composition as defined herein above.

[0560] In certain embodiments, the present disclosure provides a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid molecule or a nucleic acid composition as defined herein above.

[0561] In certain embodiments, the host cell is an isolated host cell. In certain embodiments, said host cell is a mammalian cell. In certain embodiments, said mammalian cell is a human cell. In certain embodiments, said mammalian cell is a CHO cell. In certain embodiments, said cell is a HEK cell. In certain embodiments, said cell is a PERC.6 cell. In certain embodiments, said cell is a HKB11 cell.

[0562] The skilled artisan will realize that the nucleic acid sequence or the plurality of nucleic acid sequences encoding the heavy and / or light chain of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or into the same vector.

[0563] The vectors can be introduced into the appropriate host cells such as prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cells by methods well known in the art (see e.g., “Current Protocol in Molecular Biology”, Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those ofskill in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene can be placed underthe control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as “control” elements), so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this expression construction. The coding sequence may or may not contain a signal peptide or leader sequence. Upon expression in host cells, the antibodies or antibody fragments of the present disclosure are obtained. These steps can be achieved in different ways, as will be known by the person skilled in the art. In general, such steps typically include transforming or transfecting t>3 a suitable host cell with a nucleic acid composition or vector composition or an infectious particle, which encodes the antibody, or antibody fragments. Further, such steps typically include culturing said host cells under conditions suitable for the proliferation (multiplication, growth) of said host cells and a culturingstep under conditions suitable forthe production (expression, synthesis) ofthe encoded antibody or antibody fragment. The culturing of host cells under conditions suitable for proliferation or expression is typically accomplished in the presence of media comprising components suitable for cell growth or induction of expression. In particular, embodiments, the methods forthe production of the antibodies or antibody fragments of the present disclosure further comprise the step of isolating and purifying the produced antibody or antibody fragment from the host cells or medium. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates or recovered from the cell membrane fraction. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. The antibody or antibody fragment of the present disclosure can then be purified by a number of techniques as known to the person skilled in the art.

[0564] Therapeutic use

[0565] The antibodies, antibody fragments and pair of polypeptides of the present disclosure, or pharmaceutical compositions incorporating the same, can be used for the treatment of various diseases and disorders.

[0566] The antibodies, antibody fragments and pair of molecules of the present disclosure, or pharmaceutical compositions incorporating the same, can be used for the treatment of various diseases and disorders.

[0567] In certain embodiments, the present disclosure provides the antibodies, antibody fragments and pair of polypeptides as defined herein for use in medicine. In certain embodiments, the present disclosure provides the antibodies, antibody fragments and pair of molecules as defined herein for use in medicine The therapeutic efficacy of the present disclosure can be tailored to specific indications based on its mechanism of action, targeting capabilities, and therapeutic activity.

[0568] In certain embodiments, the present disclosure provides the antibodies, antibody fragments and pair of polypeptides as defined herein for use in the treatment of cancer . In certain embodiments, the present disclosure provides the antibodies, antibody fragments and pair of molecules as defined herein for use in the treatment of cancer .In certain embodiments, said cancer is selected from a solid cancer or a hematologic cancer.

[0569] In certain embodiments, said cancer is a solid cancer. In certain embodiments, said solid cancer is selected from a carcinoma, a sarcoma or another solid tumors. In certain embodiments, said solid cancer is a carcinoma. In certain embodiments, said carcinoma is selected from breast cancer, colorectal cancer, lung cancer (including non-small cell lung cancer and small cell lung cancer), prostate cancer, gastric cancer, and pancreatic cancer. In certain embodiments, said solid cancer is a sarcoma. In certain embodiments, said sarcoma is selected from osteosarcoma, leiomyosarcoma, liposarcoma, and Ewing’s sarcoma. In certain embodiments, said solid cancer is another solid cancer. In certain embodiments, said other solid tumor is selected from melanoma, glioblastoma multiforme, hepatocellular carcinoma, and renal cell carcinoma.

[0570] In certain embodiments, said cancer is a hematologic cancer. In certain embodiments, said hematologic cancer is selected from a leukemia, a lymphoma and a plasma cell disorder. In certain embodiments, said hematologic cancer is leukemia. In certain embodiments, said leukemia is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). In certain embodiments, said hematologic cancer is lymphoma. In certain embodiments, said lymphoma is selected from nonHodgkin’s lymphoma (NHL), Hodgkin’s lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma (DLBCL). In certain embodiments, said hematologic cancer is a plasma cell disorder. In certain embodiments, said plasma cell disorder is selected from multiple myeloma and Waldenstrom’s macroglobulinemia.

[0571] In certain embodiments, the present disclosure provides the antibodies, antibody fragments pair of polypeptides or pair of molecules as defined herein for use in the treatment of an inflammatory disease. In certain embodiments, said inflammatory disease is selected from a rheumatologic condition, a dermatologic condition, and a gastrointestinal condition. In certain embodiments, said inflammatory disease is a rheumatologic condition. In certain embodiments, said rheumatologic condition is selected from rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. In certain embodiments, said inflammatory disease is a dermatologic condition. In certain embodiments, said dermatologic condition is selected from Psoriasis and hidradenitis suppurativa. In certain embodiments, said inflammatory disease is a gastrointestinal condition. In certain embodiments, said gastrointestinal condition is inflammatory bowel disease. In certain embodiments, said gastrointestinal condition is Crohn’s disease. In certain embodiments, said gastrointestinal condition is ulcerative colitis.

[0572] In certain embodiments, the present disclosure provides the antibodies, antibody fragments, pair of polypeptides or pair of molecules as defined herein for use in the treatment of an autoimmune disease. In certain embodiments, said autoimmune disease is selected from a systemic autoimmune disease and an organ-specific autoimmune disease. In certain embodiments, said autoimmune disease is a systemic autoimmune disease. In certain embodiments, said systemic autoimmune disease isselected from systemic lupus erythematosus (SLE) and systemic sclerosis. In certain embodiments, said autoimmune disease is an organ-specific autoimmune disease. In certain embodiments, said organ-specific autoimmune disease mis selected from type 1 diabetes, autoimmune thyroiditis (e.g., Hashimoto’s thyroiditis and Graves’ disease), and multiple sclerosis.

[0573] In certain embodiments, the present disclosure provides the antibodies, antibody fragments, pair of polypeptides or pair of molecules as defined herein for use in the treatment of diseases associated with aberrant immune or oncologic processes. Examples include but are not limited to neuroblastoma, retinoblastoma, and rare autoinflammatory syndromes such as familial Mediterranean fever and periodic fever syndromes.

[0574] In certain embodiments, the present disclosure provides a method forthe treatment of a disease comprising administering to a patient an antibody, anti body fragment, a pair of polypeptides or pair of molecules as disclosed herein.

[0575] In certain embodiments, the presentdisclosure provides a method forthe prevention of a disease comprising ad ministering to a patient an antibody, antibody fragment, a pair of polypeptides or pair of molecules as disclosed herein.

[0576] In an embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody, antibody fragment, pair of polypeptides or pair of molecules as disclosed herein and a pharmaceutically acceptable carrier or excipient.

[0577] The optimized binding kinetics of the heavy chain variable region (VH) of the anti-CD3 antibody to the light chain variable region (VL) provides the possibility to formulate and / or co-administer the two sets of polypeptides. EXAMPLES

[0578] Example 1: Design and generation of derivatives of UCHT

[0579] The anti-CD3 antibody UCHT has been used and tested in a split format, in which the VH domain and the VL domain are part of different molecules. The VH and the VL domain have a certain affinity to each other, which is of advantage to induce the CD3 binder restoration at the tumor, but at the same time poses the risk of pre-mature assembly in the periphery of the tumor or in healthy tissue, leading to unwanted toxicity problems. Therefore, anti-CD3 antibodies with altered kinetics of the assembly of the VH and the VL domains are needed, in order to reduce target-independent complementation while retaining on-tumor complementation of the active CD3 binder. The aim was therefore to identify amino acid substitutions with modify antigen binding affinity, decreased immunogenicity and reduced VH-VL association and / or dissociation between the amino acid chains of the same antibody variant or other antibodies.

[0580] Via a semi rational approach, several potential mutation sites within the variable heavy and the variable light chain of antibody UCHT, including those show in the following Table (numbering according to Chothia).

[0581] Amino acid sequences of certain improved UCHT derivatives are as follows (CDRs according to Chothia):

[0582] Example 2: Testing of the UCHT derivatives

[0583] To show the impact of UCHT-1 WT engineering, T cell engagers consisting of a HER2 targeting domain and a C-terminally fused scFv containing either the WT sequence (VBP-007; SEQ ID No. 76) or the variant including the G96E mutation (VBP-008; SEQ ID No. 77) were generated. Figure 11A shows the potency of T cell engagers either with the WT anti-CD3 binder (VBP-007) or the engineered version (VBP-008) in a co-culture cytotoxicity assay. As target cells SK-BR-3 cells (HER2 high) were used. The WT and the mutant version of the anti-CD3 binder induce potent toxicity on tumor cells, which was in the same range as Runimotamab. As shown in Figure 11B, a reduced binding towards CD3+ T cells could be observed for the engineered UCHT-1 variant (VBP-008) compared to the WT sequence (VBP- 007). This demonstrates that a reduced affinity to CD3 does not affect killing efficacy. At the same time the engineered version has a reduced tendency to bind to peripheral CD3+ T cells, e.g. following administration into organisms and decrease the risk of CD3-mediated drug disposition.

[0584] Example 3: Generation of BiTAC precursor molecules with CD3 binder optimization

[0585] Example 3.1 Construction of expression plasmids for the BiTAC precursors The BiTAC precursor constructs disclosed herein were produced by transiently transfecting human embryonic kidney (HEK 293) cells. For expressing a specific gene or protein, such as a full-length antibody heavy chain, a full-length antibody light chain, or a full-length heavy chain with an additional domain (e.g., a variable domain of an immunoglobulin heavy or light chain at its C-terminus), a transcription unit was designed with the following functional elements:

[0586] - The immediate early enhancer and promoter from the human cytomegalovirus (P-CMV)

[0587] - A human heavy chain immunoglobulin 5'-untranslated region (5'UTR)

[0588] - A murine immunoglobulin heavy chain signal sequence

[0589] - The target gene or protein to be expressed

[0590] - WPRE (Woodchuck posttranscriptional regulatory element) downstream of the cloning site to improve protein expression.

[0591] In addition to the expression cassette containing the target gene, the basic mammalian expression plasmid also included a pUC origin and a beta-lactamase gene.

[0592] Genes encoding the antibody heavy chain, including C-terminal fusion constructs, were engineered to contain a complete and functional antibody heavy chain sequence followed by an additional variable (V) domain, either V-heavy or V-light. This was achieved by fusing a DNA sequence encoding the respective V domain (either VH or VL) to the C-terminus of the CH3 domain of a human IgG molecule. These fusions were constructed with a flexible 4X(G4S)-linker (SEQ. ID No. 13), resulting in configurations, such as VH-CHl-hinge-CH2-CH3-linker-VH or VH-CHl-hinge-CH2-CH3-linker-VL.

[0593] Recombinant antibody molecules containing one VH or VL domain at the C-termini of one CH3 domain, were produced using the knob-into-hole technology, enabling efficient pairing of the individual chains to generate a BiTAC precursor.

[0594] Genes encoding a complete and functional antibody light chain were constructed by assembling DNA fragments that code for the relevant sequence elements (VL-CL). The transcription unit for the antibody light chain fragment contained the following functional elements:

[0595] - The immediate early enhancer and promoter from human cytomegalovirus (P-CMV)

[0596] - A 5'-untranslated region (5'UTR) from human heavy chain immunoglobulin for enhanced translation

[0597] - A murine immunoglobulin heavy chain signal sequence for secretion

[0598] - The nucleic acid sequence encoding the antibody light chain (VL-CL)

[0599] In addition to the expression cassette containing the target gene, the basic mammalian expression plasmid also included a pUC origin and a beta-lactamase gene. O Example 3.2 Expression of the BiTAC precursor molecules

[0600] The BiTAC presursor molecules were generated in transiently transfected HEK293 cells (human embryonic kidney cell line 293-derived) cultivated in Expi293™ Expression Medium (Gibco™, #A1435102). For transfection PEI MAX® Transfection Reagent (Polysciences Europe GmbH, # 24765) was used. The respective BiTAC precursor heavy- and light chain molecules as described above were expressed from individual expression plasmids. All used plasmids were synthesized by Invitrogen GeneArt services (Thermo Fisher Scientific Inc.). Transfections were performed as specified in the manufacturer's instructions. Immunoglobulin-containing cell culture supernatants were harvested five days after transfection (see also Meissner, P. et al., Biotechnol. Bioeng. 75 (2001) 197-203).

[0601] BiTACs were purified by a MabSelect SuRe (Cytiva, #11003494) (Affinity Chromatography) and followed by Superdex 200 (Cytiva, #28990944) (Size Exclusion Chromatography). The constructs generated are summarized in the following Table. Binders with specificity for SLAMF7 and CD38 were utilized as isotype controls, since SLAMF7 and CD38 are not expressed on SK-BR-3 cells.

[0602] The purification yield of BiTAC precursors is summarized in the following Table.

[0603] As can be seen, the introduced mutations into the UCHT-1 did neither decrease the production yield nor did it impact the purification process leading to monomer contents in the same range.

[0604] Example 4: Functional characterization of BiTAC precursor molecules

[0605] Figure 1 A schematically illustrates two exemplary BiTAC precursor molecules that are either directed against TAA1 or TAA2, each carrying one half of the split anti CD3 Fv, namely VH or VL. Figure IB illustrates the target-dependent complementation of an active CD3 binder on the surface of TAA1 and TAA2-expressing target cells. Thereby, CD3+ T cells are crosslinked, activated and the effector function (T cell dependent cytotoxicity (TDCC)) is induced.

[0606] Figure 3 illustrates the hypothesized process of target-independent complementation, in which two BiTAC precursors assemble into trispecific antibodies even without encountering the two target antigens. Not shown here is also the possibility of CD3 complementation if just one antibody binds to the TAA expressing cell, whereas the other BiTAC precursor is not binding competent. Both conditions would limit the therapeutic benefit of the AND-gated approach. At the bottom of Figure 3 the desired target-dependent-complementation on dual-positive target cells is shown. To mitigate the risk of target-independent complementation different point mutations were introduced into the UCHT-1 and / or VL in order to finetune the affinities between the two domains and towards their cognate antigen CD3 upon complementation as illustrated in Figure 5A. Figure 5B illustrates that the introduced point mutations still allow an efficient on-cell assembly of the two BiTAC precursors on the surface of dual-antigen positive target cells.

[0607] Example 4.1 Methods

[0608] Quantitative expression analysis of EpCAM and HER2 on target cells

[0609] EpCAM and HER2 cell surface expression was determined by antibody staining of the target cells. Cells were resuspended in cold FACS buffer (PBS + 0.5% BSA) to a final density of lxlOA6 cells / ml. AF- 488 a-hu CD340 (erbB2 / HER2) (Biolegend, #324410), APC a-hu CD326 (EpCAM) (BD Biosciences, #566842) and the corresponding Isotype control Antibodies, AF-488 Mouse IgGl-lsotype (Biolegend, #400129) and APC Mouse lgG2b-lsotype (Biolegend, #400120), were added to the cells at 100 nM and incubated for 60 min on ice in the dark. The cells were then washed three times in FACS buffer and resuspended in 200 pl FACS buffer.

[0610] To quantify the expression of EpCAM and HER2 on the cell surface, Quantum Simply Cellular (QSC) microspheres (Bangs Laboratories, #815B) were used according to the manufacturer's recommendations. In short, each of the 4 microsphere populations was incubated with 100 nM of the antibodies for 30 min at RT in the dark. The beads were then washed three times in FACS buffer and resuspended in 200 pl FACS buffer.

[0611] Flow Cytometry analysis of cells and QSC microspheres was performed on the same day with same instrument settings using a FACSymphony A3 (BD Biosciences).

[0612] The .fcs data files were analyzed using FlowJo Software version 10.8.1 (Tree Star Inc.). To calculate the number of HER2 and EpCAM molecules expressed by each cell line, the resulting median fluorescence intensity values were pasted into the Quickcal Template (Microsoft Excel, (Microsoft 365 Apps for Business, 2024), that was provided with the QSC microspheres.

[0613] Binding of BiTACs to target cells

[0614] SK-BR-3 cells were resuspended in cold FACS buffer (PBS + 0.5% BSA) to a final density of lxlOA6 c / ml. BiTACs and a human IgG Isotype control antibody (R&D Systems, #MAB002) were serially diluted (final concentrations: 0.005 - 400 nM) and added to the cells. After 60 min incubation on ice, the cells were washed three times in FACS buffer. After washing, the cells were resuspended in 100 pl FACS buffer containing secondary antibody (Goat a-hu lgG-AF488, Invitrogen #A32723, 2 pg / ml) and incubated 30 min on ice in the dark. The cells were then washed three times in FACS buffer and resuspended in 200 pl FACS buffer.

[0615] Flow Cytometry was performed by standard methods using a FACSymphony A3 (BD Biosciences) and an HTS plate-handling system. Data was analyzed using FlowJo Software version 10.8.1 (Tree Star Inc.), Microsoft Excel (Microsoft 365 Apps for Business, 2024), and GraphPad Prism version 10.2.3 (Graphpad Software, Inc.).

[0616] Binding of BiTACs to T-cells

[0617] Purified human CD3+ T-cells (Isolated with EasySep Human T-cell Isolation Kit (STEMCELL Technologies #17951)) were thawed at 37°C in RPMI 1640 (ATCC modification) medium + 10% FBS, washed once in PBS and resuspended in RPMI 1640 (ATCC modification) + 10% FBS to a final concentration of 0.5xl0A6 cells / ml. BiTACs were 5-fold serially diluted (final concentrations: 0.16 -500 nM) and added to the cells. After incubation of T-cells with different BiTAC combinations for 24h at 37°C and 5% CO2 the cells were washed twice in PBS and resuspended in FACS buffer (PBS + 0.5% BSA) containing secondary antibody (Goat a-hu lgG-AF488, Invitrogen #A32723, 2 pg / ml). Incubation was done 30 min on ice in the dark. Afterwards the cells were washed twice in FACS buffer and resuspended in 200 pl FACS buffer. Flow Cytometry was performed by standard methods using a FACSymphony A3 (BD Biosciences) and an HTS plate-handling system.

[0618] Data was analyzed using FlowJo Software version 10.8.1 (Tree Star Inc.), Microsoft Excel (Microsoft 365 Apps for Business, 2024), and GraphPad Prism version 10.2.3 (Graphpad Software, Inc.).

[0619] Cytotoxicity assay

[0620] SK-BR-3 cells were detached using trypsin, washed once with PBS and resuspended in culture medium containing 10% FBS. lxl0A4 cells per well were seeded into a 96-well flat-bottom cell culture plate (Th Geyer) and incubated for 24h at 37°C and 5% CO2. Cytotoxicity assay was performed the next day in RPMI 1640 (ATCC modification) + 5% FBS. To do so, purified human CD3+ T-cells (Isolated with EasySep Human T-cell Isolation Kit (STEMCELLTechnologies #17951) were added to the assay plate at an effector cell: target cell (E:T) ratio of 10:1. Antibodies were diluted in RPMI 1640 (ATCC modification) + 5% FBS and added to the assay plate in duplicates at the indicated concentrations. The assay plates were incubated for 48h at 37°C and 5% CO2. Cytotoxic activity was assessed from supernatants using the Cytotoxicity Detection Kit (LDH) (Roche, #11644793001) according to the manufacturer's manual. Absorbance measurements were performed using a Spark Microplate reader (Tecan).

[0621] The percentage of cell-mediated cytotoxicity was calculated considering an effector cell control, target cell control and 100% killing control (Triton-X treated target cells) as stated in the manufacturer's protocol.

[0622] (Sample value - effector cell control)-target cell control

[0623] Cytotoxicity [%] 100% killing control-target cell control

[0624] Cell viability in % was calculated from the Cytotoxicity values and normalized to a 100% alive control (target + effector cells). More precisely, the following formula was applied:

[0625] (100 / (Viability "target + effector cells'")

[0626] Cell viability [%] =

[0627] Viability "sample"

[0628] Cytokine ELISA

[0629] Supernatant samples were collected from the cytotoxicity assays to quantify the cytokine release of IL-2 induced by the BiTAC precursor combinations. Cytokine measurement was performed using human ELISA kits (RnD Systems, #DY202) according to the manufacturer's instructions. Absorbance measurements were performed using a Spark microplate reader (Tecan). Data was analyzed using Microsoft Excel (Microsoft 365 Apps for Business, 2024), and GraphPad Prism version 10.2.3 (Graphpad Software, Inc.).

[0630] Flow cytometry-based measurement of target-independent complementation

[0631] To quantify the BiTAC precursors propensity to assemble on the target cells a cell binding assay with just one targeted BiTAC precursor against HER2 in combination with a non-binding BiTAC precursor (iso) was performed by applying flow cytometry.

[0632] To do so, SK-BR-3 cells were resuspended in FACS buffer (PBS + 0.5% BSA) and lxl0A4 cells / well were seeded into a 96-well U-bottom plate. BiTACs were added in a final concentration of 100 nM and incubated for 1 hour on ice. Afterwards, the cells were washed twice in FACS buffer and a secondary antibody, either Alexa Fluor-647 anti-human Ig light chain K (Biolegend, #316514) or Alexa Fluor-647 anti-human Ig light chain A (Biolegend, #316614), was added. After incubating 30 min on ice and washing twice in FACS buffer the binding signal on the cells was analyzed in a NovoCyte 3000 Flow Cytometer (Agilent) and an HTS plate-handling system.

[0633] Data was analyzed using FlowJo Software version 10.8.1 (Tree Star Inc.).

[0634] Measurement of the VH / VL interaction via surface plasmon resonance (SPR)

[0635] Surface plasmon resonance (SPR) was used to measure the binding kinetics between the UCHT-1 variable heavy chain (as wild-type as used in VBP-002 and as G96E variant as used in VBP-003) and the UCHT-1 variable light chain (as used in VBP-001). Measured were the binding kinetics of one BiTAC molecule with a split CD3 binder to a second BiTAC molecule with a CD3 split counterpart. The assays were conducted on a Biacore system at 25 °C using a NAHLC30M neutravidin chip sourced from Xantec. The running buffer for all experiments was Phosphate-Buffered Saline (PBS) containing 0.02% Tween20.

[0636] Biotinylated ligands, specifically VBP-002 and VBP-003, were immobilized on the neutravidin-coated chip surface via direct injection. Immobilization was performed by injecting the biotin-ligands at a concentration of 25 pg / mL, with a target immobilization level of approximately 200 Response Units (RU).

[0637] Following ligand immobilization, the binding kinetics were measured by injecting a serial dilution of the BiTAC analyte VBP-001. The dilution series was prepared starting at an initial concentration of 2 pM, with a 1:5 dilution factor for subsequent concentrations. The analyte solutions were injected over the BiTAC-immobilized chip at a constant flow rate of 10 pL / min.

[0638] The sensor surface was regenerated and conditioned after each injection cycle with 50 mM NaOH /

[0639] 1 M NaCI to remove bound analyte and prepare the surface for the next measurement.

[0640] Example 4.2 Results

[0641] Example 4.2.1 Splitting of the VH and VL domains abolishes T cell binding, while retaining TDCC potency ofter complementotion on torget cells As shown in Figure 2 A exemplary BiTAC precursor molecules targeting HER2 and EpCAM carrying either the VH (VBP-002) or VL (VBP-001) of the UCHT1 clone were incubated with target and effector cells. Importantly, the single molecules did not induce significant TDCC in absence of their cognate partner, whereas the combination thereof led to strong dose-dependent killing of the target cells. As positive control the active HER2-T cell engager Runimotamab was included. The latter molecule leads to TDCC with a similar potency.

[0642] Figure 2B highlights, that BiTAC precursors (VBP-001 or VBP-002) do not bind to T cells after incubation for 24 hours at 37°C, which reveals that the splitting of VH and VL abolishes T cell binding to below a detectable limit. In contrast, the positive control Runimotamab significantly binds to the CD3+ T cells.

[0643] Example 4.2.2 Effective formation of trispecific BiTAC molecules

[0644] In order to estimate the BiTAC precursor propensity to form binding-competent CD3 binders the previously described SK-BR-3 cytotoxicity assay was used, which included an isotype-like BiTAC precursor that cannot bind the target cells (VBP-004). Figure 4A shows that the combination of the EpCAM-directed BiTAC precursor (VBP-002) with the isotype-control BiTAC precursor (VBP-004) leads to significant TDCC at concentrations >lnM. This shows, that active trispecific antibodies are formed not only on the cell surface upon encountering of two distinct targets, but also in a pre-mature manner if just one component is bound. As before, both Runimotamab and the dual targeting BiTAC precursor combinations (VBP-001+VBP-002) induced a potent dose-dependent killing which was in a similar range.

[0645] In another assay to study target-independent complementation of the CD3 binder CD3+ T cells were incubated for 24 hours with either the HER2 / EpCAM BiTAC precursor combination (VBP-001+VBP-002) or Runimotamab. T cells do neither express HER2, nor EpCAM. In case of a binding of the BiTAC precursors to T cells the binding is mediated by the reconstituted CD3 binder after target-independent complementation. Indeed, Figure 4B shows that the BiTAC precursor combination binds to T cells to similar extend as Runimotamab.

[0646] Example 4.2.3 Generation of BiTAC molecules with reduced VH / VL affinity of the anti-CD3 domain

[0647] To measure how the interface engineering affects the assembly of the split anti CD3 VH and VL, a cell-based surrogate assay for single TAA expressing cells was performed. As illustrated in Figure 6A a BiTAC precursor targeting HER2 was used (VBP-001). The respective Fab contains a kappa light chain. Additionally, it contains the anti CD3 VL. This molecule was incubated with HER2-expressing SK-BR-3 cells, followed by the addition of another BiTAC precursor containing a non-binding Fab with lambda light chain. The latter BiTAC precursor did carry a CD3 VH either as wildtype (VBP-005) or engineered version (VBP-006). By staining the cells with an anti-kappa or anti-lambda, the amount of bound molecules to the cell surface was measured and analyzed by flow cytometry. However, only if targetindependent assembly occurs, the lambda light chain-containing molecules can be detected on the surface of the cells, since it is not binding to it via the Fab. The results are shown in Figure 6B. By combining molecule VBP-001 and VBP-005 a clear signal increase in the anti-lambda staining and hence binding of VBP-005was observed. In contrast, the combination of VBP-001 and VBP-006 (mutant anti CD3 VH) led to a significantly decreased lambda signal. This reveals that by introducing mutations, the interface affinity between VH and VL in the CD3 binder was reduced.

[0648] Example 4.2.4 Variant BiTAC molecules have a reduced tendency to complement in a targetindependent manner

[0649] Next, the target-independent versus target dependent complementation of the engineered mutant was assessed. Results are shown in Figure 7A. Here, a cytotoxicity assay utilizing SK-BR-3 cells was used. EpCAM-directed precursor BiTAC carrying the anti CD3 VH mutant were combined with a non-targeted BiTAC precursor carrying the anti CD3 VL (VBP-003+VBP-004). Importantly, it was found that there are no signs of cytotoxicity observable in this target-independent setting. However, by combining the EpCAM-directed precursor molecule carrying the anti CD3 VH mutant with a HER2-targeting BiTAC precursor carrying the anti CD3 VL (VBP-003+VBP-001), a dose-dependent toxicity in the range of Runimotamab was observed.

[0650] Next it was assessed how the engineering of the anti CD3 interface affects the binding of the two BiTAC precursors (directed against HER2 and EpCAM, not expressed on T cells) to CD3+ T cells. As shown in Figure 7B, indeed, the reduced interface and / or affinity towards CD3 upon targetindependent re-constitution led to a significantly lower binding to T cells (VBP-001 + VBP-003) compared to the BiTAC precursor combination with the WT CD3 domains (VBP-001 + VBP-002). This indicates that the molecules have a reduced tendency to complement the active CD3 binder in a targetindependent manner compared to the WT anti CD3 interface.

[0651] Example 4.2.5 Variant BiTAC molecules are potent in an in vitro co-culture cytotoxicity assay on tumor cells but not on healthy surrogate cells. Next, the potency of BiTAC precursor combinations either with the WT anti CD3 interface (VBP-001 + VBP-002) or the engineered version (VBP-001 + VBP-003) was analyzed in a co-culture cytotoxicity assay. Figure 8 shows a set of four different, breast-cancer derived cell lines. These cell lines express different levels of HER2 and EpCAM. Figure 8A shows the results of SK-BR-3 cells (HER2 high, EpCAM medium), Figure 8B of JIMT-1 (EpCAM high, HER2 medium) and Figure 8C of BT-474 (HER2 high, EpCAM high). These three cell lines represent the expression of the two antigens in tumor tissue. Figure 8D shows the MDA-MB-231 cell line (HER2 low, EpCAM low) which has expression levels of the two antigens in the range of healthy tissue (Cancer Res (2023) 83 (7_Supplement): 6337. Interestingly, the WT and mutant versions induce potent toxicity on tumor cells, which was found being in the same range as Runimotamab. That highlights, that target-dependent, on-cell assembly and CD3 reconstitution is efficiently achieved. However, importantly, MDA-M B-231 cells, which are the surrogate model for healthy tissue, were spared by the engineered mutant version (VBP-001+VBP- 003). In contrast, both the combinations containing the WT anti CD3 versions and Runimotamab induced significant killing.

[0652] In the same TDCC setup BiTAC precursors either with a wild-type anti-CD3 interface (VBP-009+VBP- 010) or engineered versions at position G96 in the variable heavy chain (VBP-009+VBP-011; VBP- 009+VBP-012; VBP-009+VBP-013; VBP-009+VBP-014) were analyzed.

[0653] Figure 14A shows the results obtained with SK-BR3 cells (HER2 high, EpCAM high; expression levels correspond to tumor tissue). Figure 14B shows the results obtained with MDA-MB-231 cell line (HER2 low, EpCAM low; expression levels correspond to tumor healthy tissue (Cancer Res (2023) 83 (7_Supplement): 6337)).

[0654] The wild-type and the mutant version induce potent toxicity on tumor cells, which was found being in the same range as Runimotamab. That highlights, that target-dependent, on-cell assembly and CD3 reconstitution is efficiently achieved. However, importantly, MDA-MB-231 cells, which are the surrogate model for healthy tissue, were significantly less affected by the engineered mutant version (VBP-009+VBP-011; VBP-009+VBP-012; VBP-009+VBP-013; VBP-009+VBP-014). In contrast, both the combinations containing the wild-type anti CD3 version (VBP-010) and Runimotamab induced stronger killing.

[0655] Example 4.2.6 Variant BiTAC molecules induce a reduced IL-2 secretion on healthy surrogate tissue

[0656] Figure 9 shows a quantification of the cytokine IL-2 in the supernatant from a co-culture assay of healthy tissue surrogate MDA-MB-231 cells with BiTAC precursor combinations and CD3+ T cells. While both the BiTAC precursor combination including the WT-anti CD3 interface (VBP-001 + VBP-002) and Runimotamab induce significant IL-2 secretion, the BiTAC precursor molecules containing the engineered variant (VBP-001+ VBP-003) did not induce the secretion of significant IL-2 levels. This reduced T cell activation on healthy tissue surrogate cells (off-tumor) without IL-2 secretion reveals that a safety advantage due to reduced risk of cytokine release syndrome (CRS), in which IL-2 plays a key role, can be expected.

[0657] Example 4.2.7 Variant BiTAC molecules can be co-vialed

[0658] In Figure 10 it was assessed whether a co-administration / co-vialing could be possible with the engineered interface. It was hypothesized that at high concentrations the co-formulated BiTAC precursors form heterodimers and hence active CD3 binders are formed which pose a toxicity risk and reduce the therapeutic window. To compare the effect of co-vialing versus freshly combined BiTAC precursors on a healthy tissue surrogate, a cytotoxicity assay was performed. BiTAC precursors against HER2 and EPCAM carrying either the VH or VL of an engineered interface version (VBP-001+VBP-003) were incubated overnight at 8.5 pM before added to the plates. It was found that the cytotoxicity was not increased in the pre-incubated setting, which reveals that there was no significant presence of active trispecific complexes after dilution in the assay.

[0659] Example 4.2.8 Binding of BiTAC precursors to target cells.

[0660] Figure 12 shows the binding of molecules of the present disclosure to SK-BR-3 cells (SK-BR-3 cells are HER2 high, EpCAM medium, CD38 negative, and SLAMF7 negative). As expected HER2-directed VBP- 001 and Runimotamab (positive control) show a stronger binding to the high HER2-expressing cells in comparison to EpCAM-directed VBP-002 and VBP-003. Importantly, the UCHT-1 engineered version in VBP-003 does not affect the EpCAM targeting domain. The CD38 and SLAMF7-directed molecules (VBP- 004 and VBP-005) do not bind to the CD38 and SLAMF7-negative SK-BR-3 cells which qualify their use as non-binding controls to assess target-independent complementation as assessed in Example 4.2.3 and 4.2.4.

[0661] Example 4.2.9 Measurement of the VH / VL interaction via surface plasmon resonance (SPR)

[0662] Biotinylated BiTAC molecules, VBP-002 or VBP-003, were successfully and stably immobilized onto the NAHLC30M sensor chip surface. A control experiment confirmed no detectable non-specific binding of VBP-001 to the activated chip surface, ensuring that all subsequent binding signals were specific to the immobilized molecules.

[0663] SPR analysis was used to quantify the binding affinity of VBP-001, to immobilized VBP-002 (Figure 13A) or VBP003 (Figure 13B). The kinetic data for each interaction were fit to a 1:1 binding model to determine the binding affinity (KD). VBP-001 demonstrated a high affinity for VBP-002, with a calculated KD of approximately 0.89 nM. In contrast, its binding to VBP-003 was an order of magnitude weaker, with an estimated KD of approximately 8.1 nM. The following Table summarizes all kinetic parameters determined.

[0664] Overall, these results demonstrate that the G96E mutation introduced to VBP-002 (and which results in VBP-003) decreased the interface interaction between CD3-VH with its counterpart CD3-VL by approximately 10-fold. This mechanistically explains why target-independent assembly occurs stronger in the WT interface and is strongly reduced in the engineered version.

[0665] Example 5: Design and generation of derivatives of SP34

[0666] Similar to the anti-CD3 antibody UCHT, also the anti-CD3 antibody SP34 was derivatized in order to generate VH and VL domains that are suitable to be used in a split system. Also here the goal was to achieve an affinity between the variable heavy chain and the variable light chain which is sufficient to restore the CD3 binder at the site of the tumor, but at the same time avoids the risk of pre-mature assembly in the periphery of the tumor, in circulation or in healthy tissue. The aim was therefore to identify amino acid substitutions with modify antigen binding affinity, decreased immunogenicity and reduced VH-VL association and / or dissociation between the amino acid chains of the same antibody variant or other antibodies.

[0667] Crystal structures were used to identify amino acid residues that are involved in the pairing of the variable heavy chain with the variable light chain. Amino acid residues were identified which could by crucial for the interaction of the variable heavy chain with the variable light chain. The following mutation sites within the variable heavy chain of antibody SP34 were identified (numbering according to Chothia):

[0668] Amino acid sequences of certain improved SP34 derivatives are as follows (CDRs according to Chothia):

[0669] The SP34 based molecules shown in the following Table were generated.

[0670] The purification yield of BiTAC precursors is summarized in the following Table. The potency of the BiTAC precursor molecules with either the wild-type anti-CD3 interface (VBP- 101+VBP-102) or the engineered versions at different positions in the SP34 variable heavy chain (VBP- 101+VBP-105; VBP-101+VBP-104) was analyzed the co-culture cytotoxicity assay.

[0671] Figure 15A shows the results of the experiments with SK-BR3 cells (HER2 high, EpCAM high; representative for tumor tissue), Figure 15B the results of the experiments with MDA-MB-231 cells (HER2 low, EpCAM low; representative for healthy tissue).

[0672] Interestingly, the wild-type and mutant H95A CD3-binding domain induced potent toxicity on tumor cells with a maximum killing, comparable the HER2-TCE (VBP-007). This highlights, that targetdependent, on-cell assembly and CD3 reconstitution is efficiently achieved. The L45A mutant decreased the cytotoxicity in SK-BR3 cells significantly. This reveals that L45A decreases the interface affinity between the variable heavy chain and the variable light chain of the split CD3domain drastically. However, importantly, MDA-MB-231 cells (surrogate model for healthy tissue) were less affected by the engineered mutant versions (H95A and L45A). In contrast, the combinations containing the wild-type anti.CD3 binding domain (VBP-101) and HER2-TCE (VBP-007) induced stronger maximum killing.

[0673] The results show that the introduction of the specified mutations into the variable heavy chain of SP34 VH, in particular the H95A mutation, maintains the cytotoxic capacity on tumor cells, while having reduced cytotoxicity on healthy surrogate cells. These mutants are therefore useful for clinical applications due to their higher therapeutic index.

Claims

1. CLAIMS1. A pair of [two] molecule comprising(a) a first molecule (Ml) comprising(i) a first targeting moiety (Tl) specifically binding to a first tumor associated antigen (TAA1), and(ii) a heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), and(b) a second molecule (M2) comprising(i) a second targeting moiety (T2 ) specifically binding to a second tumor associated antigen (TAA2), and(ii) a light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said heavy chain variable region (VH) and said light chain variable region (VL) of said antibody or antibody fragment specific for cluster of differentiation 3 (CD3) are capable to form a functional anti-CD3 binding domain comprising(a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and(b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein the heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q.

2. The pair of molecules according to claim 1, wherein, wherein said heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and said light chain variable region (VL) derived from an antibody orantibody fragment specific for cluster of differentiation 3 (CD3) comprises a light chain variable region(VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ I D NO: 8.

3. The pair of molecules according to any one of claims 1 or 2, wherein said heavy chain variable region (VH) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and said light chain variable region (VL) derived from an antibody or antibody fragment specific for cluster of differentiation 3 (CD3) comprises the amino acid sequence of SEQ ID NO: 2.

4. The pair of molecules according to any one of claims 1-3, wherein said first targeting moiety (Tl) and said second targeting moiety (T2) are an antibody or an antibody fragment.

5. The pair of molecules according to any one of claims 1-4, wherein said first tumor associated antigen (TAA1) and said second tumor associated antigen (TAA2) are selected from HER2, EpCAM, CD19, MSLN, DLL3, FLT3, EGFR, CD33, CD38, M UC17, CLDN18.2, CDH3, CD70, BCMA ,PSMA, CEACAM5, HER3, STEAP- 1, TROP-2, NECTIN-4, B7-H3, B7-H4, MUC16, FOLR1, CD200, CLDN6, GPRC5D, EGFRviii, MUC1 and 5T4.

6. The pair of molecules according to any one of claims 1-5, wherein said first molecule (Ml) and said second molecule (M2) each comprise a Fc region.

7. An antibody or antibody fragment specific for cluster of differentiation 3 (CD3), wherein said antibody or antibody fragment comprises(a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 5, and(b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8, wherein said antibody or antibody fragment comprises a substitution of an amino acid selected from L45A, L45W, S95V, G96D, G96E, G96R or G96Q of the heavy chain variable region (VH).

8. The antibody or antibody fragment according to claim 2, wherein said antibody or antibody fragment comprises(a) a heavy chain variable region (VH) comprising a HCDR1 of SEQ ID No. 3, a HCDR2 of SEQ ID NO: 4 and a HCDR3 of SEQ ID NO: 12, and(b) a light chain variable region (VL) comprising a LCDR1 of SEQ ID No. 6, a LCDR2 of SEQ ID NO: 7 and a LCDR3 of SEQ ID NO: 8.

9. The antibody or antigen-binding fragment according to claim 7 or 8, wherein said antibody or antibody fragment comprises(a) a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 11, and(b) a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 2.

10. A nucleic acid molecule encoding the antibody or antibody fragment according to claims 7-9 or the pair of molecules according to claim 1-6.

11. A vector comprising the nucleic acid molecule according to claim 10.

12. A host cell comprising the nucleic acid molecule of claim 10 or the vector of claim 11.

13. The antibody or antibody fragment according to claims 7-9 or the pair of polypeptides according to claim 1-6 for use in medicine.

14. The antibody, the antibody fragment or the pair of polypeptides for use according to claim 13, wherein said used in medicine is the treatment of a cancer, an inflammatory disease or an autoimmune diseases.

Images