Cycat halfbody molecules comprising sterically occluding moieties

Abstract

The present disclosure provides CyCAT halfbody molecules and complementary pairs of such halfbody molecules which are capable of associating with each other on the surface of a cell expressing two target antigens of interest. The thus on-cell and on target formed trispecific heterodimeric antibody molecules are capable of engaging and stimulating cytotoxic T-cells for tumor cell destruction. The present disclosure further provides halfbody molecules having sterically occluding moieties attached thereto which reduce heterodimer formation of such halfbodies in the presence of only one or absence of both target antigens of interest.

Description

[0001] CYCAT HALFBODY MOLECULES COMPRISING STERICALLY OCCLUDING MOIETIES

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to CyCAT molecules and corresponding complementary pairs of CyCAT halfbody molecules of which they are composed of and which resemble and become activated on the surface of a target cell. The thus on-cell and on-target formed trispecific heteromeric antibody molecules are capable of engaging and stimulating cytotoxic T-cells for tumor cell destruction via an reconstituted aCD3-Fv antibody fragment. The present disclosure also provides strategies for further optimizing the safety profile of such CyCAT directed therapies, by attaching a sterically occluding moiety (also referred herein as “Guard-Domain”) to at least one halfbody molecule within a pair of complementary halfbody molecules. This combinatorial approach is particular useful for minimizing toxicity effects associated with antibody based therapies involving recruitment of immune cells for cancer cell destruction.

[0004] BACKGROUND OF THE INVENTION

[0005] Therapeutic concepts based on the use of multispecific antibodies usually rely on the simultaneous binding to cell surface antigens present on cancer cells and to cell surface antigens present on immune effector cells, with the aim that the immune effector cells become only activated in vicinity of a tumor cell (Miller and Kontermann, Bispecific antibodies for cancer immunotherapy: Current perspectives. BioDrugs 2010, 24(2):89- 98). One of the most explored antigens in this context is CD3, a proven T-cell stimulating antigen with strong therapeutic relevance. In this therapeutic concept, cancerous cells and cytotoxic T-cells are bridged via a bispecific antibody employing a CD3 targeting binding domain which results in the formation of an immunological synapse between the cancer cell and a cytotoxic T-cell, allowing the T-cell to directly kill the cancer cells.

[0006] One significant problem in CD3 targeted therapy relies in its dose limiting adverse effects driven by the inherent ability of CD3 binding domains to stimulate T-cells, irrespective of the presence of the target cells or by binding of the bispecific antibody to healthy tissue which may also express a cancer associated marker of interest. Strategies have been developed to provide bispecific antibodies in a “prodrug”-like approach in such a way that the CD3 binding portion is in an “inactive” state when the bispecific antibody is administrated to a patient but becomes specifically activated once the bispecific antibody reaches the envisaged place of action.

[0007] WO201 3 / 104804 provides a two component system comprising a first polypeptide with a first targeting moiety and carrying either the VH or VL domain of an aCD3-Fv binding fragment along with a second polypeptide with a second targeting moiety and carrying the complementary VL or VH domain, respectively, of the aCD3-Fv fragment. Binding of the two polypeptides to a cancer cell expressing both target antigens on its cell surface induces dimerization and functional complementation of the CD3 specific variable domains and on-cell formation and activation of a trispecific T-cell engaging antibody.

[0008] W02015 / 013671 describes a bispecific T-cell engaging approach that includes a protease cleavable masking moiety fused to a CD3 binding domain within a conventional bispecific antibody format. The coupling of the masking moiety reduces the ability of the CD3 binding domain to bind to cytotoxic T-cells. However, once the bispecific antibody binds to a cancerous cell, the masking moiety is released through the activity of cancer-specific proteases in the tumor microenvironment restoring the ability of the CD3 binding domain to co-engage T-cells.

[0009] WO201 7 / 087789 provides a two-component system similar to the one described in WO201 3 / 104804 but additionally incorporates a domain masking approach in such way that the masking moiety blocks the interaction and functional complementation of the complementary aCD3 variable domains unless cancer-specific proteases in the tumor microenvironment releases the masking moiety. However, relying on tumor-specific proteases to activate or restore CD3 binding at the site of desired action brings an additional level of complexity in the design of bispecific antibody molecules, which for instance requires the appropriate incorporation of amino acid sequences recognized by such proteases. Also, the actual type, presence and activity of tumor proteases in the tumor-microenvironment, may strongly depend on the type of cancer and also from patient to patient.

[0010] WO2022 / 051647 discloses a bispecific T-cell engaging one-component system, which makes use of a similar approach as described in WO2017 / 087789. Here, two complementary but non-binding antibody variable domains block the formation of the aCD3-scFv present on the same polypeptide unless cancer-specific proteases in the tumor microenvironment releases these inert masking moieties.

[0011] However, the common requirement to obtain a functional CD3 binding described in the prior art is the release of the masking domain(s) by means of tumor-specific proteases.

[0012] W02023 / 006809 discloses various halfbody molecules but differs from the molecules of the present disclosure in that the constructs do not encompass a Guard-Domain as described herein.

[0013] Accordingly, there is a need for improved developments in this field of T-cell redirected therapies by relying on the advantage of using a two-component system as described above with an optimized safety profile in a less complex manner as suggested in the art.

[0014] SUMMARY OF THE INVENTION

[0015] In an embodiment, the present disclosure provides a pair of halfbody molecules comprising a) a first halfbody molecule (HB1 ) comprising i. a first Fab fragment (Fab1) specific for a first antigen (AG1) ii. either a first VH (aCD3-VH1) or first VL (ocCD3-VL1) of a Fv fragment specific for CD3 (ocCD3-Fv), and iii. a first Guard-Domain (GD1), and b) a second halfbody molecule (HB2) comprising i. a second Fab fragment (Fab2) specific for a second antigen (AG2), ii. either the complementary VH (aCD3-VH2) or complementary VL (aCD3-

[0016] VL2) of the ocCD3-Fv, and iii. optionally a second Guard-Domain (GD2), wherein HB1 and HB2 are not linked via a covalent bond. In an embodiment, the present disclosure provides a pair of halfbody molecules comprising a) a first halfbody molecule (HB1 ) comprising i. a first antibody or antibody fragment (Ab1 ) specific for a first antigen (AG1 ) ii. either a first VH (aCD3-VH1) or first VL (ocCD3-VL1) of a Fv fragment specific for CD3 (ocCD3-Fv), and iii. a first Guard-Domain (GD1), and b) a second halfbody molecule (HB2) comprising i. a second antibody or antibody fragment (Ab2) specific for a second antigen (AG2), ii. either the complementary VH (aCD3-VH2) or complementary VL (aCD3-

[0017] VL2) of the ocCD3-Fv, and iii. optionally a second Guard-Domain (GD2), wherein HB1 and HB2 are not linked via a covalent bond.

[0018] In an embodiment of the present disclosure, said pair of halfbody molecules is a complementary pair of halfbody molecules. In an embodiment of the present disclosure, HB1 and HB2 are capable of forming a heterodimer. In an embodiment, HB1 and HB2 are capable of forming a heterodimer with each other. In an embodiment, HB1 and HB2 are capable of forming a heterodimer when mixed in solution. In an embodiment, HB1 and HB2 are capable of forming a heterodimer on the surface of a cell. In an embodiment, HB1 and HB2 are capable of forming a heterodimer on the surface of a cell expressing AG1 and AG2 on its cell surface.

[0019] In an embodiment, the formation of the heterodimer of HB1 and HB2 occurs via dimerization of aCD3-VH1 and aCD3-VL2 or of ocCD3-VL1 and ocCD3-VH2. In an embodiment, the dimerization of aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3- VH2 results in the formation of the aCD3-Fv. In an embodiment, the dimerization of aCD3- VH1 and ocCD3-VL2 or of ocCD3-VL1 and ocCD3-VH2 results in the formation of the T-cell engaging trispecific antibody. In an embodiment, the heterodimerization of HB1 and HB2 results in the formation of the T-cell engaging trispecific antibody.

[0020] In an embodiment of the present disclosure, GD1 has no substantial binding to aCD3- VH1 , ocCD3-VL1 or HB1. In an embodiment, GD2 has no substantial binding to aCD3- VH2, ocCD3-VL2 or HB2. In an embodiment, GD1 has no binding to ocCD3-VH1 , ocCD3- VL1 or to HB1 and wherein GD2 has no binding to aCD3-VH2 or aCD3-VL2 or to HB2.

[0021] In an embodiment of the present disclosure, each of GD1 and GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein or a soluble globular polypeptide. In an embodiment, GD1 is a soluble protein, a soluble polypeptide, a soluble globular protein or a soluble globular polypeptide. In an embodiment, GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein or a soluble globular polypeptide.

[0022] In an embodiment of the present disclosure, each of GD1 and GD2 has a molecular size of less than 70 kDa. In an embodiment, GD1 has a molecular size of less than 70 kDa. In an embodiment, GD2 has a molecular size of less than 70 kDa.

[0023] In an embodiment of the present disclosure, each of GD1 and GD2 is selected from the group of: albumin, fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulin domain or a fragment therefrom. In an embodiment, said albumin is human serum albumin or a fragment of human serum albumin. In an embodiment of the present disclosure, said fragment of human serum albumin is Domain 1 , Domain 2 or Domain 3 of human serum albumin.

[0024] In an embodiment of the present disclosure, said immunoglobulin domain is an IgG CH1 , CH2 or CH3 domain or an antibody variable domain. In an embodiment, said immunoglobulin domain or antibody variable domain is a VH domain or a VL domain. In an embodiment, GD1 and / or GD2 is a VH domain (GDVH) or a VL domain (GDVL). In an embodiment of the present disclosure, GD1 does not comprise the amino acid sequence of an antibody variable domains comprised in aCD3-Fv, Fab1 and / or Fab2.

[0025] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein if HB1 comprises an aCD3-VH1 than GD1 may be selected of being a VH domain (GDVH) but not a VL domain (GDVL) and wherein if HB1 comprises an aCD3- VL1 than GD1 may be selected of being a VL domain (GDVL) but not a VH domain (GDVH).

[0026] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein if HB2 comprises an aCD3-VH2 than GD2 may be selected of being a VH domain (GDVH) but not a VL domain (GDVL) and if HB2 comprises an ocCD3-VL2 than GD2 may be selected of being a VL domain (GDVL) but not a VH domain (GDVH).

[0027] In an embodiment of the present disclosure, if said HB1 comprises an aCD3-VH1 then GD1 is not a GDVL and if said HB1 comprises an ocCD3-VL1 then GD1 is not a GDVH. In an embodiment of the present, if said HB2 comprises an aCD3-VH2 then GD2 is not a GDVL and if HB2 comprises an ocCD3-VL2 then GD2 is not a GDVH.

[0028] In an embodiment of the present disclosure GDVH is not capable of forming a functional Fv fragment with GDVL. In an embodiment GDVH is not capable of forming a functional Fv fragment with aCD3-VL1 . In an embodiment, GDVH is not capable of forming a functional Fv fragment with aCD3-VL2. In an embodiment of the present disclosure, GDVL is not capable of forming a functional Fv fragment with aCD3-VH1 . In an embodiment, GDVL is not capable of forming a functional Fv fragment with aCD3-VH2.

[0029] In an embodiment of the present disclosure, each of GD1 and GD2 cannot be cleaved off from HB1 or HB2, respectively, by a tumor specific protease. In an embodiment, each of GD1 and GD2 does not comprise a cleavage site for a tumor specific protease.

[0030] In an embodiment of the present disclosure, GD1 and aCD3-VH1 or GD1 and aCD3-VL1 are positioned adjacent and parallel to each other on HB1. In an embodiment, GD2 and aCD3-VH2 or GD2 and aCD3-VL2 are positioned adjacent and parallel to each other on HB2.

[0031] In an embodiment of the present disclosure, GD1 and aCD3-VH1 or GD1 and aCD3-VL1 of HB1 are present on two different polypeptides. In an embodiment, GD2 and aCD3-VH2 or GD2 and aCD3-VL2 of HB2 are present on two different polypeptides.

[0032] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in absence of AG1 and / or AG2, the presence of GD1 on HB1 i. inhibits the dimerization of aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3-VH2, respectively, ii. inhibits the formation of the functional aCD3-Fv, iii. inhibits the dimerization of HB1 with HB2, and / or iv. inhibits the formation of the T-cell engaging trispecific antibody molecule.

[0033] In an embodiment of the present disclosure said inhibition is compared to the inhibition determined in absence of AG1 and / or AG2 and absence of GD1 on HB1.

[0034] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules as described herein, wherein in presence of AG1 and AG2, the presence of GD1 i. does not inhibit the binding of Fab1 to AG1 or of Fab2 to AG2, ii. does not inhibit the dimerization of aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3-VH2, iii. does not inhibit the formation of the functional aCD3-Fv, iv. does not inhibit the binding of the aCD3-Fv to CD3, v. does not inhibit the dimerization of HB1 with HB2, vi. does no inhibit the formation of the T-cell engaging trispecific antibody molecule, and / or vii. does no inhibit the activity of the trispecific antibody molecule to mediate T-cell redirected killing of cells having AG1 and AG2 on their cell surface.

[0035] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules as described herein, wherein in presence of AG1 and AG2, the presence of GD1 does not inhibit the binding of Ab1 to AG1 or of Ab2 to AG2,

[0036] In an embodiment of the present disclosure, said inhibition is compared to the inhibition determined in the presence of AG1 and AG2 but in the absence of GD1 . In an embodiment, the present disclosure provides a complementary pair of halfbody molecules as described herein, wherein the activity of the trispecific antibody molecule to mediate T-cell redirected killing of cells having AG1 and AG2 on their cell surface is at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, at least 10 fold, higher when compared to its activity under conditions in which cells having either AG1 or AG2 on their cell surface.

[0037] In an embodiment, said activity is determined in an in vitro T-cell cytotoxicity assay as described herein.

[0038] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the ICso concentration for the trispecific antibody molecule to mediate T-cell redirected killing of cells expressing AG1 and AG2 on their cell surface is at least 2 fold lower, 3 fold lower, 4 fold lower, 5 fold lower, 6 fold lower, 7 fold lower, 8 fold lower, 9 fold lower, or at least 10 fold lower, compared to the ICso concentration determined under conditions in which cells expressing AG1 and / or AG2 are absent.

[0039] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of GD1 on HB1 or of GD2 on HB2, the ratio of the ICso concentration determined for the halfbody pair induced T cell mediated killing of cells expressing either AG1 or AG2 on their cell surface to the ICso concentration determined for the halfbody pair induced T cell mediated killing of cells expressing AG1 and AG2 on their cell surface, is increased, as compared to the same ratio determined for the complementary pair of halfbody molecules lacking GD1 on HB1 or lacking GD2 on HB2.

[0040] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the presence of GD1 on HB1 or of GD2 on HB2 increases the ratio of the ICso (AG1 or AG2) I ICso (AG1 and AG2) as compared to the ICso (AG1 or AG2) I ICso (AG1 and AG2) in the absence of GD1 or GD2, respectively, wherein said ratio of the ICso (AG1 or AG2) I ICso (AG1 and AG2) is defined as the ratio of the ICso concentration determined for the halfbody pair induced T-cell mediated killing of cells expressing either AG1 or AG2 on their cell surface to the ICso concentration determined for the halfbody pair induced T-cell mediated killing of cells expressing AG1 and AG2 on their cell surface. In an embodiment of the present disclosure, said ratio of the IC50 concentrations is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400- fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold.

[0041] In an embodiment of the present disclosure, HB1 , HB2, aCD3-VH1 , aCD3-VH2, ocCD3- VL1 and / or aCD3-VL2 by themselves are not capable of binding to CD3.

[0042] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein aCD3-VH1 and / or aCD3-VH2 comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12.

[0043] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein aCD3-VL1 or aCD3-VL2 comprise the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0044] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein AG1 and AG2 are identical antigens or wherein AG1 and AG2 are different antigens. In an embodiment, AG1 and AG2 are present on the surface of a cell. In an embodiment, AG1 and AG2 are present on the surface of the same cell. In an embodiment, said cell is a cancer cell or a tumor cell. In an embodiment, AG1 and / or AG2 is a cancer associated antigen. In an embodiment, AG1 and / or AG2 is / are expressed on the surface of tumor cells or on the surface of progenitor / precursor cells of a tumor. In an embodiment of the present disclosure, the combination of AG1 and AG2 is only found on cancerous cells, but not on cells that are not cancerous. In an embodiment, the combination of AG1 and AG2 are present on the surface of the same cell.

[0045] In an embodiment of the present disclosure, HB1 further comprises a first Fc region and / or HB2 further comprises a second Fc region. In an embodiment, HB1 further comprises a first Fc region. In an embodiment, HB2 further comprises a second Fc region. In an embodiment, the first and / or the second Fc region comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to a Fc receptor and / or to C1q and / or reduces its effector function.

[0046] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein Fab1 , aCD3-VH1 or aCD3-VL1 , GD1 and the first Fc region of HB1 are fused to each other via peptide linkers. In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein Fab2, aCD3-VH2 or aCD3-VL2, GD2 and the second Fc region of HB2 are fused to each other via peptide linkers.

[0047] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein Ab1 , aCD3-VH1 or aCD3-VL1 , GD1 and the first Fc region of HB1 are fused to each other via peptide linkers. In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein Ab2, aCD3-VH2 or aCD3- VL2, GD2 and the second Fc region of HB2 are fused to each other via peptide linkers.

[0048] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in absence of AG1 and / or AG2 and in the presence of GD1 on HB1 , the dissociation constant (KD) between aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3-VH2 is at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or at least 10 fold greater compared to the KD determined in absence of GD1 on HB1 .

[0049] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in absence of AG1 and / or AG2 and in the presence of GD1 on HB1 , the amount of heterodimer formed between HB1 and HB2 is at least 2 fold lower, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or at least 10 fold lower compared to the amount determined in absence of AG1 and / or AG2 and in absence of the GD1 on HB1.

[0050] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 the dissociation constant (KD) between aCD3-VH1 on HB1 and ocCD3-VL2 on HB2, or of ocCD3-VL1 on HB1 and ocCD3-VH2 on HB2, is about the same compared to the dissociation constant (KD) determined in presence of AG1 and AG2 but the absence of GD1.

[0051] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 , the dissociation constant (KD) between aCD3-VH1 on HB1 and ocCD3-VL2 on HB2, or of ocCD3-VL1 on HB1 and ocCD3-VH2 on HB2, is within 2 fold of the KD determined in presence of AG1 and AG2 but the absence of GD1 . In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 the amount of heterodimerized HB1 and HB2 is about the same compared to the amount determined in presence of AG1 and AG2 but in the absence of GD1 on HB1.

[0052] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 the amount of heterodimerized HB1 and HB2 is within 2 fold of the amount determined in presence of AG1 and AG2 but in the absence of GD1 on HB1.

[0053] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of either AG1 orAG2 and in the presence of GD1 on HB1 , the activity of the formed trispecific antibody molecule to mediate T-cell redirected killing of a cell expressing either said AG1 or AG2 on its cell surface is at least 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or at least 10 fold weaker compared to its activity determined in presence of either AG1 or AG2 but absence of GD1 on HB1 , as determined in an in vitro T-cell cytotoxicity assay.

[0054] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 , the activity of the trispecific antibody to mediate T-cell redirected killing of a cell expressing AG1 and AG2 on its cell surface is about the same when compared to the activity determined in presence of AG1 and AG2 but absence of GD1 on HB1 , as determined in an in vitro T-cell cytotoxicity assay.

[0055] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in presence of AG1 and AG2 and in the presence of GD1 on HB1 , the activity of the trispecific antibody to mediate T-cell redirected killing of a cell expressing AG1 and AG2 on its cell surface is within 5 fold of the activity determined in presence of AG1 and AG2 and in the absence of GD1 on HB1 , as determined in an in vitro T-cell cytotoxicity assay.

[0056] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in the absence of AG1 and / or AG2, the presence of GD1 on HB1 reduces the ability of aCD3-VH1 on HB1 to dimerize with aCD3-VL2 on HB2, or of ocCD3- VL1 on HB1 to dimerize with aCD3-VH2 on HB2, by at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, compared to respective ability in absence of GD1 on HB1.

[0057] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein in the presence of AG1 and AG2, the presence of GD1 on HB1 reduces the ability of aCD3-VH1 on HB1 to dimerize with aCD3-VL2 on HB2; or of ocCD3- VL1 on HB1 to dimerize with aCD3-VH2 on HB2, by not more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or not more than 10%, as compared to the respective ability to dimerize in absence of GD1 on HB1. In an embodiment, said ability to dimerize is assayed in vitro by surface plasmon resonance or biolayer interferometry assay. In an embodiment, said ability to dimerize is assayed by size exclusion chromatography.

[0058] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the amount of the heterodimer formed between HB1 and HB2 under conditions where AG1 and AG2 are present, is at least 2 fold higher, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or at least 10 fold higher compared to the amount of heterodimer formed under conditions in which AG1 and / or AG2 is / are absent.

[0059] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein GD1 and / or GD2 comprise an amino sequence selected from the group of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0060] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the aCD3-Fv fragment (aCD3-Fv) specifically binds to human CD3.

[0061] In an embodiment, said aCD3-Fv specifically binds to CD3 epsilon. In an embodiment, said CD3 is CD3 epsilon. In an embodiment, said aCD3-Fv cross-reactively binds to cynomolgus CD3.

[0062] In an embodiment of the present disclosure, said aCD3-VH1 or aCD3-VH2 comprise an HCDR1 region comprising the amino sequence of SEQ ID NO: 1 or SEQ ID NO: 9, an HCDR2 region comprising the amino sequence of SEQ ID NO: 2 or SEQ ID NO 10: and an HCDR3 region comprising the amino sequence of SEQ ID NO: 3. In an embodiment of the present disclosure, said aCD3-VL1 or aCD3-VL2 comprise a LCDR1 region comprising the amino sequence of SEQ ID NO: 4 or SEQ ID NO: 11 , a LCDR2 region comprising the amino sequence of SEQ ID NO: 5, and a LCDR3 region comprising the amino sequence of SEQ ID NO: 6.

[0063] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of a first and second polypeptides, wherein

[0064] (a) the first polypeptide comprises from its N-terminus to its C-terminus the heavy chain of Fab1 and either aCD3-VH1 or aCD3-VL1 and the second polypeptide comprises from its N-terminus to its C-terminus the light chain of Fab1 and GD1 ; or

[0065] (b) the first polypeptide comprises from its N-terminus to its C-terminus the heavy chain of Fab1 and GD1 and the second polypeptide comprises from its N-terminus to its C- terminus the light chain of Fab1 and either aCD3-VH1 or aCD3-VL1 .

[0066] In an embodiment of the present disclosure, HB1 further comprises a first Fc region. In an embodiment, said first Fc region is composed of a first and second Fc region subunit. In an embodiment, each Fc region subunit is composed of a CH2 and CH3 domain.

[0067] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the first Fc region is positioned between the C-terminus of the heavy chain of Fab1 and the N-terminus of aCD3-VH1 or aCD3-VL1. In an alternative embodiment, the first Fc region is positioned at the C-terminus of aCD3-VH1 or aCD3- VL1.

[0068] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of a first, second, and third polypeptide, wherein

[0069] (a) the first polypeptide comprises from its N-terminus to its C-terminus

[0070] (i) the heavy chain of Fab1 ,

[0071] (ii) either aCD3-VH1 or aCD3-VL1 ,

[0072] (iii) a first Fc region subunit, and

[0073] (b) the second polypeptide comprises from its N-terminus to its C-terminus, (i) GD1

[0074] (ii) a second Fc region subunit, and

[0075] (c) the third polypeptide comprises the light chain of Fab1 .

[0076] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of a first, second, and third polypeptide, wherein

[0077] (a) the first polypeptide comprises from its N-terminus to its C-terminus

[0078] (i) the heavy chain of Fab1 ,

[0079] (ii) GD1 ,

[0080] (iii) a first Fc region subunit, and

[0081] (b) the second polypeptide comprises from its N-terminus to its C-terminus,

[0082] (i) either aCD3-VH1 or aCD3-VL1

[0083] (ii) a second Fc region subunit, and

[0084] (c) the third polypeptide comprises the light chain of Fab1 .

[0085] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of a first, second, and third polypeptide, wherein

[0086] (a) the first polypeptide comprises from its N-terminus to its C-terminus

[0087] (i) the heavy chain of Fab1 ,

[0088] (ii) a first Fc region subunit,

[0089] (iii) either aCD3-VH1 or ocCD3-VL1 , and

[0090] (b) the second polypeptide comprises from its N-terminus to its C-terminus,

[0091] (i) a second Fc region subunit,

[0092] (ii) GD1 , and

[0093] (c) the third polypeptide comprises the light chain of Fab1 . In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 further comprises a third Fab fragment (Fab3) specific for the first antigen (AG1 ).

[0094] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of a first, second, third and fourth polypeptide, wherein

[0095] (a) the first polypeptide comprises from its N-terminus to its C-terminus

[0096] (i) the heavy chain of Fab1 ,

[0097] (ii) either aCD3-VH1 or aCD3-VL1 ,

[0098] (iii) a first Fc region subunit, and

[0099] (b) the second polypeptide comprises from its N-terminus to its C-terminus

[0100] (i) the heavy chain of Fab3,

[0101] (ii) GD1 ,

[0102] (iii) the second Fc region subunit, and

[0103] (c) the third polypeptide comprises the light chain of Fab1 , and

[0104] (d) the fourth polypeptide comprises the light chain of Fab3.

[0105] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB1 is composed of four polypeptides, wherein

[0106] (a) the first polypeptide comprises from its N-terminus to its C-terminus

[0107] (i) the heavy chain of Fab1 ,

[0108] (ii) a first Fc region subunit,

[0109] (iii) either ocCD3-VH1 or aCD3-VL1 , and

[0110] (b) the second polypeptide comprises from its N-terminus to its C-terminus

[0111] (i) the heavy chain of Fab3,

[0112] (ii) the second Fc region subunit (iii) GD1 , and

[0113] (c) the third polypeptide comprises the light chain of Fab1 , and

[0114] (d) the fourth polypeptide comprises the light chain of Fab3.

[0115] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein GD1 and aCD3-VH1 or GD1 and aCD3-VL1 , are positioned adjacent and parallel to each other on the first and second polypeptide.

[0116] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of a fifth and sixth polypeptide, wherein

[0117] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus the heavy chain of Fab2 and either aCD3-VH2 or aCD3-VL2 and the sixth polypeptide comprises from its N-terminus to its C-terminus the light chain of Fab2 and optionally GD2 or

[0118] (b) the fifth polypeptide comprises from its N-terminus to its C-terminus the heavy chain of Fab2 and optionally GD2 and the sixth polypeptide comprises from its N-terminus to its C-terminus the light chain of Fab2 and either aCD3-VH2 or aCD3-VL2.

[0119] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 further comprises a second Fc region composed of a third and fourth Fc region subunit, wherein each Fc region subunit is composed of an CH2 and CH3 domain.

[0120] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein the second Fc region is positioned between the C-terminus of the heavy chain of Fab2 and the N-terminus of aCD3-VH2 or aCD3-VL2; or is positioned at the C-terminus of aCD3-VH2 or aCD3-VL2, respectively.

[0121] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of a fifth, sixth and seventh polypeptide, wherein

[0122] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0123] (i) the heavy chain of Fab2,

[0124] (ii) either ocCD3-VH2 or ocCD3-VL2, (iii) a third Fc region subunit, and

[0125] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus,

[0126] (i) the optional GD2,

[0127] (ii) the fourth Fc region subunit, and

[0128] (c) the seventh polypeptide comprises the light chain of Fab2.

[0129] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of a fifth, sixth and seventh polypeptide, wherein

[0130] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0131] (i) the heavy chain of Fab2,

[0132] (ii) GD2,

[0133] (iii) a third Fc region subunit, and

[0134] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus,

[0135] (i) either ocCD3-VH2 or ocCD3-VL2,

[0136] (ii) the fourth Fc region subunit, and

[0137] (c) the seventh polypeptide comprises the light chain of Fab2.

[0138] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of three polypeptides, wherein

[0139] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0140] (i) the heavy chain of Fab2,

[0141] (ii) a third Fc region subunit,

[0142] (iii) either ocCD3-VH2 or ocCD3-VL2,

[0143] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus,

[0144] (i) a fourth Fc region subunit,

[0145] (ii) optionally GD2, and (c) the seventh polypeptide comprises the light chain of Fab2.

[0146] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 further comprises a fourth Fab fragment (Fab4) specific for the second antigen (AG2).

[0147] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of four polypeptides, wherein

[0148] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0149] (i) the heavy chain of Fab2,

[0150] (ii) either ocCD3-VH2 or ocCD3-VL2,

[0151] (iii) the third Fc region subunit, and

[0152] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus

[0153] (i) the heavy chain of Fab4,

[0154] (ii) the fourth Fc region subunit, and

[0155] (c) the seventh polypeptide comprises the light chain of Fab2, and

[0156] (d) the eight polypeptide comprises the light chain of Fab4.

[0157] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of four polypeptides, wherein

[0158] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0159] (i) the heavy chain of Fab2,

[0160] (ii) either ocCD3-VH2 or ocCD3-VL2,

[0161] (iii) the third Fc region subunit, and

[0162] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus

[0163] (i) the heavy chain of Fab4,

[0164] (ii) either aCD3-VH2 or aCD3-VL2, and (iii) the fourth Fc region subunit,

[0165] (c) the seventh polypeptide comprises the light chain of Fab2, and

[0166] (d) the eights polypeptide comprises the light chain of Fab4, wherein if the fifth polypeptide comprises an aCD3-VH2 then the sixth polypeptide comprises an aCD3-VH2, or wherein if the fifth polypeptide comprises an aCD3-VL2 then the sixth polypeptide comprises an aCD3-VL2.

[0167] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, wherein HB2 is composed of four polypeptides, wherein

[0168] (a) the fifth polypeptide comprises from its N-terminus to its C-terminus

[0169] (i) the heavy chain of Fab2,

[0170] (ii) the third Fc region subunit,

[0171] (iii) either aCD3-VH2 or ocCD3-VL2, and

[0172] (b) the sixth polypeptide comprises from its N-terminus to its C-terminus

[0173] (i) the heavy chain of Fab4,

[0174] (ii) the fourth Fc region subunit,

[0175] (iii) optionally either aCD3-VH2 or aCD3-VL2; or optionally GD2, and

[0176] (c) the seventh polypeptide comprises the light chain of Fab2, and

[0177] (d) the eights polypeptide comprises the light chain of Fab4, wherein if the fifths polypeptide comprises an aCD3-VH2 then the sixth polypeptide comprises an aCD3-VH2 or GD2, or wherein if the fifth polypeptide comprises an ocCD3-VL2 the sixth polypeptide comprises an aCD3-VL2 or GD2.

[0178] In an embodiment of the present disclosure, the first and / or the second Fc region comprises one or more amino acid modification promoting the association of the first and second Fc region subunit and / or of the third and fourth Fc region subunit.

[0179] In an embodiment of the present disclosure, the CH3 domain of first and / or third Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) and in the CH3 domain of the second and / or fourth Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A) and the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C); or vice versa, with numbering according EU index.

[0180] In an embodiment of the present disclosure, the first, the second, the third and the fourth Fc region subunit comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces its effector function, wherein said one or more amino acid mutations are selected from the group consisting of L234A, L235E, G237A, A330S and P331 S (EU numbering).

[0181] In an embodiment of the present disclosure Fab1 , aCD3-VH1 , aCD3-VL1 , GD1 , the first Fc region, and Fab3 on HB1 are linked to each other via peptide linkers. In an embodiment, Fab2, aCD3-VH2, ocCD3-VL2, GD2, the second Fc region, and Fab4 on HB2 are linked to each other via peptide linkers.

[0182] In an embodiment of the present disclosure, the peptide linker according to the present disclosure is an unstructured and / or flexible peptide linker. In an embodiment, said peptide linker is a non-cleavable peptide linker. In an embodiment, said peptide linker does not comprise a protease cleavage site, in particular a protease cleavage site cleavable by a cancer associated protease. In an embodiment, said peptide linker comprises an immunoglobulin hinge derived sequence. In an embodiment, said peptide linker is composed of the amino acid residues selected from the group of A, Q, D, P, H, G, S, E, T, K, and C. In an embodiment of the present disclosure, the peptide linker has a length of 1 - 50 amino acid residues.

[0183] In an embodiment, the present disclosure provides a complementary pair of halfbody molecules for the use as a medicament. In an embodiment, the present disclosure provides a complementary pair of halfbody molecules, for the use in the treatment of a disease associated with the undesired presence of AG1 and AG2. In an embodiment, the present disclosure provides a pharmaceutical composition comprising HB1 and a pharmaceutical acceptable carrier. In an embodiment, the present disclosure provides a pharmaceutical composition comprising HB2 and a pharmaceutical acceptable carrier. In an embodiment, the present disclosure provides a kit comprising the pharmaceutical composition comprising HB1 and the pharmaceutical composition comprising HB2.

[0184] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0185] Figure 1 : Complementary CyCAT halfbody molecules in the B027 format with a Fab fragment fused at the C-terminus of its heavy chain to the N-terminus to a single aCD3- variable domain (aCD3-SVD, either aCD3-VH or aCD-VL).

[0186] Figure 2: Fig. 2A - 2F’: Schematic representation of various CyCAT halfbody formats according to the present disclosure lacking a Guard-Domain (GD). Each halfbody format is shown either carrying aCD3-VH domain(s) or aCD3-VH domain(s).

[0187] Fig. 2A: CyCAT halfbody molecules in the B036 format comprising a Fab fragment, a single aCD3-variable domain (aCD3-SVD) and an Fc region comprising Knobs-into-Holes (KiH) mutations. The halfbody molecule has a main chain in which the single aCD3-VH domain (Fig. 2A’) or single aCD3-VL domain (Fig. 2A) is fused at its N-terminus to the heavy chain of the Fab and at its C-terminus to the Fc region subunit carrying the Knobmutations.

[0188] Fig. 2B: CyCAT halfbody molecules in the B063 format comprising two identical Fab fragments, a single aCD3-SVD and a Fc region comprising KiH mutations. The halfbody molecule has a main chain in which the aCD3-VH (B’) or aCD3-VL domain (B) is fused at its N-terminus to the heavy chain of one Fab, and at its C-terminus to the Fc region subunit carrying the Knob-mutations. The second Fc region subunit carrying the Hole mutations in its CH3 domain is coupled at its N-terminus to the heavy chain of the second Fab.

[0189] Fig. 2C: CyCAT halfbody molecules in the B039 format comprising two identical Fab fragments, two identical aCD3-SVDs and one Fc region. The halfbody molecule has two identical main chains, in which one aCD3-VH domain (C’) or one aCD3-VL domain (C) is fused at its N-terminus to the heavy chain of one Fab and at its C-terminus to one Fc region subunit. In addition, a second identical aCD3-VH domain (C’) or second identical aCD3-VL domain (C), respectively, is fused at its N-terminus to the heavy chain of the second Fab and at its C-terminus to the second Fc region subunit. Based on its symmetrical structure, this format is void of Knob-into-Hole (KiH) mutations in its Fc region.

[0190] Fig. 2D: CyCAT halfbody molecules in the B038 format comprising one Fab fragment, one Fc region comprising KiH mutations and one single aCD3-SVD. This halfbody molecule has a main chain in which the Fc region subunit carrying the Knob mutations is fused at its N-terminus to the heavy chain of the Fab fragment and at its C-terminus to the single aCD3-VH domain (D’) or single aCD3-VL domain (D).

[0191] Fig. 2E: CyCAT halfbody molecules in the B064 format comprising two identical Fab fragments, one Fc region comprising KiH mutations and a single aCD3-SVD. The halfbody molecule has one main chain in which the Fc region subunit carrying the Knob mutations is coupled at its N-terminus to the heavy chain of one Fab and at its C-terminus to the single aCD3-VH domain (E’) or single aCD3-VL domain (E), respectively. The Fc region subunit carrying the Hole mutations is fused at its N-terminus to the heavy chain of the second Fab fragment.

[0192] Fig. 2F : CyCAT halfbody molecules in the B050 format comprising two identical Fab fragments, one Fc region and two identical aCD3-SVDs. The halfbody molecule has two identical main chains, in which one Fc region subunit is fused at its N-terminus to the heavy chain of one Fab and at its C-terminus to either one aCD3-VH domain (F’) or one aCD3-VL domain (F) and in which the other Fc region subunit is coupled at its N-terminus to the heavy chain of the second Fab fragment and at its C-terminus to the second identical aCD3-VH domain (F’) or second identical aCD3-VL domain (F), respectively. Based on its symmetrical structure, this format is void of KiH mutations in its Fc region.

[0193] Figure 3: Schematic representation of various CyCAT halfbody formats according to the invention comprising a Guard-Domain (GD). In the present examples, inactive antibody variable domains (GDVH, GDvH-Knob or GDVL) or HSA (GDHSAWO or Domain III of HSA (GDHSA-DIII) were used as Guard-Domains. In case, a Guard-Domain was selected of being an inactive antibody variable domain, it was selected of being of the same variable chain type as the aCD3-SVDs, i.e. in case of an aCD3-VL domain a GDVL was used; and in case of a aCD3-VH domain, a GDVH was used.

[0194] Fig. 3A: CyCAT halfbody molecules in the B073 format, encompassing a Fab fragment, an aCD3-SVD, a single Guard-Domain and an Fc region comprising Knob-into-Hole mutations. The halfbody molecule has a main chain in which the aCD3-VH domain (A’) or aCD3-VL domain (A) is coupled via its N-terminus to the heavy chain of a Fab fragment and via its C-terminus to the Fc region subunit carrying the Knob mutations. The Guard- Domain is coupled via its C-terminus to the second Fc region subunit carrying the Hole mutations, so that the respective aCD3-SVD and the GD are oriented parallel and adjacent to each other.

[0195] Fig. 3B: CyCAT halfbody molecules in the B099 format as an alternative embodiment to the B073 format. The aCD3-SVD domains and the single GD are swapped for each other on the two polypeptide chains. The halfbody molecule has a main chain in which the GD is coupled via its N-terminus to the heavy chain of a Fab fragment and via its C-terminus to the Fc region subunit carrying the Hole mutations. The aCD3-VH domain (B’) or aCD3- VL domain (B) is in turn coupled via its C-terminus to the second Fc region subunit carrying the Knob mutations.

[0196] Fig. 3C: CyCAT halfbody molecules in the B077 format comprising two Fab fragments, a single aCD3-SVD, a single Guard-Domain, and one Fc region. The halfbody molecule has two non-identical main chains, in which one aCD3-VH domain (C’) or one aCD3-VL domain (C) is coupled via its N-terminus to the heavy chain of one Fab fragment and via its C-terminus to one Fc region subunit carrying the Knob-Mutations. The single GD is coupled via its N-terminus to the heavy chain of the second Fab fragment and via its C- terminus to the second Fc region subunit carrying the Hole mutations, so that the respective aCD3-SVD and the GD are oriented parallel and adjacent to each other.

[0197] Fig. 3D: CyCAT halfbody molecules in the B103 format comprising one Fab fragment, an Fc region, a single aCD3-SVD and a single Guard-Domain (GD). The halfbody molecule has one main chain in which the Fc region subunit carrying the Knob mutations is coupled via its N-terminus to the heavy chain of the Fab fragment and via its C-terminus to the single aCD3-VH domain (D’) or single aCD3-VL domain (D). The GD is coupled via its N- terminus to the C-terminus of the Fc region subunit carrying the Hole mutations, so that the single aCD3-SVD and the GD are oriented parallel and adjacent to each other.

[0198] Fig. 3E: CyCAT halfbody molecules in the B101 format comprising two Fab fragments, one Fc region, a single aCD3-SVD and a single Guard-Domain. The halfbody molecule has two main chains, in which within one main chain, the Fc region subunit carrying the Knob mutations is coupled via its N-terminus to the heavy chain of one Fab fragment and via its C-terminus to the single aCD3-VH domain (E’) or single aCD3-VL domain (E), respectively. Within the second main chain, the Fc region subunit carrying the Hole mutations is coupled via its N-terminus to the heavy chain of the second Fab fragment and via its C-terminus to the Guard-Domain, so that the aCD3-SVD and the GD are oriented parallel and adjacent to each other.

[0199] Figure 4: Illustration of complex formation of a pair of two complementary CyCAT halfbody molecules, wherein one of the two halfbody molecules comprises a Guard- Domain. Co-localization of two complementary halfbody molecules on a target cell results in displacement of the GD and formation of a functional Fv binding fragment being able to bind to CD3 on the surface of a T-cell.

[0200] Figure 5: Exemplary results of cytotoxicity assays to determine the assay window for a Dual-Targeting halfbody pair. Shown are results according to Table 8 for Dual Targeting Halfbody Pair: 1 , Mono-Targeting Halfbody Pair: 2, Dual Targeting Halfbody Pair: 3 and Mono-Targeting Halfbody Pair: 4.

[0201] DETAILED DESCRITPION OF THE INVENTION

[0202] Definitions

[0203] As used herein, the terms “first”, “second”, “third”, “fourth”, “fifth”, “sixth” “seventh”, “eights” and so on, with respect to an target antigen, halfbody, antibody, antibody fragment, Fab, Fv region, Fc region, Fc region subunit, peptide linker, or polypeptide are used for distinguishing when there is more than one of each type of a component. Use of these terms is not intended to confer a specific order or orientation unless explicitly so stated.

[0204] “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.

[0205] 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.

[0206] The term “antigen” or “target antigen” as used herein refers to any molecule of interest that can be bound by one of the binding sites present in an antibody. Typically, an antigen is a peptide, a protein or any other proteinaceous molecule. Alternatively, an antigen may be any other organic or inorganic molecule, such as carbohydrate, fatty acid, lipid, dye or fluorophore.

[0207] As used herein, “amino acid residues” or “amino acid” will be indicated either by their full name or according to the standard three-letter or one-letter amino acid code. “Natural occurring amino acids” means the following amino acids: Table 1 : Natural occurring amino acids

[0208] The term “antibody” molecule or “immunoglobulin” (Ig) molecule used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains, which interacts with an antigen. Each heavy chain (HC) is comprised of a heavy chain variable domain (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain (LC) is comprised of a light chain variable domain (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 domains 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 N-terminus to C-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the heavy and light chains (VH and VL) contain or form a “binding site” or “antigen binding site” that selectively interacts with or binds to 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., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology. 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, 5thedit., 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. The term “antibody” as used herein is intended to include antibody fragments, CyCAT molecules, halfbody molecules, monospecific specific antibodies as well as bispecific and multispecific antibodies.

[0209] The term “antibody fragment” of an antibody, 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 antibody fragments include, but are not limited to, a Fab fragment (“Fab”), a monovalent fragment consisting of the VL, VH, CL and CH1 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 CH1 domains; a Fv fragment (“Fv”) 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 domains 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”. 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 23:1126-1136). 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 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).

[0210] As used herein, the term “binding site” or “antigen binding site” or “antigen binding region” refer to a structure formed by a protein that is capable of binding or specifically binding to an antigen. The binding site need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain. For example, in a Fv produced from two different polypeptide chains the “binding site” is made up of a series of amino acids of a VL and a VH that interact with the antigen and that are generally, however not always, in the one or more of the CDRs in each variable region. In certain embodiments, a “binding site” is or comprises or is formed by a complementary antibody variable heavy (VH) and light chain (VL) pair. The VH and the V which form the binding site can be in a single polypeptide chain or in different polypeptide chains. In preferred embodiments, the binding site is or comprises or is formed by a VH present on a first halfbody molecule according to the present disclosure and the complementary VL is present on the second halfbody molecule according to the present disclosure, or vice versa. In some embodiments, the “binding site” has one VH and one VL. In certain embodiments, the binding site comprises one or more CDRs of an antibody. In other embodiments, a binding site is derived from an antibody mimetic, such as for instance from an affibody molecule, alphabody, anticalin, avimer, DARPin, fynomer, Kunitz domain peptide, helix-turn-helix peptide, or monobody. As used herein the term “binds specifically to”, “specifically binds to”, is “specific to / for” or “specifically recognizes”, or the like, refers to measurable and reproducible interactions such as binding between a target antigen and an antibody, antibody fragment or halfbody molecule disclosed herein, which is determinative of the presence of the target antigen in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody, antibody fragment or halfbody molecule disclosed herein that specifically binds to a target antigen (which can be an antigen or an epitope of an antigen) is an antibody, antibody fragment, or halfbody molecule that binds this target with greater affinity, avidity, more readily, and / or with greater duration than it binds to other target antigens. In certain embodiments, an antibody, antibody fragment or halfbody molecule specifically binds to an epitope on a protein that is conserved among the protein from different species. In another embodiment, specific binding can include, but does not require exclusive binding. The antibodies, antibody fragments or halfbody molecules disclosed herein specifically bind to antigens. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, a standard ELISA assay. The scoring may be carried out by standard color development (e.g. secondary antibody with horseradish peroxide and tetramethyl benzidine with hydrogen peroxide). The reaction in certain wells is scored by the optical density, for example, at 450 nm. Typical background (=negative reaction) may be 0.1 OD; typical positive reaction may be 1 OD. This means the difference positive / negative can be more than 5-fold. Typically, determination of binding specificity is performed by using not a single reference antigen, but a set of three to five unrelated antigens, such as milk powder, BSA, transferrin or the like.

[0211] “CD3” refers to an antigen which is expressed on T cells as part of the multimolecular T cell receptor (TCR) and which consists of a homodimer or heterodimer formed from the association of two of four receptor chains: CD3epsilon (CD3e), CD3delta, CD3zeta, and CD3gamma.

[0212] Human CD3epsilon (or human CD3e) has the amino acid sequence of UniProt P07766: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYP GSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPE DANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWS A / R A A PVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 19) (signal sequence underlined, intracellular region italic, transmembrane region bold).

[0213] The mature extracellular domain of human CD3epsilon without signal sequence comprises the amino acid sequence of:

[0214] QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNI GSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 20)

[0215] Cynomolgus monkey CD3epsilon (or cynomolgus CD3e or cyno CD3e) has the amino acid sequence of UniProt Q95LI5:

[0216] MQSGTRWRVLGLCLLSIGVWGQDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQ WQHNGKNKEDSGDRLFLPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCM EMDVMAVATIVIVDICITLGLLLLVYYWSKA / RKAKAKPYTRGAGAGGRQRGQA / KERPP PVPNPDYEPIRKGQQDLYSGLNQRRI (SEQ ID NO: 21 ) (signal sequence underlined, intracellular region italic, transmembrane region bold).

[0217] The mature extracellular region of cynomolgus monkey CD3 epsilon without the signal sequence comprises has the amino acid sequence of:

[0218] QDGNEEMGSITQTPYQVSISGTTVILTCSQHLGSEAQWQHNGKNKEDSGDRLF LPEFSEMEQSGYYVCYPRGSNPEDASHHLYLKARVCENCMEMDVMAVATIVIV DICITLGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYE PIRKGQQDLYSGLNQRRI (SEQ ID NO: 22).

[0219] The term “chimeric antibody” or “chimeric antibody fragment” is defined herein as an antibody 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. The term “CyCAT halfbody molecule”, “halfbody molecule” or “HB”, or “halfbody” as used herein, refers in its broadest sense to an antigen binding molecule that specifically binds to at least one target antigen and which is composed of an antibody fragment, such as a Fab fragment and either the VH or VL of an antibody Fv fragment with specificity for CD3. Accordingly, an halfbody molecule according to the preset disclosure incorporates one half antibody Fv binding fragment with specificity for CD3. The essential features of halfbody molecules according to the present disclosure are described in WO2013 / 104804 (JULIUS-MAXIMILIANS-UNIVERSITAT WURZBURG), which is incorporated herein in its entirety.

[0220] The term “CyCAT molecule” as used herein refers to a functional complex of two complementary CyCAT halfbody molecules as described herein wherein each halfbody body molecule carries one half of an antibody Fv fragment with specific for CD3 and wherein only said complex is functional in respect to the function of said Fv fragment but not the individual CyCAT halfbody molecules by themselves.

[0221] “Complementary” CyCAT halfbody molecule(s) as used herein, refers to any complex or pair of two CyCAT halfbody molecules as described herein; provided that one CyCAT halfbody molecule carries at least one single aCD3-VH or aCD3-VL domain and the second CyCAT halfbody molecule carries at least the complementary unpaired aCD3-VL or aCD3-VH domain, respectively, of an aCD3-Fv fragment. Dimerization and functional complementation of the aCD3-VH and aCD3-VL domain occurs upon binding of the two complementary CyCAT halfbody molecules to their target antigen present on the surface of the same target cell.

[0222] As used herein, “covalent bond” refers to an interatomic bond characterized by sharing of electrons.

[0223] The term “ECso” as used herein, refers to the concentration of an antibody or an antibody fragment or CyCAT molecule which induces a response in an assay half way between the baseline and maximum. It therefore represents the concentration at which 50% of the maximal effect is observed.

[0224] A “human antibody” or “human antibody fragment” as used herein, includes antibodies and antibody fragments 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). 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, 5thedit., 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. Human antibodies and human variable regions can also be isolated from synthetic libraries or from transgenic mice (e.g. xenomouse) 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.

[0225] A “humanized antibody” or “humanized antibody fragment” is defined herein as an antibody 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. Humanization may be achieved by various methods including, but not limited to (a) grafting the non-human (e.g., donor antibody) CDRs onto human (e.g. recipient antibody) framework and constant regions with or without retention of critical framework residues (e.g. those that are important for retaining good antigen binding affinity or antibody functions), (b) grafting only the non-human specificity-determining regions (SDRs or a-CDRs; the residues critical for the antibody-antigen interaction) onto human framework and constant regions, or (c) transplanting the entire non-human variable domains, but “cloaking” them with a human-like section by replacement of surface residues. Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); US Patent Nos. 5,821 ,337, 7,527,791 , 6,982,321 , and 7,087,409; Jones et al., Nature 321 , 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81 , 6851 -6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al, Science 239, 1534-1536 (1988); Padlan, Molec Immun 31 (3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991 ) (describing “resurfacing”); Dall’Acqua et al, Methods 36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al, Methods 36, 61 -68 (2005) and Klimka et al, Br J Cancer 83, 252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0226] The term “ICso” as used herein, refers to the concentration of an antibody or antibody fragment or CyCAT molecule that inhibits a response in an assay half way between the maximal response and the baseline. It therefore represents the concentration that reduces a given response by 50%.

[0227] The term “isolated” refers to a compound, which can be e.g. an antibody, antibody fragment or halfbody molecule, that is substantially free of other antibodies, antibody fragments or halfbody molecules having different antigenic specificities. Moreover, an isolated antibody, antibody fragment or halfbody molecule may be substantially free of other cellular material and / or chemicals. Thus, in some embodiments, the antibodies, antibody fragments or halfbody molecules provided herein are isolated antibodies, antibody fragments or halfbody molecules that have been separated from antibodies or halfbody molecules with a different specificity. An isolated antibody or halfbody molecule may be a monoclonal antibody, antibody fragment or halfbody molecule. An isolated antibody, antibody fragments or halfbody molecule may be a recombinant monoclonal antibody, antibody fragment or halfbody molecule. An isolated antibody, antibody fragment or halfbody molecule that specifically binds to an epitope, isoform or variant of a target may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., species homologs).

[0228] The terms “inhibition” or “inhibit” or “reduction” or “reduce” or “neutralization” or “neutralize” refer to a decrease or cessation of any phenotypic characteristic (such as binding, a biological activity or function) or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. The “inhibition” needs not to be complete as long as it is detectable using an appropriate assay. In some embodiments, by “reduce” or “inhibit” is meant the ability to cause a decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause a decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.

[0229] The term “KD”, as used herein, refers to the dissociation constant, which is obtained from the ratio of Kd to Ka(i.e. Kd / Ka) and is expressed as a molar concentration (M). KD values for antigen binding moieties like e.g. monoclonal antibodies can be determined using methods well established in the art. Methods for determining the KD of an antigen binding moiety like e.g. a monoclonal antibody are SET (soluble equilibrium titration), surface plasmon resonance using a biosensor system such as a Biacore® system or Biolayer Interferometry (BLI).

[0230] As used herein, the term “monoclonal antibody”, “monoclonal antibody fragment” or ’’monoclonal halfbody molecule” refers to an antibody, antibody fragment or halfbody molecule disclosed herein that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies or antibody fragments may be made by the hybridoma method as described in Kohler et a / .; Nature, 256:495 (1975) or may be isolated from phage libraries. Other methods for the preparation of clonal cell lines and monoclonal antibodies or halfbody molecule as disclosed herein expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5thEd., Ausubel et al., eds., John Wiley and Sons, New York).

[0231] The term “multispecific” means that an antibody, halfbody molecule or CyCAT molecule or is able to specifically bind to two or more different antigens. Typically, a multispecific antibody, halfbody molecule or CyCAT molecule comprises of two or more antigen binding sites, each of which is specific for a different antigen or epitope. The term “bispecific” means that an antibody, halfbody molecule or CyCAT molecule is able to specifically bind to two different antigens. Typically, a bispecific antibody or halfbody molecule or CyCAT molecule comprises two antigen binding sites, each of which is specific for a different antigen or epitope. The term “trispecific” means that an antibody or halfbody molecule or CyCAT molecule is able to specifically bind to three different antigens. Typically, a trispecific antibody, halfbody molecule or CyCAT molecule comprises three antigen binding sites, each of which is specific for a different antigen or epitope.

[0232] The term "pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical composition, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0233] As used herein, “non-covalent association” refers to molecular interactions that do not involve an interatomic bond. Noncovalent interactions involve, for example, ionic bonds, hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0234] The term "pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

[0235] The term "polypeptide” as used herein refer to a polymer of amino acid residues and does not refer to a specific length of a product. The term applies to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular amino acid sequence of a polypeptide also implicitly encompasses conservatively modified variants thereof (e.g. by replacing an amino acid residue with another amino acid residue having similar structural and / or chemical properties). A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including chemical synthesis. A polypeptide may also comprise one or more disulfide bonds.

[0236] The term “recombinant antibody”, “recombinant antibody fragment” or “recombinant halfbody molecule”, as used herein, includes all antibodies, antibody fragments or halfbody molecules according to the present disclosure that are prepared, expressed, created or segregated by means not existing in nature. For example, antibodies or halfbody molecules isolated from a host cell transformed to express the antibody or halfbody molecule, 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 or halfbody molecules 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 or halfbody molecules 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 or halfbody molecules 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 or halfbody molecule may be a recombinant monoclonal antibody or a recombinant monoclonal halfbody molecule. In an embodiment, the antibodies and antibody fragment disclosed herein are isolated from the Ylanthia® antibody library as disclosed in US 13 / 321 ,564 or US 13 / 299,367, which both herein are incorporated by reference.

[0237] “Species”, as used in herein, refers to any mammal, including rodents, such as mouse or rat, and primates, such as cynomolgus monkey (Macaca fascicularis), rhesus monkey (Macaca mulatta) or humans (Homo sapiens). Preferably the subject is a primate, most preferably a human.

[0238] A “therapeutically effective amount” or “effective amount” of an agent, e g. a pharmaceutical composition, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of an agent for example eliminates, decreases, delays, minimizes or prevents adverse effects of a disease.

[0239] As used herein, “treatment”, “treat” or “treating” or the like refers to clinical intervention in an attempt to alter the natural course of a disease in the individual 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, halfbody molecules according to the preset disclosure are used to delay development of a disease or to slow the progression of a disease.

[0240] Embodiments

[0241] The present invention is directed to methods of reducing the toxicity and on or off-target side effects of bispecific or multispecific antibodies (including antibody-like functional proteins or biologies) that co-engage T-cells for tumor cell destruction by activating their CD3 binding capabilities only in the vicinity of the cancerous tissue, to avoid off- and on- target interactions. The combinatorial approach of the present invention uses two complementary bifunctional halfbody molecules, each of them targeting two different tumor-associated antigens, each of the two CyCAT halfbody molecules equipped with one half of an aCD3-Fv binding fragment, more particular, with its VH or VL, respectively.

[0242] Complementary CyCAT halfbody molecules as used herein, refers to any pair of two CyCAT halfbody molecules as described herein; provided that one CyCAT halfbody molecule carries at least one single aCD3-VH or aCD3-VL domain and the second CyCAT halfbody molecule carries at least one single complementary unpaired aCD3-VH or aCD3-VL domain, respectively, of an aCD3 Fv fragment, which upon dimerization form a functional aCD3-Fv binding fragment. Only when both complementary CyCAT halfbody molecules bind to the same target cell expressing both target antigens on its cell surface, a functional VHA / L CD3 specificity is generated by on cell complementation. This combinatorial principle enhances the specificity of the targeting- as well as of the effectorprocess and therefore solves the central problem of T cell activating bispecific antibodies.

[0243] Accordingly, the present invention is directed to complementary pairs of halfbody molecules (each of such halfbody molecule also referred herein as to an “CyCAT halfbody molecule” or “CyCAT halfbody” or “halfbody” or “halfbody molecule” or just “HB”). Each halfbody molecule is capable of binding to a target antigen expressed on the surface of a cell (such as a tumor associated antigen expressed on a cancerous cell) but by themselves are not able to recruit T-cells for tumor cell destruction, as they carry only one half of a functional aCD3-Fv antibody fragment. Each halfbody according to the present disclosure has a number of functional protein components which may be fused to each other via peptide linkers. Another component is one single or two identical antibody variable domain(s) (such as a one single VH domain or two identical VH domains or one single VL domain or two identical VL domains) of an CD3 specific antibody, that would bind to CD3 when it gets in close proximity of its complementary or cognate antibody variable domain to form the functional aCD3 Fv binding domain. In this respect the active CD3 binding domain (and thus the trispecific antibody of the present disclosure) is formed on the surface of a tumor cell expressing both targeted tumor antigens. A further component of such halfbody molecule may comprise an IgG Fc region which allows for an extended half-life. The IgG Fc region may further comprise an immunoglobulin hinge region at its C-terminus which further stabiles the heterodimeric Fc region by formation of disulfide bond linkage.

[0244] Guard-Domains and Halfbodies comprising such Guard-Domains

[0245] To increase the therapeutic safety profile for the CyCAT approach described herein, the present invention aims to minimize the amount of any complex formation between two complementary halfbody molecules in absence of a target cell having both target antigens of interest on their cell surface. Even small amounts of such complexes or aggregates may lead to on-target as well as off-target T-cell activation. It is therefore of importance to avoid any such unwanted heterodimerization by the selection of suitable halfbody structures. Such on-target and off-target induced heterodimerization may be caused by the intrinsic low affinity binding (hydrophobic interaction) of antibody VH and VL domains under local high concentration conditions of complementary CyCAT halfbody molecules. Such a condition may occur for instance right after or during simultaneous administration of highly concentrated halfbody compositions. To accomplish such increase of the “assay window” or “therapeutic window”, the present invention utilizes at least one Guard-Domain (GD) which is covalently fused to at least one of the two complementary halfbody molecules.

[0246] A “Guard-Domain” us used in the context of the present disclosure is positioned in such way on a halfbody that it sterically blocks or shields the unpaired aCD3-VH or aCD3-VL domain, from binding to its complementary unpaired aCD3-SVDs (aCD3-VH or aCD3- VL) present on the second halfbody molecule, in the absence of a cell expressing both target antigens of interest on its cell surface. However, this shielding effect of a Guard- Domain as described herein is only effective as long as no target cells expressing both target antigens are present; but becomes ineffective once two complementary CyCAT halfbody molecules bind to their target antigens on the surface of such target cell. Based on the evidence provided in the examples herein, it is suggested that a GD is displaced from the shielded aCD3-SVD once two complementary halfbody molecules come in close proximity to each other on the surface of a cell expressing both target antigens, thus allowing the formation of a functional aCD3-Fv domain (see also Figure 4 of the present application). In this sense, a GD does not function as a specific binding or mispairing partner for an aCD3-SVD to be shieled and thus does not require its entire release from the halfbody molecule, for instance by proteolytic cleavage.

[0247] The halfbody molecules according to the present disclosure are formatted in such way that the complementary aCD3-VH and aCD3-VL domains show minimal association / dimerization in solution in absence of a target cell over a broad concentration range, but unhindered association / dimerization in presence of such target cell without the need of releasing the GD domain from the halfbody molecule(s). As mentioned above, this is achieved in a manner other than by binding of the Guard Domain to the unpaired aCD3-SVD, such as by virtue of its size and proximity.

[0248] Accordingly, a Guard-Domain according to the present disclosure may be any protein or polypeptide that is of sufficient molecular size to provide steric blockade of the aCD3-SVD when positioned to a site near to such aCD3-SVD to be shielded, but neither inhibits or interferes with the specific binding of the targeting Fab fragment nor does the GD binds to any other component of a halfbody molecule to which it is attached to. In particular, it neither binds to or associates with an aCD3-SVD present on the same halfbody molecule which is to be shielded, nor does it form a functional Fv domain with any other component present on either of the two complementary halfbody molecules.

[0249] In an embodiment of the present disclosure, the GD of the present disclosure does not bind to the either VH (aCD3-VH) or VL (ocCD3-VL) of a CyCAT halfbody molecule as described herein. In an embodiment, a GD does not specifically bind to the aCD3-VH or aCD3-VL of the Fv specific for CD3 present on the same CyCAT halfbody molecule. In an embodiment of the present disclosure, the GD does not bind to an aCD3-SVD. In an embodiment, the GD does not specifically bind to an aCD3-SVD. In an embodiment, the GD does not specifically bind to an aCD3-VH or aCD3-VL. By does not bind, the present disclosure does not exclude nonspecific binding or low levels of residual binding (for example, < 1 %, <5%, <10%).

[0250] In an embodiment of the present disclosure, the GD is present in, part of, or fused to one of the two complementary halfbody molecules according to the present disclosure. In an embodiment, said GD is present in, part of, or fused to each one of the two complementary halfbody molecules according to the present disclosure. In an embodiment, the GD does not bind to any component of a CyCAT halfbody molecule. In an embodiment, the GD does not bind to any component of an halfbody molecule to which it is fused to. In an embodiment, said component is selected from the group of: the targeting moiety, such as a Fab fragment, an antibody variable region, an antibody constant domain, a Fc region, a Fc region subunit, a peptide linker.

[0251] Guard-Domains according to the present disclosure are used in several different ways, depending on the structure of a CyCAT halfbody molecule, as is described herein and as shown in the Figure 3.

[0252] In case a CyCAT halfbody molecule is intended to be used in humans, a GD is preferably selected of being a human protein or is mainly composed of a human protein, in order to minimize the risk of an immunogenic reaction in humans.

[0253] In some embodiments, a GD domain according to the present disclosure is a protein. In some embodiments, a GD is a polypeptide. In some embodiments, a GD is a human protein or human polypeptide or is a protein or polypeptide of human origin or is derived from a human protein or human polypeptide or is composed of mainly a human protein or human polypeptide.

[0254] In some embodiments, the GD according to the present disclosure has a molecularweight of between 10 kDa to 110 kDa, such as 20 kDa - 100 kDa, such as 25 kDa to 90 kDa, 30 kDa to 85 kDa, 35 kDa to 75 kDa, 40 kDa to 70 kDa. In some embodiments, the GD has a molecular weight of less than 100 kDa, such as 95 kDa, 90 kDa, 85 kDa, 80 kDa, 75 kDa, 70 kDa, 65 kDa, 60 kDa, 55 kDa, 50 kDa, 45 kDa, 40 kDa, 35 kDa, 30 kDa, 25 kDa, 20 kDa, 15 kDa or 10 kDa. In an embodiment, a GD of the present disclosure has a molecular weight of less than 70 kDa. In an embodiment, a GD of the present disclosure has a molecular weight of about 10kDa to about 70 kDa.

[0255] Examples for Guard-Domains

[0256] Examples of GDs suited for the present purpose of shielding single aCD3-VH or aCD3- VL domains from binding to each other, include globular proteins, such as serum albumin, transferrin (Trf), and immunoglobulin domains.

[0257] In an embodiment, the GD is a water soluble globular protein or spheroprotein. In an embodiment, the GD is selected from the group of but not limited to: serum albumin (SA), transferrin (Trf), portions or fragments of serum albumin or transferrin, and an immunoglobulin domain. In an embodiment, said immunoglobulin domain is an antibody variable domain, such as an VH or VL domain, or an IgG constant domain, such as a CH1 , CH2, CH3 or CH4 domain. In an embodiment, said GD is an antibody variable domain. In an embodiment, said GD is serum albumin, preferably, human serum albumin (HSA) or a fragment thereof.

[0258] Antibody variable domains as Guard-Domains

[0259] In an embodiment, the GD according to the present disclosure is an antibody variable domain (GDVD). There are a number of GDVDS that may find use in the present invention.

[0260] A GD or GDVD may comprise either a VH or a VL with framework regions, preferably human framework regions or human consensus framework regions, and “inactive” CDRs, which when paired with a complementary antibody variable domain, would not form an functional antibody Fv domain being able to specifically bind to any target antigen. In an embodiment, the GD is an inactive antibody variable domain (GDVD). In an embodiment, the GD is an inactive antibody variable heavy chain domain (GDVH). In an embodiment, the GD is an inactive antibody variable light chain domain (GDVL). In order to prevent the intramolecular formation of inactive Fv fragments (i.e. unable of specifically binding to a target antigen) via dimerization of an aCD3-VH with an GDVL or of an aCD3-VL with an GDVH, one halfbody molecule preferably utilizes the same chain type of antibody variable domains. Possible combinations are thus of the type: aCD3-VH with GDVH or aCD3-VL with GDVL. In some embodiments, when either the VH (aCD3-VH) or either the VL (aCD3-VL) domain of the Fv specific for CD3 (ocCD3-Fv) is a ocCD3-VH then the GD is a GDVH; and when the ocCD3-VH or ocCD3-VL is a VL, then the GD is a GDVL. In some embodiments, when the ocCD3-SVD is a ocCD3-VH, the GD is a GDVH and when the ocCD3-SVD is a ocCD3-VL, the GD is a GDVL. In case a GD is selected of being a GDVD, it may have the same sequence or a different sequence than the aCD3-VH oraCD3-VL to be shielded. Preferably, a GDVD is selected of having a different sequence than the aCD3-SVD. In an embodiment, the GDVD has the same sequence as the ocCD3-SVD. In an embodiment, the GDVD has a different sequence than the aCD3-SVD.

[0261] Accordingly, a GDVH and / or GDVL each independently may comprise one or more mutations that prevent specific binding to a target antigen of such Guard-Domain, either by (i) themselves or (ii) when paired with a complementary inactive variable domain, or (iii) when paired with an active aCD3-SVD.

[0262] The Fv fragment formed as described under (i) to (iii) above may be referred herein as an “inactive Fv fragment” or “Fvinactive; that is an Fv fragment formed between (a) a GDVH and a GDVL, (b) a GDVH and an ocCD3-VL, or (c) an ocCD3-VH and a GDVL.

[0263] Due to the combinatorial use of the same type of antibody variable chains for a aCD3- SVD and GD on the same halfbody molecule (i.e. aCD3-VH with GDVH or OCCD3VL with GD-VL) in conjunction of using inactive CDRs within a GDVD, a halfbody molecule according to the present disclosure by itself could not bind to CD3.

[0264] In addition, a GDVH domain present on one halfbody molecule and a GDVL present on a complementary halfbody molecule could only form an inactive Fv domain incapable of binding to a target antigen when associated with each other. Preferred GDVH and GDVL according to the present disclosure are formed by mutation of a wild type or parental VH or VL domain sequence as more outlined below. Exemplary mutations are within CDR1 , CDR2 or CDR3 regions of the respective VH or VL domain.

[0265] In a preferred embodiment, the sequence of the framework region of a GDVD is identical or aligns to the sequence of the framework region of the aCD3-SVD to be shielded. That is for example, if the framework region of an aCD3-VH comprise germline protein sequences of VH1-18 then the GDVH framework region also comprises the germline protein sequence of VH1-18. The “germline protein sequences” or “germline amino acid sequences” of antibodies encoded by the germline genes are for example disclosed in the following publications: for VH: Tomlinson et al., (1992), “The Repertoire of Human Germline Vh Sequences Reveals about Fifty Groups of Vh Segments with Different Hypervariable Loop” J. Mol. Biol. 227, 776-798; Matsuda et al. (1998); “The complete nucleotide sequence of the human immunoglobulin heavy chain variable region locus” J Exp Med 188(11 ):2151-62; and LeFranc MP (2001 ) “Nomenclature of the human immunoglobulin heavy (IGH) genes.” Exp Clin Immunogenet. 18(2):100-16; for VA: Kawasaki et al., (1997) “One-Megabase Sequence Analysis of the Human immunoglobulin lambda Gene Locus” Genome Research 7(3):250-61 ; Frippiat et al., (1995) “Organization of the human immunoglobulin lambda light-chain locus on chromosome 22q11.2” Hum. Mol. Genet., 4, 983-991 ; and LeFranc MP (2001 ) “Nomenclature of the human immunoglobulin lambda (IGL) genes. Exp Clin Immunogenet.; 18:242-254; and for VK: Schable and Zachau (1993), “The variable genes of the human immunoglobulin kappa locus,” Biol. Chem Hoppe Seyler. 374(11 ):1001-22; Brensing-Kuppers et al. (1997), “The human immunoglobulin kappa locus on yeast artificial chromosomes (YACs)” Gene. 191 (2): 173-81 ; Kawasaki et al. (2001), “Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes” Eur J Immunol 31 (4): 1017-28; and Lefranc MP (2001) “Nomenclature of the human immunoglobulin kappa (IGK) genes” Exp Clin Immunogenet., 18, 161-174, which are all hereby incorporated by reference in their entireties.

[0266] In some embodiments, the generation of inactive GDVD according to the present disclosure is generally done by altering one or more of the CDRs in the VH and / or VL of an antibody, such as of an aCD3 antibody, including making changes in one or more of the three CDRs of an aCD3-VH or aCD3-VL or any other functional variable domain. This can be done by making one or more amino acid substitutions at functionally important residues in one or more CDRs, replacing some or all CDR residues with random sequences, replacing one or more CDRs with tag or flag sequences, and / or swapping CDRs and / or variable regions with those from an irrelevant antibody (one directed to a different organism’s protein for example, such as chicken egg lysozyme). In some cases, only one of the CDRs in a variable region can be altered to render it inactive, although other embodiments include alterations in one, two, three, four, five or six CDRs.

[0267] Inactive GDVL of the present invention

[0268] In the present examples, inactive GDVLS were identified through alanine scanning of the LCDR3 region of the CD3 specific antibody “ocCD3-Fv”. Generation of aCD3-Fv is described in WO 2022 / 063819 which is incorporated herein in its entirety.

[0269] In an embodiment, the GDVL according to the present disclosure comprises a VL comprising the amino acid sequence of:

[0270] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSG VPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 14).

[0271] Inactive GDVH of the present invention

[0272] In the present disclosure, inactive GDVHS were identified through affinity maturation and deimmunization of aCD3-Fv as described in unpublished European Application Number EP22163663.2, which is incorporated here in its entirety.

[0273] In an embodiment, the GDVH according to the present disclosure comprises a VH comprising the amino acid sequence of:

[0274] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWVRQAPGKGLEWVANINQIGYS SYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWG QGTLVTVSS (SEQ ID NO: 15).

[0275] Guard-Knob Variable Domains (GDvo-Knob)

[0276] In the present disclosure, the germline framework regions of an GDVD can be further modified so as to introduce a protuberance into its interface region with the aim to further lower the ratio of heterodimer formation of two complementary CyCAT halfbody molecules in solution and in absence of a target cell.

[0277] One skilled in the art will appreciate that the “interface” of the engineered GDVD disclosed herein includes those contact amino acid residues in the GDVD, which interact or would contact with one or more contact amino acid residues in the interface of a complementary antibody variable domain.

[0278] A “protuberance” as used herein refers to at least one amino acid side chain which projects from the interface of the GDVD and thus destabilizes a potential heterodimer. Protuberances are constructed by replacing small amino acid side chains from the interface of the GDVD with larger side chains (e.g., tyrosine (Y) or tryptophan (W)). In some embodiments, the protuberance includes an amino acid residue substituted into the interface of the GDVD, and wherein the substituted amino acid residue is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some embodiments, the amino acid residue having a larger side chain volume than the original amino acid residue is selected from R, F, Y and W. In some embodiments, the original amino acid residue having a smaller side chain volume is selected from: A, S, T and V.

[0279] In some embodiments, at least one amino acid residue is substituted by an amino acid residue having a larger side chain volume than the original amino acid residue, thereby generating a protuberance within the interface, wherein the protuberance is located in the antibody variable heavy chain domain (VH).

[0280] In some embodiments, the amino acid residue is substituted into the interface of the GDVH at position 37, 39, 44, 45 and / or 47 according Chothia annotation. In some embodiments, the amino acid residue is substituted into the interface of the GDVH at position 37 according Chothia annotation. In some embodiments, V is substituted into the interface of the GDVH at position 37 according Kabat to R, F, Y or W. In some embodiments, V is substituted into the interface of the GDVH at position 37 according Kabat to F. In some embodiments, the amino acid residue is substituted into the interface of the GDVH having SEQ ID NO: 15 at position 37 according Kabat.

[0281] In an embodiment, the GDvH-Knob according to the present disclosure comprises a VH comprising the amino acid sequence of:

[0282] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEWVANINQIGYS SYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWG QGTLVTVSS (SEQ ID NO: 16). In an embodiment, said GDVH is a GDvH-Knob according to the present disclosure.

[0283] HSA Guard-Domains

[0284] The CyCAT halfbody molecules of the present disclosure may include a half-life extension domain as a Guard-Domain, such as human serum albumin (HSA) or domain 1 , domain 2 or domain 3 of HSA.

[0285] Human serum albumin (HSA; Uniprot: P02768) (molecular mass ~67 kDa) is the most abundant protein in human plasma and has a half-life of around 20 days in humans. Noncovalent association with albumin extends the elimination half-time of short-lived proteins.

[0286] In an embodiment, the GD of the present disclosure is or comprises human serum albumin (GDHSA-wt) or a fragment or portion or a domain of human serum albumin (GDHSA-frag.). In an embodiment, a GD according to the present disclosure comprises HSA or a truncation of HSA. In an embodiment, a GD according to the present disclosure comprises HSA having the amino acid sequence of Uniprot: P02768 (without signal sequence):

[0287] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESA ENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKA ACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSK LVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEV ENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAK TYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQT ALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 17).

[0288] In some embodiments, a halfbody molecule described herein comprises a truncated and / or variant versions of HSA, preferably as long as the pH sensitive binding to FcRn is retained. Binding to FcRn can be assessed by binding assays such as Biolayer interferometry (BLI, Octet) or surface plasmon resonance (SPR, BIAcore).

[0289] In some embodiments, the truncated version of HSA comprises the Domain III of HSA (HSA-DII I) having the amino acid sequence of: LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCC KHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDET YVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGKKLVA (SEQ ID NO: 18)

[0290] Other suitable HSA truncations and HSA variants are known in the art. See, for example, US10, 711 ,050 and Sand et al., JBC 289(5):34583 (2014), both incorporated herein by reference in their entirety.

[0291] In some embodiments, the GD according to the present disclosure has at least 90%, such as 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, sequence identity to the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the GD according to the present disclosure has the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 , SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the HSA is a variant HSA comprising the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 18 and one or more, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, amino acid modifications, such as substitution, addition and / or deletion.

[0292] The constructs and formats of the present invention are variations or improvements over inventions described in WO2013 / 156178, WO2022 / 248662, W02023 / 006809 and hereby expressly incorporated by reference in their entirety.

[0293] CyCAT halfbody molecules

[0294] The CyCAT halfbody molecules according to the present disclosure and as illustrated in Figures 1 - 3 may be freely combined to pairs of complementary halfbody molecules as long as one halfbody carries at least one unpaired aCD3-VH domain and the other halfbody carries at least one unpaired aCD3-VL domain.

[0295] As such, possible complementary pairs of halfbody molecule according to the present disclosure are those denoted as A / A’, B / B’, C / C’, D / D’, E / E’, F / F’ in Figures 1 - 3 or any other combination of halfbody molecules such as A / B’, A / C’, A / D’, A / E’, A / F’, B / A’, B / C’, B / D’, B / E’, B / F’,C / A’, C / B’, C / D’, C / E’, C / F’,D / A’, D / B’, D / C’, D / E’, D / F’, E / A’, E / B’, E / C’, E / D’, E / F’, F / A’, F / B’, F / C’, F / D’, F / E’. CyCAT halfbodies lacking a Guard-Domain

[0296] In an embodiment, the present disclosure provides a CyCAT halfbody molecule lacking a Guard-Domain as described herein.

[0297] In an embodiment, the present disclosure pertains to a halfbody molecule comprising a) a first Fab specific for a first antigen, b) a first peptide linker, c) either a first VH (ocCD3-VH1) or first VL (ocCD3-VL1) of a Fv specific for CD3 (ocCD3- Fv), wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the ocCD3-VH1 or ocCD3-VL1 via the first peptide linker.

[0298] In an embodiment, said halfbody molecule has a structure as shown in Figure 1A or 1A’.

[0299] In some embodiments, the halfbody molecule according to the present disclosure further comprises d) a second peptide linker, e) a Fc region composed of a first and second Fc region subunit, wherein each Fc region subunit is composed of an CH2 and CH3 domain, f) a third peptide linker, and wherein the C-terminus of the aCD3-VH1 or aCD3-VL1 is fused to the N-terminus of the first Fc region subunit via the second peptide linker and wherein the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0300] In an embodiment, said halfbody molecule has a structure as shown in Figure 2A or 2A’.

[0301] In some embodiments, the halfbody molecule according to the present disclosure further comprises a second Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0302] In an embodiment, said halfbody molecule has a structure as shown in Figure 2B or 2B’.

[0303] In an alternative embodiment, the halfbody molecule according to the present disclosure further comprises g) a second Fab specific for the first antigen, h) a third peptide linker i) a second aCD3-VH1 or second aCD3-VL1 , j) a fourth peptide linker, and wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second aCD3-VH1 or second aCD3-VL1 via the third peptide linker, and wherein the C-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker, wherein if the first aCD3-VH1 or first aCD3-VL1 is an ocCD3-VH then the second ocCD3-VH1 or second ocCD3-VL1 is an ocCD3-VH, or if the first ocCD3-VH1 or first ocCD3-VL1 is an ocCD3-VL then the second ocCD3-VH1 or second ocCD3-VL1 is an ocCD3-VL.

[0304] In an embodiment, the first aCD3-VH1 and the second aCD3-VH1 are identical. In an embodiment, the first aCD3-VL1 and the second aCD3-VL1 are identical. In an embodiment, said halfbody molecule has a structure as shown in Figure 2C or 2C’.

[0305] In some embodiments, the present disclosure pertains to a halfbody molecule comprising a) a first Fab specific for a first antigen b) a first peptide linker, c) a Fc region composed of a first and second Fc region subunit, d) a second peptide linker, e) either a first aCD3-VH1 or first aCD3-VL1 of an ocCD3-Fv, and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc region subunit via the first peptide linker, and wherein the N-terminus of the first aCD3-VH1 or first aCD3-VL1 , respectively, is fused to the C-terminus of the first Fc region subunit via the second peptide linker.

[0306] In an embodiment, said halfbody molecule has a structure as shown in Figure 2D or 2D’.

[0307] In some embodiments, the halfbody molecule according to the present disclosure further comprises f) a second Fab specific for the first antigen, g) a third peptide linker, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0308] In an embodiment, said halfbody molecule has a structure as shown in Figure 2E or 2E’.

[0309] In an alternative embodiment, the halfbody molecule according to the present disclosure molecule further comprises either a second aCD3-VH1 or a second aCD3-VL1 , wherein the N-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused to the C-terminus of the second Fc region subunit via the fourth peptide linker, and wherein if the either first ocCD3-VH1 or first ocCD3-VL1 is an ocCD3-VH then the either second ocCD3-VH1 or second aCD3-VL1 is an ocCD3-VH1 , or if the either first aCD3-VH1 or first ocCD3-VL1 is a VL then the either second aCD3-VH1 or second aCD3-VL1 is an aCD3-VL1 .

[0310] In an embodiment, the first aCD3-VH1 and the second aCD3-VH1 are identical. In an embodiment, the first aCD3-VL1 and the second aCD3-VL1 are identical.

[0311] In an embodiment, said halfbody molecule has a structure as shown in Figure 2F or 2F’.

[0312] Complementary pairs of CyCAT halfbodies lacking Guard-Domains

[0313] In an embodiment, the present disclosure pertains to a pair of complementary CyCAT halfbody molecules comprising

[0314] I. a first halfbody molecule comprising a a) a first Fab specific for a first antigen, b) a first peptide linker, c) either a first VH (aCD3-VH1) or first VL (ocCD3-VL1) of a Fv specific for CD3, wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first aCD3-VH1 or first aCD3-VL1 via the first peptide linker, and

[0315] II. a second halfbody molecules comprising a d) a second Fab specific for a second antigen, e) a fifth peptide linker, f) either the complementary first VH (aCD3-VH2) or complementary first VL (aCD3- VL2), respectively of the Fv specific for CD3, and wherein the C-terminus of the heavy chain of the second Fab is fused to the N- terminus of the complementary first aCD3-VH2 or complementary first aCD3-VL2 via the fifth peptide linker.

[0316] In an embodiment, the first halfbody molecule according to the present disclosure further comprises g) a second peptide linker, h) a first Fc region composed of a first and second Fc region subunit, i) a third peptide linker wherein the C-terminus of the first aCD3-VH1 or first aCD3-VL1 is fused to the N- terminus of the first Fc region subunit via the second peptide linker, and wherein the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0317] In an embodiments, the first halfbody molecule according to the present disclosure further comprises a third Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0318] In an alternative embodiment, the first halfbody molecule according to the present disclosure further comprises g) a third Fab specific for the first antigen, h) a third peptide linker, i) a second aCD3-VH1 or second aCD3-VL1 , j) a fourth peptide linker wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second aCD3-VH1 or second aCD3-VL1 ,respectibly, via the third peptide linker, wherein the C-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker, and wherein if the first aCD3-VH1 or first ocCD3-VL1 is a ocCD3-VH then the second ocCD3-VH1 or second aCD3-VL1 is a ocCD3-VH, or if the first ocCD3-VH1 or first ocCD3-VL1 is an ocCD3-VL then the second ocCD3-VH1 or ocCD3-VL1 is a ocCD3-VL. In further embodiments, the second halfbody molecule according to the present disclosure further comprises g) a sixth peptide linker, h) a second Fc region composed of a third and fourth Fc region subunit, i) a seventh peptide linker, and wherein the C-terminus of the complementary first aCD3-VH2 or complementary first aCD3-VL2 is fused to the N-terminus of the third Fc region subunit via the sixth peptide linker and wherein the C-terminus of the seventh peptide linker is fused to the N- terminus of the fourth Fc region subunit.

[0319] In an embodiment, the second halfbody molecule according to the present disclosure further comprises a fourth Fab specific for the second antigen, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the second Fc region subunit via the seventh peptide linker.

[0320] In an alternative embodiment, the second halfbody molecule according to the present disclosure further comprises j) a fourth Fab specific for the second antigen, k) a seventh peptide linker, l) a complementary second aCD3-VH2 or complementary second aCD3-VL2, and m) an eight peptide linker, and wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the complementary second aCD3-VH2 or complementary second aCD3-VL2 via the seventh peptide linker, wherein the C-terminus of the complementary second aCD3-VH2 or complementary second aCD3-VL2 is fused to the N-terminus of the fourth Fc region subunit via the eights peptide linker, wherein if the complementary first ocCD3-VH2 or complementary first aCD3-VL2 is a complementary first aCD3-VH2 then the complementary second aCD3-VH2 or complementary second aCD3-VL2 is a complementary second aCD3-VH, and if the complementary first aCD3-VH2 or complementary first aCD3-VL2 is a complementary first aCD3-VL2 then the complementary second aCD3-VH2 or complementary second aCD3-VL2 is a complementary second aCD3-VL. In alternative embodiments, the second halfbody molecule comprises a a) a second Fab specific for a second antigen b) fifth peptide linker, c) a second Fc region composed of a third and fourth Fc region subunit, d) a sixth peptide linker, e) a seventh peptide linker, f) either a complementary first aCD3-VH2 or complementary first aCD3-VL2 of the Fv specific for CD3, and wherein the N-terminus of the complementary first aCD3-VH2 or complementary first aCD3-VL2 is fused to the C-terminus of the third Fc region subunit via the sixth peptide linker, and wherein the C-terminus of the seventh peptide linker is fused to the N- terminus of the second Fc region.

[0321] In a further embodiment, the second halfbody molecule further comprises a fourth Fab specific for a second antigen wherein the N-terminus of the fourth Fc region subunit is fused to the C-terminus of the heavy chain of the second Fab via the seventh peptide linker.

[0322] In a further embodiment, the second halfbody molecule further comprises an eight peptide linker and a complementary second aCD3-VH2 or complementary second aCD3-VL2, wherein the N-terminus of the complementary second aCD3-VH2 or complementary second aCD3-VL2 is fused the C-terminus of the fourth Fc region subunit via the eight peptide linker.

[0323] CyCAT halfbody molecules comprising a Guard-Domain

[0324] In an embodiment, the present disclosure pertains to an halfbody molecule comprising: a) a first Fab specific for a first antigen, b) a first peptide linker, c) either a first VH (aCD3-VH) or first VL (ocCD3-VL) of a Fv specific for a CD3 (ocCD3- Fv), d) a second peptide linker, e) a first Fc region composed of a first and second Fc region subunit, f) a third peptide linker, g) a first Guard-Domain (GD), and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first aCD3-VH or first aCD3-VL via the first peptide linker, and wherein the C- terminus of the first aCD3-VH or first aCD3-VL is fused to the N-terminus of the first Fc region subunit via the second peptide linker, and wherein the C-terminus of the GD is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0325] In an embodiment, the halfbody molecule has a structure as shown in Figure 3A or 3A’.

[0326] In an embodiment, the halfbody molecule further comprises h) a second Fab specific for the first antigen and a fourth peptide linker, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the GD via the fourth peptide linker.

[0327] In an embodiment, the halfbody molecule as a structure has shown in Figure 3C or 3C’.

[0328] In alternative embodiments, the present disclosure pertains to a halfbody molecule comprising: a) a first Fab specific for a first antigen, b) a first peptide linker, c) a first Guard-Domain d) a second peptide linker, e) a first Fc region composed of a first and second Fc region subunit, f) a third peptide linker, g) either the first VH (aCD3-VH1 ) or first VL (ocCD3-VL1 ) of a Fv specific for CD3 (ocCD3-Fv), and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first GD via the first peptide linker, wherein the C-terminus of the GD is fused to the N-terminus of the first Fc region subunit via the second peptide linker, and wherein the C-terminus of the first aCD3-VH1 or first aCD3-VL1 is fused to the N-terminus of the second Fc region subunit via the third peptide linker. In an embodiment, said halfbody molecule has a structure as shown in Figure 3B or 3B’.

[0329] In alternative embodiments, the present disclosure pertains to a halfbody molecule comprising a) a first Fab specific for a first antigen b) a first peptide linker, c) a first Fc region composed of a first and second Fc region subunit, d) a second peptide linker, e) either the first VH (aCD3-VH1 ) or first VL (ocCD3-VL1 ) of a Fv specific for CD3 (ocCD3-Fv), f) a third peptide linker, g) a fourth peptide linker, h) a Guard-Domain (GD), and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc region subunit via the first peptide linker, wherein the N-terminus of either the first aCD3-VH1 or first aCD3-VL1 is fused to the C-terminus of the first Fc region subunit via the second peptide linker, wherein the N-terminus of the GD is fused to the C-terminus of the second Fc region subunit via the third peptide linker, and wherein the N-terminus of the second Fc region subunit is fused to the fourth peptide linker.

[0330] In an embodiment, the said molecule has a structure as shown in Figure 3D, 3D’.

[0331] In an further embodiment, said halfbody molecule further comprises a second Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker

[0332] In an embodiment, said halfbody molecule has a structure as shown in 3E or 3E’.

[0333] Complementary pairs of CyCAT halfbodies both comprising Guard-Domains

[0334] In an embodiment, the present disclosure pertains to a pair of complementary halfbody molecules comprising,

[0335] I) a first halfbody molecule comprising a) a first Fab specific for a first antigen, b) a first peptide linker, c) either the first aCD3-VH1 or first aCD3-VL1 of an ocCD3-Fv, d) a second peptide linker, e) a Fc region composed of a first and second Fc region subunit, f) a third peptide linker g) a first Guard-Domain (GD), and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first aCD3-VH1 or first aCD3-VL1 via the first peptide linker, wherein the C- terminus of the first aCD3-VH or first aCD3-VL1 is fused to the N-terminus of the first Fc region subunit via the second peptide linker, and wherein the C-terminus of the first GD is fused to the N-terminus of the second Fc region subunit via the third peptide linker; and

[0336] II) a second halfbody molecule comprising a) a second Fab specific for a second antigen, b) a fifth peptide linker, c) either a complementary first aCD3-VH2 or complementary first aCD3-VL2 of the ocCD3-Fv, d) a sixth peptide linker and e) a second Fc region composed of a third and fourth Fc region subunit, f) a seventh peptide linker g) a second Guard-Domain (GD), wherein the C-terminus of the heavy chain of the second Fab is fused to the N- terminus of the complementary first aCD3-VH2 or complementary first aCD3-VL2 via the fifth peptide linker, wherein the C-terminus of the complementary first ocCD3-VH2 or complementary first aCD3-VL2 is fused to the N-terminus of the third Fc region subunit via the sixth peptide linker, wherein the C-terminus of the second GD is fused to the N-terminus of the fourth Fc region subunit via the seventh peptide linker. In a further embodiment, the first halfbody molecule further comprises a third Fab specific for the first antigen and a fourth peptide linker, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the first GD via the fourth peptide linker.

[0337] In an embodiment, the second halfbody molecule further comprises a fourth Fab specific for the second antigen and an eight peptide linker, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the second GD via the eight peptide linker.

[0338] In alternative embodiments, the present disclosure pertains to a pair of halfbody molecules comprising:

[0339] I) a first halfbody molecule comprising a) a first Fab specific for a first antigen, b) a first peptide linker, c) a first Guard-Domain (GD) d) a second peptide linker, e) a first Fc region composed of a first and second Fc region subunit, f) a third peptide linker, and g) either a first ocCD3-VH1 or first aCD3-VL1 , wherein the C-terminus of the first Fab heavy chain is fused to the N-terminus of the first GD via the first peptide linker, wherein the C-terminus of the first GD is fused to the N-terminus of the first Fc region subunit via the second peptide linker, and wherein the C-terminus of the first aCD3-VH1 or first aCD3-VL1 is fused to the N- terminus of the second Fc region subunit via the third peptide linker; and

[0340] II) a second halfbody molecule comprising a) a second Fab specific for a second antigen, b) a fourth peptide linker, c) a second Guard-Domain (GD) d) a fifth peptide linker, e) a second Fc region composed of a third and fourth Fc region subunit, f) a sixth peptide linker, and g) either the complementary first aCD3-VH2 or complementary first aCD3-VL2, wherein the C-terminus of the heavy chain of the second Fab is fused to the N- terminus of the second GD via the fourth peptide linker, wherein the C-terminus of the second GD is fused to the N-terminus of the third Fc region subunit via the fifth peptide linker, and wherein the C-terminus of the complementary first aCD3-VH2 or complementary first aCD3-VH2 is fused to the N-terminus of the fourth Fc region subunit via the sixth peptide linker.

[0341] In an embodiment, the present disclosure pertains to pair of halfbody molecules comprising

[0342] I) a first halfbody molecule comprising a) a first Fab comprising specific for a first antigen, b) a first peptide linker, c) a first Fc region composed of a first and second Fc region subunit, d) a second peptide linker, e) either the first ocCD3-VH1 or first aCD3-VL1 an ocCD3-Fv, f) a third peptide linker, g) a fourth peptide linker, and h) a first Guard-Domain (GD), and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first Fc region subunit via the first peptide linker, wherein the N-terminus of the first aCD3-VH1 or first aCD3-VL1 is fused to the C-terminus of the first Fc region subunit via the second peptide linker, wherein the N-terminus of the first GD is fused to the C- terminus of the second Fc region subunit via the third peptide linker, and wherein the N-terminus of the second Fc region subunit is fused to the fourth peptide linker; and

[0343] II) a second halfbody molecule comprising a) a second Fab comprising specific for a second antigen b) a fifth peptide linker, c) a second Fc region composed of a third and fourth Fc region subunit, d) a sixth peptide linker, e) either the complementary first aCD3-VH2 or complementary first aCD3-VL2 of an ocCD3-Fv, f) a seventh peptide linker, g) an eight peptide linker, and h) a second Guard-Domain (GD2), wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the third Fc region subunit via the fifth peptide linker, wherein the N-terminus of the complementary first aCD3-VH2 or complementary first aCD3-VL2 is fused to the C- terminus of the third Fc region subunit via the second peptide linker, wherein the N- terminus of the second GD is fused to the C-terminus of the fourth Fc region subunit via the seventh peptide linker, and wherein the N-terminus of the fourth Fc region subunit is fused to the eight peptide linker.

[0344] In some embodiment, the first halfbody molecule further comprises a third Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via the fourth peptide linker.

[0345] In some embodiment, the second halfbody molecule further comprises a fourth Fab specific for the second antigen, wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the fourth Fc region subunit via the eight peptide linker.

[0346] Complementary pairs of CyCAT halfbodies with one halfbody comprising a Guard- Domain

[0347] In an embodiment, the present disclosure pertains to a pair of halfbody molecules comprising

[0348] I) a first halfbody molecules comprising a a) a first Fab specific for a first antigen, b) a first peptide linker, c) either a first VH (ocCD3-VH1) or first VL (ocCD3-VL1) of an ocCD3-Fv, and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first aCD3-VH1 or first aCD3-VL1 via the first peptide linker; and

[0349] II) a second halfbody molecule comprising a) a second Fab specific for a second antigen, b) a fifth peptide linker, c) either the first complementary VH (aCD3-VH2) or first complementary VL (aCD3- VL2) of an ocCD3-Fv, d) a sixth peptide linker, e) a second Fc region composed of a third and fourth Fc region subunit, f) a seventh peptide linker, and g) a first Guard-Domain (GD), wherein the C-terminus of the second Fab heavy chain is fused to the N-terminus of the first aCD3-VH2 or first aCD3-VL via the fifth peptide linker, wherein the C- terminus of the first aCD3-VH2 or first aCD3-VL2 is fused to the N-terminus of the third Fc region subunit via the sixth peptide linker, and wherein the C-terminus of the first Guard-Domain is fused to the N-terminus of the second Fc region subunit via the seventh peptide linker.

[0350] In some embodiments, the first halfbody molecule further comprises a) a second peptide linker, b) a first Fc region composed of a first and second Fc region subunit, c) a third peptide linker wherein the C-terminus of the first aCD3-VH1 or first aCD3-VL1 is fused to the N- terminus of the first Fc region subunit via the second peptide linker and wherein the C- terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit. In some embodiments, the first halfbody molecule according to the present disclosure further comprises a third Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0351] In an alternative embodiment, the first halfbody molecule according to the present disclosure further comprises a second aCD3-VH1 or second aCD3-VL1 and a fourth peptide linker, and wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second aCD3-VH1 or second aCD3-VL1 via the fourth peptide linker, wherein the C-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused to the N-terminus of the second Fc region subunit via the third peptide linker, and wherein if the first aCD3-VH1 or first ocCD3-VL1 is an ocCD3-VH then the second ocCD3-VH1 or second aCD3-VL1 is a ocCD3-VH, or if the first ocCD3-VH1 or first ocCD3-VL1 is an ocCD3- VL then the second aCD3-VH1 or second aCD3-VL1 is a ocCD3-VL.

[0352] In an embodiment of the present disclosure, the first aCD3-VH1 and second aCD3-VH1 or the first aCD3-VL1 and second aCD3-VL1 are identical.

[0353] In some embodiments, the second halfbody molecule further comprises a fourth Fab specific for the second antigen and an eight peptide linker, and wherein the C-terminus of the heavy chain of the fourth Fab is fused to the N-terminus of the first Guard-Domain via the eight peptide linker.

[0354] In an embodiment, the present disclosure pertains to a pair of halfbody molecules comprising

[0355] I) a first halfbody molecules comprising a a) a first Fab specific for a first antigen, b) a first peptide linker, c) either a first VH (ocCD3-VH1) or first VL (ocCD3-VL1) of an ocCD3-Fv, and wherein the C-terminus of the heavy chain of the first Fab is fused to the N-terminus of the first aCD3-VH1 or first aCD3-VL1 via the first peptide linker; and

[0356] II) a second halfbody molecule comprising a) a second Fab specific for a second antigen, b) a fifth peptide linker, c) a first Guard-Domain d) a sixth peptide linker, e) a second Fc region composed of a third and fourth Fc region subunit, f) a seventh peptide linker, and g) either the complementary first VH (aCD3-VH2) or complementary first VL (ocCD3-VL2) of the ocCD3-Fv, wherein the C-terminus of the heavy chain of the second Fab is fused to the N-terminus of the first Guard-Domain via the fifth peptide linker, wherein the C-terminus of the first Guard-Domain is fused to the N-terminus of the third Fc region subunit via the sixth peptide linker, and wherein the C-terminus of the first aCD3-VH2 or first aCD3-VH2 is fused to the N-terminus of the fourth Fc region subunit via the seventh peptide linker.

[0357] In some embodiments, the first halfbody molecule further comprises a) a second peptide linker, and b) a first Fc region composed of a first and second Fc region subunit, and c) a third peptide linker, wherein the C-terminus of the first aCD3-VH1 or first aCD3- VL1 is fused to the N-terminus of the first Fc region subunit via the second peptide linker and wherein the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0358] In some embodiments, the first halfbody molecule further comprises a third Fab specific for the first antigen, wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second Fc region subunit via the third peptide linker.

[0359] In an alternative embodiment, the first halfbody molecule further comprises d) a third Fab specific for the first antigen, f) a second aCD3-VH1 or second aCD3-VL1 , g) a fourth peptide linker, and wherein the C-terminus of the heavy chain of the third Fab is fused to the N-terminus of the second aCD3-VH1 or second aCD3-VL1 via the third peptide linker, wherein the C-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused to the N- terminus of the second Fc region subunit via the fourth peptide linker, wherein if the first ocCD3-VH1 or first ocCD3-VL1 is a ocCD3-VH then the second ocCD3-VH1 or second aCD3-VL1 is a ocCD3-VH, or if the first ocCD3-VH1 or first ocCD3-VL1 is an ocCD3-VL then the second ocCD3-VH1 or second ocCD3-VL1 is a ocCD3-VL.

[0360] In an embodiment of the present disclosure, the first aCD3-VH1 and second aCD3-VH1 are identical or wherein the first aCD3-VL1 and second aCD3-VL1 are identical.

[0361] In alternative embodiments, the first halfbody molecule comprises a a) a first Fab specific for a first antigen b) first peptide linker, c) a first Fc region composed of a first and second Fc region subunit, d) a second peptide linker e) a third peptide linker f) either a first aCD3-VH1 or first aCD3-VL1 of an ocCD3-Fv, wherein the N-terminus of the aCD3-VH1 or aCD3-VL1 is fused to the C-terminus of the first Fc region subunit via the second peptide linker and wherein the C-terminus of the third peptide linker is fused to the N-terminus of the second Fc region subunit.

[0362] In some embodiment, the first halfbody molecule further comprises a third Fab specific for the first antigen, wherein the N-terminus of the second Fc region subunit is fused to the C-terminus of the heavy chain of the third Fab via the third peptide linker.

[0363] In some embodiments, the first halfbody molecule further comprises a fourth peptide linker and a second aCD3-VH1 or second aCD3-VL1 , wherein the N-terminus of the second aCD3-VH1 or second aCD3-VL1 is fused the C-terminus of the third Fc region subunit via the fourth peptide linker, and wherein if the first aCD3-VH1 or first aCD3-VL1 is a aCD3- VH then the second aCD3-VH1 or second aCD3-VL1 is a ocCD3-VH, or if the first ocCD3- VH1 or first ocCD3-VL1 is an ocCD3-VL then the second ocCD3-VH1 or ocCD3-VL1 is a ocCD3-VL. In an embodiment of the present disclosure, the first aCD3-VH1 and second aCD3-VH1 are identical or wherein the first aCD3-VL1 and second aCD3-VL1 are identical.

[0364] Peptide Linkers

[0365] A CyCAT halfbody molecule according to the present disclosure can be designed such that its individual components (such as the targeting Fab fragment or the unpaired aCD3- SVD) are fused to each other either directly or indirectly through a linker. In an embodiment, the linker is a peptide linker. In certain embodiments, the individual components of an CyCAT halfbody molecule are genetically fused to each other. Such fusion can be achieved by a number of strategies, which include, but are not limited to peptide or polypeptide fusions between the N- and C-terminus of peptides or polypeptides, fusion via disulfide bonds, and fusion via chemical cross-linking reagents.

[0366] In an embodiment of the present disclosure, the linker is a peptide linker comprising one or more amino acid residues, joined by peptide bonds that are known in the art. The composition and length of a peptide linker may be determined in accordance with methods well known in the art and may be tested for efficacy.

[0367] The peptide linker should have a length that is adequate to fuse the two components in such a way that they assume the correct conformation relative to one another so that they retain or obtain the desired activity or functionality.

[0368] In an embodiment, a peptide linker according to the present disclosure is composed of only naturally occurring amino acid residues. In an embodiment, the peptide linker is non- immunogenic. In an embodiment, the peptide linker is an unstructured peptide linker. In an embodiment, said peptide linker is a flexible peptide linker. In an embodiment, said peptide linker does not comprise a protease cleavage site.

[0369] In an embodiment, the peptide linker may comprise G-A polymers, A-S polymers, P-A polymers or P-A-S polymers, such as (GS)n (SEQ ID NO: 23), (G4S)n (SEQ ID NO: 24),

[0370] (SG4)n (SEQ ID NO: 25), (GSGGS)n (SEQ ID NO: 26), (GGGS)n (SEQ ID NO: 27), G4(SG4)n (SEQ ID NO: 28), (GGSG)n (SEQ ID NO: 29), (GGSGG)n (SEQ ID NO: 30), (GSGSG)n (SEQ ID NO: 31 ), (GSGGG)n (SEQ ID NO: 32), (GGGSG)n (SEQ ID NO: 33), and (GSSSG)n (SEQ ID NO: 34), wherein n is an integer between 1 and 10, typically between 2 and 4.

[0371] Suitable peptide linkers can be also derived from immunoglobulin light or heavy chain constant domains, such as CLK or CLX domains or the CH1 domain, but not all residues of such a constant domain, for example only the first 5 - 12 amino acid residues. In an embodiment, the peptide linker according to the present disclosure comprises an amino acid sequence of: QPKAAP (SEQ ID NO: 35) or ASTKGP (SEQ ID NO: 36).

[0372] A peptide linker may also comprise an immunoglobulin hinge (e.g. a human lgG1 hinge or part thereof) or any peptide derived from such hinge. Preferably, where only a part or portion of an immunoglobulin hinge is used, the truncated hinge may still include one or more of its interchain cysteines. The presence of the interchain cysteines allows for the formation of a dimeric peptide linker (or hinge region) by disulphide bridges, in situations where two of such hinge peptide linkers are used in two neighbouring polypeptides. The presence of a dimeric peptide linker or hinge region additionally promotes and stabilizes the dimerization of the two Fc region subunits which may be present in an CyCAT halfbody molecule according to the present disclosure.

[0373] In an embodiment, the peptide linker according to the present disclosure comprises an amino acid sequence selected from the group of: EPKSCDKTHTCPPCP (SEQ ID NO: 37), DKTHTCPPCP (SEQ ID NO: 38), KTHTCPPCP (SEQ ID NO: 39) and KTHT (SEQ ID NO: 40). In a further embodiment, a peptide linker according to the present disclosure comprises an amino acid sequence selected from the group of: QPKAAPDKTHTCPPCP (SEQ ID NO: 41 ); ASTKGPDKTHTCPPCP (SEQ ID NO: 42), QPKAAPKTHTCPPCP (SEQ ID NO: 43) or ASTKGPKTHTCPPCP (SEQ ID NO: 44).

[0374] In an embodiment, a peptide linker according to the present disclosure is composed of the amino acid residues A, Q, D, P, H, G, S, E, T, K, and C. In an embodiment, the peptide linker according to the present disclosure is composed of amino acid residues selected from the group of: A, Q, D, P, H, and G. In an embodiment, the peptide linker according to the present disclosure comprises an amino acid sequence selected from the group of: GQPSG (SEQ ID NO: 45), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 46), PAAPAP (SEQ ID NO: 47), PAAPAPDA (SEQ ID NO: 48), AQPAAPAPAE (SEQ ID NO: 49), DQPAAPAPDA (SEQ ID NO: 50), AQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 51), DQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 52),

[0375] AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 53), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGS (SEQ ID NO: 54), AQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 55), DQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 56), DQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 57), and

[0376] AQPAAPAPDAHEAPAPAQGADQPAAPAPDAHEAPAPAQGSKTHTCPPCP (SEQ ID NO: 58).

[0377] In some embodiments, the peptide linker according to the present disclosure has a length of about 4 to 100, 4 to 50, or 4 to 30 amino acids residues. In some embodiment, the peptide linker comprises 1 to 200, 1 to 100, 1 to 70, 1 to 65, 1 to 50, 1 to 25 or 1 to 20 amino acids. In some embodiments, the peptide linker has a length of at least 4, of at least 5, of at least 10, at least 15, or at least 20 amino acids residues. In some embodiments, the peptide linker has a length of not more than 50, not more than 60, not more than 70, not more than 80, not more than 90, or not more than 100 amino acids residues. In an embodiment, the peptide linker according to the present disclosure has a length of between 5 and 50 amino acid residues, such as 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acid residues. In an embodiment, the peptide linker has a length of 5 to 50 amino acids residues. In an embodiment, a peptide linker according to the present disclosure has a length of 5 to 50 amino acids residues, 5 to 45 amino acid residues, 5 to 40 amino acid residues, 5 to 35 amino acid residues, 5 to 30 amino acid residues, 5 to 25 amino acid residues, 5 to 20 amino acid residues, 5 to 15 amino acid residues, or 5 to 10 amino acid residues. In an embodiment, the peptide linker has a length of 5 to 40 amino acid residues. In an embodiment, the peptide linker has a length of 9 to 29 amino acid residues. In an embodiment, the peptide linker has a length of 5 to 29 amino acid residues. In an embodiment, the peptide linker has a length of 5 amino acids residues, 8 amino acids residues, 9 amino acid residues, 10 amino acids residues, 15 amino acids residues, 20 amino acids residues, 29 amino acids residues, 40 amino acids residues, or 49 amino acid residues.

[0378] In an embodiment, the peptide linker has a length of 5 amino acids residues. In an embodiment, the peptide linker has a length of 8 amino acids residues. In an embodiment, the peptide linker has a length of 9 amino acids residues. In an embodiment, the peptide linker has a length of 10 amino acids residues. In an embodiment, the peptide linker has a length of 15 amino acids residues. In an embodiment, the peptide linker has a length of 19 amino acids residues. In an embodiment, the peptide linker has a length of 20 amino acids residues. In an embodiment, the peptide linker has a length of 25 amino acids residues. In an embodiment, the peptide linker has a length of 29 amino acids residues. In an embodiment, the peptide linker has a length of 40 amino acids residues. In an embodiment, the peptide linker has a length of 45 amino acids residues. In an embodiment, the peptide linker has a length of 49 amino acids residues.

[0379] It is understood that a peptide linker as used herein is not limited to only one of the aforementioned and exemplified peptide linkers but may comprise any combination of two or more such linker which are fused to each other. For instance, a peptide linker as used herein may be built from a G-S polymer and an immunoglobulin hinge derived sequence. In an embodiment, a peptide linker as used herein does not comprise an IgG Fc region. In an embodiment, a peptide linker does not comprise a monomer of an IgG Fc region subunit. In an embodiment, the peptide linkers present in a CyCAT halfbody molecules according to the present disclosure are identical. In an embodiment, the peptide linkers are different. In an embodiment, the peptide linkers are of identical length. In an embodiment, the peptide linkers are of different length.

[0380] Preferred Peptide Linkers

[0381] Preferably, a Guard-Domain according to the present disclosure is fused to a halfbody molecule in a “non-cleavable” fashion.

[0382] By “non-cleavable” is meant the inability to be cleaved and released from a CyCAT halfbody molecule through the activity of an enzyme expressed by a cancer cell to which such CyCAT halfbody molecules binds to. For example, a GD of the present disclosure may be fused to halfbody molecule through a peptide linker, in particular, by a non- cleavable peptide linker.

[0383] By “non cleavable peptide linker” herein is meant an amino acid sequence that cannot be cleaved by a human protease under normal physiological conditions. Accordingly, a GD according to the present disclosure and / or the peptide linker which fuses a GD to a halfbody molecule does not comprise a protease cleavage site. Accordingly, a GD according to the present disclosure and / or the peptide linker according to the present disclosure is not cleavable by a protease. In an embodiment, said protease comprises a tumor specific protease. In an embodiment, said protease comprises a matrix metalloprotease (MMP) or a serine protease. In an embodiment, said matrix metalloprotease comprises MMP2, MMP7, MMP9, MMP13, or MMP14. In an embodiment, said serine protease comprises matriptase, urokinase, or hepsin.

[0384] A “protease cleavage site" as meant herein, is an amino acid sequence that can be cleaved by a protease, such as, for example, a matrix metalloproteinase or a furin. Examples of such sites include: Gly-Pro-Leu-Gly-lle-Ala-Gly-GIn (SEQ ID NO: 59) or Ala- Val-Arg-Trp-Leu-Leu-Thr-Ala (SEQ ID NO: 60), which can be cleaved by metalloproteinases, or Arg-Arg-Arg-Arg-Arg-Arg (SEQ ID NO: 61 ), which is cleaved by a furin.

[0385] In therapeutic applications, the protease cleavage site could be cleaved by a protease that is produced by target cells, for example cancer cells or infected cells, or pathogens. Cancer cells are known to express proteases such as matrix-metalloproteinases and proteases associated with cancer as well as corresponding protease cleavage sites (i.e. an particular amino acid sequence recognized by such protease) are well known in the art. Cancer associated protease families may include metalloproteases, serine proteases, cysteine proteases, aspartic proteases and threonine proteases. Protease databases include PMAP (www.proteolysis.orq), ExPASy Peptide Cutter (ca.expasy.org / tools / peptidecutter) and PMAP. Cut DB (cutdb.burnham.org). Further databases which helps to identify particular protease cleavage sites are Oncomine (www.oncomine.org), the European Bioinformatic Institute (www.ebi.ac.uk), in particular (www.ebi.ac.uk / gxa). In an embodiment, a GD according to the present disclosure does not comprise a protease cleavage site. In an embodiment, a GD according to the present disclosure does not comprise a protease cleavage site, in particular a cancer associated protease cleavage site. In an embodiment, the peptide linker which fuses a GD to a CyCAT halfbody molecule according to the present disclosure does not comprises a protease cleavage site. In an embodiment, the peptide linker which fuses a GD to a CyCAT halfbody molecule according to the present disclosure does not comprises a protease cleavage site, in particular a protease cleavage site cleaved or cleavable by a cancer associated protease. In one such embodiment, the GD is fused to one Fc region subunit of a CyCAT halfbody molecule. In an embodiment, a GD according to the present disclosure is not released from a CyCAT halfbody molecule. In an embodiment, a GD domain is not released from a CyCAT halfbody molecule through proteolytic cleavage.

[0386] In an embodiment, a GD domain according to the present disclosure is not released from a halfbody molecule after administration to a subject. In an embodiment, a GD domain is not released from a halfbody molecule after administration to a subject through proteolytic cleavage. In an embodiment, a GD domain is not released from a halfbody molecule after administration to a subject through proteolytic cleavage by a protease. In an embodiment, a GD domain is not released from a halfbody molecule after administration to a subject through proteolytic cleavage by a cancer associated protease.

[0387] Antibodies

[0388] The antibodies or antibody fragments as well as the VH and VL domains used in the CyCAT halfbody molecules according to the present disclosure can be of any animal species origin, such as murine, rat, human or non-human primate. Preferably, the origin is human or may be also obtained by humanization approaches.

[0389] CD3 Binding Domains

[0390] There are a number of suitable CD3 specific antibodies and / or corresponding VH and VL domains or CDRs, that are known in the art that find use in the present disclosure.

[0391] For example, the CDRs and / or VH and VL domains are derived from known anti-CD3 antibodies, such as, for example, muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031 ), visilizumab (Nuvion), SP34 or any humanized variants of SP34, I2C, H2C, TR-66 or X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, Fl 11-409, CLB-T3.4.2, WT32, SPv-T3b, 11 D8, XIII-141 , XIII-46, XIII- 87, 12F6, T3 / RW2- 8C8, T3 / RW2-4B6, OKT3D, M-T301 , SMC2, F101.01 , UCHT-1 and WT-31.

[0392] Preferably, the VH and VL domain that form an active CD3 specific Fv fragment that are those described in published International Application Number PCT / EP2021 / 076052, which is incorporated herein by reference in its entirety. In an embodiment of the present disclosure, the VH and VL of the Fv specific for CD3 according to the present disclosure originates from or is a derivative or variant of any one of the CD3 specific antibodies disclosed in International Application No. PCT / EP2021 / 076052. In an embodiment, the aCD3-Fv according to the present disclosure competes with an antibody specific for CD3 for binding to an epitope on CD3, in particular CD3 epsilon. In an embodiment, the aCD3- Fv according to the present disclosure competes with any one of the antibodies specific for CD3 disclosed herein. In an embodiment, the aCD3-Fv according to the present disclosure competes with any one of the antibodies specific for CD3 disclosed in International Application No. PCT / EP2021 / 076052. In an embodiment of the present disclosure, the aCD3-Fv comprises any of the VH and / or VL domains disclosed in PCT / EP2021 / 076052. In an embodiment, the ocCD3-Fv competes with any one of the antibodies specific for CD3 disclosed in Table 2 or Table 3 of the present specification for binding to an epitope on CD3.

[0393] In an embodiment, the aCD3-Fv according to the present disclosure competes with an antibody comprising a aCD3-VH having an amino acid sequence of SEQ ID NO: 7 or SEQ ID: 12 and a ocCD3-VL having an amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13 for binding to an epitope on CD3.

[0394] In an embodiment, the VH and VL domains and / or their corresponding CDR regions incorporated in the CyCAT halfbody molecule according to the present disclosure that are capable of forming an active anti-CD3 Fv domain (aCD3-Fv) that binds to human CD3 are shown in Tables 2 and Table 3 of the present specification. Table 2: VH, VL and CDR amino acid sequences of the low-affinity aCD3epsilon antibody which

[0395] In an embodiment, the aCD3-VH and aCD3-VL domains (collectively the “split variable domains” or “ocCD3-SVD”) form the active anti-CD3 Fv domain (aCD3-Fv). In an embodiment, said aCD3-Fv is composed of consists of an aCD3-VL and aCD3-VH domain as disclosed herein. In an embodiment, said aCD3-VH and aCD3-VL are complementary to each other. In an embodiment, said aCD3-VH and aCD3-VL are capable of non-covalently dimerization with each other. In an embodiment, said aCD3- VH and aCD3-VL are capable of non-covalently associating with each other. In an embodiment, said non-covalent association results in the formation of the aCD3-Fv. In an embodiment, said dimerization or non-covalent association occurs in solution. In an embodiment, said dimerization or non-covalent association occurs in solution in the presence of a target cell. In an embodiment of the present disclosure, the aCD3-VH and said aCD3-VL are not linked by a covalent bond. In an embodiment of the present disclosure, said aCD3-VH by itself is not capable of binding to CD3. In an embodiment of the present disclosure, said aCD3-VL by itself is not capable of binding to CD3. In an embodiment of the present disclosure, neither the aCD3-VH by itself nor the aCD3-VL by itself of the aCD3-Fv is capable of binding to CD3.

[0396] In an embodiment, the aCD3-Fv according to the present disclosure binds to human CD3. In an embodiment, the aCD3-Fv according to the present disclosure binds to human CD3 and cynomolgus monkey CD3. In an embodiment, the aCD3-Fv according to the present disclosure binds to human CD3 and cross-reactively binds to cynomolgus CD3. In an embodiment, said binding of the aCD3-Fv to CD3 is specific.

[0397] In an embodiment, said aCD3-Fv binds to immune cells expressing CD3. In an embodiment, the aCD3-Fv binds to T-cells expressing CD3. In an embodiment, the aCD3- Fv binds to cytotoxic T-cells expressing CD3. In an embodiment, the aCD3-Fv binds to human cells expressing human CD3. In an embodiment, the aCD3-Fv binds to cynomolgus monkey cells expressing cynomolgus monkey CD3. In an embodiment, the aCD3-Fv binds to human cells expressing human CD3 and to cynomolgus monkey cells expressing cynomolgus monkey CD3.

[0398] In an embodiment of the present disclosure, the CD3 is CD3 epsilon. In an embodiment, the CD3 is human CD3 epsilon. In an embodiment, the human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 19. In an embodiment, the CD3 is cynomolgus CD3 epsilon. In an embodiment, the cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 21. In an embodiment, the aCD3-Fv according to the present disclosure binds to the extracellular region of CD3 epsilon. In an embodiment, the aCD3- Fv binds to the extracellular region human CD3 epsilon. In an embodiment, the aCD3-Fv binds to the extracellular region cynomolgus CD3 epsilon. In an embodiment, the aCD3- Fv binds to the extracellular region human and cynomolgus CD3 epsilon. In an embodiment, the extracellular region of human CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 20. In an embodiment, the extracellular region of cynomolgus CD3 epsilon comprises the amino acid sequence of SEQ ID NO: 22. In an embodiment, the aCD3-Fv binds to a human CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20.

[0399] In an embodiment, the aCD3-Fv according to the present binds to a cynomolgus CD3 epsilon polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 22. In an embodiment, the ocCD3-Fv according to the present disclosure specifically binds to a polypeptide comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0400] In an embodiment, the aCD3-VH according to the present disclosure comprises the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3.

[0401] In an embodiment of the present disclosure, the aCD3-VH comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 12. In an embodiment of the present disclosure, the aCD3-VH comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12.

[0402] In an embodiment, the aCD3-VL according to the present disclosure comprises the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO 11 , the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6. In an embodiment of the present disclosure, the aCD3-VL comprises an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO 13. In an embodiment of the present disclosure, the ocCD3-VL comprises the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13. In an embodiment, the aCD3-Fv according to the present disclosure comprises a) an aCD3-VH comprising the HCDR1 region comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9, the HCDR2 region comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10, and the HCDR3 region comprising the amino acid sequence of SEQ ID NO: 3 and b) an aCD3-VL comprising the LCDR1 region comprising the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 11 , the LCDR2 region comprising the amino acid sequence of SEQ ID NO: 5, and the LCDR3 region comprising the amino acid sequence of SEQ ID NO: 6.

[0403] In an embodiment of the present disclosure, the aCD3-Fv comprises an aCD3-VH comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence SEQ ID NO: 7 or SEQ ID NO: 12 and an aCD3-VL comprising an amino acid sequence that is at least 90%, 91 %, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0404] In an embodiment of the present disclosure, the aCD3-Fv comprises an aCD3-VH comprising an amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 12 and an aCD3- VL comprising the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 13.

[0405] In an embodiment, the aCD3-Fv according to the present disclosure is an isolated antibody fragment. In an embodiment, the aCD3-Fv is a human antibody fragment. In an embodiment, the aCD3-Fv is a recombinant antibody fragment. In an embodiment, the aCD3-Fv is a synthetic antibody fragment. In an embodiment, the aCD3-Fv is a chimeric antibody fragment. In an embodiment, the aCD3-Fv is a monoclonal antibody fragment.

[0406] Fc region

[0407] The Fc region of a CyCAT halfbody molecule may comprise or consists of a pair of polypeptides comprising heavy chain constant domains of a regular immunoglobulin. The Fc region of a regular IgG exists as a dimer, each subunit of which comprises the CH2 and CH3 IgG heavy chain constant domains. The two Fc region subunits are capable of stable association with each other. Accordingly, in an embodiment, the two Fc region subunits of an CyCAT halfbody molecule according to the present disclosure are capable of stable association with each other.

[0408] In an embodiment, the Fc region of an CyCAT halfbody molecule according to the present disclosure is an IgG Fc region. In an embodiment, the Fc region is an lgG1 Fc region. In an embodiment, the Fc region is human. In an embodiment, the Fc region is a human lgG1 Fc region.

[0409] The heterodimeric Fc region

[0410] The two Fc region subunits of an CyCAT halfbody molecule according to the present disclosure may be comprised in two non-identical polypeptide chains. To improve the yield and purity of the halfbody molecule in recombinant production, it is advantageous to introduce in the Fc region one or more modifications promoting the association of the two non-identical polypeptides forming the Fc region subunits. Accordingly, in certain embodiments, the present disclosure provides heterodimeric CyCAT halfbody molecules that each rely on the use of two different variant Fc region subunits that will self-assemble to form a heterodimeric molecule.

[0411] In an embodiment, the Fc region of an CyCAT halfbody molecule according to the present disclosure comprises one or more modifications promoting the association of the first and the second Fc region subunit and / or of the third and the fourth Fc region subunit. In an embodiment, the first and second Fc region subunit and / or the third and the fourth Fc region subunit of a CyCAT halfbody molecule may comprise one or more modification promoting the association of the first and the second Fc region subunit and / or of the third and the fourth Fc region subunit, respectively.

[0412] In an embodiment, the first Fc region subunit and second Fc region subunit and / or the third and the fourth Fc region subunit comprise one or more modifications that reduce homodimerization or reduce homodimer formation between two identical polypeptide chains comprising the same Fc region subunit.

[0413] In an embodiment, the first and second Fc region subunit and / or the third and the fourth Fc region subunit comprises different amino acid modifications, such that the heterodimeric Fc region is more stable than the homodimeric Fc region. In an embodiment, the first and second Fc region subunit and / or the third and the fourth Fc region subunit comprise different amino acid modification, such that the association of the first and second Fc region subunit and / or of the third and the fourth Fc region subunit is promoted. A modification may be present in the first Fc region subunit and / or the second Fc region subunit and / or the third and / or the fourth Fc region subunit. In an embodiment, such modification is present in the first and second Fc region subunit. In an embodiment, such modification is present in the third and fourth Fc region subunit. In an embodiment, such modification occurs in the CH3 domain of each Fc region subunit. A modification can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis.

[0414] Typically, in the heterodimerization approaches known in the art, the CH3 domain of one polypeptide chain (e.g. immunoglobulin heavy chain) and the CH3 domain of the other polypeptide chain are both engineered in a complementary manner so that the polypeptide comprising one engineered CH3 domain can no longer homodimerize with another polypeptide chain of the same structure. Thereby the polypeptide comprising one engineered CH3 domain is forced to heterodimerize with the other polypeptide comprising the CH3 domain, which is engineered in a complementary manner. Several approaches for CH3 modifications in order to promote heterodimerization have been described, for example in WO96 / 27011 , W098 / 050431 , EP1870459, W02007 / 110205,

[0415] W02007 / 147901 , W02009 / 089004, WO201 0 / 129304, WO201 1 / 90754,

[0416] WO20 11 / 143545, WO2012 / 058768, WO2013 / 157954, WO2013 / 096291 , which are herein incorporated by reference.

[0417] One of these heterodimerization approaches known in the art is the so-called "knobs-into- holes" (KiH) technology, which is described in detail providing several examples in e.g. WO 96 / 027011 , Ridgway, J.B., et al, Protein Eng. 9 (1996) 617-621 ; Merchant, A.M., et al, Nat. Biotechnol. 16 (1998) 677-681 ; US 5,731 ,168; US 7,695,936; WO 98 / 050431 , Carter, J Immunol Meth 248, 7-15 (2001) which are incorporated herein by reference. The "knobs-into-holes" technology broadly involves: (1) mutating the CH3 domains in each Fc region subunit to promote heterodimerization; and (2) combining the mutated Fc region subunits under conditions that promote heterodimerization. "Knobs" or "protuberances" are typically created by replacing a small amino acid in a parental antibody with a larger amino acid (e.g., T366Y or T366W); "Holes" or "cavities" are created by replacing a larger residue in a parental antibody with a smaller amino acid (e.g., Y407T, T366S, L368A and / or Y407V) with numbering according EU index.

[0418] In an embodiment, the modification present in the Fc region of a CyCAT halfbody molecule according to the present disclosure is a "knobs-into-holes" modification, comprising "knob mutations” in one of the two Fc region subunits and "hole mutations” in the other complementary Fc region subunit. The knob modifications and hole modifications can be made by altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In an embodiment, the CH3 domain of each Fc region subunit is modified according to the knobs-into-holes technology.

[0419] In an embodiment, in the CH3 domain of the first and / or third Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and in the CH3 domain of the second Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V) with numbering according EU index. In an embodiment, in the CH3 domain of the second or fourth Fc region subunit, the threonine residue at position 366 is replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A) with numbering according EU index. In an embodiment, in the CH3 domain of the first and / or third Fc region subunit, the serine residue at position 354 is replaced with a cysteine residue (S354C), and in the CH3 domain of the second and / or fourth Fc region subunit the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) with numbering according EU index based. Introduction of these two cysteine residues results in formation of a disulfide bridge between the two Fc region subunits, further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

[0420] In a more specific embodiment, the present disclosure provides an CyCAT halfbody molecule, wherein in the CH3 domain of first and / or third Fc region subunit, the threonine residue at position 366 is replaced with a tryptophan residue (T366W) and the serine residue at position 354 is replaced with a cysteine residue (S354C) and in the CH3 domain of the second and / or fourth Fc region subunit the tyrosine residue at position 407 is replaced with a valine residue (Y407V), the threonine residue at position 366 is replaced with a serine residue (T366S), the leucine residue at position 368 is replaced with an alanine residue (L368A) and the tyrosine residue at position 349 is replaced by a cysteine residue (Y349C) with numbering according EU index.

[0421] In an embodiment, the present disclosure provides an CyCAT halfbody molecule, wherein the aCD3-VH or aCD3-VL domain and the Fc region subunit comprising the knobmutations are present on the same polypeptide chain. In alternative embodiments, the present disclosure provides an CyCAT halfbody molecule, wherein the aCD3-VH or aCD3-VL domain and the Fc region subunit comprising the hole-mutations are present on the same polypeptide chain. In an embodiment, the Fab heavy chain and the aCD3-VH or aCD3-VL domain and the Fc region subunit comprising the knob-mutations are present on the same polypeptide chain.

[0422] In an alternative embodiment, the Fab heavy chain and the Fc region subunit comprising the knob-mutations are present on the same polypeptide chain and the Fc region subunit comprising the hole-mutations and the aCD3-VH or aCD3-VL domain are present on a second different polypeptide chain.

[0423] Fc receptor binding and / or effector function

[0424] For certain therapeutic situations, it may be desirable to reduce or inhibit the normal or wildtype binding of an IgG Fc region to one or more or all of the Fc receptors and / or binding to a complement component, such as C1q. For instance, it may be desirable to reduce or prevent the binding of an Fc region to one or more or all of the Fey receptors (e.g. FcyRI, FcyRlla, FcyRllb, FcyRllla). In particular, when a pair of complementary halfbody molecules according to the present disclosure co-engages a receptor of an immune effector cell (such as the TCR), it is advisable to prevent FcyRllla binding to abolish or significantly reduce ADCC activity and / or to prevent C1q binding to eliminate or significantly reduce CDC activity. The reduced or abolished effector function can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced or abolished antibody-dependent cell-mediated cytotoxicity (ADCC), reduced or abolished antibody-dependent cellular phagocytosis (ADCP). In certain embodiments, the reduced or abolished effector function is one or more selected from the group consisting of CDC, ADCC and ADCP. In an embodiment, the reduced or abolished effector function is ADCC. In an embodiment, the reduced or abolished effector function is CDC. In an embodiment, the reduced or abolished effector function is ADCP. In an embodiment, the reduced or abolished effector function is CDC, ADCC and ADCP.

[0425] In an embodiment, the Fc region of an halfbody according to the present disclosure is engineered to have a reduced binding affinity to an Fc receptor and / or to C1 q and / or to have reduced effector function when compared to a non-engineered Fc region. In an embodiment, the Fc region of an halfbody according to the present disclosure is engineered to have reduced effector function when compared to a non-engineered Fc region. In an embodiment, the Fc region of an halfbody according to the present disclosure comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces its effector function. In general, the same one or more amino acid mutation(s) is present in each one of the two Fc region subunits forming the Fc region. In an embodiment, the one or more amino acid mutations reduces the binding affinity of the Fc region to an Fc receptor. In an embodiment, the engineered Fc region does not bind substantially to an Fc receptor and / or C1q and / or induce effector function. In an embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In an embodiment, the Fc receptor is an Fey receptor. In an embodiment, the Fc receptor is an activating human Fey receptor, more specifically human FcyRllla, FcyRI or FcyRlla, most specifically human FcyRllla. In an embodiment, the binding affinity of the Fc region to a complement component, in particular the binding affinity to C1q, is reduced or abolished. In an embodiment, the reduced or abolished effector function is one or more selected from the group of reduced or abolished CDC, reduced or abolished ADCC and reduced or abolished ADCP. In a particular embodiment, the reduced or abolished effector function is reduced ADCC, CDC, and ADCP. In an embodiment, the Fc region of HB1 and / or HB2 according to the present disclosure comprises one or more amino acid mutation(s) that reduce(s) the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces the effector function.

[0426] In an embodiment, the amino acid mutation is an amino acid substitution. In an embodiment, the Fc region of an halfbody according to the present disclosure comprises one or more amino acid mutations that reduces the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces its effector function, wherein each Fc region subunit comprises an amino acid substitution at a position selected from the group of 234, 235, 237, 330 and 331 with numbering according EU index.

[0427] In an embodiment, each Fc region subunit of an halfbody according to the present disclosure comprises an amino acid substitution at a position selected from the group of L234, L235 and G237 (numbering according EU index). In an embodiment, each Fc subunit comprises the amino acid substitutions L234A and L235E with numbering according EU index. In an embodiment, each Fc region subunit comprises the amino acid substitutions L234A, L235E and G237A with numbering according EU index. In an embodiment, each Fc region subunit comprises an amino acid substitution at a position selected from the group of 330 and 331 with numbering according EU index. In an embodiment, each Fc region subunit comprises an amino acid substitution at the positions 330 and 331 with numbering according EU index. In an embodiment, the amino acid substitution is A330S or P331 S. In an embodiment, the Fc region of an halfbody according to the present disclosure comprises one or more amino acid mutations in each Fc region subunit that reduces the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces the effector function, wherein said one or more amino acid mutations are L234A, L235E, G237A, A330S and P331 S. In an embodiment, the Fc region of an halfbody according to the present disclosure consists of one or more amino acid mutation in each Fc region subunit that reduces the binding affinity of the Fc region to an Fc receptor and / or to C1q and / or reduces the effector function, wherein the one or more amino acid mutations are L234A, L235E, G237A, A330S and P331 S. In an embodiment, the Fc region is an lgG1 Fc region, particularly a human lgG1 Fc region. Mutant Fc regions or Fc region subunits can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, and the like. The correct nucleotide changes can be verified for example by sequencing.

[0428] In an embodiment, the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-Knob):

[0429] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPC REEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

[0430] DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 62)

[0431] In an embodiment, the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS-hole): PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPS REEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 63)

[0432] In an embodiment, the Fc region subunit according to the present disclosure comprises the amino acid sequence of (Fc-AEASS):

[0433] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 64)

[0434] Functionality

[0435] A pair of complementary CyCAT halfbody molecules according to the present disclosure may be used for the prevention and treatment of diseases, which are mediated by biological pathways in which target antigens of interest are involved. This may be preferably achieved by recruiting cytotoxic immune cells, such as T-cells, to cells expressing the target antigens, preferably the tumor or cancer associated antigens (TAAs or CAAs, respectively).

[0436] The biological activity of a pair of complementary CyCAT halfbody molecules according to the present disclosure can be measured by various assays known in the art, including those described in the examples in the present application. Methods for assaying functional activity may utilize binding assays, such as the enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS) and other methods that are well known in the art (see Hampton, R. et al. (1990; Serological Methods a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al. (1983; J. Exp. Med. 158:1211 -1216). Alternatively, assays may test the ability of a pair of complementary CyCAT halfbody molecules according to the present disclosure in eliciting a biological response because of binding to a set of biological target antigens, either in vivo or in vitro. Biological activities may for example include the induction of proliferation of T-cells, the induction of signaling in T-cells, the induction of expression of activation markers in T-cells, the induction of cytokine secretion by T-cells, the inhibition of signaling in target cells such as tumor cells or cells of the tumor stroma, the inhibition of proliferation of target cells, the induction of lysis of target cells, and the induction of tumor regression and / or the improvement of survival.

[0437] In an embodiment, the present disclosure provides a method for inducing lysis of a target cell, such as a tumor cell, comprising contacting said cell in the presence of a cytotoxic T- cell with a pair of complementary CyCAT halfbody molecules according to the present disclosure.

[0438] In an embodiment, the present disclosure provides a method for inhibition of proliferation of a target cell, such as a tumor cell, comprising contacting said cell in the presence of a cytotoxic T-cell with a pair of complementary CyCAT halfbody molecules according to the present disclosure.

[0439] In an embodiment, the present disclosure provides a method for inducing a cellular response in cytotoxic T cells, comprising contacting said cytotoxic T-cell in the presence of a target cell, such as a tumor cell, with a pair of complementary CyCAT halfbody molecules according to the present disclosure. In an embodiment, said cytotoxic T cell is a cytotoxic CD8+ T cell.

[0440] In an embodiment, said cellular response is selected from the group consisting of: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, and expression of activation markers.

[0441] In an embodiment, the present disclosure provides a method for inducing human T-cell proliferation in the presence of a target cell, such as a tumor cell, comprising contacting said cell in the presence of a T-cell with a pair of complementary CyCAT halfbody molecules according to the present disclosure.

[0442] In an embodiment, the present disclosure provides a method for stimulating a primary T- cell response in the presence of a target cell, such as a tumor cell, comprising contacting said cell in the presence of said T-cell with a pair of complementary CyCAT halfbody molecules according to the present disclosure.

[0443] In an embodiment, the present disclosure provides a method for re-directing cytotoxic activity of a T-cell to a target cell, such as a tumor cell, comprising contacting said cancer cells in the presence of said T-cell with a pair of complementary CyCAT halfbody molecules according to the present disclosure.

[0444] In an embodiment, the present disclosure provides the use of a pair of complementary CyCAT halfbody molecules according to the present disclosure for the treatment of cancer that is positive for at least two tumor associated antigen (TAA) in a subject, comprising:

[0445] (a) selecting a subject who is afflicted with a cancer,

[0446] (b) collecting one or more biological samples from the subject,

[0447] (c) identifying the at least two tumor associated antigens expressing cancer cells in the one or more samples, and

[0448] (d) administering to the subject an effective amount of a pair of CyCAT halfbody molecules according to the present disclosure.

[0449] In an embodiment, said cancer cell expresses a first and second TAA. In a preferred embodiment, said first and second TAA are different. In an embodiment, in the pair of complementary CyCAT halfbody molecule according to the present disclosure, HB1 binds to the first TAA and HB2 binds to the second TAA.

[0450] Fusion molecules

[0451] A halfbody molecule according to the present disclosure may be fused to one or more further moieties. Such a fusion protein may be prepared in any suitable manner, including genetically or chemically approaches. Said linked moieties may contain secretory or leader sequences, sequences that aid detection, expression, separation or purification, or sequences that confer to increased protein stability, for example, during recombinant production. Non-limiting examples of potential moieties include beta-galactosidase, glutathione-S-transferase, luciferase, a T7 polymerase fragment, a secretion signal peptide, a toxin, an antibody or antibody fragment, a reporter enzyme, a moiety being capable of binding a metal ion like a poly-histidine tag, a tag suitable for detection and / or purification, a moiety which increases solubility of a protein, or a moiety which comprises an enzymatic cleavage site. It should be clear that such further moieties may or may not provide further functionality to an halfbody molecule according to the present disclosure and may or may not modify the properties of HB1 and / or HB2. The halfbody molecules and moieties according to the present disclosure may be fused by peptide linkers as described herein.

[0452] Production

[0453] Methods to produce CyCAT halfbody molecules as disclosed herein are well known in the art (see e.g. Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). An CyCAT halfbody molecule according to the present disclosure may be obtained, for example, by solid-state peptide synthesis or recombinant production. For recombinant production, one or more nucleic acid sequences encoding an CyCAT halfbody molecule are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Complementary CyCAT halfbody molecules according the present disclosure a preferably produced separately.

[0454] Methods which are well known to those skilled in the art can be used to construct expression vectors containing the coding sequences for a CyCAT halfbody molecule along with appropriate transcriptional / translational control signals. Such methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. See, for example, the techniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, N.Y (1989). 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 of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. The coding sequences can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator, so that the DNA sequence encoding the desired protein or polypeptide is transcribed into RNA in the host cell transformed by a vector or vectors containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Depending on the expression system and host cell selected, a halfbody molecule according to the present disclosure is produced by growing host cells transformed by expression vectors described before under conditions whereby the halfbody molecule of interest is expressed. The halfbody molecule is then isolated from the host cells and purified. If the expression system secretes the halfbody molecule into growth media, the protein can be purified directly from the media. If the CyCAT halfbody molecule 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. It should be noted that an CyCAT halfbody molecule according to the present disclosure is not a naturally occurring protein. Typically, a halfbody molecule according to the present disclosure is a recombinant, synthetic or semisynthetic protein. In an embodiment, a method of producing an halfbody molecule according to the present disclosure is provided, wherein the method comprises culturing a host cell comprising a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid sequence or plurality of nucleic acid sequences encoding an CyCAT halfbody molecule according to the present disclosure, under conditions suitable for expression of the CyCAT halfbody molecule, and recovering the CyCAT halfbody molecule from the host cell or host cell culture medium. In embodiments, the methods for the production of an CyCAT halfbody molecule according to the present disclosure further comprise the step of isolating the produced CyCAT halfbody molecule from the host cells or medium. An halfbody molecule recovered as described herein may be purified techniques know in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The conditions used to purify a particular halfbody molecule will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity etc., and will be apparent to those having skill in the art. For affinity chromatography purification an antibody, ligand, receptor or antigen can be used to which an halfbody molecule binds. For example, for affinity chromatography purification of CyCAT halfbody molecules comprising a IgG Fc region, a matrix with protein A or protein G may be used. The purity of an CyCAT halfbody molecule can be determined by any of a variety of well-known analytical methods including gel electrophoresis, high-pressure liquid chromatography, and the like.

[0455] Therapeutic Methods

[0456] A pair of complementary CyCAT halfbody molecules according to the present disclosure may be used in therapeutic methods. In an embodiment, the present disclosure provides a pair of CyCAT halfbody molecules according to the present disclosure for the treatment of a disease. In an embodiment, the present disclosure provides a pair of complementary CyCAT halfbody molecules for use in the treatment of a disease in a subject in need thereof. The pair of CyCAT halfbody molecules according to the present disclosure may be used for the treatment of cancer. In an embodiment, the present disclosure provides the use of a pair of CyCAT halfbody molecules for the manufacture of a medicament. In an embodiment, the present disclosure provides a pair of CyCAT halfbody molecules for use as a medicament. In an embodiment, the present disclosure provides a pair of CyCAT halfbody molecules for use as a medicament for the treatment of a disease in a subject in need thereof. In an embodiment, the disease is associated with the undesired presence of an antigen. In a preferred embodiment, the disease is associated with the undesired presence of two antigens.

[0457] In an embodiment, the disease to be treated is a proliferative disease. In a particular embodiment, the disease is a cancer or a tumor. Non-limiting examples of cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer.

[0458] In an embodiment, the present disclosure provides a pair of complementary halfbody molecules according to the present disclosure for use in a method of treating a subject having a disease comprising administering to the subject a therapeutically effective amounts of the pair of complementary CyCAT halfbody molecules.

[0459] In an embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more specifically a human. For use in therapeutic methods, a pair of complementary halfbody molecules according to the present disclosure would be formulated, dosed, and administered in a way consistent with good medical practice. In an embodiment, in the pair of complementary halfbody molecules according to the present disclosure, the two complementary halfbody molecules are administered separately. In an embodiment, the two complementary halfbody molecules are administered consecutively. In an embodiment, the two complementary halfbody molecules are administered one after the other.

[0460] In an embodiment, the present disclosure provides a method for induction of tumor regression in a patient who has cancer, comprising administering to said subject, a therapeutically effective amount of a pair of CyCAT halfbody molecule. In an embodiment, the present disclosure provides a method for improving survival of a subject who has cancer, comprising administering to said subject a therapeutically effective amount of a pair of complementary halfbody molecule. In an embodiment, the present disclosure provides a method for eliciting, stimulating or inducing an immune response in a subject who has cancer, comprising administering to said subject, a therapeutically effective amount of a pair of complementary halfbody molecules according to the present disclosure. In an embodiment, the present disclosure provides a method for enhancing or inducing anti-cancer immunity in a subject who has cancer, comprising administering to said subject, a therapeutically effective amount of a pair of complementary halfbody molecule according to the present disclosure.

[0461] In an embodiment, the present disclosure provides a method for treating cancer in a subject caused by a cancerous cell expressing a first and a second antigen on its cell surface, wherein a first halfbody molecule comprising a Guard-Domain, either the aCD3- VH or aCD3-VL and a Fab that binds to the first antigen; and a second halfbody molecule comprising either the complementary aCD3-VH or aCD3-VL, respectively, and a Fab that binds to the second antigen, are administered simultaneously or sequentially to a subject having that cancerous cell that expresses the first antigen and the second antigen on its cell surface. In an embodiment, said first halfbody molecule and said second halfbody molecule are not linked by a covalent bond. In an embodiment, said first halfbody molecule and said second halfbody molecule are not linked by a covalent bond before and after administration but form a heterodimer on the surface of said cancerous cell. Pharmaceutical Compositions

[0462] In an embodiment, the present disclosure provides a pharmaceutical composition comprising an halfbody molecule according to the present disclosure and at least one pharmaceutically acceptable carrier. In an embodiment, the present disclosure provides a pharmaceutical composition comprising HB1 and HB2 according to the present disclosure, said pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In further embodiments, HB1 and HB2 may be formulated or comprised in a separate pharmaceutical composition.

[0463] In an embodiment, the present disclosure provides a first pharmaceutical composition comprising HB1 according to the present disclosure and at least one pharmaceutically acceptable carrier. In an embodiment, the present disclosure provides a second pharmaceutical composition comprising HB2 according to the present disclosure and at least one pharmaceutically acceptable carrier. The pharmaceutical compositions may further comprise at least one other pharmaceutically active compound according to the present disclosure. The pharmaceutical compositions according to the present disclosure can be used in the diagnosis, prevention and / or treatment of diseases associated with the presence of AG1 and / or AG2.

[0464] In particular, the present disclosure provides (a) a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, suited for prophylactic, therapeutic and / or diagnostic use in a mammal, more particular in a human.

[0465] In an embodiment, the present disclosure provides a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2, according to the present disclosure, for use in the prevention and / or treatment of a disease associated with the undesired presence of AG1 and AG2.

[0466] In an embodiment, the present disclosure provides a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2 for use as a medicament. In an embodiment, the present disclosure provides a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2 for use in the prevention and / or treatment of a disease. In an embodiment, the present disclosure provides a method for the treatment of a disease in a subject in need thereof using a pharmaceutical composition comprising HB1 and a pharmaceutical composition comprising HB2.

[0467] In an embodiment, said disease is an autoimmune disease, inflammatory disease, cancer, vascular disease, infectious disease, thrombosis, myocardial infarction, and / or diabetes. In an embodiment, the disease is a proliferative disease. In a particular embodiment, the disease is a cancer or a tumor. In an embodiment, said disease is cancer. In an embodiment, said cancer is a cancer expressing AG1. In an embodiment, said cancer is a cancer expressing AG2. In an embodiment, said cancer is a cancer expressing AG1 and AG2.

[0468] Pharmaceutical compositions according to the present disclosure may comprise a therapeutically effective amount of HB1 and HB2, dissolved in a pharmaceutically acceptable carrier. In an embodiment, the present disclosure provides a kit comprising HB1 and HB2 according to the present disclosure. In an embodiment, the present disclosure provides a kit comprising a first pharmaceutical composition comprising HB1 and a second pharmaceutical composition comprising HB2. In an embodiment, the present disclosure provides a method for the treatment of an autoimmune disease, inflammatory disease, cancer, vascular disease, infectious disease, thrombosis, myocardial infarction, and / or diabetes in a subject in need thereof using a first pharmaceutical composition comprising a HB1 and a second pharmaceutical composition comprising HB2.

[0469] Dosing

[0470] For the prevention or treatment of a disease, the appropriate dosage of HB1 and HB2 according to the present disclosure, needs to ensure effective amounts for the purpose intended and will depend on the type of disease to be treated, the route of administration, the body weight of the subject, the seventy and course of the disease, whether the HB1 and HB2 are administered for preventive or therapeutic purposes, previous or concurrent therapeutic interventions, the subject's clinical history and response to the pair of complementary halfbody molecules, and the discretion of the attending physician. Effective dosages and schedules for administering pharmaceutical compositions comprising HB1 and HB2 of the present disclosure may be determined empirically; for example, patient progress can be monitored by periodic assessment, and the dose adjusted accordingly. Moreover, interspecies scaling of dosages can be performed using well-known methods in the art (e.g., Mordenti et al., 1991 , Pharmaceut. Res. 8:1351). In an embodiment, the present disclosure provides HB1 and HB2 according the present disclosure, wherein said HB1 and HB2 are administered at a dose sufficient to achieve a therapeutically effective serum level. The administration of HB1 and HB2 according to the present disclosure encompass separate administration, in which case, administration of HB1 occurs prior to, simultaneously, and / or following, administration of the HB2 or vice versa. In certain embodiments, HB1 and HB2 are administered intravenously. In certain embodiments, HB1 and HB2 are administered subcutaneously.

[0471] Combination Therapies

[0472] HB1 and HB2 according to the present disclosure may be administered in combination with one or more other therapeutic agents. "Therapeutic agent" encompasses any agent administered to treat a symptom or disease in a subject in need of such treatment. In certain embodiments, an additional therapeutic agent is an immunomodulatory agent, a cytostatic agent, an inhibitor of cell adhesion, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptotic inducers. Such other therapeutic agents are suitably present in combination in amounts that are effective for the purpose intended. Combination therapies encompass combined administration (where two or more therapeutic agents are included in the same or separate compositions), and separate administration, in which case, administration of HB1 and HB2 according to the present disclosure can occur prior to, simultaneously, and / or following, administration of the additional therapeutic agent. HB1 and HB2 according to the present disclosure can also be used in combination with radiation therapy.

[0473] Diagnostics

[0474] In an embodiment, the present disclosure provides the use of HB1 and HB2 according to the present disclosure for the diagnosis of a disease. In an embodiment, the present disclosure provides the use of HB1 and HB2 for the detection of AG1 and / or AG2. In an embodiment, the present disclosure provides a method for detecting AG1 and / or AG2 in a subject or a sample, comprising the step of contacting said subject or sample with the complementary pair of halfbody molecules according to the present. In an embodiment, the present disclosure provides a method for diagnosing a disease in a subject, comprising the step of contacting said subject or sample with a complementary pair of halfbody molecules according to the present disclosure.

[0475] WORKING EXAMPLES

[0476] The following are examples of molecules and methods according to the present disclosure. It is understood that various other embodiments may be practiced, given the general description provided herein. Standard methods were used to manipulate DNA as described in Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91 -3242.

[0477] Binding domains:

[0478] For human HER2 (UniPROT: P04626) binding, nucleotide sequences encoding the VH and VL domains of Trastuzumab (HERCEPTIN®) as described by Baselga et al. 1998, Cancer Res 58(13): 2825-2831) were used. Trastuzumab and its method of preparation are disclosed in US patent US 5,821 ,337.

[0479] For human EGFR (UniPROT: P00533) binding, nucleotide sequences encoding the VH and VL domains from Cetuximab (Erbitux®) were used. Cetuximab and its method of preparation are described in US patent US7,060,808.

[0480] For hen egg lysozyme binding, nucleotide sequences encoding the VH and VL domains from an in-house control antibody MOR03207 were used.

[0481] For CD3 binding, nucleotide sequences encoding the VH and VL domains of the human anti-CD3 antibodies according to Table 2 and Table 3 of the present application were used. The anti-CD3 antibodies and their method of preparation are described in WO2022 / 063819. A summary of the amino acid sequences of produced CyCAT halfbody molecules made in accordance with the examples described herein are set forth in the following sections.

[0482] Construction of CyCAT halfbody molecules

[0483] CyCAT halfbody formats lacking a GUARD-Domain

[0484] Various CyCAT halfbody molecules comprising a Fab fragment and an unpaired aCD3 antibody variable domain (“aCD3-SVD”), such as an aCD3-VH oraCD3-VL domain, were designed and produced.

[0485] B027 format

[0486] The structure of CyCAT halfbody molecule in the B027 format is provided in Figure 1 . This halfbody molecule is composed of a Fab fragment as a targeting moiety fused at the C- terminus of its Fab heavy chain to the N-terminus of a single aCD3-VL domain (Figure 1A) or single aCD3-VH domain (Figure 1A’) via a short peptide linker. Each halfbody molecule is composed of two polypeptide chains, a main chain comprising the Fab heavy chain and the aCD3-SVD and a second polypeptide comprising the Fab light chain of either Trastuzumab or Cetuximab as the context requires. Exemplary sequences of CyCAT halfbody molecules in the B027 format as used in the present examples are summarized below.

[0487] Halfbody 1 (HB-1): (Fab aHER2 I O.CD3VLIOW)

[0488] Main chain: VH(Trast )- CH1 - Linker(GQPSG (SEQ ID NO: 45))- VL(acD3iow) QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 65)

[0489] Halfbody 2 (HB-2): (Fab«HER2 1 OCCD3VHIOW)

[0490] Main chain: VH(Trast )- CH1 - Linker(GQPSG (SEQ ID NO: 45))- VH(acD3iow)

[0491] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFTFKSYYMSVWRQAPGKGLEW VANIDYQSQHAYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQG TLVTVSS (SEQ ID NO: 66) Halfbody 3 (HB-3): (Fab aEGFR I CD3VLIOW)

[0492] Main chain: VH(cetuxi ) - CH1 - Linker(GQPSG (SEQ ID NO: 45)) - VL(aCD3iow)

[0493] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0494] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0495] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0496] NTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRN

[0497] NQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID

[0498] NO: 67)

[0499] Halfbody 4 (HB-4): (Fab aEGFR I aCD3vHlow)

[0500] Main chain: VH(cetuxi.) - CH1 - Linker(GQPSG (SEQ ID NO: 45)) - VH( CD3iow)

[0501] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0502] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0503] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0504] NTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFTFKSYYMSVWRQAPGKGLEVWA

[0505] NIDYQSQHAYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTL

[0506] VTVSS (SEQ ID NO: 68)

[0507] Fab light chain of Trastuzumab

[0508] Light chain: VL(Trast ) - CL

[0509] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD

[0510] FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR

[0511] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0512] GEC (SEQ ID NO: 69)

[0513] Fab light chain of Cetuximab

[0514] Light chain: VL(cetuxi.) - CL

[0515] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0516] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0517] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

[0518] C (SEQ ID NO: 70)

[0519] B036 format

[0520] The structure of a CyCAT halfbody molecule in the B036 format is provided in Fig. 2A & Fig. 2A’. This halfbody format incorporates a full human lgG1 Fc region. The fusion of the Fab fragment to the single unpaired aCD3-VL (Fig. 2A) or aCD3-VH domain (Fig. 2A’) is achieved by a first peptide linker. The unpaired aCD3-SVD is then in turn fused to the Fc region subunit carrying the Knob-mutations via a second peptide linker which comprises a short stretch of an IgG hinge sequence. The second Fc region subunit carrying the Holemutations also comprises a short IgG hinge linker sequence at its N-terminus. The use of cysteine residues in the linker sequences allows for a further stabilization of the Fc region via the formation of interchain-disulfide bridges. Exemplary sequences of halfbody molecules in the B036 format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of three polypeptide chains, a first main chain comprising the Fab heavy chain, the aCD3-SVD and the first Fc region subunit; a second main chain comprising the 2ndFc region subunit; and a third chain comprising the Fab light chain of either Trastuzumab, Cetuximab, or aLysozyme as the context requires.

[0521] Halfbody 5 (HB-5): (FabaHER2 / aCD3vi_high)

[0522] Main chain 1 : VH(Trast )- CH1 - Linker(G4S)4(SEQ ID NO: 46) - VL(acD3VLhigh) - LinkeqpAPDA-

[0523] Hinge trunc,) - CH2(AEASS) -CH3(knob)

[0524] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0525] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0526] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0527] PSNTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGAN

[0528] YVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVF

[0529] GGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVT CVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[0530] DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)

[0531] Halfbody 6 (HB-6): (FabaEGFR / aCD3vHhigh)

[0532] Main chain 1 : VH(cetuxi.)- CH1 - Linker(G4S)4(SEQ ID NO: 46) - VH(acD3high) - Linker(PAPDA-mnge trunc.) - CH2(AEASS) - CH3(knob)

[0533] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHY MTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSA

[0534] EFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72)

[0535] Halfbody 7 (HB-7): (FabaEGFR / CD3vi_high)

[0536] Main chain 1 : VH(cetuxi.)- CH1 - Linker(G4S)4 (SEQ ID NO: 46) - VL(acD3high) - Linker(PAPDA-mnge trunc.) - CH2(AEASS) - CH3(knob)

[0537] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0538] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYV

[0539] YWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGG GTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCV

[0540] WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPS SIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0541] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73)

[0542] Halfbody 8 (HB-8): B036 (ccLysozyme / aCD3vi_high)

[0543] Main chain 1 : VH(Lysoz ) - CH1 - Linker(G4S)4 (SEQ ID NO: 46) - VL(acD3high) - Linker(PAPDA-Hinge trunc.) - CH2(AEASS) - CH3(knob)

[0544] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY

[0545] ICNVNHKPSNTKVDKKVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGS

[0546] SSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDR

[0547] HSHGAVFGGGTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLM ISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC KVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNY

[0548] KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74)

[0549] Main chain 2: Linker(Hinge trunc.) - CH2(AEASS) - CH3(hoie)

[0550] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0551] REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKN

[0552] QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMH

[0553] EALHNHYTQKSLSLSPGK (SEQ ID NO: 75)

[0554] Fab light chain of Trastuzumab

[0555] Light chain: VL(Trast ) - CL

[0556] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD

[0557] FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR

[0558] EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR

[0559] GEC (SEQ ID NO: 69)

[0560] Fab light chain of Cetuximab

[0561] Light chain: VL(cetuxi.) - CL

[0562] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0563] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0564] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE

[0565] C (SEQ ID NO: 70)

[0566] Fab light chain of aLysozyme

[0567] Light chain: VL(i_y soz.) - CL

[0568] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTA

[0569] TLTISGTQAEDEADYYCASWDPSSGWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFY

[0570] PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT

[0571] ECS (SEQ ID NO: 76) B039 format

[0572] The structure of a CyCAT halfbody molecule in the B039 format is provided in Fig. 2C & Fig. 2C’. This halfbody format is composed of two identical Fab fragments as targeting moieties as well as two identical neighboring aCD3-SVDs (e.g. two identical aCD3-VH (Fig. 2C’) or two identical aCD3-VL domains (Fig. 2C)). The ocCD3-SVDs are centrally located between the two Fab arms and the Fc region. The fusion of each one of the two Fab fragments to either one of the two identical aCD3-SVDs is achieved by peptide linkers. Each of the two identical aCD3-SVDs are in turn fused to one of the two Fc region subunits by using two additional peptide linkers comprising a short stretch of an IgG hinge sequence. Based on its symmetrical structure this format is void of Knob-into-Hole mutations in its Fc region. Exemplary sequences of halfbody molecules in the B039 format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of four polypeptide chains, two identical main chains each comprising one Fab heavy chain, the aCD3-SVD and the first Fc region subunit; and two identical polypeptide chains comprising the Fab light chain of Cetuximab.

[0573] Halfbody 9 (HB-9): B039 (FabaEGFR / aCD3vHhigh)

[0574] Main chains: VH(cetuxi )- CH1 - Linker(G4S)4(SEQ ID NO: 46) - VH(acD3high) - Linker(PAPDA-Hinge trunc.) - CH2(AEASS) - CH3

[0575] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0576] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0577] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0578] NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHY

[0579] MTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSA

[0580] EFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKD

[0581] TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE

[0582] NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77)

[0583] Halfbody 10 (HB-10): B039 (FabaEGFR / aCD3vi_high)

[0584] Main chains: VH(cetuxi.) - CH1 - Linker(G4S)4 (SEQ ID NO: 46) - VL(acD3high) - Linker(PAPDA-Hinge trunc.) - CH2(AEASS) - CH3

[0585] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0586] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0587] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0588] NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYV

[0589] YWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGG GTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPS SIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78)

[0590] Fab light chain of Cetuximab

[0591] Light chain: VL(cetuxi.) - CL

[0592] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0593] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0594] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[0595] B063 format

[0596] The structure of CyCAT halfbody molecule in the B063 format is provided in Fig. 2B and Fig. 2B’. This halfbody format is composed of two identical Fab fragments as targeting moieties and one single aCD3-SVD (e.g. one aCD3-VH (Fig. 2B’) or one aCD3-VL (Fig. 2B)) which are located between one of the two Fab fragments and one Fc region subunit. The fusion of second Fab fragment to the Fc region subunit carrying the Hole-mutations is achieved by the short hinge derived sequence. The aCD3-SVD in turn is fused to the Fc region subunit carrying the Knob-mutations. Exemplary sequences of CyCAT halfbody molecules in the B063 format as used in the examples of the present disclosure are summarized in below. Each halfbody molecule is composed of four polypeptide chains, a first main chain comprising the first Fab heavy chain, the single aCD3-SVD and the first Fc region subunit; a second main chain comprising the second Fab heavy chain and the second Fc region subunit, and two identical polypeptides each comprising the Fab light chain of Cetuximab.

[0597] Halfbody 11 (HB-11): B063 (FabaEGFR / aCD3vHhigh)

[0598] Main chain 1 : VH(cetuxi.)- CH1 - Linker(G4S)4(SEQ iD NO: 46) - VH(acD3high) - Linker(PAPDA-Hinge trunc.) - CH2(AEASS) - CH3(knob)

[0599] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHY MTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSA EFAHRSGLDVWGQGTLVTVSSAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKD TLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72) Halfbody 12 (HB-12): B063 (FabaEGFR / aCD3vi_high)

[0600] Main chain 1 : VH(cetuxi ) - CH1 - Linker(G4S)4 (SEQ ID NO: 46) - VL(acD3high) - Linker(PAPDA-Hinge trunc.) - CH2(AEASS) - CH3(knob)

[0601] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0602] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYV YWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGG

[0603] GTKLTVLGQAQPAAPAPDAHEAPAPAQGSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCV WDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPS

[0604] SIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS

[0605] DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73)

[0606] Second main chain for the halfbody molecules in the B063 format

[0607] Main chain 2: VH(cetuxi ) — CH1 — Linkeqninge trunc.) — CH2(AEASSJ — CH3(hoie)

[0608] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0609] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0610] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0611] NTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW

[0612] YVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQ

[0613] VCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR

[0614] WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 79)

[0615] Fab light chain of Cetuximab

[0616] Light chain: VL(cetuxi.) - CL

[0617] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0618] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0619] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[0620] B038 format

[0621] The structure of CyCAT halfbody molecule in the B038 format is provided in Fig. 2D and Fig. 2D’. This halfbody format is composed of an human lgG1 Fc region centrally located between one Fab arm and one single unpaired aCD3-VL (Fig. 2D) or aCD3-VH (Fig. 2D’) domain. The fusion between the C-terminus of the Fab heavy chain and the N-terminus of one of the Fc region subunits carrying the Knob-mutations is achieved by using a short peptide linker comprising a hinge derived amino acid sequence . The second Fc region subunit comprising the Hole-mutations also comprises a short hinge derived peptide linker at its N-terminus. Exemplary sequences of CyCAT halfbody molecules in the B038 format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of three polypeptide chains, a first main chain comprising the Fab heavy chain of either Trastuzumab or aLysozyme, the first Fc region subunit and the single aCD3-SVD and; a second main chain comprising the second Fc region subunit; and a third polypeptide comprising the Fab light chain of either Trastuzumab or aLysozyme, as the context requires.

[0622] Halfbody 13 (HB-13): B038 (FabaHER2 / aCD3vi_high)

[0623] Main chain: VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker((G4S)4) (SEQ iD NO:

[0624] 46) - VL(aCD3high)

[0625] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASG TPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSE DEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 80)

[0626] Halfbody 14 (HB-14): B038 (FabaHER2 / <XCD3VLIOW)

[0627] Main chain: VH(Trast ) - CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob) - Linker(G4S)4 (SEQ ID NO:

[0628] 46) - VL(aCD3low)

[0629] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0630] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0631] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASG TPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSE

[0632] DEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 81)

[0633] Halfbody 15 (HB-15): B038 (FabaHER2 / aCD3vHhigh)

[0634] Main chain: VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker((G4S)4) (SEQ iD NO:

[0635] 46) - VH(aCD3high)

[0636] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0637] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0638] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLV

[0639] QPGGSLRLSCAASGFTFRSHYMTWVRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 82) Halfbody 16 (HB-16): B038 (a Lysozyme / aCD3vi_high)

[0640] Main chain: VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker((G4S)4) (SEQ ID NO:

[0641] 46) - VL(aCD3high)

[0642] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA

[0643] KGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQP PSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 83)

[0644] Halfbody 17 (HB-17): B038 (aLysozyme / aCD3vuow)

[0645] Main chain: VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker((G4S)4) (SEQ ID NO:

[0646] 46) - VL(aCD3Vllow)

[0647] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA

[0648] KGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSQSVLTQP PSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 84)

[0649] Halfbody 18 (HB-18): B038 (aLysozyme / aCD3vHhigh)

[0650] Main chain: VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker((G4S)4) (SEQ ID NO:

[0651] 46) - VH(aCD3VHhigh)

[0652] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL

[0653] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHE

[0654] DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKA KGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVES

[0655] GGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNA KNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 85)

[0656] Second main chain for halfbody molecules in the B038 format

[0657] 2ndMain Chain: LinkeF(Hingetrunc.)- CH2(AEASS) - CH3(hoie)

[0658] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKP

[0659] REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKN

[0660] QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMH

[0661] EALHNHYTQKSLSLSPGK (SEQ ID NO: 75) Fab light chain of aLysozyme

[0662] Light chain: VL(Lysozyme)—CL

[0663] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTA

[0664] TLTISGTQAEDEADYYCASWDPSSGWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFY

[0665] PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT

[0666] ECS (SEQ ID NO: 76)

[0667] Fab light chain of Trastuzumab

[0668] Light chain: VL(Trast ) - CL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69)

[0669] B064 format

[0670] The structure of a CyCAT halfbody molecule in the B064 is provided in Fig. 2E and Fig. 2E’. As an alternative embodiment to the B038 format, this halfbody format is composed of two identical Fab fragments allowing for bivalent targeting. Exemplary sequences of halfbody molecules in the B064 format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of four polypeptide chains, a first main chain comprising the first Fab heavy chain of either Trastuzumab or aLysozyme, the first Fc region subunit comprising the Knob-mutations, and the single aCD3-SVD and; a second main chain comprising the second Fab heavy chain of either Trastuzumab or aLysozyme, and the second Fc region subunit comprising the Holemutations; and two identical polypeptides each comprising the Fab light chain of either Trastuzumab or aLysozyme, as the contest requires.

[0671] Halfbody 19 (HB-19): B064 (Fab«HER21 aCD3vLhigh)

[0672] Main chain 1 : VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker(G4S)4(SEQ iD

[0673] NO: 46) - VL(aCD3high) QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGT PGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED EADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86) Halfbody 20 (HB-20): B064 (Fab aHER2 I aCD3vLlow)

[0674] Main chain 1 : VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker(G4S)4(SEQ iD

[0675] NO: 46) - VL(aCD3low)

[0676] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0677] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0678] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0679] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0680] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[0681] EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0682] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGT

[0683] PGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED

[0684] EADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 87)

[0685] Halfbody 21 (HB-21): B064 (Fab aHER2 I O(.CD3vHhigh)

[0686] Main chain 1 : VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob)- Linker(G4S)4(SEQ iD

[0687] NO: 46) - VH(aCD3high)

[0688] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0689] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0690] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0691] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0692] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[0693] EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[0694] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLV

[0695] QPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYL

[0696] QMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[0697] Halfbody 22 (HB-22) (B064) (aLysozyme I aCD3vi_high)

[0698] Main chain 1 : VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob) -Linker(G4S)4(SEQ iD

[0699] NO: 46) - VL(aCD3high)

[0700] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0701] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQP PSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS

[0702] GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 89)

[0703] Halfbody 23 (HB-23) (B064) (aLysozyme I aCD3vuow)

[0704] Main chain 1 : VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob) - Linker(G4S)4(SEQ iD

[0705] NO: 46) - VL(«CD3IOW)

[0706] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0707] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQP PSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS

[0708] GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 90)

[0709] Halfbody 24 (HB-24) (B064) (aLysozyme I CD3vHhigh)

[0710] Main chain 1 : VH(Lysoz)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(knob) -Linker(G4S)4(SEQ iD

[0711] NO: 46) - VH(«CD3high)

[0712] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0713] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNA

[0714] KNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 91)

[0715] Second main chain for halfbody molecules in the B064 format

[0716] Main chain 2: VH(Trast)- CH1 - Linker(hinge) - CH2(AEASS) - CH3(hoie)

[0717] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0718] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0719] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0720] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0721] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[0722] EPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK

[0723] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 92)

[0724] Main chain 2: VH(Lysoz) - CH1 - LinkeC(hinge) - CH2(AEASS) - CH3(hole)

[0725] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS

[0726] RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL

[0727] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY

[0728] ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0729] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK

[0730] AKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV

[0731] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 93)

[0732] Fab light chain of aLysozyme

[0733] Light chain: VL(i_ysozyme.)—CL

[0734] DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTA

[0735] TLTISGTQAEDEADYYCASWDPSSGWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFY

[0736] PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS (SEQ ID NO: 76)

[0737] Fab light chain of Trastuzumab:

[0738] Light chain: VL(Trast ) - CL

[0739] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69) B050 format

[0740] The structure of a CyCAT halfbody molecule in the B050 is provided in Fig. 2F and Fig. 2F’. As an alternative embodiment to the B064 format, this halfbody is composed of two identical neighboring aCD3-SVDs (e.g. either two identical aCD3-VH domains (Fig. 2F’) or two identical aCD3-VL domains (Fig. 2F)) present at the C-terminus of the halfbody. Because of its symmetrical structure and the presence of two identical main chains, this format is void of Knob-into-Hole mutations in its Fc region. Exemplary sequences of CyCAT halfbody molecules in the B050 format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of four polypeptide chains, two identical main chains comprising the Fab heavy chains of either Trastuzumab or aLysozyme, the Fc region subunits, and a aCD3-SVD; and two identical polypeptides each comprising the Fab light chain of either Trastuzumab or aLysozyme, as the context requires.

[0741] Halfbody 25 (HB-25): B050 (FabaHER21 aCD3vi_high)

[0742] Main chains: VH(Trast )- CH1 - Linker(hinge) - CH2(AEASS) - CH3 - Linker(G4S)4(SEQ iD NO: 46)

[0743] - VL(aCD3high)

[0744] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTP GQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 94)

[0745] Halfbody 26 (HB-26): B050 (Fab aHER2 I aCD3vLlow)

[0746] Main chains: VH(Trast )- CH1 - Linker(hinge) - CH2(AEASS) - CH3 - Linker(G4S)4(SEQ iD NO: 46)

[0747] - VL(aCD3low)

[0748] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE VARIYPTNGYTRYADSVKGRFTI

[0749] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0750] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0751] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[0752] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[0753] EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0754] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTP

[0755] GQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE

[0756] ADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 95) Halfbody 27 (HB-27): B050 (Fab <xHER2 I OtCD3vHhigh)

[0757] Main chains: VH(Trast )- CH1 - Linker(hinge) - CH2(AEASS) - CH3- Linker(G4S)4(SEQ iD NO: 46)

[0758] - VH(aCD3high)

[0759] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK

[0760] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGS

[0761] EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO:96)

[0762] Halfbody 28 (HB-28): B050 (aLysozyme I aCD3vLhigh)

[0763] Main chains: VH(Lysoz )- CH1 - Linker(hinge) - CH2(AEASS) - CH3 -Linker(G4S)4(SEQ iD NO: 46)

[0764] - VL(aCD3high)

[0765] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0766] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQP PSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS

[0767] GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 97)

[0768] Halfbody 29 (HB-29): B050 (aLysozyme I aCD3vuow)

[0769] Main chains: VH(Lysoz) - CH1 - Linker(hinge) - CH2(AEASS) - CH3 - Linker(G4S)4 (SEQ iD NO: 46)

[0770] - VL(aCD3low)

[0771] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS

[0772] RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL

[0773] APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY

[0774] ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0775] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK

[0776] AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0777] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQP

[0778] PSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAIS

[0779] GLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 98)

[0780] Halfbody 30 (HB-30): B050 (aLysozyme I aCD3vHhigh)

[0781] Main chains: VH(Lysoz )- CH1 - Linker(hinge) - CH2(AEASS) - CH3 - Linker(G4S)4(SEQ iD NO: 46)

[0782] - VH(aCD3high)

[0783] QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWSWIRQSPGRGLEWLGRIYYRSKWYNDYAVSVKS RITINPDTSKNQFSLQLNSVTPEDTAVYYCARLDHRYHEDTVYPGMDVWGQGTLVTVSSASTKGPSVFPL APSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTY ICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSH

[0784] EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY

[0785] SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVES GGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNA

[0786] KNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 99)

[0787] Fab light chain of aLysozyme

[0788] Light Chain: VL(Lysozyme)—CL DIELTQPPSVSVAPGQTARISCSGDNLPAYTVTWYQQKPGQAPVLVIYDDSDRPSGIPERFSGSNSGNTA TLTISGTQAEDEADYYCASWDPSSGWFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFY PGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS (SEQ ID NO: 76)

[0789] Fab light chain of Trastuzumab

[0790] Light Chain: VL(Trast ) - CL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69)

[0791] CyCAT halfbody formats comprising Guard-Domains (HBGD)

[0792] A Guard-Domain (GD) attached to a CyCAT halfbody as described herein sterically occludes an unpaired aCD3-SVD present on the same halfbody from binding to a complementary aCD3-SVD present on a second halfbody molecule in the absence of target cells expressing both target antigens or only one of the two target antigen of interest. Preferably, the GD of the present disclosure is not able to bind or interact with the unpaired aCD3-SVD present on the same halfbody molecule to be shielded.

[0793] In the present examples, this is exemplified by either using human serum albumin (HSA) (SEQ ID NO: 17), Domain III of HSA (SEQ ID NO: 18), an inactive VL Guard-Domain (such as having the amino acid sequence of SEQ ID NO: 14), an inactive VH Guard- Domain (such as having the amino acid sequence of SEQ ID NO: 15) or an inactive VH Knob-Guard-Domain (such as having the amino acid sequence of SEQ ID NO: 16).

[0794] Negative Controls: Guard-Domain Halfbodies lacking an aCD3-SVD

[0795] Amino acid sequences of halfbodies based on the B027 format as described above comprising a Fab fragment with specificity for HER2 or EGFR fused to an VL Guard- Domain (GDVL) or an VH Guard-Domain (GDVH) or VH Knob Guard-Domain (GDvH-Knob). Each halfbody molecule is composed of two polypeptide chains, a main chain comprising the Fab heavy chain of either Trastuzumab or Cetuximab and a Guard-Domain; and a second polypeptide comprising the Fab light chain of either Trastuzumab or Cetuximab, as the context requires.

[0796] GD-Halfbody 1 (HBGD-1): B027 (Fab aHER2 I GDVL)

[0797] Main chain: VH(Trast )- CH1 - Linker(GQPSG (SEQ ID NO: 45))- GDVL

[0798] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIY RNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 100)

[0799] GD-Halfbody 2 (HBGD-2): B027 (Fab«HER21 GDVH)

[0800] Main chain: VH(Trast ) - CH1 - LinkeqcopsG (SEQ ID NO: 45)) - GDVH

[0801] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0802] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0803] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0804] PSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSVWRQAPGKGLEW

[0805] VANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQG

[0806] TLVTVSS (SEQ ID NO: 101)

[0807] GD-Halfbody 3 (HBGD-3): B027 (Fab aHER2 I GDvH-Knob)

[0808] Main chain: VH(Trast ) - CH1 - LinkeqcopsG (SEQ ID NO: 45)) - GDvH-Knob

[0809] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[0810] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[0811] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[0812] PSNTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEW

[0813] VANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQG

[0814] TLVTVSS (SEQ ID NO: 102)

[0815] GD-Halfbody 4 (HBGD-4): B027 (Fab aEGFR I GDVL)

[0816] Main chain: VH(cetuxi.) - CH1 - Linker(GQPSG (SEQ ID NO: 45)) - GDVL

[0817] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0818] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0819] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0820] NTKVDKKVEPKSCGQPSGQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRN

[0821] NQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO:

[0822] 103)

[0823] GD-Halfbody 5 (HBGD-5): B027 (Fab aEGFR I GDVH)

[0824] Main chain: VH(cetuxi.) - CH1 - Linker(GQPSG (SEQ ID NO: 45)) - GDVH

[0825] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0826] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0827] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0828] NTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSVWRQAPGKGLEVWA NINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLV

[0829] TVSS (SEQ ID NO: 104)

[0830] GD-Halfbody 6 (HBGD-6): B027 (Fab aEGFR I GDvH-Knob)

[0831] Main chain: VH(cetuxi.) - CH1 - Linker(GQPSG (SEQ DI NO: 45)) - GDvH-Knob

[0832] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0833] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0834] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0835] NTKVDKKVEPKSCGQPSGEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEVWA

[0836] NINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLV

[0837] TVSS (SEQ ID NO: 105)

[0838] Fab light chain of Trastuzumab

[0839] Light chain: VL(Trast ) - CL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69)

[0840] Fab light chain of Cetuximab

[0841] Light chain: VL(cetuxi.) - CL DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[0842] B073 Guard-Domain Halfbodies

[0843] The structure of a halfbody molecules in the B073 Guard-Domain format is provided in Fig. 3A and Fig. 3A’. In difference to the B036 non Guard-Domain format, one single Guard-Domain is attached at its C-terminus to the N-terminus of the second Fc region subunit comprising the Hole-mutations. The fusion of the GD to the Fc region subunit is achieved via a short hinge sequence. By way of this fusion, the GD is positioned adjacent and parallel to the either aCD3-VH domain (Fig. 3A’) or aCD3-VL domain (Fig. 3A), which allows for an optimal shielding effect. Sequences of halfbody molecules in the B073 format either using a GDVH, a GDvH-Knob, a GDVL, a GDHSAWI, or a GDHSA-DIII as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of three polypeptide chains: a first main chain comprising the Fab heavy chain of Cetuximab, the aCD3-SVD and the first Fc region subunit; a second main chain comprising the Guard-Domain and the 2ndFc region subunit, and a third polypeptide chain comprising the Fab light chain of Cetuximab. GD-Halfbody 7 (H BGD-7): B073 (Fab aEGFR I OtCD3vHhigh I GDVH)

[0844] Main chain 1 : VH(cetuxi )— CH1 — Linker(APAE_ioj — VH(aCD3high) — Linker(Hinge trunc,j —

[0845] CH2(AEASS) - CH3(knob)

[0846] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0847] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0848] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0849] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[0850] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[0851] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[0852] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[0853] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0854] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0855] Main chain 2: GDVH - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hoie)

[0856] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSVWRQAPGKGLEVWANINQIGYSSYYVESVKGRF

[0857] TISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAE

[0858] GAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS

[0859] VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYP

[0860] SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

[0861] SPGK (SEQ ID NO: 107)

[0862] GD-Halfbody 8 (HBGD-8): B073 (Fab aEGFR I aCD3vHhigh I GDvH-Knob)

[0863] Main chain 1 : VH(cetuxi.)- CH1 - Linker(APAE_ ) - VH(aCD3high) - Linker - CH2(AEASS) -

[0864] CH3(knob)

[0865] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0866] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0867] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0868] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[0869] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[0870] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[0871] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[0872] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0873] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0874] Main-chain 2: GDvH-Knob — Linker(Hinge trunc,j — CH2(AEASSJ — CH3(hoie)

[0875] EVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGLEVWANINQIGYSSYYVESVKGRF

[0876] TISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAE

[0877] GAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS

[0878] VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYP

[0879] SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

[0880] SPGK (SEQ ID NO: 108)

[0881] GD-Halfbody 9 (HBGD-9): B073 (Fab EGFR I CD3vi_high I GDVL)

[0882] Main chain 1 : VH(cetuxi )— CH1 — Linker(APAE_ioj — VL( CD3high) — Linker(Hinge trunc,j —

[0883] CH2(AEASS) - CH3(knob)

[0884] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0885] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0886] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK

[0887] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH

[0888] TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0889] QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS

[0890] LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0891] HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0892] Main chain 2: GDVL - Linker(Hinge trunc,) - CH2(AEASS) - CH3(hoie)

[0893] QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKPK DTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGK EYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:

[0894] 110)

[0895] GD-Halfbody 10 (HBGD-10): B073 (Fab aEGFR I CD3vHhigh I GDHSA-DII|)

[0896] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VH(aCD3high) — Linksr(Hinge trunc,) —

[0897] CH2(AEASS) - CH3(knob)

[0898] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0899] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0900] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0901] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[0902] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[0903] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[0904] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[0905] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0906] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[0907] Main chain 2: GDHSA-DIII - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hoie)

[0908] LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCA

[0909] EDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE

[0910] RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEA

[0911] EGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW

[0912] SVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFY

[0913] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

[0914] LSPGK (SEQ ID NO: 1 11 )

[0915] GD-Halfbody 11 (HBGD-11 ): B073 (Fab aEGFR I aCD3vHhigh I GDHSAwt )

[0916] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VH( CD3high) — Linksr(Hinge trunc,) —

[0917] CH2(AEASS) - CH3(knob)

[0918] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0919] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0920] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0921] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[0922] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[0923] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[0924] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[0925] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[0926] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106) Main chain 2: GDHSAWI - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hoie)

[0927] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF GDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER

[0928] AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP

[0929] TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS

[0930] HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112)

[0931] GD-Halfbody 12 (HBGD-12): B073 (Fab aEGFR I CD3vLhigh I GDHSAwt)

[0932] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VL(aCD3high) — Linksr(Hinge trunc,) —

[0933] CH2(AEASS) - CH3(knob)

[0934] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0935] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0936] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0937] NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK

[0938] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH

[0939] TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[0940] QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS

[0941] LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0942] HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0943] Main chain 2: GDHSAWI - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hoie)

[0944] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLF GDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYL YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGER

[0945] AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECC EKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLA KTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTP

[0946] TLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSA LEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVS

[0947] HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTIS KAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL VSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 112)

[0948] GD-Halfbody 13 (HBGD-13): B073 (Fab aEGFR I aCD3vLhigh I GDHSA-DII|)

[0949] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VL( CD3high) — Linksr(Hinge trunc,) —

[0950] CH2(AEASS) - CH3(knob)

[0951] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0952] NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK

[0953] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[0954] HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[0955] Main chain 2: GDHSA-DIII - Linker(Hingetrunc,) -CH2(AEASS) - CH3(hoie)

[0956] LVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCA

[0957] EDYLSWLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE

[0958] RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAKTHTCPPCPAPEA

[0959] EGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRW

[0960] SVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFY

[0961] PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS

[0962] LSPGK (SEQ ID NO: 1 11 )

[0963] Fab light chain Cetuximab:

[0964] Light Chain: VL(Cetuxi.) - CL

[0965] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0966] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[0967] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[0968] B099 Guard-Domain Halfbodies

[0969] The structure of a halfbody molecule in the B099 format is provided in Fig. 3B and Fig. 3B’. In difference to the B073 format, the Guard-Domain and the aCD3-SVD are swapped in such way that the GD is present on the polypeptide comprising the Fab heavy chain and the Fc region subunit having the Hole-mutations and the aCD3-SVD is present on the polypeptide comprising the second Fc region subunit having the Knob mutations. Exemplary sequences of CyCAT halfbody molecule in the B099 Guard-Domain format as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of three polypeptide chains, a first main chain comprising the Fab heavy chain of Cetuximab, the Guard-Domain and the first Fc region subunit; a second main chain comprising the aCD3-SVD and the 2ndFc region subunit; and a third polypeptide chain comprising the Fab light chain of Cetuximab.

[0970] GD-Halfbody 14 (HBGD-14): B099 (Fab aEGFR I GDVL I O(.CD3vLhigh)

[0971] Main chain 1 : VH(cetuxi.)- CH1 - LinkeqAPAE o) - GDVL - Linker(Hinge trunc,) - CH2(AEASS) - CH3(hole)

[0972] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHT CPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVS LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH

[0973] NHYTQKSLSLSPGK (SEQ ID NO: 1 13)

[0974] Main chain 2: VL(aCD3high) - Li nkeTfHinge trunc,) - CH2(AEASS) — CH3(knob)

[0975] QSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSG TSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTHTCPPCPAPEAEGAPSVFLFPPKP KDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNG

[0976] KEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQ

[0977] PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 114)

[0978] GD-Halfbody 15 (HBGD-15): B099 (Fab aEGFR I GDVH I O(.CD3vHhigh)

[0979] Main chain 1 : VH(cetuxi.)- CH1 - Linker(APAE_ ) - GDVH - Linker(Hingetrunc,) - CH2(AEASS) -

[0980] CH3(hole)

[0981] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[0982] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[0983] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[0984] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSVWRQAPGKGL

[0985] EVWANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWG

[0986] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLP

[0987] PSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQG

[0988] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 115)

[0989] Main chain 2: VH(acD3high) - Linker(Hingetrunc,) - CH2(AEASS) - CH3(knob)

[0990] EVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRF

[0991] TISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSSKTHTCPPCPAPEAE

[0992] GAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWS

[0993] VLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYP

[0994] SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

[0995] SPGK (SEQ ID NO: 116)

[0996] Fab light chain Cetuximab:

[0997] Light Chain: VL(cetuxi.) - CL

[0998] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[0999] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[1000] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[1001] B077 Guard-Domain Halfbodies

[1002] The structure of halfbody molecules in the B077 format is provided in Fig. 3C and Fig. 3C’. In difference to the B039 non Guard-Domain format, one of the two identical aCD3- SVD has been substituted for a Guard-Domain, which thus requires the introduction of Knob-into-Hole mutations in the Fc region. In the B077 format, a single Guard-Domain is fused at its N-terminus to the C-terminus of the second Fab heavy chain and at its C- terminus to the N-terminus of the Fc-region subunit carrying the Hole-mutations. By way of this fusion, the GD is positioned adjacent and parallel to the OCCD3SVD domain. Exemplary sequences of halfbody molecules in the B077 Guard-Domain format using either a GDVH, a GDvH-Knob, a GDHSAWI or a GDHSA-DIII are summarized below. Each halfbody molecule is composed of four polypeptide chains: one main chain comprising the Fab heavy chain of Cetuximab, the aCD3-SVD and the first Fc region subunit comprising the Knob-mutations; a second main chain comprising the Fab heavy chain of Cetuximab, the GD and the second Fc region subunit comprising the Hole-mutations; and two identical polypeptides each comprising the Fab light chain of Cetuximab.

[1003] GD-Halfbody 16 (HBGD-16): B077 (Fab aEGFR I O(.CD3vHhigh I GDVH)

[1004] Main chain 1 : VH(cetuxi ) — CH1 — Linker(APAE_io) — VH(CD3high) — Linkeqninge trunc,) — CH2(AEASS) - CH3(knob) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[1005] Main chain 2: VH(cetuxi.)- CH1 - LinkeqAPAE o) - OCGDVH - CH2(AEASS) - CH3(hoie) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSVWRQAPGKGL EVWANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWG QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 117)

[1006] GD-Halfbody 17 (HBGD-17): B077 (Fab aEGFR I aCD3vHhigh I GDvH-Knob)

[1007] Main chain 1 : VH(cetuxi ) — CH1 — Linker(APAE_io) — VH( CD3high) — Linker(Hinge trunc,j —

[1008] CH2(AEASS) - CH3(knob)

[1009] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1010] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1011] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[1012] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[1013] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[1014] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[1015] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[1016] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[1017] Main chain 2: VH(cetuxi.)- CH1 - Linker(APAE_ ) - GD(VH-Knob) - CH2(AEASS) - CH3(hoie) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFSFGSHYMSWFRQAPGKGL EVWANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGASAEFAHRSGLDVWG QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 118)

[1018] GD-Halfbody 18 (HBGD-18): B077 (Fab aEGFR I O(.CD3vLhigh I GDVL)

[1019] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VL(aCD3high) — Linksr(Hinge trunc,) —

[1020] CH2(AEASS) - CH3(knob)

[1021] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[1022] Main chain 2: VH(cetuxi.)- CH1 - Linker(APAE_ ) - GDVL - Linker(Hinge trunc,) -CH2(AEASS) -

[1023] CH3(hole)

[1024] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1025] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1026] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[1027] NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPK

[1028] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAVFGGGTKLTVLGQKTHT

[1029] CPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[1030] QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVS

[1031] LSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH

[1032] NHYTQKSLSLSPGK (SEQ ID NO: 1 13)

[1033] GD-Halfbody 19 (HBGD-19): B077 (Fab aEGFR I aCD3vHhigh I G DHSAwt)

[1034] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VH( CD3high) — Linksr(Hinge trunc,) —

[1035] CH2(AEASS) - CH3(knob)

[1036] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1037] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1038] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[1039] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[1040] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[1041] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[1042] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[1043] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[1044] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106) Main chain 2: VH(cetuxi )- CH1 - Linker(APAE_ ) - GDHSAWI - CH2(AEASS) - CH3(hoie)

[1045] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADD RADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFL GMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQL GEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKT PVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPK ATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFP

[1046] PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 1 19)

[1047] GD-Halfbody 20 (HBGD-20): B077 (Fab aEGFR I O(.CD3vHhigh I GDHSA-DII|)

[1048] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VH(aCD3high) — Linksr(Hinge trunc,) —

[1049] CH2(AEASS) - CH3(knob)

[1050] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1051] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1052] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[1053] NTKVDKRVEPKSCAQPAAPAPAEEVQLVESGGGLVQPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGL

[1054] EVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWG

[1055] QGTLVTVSSKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGV

[1056] EVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLP

[1057] PCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG

[1058] NVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 106)

[1059] Main chain 2: VH(cetuxi.)- CH1 - Linker(APAE_ ) - GDHSA-DIII - CH2(AEASS) - CH3(hoie) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKET CFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYT LPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 120)

[1060] GD-Halfbody 21 (HBGD-21 ): B077 (Fab aEGFR I aCD3vLhigh I G DHSAwt)

[1061] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VL( CD3high) — Linksr(Hinge trunc,) —

[1062] CH2(AEASS) - CH3(knob)

[1063] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1064] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1065] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK

[1066] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH

[1067] TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[1068] QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS

[1069] LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[1070] HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[1071] Main chain 2: VH(Cetuxi.)- CH1 - Linker(APAE_ ) - GD(HSAwt) - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hole)

[1072] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAEDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVT EFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLV RPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADD RADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFL GMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQL GEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKT PVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPK ATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLKTHTCPPCPAPEAEGAPSVFLFP PKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDW LNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 121 )

[1073] GD-Halfbody 22 (H BGD-22): B077 (Fab aEGFR I O(.CD3vLhigh I GDHSA-DII|)

[1074] Main chain 1 : VH(cetuxi )— CH1 — Linksr(APAE_io) — VL(aCD3high) — Linksr(Hinge trunc,) —

[1075] CH2(AEASS) - CH3(knob)

[1076] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI

[1077] NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

[1078] GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS

[1079] NTKVDKRVEPKSCAQPAAPAPAEQSVLTQPPSASGTPGQRVTISCSGSSSNIGANYVYWYQQLPGTAPK

[1080] LLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDRHSHGAVFGGGTKLTVLGQKTH

[1081] TCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREE

[1082] QYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPCREEMTKNQVS

[1083] LWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL

[1084] HNHYTQKSLSLSPGK (SEQ ID NO: 109)

[1085] Main chain 2: VH(cetuxi.)- CH1 - Linker(APAE_ ) - GDHSA-DIII - Linker(Hinge trunc,) -CH2(AEASS) - CH3(hole)

[1086] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHVWRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPS NTKVDKRVEPKSCAQPAAPAPAELVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEV SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKET CFAEEGKKLVAKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCT LPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 122) Fab light chain Cetuximab:

[1087] Light chain: VL(cetuxi ) - CL

[1088] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTL

[1089] SINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREA

[1090] KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C (SEQ ID NO: 70)

[1091] B103 Guard-Domain halfbodies

[1092] The structure of a halfbody molecule in the B103 format is provided in Fig. 3D and Fig. 3D’. In difference to the B038 non Guard-Domain format, one single Guard-Domain is fused at its N-terminus to the C-terminus of the second Fc region subunit comprising the Hole-mutations. By way of this fusion, the GD is positioned adjacent and parallel to the aCD3-SVD domain. Exemplary sequences of halfbody molecules in the B103 Guard- Domain format using either a GDVH or a GDVL as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of three polypeptide chains: one main chain comprising the Fab heavy chain Trastuzumab, the first Fc region subunit comprising the Knob-mutations and the aCD3-SVD; a second main chain comprising the second Fc region subunit comprising the Hole-mutations and the Guard-Domain, and two identical polypeptides each comprising the Fab light chain of Trastuzumab.

[1093] GD-Halfbody 23 (HBGD-23): B103 (FabaHER21 GDVL I CD3vLhigh)

[1094] Main Chain 1 : VH(Trast ) - CH1 - CH2(AEASS) - CH3(knob) - Linker(G4S)4(SEQ iD NO: 46) - VL(CD3high) QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGT PGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED EADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86)

[1095] Main Chain 2: Linker(Hinge trunc,) -CH2(AEASS) - CH3(hoie) - Linker(G4S)4 (SEQ ID NO: 46) - GDVL

[1096] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

[1097] REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKN

[1098] QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMH

[1099] EALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGS

[1100] NYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAADHHRAGAV

[1101] FGGGTKLTVLGQ (SEQ ID NO: 123) GD-Halfbody 24 (HBGD-24): B103 (FabaHER21 GDVH I ocCD3vHhigh)

[1102] Main Chain 1 : VH(Trast ) - CH1 - CH2(AEASS) - CH3(knob) - Linker(G4S)4(SEQ ID NO: 46) -

[1103] VH(CD3high)

[1104] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[1105] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[1106] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[1107] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[1108] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[1109] EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[1110] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYL

[1111] QMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[1112] Main Chain 2: Linker(Hinge trunc,) - CH2 (AEASS) - CH3(hoie) - Linker(G4S)4 (SEQ ID NO: 46) - GDVH

[1113] KTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKP

[1114] REEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVCTLPPSREEMTKN

[1115] QVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMH

[1116] EALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFSFG SHYMSVWRQAPGKGLEVWANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARG

[1117] ASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 124)

[1118] Fab light chain of Trastuzumab:

[1119] Light chain: VL(Trast ) - CL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69)

[1120] B101 Guard-Domain halfbodies

[1121] The structure of a halfbody molecule in the B101 Guard-Domain format is provided in Fig. 3E and Fig. 3E’. In difference to the B064 non Guard-Domain format, one single GD is fused to the C-terminus of the second Fc region subunit comprising the Hole-mutations. By way of this fusion, the GD is positioned adjacent and parallel to the aCD3-SVD domain. Exemplary sequences of halfbody molecules in the B101 format either using a GDVH or a GDVL as used in the examples of the present disclosure are summarized below. Each halfbody molecule is composed of four polypeptides: a first main chain comprising the first Fab heavy chain of Trastuzumab, the first Fc region subunit comprising the Knobmutations and the aCD3-SVD; a second main chain comprising the Fab heavy chain of Trastuzumab, the second Fc region subunit comprising the Hole-mutations and the GD; and two identical polypeptides each comprising the Fab light chain of Trastuzumab. GD-Halfbody 25 (HBGD-25): B101 (Fab aHER2 I GDVL I OtCD3vLhigh)

[1122] Main chain 1 : VH(Trast ) - CH1- CH2(AEASS) - CH3(knob) - Linker(G4S)4(SEQ ID NO: 46) -

[1123] VL(aCD3high)

[1124] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[1125] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[1126] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[1127] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[1128] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[1129] EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGT

[1130] PGQRVTISCSGSSSNIGANYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSED

[1131] EADYYCAAWDRHSHGAVFGGGTKLTVLGQ (SEQ ID NO: 86)

[1132] Main chain 2: VH(Trast ) - CH1 - CH2(AEASS) - CH3(hoie)- Linker(G4S)4(SEQ iD NO: 46) - GD(VL)

[1133] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[1134] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[1135] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[1136] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[1137] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[1138] EPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK

[1139] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSQSVLTQPPSASGTP GQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE

[1140] ADYYCAAADHHRAGAVFGGGTKLTVLGQ (SEQ ID NO: 125)

[1141] GD-Halfbody 26 (HBGD-26): B101 (FabaHER2 / GDVH I ocCD3vHhigh)

[1142] Main chain 1 : VH(Trast ) - CH1 - CH2(AEASS) - CH3(knob) - Linker(G4S)4(SEQ ID NO: 46) - VH(acD3)

[1143] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[1144] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[1145] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[1146] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[1147] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[1148] EPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD

[1149] KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLV QPGGSLRLSCAASGFTFRSHYMTVWRQAPGKGLEVWANIDYEGTRTYYAESVKGRFTISRDNAKNSLYL

[1150] QMNSLRAEDTAVYYCARGYSAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 88)

[1151] Main chain 2: VH(Trast ) - CH1- CH2(AEASS) - CH3(hoie) - Linker(G4S)4(SEQ ID NO: 46) - GD(VH)

[1152] QVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHVWRQAPGKGLEVWARIYPTNGYTRYADSVKGRFTI

[1153] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST

[1154] SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK

[1155] PSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKF

[1156] NWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPR

[1157] EPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDK

[1158] SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQ PGGSLRLSCAASGFSFGSHYMSVWRQAPGKGLEVWANINQIGYSSYYVESVKGRFTISRDNAKNSLYLQ

[1159] MNSLRAEDTAVYYCARGASAEFAHRSGLDVWGQGTLVTVSS (SEQ ID NO: 126) Fab light chain of Trastuzumab:

[1160] Light chain: VL(Trast ) - CL DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWFQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC (SEQ ID NO: 69)

[1161] General components

[1162] For CyCAT halfbody molecules employing two non-identical main chains, the Fc region was modified by introducing mutations into the CH3 domain of each Fc region subunit according to the knob-into-holes technology. Thereby, the Fc region subunit comprising one mutated CH3 domain is forced to heterodimerize with the other Fc region subunit comprising the other CH3 domain, which is engineered in a complementary manner. All Fc region subunits were additionally modified by introducing mutations into the CH2 domain in order to abolish its ability to mediate effector function, such as ADCC, CDC, ADCP and / or C1q binding (referred herein as to “AEASS” mutations).

[1163] Methods

[1164] Cloning of CyCAT halfbody molecules

[1165] All nucleic acid sequences or desired gene segments were generated by PCR using appropriate templates or were gene synthesized as linear DNA fragments with appropriate flanking regions (e.g. suitable restriction enzyme recognition sites, linker sequences) inhouse or by an external provider. The nucleic acid sequences or gene segments flanked by singular restriction endonuclease cleavage sites were cloned into respective mammalian expression vectors using standard molecular biology methods.

[1166] Production of CyCAT halfbody molecules

[1167] For expression of halfbody molecules, exponentially growing eukaryotic HEK293-6E cells were (co-)transfected with a mammalian expression vector encoding all components of a halfbody molecule, resulting in heteromeric molecules comprising either 1 :1 , 1 :1 :1 , or 1 :1 :1 :1 polypeptide chains. Cell culture supernatants were harvested on day 6 post transfection and subjected to anti-CH1 affinity chromatography or Protein A affinity chromatography, respectively. Buffer exchange was performed to 1x Dulbcecco's PBS (pH 7.2 | Invitrogen) and samples were sterile filtered (0.2 pm pore size). Protein concentrations were determined by UV-spectrophotometry and purities of the constructs were analyzed under denaturing, reducing and non-reducing conditions using CE-SDS (LabChip GX Touch | Perkin Elmer | USA). UHP-SEC was performed to analyze halfbody preparations in native state. The ability of the produced halfbodies to bind to their target antigen was confirmed, i.e. by standard ELISA using soluble ectodomains of human HER2 or human EGFR, respectively.

[1168] Isolation of human T-cells

[1169] Human whole blood from healthy donors was collected e.g. in Li-Heparin containing S- Monovette containers (Sarstedt). 20 mL blood were transferred to 50 mL conical tubes, mixed with 1 mL of RosetteSep Human CD8+ Enrichment Cocktail (Stemcell Technologies, #15063) and incubated for 20 min at room temperature. Blood containing RosetteSep human CD8+ enrichment cocktail was diluted with an equal volume of PBS containing 2% fetal bovine serum (Sigma, #F7524) and 2 mM EDTA. Diluted blood was transferred to SepMate-50 tubes (Stemcell Technologies, #85450) containing 15 mL of Lymphoprep density gradient medium (Stemcell Technologies, #07811) and centrifuged for 20 min at 1200 x g at room temperature. Supernatant was transferred into a 50 mL conical tube, diluted to 45 mL with PBS containing 2% fetal bovine serum and 2 mM EDTA and centrifuged for 5 min at 800 x g. The supernatant was discarded, and the cell pellet resuspended in 1 mL PBS containing 2 % fetal bovine serum. Cell suspensions were pooled and transferred to a 50 mL tube and diluted to 30 mL PBS containing 2 % fetal bovine serum. Cells were pelleted by centrifugation for 5 min at 800 x g. The cell pellet was resuspended in 2 mL of 1x Pharm Lyse Red Blood Cell lysing buffer (BD, #555899) and incubated at 4°C for 10 min. PBS containing 2 % fetal bovine serum was added to a final volume of 15 mL. Cells were pelleted for 10 min at 120 x g and the supernatant decanted. The cells were washed twice with PBS containing 2 % fetal bovine serum and counted (CASY TT device, Beckmann Coulter).

[1170] In vitro cytotoxicity assays

[1171] HER2 I EGFR double positive SKOV-3 ovarian cancer cells (ATCC® HTB-77™) were suspended in culture medium supplemented with 10% FCS, seeded in black 96 well assay plates (Coming) and incubated over night at 37°C and 5% CO2 and humidity. Halfbody molecules were separately serially diluted in assay medium (RPMI 1640 w / o Phenol red (Gibco, #32404-014); GlutaMAX (Gibco 35050-038); 10% fetal bovine serum), before being mixed in solution resulting in a final CyCAT molecule concentration of 0.00001 - 1000 nM. CellToxGreen dye (Promega, #G8731 ), serially diluted (and pre-mixed pairs of) halfbody molecules as well as purified human T cells (E:T ratio 10:1) were added to the cancer cell line and incubated for 48 hrs or 72 hrs at 37°C and 5% CO2 and humidity. Cytotoxic activity was assessed by measuring incorporated CellToxGreen fluorescence at 485 nm excitation and 535 nm emission using a Tecan Infinite F500 device.

[1172] Example 1 : Control Experiments

[1173] A) Halfbody molecules carrying a single aCD3vn, aCD3vL, GDVH, GDvH-Knob or a GDVL domain by themselves are non-functional in respect of binding to CD3 and / or recruiting T- cells for tumor cell killing.

[1174] Results of the Control Experiment 1

[1175] Table 4 summarizes the results of the Control Experiment 1. As expected, unpaired halfbody molecules were not able to mediate killing of SKOV-3 in presence of human T cells (see Table 4; Halfbody Pairs: 3 - 12).

[1176] B) Combining half bodies comprising a single aCD3-SVD domain with half bodies comprising a Guard-Doman (GDVD) but lacking a complementary aCD3-SVD does not result in the formation of a functional aCD3-Fv fragment.

[1177] Since the employed GDVH, GDvH-Knob, and GDVL domains originate from a functional ocCD3 antibody, it was further tested if a combination of a a) halfbody carrying an «CD3-VH with a halfbody molecule carrying a GDVL, b) halfbody carrying an oCD3-VL with a halfbody carrying a GDVH, and c) halfbody carrying GDVH with a halfbody carrying an GDVL would lead to the formation of a functional aCD3-Fv fragment capable of binding to CD3 and to redirect T-cells for tumor cell killing. The ability of the tested combinations to mediate T-cell redirected killing of the cancer cell line SKOV-3 was assessed as described above. Results of the Control Experiment 2

[1178] Table 4 summarizes the results of Control Experiment 2. As expected, combining halfbodies with specificity for HER2 and EGFR and carrying a complementary pair of tzCD3-SVDs resulted in the formation of a functional aCD3-Fv binding fragment and T- cell redirected cell killing of SKOV-3 cells with potencies in the single digit picomolar range (see Table 4; Halfbody Pair: 1 and Halfbody Pair: 2).

[1179] Combining a halfbody molecule carrying an oCD3-VL domain with an halfbody molecule carrying a GDVH or GDvH-Knob domain but lacking a complementary zCD3-VH domain did not resulted in any cell killing activity. The same result was observed for a combination of a halfbody molecule carrying an zCD3-VH and a halfbody molecule carrying a GDVL. Finally, also the combination of a halfbody carrying a single GDVL with a halfbody carrying a GDVH or a GDvH-Knob revealed no cell killing activity, (see Table 2; Halfbody Pairs: 13 - 22 ). In sum, the control experiments confirmed that the Guard-Domains of the present disclosure are not able to form a functional aCD3-Fv binding fragment.

[1180] Table 4: Results of Control Experiments 1 & 2

[1181] Example 2: Combination of halfbody molecules lacking a Guard-Domain with halfbody molecules carrying a Guard-Domain

[1182] The CyCAT approach relies on the on-cel l / on-target functional complementation of an VH domain and a complementary VL domain of an aCD3 antibody, each of them present on one of two complementary CyCAT halfbody molecules. However, because of the intrinsic property of complementary antibody variable domains to associate with each other, a residual unwanted functional complementation may occur in solution and in absence of cells expressing both target antigens of interest or on cells expressing only one of the two targets of interest (such as on healthy tissue). Such unwanted heteroassociation relies, amongst others factors, on the local concentration of the two complementary halfbody molecules and the interface affinity between the two complementary aCD3 variable domains.

[1183] Methods to determine the assay window for a given CyCAT halfbodv pair (“DualTargeting” vs. “Mono-Targeting”

[1184] To determine the contribution of the aforementioned unwanted formation of functional halfbody complexes (“unwanted heteroassociation”) to the overall killing activity of a halfbody pair in presence of both target antigens, two parallel assay settings were performed.

[1185] The maximum achievable killing potency on SKOV-3 cells (expressing EGFR and HER2) was determined by the regular “Dual-Targeting” approach by combining two complementary halfbody pairs, each halfbody comprising a Fab targeting moiety with specificity for either HER2 or EGFR and either an OCCD3VH or OCCD3VL domain.

[1186] To determine the contribution of CyCAT halfbody complexes formed by unwanted heteroassociation to the determined cell killing activity of the Dual-Targeting approach, the Fab target specificity of one complementary CyCAT halfbody molecule was switched to an irrelevant antigen not present on SKOV-3 cells; in the present example to hen egg lysozyme. This approach is referred to here as the “Mono-Targeting” approach.

[1187] As one of the halfbody partners is not able to bind to the cancer cell line, any determined cell killing activity can be directly attributed to unwanted heteroassociation.

[1188] The quotient of the potencies determined for the two aforementioned parallel assay approaches (“Dual-Targeting” vs. “Mono Targeting”) defines the assay window or therapeutic window for the given Dual-Targeting CyCAT halfbody pair. A larger assay window indicates that a provided halfbody pair can be mixed or can be present at higher local concentration without forming significant amounts of undesired functional complexes and therefore provides a preferable and particular safe combination of such halfbody molecules.

[1189] Results for Example 2

[1190] The results of the experiments for the combinatorial approaches for the different CyCAT halfbody pairs utilizing different halfbody formats are summarized in Tables 5 - 10.

[1191] In general, and as expected, co-cultivation of human T cells and EGFR / HER double positive SKOV-3 cells in presence of complementary pairs of EGFR / HER2 Dual-Targeting CyCAT halfbody molecules lacking any Guard-Domain, induced potent killing of SKOV-3 cells in a dose dependent manner.

[1192] Furthermore, co-cultivation of human T cells and SKOV-3 cells in presence of complementary pairs of EGFR / HER2 Dual-Targeting halfbody molecules, where one cognate halfbody partner comprised at least a Guard-Domain, also induced potent killing of SKOV-3 cells in a dose dependent manner. This result confirmed that the presence of a Guard-Domain is not detrimental for the functional complementation of the aCD3-SVD domains and also confirmed that the presence of a Guard-Domain does not interfere with target binding.

[1193] However, when for one halfbody partner, the target specificity was altered from HER2 to Lysozyme (Mono-Targeting), T cell mediated killing of SKOV-3 cells still occurred, although with a weaker potency compared to the Dual Targeting approach. This finding pointed out to unwanted halfbody complex formation.

[1194] However, it was surprisingly found that when in the Mono-Targeting scenario, one halfbody partner additionally comprised a Guard-Domain, the undesired cell killing activity was strongly reduced. These finding confirmed that the presence of a Guard-Domain effectively inhibited unwanted heteroassociation of complementary CyCAT halfbody molecules in the presence of only one target antigen but at the same time did not interfere with the on-cell formation of functional trispecific T cell engaging antibodies in the presence of both target antigens of interest.

[1195] Results for one halfbody in the B036 format

[1196] Complementary pairs of Dual-Targeting halfbodies in the B036 format (Table 5; Halfbody Pair: 1) mediated T cell killing of SKOV-3 cells with potencies in the triple digit picomolar range. However, a corresponding Mono-Targeting halfbody pair (Halfbody pair: 2) in which for the first halfbody the target specificity was altered from HER2 to Lysozyme, still mediated killing of SKOV-3 cells with potencies in the low double digit nanomolar range, resulting in an assay window of 118 fold. Dual-Targeting of halfbodies in the B036 non Guard-Domain format paired with halfbodies in the structurally related B073 (Halfbody Pair: 3) or B099 (Halfbody Pair: 5) Guard-Domain formats revealed comparable cell killing activities compared to Halfbody Pair: 1 but with a significant larger assay window (914 fold for Halfbody Pair 3 and 1300 fold for Halfbody Pair 5). These findings clearly demonstrate the ability of the Guard-Domains of the present invention to inhibit unwanted heteroassociation in the absence of both target antigens but at the same time to not interfere with the desired heteroassociation of the halfbody molecules in presence of both target antigens of interest.

[1197] Table 5: Cell killing activity and assay window determined for halfbodies in the B036 non Guard-

[1198] Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B036 non Guard-Domain format or the B073 & B099 Guard-Domain format, with specificity for EGFR.

[1199] Results for one halfbody in the B038 format

[1200] Complementary pairs of Dual-Targeting halfbodies combining the B038xB036 non Guard- Domain formats (see Table 6; Halfbody Pair: 1 and Halfbody Pair: 11 ) mediated SKOV-3 cell killing with potencies in the double digit picomolar range. Corresponding MonoTargeting halfbody pairs were still able to mediate cell killing with potencies in the double digit nanomolar range, resulting in an assay window of 418 fold or 950 fold, respectively. Dual-Targeting of SKOV-3 cells by combining halfbodies in the B038 non Guard-Domain format with halfbodies in the B073 Guard-Domain format using inactive antibody variable domains as Guard-Domains (see Halfbody Pairs: 3, 5, and 13) resulted in similar killing activities as the aforementioned Dual-Targeting halfbodies in the B038xB036 non-Guard formats but at the same time revealed an up to 10 fold increased assay window (see for example Halfbody Pair: 5 with an determined assay window of 4416 fold).

[1201] Table 6: Cell killing activity and assay window determined for halfbodies in the B038 non Guard-

[1202] Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B036 non Guard-Domain format or B073 Guard-Domain format with specificity for EGFR.

[1203] Interestingly, using human serum albumin (HSA-wt) or Domain III of human serum albumin (HSA-DIII) resulted in a decreased cell killing activity and decreased assay window for the Dual-Targeting halfbody pairs (see Halfbody Pair: 7, 9, 15, and 17 with an assay window of 285 fold, 178 fold, 136 fold and 421 fold, respectively). Results for one halfbodv in the B038 format

[1204] Dual-Targeting of SKOV-3 cells with halfbodies by combining the B038xB063 (see Table 7; Halfbody Pairs: 1 & 13) or B038xB039 (Halfbody Pairs: 3 & 15) non Guard-Domain formats resulted in cell killing with potencies in the low picomolar range and assay windows of ranging from 3565 fold for Halfbody Pair 3 to >11000 fold for Halfbody Pair: 15).

[1205] Dual-Targeting of SKOV-3 cells by combining halfbodies in the B038 non Guard- Domain format with halfbodies in the B077 Guard-Domain format utilizing different types of Guard- Domains (i.e. GDVH, GDVH-KNOB or GDHSAWO resulted in assay windows ranging from of 3324 fold for Halfbody Pair 20 to >133.333 fold for example Halfbody Pair: 17.

[1206] Table 7: Cell killing activity and assay window determined for halfbodies in the B038 non Guard-

[1207] Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B036 non-Guard-Domain format or B077 Guard-Domain format with specificity for EGFR. Results for one halfbodv in the B064 format

[1208] Cell killing activity of halfbodies in the B064 non Guard-Domain format paired with halfbodies in the B073 Guard-Domain format or the B064 format paired with halfbodies in the B099 Guard-Domain format (comprising inactive GDVH, inactive GDvH-Knob or inactive GDVL) was comparable to the killing activity of halfbodies in the B064 format paired with halfbodies in the B036 non Guard-Domain format but revealed significant larger assay windows (see Table 8; Halfbody Pairs: 3, 5, 9, 17 and 23). Assay windows for these Halfbody Pairs ranged from 6104 fold for Halfbody Pair 23 to >40.000 fold for Halfbody Pair: 3.

[1209] Figure 5 shows exemplary results of the cytotoxicity assays to determine the assay window for Halfbody Pairs: 1 & 2 and Halfbody Pairs: 3 & 4 according to Table 8.

[1210] Table 8: Cell killing activity and assay window determined of halfbodies in the B064 non-Guard-

[1211] Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B036, B073 or B099 formats with specificity for EGFR.

[1212] Results for one halfbody in the B064 format

[1213] Cell killing activity of halfbodies in the B064 non Guard-Domain format paired with halfbodies in the B077 Guard-Domain format was comparable to the activity of Dual Targeting halfbody pairs in the B064xB039 or B064xB063 non Guard-Domain formats (see Table 9).

[1214] However, significant larger assay windows were observable for halfbody pairs utilizing GDVH, GDvH-Knob, or GDVL Guard-Domains (such as Halfbody Pairs: 13, 15, 19 and 21 as shown in Table 9) when compared to the assay windows of the corresponding halfbody pairs not utilizing any Guard-Domain (such as Halfbody Pairs: 1 , 11 , 17 and 19).

[1215] Table 9: Cell killing activity and assay window determined for halfbodies in the B064 non Guard-

[1216] Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B039 or B063 non Guard-Domain format or B077 Guard-Domain format with specificity for EGFR. Results for one halfbody in the B050 format

[1217] Cell killing activity of halfbodies in the B050 non Guard-Domain format paired with halfbodies in the B073 or B077 Guard-Domain formats was comparable to the activity of Dual Targeting halfbody pairs in the related B050xB036 or B050xB039 non Guard- Domain formats (see Table 10). However, the determined assay windows for halfbody pairs utilizing Guard-Domains were significant larger (such as for Halfbody Pairs: 3, 5, 9, 15, 17, 21 and 23 as shown in Table 10) when compared to the assay window determined for their corresponding halfbody pairs not utilizing any Guard-Domain (such as for Halfbody Pairs: 1 , 7, 13 and 19).

[1218] Table 10: Cell killing activity and assay window determined for halfbodies in the B050 non Guard- Domain format with specificity for HER2 or Lysozyme paired with halfbodies in the B036 or B039 non Guard-Domain formats or the B073 or B077 Guard-Domain formats with specificity for EGFR.

Claims

WE CLAIM1 . A complementary pair of halfbody molecules comprising a) a first halfbody molecule (HB1 ) comprising i. a first Fab fragment (Fab1) specific for a first antigen (AG1), ii. either a first VH (aCD3-VH1) or first VL (ocCD3-VL1) of a Fv fragment specific for CD3 (ocCD3-Fv), and iii. a first Guard-Domain (GD1), and b) a second halfbody molecule (HB2) comprising i. a second Fab fragment (Fab2) specific for a second antigen (AG2), ii. either a complementary first VH (aCD3-VH2) or complementary first VL (ocCD3-VL2) of the ocCD3-Fv, and iii. optionally a second Guard-Domain (GD2), wherein HB1 and HB2 are not linked via a covalent bond, wherein HB1 and HB2 are capable of forming a heterodimer, wherein said formation of a heterodimer of HB1 and HB2 occurs via dimerization of ocCD3-VH1 and ocCD3-VL2 or of ocCD3-VL1 and ocCD3-VH2, wherein said dimerization of aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3-VH2 results in the formation of the aCD3-Fv, and wherein said heterodimerization of HB1 and HB2 results in the formation of a T- cell engaging trispecific antibody.

2. The complementary pair of halfbody molecules according to claim 1 , wherein GD1 has no binding to aCD3-VH1 , aCD3-VL1 or HB1 and wherein GD2 has no binding to ocCD3-VH2, ocCD3-VL2 or HB2.

3. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein each of GD1 and GD2 is a soluble protein, a soluble polypeptide, a soluble globular protein or a soluble globular polypeptide.

4. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein each of GD1 and GD2 has a molecular size of less than 70 kDa.

5. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein each of GD1 and GD2 is selected from the group comprising albumin, fibrinogen, fibronectin, hemoglobin, transferrin, an immunoglobulin domain or a fragment therefrom.

6. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein if HB1 comprises an aCD3-VH1 than GD1 may be selected of being a VH domain (GDVH) but not a VL domain (GDVL) and if HB1 comprises a aCD3-VL1 than GD1 may be selected of being a VL domain (GDVL) but not a VH domain (GDVH); and if HB2 comprises a aCD3-VH2 than GD2 may be selected of being a VH domain (GDVH) but not a VL domain (GDVL) and if HB2 comprises a ocCD3-VL2 than GD2 may be selected of being a VL domain (GDVL) but not a VH domain (GDVH).

7. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein each of GD1 and GD2 is not released from HB1 or HB2, respectively, after administration to a subject.

8. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein each of GD1 and GD2 cannot be cleaved off from HB1 or HB2, respectively, by a tumor specific protease.

9. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein GD1 and aCD3-VH1 or GD1 and aCD3-VL1 are positioned adjacentand parallel to each other and wherein GD2 and aCD3-VH2 or GD2 and aCD3-VL2 are positioned adjacent and parallel to each other.

10. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein GD1 and aCD3-VH1 or GD1 and aCD3-VL1 of HB1 are present on two different polypeptides and wherein GD2 and aCD3-VH2 or GD2 and aCD3-VL2 of HB2 are present on two different polypeptides.11 . The complementary pair of halfbody molecules according to any one of the preceding claims, wherein in absence of AG1 and / or AG2, the presence of GD1 on HB1 i. inhibits the dimerization of aCD3-VH1 and aCD3-VL2 or of aCD3-VL1 and ocCD3-VH2, respectively, ii. inhibits the formation of the aCD3-Fv, iii. inhibits the dimerization of HB1 with HB2, and / or iv. inhibits the formation of the T-cell engaging trispecific antibody.

12. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein in presence of AG1 and AG2, the presence of GD1 i. does not inhibit the binding of Fab1 to AG1 or of Fab2 to AG2, ii. does not inhibit the dimerization of aCD3-VH1 with aCD3-VL2 or of aCD3-VL1 with ocCD3-VH2, iii. does not inhibit the formation of the aCD3-Fv, iv. does not inhibit the binding of the aCD3-Fv to CD3, v. does not inhibit the dimerization of HB1 with HB2, vi. does no inhibit the formation of the T-cell engaging trispecific antibody, and / or vii. does no inhibit the activity of the trispecific antibody to mediate T-cell redirected killing of cells having AG1 and AG2 on their cell surface.

13. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein the activity of the trispecific antibody to mediate T-cell redirected killingof cells having AG1 and AG2 on their cell surface is at least two fold higher when compared to its activity under conditions in which cells having either AG1 or AG2 on their cell surface.

14. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein in presence of GD1 on HB1 or of GD2 on HB2, the ratio of the IC50 concentration determined for the halfbody pair induced T cell mediated killing of cells expressing either AG1 or AG2 on their cell surface to the IC50 concentration determined for the halfbody pair induced T cell mediated killing of cells expressing AG1 and AG2 on their cell surface, is increased, as compared to the same ratio determined for the complementary pair of halfbody molecules lacking GD1 on HB1 or lacking GD2 on HB2.

15. The complementary pair of halfbody molecules according to claim 14, wherein the ratio of the ICso concentrations is increased by at least 1.5-fold.

16. The complementary pair of halfbody molecules according to any one of the preceding claims, where HB1 , HB2, aCD3-VH1 , ocCD3-VH2, ocCD3-VL1 and ocCD3-VL2 by themselves are not capable of binding to CD3.

17. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein AG1 and AG2 are present on the surface of the same cell.

18. The complementary pair of halfbody molecules according to any one of the preceding claims, wherein HB1 further comprises a first Fc region and / or wherein HB2 further comprises a second Fc region.

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