FIELD OF THE INVENTION

[1] The present invention relates to a bispecific antibody comprising a first binding domain capable of binding to a first target and a second binding domain capable of binding to a second target, wherein (i) said second binding domain comprises a V _H and a V _L domain capable of binding a second target and wherein (ii) said first binding domain comprises a single chain F _v capable of binding a first target which is coupled to the constant domain of the light chain of said second binding domain.

BACKGROUND

[2] Immunoglobulins are proteins which are composed of two heavy (H) and two light (L) chains connected by disulfide bonds. They are being separated functionally into a F _ab (fragment, antigen-binding) region capable of binding to antigens and into the F _c (fragment, crystallizable) region that specifies effector functions such as activation of complement or binding to Fc receptors. By binding to a specific class of Fc receptors, this F _c region can influence the type of ensueing immune response.

[3] In general, five types of mammalian Ig heavy chain denoted as α, δ, ε, γ, and μ are known. The type of heavy chain defines the class of antibody such as IgA, IgD, IgE, IgG, and IgM antibodies, respectively. Normally, a γ, a and δ heavy chain comprises one variable (V _H ) domain followed by a constant domain (CH-i), a hinge region, and two more constant (CH ₂ and CH ₃ ) domains.

[4] The light chain in turn comprises one variable (V _L ) and one constant (C _L ) domain. In mammals there are two types of immunoglobulin light chain, which are called lambda (λ) and kappa (κ), whereas only one type of light chain, either κ or λ, is present per antibody. Further, the amino terminal end of an antibody, the variable domains of the heavy and the light chain form the paratope of an antibody, a small region (5 to 10 amino acids) of an antibody which recognizes and binds one particular epitope on an antigen. The paratope is part of the antibody's F _v region.

[5] The F _v region belongs to the F _ab region that contains one complete light chain and the V _H and CH-i domain of one heavy chain. However, the F _ab region can also be divided into said F _v region composed of the V _H and the V _L domain, and a F _b region composed of the C _L and the CH-i domain. [6] A single chain F _v region (scF _v region) only comprises the variable domain of the heavy (V _H ) and the light chain (V _L ). They may be genetically engineered but unmodified sequences may also be used to form scF _v regions. ScF _v regions recapitulate the monovalent antigen binding characteristics of the original, parent antibody, despite removal of the constant regions and the introduction of a linker (e.g. GS-linkers). Said linker connects the N-terminus of the V _H with the C-terminus of the V _L , or the C-terminus of the V _H with the N-terminus of the V _L . In the scF _v construction, the order of the domains can be either V _H -linker-V _L or V _L -linker-V _H , mostly the scF _v regions are constructed in a V _H -linker-V _L orientation (Schroeder and Cavacini, 2010, "Structure and Function of Immunoglobulins", The Journal of allergy and clinical immunology; 125(2 0 2):S41 -S52; Charles Janeway, 2001 , Immunobiology (5th ed.), Garland Publishing; Smith et al., 2004, 'Demystified... recombinant antibodies", J Clin Pathol; 57:912-917; Putnam et al., 1979, "Primary structure of a human lgA1 immunoglobulin. IV. Streptococcal lgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain", J Biol Chem. 254 (8): 2865-74; Dai et al., 2003, "Generation and characterization of recombinant single chain F _v antibody that recognizes platelet glycoprotein Iba", Thrombosis Research, vol. 109, no. 2-3, pp. 137—144

[7] Based on the antibody structure mentioned above, it is common in the literature to create new formats of antibody arms derived from the general structure, which is known in the prior art thereby allowing for greater flexibility and improvement of the function of designed and engineered IgG-like bispecific antibodies.

[8] New formats of IgG-like bispecific antibodies may often include coupling of scF _v regions to an antibody. In general, they can be linked to the N-termini or the C-termini of IgGs.

[9] In view of new antibody formats, it is known in the prior art that the antibody affinity is increased upon bivalent binding of the antibody, however bivalent binding is lost with single chain F _v antibodies, since this scF _v format only exhibits monovalent binding to one antigen. Vice versa the person skilled in the art is aware of the fact that a single chain F _v antibody has therefore reduced affinity (Pardridge, 2001 , "Brain Drug Targeting. The future of brain drug development", Cambridge University Press, pp. 147-148). Other researchers also identified that an improper conformational arrangement of the variable regions results in the conversion of the scF _v region into the IgG, thus resulting in a loss of affinity, which cannot be compensated by avidity (Menzel et al., 2008, "Human antibody RNase fusion protein targeting CD30 ⁺ lymphomas", Blood 1 1 1 :3830-3837).

[10] Further, the arrangement of the V _H and the V _L domain of the scF _v via short linkers, preferably short GS-linkers, is considered as being not sufficiently conformational stable in order to recreate the binding site of the parent antibody (Yuan et al., 1997, "Molecular cloning, expression and characterization of a functional single-chain F _v antibody to the mycotoxin zearalenone", Applied and Environmental Microbiology, 63, 263-269). [11] Considering the above, a bispecific antibody format in which scF _v regions are fused to the antibody in any way, may be associated with difficulties in view of reduced affinity and/or conformational hurdles.

[12] The object of the present invention is to provide an improved antibody, which is able to bridge any difficulties associated with antibody manufacturing and scF _v fusion.

SUMMARY OF THE INVENTION

[13] The present invention provides a bispecific antibody comprising a first binding domain capable of binding to a first target and a second binding domain capable of binding to a second target, wherein (i) said second binding domain comprises a V _H and a V _L domain capable of binding a second target and wherein (ii) said first binding domain comprises a single chain F _v region capable of binding a first target, which is coupled to the constant domain of the light chain of said second binding domain. The first target of an antibody of the present invention is preferably an immune checkpoint protein or a cell surface molecule and the second target of an antibody of the present invention is preferably a cancer antigen. Preferably, said immune checkpoint protein is PD-L1 and said cell surface molecule is CD3. Preferably, said cancer antigen is TA-MUC1 or HER2. In particular, said antibody may bind to TA-MUC1 and PD-L1 or TA-MUC1 and CD3 or HER2 and CD3.

[14] The present invention also contemplates an antibody of the present invention, wherein the antibody may be obtainable from prokaryotic or eukaryotic cells, preferably mammalian cell lines, more preferably human cell lines. An antibody of the present invention is preferably obtainable from a cell line selected from the group consisting of NM-F9 (DSM ACC 2606), NM- D4 (DSM ACC2605), GT-2X (DSM ACC2858), NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8 (DSM ACC2806), NM-H9D8-E6 (DSM ACC2807), NM-H9D8-E6Q12 (DSM ACC2856), GT-5s (DSM ACC 3078) or a cell or cell line derived therefrom.

[15] Further, an antibody of the present invention may be capable of binding to FcYRIIIa and may comprise an F _c region.

[16] Additionally, an antibody of the present invention may be capable of mediating enhanced ADCC and/or T cell activation.

[17] The present invention also envisages an antibody of the present invention, wherein the antibody may comprise a second binding domain preferably capable of binding to TA-MUC1 or HER2, wherein said second binding domain may comprise a V _H and a V _L domain.

[18] The present invention may further contemplate an antibody of the present invention for use in therapy. Further, the present invention may provide an antibody of the present invention for use in a method for enhancing ADCC activity and/or T cell activation, wherein the enhancement of ADCC activity and/or T cell activation may be for the treatment of cancer disease, inflammatory disease, virus infectious disease and autoimmune disease. Cancer disease may be selected from Melanoma, Carcinoma, Lymphoma, Sarcoma, and Mesothelioma including Lung Cancer, Kidney Cancer, Bladder Cancer, Gastrointestinal Cancer, Skin Cancer, Breast Cancer, Ovarian Cancer, Cervical Cancer, and Prostate Cancer, whereas inflammatory disease may be selected from Inflammatory Bowel Disease (IBD), Pelvic Inflammatory Disease (PID), Ischemic Stroke (IS), Alzheimer's Disease, Asthma, Pemphigus Vulgaris, Dermatitis/Eczema. Virus infectious disease may be selected from Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Epstein Barr Virus (EBV), Influenza Virus, Lymphocytic Choriomeningitis Virus (LCMV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), whereas autoimmune disease may be selected from Diabetes Mellitus (DM), Type I, Multiple Sclerosis (MS), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA), Vitiligo, Psoriasis and Psoriatic Arthritis, Atopic Dermatitis (AD), Scleroderma, Sarcoidosis, Primary Biliary Cirrhosis, Guillain-Barre Syndrome, Graves' Disease, Celiac Disease, Autoimmune Hepatitis, Ankylosing Spondylitis (AS).

BRIEF DESCRIPTION OF THE FIGURES

[19] Fig. 1 : Measuring core fucosylation.

Ck- and CH ₃ -variant of normal-fucosylated bi-specific anti-PD-L1/TA-MUC1 antibodies have comparable amounts of core fucosylated N-glycans; same results are seen for fucose-reduced bi-specific anti-PD-L1/TA-MUC1 antibodies: The relative molar amounts of core fucosylated N- glycans show that PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ have high levels, preferably 91 % core fucosylated N-glycans for both variants, whereas PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ contain low percentages of core fucosylated N-glycans, preferably 1 % for the PM-PDL-GEX Fuc-/Ck variant and 0% core fucosylated N-glycans for the PM-PDL- GEX Fuc-/CH ₃ variant. This is described in Example 1.

[20] Fig. 2: PD-L1 binding and blocking capacity of Ck- and CH ₃ -variants of PM-PDL- GEX.

Ck- and CH ₃ -variants of PM-PDL-GEX show comparable PD-L1 binding and blocking capacity: A) All tested antibodies (PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc- /Ck and PM-PDL-GEX Fuc-/CH ₃ ) compared to each other in the PD-L1 antigen ELISA showed a comparable concentration-dependent antigen binding. B) Concentration-dependent blocking of PD-1 binding was detected for PM-PDL-GEX H9D8/Ck compared to PM-PDL-GEX H9D8/CH ₃ in the PD-L1/PD-1 blocking ELISA, but no difference was seen between the Ck- and CH ₃ -variant. C) PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ also showed no obvious difference to each other. This is described in Example 2. [21] Fig. 3: Binding to FcYRIIIa of Ck- and CH ₃ -variants of PM-PDL-GEX.

Ck- and CH ₃ -variants of PM-PDL-GEX show comparable binding to FcYRIIIa: The different variants of PM-PDL-GEX were quantitatively compared by calculation of a relative potency compared to a normal-fucosylated reference antibody (EC50 of reference antibody divided by EC50 of antibody variant).

For the normal-fucosylated variants PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ relative potencies of 1 .9 and 1 .6 were determined. In contrast, the relative potency of fucose- reduced PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ was determined as 10.4 and 13.2. This is described in Example 3.

[22] Fig 4: Cell binding of Ck- and CH ₃ -variants of PM-PDL-GEX.

Ck- and CH ₃ -variants of PM-PDL-GEX show comparable cell binding: A) The breast cancer cell line ZR-75-1 strongly expressed TA-MUC1 , whereas the prostate carcinoma cell line Du-145 showed only weak expression of TA-MUC1. On ZR-75-1 the PD-L1 expression was drastically lower compared to TA-MUC1 , whereas on Du-145 the PD-L1 expression was higher compared to TA-MUC1 . B) PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ showed comparable binding to the cell lines ZR-75-1 . C) PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ showed comparable binding to Du-145. D) The Ck- and CH ₃ -variant of PM-PDL-GEX Fuc- were directly compared to each other using Du-145, also showing comparable binding. This is described in Example 4.

[23] Fig. 5: Measuring ADCC activity of Ck- and CH ₃ -variants of PM-PDL-GEX.

CH ₃ -variants of PM-PDL-GEX show lower ADCC activity against certain target cells lines compared to Ck-variants: A) The breast cancer cell line T47-D showed high expression of TA- MUC1 and in comparison the PD-L1 expression was lower. In comparison to T-47D, the TA- MUC1 expression of the head and neck squamous cell carcinoma cell line HSC-4 was on a very low level. The TA-MUC1 expression on HSC-4 was slightly higher compared to PD-L1 , but it is to mention that the difference between TA-MUC1 and PD-L1 expression was not as drastic as observed for ZR-75-1 and T47-D. B) PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ showed strongly enhanced ADCC activity compared to PM-PDL-GEX H9D8/Ck and PM-PDL- GEX H9D8/CH ₃ when using ZR-75-1 as target cells _. C) PM-PDL-GEX H9D8/Ck and PM-PDL- GEX H9D8/CH ₃ were tested in an ADCC against T-47D. Both antibodies mediated comparable ADCC activity, hence no difference between Ck- and CH ₃ -variants were seen. D) PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were tested in an ADCC assay against Du-145. As expected, the fucose-reduced PM-PDL-GEX variants showed strongly enhanced ADCC compared to the normal-fucosylated variants. Different ADCC activity of the Ck- and CH ₃ -varaints was observed. In presence of PM-PDL- GEX Fuc-/Ck the ADCC effect was increased compared to PM-PDL-GEX Fuc-/CH ₃ . In line with that, PM-PDL-GEX H9D8/Ck showed ADCC activity, whereas no ADCC activity was observed using PM-PDL-GEX H9D8/CH ₃ . E) The different PM-PDL-GEX variants were tested against HSC-4 for their capacity to mediate ADCC. Similar to the results with Du-145 as target cells, drastically differences between the Ck- and CH ₃ -variants were seen. PM-PDL-GEX Fuc-/Ck mediated increased ADCC compared to PM-PDL-GEX Fuc-/CH ₃ and PM-PDL-GEX H9D8/Ck mediated ADCC was stronger compared to PM-PDL-GEX H9D8/CH ₃ . This is described in Example 5.

[24] Fig. 6: Measuring T cell activation of Ck- and CH ₃ -variants of PM-PDL-GEX.

CH ₃ -variants of PM-PDL-GEX show less T cell activation compared to Ck-variants: In a first allogeneic MLR PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ were compared to each other and showed activation of CD8 T cells (CD3 ⁺ CD8 ⁺ cells) measured via the activation marker CD25 (A) and the co-stimulatory molecule CD137 (B) compared to the medium control. However, the Ck-variant showed increased activation compared to the CH ₃ -variant.

In a second allogeneic MLR, serial dilutions of PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc- /CH ₃ were compared. PM-PDL-GEX Fuc-/Ck showed a strong concentration-dependent activation of T cells measured by CD25 (C) and CD137 expression (D), whereas incubation with PM-PDL-GEX Fuc-/CH ₃ resulted only in a slightly increased activation compared to the medium control. This is described in Example 6.

[25] Fig. 7: Lower NK cell-mediated ADCC activity of CH ₃ -varaints of a bispecific anti- TA-MUC1/CD3 antibody and of a bispecific anti-HER2/CD3 antibody.

The Ck-variants of both, the bispecific anti-TA-MUC1/CD3 (PM-CD3-GEX) antibody (A) and the bispecific anti-HER2/CD3 (TM-CD3-GEX) antibody (B) show increased ADCC-activity compared to the respective CH ₃ -variants. This is described in Example 7.

[26] Fig. 8: PM-PDL-GEX CDR mutants of the Ck-variant show comparable binding and blocking capacity compared to the non-mutated counterpart.

A) Fucose-reduced PM-PDL-GEX Ck-variants having different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1 , such as PM-PDL-GEX Fuc- CDRmut a (SEQ ID NO. 75), or PM-PDL-GEX Fuc- CDRmut b (SEQ ID NO. 77 + SEQ ID NO. 83), show comparable PD-L1 binding capacity to the non-mutated PM-PDL-GEX Fuc- using PD-L1 antigen ELISA. B) The CDR mutants of the fucose-reduced PM-PDL-GEX /Ck-variants also show comparable blocking capacity to the non-mutated PM-PDL-GEX Fuc-/Ck using PD- L1/PD1 blocking ELISA. C) Fucose-reduced PM-PDL-GEX/Ck-variants having different mutations in the CDRs of the V _H domain show comparable TA-MUC1 binding capacity to the non-mutated PM-PDL-GEX Fuc-/Ck using TA-MUC1 expressing T-47D and flow cytometric analysis. This is described in Example 8.

[27] Fig. 9: PM-PDL-GEX CDR mutants of the Ck-variant show comparable enhanced activation of CD8 T cells to the non-mutated counterparts.

Fucose-reduced PM-PDL-GEX/Ck having different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1 , such as PM-PDL-GEX Fuc- CDRmut a (SEQ ID No. 75), or PM-PDL-GEX Fuc- CDRmut b (SEQ I D NO. 77 + SEQ ID NO. 83) show comparable enhanced CD8 T cell activation (CD25+ cells of CD8 T cells) to the non-mutated PM-PDL-GEX Fuc-/Ck. The CDR mutated PM-PDL-GEX H9D8 variants activated CD8 T cells comparable to non- mutated PM-PDL-GEX H9D8. This is described in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

[28] The solution of the present invention is described in the following, exemplified in the appended examples, illustrated in the Figures and reflected in the claims.

[29] The present invention provides an antibody comprising a first binding domain capable of binding to a first target and a second binding domain capable of binding to a second target, wherein (i) said second binding domain comprises a variable domain (V _H ) of the heavy chain and a variable domain (V _L ) of the light chain capable of binding a second target and wherein (ii) said first binding domain comprises a single chain F _v region capable of binding a first target, which is coupled to the constant domain of the light chain of said second binding domain.

[30] An antibody of the present invention is bispecific. It is preferably capable of binding as a first target an immune checkpoint protein, wherein said immune checkpoint protein is preferably PD-L1 . Further an antibody of the present invention is preferably capable of binding as a first target to a cell surface molecule, wherein said cell surface molecule is preferably CD3. Additionally, an antibody of the present invention may bind as a second target a cancer antigen, wherein said cancer antigen is preferably TA-MUC1 or HER2. Thus, the antibody of the present invention may bind preferably to TA-MUC1 and PD-L1 or TA-MUC1 and CD3 or HER2 and CD3.

[31] PD-L1 is known as the Programmed death-ligand 1 (PD-L1 ), the cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1 ), a protein that in humans is encoded by the CD274 gene and preferably refers to an immune checkpoint protein. PD-L1 may constitutively be expressed on mouse T and B cells, dendritic cells (DCs), macrophages, mesenchymal stem cells and bone marrow-derived mast cells (Yamazaki et al., 2002, J. Immunol. 169: 5538-45). According to Keir et al. (2008), Annu. Rev. Immunol. 26: 677-704, PD-L1 can also be expressed on a wide range of non-hematopoietic cells such as cornea, lung, vascular epithelium, liver non-parenchymal cells, mesenchymal stem cells, pancreatic islets, placental synctiotrophoblasts, keratinocytes, etc. Further, upregulation of PD-L1 is achieved on a number of cell types after activation of said cells.

[32] PD-L1 binds to the programmed death-1 receptor (PD-1 ) (CD279), which provides an important negative co-stimulatory signal regulating T cell activation. In general, the binding of PD-L1 to PD-1 transmits an inhibitory signal which reduces the proliferation of CD8 ⁺ T cells. PD- L1 is also considered as a binding partner for B7.1 (CD80) (Butte et al., 2007, Immunity 27: 1 1 1 -22). Chemical crosslinking studies suggest that PD-L1 and B7.1 can interact through their IgV-like domains. Moreover, B7.1 :PD-L1 interactions can induce an inhibitory signal into T cells.

[33] When T cells lacking all known receptors for PD-L1 (i.e., no PD-1 and B7.1 ), T cell proliferation is no longer impaired. In other words, an impairment of the engagement of PD-L1 with its receptor PD-1 on T cells leads to T cell receptor-mediated activation of IL-2 production and T cell proliferation. Thus, PD-L1 plays a specific role in inhibiting T cells either through B7.1 or PD-1 .

[34] Cancer cells may also upregulate PD-L1 as well, thus allowing cancers to evade the host immune system. PD-L1 is expressed on a variety of different cancer types including, but not limited to carcinomas, sarcomas, lymphomas and leukemia, germ cell tumors and blastomas.

[35] For an even more specific cancer cell targeting, an antibody of the present invention may bind, as described above, as a second target to the cancer antigen "the tumor-associated mucin 1 epitope TA-MUC1 ". TA-MUC1 is present on tumor cells but not on normal cells and/or it is only accessible by antibodies in the host's circulation when present on tumor cells but not when present on normal cells. Targeting TA-MUC1 may provide specific direction and accumulation into the tumor.

[36] Further, an antibody of the present invention may comprise a linker, which couples scF _v regions either to the constant domain of the light chains or to the CH ₃ domain of the heavy chain of said antibody. The linker may in principle have any number of amino acids and any amino acid sequence. The linker may comprise at least 3, 5, 8, 10, 15 or at least 20 amino acids, preferably at least 5 amino acids. Further, the linker may comprise less than 50 or less than 40, 35, 30, 25, 20 amino acids, preferably less than 45 amino acids. In particular, the linker may comprise from 5 to 20 amino acids. Preferably, the linker may consist of glycine and serine residues. Glycine and serine may be present in the linker in a ratio of 2 to 1 , 3 to 1 , 4 to 1 or 5 to 1 (number of glycine residues to number of serine residues). For example, the linker may comprise a sequence of four glycine residues followed by one serine residue, and in particular at least 1 , 2, 3, 4, 5 or at least 6 repeats of this sequence. Preferably, linkers consisting of 2 repeats of the amino acid sequence refer to (GGGGS) ₂ , 4 repeats of the amino acid sequence may refer to (GGGGS) ₄ and 6 repeats of the amino acid sequence may refer to (GGGGS) _6. Especially, linkers consisting of 4 repeats of the amino acid sequence (GGGGS) ₄ may be preferred. An antibody of the present invention, which couples scF _v regions to the constant domain of the light chains or to the CH ₃ domain of the heavy chain, may prefer a so called GS- linker. Additionally, the linker may comprise sequences, which may show no or only minor immunogenic potential in humans, preferably sequences, which are human sequences or naturally occurring sequences. Further, the linkers and the adjacent amino acids may show no or only minor immunogenic potential.

[37] An antibody of the present invention may refer to a Ck-variant, no matter if said variant is normal-fucosylated or fucose-reduced. Thus, said variant may be obtainable from prokaryotic or eukaryotic cells, preferably mammalian cell lines, more preferably human cell lines. Mammalian cell lines may refer to NS0, SP2/0, CHO-K1 , CHOdhfr-, COS-1 , COS-7, and BHK. Human cell lines may refer to HEK293, HT-1080, K562, Namalwa, Percy 6, HKB-1 1 , CAP and HuH-7. Thus, an antibody of the present invention may be obtainable from prokaryotic or eukaryotic cells, preferably from mammalian cell lines such as NSO, SP2/0, CHO-K1 , CHOdhfr-, COS-1 , COS-7, and BHK, more preferably from human cell lines such as HEK293, HT-1080, K562, Namalwa, Percy 6, HKB-1 1 , CAP and HuH-7. An antibody of the present invention is preferably obtainable from a cell line selected from the group consisting of NM-F9 (DSM ACC 2606), NM-D4 (DSM ACC 2605), GT-2X (DSM ACC 2858), NM-H9, NM-E-2F9, NM-C-2F5, NM-H9D8 (DSM ACC 2806), NM-H9D8-E6 (DSM ACC 2807), NM-H9D8-E6Q12 (DSM ACC 2856), GT-5s (DSM ACC 3078) or a cell or cell line derived therefrom. An antibody of the present invention, which may comprise scF _v regions being coupled to the constant domain of the light chains, may be produced from one or more of the cell lines mentioned above. Preferably, an antibody of the present invention is preferably obtainable from NM-H9D8-E6 (DSM ACC 2807) or NM-H9D8- E6Q12 (DSM ACC 2856), thus being a fucose-reduced antibody.

[38] In the most preferred embodiment of the invention the host cell of the invention is preferably the cell line NM-F9 (DSM ACC 2606), NM-D4 (DSM ACC 2605), NM-E-2F9, NM-C- 2F5, NM-H9D8 (DSM ACC 2806), GT-2X (DSM ACC 2858), or NM-H9D8-E6 (DSM ACC 2807), or NM M9D8-E6Q12 (DSM ACC 2856), which grow and produce an antibody of the present invention, wherein scF _v regions may be coupled to the constant domain of the light chains, under serum-free conditions. Also it may be preferred hereunder cells growing under serum-free conditions, wherein the nucleic acid encoding an antibody of the present invention may be introduced in these cells and wherein an antibody of the present invention may be isolated under serum-free conditions.

[39] First, a bi-specific anti-PD-L1/TA-MUC1 antibody was engineered in four different forms including said Ck-variant of the present invention, one being normal-fucosylated and one being fucose-reduced, and a reference variant, which refers to the CH ₃ -variant. Each variant was tested and compared to each other with regard to core-fucosylation, PD-L1 binding and blocking capacity, binding to FcYRIIIa, binding to cells expressing TA-MUC1 and/or PD-L1 , ADCC activity and T cell activation. The four different forms of each antibody variant are described as follows: I) a normal-fucosylated hlgG1 with specificity towards TA-MUC1 and with an anti-PD-L1 scF _v region coupled to the constant domain of the light chain termed PM-PDL-GEX H9D8/Ck; II) a normal- fucosylated hlgG1 with specificity towards TA-MUC1 and with an anti-PD-L1 scF _v region coupled to the CH ₃ domain of the heavy chain termed PM-PDL-GEX H9D8/CH ₃ , III) a fucose- reduced hlgG1 with specificity towards TA-MUC1 and with an anti-PD-L1 scF _v region coupled to the constant domain of the light chain termed PM-PDL-GEX Fuc-/Ck and IV) a fucose-reduced hlgG1 with specificity towards TA-MUC-1 and with an anti-PD-L1 scF _v region coupled to the CH ₃ domain of the heavy chain termed PM-PDL-GEX Fuc-/CH ₃ . The CH ₃ -variant may be used as a reference antibody variant for said Ck-variant, which may refer to the preferred antibody of the present invention.

[40] First, N-glycosylation of all four antibodies (PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ ) was analyzed by HILIC-UPLC- HiResQToF MSMS (Fig. 1 ). The relative molar amounts of core fucosylated N-glycans show that PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ may have high levels of core fucosylated N-glycans, whereas PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ may contain low percentages of core fucosylated N-glycans.

[41] The normal-glycosylated bispecific PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ may contain more than 80% core fucosylated N-glycans (core-fucosylation). The present invention envisages normal-glycosylated antibodies containing preferably more than 80% less than 100% core fucosylated N-glycans. The normal-glycosylated antibodies of the present invention may preferably contain about 81 % to 100%, 85% to 95% fucosylated N- glycans or 90% to 95 % fucosylated N-glycans. The normal-fucosylated antibodies of the present invention may contain more than 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% fucosylated N- glycans, preferably about 91 % core fucosylated N-glycans for the PM-PDL-GEX H9D8/Ck variant and about 91 % core fucosylated N-glycans for the PM-PDL-GEX H9D8/CH ₃ variant. These antibodies having more than 80% core fucosylated N-glycans may therefore refer to normal-fucosylated antibodies.

[42] The fucose-reduced bispecific PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ may contain only low percentages of core fucosylated N-glycans. The present invention provides fucose-reduced antibodies preferably being from 0% to 80% fucosylated. The fucose-reduced antibodies of the present invention may preferably contain about 0% to 70%, 0% to 60%, 0% to 50 %, 0% to 40 %, 0% to 30 %, 0% to 20 %, 0% to 10 % or 10% to 50%, 15% to 50%, 20% to 50%, 25% to 50%, 30% to 50%, 35% to 50%, 40% to 50%, 45% to 50% or 1 % to 20%, 1 % to 15%, 1 % to 10%, 1 % to 5% or 5% to 30%, 5% to 20%, 5% to 15% fucosylated N-glycans. The fucose-reduced antibodies of the present invention may preferably contain 0%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20.0%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 41 %, 42%, 43%, 44%, 45.0%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61.0%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or even 80% core fucosylated N-glycans, preferably about 1 % core fucosylated N- glycans for the PM-PDL-GEX Fuc-/Ck variant and about 0% core fucosylated N-glycans for the PM-PDL-GEX Fuc-/CH ₃ variant. These antibodies being from 0% to 80% fucosylated may therefore refer to fucose-reduced antibodies.

[43] Further, different ELISAs were developed to analyze PD-L1 binding and PD-L1/PD-1 blocking capacity of all four antibodies, since the PD-L1/PD-1 blocking ELISA is considered as the most relevant ELISA for antibodies being capable of binding to PD-L1 by depicting the blocking situation between PD-1 and PD-L1 . [44] First, PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared in the PD-L1 antigen ELISA. All tested antibodies showed a comparable concentration-dependent antigen binding (Fig. 2A).

Second, PM-PDL-GEX H9D8/Ck was compared to PM-PDL-GEX H9D8/CH ₃ in the PD-L1/PD-1 blocking ELISA (Fig. 2B). Concentration-dependent blocking of PD-1 binding was detected, but no difference was seen between the Ck- and CH ₃ -variant. In addition, PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared also showing no obvious difference to each other (Fig. 2C).

[45] Additionally, it was further shown that fucose-reduced PM-PDL-GEX/Ck having different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1 , preferably having the amino acid sequence as shown in SEQ ID NO. 75 (having a mutation of glycine to alanine at position 26 according to Kabat-numbering and having a mutation of aspartic acid to glutamic acid at position 31 according to Kabat-numbering in the CDR1 of the V _H domain) or having the amino acid sequences as shown in SEQ ID NO. 77 (having a mutation of threonine to serine at position 28 according to Kabat-numbering in the CDR1 of the V _H domain) and SEQ ID NO. 83 (having a mutation of serine to threonine at position 62 according to Kabat-numbering in the CDR2 of the V _H domain), may show comparable PD-L1 binding capacity, comparable blocking capacity of PD-L1/PD1 interaction and comparable TA-MUC1 binding capacity to the non- mutated PM-PDL-GEX/Ck (Fig. 8A, B and C).

[46] These data clearly demonstrate that PD-L1 binding and PD-L1/PD-1 blocking is unaffected by the location of the anti-PD-L1 scF _v , whether it is fused to the constant domain of the light chain or to the CH ₃ domain of the heavy chain. Further, these data reveal that targeting tumor cells expressing TA-MUC1 may also be achieved with fucose-reduced Ck-variants having different CDR mutations in the V _H domain of the scF _v region binding to PD-L1 of said antibodies of the present invention preferably having the amino acid sequence as shown in SEQ ID NO. 75 or having the amino acid sequences as shown in SEQ ID NO. 77 and 83 as indicated above.

[47] In order to characterize binding of the F _c part of said antibodies to FcYRIIIa on a molecular level, a new assay using a bead-based technology of Perkin Elmer (AlphaScreen®) was developed. The different variants of PM-PDL-GEX were quantitatively compared by calculation of a relative potency to a normal-fucosylated reference antibody (relative potency=EC50 of reference antibody divided by EC50 of antibody variant). For the normal- fucosylated variants PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ relative potencies of 1 .9 and 1 .6 were determined. In contrast, the relative potency of fucose-reduced PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were determined as 10.4 and 13.2 (Fig. 3), thus having an increased binding preferably to FcYRIIIa for the fucose-reduced PM-PDL-GEX Fuc-/Ck and PM- PDL-GEX Fuc-/CH ₃ variant. [48] The results from the experiment described in Example 3 were expected since it is known that reduced fucosylation of hlgG1 may lead to better FcYRIIIa-binding. No obvious difference between the Ck- and the CH ₃ -variants of the fucose-reduced and the normal- fucosylated antibody, respectively, was detected. An antibody of the present invention is preferably capable of binding to FcyRs, more preferably to FcyRllla. If this particular Ck-variant is further fucose-reduced, FcyRllla-binding may be even more enhanced. In view of the data above that there is no obvious difference in binding to FcyRllla between the Ck- and the CH ₃ - variants the other CH ₃ -variant may also be preferred in the present invention. Similarly, if this particular CH ₃ -variant may be further fucose-reduced, FcyRllla-binding may be even more enhanced.

[49] In addition, cell binding properties of the different variants of PM-PDL-GEX were analyzed using the mamma carcinoma cell line ZR-75-1 and prostate carcinoma cell line DU- 145. Both cell lines were characterized for their expression of TA-MUC1 and PD-L1. The breast cancer cell line ZR-75-1 strongly expressed TA-MUC1 , whereas the prostate carcinoma cell line Du-145 showed only weak expression of TA-MUC1. On ZR-75-1 , in comparison to TA-MUC1 the PD-L1 expression was drastically lower, whereas on Du-145 the PD-L1 expression was higher compared to TA-MUC1 (Fig. 4A).

[50] In order to test binding of the different PM-PDL-GEX variants, target cells were incubated with indicated antibodies in serial dilutions. PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ showed comparable binding to the cell lines ZR-75-1 (Fig. 4B).

Binding of the tested antibodies is most likely due to specificity towards TA-MUC1 since ZR-75- 1 shows only weak expression of PD-L1 compared to TA-MUC1. Therefore, it can be concluded that whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or to the CH ₃ domain of the heavy chain has no influence on binding to TA-MUC-1 .

[51] Similar to results with ZR-75-1 , PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM- PDL-GEX H9D8/CH ₃ showed comparable binding to Du-145 (Fig. 4C). In addition, the Ck- and CH ₃ -variant of PM-PDL-GEX Fuc- were directly compared to each other using Du-145, also showing comparable binding (Fig. 4D).

[52] In summary, whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or whether it is coupled to the CH ₃ domain of the heavy chain has no influence on binding to cell lines expressing TA-MUC1 and/or PD-L1 .

[53] The capacity to mediate ADCC against tumor cells was analyzed using a europium release assay. Four different cancer cell lines were used for the ADCC assays: I) the breast cancer cell line ZR-75-1 , II) the breast cancer cell line T47-D, III) the prostate carcinoma cell line DU-145 and IV) the head and neck squamous cell carcinoma cell line HSC-4. Specific cytotoxicity was calculated as: % specific lysis = (experimental release - spontaneous release) / (maximal release - spontaneous release) x100.

[54] Similar to ZR-75-1 (see Example 4), T47-D showed high expression of TA-MUC1 and in comparison the PD-L1 expression was lower. In comparison to T-47D, the TA-MUC1 expression of HSC-4 was on a very low level. The TA-MUC1 expression on HSC-4 was slightly higher compared to PD-L1 , but it is to mention that the difference between TA-MUC1 and PD- L1 expression was not as drastic as observed for ZR-75-1 and T47-D (Fig. 5A).

[55] First, the capacity to mediate ADCC activity of the different PM-PDL-GEX variants was analyzed against ZR-75-1 . PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ showed strongly enhanced ADCC activity compared to PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ (Fig. 5B). This was expected since PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ are fucose-reduced (see Example 1 ) and therefore may have higher affinity to FcyRs, preferably to FcyRllla (see Example 3). No obvious difference between the fucose-reduced Ck- and CH _3- variants was seen. The ADCC mediated by PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX Fuc-/CH ₃ , PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ against ZR-75-1 was most likely primarily due to TA-MUC1 specificity since a fucose-reduced mono-specific anti-PD-L1 antibody (PDL- GEX Fuc-) induced no ADCC effect.

[56] Second, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ were tested in an ADCC against T-47D (Fig. 5C). Both antibodies mediated comparable ADCC activity hence no difference between normal-fucosylated Ck- and CH ₃ -variants was seen. As mentioned for ZR- 75-1 , the ADCC due to PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ was probably mediated via the TA-MUC1 specificity because no ADCC effect was detected using a fucose- reduced mono-specific anti-PD-L1 antibody (PDL-GEX Fuc-).

[57] Third, PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were tested in an ADCC assay against Du-145 (Fig. 5D). As expected, the fucose-reduced PM-PDL-GEX variants showed strongly enhanced ADCC compared to the normal-fucosylated variants. However, the present inventors surprisingly found that using Du- 145 as a target cell line resulted in different ADCC activity of the Ck- and CH ₃ -varaints. In presence of PM-PDL-GEX Fuc-/Ck the ADCC effect was increased compared to PM-PDL-GEX Fuc-/CH ₃ . In line with that, PM-PDL-GEX H9D8/Ck showed ADCC activity, whereas no ADCC activity was observed using PM-PDL-GEX H9D8/CH ₃ . In contrast to ZR-75-1 and T-47D, the ADCC of PM-PDL-GEX variants against Du-145 was primarily mediated due to the PD-L1 specificity since an anti-TA-MUC1 hlgG1 only mediated minimal ADCC activity.

[58] Fourth, the different PM-PDL-GEX variants were tested against HSC-4 for their capacity to mediate ADCC (Fig. 5E). Similar to the results with Du-145 as target cells, drastically differences between the Ck- and CH ₃ -variants were seen. PM-PDL-GEX Fuc-/Ck mediated increased ADCC compared to PM-PDL-GEX Fuc-/CH ₃ and PM-PDL-GEX H9D8/Ck mediated ADCC was stronger compared to PM-PDL-GEX H9D8/CH ₃ . A mono-specific anti-TA-MUC1 hlgG1 showed only minimal ADCC activity, therefore it can be concluded that ADCC mediated by the PM-PDL-GEX antibody is mainly due to PD-L1 specificity.

[59] In summary, whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or whether it is coupled to the CH ₃ domain of the heavy chain has no influence on antigen binding to PD-L1 (see Example 2) and TA-MUC1 (see Example 4), the blocking of PD-L1/PD-1 interaction (see Example 2) or the FcYRIIIa binding (see Example 3). Here, no obvious difference between the Ck- and the CH ₃ -variants of the fucose-reduced and the normal- fucosylated antibody, respectively, was detected. Instead, the ADCC against particular cancer cells may be affected. This may also be confirmed by the Ck-variant being a PankoMab- antiCD3-GEX (PM-CD3-GEX) having scF _v regions being preferably capable of binding to CD3 or by the Ck-variant being a Trastuzumab-antiCD3 (TM-CD3-GEX) also having scF _v regions being preferably capable of binding to CD3, when comparing ADCC to a corresponding CH ₃ - variant against CD3 positive Jurkat cells. In particular, it was shown that Ck-variants of a bispecific anti-TA-MUC1/CD3 antibody (PM-CD3-GEX) and of a bispecific anti-HER2/CD3 antibody (TM-CD3-GEX) showed increased NK cell-mediated ADCC activity compared to the respective CH ₃ -variants (Fig. 7A and B).

[60] These findings demonstrate that the activation of FcYRIIIa (or other FcyRs) may be surprisingly impaired when the anti-PDL1 or the anti-CD3 scF _v is fused to the CH ₃ domain of the heavy chain, despite the fact, that binding to FcyRllla has been shown to be unaffected. Thus, the Ck-variant may be considered as being a promising variant for mediating ADCC activity against all kinds of cells, preferably against cancer cells, endothelial cells, virus-infected immune cells, virus particles, altered brain cells, most preferably cancer cells. If this particular Ck-variant may further be fucose-reduced, ADCC activity may be even more enhanced. An antibody of the present invention is therefore preferably capable of mediating ADCC activity.

[61] Therefore, the present invention may provide bispecific antibodies with enhanced ADCC activity, wherein the first binding domain of an antibody variant is fused to the C-kappa domain of the light chain in comparison to an antibody variant, wherein the first binding domain is fused to the CH ₃ domain of the heavy chain. When reduced ADCC activity is preferred the bispecific antibody variant, wherein the first binding domain is fused to the CH ₃ domain of the heavy chain is selected.

[62] To complete the findings above, a mixed lymphocyte reaction (MLR) was additionally used in Example 6. MLR is a functional assay which was established to analyze the effect of PD-L1 blocking antibodies on the suppression of PD-1 expressing T cells by PD-L1 expressing antigen presenting cells. The assay measures the response of T cells from one donor as responders to monocyte-derived dendritic cells (moDCs) from another donor as stimulators (= allogenic MLR). [63] The present inventors also surprisingly found that in a first allogeneic MLR when comparing PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ to each other activation of CD8 T cells (CD3 ⁺ CD8 ⁺ cells) measured via the activation marker CD25 (Fig. 6A) and the co- stimulatory molecule CD137 (Fig. 6B) was shown. However, the Ck-variant again showed increased T cell activation compared to the CH ₃ -variant.

[64] In a second allogeneic MLR, serial dilutions of PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared. PM-PDL-GEX Fuc-/Ck showed a strong concentration-dependent activation of T cells measured by CD25 (Fig. 6C) and CD137 expression (Fig. 6D), whereas incubation with PM-PDL-GEX Fuc-/CH ₃ resulted only in a slightly increased activation compared to the medium control.

[65] Finally, the fucose-reduced Ck-variants having different CDR mutations in the V _H domain of the scF _v region binding to PD-L1 , preferably having the amino acid sequence as shown in SEQ ID NO. 75 (having a mutation of glycine to alanine at position 26 according to Kabat- numbering and having a mutation of aspartic acid to glutamic acid at position 31 according to Kabat-numbering in the CDR1 of the V _H domain) or having the amino acid sequences as shown in SEQ ID NO. 77 (having a mutation of threonine to serine at position 28 according to Kabat- numbering in the CDR1 of the V _H domain) and 83 (having a mutation of serine to threonine at position 62 according to Kabat-numbering in the CDR2 of the V _H domain) as indicated elsewhere herein, may further show comparable enhanced CD25 T cell activation to the non- mutated PM-PDL-GEX Fuc-/Ck (Fig. 9). These data reveal that fucose-reduced Ck-variants of the present invention and/or fucose-reduced Ck-variants having different CDR mutations in the V _H domain of the scF _v region binding to PD-L1 , preferably having the amino acid sequence as shown in SEQ ID NO. 75 or having the amino acid sequences as shown in SEQ ID NO. 77 and 83 may enhance T cell activation.

[66] Whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or whether it is coupled to the CH ₃ domain of the heavy chain has no influence on antigen binding to PD-L1 (see Example 2) and TA-MUC1 (see Example 4), the blocking of PD-L1/PD-1 interaction (see Example 2) or the FcyRllla binding (see Example 3), but the activation of T cells may be affected. T cell activation may refer to cytotoxic T cells such as CD8 ⁺ T cells or helper T cells such as CD4 ⁺ T cells, preferably it may be CD8 ⁺ T cells. Thus, the Ck-variant may now be even more considered as being a promising variant for enhancing T cell activation. An antibody of the present invention is therefore preferably capable of mediating enhanced T cell activation.

In conclusion, a bispecific normal-fucosylated or fucose-reduced antibody of the present invention having anti-PD-L1 scF _v regions fused to the constant domain of the light chains, may have higher activity in relevant mode of actions such as ADCC and T cell activation compared to the counterparts with the scF _v regions coupled to the CH ₃ domain of the heavy chain. The present invention may further envisage a bispecific normal-fucosylated or fucose-reduced antibody having anti-CD3 scF _v regions fused to the constant domain of the light chains, which may have higher activity in relevant mode of actions such as ADCC and T cell activation compared to the counterparts with the scF _v regions coupled to the CH ₃ domain of the heavy chain. Thus, an antibody of the present invention is preferably capable of mediating enhanced ADCC and/or T cell activation in comparison to a bispecific reference antibody, wherein scF _v regions may be coupled to the CH ₃ domain of the heavy chain of said reference antibody.

[67] This surprising and promising finding of an bispecific trifunctional antibody presented by the invention, wherein scF _v regions are coupled to the constant domain of the light chains, exhibiting tetravalent binding enriches the prior art. Additionally, the prior art lacks engineered antibodies coupled to scF _v regions, which are even associated with T cell activation, which may be seen as a very encouraging approach for all kinds of diseases.

[68] To this end and in view of enhancing ADCC activity and/or T cell activation with the Ck- variant, the present invention may further encompass an antibody of the present invention for use in therapy. Further, the present invention provides an antibody of the present invention for use in a method for enhancing ADCC activity and/or T cell activation in comparison to a reference antibody, wherein scF _v regions may be coupled to the CH ₃ domain of the heavy chain of said reference antibody. The enhancement of ADCC activity and/or T cell activation is preferably for the treatment of cancer disease, inflammatory disease, virus infectious disease and autoimmune disease. Preferably, enhancement of ADCC activity and/or T cell activation is useful for the treatment of cancer disease.

[69] Cancer disease may be selected from Thymic Carcinoma, Lymphoma incl. Hodgkin's Lymphoma, Malignant Solitary Fibrous Tumor of the Pleura (MSFT), Penile Cancer, Anal Carcinoma, Thyroid Carcinoma, Head and Neck Squamous Carcinoma (HNSC), Non-small cell lung cancer (NSCLC), Small Cell Lung Cancer (SCLC), Vulvar Cancer (squamous cell carcinoma), Bladder Cancer, Cervical Cancer, Non-Melanoma Skin Cancer, (Retro-) Peritoneal Carcinoma, Melanoma, Gastrointestinal Stromal Tumor (GIST), Malignant Pleural Mesothelioma, Renal Cell Carcinoma (RCC), Kidney Cancer, Hepatocellular Carcinoma (HCC), Esophageal and Esophagogastric Junction Carcinoma, Extrahepatic Bile Duct Adenocarcinoma, Male Genital Tract Malignancy, Small Intestinal Malignancy, Sarcoma, Pancreatic Adenocarcinoma, Stomach Cancer (Gastric Adenocarcinoma), Breast Carcinoma, Colorectal Cancer (CRC), Malignant Mesothelioma, Merkel Cell Carcinoma, Squamous Cell Cancer, Advanced Carcinoma, Prostate Cancer, Ovarian Cancer, Endometrial Cancer, Urothelial Carcinoma (UCC), Lung Cancer. Preferably, cancer disease may be selected from Melanoma, Carcinoma, Lymphoma, Sarcoma, and Mesothelioma including Lung Cancer, Kidney Cancer, Bladder Cancer, Gastrointestinal Cancer, Skin Cancer, Breast Cancer, Ovarian Cancer, Cervical Cancer, and Prostate Cancer, most preferably cancer disease may be Breast Cancer.

[70] Further, the present invention may envisage the use of an antibody of the present invention, wherein a scF _v region is coupled to the constant domain of the light chain, for the manufacture of a medicament for therapeutic application in cancer disease, inflammatory disease, virus infectious disease and autoimmune disease. Further, the present invention may encompass the use of an antibody of the present invention, wherein a scF _v region is coupled to the constant domain of the light chain, for the manufacture of a medicament for enhancing ADCC activity and/or T cell activation.

[71] Additionally, the present invention may include a method for enhancing ADCC activity and/or T cell activation in a subject comprising administering an effective amount of said antibody variant, wherein a scF _v region is coupled to the constant domain of the light chain, to a subject in need thereof.

[72] The present invention may further contemplate an antibody of the present invention, wherein a scF _v region is coupled to the constant domain of the light chain, for use in a method for enhancing ADCC activity and/or T cell activation in a subject. An antibody of the present invention is administered to a subject suffering from cancer disease and/or inflammatory disease and/or virus infectious disease and/or autoimmune disease. The subject may be any subject as defined herein, preferably a human subject. The subject is preferably in need of the administration of an antibody of the present invention. Preferably, the subject may be an animal, including birds. The animal may be a mammal, including rats, rabbits, pigs, mice, cats, dogs, sheep, goats, and humans. Most preferably, the subject is a human. In one embodiment, the subject is an adult.

[73] Definitions:

[74] The term "Ck-variant" refers to a bispecific antibody comprising a first binding domain capable of binding to a first target and a second binding domain capable of binding to a second target, wherein (i) said second binding domain comprises a V _H and a V _L domain capable of binding a second target and wherein (ii) said first binding domain comprises a single chain F _v region capable of binding a first target, which is coupled to the constant domain of the light chain of said second binding domain. The first target of a Ck-variant of the present invention is preferably an immune checkpoint protein, wherein said immune checkpoint protein is preferably PD-L1 or preferably a cell surface molecule, wherein said cell surface molecule is preferably CD3. The second target of a Ck-variant of the present invention is preferably a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. The present invention may further provide a Ck-variant of the present invention being preferably capable of binding to TA-MUC1 and PD- L1 . Further, a Ck-variant preferably binds to TA-MUC1 and CD3 or HER2 and CD3. The Ck- variant may be preferred by the present invention. The present invention may also contemplate a Ck-variant, wherein the Ck-variant is preferably capable of binding as a first target to a cancer antigen, preferably TA-MUC1 and as a second target preferably to an immune checkpoint protein, preferably PD-L1. Here, the term "Ck" refers to the constant domain of the kappa light chain of Ck-variant. The Ck-variant may refer either to SEQ ID No. 13 (or SEQ ID NO. 41 ) and 14 and/or to SEQ ID No. 15 (or SEQ ID NO. 42) and 16.

[75] If a Ck-variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region is addressed in the present invention having different mutations in the CDRs of the V _H domain of the scF _v region, said antibody may also have any one of the amino acid sequences as shown in SEQ ID NOs. 46-49 and 14. Herein, SEQ ID NOs. 46-49 refer to a light chain coupled to a scF _v region capable of binding to PD-L1 and comprising two 4 GS-linkers, said light chain coupled to scF _v region additionally comprising different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1. Preferably, said Ck-variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region having different mutations in the CDRs of the V _H domain of the scF _v region and comprising two 4 GS-linkers, has the amino acid sequences as shown in SEQ ID NO. 47 or 48.

[76] Also comprised by the present invention is a Ck-variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region having different mutations in the CDRs of the V _H domain of the scF _v region, wherein said antibody may also have any one of the amino acid sequences as shown in SEQ ID NOs. 50-53 and 16. Herein, SEQ ID NOs. 50-53 refer to a light chain coupled to a scF _v region capable of binding to PD-L1 and comprising one 4 GS-linker and one 6 GS- linker, said light chain coupled to scF _v region additionally comprising different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1. Preferably, said Ck-variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region having different mutations in the CDRs of the V _H domain of the scF _v region and comprising one 4 GS-linker and one 6 GS-linker, has the amino acid sequences as shown in SEQ ID NO. 51 or 52.

[77] The term "CH ₃ -variant" refers to a bispecific antibody comprising a first binding domain capable of binding to a first target and a second binding domain capable of binding to a second target, wherein (i) said second binding domain comprises a V _H and a V _L domain capable of binding a second target and wherein (ii) said first binding domain comprises a single chain F _v region capable of binding a first target, which is coupled to the CH ₃ -domain of the heavy chain. The first target of the CH ₃ -variant is preferably an immune checkpoint protein, preferably said immune checkpoint protein is PD-L1 or preferably a cell surface molecule, wherein said cell surface molecule is preferably CD3. The second target of a CH ₃ -variant is preferably a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. The CH ₃ -variant preferably binds to TA-MUC1 and PD-L1 . Further, the CH ₃ -variant may bind to TA-MUC1 and CD3 or HER2 and CD3. Additionally, the present invention may also contemplate a CH ₃ -variant, wherein the CH ₃ - variant is preferably capable of binding as a first target to a cancer antigen, preferably TA- MUC1 and as a second target preferably to an immune checkpoint protein, preferably PD-L1. Here, the term "CH ₃ " refers to the third constant domain called CH ₃ -domain of the heavy chain of the CH ₃ -variant. The CH ₃ -variant may also be preferred in the present invention. The CH ₃ - variant may refer either to SEQ ID No. 17 and 18 (or SEQ ID NO. 43) and/or to SEQ ID No. 19 and 20 (or SEQ ID NO. 44).

[78] If a CH ₃ -variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region is addressed in the present invention having different mutations in the CDRs of the V _H domain of the scF _v region, said antibody may also have any one of the amino acid sequences as shown in SEQ ID NOs. 54-57 and 18 (or SEQ ID NO. 43). Herein, SEQ ID NOs. 54-57 refer to a heavy chain coupled to a scF _v region capable of binding to PD-L1 and comprising two 4 GS-linkers, said heavy chain coupled to scF _v region additionally comprising different mutations in the CDRs of the VH domain of the scF _v region binding to PD-L1.

[79] Also comprised by the present invention is a CH ₃ -variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region having different mutations in the CDRs of the V _H domain of the scF _v region, wherein said antibody may also have any one of the amino acid sequences as shown in SEQ ID NOs. 58-61 and 20 (or SEQ ID NO. 44). Herein, SEQ ID NOs. 58-61 refer to a heavy chain coupled to a scF _v region capable of binding to PD-L1 and comprising one 4 GS- linker and one 6 GS-linker, said heavy chain coupled to scF _v region additionally comprising different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1 .

[80] The term "PM-PDL-GEX" refers to a PankoMab antibody combined with PD-L1 specificity, also called a bispecific PankoMab-antiPDL1 -GEX antibody or anti-PD-L1/TA-MUC1 hlgG1 antibody". A PM-PDL-GEX antibody is developed by Glycotope GmbH. Here, the PankoMab antibody with PD-L1 specificity is trifunctional bispecific. Further, the anti-PD-L1 part as a scF _v region of the PankoMab-anti-PD-L1 -GEX antibody may comprise an antagonistic effect.

[81 ] The term "PM-CD3-GEX" refers to a PankoMab antibody combined with CD3 specificity, also called a bispecific PankoMab-antiCD3-GEX antibody. A PM-CD3-GEX antibody is developed by Glycotope GmbH. Here, the PankoMab antibody with CD3 specificity is trifunctional bispecific.

[82] The term "TM-CD3-GEX" refers to a Trastuzumab antibody combined with CD3 specificity, also called a bispecific Trastuzumab-antiCD3-GEX antibody. A TM-CD3-GEX antibody is developed by Glycotope GmbH. Here, the Trastuzumab antibody with CD3 specificity is trifunctional bispecific.

[83] The term "PankoMab" refers to a humanized monoclonal antibody recognizing the tumor-specific epitope of mucin-1 (TA-MUC1 ), enabling it to differentiate between tumor MUC1 and non-tumor MUC1 epitopes. It is developed by Glycotope GmbH. Here, the PankoMab with PD-L1 or anti-CD3 specificity is trifunctional bispecific. A PankoMab antibody of the present invention is preferably being capable of binding to a cancer antigen, preferably TA-MUC1 and is preferably combined with PD-L1 specificity, thus being capable of binding with its scF _v regions preferably to an immune checkpoint protein, preferably PD-L1. Further, a PankoMab antibody of the present invention may also be combined with a CD3 specificity, thus being capable of binding with its scF _v regions preferably to a cell surface molecule, preferably CD3.

[84] As it is well known in the art, an "antibody" is an immunoglobulin molecule capable of specific binding to a target (epitope) through at least one epitope recognition site, located in the variable region of the immunoglobulin molecule. The term "antibody" as used herein may comprise monoclonal and polyclonal antibodies, as well as (naturally occurring or synthetic) fragments or variants thereof, including fusion proteins comprising an antibody portion with an antigen-binding fragment of the required specificity and any other modified configuration of the antibody that comprises an antigen-binding site or fragment (epitope recognition site) of the required specificity. Illustrative examples of the antibody fragments or antibodies may include dAb, F _ab , F _ab ', F(ab') ₂ , F _v , single chain F _v s (scF _v ), single chain F _v s (scF _v s) coupled to the constant domain of the kappa light chains or to the CH ₃ domain of the heavy chains, diabodies, and minibodies. The term "antibody" can be used interchangeably with the term "variant". The antibody of the present invention when referred to herein may also be a composition comprising a plurality of antibodies.

[85] The term "plurality of antibodies" refers to the amount of antibodies which is preferably required for glycan analysis, preferably ^g.

[86] The antibody of the present invention may be a humanized antibody (or antigen-binding variant or fragment thereof). The term "humanized antibody" refers to an antibody containing a minimal sequence derived from a non-human antibody. In general, humanized antibodies are human immunoglobulins comprising residues from a hypervariable region of an immunoglobulin derived from non-human species such as mouse, rat, rabbit or non-human primate ("donor antibody") grafted onto the human immunoglobulin ("recipient antibody"). In some instances, frame work region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are neither found in the recipient antibody nor in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody may comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also may comprise at least a portion of an immunoglobulin constant region (F _c ), typically that of a human immunoglobulin.

[87] The term "bispecific antibody" is in the context of the present invention to be understood as an antibody with two different antigen-binding regions (based on sequence information). This can mean different target binding but includes as well binding to different epitopes in one target. In particular, a bispecific antibody of the present invention may refer to a Ck-variant capable of binding with its scF _v region coupled to the constant domain of the light chain of said second binding domain preferably to an immune checkpoint protein, preferably said immune checkpoint protein is PD-L1 or preferably to a cell surface molecule, preferably said cell surface molecule is CD3. Further, a bispecific antibody of the present invention referring to a Ck-variant is preferably capable of binding with its V _H and V _L domain to a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. The bispecific antibody of the present invention having the structure of a Ck-variant is a trifunctional bispecific antibody, which may exhibit tetravalent binding. The bispecific antibody of the present invention may also refer to a CH ₃ -variant capable of binding with its scF _v region coupled to the CH ₃ -domain of the heavy chain preferably to an immune checkpoint protein, preferably said immune checkpoint protein is PD-L1 or preferably to a cell surface molecule, preferably said cell surface molecule is CD3. Further, a bispecific antibody of the present invention referring to a CH ₃ -variant is preferably capable of binding with its V _H and V _L domain to a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. The bispecific antibody having the structure of a CH ₃ -variant is a trifunctional bispecific antibody, which may exhibit tetravalent binding.

The present invention may also comprise an antibody comprising polypeptide chains, wherein each of the polypeptide chain may have at least 50 % sequence identity to any one of SEQ ID No. 13 (or SEQ ID NO. 41 ) and 14 as well as 15 (or SEQ ID NO. 42) and 16. An antibody of the present invention may comprise polypeptide chains, wherein each of the polypeptide chain may have at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to any one of SEQ ID No. 13 (or SEQ ID NO. 41 ) and 14 as well as 15 and 16. The present invention may envisage an antibody comprising a light chain coupled to a scF _v region capable of binding to PD-L1 and comprising two 4 GS-linkers, having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID No. 13 (or SEQ ID NO. 41 ) and a heavy chain having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 14. The present invention may contemplate an antibody with two light chains coupled to scF _v regions capable of binding to PD-L1 and comprising two 4 GS-linkers having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 13 (or SEQ ID NO. 41 ) and two heavy chains having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 14. The present invention may also include an antibody comprising a light chain coupled to a scF _v region capable of binding to PD-L1 and comprising one 4 GS-linker and one 6 GS-linker having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID No. 15 (or SEQ ID NO. 42) and a heavy chain having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 16. The present invention may contemplate an antibody with two light chains coupled to scF _v regions capable of binding to PD-L1 and comprising one 4 GS-linker and one 6 GS-linker having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 15 (or SEQ ID NO. 42) and two heavy chains having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 16. An antibody of the present invention comprising polypeptide chains, wherein each of the polypeptide chain may have at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to any one of SEQ ID No. 13 (or SEQ ID NO. 41 ) and 14 as well as 15 (or SEQ ID NO. 42) and 16 may also be capable of binding to PD-L1 and TA-MUC1.

The present invention may also comprise an antibody comprising polypeptide chains, wherein each of the polypeptide chain may have at least 50 % sequence identity to any one of SEQ ID No. 17 and 18 (or SEQ ID NO. 43) as well as 19 and 20 (or SEQ ID NO. 44). An antibody of the present invention may comprise polypeptide chains, wherein each of the polypeptide chain may have at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to any one of SEQ ID No. 17 and 18 (or SEQ ID NO. 43) as well as 19 and 20 (or SEQ ID NO. 44). The present invention may envisage an antibody comprising a heavy chain coupled to a scF _v region capable of binding to PD-L1 and comprising two 4 GS-linkers, having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID No. 17 and a light chain having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 18 (or SEQ ID NO. 43). The present invention may contemplate an antibody with two heavy chains coupled to scF _v regions capable of binding to PD-L1 and comprising two 4 GS-linkers having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 17 and two light chains having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 18 (or SEQ ID NO. 43).

The present invention may also include an antibody comprising a heavy chain coupled to a scF _v region capable of binding to PD-L1 and comprising one 4 GS-linker and one 6 GS-linker having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID No. 19 and a light chain having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 20 (or SEQ ID NO. 44). The present invention may contemplate an antibody with two heavy chains coupled to scF _v regions capable of binding to PD-L1 and comprising one 4 GS-linker and one 6 GS-linker having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 19 and two light chains having at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to SEQ ID NO. 20 (or SEQ ID NO. 44). An antibody of the present invention comprising polypeptide chains, wherein each of the polypeptide chain may have at least 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 %, 97 %, 98 %, or at least 99 % sequence identity to any one of SEQ ID No. 17 and 18 (or SEQ ID NO. 43) as well as 19 and 20 (or SEQ ID NO. 44) may also be capable of binding to PD-L1 and TA-MUC1.

[88] The term "Afunctional bispecific antibody" may refer to an antibody of the present invention wherein, the F _c region may bind to an FcyR receptor, preferably to FcyRllla and the V _H and V|_ domains may bind to a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. Further, said trifunctional bispecific antibody capable of binding to TA-MUC1 or HER2 may further have scF _v regions, which may bind to an immune checkpoint protein, preferably said immune checkpoint protein is PD-L1 or to a cell surface molecule, preferably said cell surface molecule is CD3.

[89] The term„f irst binding domain" refers to a scF _v region, which is coupled either to the constant domain of the light chain or to the CH ₃ domain of the heavy chain and capable of binding to a first target. In this context, the scF _v region or the first binding domain, which can be used interchangeably, may be capable of binding to a first target. The antibody of the present invention may have two first binding domains, which may both comprise a scF _v region.

[90] The term„second binding domain" refers to the V _H domain and the V _L domain capable of binding to a second target. In this context, the V _H and V _L domain or the second binding domain, which can be used interchangeably, are capable of binding to a second target. An antibody may have two second binding domains, which may both comprise a V _H and a V _L domain.

[91] The term "GS-linker" refers to a peptide linker or a sequence with stretches of glycine (Gly/G) and serine (Ser/S) residues. A GS-linker may contain at least 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or at least 25 or more than 25 amino acids, preferably about 5 amino acids, which may couple the scF _v regions capable of binding said first target to the constant domain of the light chains of said second binding domain. The length of said GS-linker may allow sufficiently conformational stability to the antibody fused with scF _v . The present invention comprises an antibody of the present invention, which may use the common (G4S) 4 linker repeat (here called as 4 GS-linker - "GGGGS-GGGGS- GGGGS- GGGGS") or (G6S) 6 linker peptide (here called as 6 GS-linker - "GGGGS-GGGGS- GGGGS- GGGGS-GGGGS-GGGGS"). A 4 GS-linker and/or a 6 GS-linker may both be considered as linkers, which may allow sufficiently conformational stability to the antibody fused with scF _v regions.

The Ck-variant of the present invention may comprise a GS-linker containing at least 5, 10, 15, 20, or at least 25 or more than 25 amino acids, preferably 5 amino acids. Further, the Ck-variant may comprise either a 4 GS-linker or a 6 GS-linker. The Ck-variant may comprise two 4 GS- linkers, whereby the first 4 GS-linker may couple the V _H - domain of the scF _v region to the constant domain of the light chain of the Ck-variant and the second 4 GS-linker couples the VH- domain to the V _L -domain of the scF _v region, having a V _H -linker-V _L orientation. Said light chain coupled to one scF _v region, being able to bind PD-L1 , and comprising two 4 GS-linkers (SEQ ID No. 13 or SEQ ID NO. 41 ) as well as the heavy chain of the Ck-variant (SEQ ID No. 14) may refer to the antibody called hPM-Ck-4GS-PDLscF _v 4GS. The Ck-variant of the present invention may further comprise one 4 GS-linker, whereby said 4 GS-linker may couple the V _H -domain of the scF _v region to the constant domain of the light chain of said variant and another 6 GS-linker, which may couple the V _H -domain to the V _L -domain of the scF _v region, having again a VH-linker- VL orientation. Said light chain coupled to one scF _v region, being able to bind to PD-L1 , and comprising one 4 GS-linker and one 6 GS-linker (SEQ ID No. 15 or SEQ ID NO. 42) as well as the heavy chain of said Ck-variant (SEQ ID No. 16) may refer to the antibody called hPM-Ck- 4GS-PDLscF _v 6GS. The Ck-variant comprising a light chain coupled to the scF _v region having two 4 GS-linkers may be preferred in the present invention. The Ck-variant comprising a light chain coupled to the scF _v region having one 4 GS-linker and another 6 GS-linker may also be preferred in some in the present invention.

The CH ₃ -variant of the present invention may comprise a GS-linker containing at least 5, 10, 15, 20, or at least 25 or more than 25 amino acids, preferably 5 amino acids. Further, the CH ₃ - variant may comprise either a 4 GS-linker or a 6 GS-linker. The CH ₃ -variant may comprise two 4 GS-linkers, whereby the first 4 GS-linker may couple the V _H -domain of the scF _v region to the CH ₃ domain of the heavy chain of said variant and the second 4 GS-linker may couple the V _H - domain to the V _L -domain of the scF _v region, having a V _H -linker-V _L orientation. Said heavy chain coupled to one scF _v region, being able to bind to PD-L1 , and comprising two 4 GS-linkers (SEQ ID No. 17) as well as the light chain of said CH ₃ -variant (SEQ ID No. 18 or SEQ ID NO. 43) may refer to the antibody called hPM-CH ₃ -4GS-PDLscF _v 4GS. The CH ₃ -variant of the present invention may further comprise one 4 GS-linker, whereby said 4 GS-linker may couple the V _H - domain of the scF _v region to the CH ₃ domain of the heavy chain of said variant and another 6 GS-linker, which may couple the V _H -domain to the V _L -domain of the scF _v region, having a V _H - linker-V|_ orientation. Said heavy chain coupled to one scF _v region, being able to bind to PD-L1 , and comprising one 4 GS-linker and one 6 GS-linker (SEQ ID No. 19) as well as the light chain of said CHs-variant (SEQ ID No. 20 or SEQ ID NO. 44) may refer to the antibody called hPM- CH ₃ -4GS-PDLscF _v 6GS.

[92] The term "scF _v region" refers to the term single-chain fragment variable region comprising a variable domain of the heavy chain (V _H domain) and a variable domain of the light chain (V _L domain). scF _v regions may be coupled symmetrically to the constant domain of the light chain ("C-terminal-fusion") of said antibody or to the CH ₃ domain of the heavy chain of said antibody ("C-terminal- fusion") by linkers, preferably by GS-linkers. Here, a scF _v region may refer to the first binding domain of said antibody, which is preferably capable of binding a first target, wherein the first target may be an immune checkpoint protein, preferably PD-L1 or a cell surface molecule, preferably CD3. Further, a scF _v region may also be capable of binding to TA- MUC1.

ScF _v regions when referred to herein may be coupled by GS-linkers, preferably by a 4 GS-linker, either to the constant domain of the light chains or to the CH ₃ domain of the heavy chains of an antibody of the present invention. A scF _v region may consist of one V _H (SEQ ID No. 21 ) and one V _L (SEQ ID No. 22) domain, connected by linkers as well, preferably by GS-linkers. Here, the GS-linker may either be a 4 GS-linker or a 6 GS-linker, preferably a 4 GS-linker. The present invention may comprise an antibody having two scF _v regions, one per light chain, which may be coupled to the constant domain of the light chains. The present invention may further envisage an antibody having two scF _v regions, one per heavy chain, which may be coupled to the CH ₃ domain of the heavy chains.

Also comprised by the present invention may be a scF _v region consisting of one mutated V _H domain, preferably having any one of amino acid sequences as shown in SEQ ID NOs. 62-70 and of one non-mutated V _L domain as shown in SEQ ID No. 22, if a Ck-variant or a CH ₃ - variant,which comprises different mutations in the CDRs of the V _H domain of the scF _v region binding to PD-L1 , is addressed in the present invention.

An antibody of the present invention, no matter if it is the Ck-variant or the CH ₃ -variant, may comprise the following V _H and V _L domain CDRs having the amino acid sequence shown in SEQ ID Nos. 1 -6, which preferably confer binding to PD-L1. SEQ ID Nos. 1 -3 may refer to the V _H domain CDRs of the scF _v region, whereas SEQ ID Nos. 4-6 may refer to the V _L domain CDRs of the scF _v region:

SEQ ID No. 1 : Gly Phe Thr Phe Ser Asp Ser Trp lie His (CDR1 in the V _H domain of the PD-L1 binding site)

SEQ ID No. 2: Ala Trp lie Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly (CDR2 in the V _H domain of the PD-L1 binding site),

SEQ ID No. 3: Arg His Trp Pro Gly Gly Phe Asp Tyr (CDR3 in the V _H domain of the PD-L1 binding site). SEQ ID No. 4: Arg Ala Ser Gin Asp Val Ser Thr Ala Val Ala (CDR1 in the V _L domain of the PD- L1 binding site),

SEQ ID No. 5: Ser Ala Ser Phe Leu Tyr Ser (CDR2 in the V _L domain of the PD-L1 binding site), SEQ ID No. 6: Gin Gin Tyr Leu Tyr His Pro Ala Thr (CD3 in the V _L domain of the PD-L1 binding site).

The present invention may also comprise an antibody, wherein the V _H domain CDR1 of the scF _v region capable of binding to PD-L1 may have 1 , 2, 3, 4, or 5 mutations as compared to SEQ ID No. 1 . Further, the present invention may comprise an antibody, wherein the V _H domain CDR2 of the scF _v region capable of binding to PD-L1 may have 1 , 2, 3, 4, 5, 6, 7, 8, or 9 mutations as compared to SEQ ID No. 2. Additionally, the invention may contemplate an antibody, wherein the VH domain CDR3 of the scF _v region capable of binding to PD-L1 may have 1 , 2, 3, 4, or 5 mutations as compared to SEQ ID No. 3. Further, the present invention may envisage an antibody, wherein the V _H domain frame work region 1 of the scF _v region may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 mutations compared to frame work region 1 of SEQ ID No. 25. Further, the present invention may envisage an antibody, wherein the V _H domain frame work region 2 of the scF _v region may have 1 , 2, 3, 4, 5 or 6 mutations compared to frame work region 2 of SEQ ID No. 26. Additionally, the present invention may envisage an antibody, wherein the V _H domain frame work region 3 of the scF _v region may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 mutations compared to frame work region 3 of SEQ ID No. 27. The present invention may envisage an antibody, wherein the V _H domain frame work region 4 of the scF _v region may have 1 , 2, 3, 4, or 5 mutations compared to frame work region 4 of SEQ ID No. 28.

The present invention may also envisage an antibody, wherein the V _L domain CDR1 of the scF _v region capable of binding to PD-L1 may have 1 , 2, 3, 4, or 5 mutations as compared to SEQ ID No. 4. The present invention may include an antibody having 1 , 2, or 3 mutations in the V _L domain CDR2 of the scF _v region capable of binding to PD-L1 as compared to SEQ ID No. 5. The present invention may also encompass an antibody having 1 , 2, 3, or 4 mutations in the V _L domain CDR3 of the scF _v region capable of binding to PD-L1 as compared to SEQ ID No. 6. Further, the present invention may envisage an antibody, wherein the V _L domain frame work region 1 of the scF _v region may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 mutations compared to frame work region 1 of SEQ ID No. 29. Further, the present invention may envisage an antibody, wherein the V _L domain frame work region 2 of the scF _v region may have 1 , 2, 3, 4, 5, 6, or 7 mutations compared to frame work region 2 of SEQ ID No. 30. Additionally, the present invention may envisage an antibody, wherein the V _L domain frame work region 3 of the scF _v region may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 mutations compared to frame work region 3 of SEQ ID No. 31. The present invention may envisage an antibody, wherein the V _L domain frame work region 4 of the scF _v region may have 1 , 2, 3, 4, or 5 mutations compared to frame work region 4 of SEQ ID No. 32. Further, an antibody of the present invention having one or more V _H and V _L domain CDRs having said mutations, may also confer binding to PD-L1. Additionally, the present invention may also contemplate an antibody comprising V _H and V _L domain CDRs of scF _v regions, which may be capable of binding a cancer antigen, preferably TA-MUC1.

[93] If a Ck-variant binding to TA-MUC1 and binding to PD-L1 with its scF _v region having different mutations in the CDRs of the V _H domain of the scF _v region is addressed in the present invention, said Ck-variant may also have the amino acid sequences as shown in SEQ ID NO. 71 (having a mutation of phenylalanine to isoleucine at position 29 according to Kabat- numbering in the CDR1 of the V _H domain) and 79 (having a mutation of serine to threonine at position 52 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 73 (having a mutation of glycine to alanine at position 26 according to Kabat-numbering in the CDR1 of the V _H domain) and 80 (having a mutation of alanine to glycine at position 49 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 74 (having a mutation of isoleucine to methionine at position 34 according to Kabat-numbering in the CDR1 of the V _H domain) and 81 (having a mutation of isoleucine to leucine at position 51 according to Kabat- numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 75 (having a mutation of glycine to alanine at position 26 according to Kabat- numbering and having a mutation of aspartic acid to glutamic acid at position 31 according to Kabat-numbering in the CDR1 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 76 (having a mutation of aspartic acid to glutamic acid at position 31 according to Kabat-numbering in the CDR1 of the V _H domain) and 82 (having a mutation of valine to leucine at position 63 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 77 (having a mutation of threonine to serine at position 28 according to Kabat-numbering in the CDR1 of the V _H domain) and 83 (having a mutation of serine to threonine at position 62 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 74 (having a mutation of isoleucine to methionine at position 34 according to Kabat-numbering in the CDR1 of the V _H domain) and 83 (having a mutation of serine to threonine at position 62 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 78 (having a mutation of serine to threonine at position 32 according to Kabat-numbering in the CDR1 of the V _H domain) and 85 (having a mutation of serine to threonine at position 56 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequences as shown in SEQ ID NO. 74 (having a mutation of isoleucine to methionine at position 34 according to Kabat-numbering in the CDR1 of the V _H domain) and 79 (having a mutation of serine to threonine at position 52 according to Kabat- numbering in the CDR2 of the V _H domain), or having the amino acid sequence as shown in SEQ ID NO. 72 (having a mutation of phenylalanine to isoleucine at position 29 according to Kabat-numbering in the CDR1 of the V _H domain and having a mutation of aspartic acid to glutamic acid at position 31 according to Kabat-numbering in the CDR1 of the V _H domain), or having the amino acid sequence as shown in SEQ ID NO. 84 (having a mutation of glycine to alanine at position 65 according to Kabat-numbering in the CDR2 of the V _H domain), or having the amino acid sequence as shown in SEQ ID NO. 86 (having a mutation of tyrosine to serine at position 53 according to Kabat-numbering in the CDR2 of the V _H domain.

Preferably, said Ck-variant may comprise the following V _H CDRs which preferably confer binding to PD-L1 : SEQ ID NO. 75 having a mutation of glycine to alanine at position 26 in the CDR1 of the V _H domain according to Kabat-numbering and having a mutation of aspartic acid to glutamic acid at position 31 in the CDR1 of the V _H domain according to Kabat-numbering or SEQ ID NO. 77 having a mutation of threonine to serine at position 28 in the CDR1 of the V _H domain according to Kabat-numbering and SEQ ID NO. 83 having a mutation of serine to threonine at position 62 according to Kabat-numbering in the CDR2 of the V _H domain as indicated elsewhere herein.

[94] The term "V _H and V _L domain" may refer to the variable domain of the heavy chain and the variable domain of the light chain of the F _ab region of an antibody of the present invention. A VH and V _L domain may consist of one V _H (SEQ ID No. 23) and one V _L (SEQ ID No. 24 or SEQ ID NO. 45) domain. Is the variable domain of the heavy chain and the variable domain of the light chain of the scF _v region addressed in the present invention, the term "V _H and V _L domain of the scF _v region" may be used. Said V _H and V _L domains may refer to the second binding domain of an antibody of the present invention, which is preferably capable of binding a second target, wherein the second target is a preferably a cancer antigen, preferably said cancer antigen is TA-MUC1 or HER2. The present invention comprises an antibody having two V _H and V _L domains, thus one light and one heavy chain of said antibody comprise together a V _H and a V _L domain, whereas the other light and other heavy chain of said antibody also comprise together a V _H and a V _L domain. An antibody of the present invention may comprise the following V _H and V _L domain CDRs having the amino acid sequence shown in SEQ ID Nos. 7-12, which preferably confer binding to TA-MUC1. SEQ ID Nos. 7-9 may refer to the V _H domain CDRs, whereas SEQ ID Nos. 10-12 may refer to the V _L domain CDRs:

SEQ ID No. 7: Asn Tyr Trp Met Asn (CDR1 in the V _H domain of the TA-MUC1 binding site), SEQ ID No. 8: Glu lie Arg Leu Lys Ser Asn Asn Tyr Thr Thr His Tyr Ala Glu Ser Val Lys Gly (CDR2 in the V _H domain of the TA-MUC1 binding site),

SEQ ID No. 9: His Tyr Tyr Phe Asp Tyr (CDR3 in the V _H domain of the TA-MUC1 binding site). SEQ ID No. 10: Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly lie Thr Tyr Phe Phe (CDR1 in the V|_ domain of the TA-MUC1 binding site), SEQ ID No. 1 1 : Gin Met Ser Asn Leu Ala Ser (CDR2 in the V _L domain of the TA-MUC1 binding site),

SEQ ID No. 12: Ala Gin Asn Leu Glu Leu Pro Pro Thr (CDR3 in the V _L domain of the TA-MUC1 binding site).

The present invention may also comprise an antibody, wherein the V _H domain CDR1 region may have 1 , 2, or 3 mutations as compared to SEQ ID No. 7. Further, the present invention may comprise an antibody, wherein the V _H domain CDR2 may have 1 , 2, 3, 4, 5, 6, 7, 8, or 9 mutations as compared to SEQ ID No. 8. Additionally, the invention may contemplate an antibody, wherein the V _H domain CDR3 may have 1 , 2, or 3 mutations as compared to SEQ ID No. 9. Further, the present invention may envisage an antibody, wherein the V _H domain frame work region 1 may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 mutations compared to frame work region 1 of SEQ ID No. 33. Further, the present invention may envisage an antibody, wherein the V _H domain frame work region 2 may have 1 , 2, 3, 4, 5, 6, or 7 mutations compared to frame work region 2 of SEQ ID No. 34. Additionally, the present invention may envisage an antibody, wherein the V _H domain frame work region 3 may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 mutations compared to frame work region 3 of SEQ ID No. 35 The present invention may envisage an antibody, wherein the V _H domain frame work region 4 may have 1 , 2, 3, 4, or 5 mutations compared to frame work region 4 of SEQ ID No. 36.

The present invention may also envisage an antibody, wherein the V _L domain CDR1 may have 1 , 2, 3, 4, 5, 6, 7, or 8 mutations as compared to SEQ ID No. 10. The present invention may include an antibody having 1 , 2, or 3 mutations in the V _L domain CDR2 as compared to SEQ ID No. 1 1 . The present invention may also encompass an antibody having 1 , 2, 3, or 4 mutations in the V|_ domain CDR3 as compared to SEQ ID No. 12. Further, the present invention may envisage an antibody, wherein the V _L domain frame work region 1 may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 mutations compared to frame work region 1 of SEQ ID No. 37. Further, the present invention may envisage an antibody, wherein the V _L domain frame work region 2 may have 1 , 2, 3, 4, 5, 6, or 7 mutations compared to frame work region 2 of SEQ ID No. 38. Additionally, the present invention may envisage an antibody, wherein the V _L domain frame work region 3 may have 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 mutations compared to frame work region 3 of SEQ ID No. 39. The present invention may envisage an antibody, wherein the V _L domain frame work region 4 may have 1 , 2, 3, 4, 5, or 6 mutations compared to frame work region 4 of SEQ ID No. 40.

Further, an antibody of the present invention having one or more V _H and V _L domain CDRs having said mutations, may also confer binding to TA-MUC1. The present invention may also contemplate an antibody comprising V _H and V _L domain CDRs, which may be capable of binding an immune checkpoint protein, preferably PD-L1 . [95] The term "frame work region" refers to the amino acid region before and after a CDR and inbetween CDRs either in the V _H and V _L domain or in the V _H and V _L domain of the scF _v regions.

[96] The term "CDRs" refers to complementarity-determining regions, which refer to variable loops of β-strands, three each on the variable domains of the light (V _L ) and heavy (V _H ) chains in immunoglobulins (antibodies) generated by B-cells respectively or in single chain F _v regions coupled to an immunoglobulin being responsible for binding to the antigen. Unless otherwise indicated CDRs sequences of the disclosure follow the definition by Maass 2007 (Journal of Immunological Methods 324 (2007) 13-25. Other standards for defining CDRs exist as well, such as the definition according to Kabat CDRs, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991 ), eds. Kabat et al. Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia (see, e.g., Chothia, et al. (1992); J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638). Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modelling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). It is understood that embodiments described with respect to the CDR definition of Maass, can alternatively be implemented using similar described relationships such as with respect to Kabat CDRs, Chothia hypervariable loops or to the AbM-defined loops.

[97] The term "mutation" refers to substitution, insertion and/or deletion. Mutations may occur in the V _H and V _L domain CDRs and/or in the corresponding frame work region of the V _H and V _L domains. Mutations may also occur in the V _H and V _L domain CDRs of the scF _v regions and/or in the corresponding frame work region of the V _H and V _L domains of the scF _v regions.

[98] The term "affinity" refers to the strength with which an antibody molecule binds an epitope. Affinity measures the strength of any given bond between an epitope and an antibody's antigen binding site (also called paratope). High affinity antibodies may bind quickly to the antigen, permit greater sensitivity in assays and maintain this bond more readily under difficult conditions. Low affinity antibodies, by contrast, may bind weakly to the antigen and often do not detect the antigen in vivo or in assays. The affinity of monoclonal antibodies can be measured accurately because they are homogeneous and selective for a single epitope. Polyclonal antibodies are heterogeneous and will contain a mixture of antibodies of different affinities recognizing several epitopes - therefore only an average affinity can be determined.

[99] The term "epitope" may refer to a fragment of a polypeptide or protein or a non-protein molecule having antigenic or immunogenic activity in an animal, preferably in a mammal, and most preferably in a human. An epitope having immunogenic activity may be a fragment of a polypeptide or protein that may elicit an antibody response in an animal. An epitope having antigenic activity may be a fragment of a polypeptide or protein to which an antibody immunospecifically may bind as determined by any method well-known to one of skill in the art, for example by immunoassays. Antigenic epitopes may not necessarily be immunogenic.

[100] The term "tetravalent binding" refers to the binding to four antigenic determinants (antigens) by an individual antibody molecule. Tetravalent antibodies may have two binding sites for each antigen. Here, the bispecific antibody of the present invention may have two binding sites for each antigen, two scF _v regions being capapble of binding to PD-L1 or CD3 and two V _H and V _L domains being capable of binding to TA-MUC1 or HER2.

[101] The term "immune checkpoint protein" refers to a protein molecule in the immune system which modulates immune response, either anti-inflammatory or pro-inflammatory. They monitor the correct function of the immune response by either turning up a signal (co-stimulatory molecules) or turning down a signal. There are inhibitory (anti-inflammatory) immune checkpoint proteins such as A2AR, B7-H3 (CD276), B7-H4 (VTCN1 ), BTLA, CTLA-4, IDO, KIR, LAG 3, PD- 1 , PD-L1 , TIM-3, VISTA (protein) and pro-inflammatory immune checkpoint proteins such as CD27, CD40, OX40, GITR, CD137 (4-1 BB), ICOS (CD278), CD40 Ligand (CD154), HVEM (CD270) and LIGHT (CD258). The present invention may prefer the inhibitory immune checkpoint proteins. Here, the immune checkpoint protein preferably refers to PD-L1 .

[102] The term "cell surface molecule" refers to a molecule naturally associated with the cell surface. Herein, the cell surface molecule may be the cluster of differentiation 3 (CD3) T-cell co- receptor, which may help to activate cytotoxic T-cells. CD3 refers to a protein complex, consisting of four distinct chains. In general, it contains CD3y chain, a CD35 chain, and two CD3e chains, associating with the T-cell receptor (TCR) and the ζ-chain (zeta-chain) to generate an activation signal in T lymphocytes.

[103] The term "cancer antigen" refers to an antigenic substance produced in cancer cells. Cancer antigens, due to their relative abundance in cancer cells are useful in identifying specific cancer cells. Certain cancers have certain cancer antigens in abundance. Cancer-associated antigens may include, but are not limited to HER2, EGFR, VEGF, TA-MUC1 , PSA. Here, the cancer antigen preferably refers to TA-MUC1 or HER2. The term "tumor antigen" can be used interchangeably.

[104] The term "derived from" or "derived therefrom" may be used interchangeably with the term "originated from" / "originated therefrom" or "obtained from" / "obtained therefrom". For example, a cell or cell line may originate from another cell or a cell line mentioned in the present invention.

[105] The term "normal-fucosylated antibody" may refer to an antibody, either a Ck-variant or a CH ₃ -variant, which may have a normal FcyR-binding capacity, preferably FcyRllla-binding capacity. The normal-fucosylated antibodies of the present invention are glycosylated, having two N-linked sugar chains bound to the F _c region, wherein among the total complex N-linked sugar chains bound to the F _c region, the content of 1 ,6-core-fucose may be more than 80%. The normal-fucosylated antibodies of the present invention may contain more than 80% less than 100% core fucosylated N-glycans. The normal-glycosylated antibodies of the present invention may preferably contain about 81 % to 100%, 85% to 95% fucosylated N-glycans or 90% to 95 % fucosylated N-glycans. The normal-fucosylated antibodies of the present invention may contain more than 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% fucosylated N-glycans. The term "normal- fucosylated antibody" may refer to the term "glycosylated, normal-fucosylated antibody". Here, a normal-fucosylated antibody may refer to a trifunctional bispecific PM-PDL-GEX-H9D8/Ck as well as a PM-PDL-GEX H9D8/CH ₃ antibody.

[106] The term "core fucosylated N-glycans" refers to N-glycans of a plurality of antibodies, which are core fucosylated. The molar amount of core fucosylated N-glycans relative to the molecular amount of total N-glycans of a plurality of antibodies may be more than 80 % or from 0% to 80 %. The content of more than 80 % core fucosylated N-glycans as it is described for said normal-fucosylated antibodies of the present invention is preferably be determined from a plurality of antibodies, wherein more than 80 % of the molecular amount of total N-glycans of a plurality of antibodies may be core a1 ,6-fucosylated. The content of 0% to 80% core fucosylated N-glycans as it is described for said fucose-reduced antibodies of the present invention may also be determined preferably from a plurality of antibodies, wherein 0% to 80% of molecular amount of N-glycans of a plurality of antibodies may be core a1 ,6-fucosylated. Core- fucosylation of the N-glycans is determined in Example 1. Fucose addition or reduction may be catalyzed by alpha-(1 .6)-fucosyltransferase (FUT8), which is an enzyme that in humans is encoded by the FUT8 gene.

[107] The term "core-fucose" or "core-fucosylated" refers to the monosaccharide fucose, which is attached at position a-1 ,6 being the first N-acetylglucosamine (GlcNac), which is part of the mannosyl-chitobiose core (Man3GlcNAc2-Asn), which is bound to each conserved amino acid asparagine N297 in the CH ₂ domains of the F _c region.

[108] The term "content of a-1 ,6-core-fucose" refers to the amount of core-fucose, which is being attached onto the first N-acetylglucosamine (GlcNac) being part of the mannosyl- chitobiose core (Man3GlcNAc2-Asn), which is bound to each conserved amino acid asparagine N297 in the CH ₂ domains of the F _c region. Among the total complex N-linked sugar chains bound to the F _c region, the content of a-1 ,6-core-fucose may be more than 80 % for said normal-fucosylated antibodies or from 0 % to 80 % for said fucose-reduced antibodies. The content of a-1 ,6-core-fucose may be determined preferably by a plurality of antibodies. Preferably, the content of a-1 ,6-core-fucose, thus the content of a-1 ,6-core-fucose of the N- glycans with regard to the plurality of antibodies, may be analyzed by HILIC-UPLC-HiResQToF MSMS (see Example 1 ). [109] The term "fucose-reduced antibody" may refer to an antibody, either a Ck-variant or a CH ₃ -variant, which may have an increased FcyR-binding capacity, preferably FcyRllla-binding capacity. Fucose-reduced antibodies of the present invention contain two N-linked oligosaccharides at N297, thus being glycosylated. Further, fucose-reduced antibodies of the present invention may comprise from 0% to 80% a-1 ,6-core fucosylation. In particular, fucose- reduced antibodies of the present invention comprise an F _c region and have two complex N- linked sugar chains bound to the F _c region, wherein among the total complex N-linked sugar chains bound to the F _c region, the content of 1 ,6-core-fucose may be from 0% to 80%. The fucose-reduced antibodies of the present invention may preferably contain about 0% to 70%, 0% to 60%, 0% to 50 %, 0% to 40 %, 0% to 30 %, 0% to 20 %, 0% to 10 % or 10% to 50%, 15% to 50%, 20% to 50%, 25% to 50%, 30% to 50%, 35% to 50%, 40% to 50%, 45% to 50% or 1 % to 20%, 1 % to 15%, 1 % to 10%, 1 % to 5% or 5% to 30%, 5% to 20%, 5% to 15% fucosylated N-glycans. The fucose-reduced antibodies of the present invention may preferably contain 0%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20.0%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 41 %, 42%, 43%, 44%, 45.0%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61.0%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or even 80% fucosylated N-glycans. The term "fucose- reduced antibody" may refer to the term "glycosylated antibody being fucose-reduced". Here, a fucose-reduced antibody of the present invention may refer to a trifunctional bispecific PM-PDL- GEX-Fuc-/Ck as well as a PM-PDL-GEX Fuc-/CH ₃ antibody.

[110] The term "fucose-reduced" refers to the reduction of the content of a-1 ,6-core fucose, which is attached onto the first N-acetylglucosamine (GlcNac), which is part of the mannosyl- chitobiose core (Man3GlcNAc2-Asn), which is bound to each conserved amino acid asparagine N297 in the CH ₂ domains of the F _c region. The term "fucose-reduced" may also be used interchangeably with the term "de-fucosylated/essentially de-fucosylated" or nouns such as "de- fucosylation" thereof.

[111] The term "glycosylated antibody" refers to an antibody, either a Ck-variant or a CH ₃ - variant, containing two N-linked oligosaccharides at N297. Further, a glycosylated antibody may comprise either more than 80% a-1 ,6-core fucosylation, thus referring to a glycosylated antibody, being normal-fucosylated or from 0% to 80% a-1 ,6-core fucosylation, thus referring to a glycosylated antibody, being fucose-reduced. Modifications of the oligosaccharide structure in the F _c region (F _c /V-glycosylation) may predominantly influence binding of antibodies to the Fc receptor and are an established approach for enhancing ADCC activity. In general, glycosylation itself and variations in glycoforms are being known to play an important role by affecting biological functions of IgG antibodies.

In general, glycosylated antibodies may comprise N-linked oligosaccharides at the conserved asparagine 297 (N297), according to EU-nomenclature, in the CH ₂ domain. Typical N-glycans of the present invention attached to N297 of the antibody may be of the complex type but also highmannose or hybride type N-glycans may be linked to N297 of the antibody. The complex type N-glycosylation of the antibodies of the present invention may be characterized by a mannosyl-chitobiose core (Man3GlcNAc2-Asn) with variations in the presence/absence of bisecting N-acetylglucosamine and core-fucose, which may be a-1 .6-linked to the N- acetylglucosamine that is attached to the antibodies. Furthermore, the complex type N- glycosylation of the antibodies of the present invention may be characterized by antennary N- acetylglucosamine linked to the mannosyl-chitobiose core (Man3GlcNAc2-Asn) with optional extension of the antenna by galactose and sialic acid moieties. Additionally, antennary fucose and/or N-acetylgalactosamine may be part of the extension of antenna. The core fucose of the present invention may be a-1.6-linked to the N-acetylglucosamine (GlcNac) of the N-linked oligosaccharide structure.

Here, a glycosylated antibody may refer to a trifunctional bispecific PM-PDL-GEX-H9D8/Ck as well as a PM-PDL-GEX H9D8/CH ₃ antibody, being also normal-fucosylated, or a PM-PDL-GEX- Fuc-/Ck as well as a PM-PDL-GEX Fuc-/CH ₃ antibody, being fucose-reduced.

[112] The term "N-linked oligosaccharides" refers to N-linked sugar chains/N-glycans bound to the F _c region, more specific it refers to N-linked sugar chains/N-glycans, which are bound to both CH ₂ domains of the F _c region, preferably attached onto each N297 in both CH ₂ domains of the F _c region. In total, the present invention comprises two N-linked oligosaccharides.

[113] The term "ADCC activity" refers to antibody-dependent cell cytotoxicity (ADCC), the ability to mediate cellular cytotoxic effector functions which is a promising means to enable the enhancement of the antitumor potency of antibodies. In general, for IgG class antibodies ADCC as well as ADCP (antibody-dependent cellular phagocytosis) are mediated by engaging of the F _c region with specific so called Fc gamma receptors (FcyRs). There are three classes of receptors in humans: the FcyRI (CD64), FcyRII (CD32) with its isoforms FcyRlla, FcyRllb and FcyRllc, and FcyRIII (CD16) with its isoforms FcyRllla and FcyRlllb. The same region on IgG F _c is bound by all FcyRs, only differing in their affinities with FcyRI having a high affinity and FcyRII and FcyRIII having a low affinity. Therefore, an antibody with an optimized FcyR affinity may result in an altered functionality, for example a better cellular cytotoxicity against targeted cancer cells in therapy. ADCC is a mechanism whereby the antibody binds with its F _ab region to a target cell antigen and recruits effector cells by binding of its F _c part to Fc receptors on their surface of these cells, resulting in the release of cytokines such as IFN-γ and cytotoxic granules containing perforin and granzymes that enter the target cell and promote cell death. It was found that in particular the FcyRllla plays the most crucial role in mediating ADCC activity to targeted cancer cells. ADCC activity against TA-MUC1 ⁺ cancer cells and PD-L1 ⁺ cancer cells may be even more enhanced for an antibody of the present invention due to de-fucosylation of oligosaccharides in the F _c region at the a-1 .6 position, enhancing the engagement of the F _c region to FcyRs, in particular FcYRIIIa on natural killer cells (NKs) for strong NK cell ADCC and strong phagocytosis (ADCP).

[114] The term "T cell activation" refers to the process, wherein T cells encounter their specific antigen in the form of a peptide:MHC complex on the surface of an activated antigen-presenting cell (APC) and become activated. The most important antigen-presenting cells are the highly specialized dendritic cells (DCs), functioning through ingesting and presenting antigens. Tissue dendritic cells ingest antigen at sites of infection and are activated as part of the innate immune response. DCs migrate then to local lymphoid tissue and mature into cells that are highly effective at presenting antigen to recirculating T cells. The characterization of these mature dendritic cells is based on surface molecules, known as co-stimulatory molecules that synergize with antigen in the activation of naive T cells into effector T cells. Depending on the peptide antigens (e.g. intracellular and extracellular) presented by the DCs to T cells, different T cells are being activated. Intracellular antigens are carried to the cell surface by MHC class I molecules and presented to CD8 T cells. After differentiation into cytotoxic T cells they kill infected target cells. Extracellular peptides are carried to the cell surface by MHC class II molecules and presented to CD4 T cells. Amongst others, two major types of effector T cells, called T _H 1 and T _H 2 are differentiated thereof (Janeway CA Jr, Travers P, Walport M, et al., 2001 , "Immunobiology: The Immune System in Health and Disease", Garland Science, 5th edition). It was surprisingly found that the F _c region of an antibody of the present invention, preferably said Ck-variant may bind to the FcYRIIIa on DCs potentially leading to an enhanced T cell activation via FcYRIIIa-dependent maturation of DCs.

An antibody of the present invention, wherein a scF _v region is coupled to the constant domain of the light chain synergizing different functions such as ADCC activity and PD-1/PD-L1 blockade, which is even capable of enhancing T cell activation, is not yet known by the prior art.

* * * *

[115] It is noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[116] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention. [117] The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[118] The term "less than" or in turn "more than" does not include the concrete number. For example, less than 20 means less than the number indicated. Similarly, more than or greater than means more than or greater than the indicated number, f.e. more than 80% means more than or greater than indicated number of 80 %.

[119] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having". When used herein "consisting of" excludes any element, step, or ingredient not specified.

[120] The term "including" means "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[121] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[122] All publications cited throughout the text of this specification (including all patents, patent application, scientific publications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[123] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.

[124] A better understanding of the present invention and of its advantages will be had from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way. EXAMPLES

[125] Hereinafter, the present invention is described in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention.

[126] Example 1 : Ck- and CH ₃ -variant of normal-fucosylated and/or fucose-reduced bi- specific anti-PD-L1/TA-MUC1 antibodies have comparable amounts of core fucosylated N-glycans.

[127] PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ have high levels of core fucosylated N-glycans, whereas PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ contain low percentages of core fucosylated N-glycans (Fig. 1 ).

[128] /V-glycosylation of PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ was analyzed by HILIC-UPLC-HiResQToF MSMS (hydrophilic interaction ultra-performance chromatography coupled to high resolution quadrupole time-of-flight tandem mass spectrometry). Briefly, the antibody was denatured by RapiGest SF® (Waters Inc.) and tris-(2-carboxyethyl)phosphine (120 min, 95 °C). N-Glycans were released by Rapid PNGase F® (10 min, 55 °C; Waters Inc.), followed by fluorescence tagging with RapiFluor MS® reagent in dimethylformamide for 5min at room temperature. For clean-up of tagged glycans a μΕΙυίίοη Plate (HILIC SPE) was used. Labeled N-glycans were separated on a HILIC phase (UPLC BEH GLYCAN 1 .7 150 mm, Waters Inc.) employing an ultra-performance chromatography device (l-Class, Waters Inc.) including a fluorescence detector. RapiGest SF® tagged N-glycans were detected at 265 nm excitation wavelength and 425 nm emission wavelength. Fluorescence signals were employed for glycan quantification. In series to the fluorescence detector a high resolution mass spectrometer was coupled (Impact HD, Bruker Daltonik GmbH). Precursor in combination with a series of fragment masses allowed for unambiguous identification of glycan structures.

[129] Example 2: Ck- and CH ₃ -variants of PM-PDL-GEX show comparable PD-L1 binding and blocking capacity.

[130] First, PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared in the PD-L1 antigen ELISA (Fig. 2A). All tested antibodies showed a comparable concentration-dependent antigen binding.

[131] Second, PM-PDL-GEX H9D8/Ck was compared to PM-PDL-GEX H9D8/CH ₃ in the PD- L1/PD-1 blocking ELISA. Concentration-dependent blocking of PD-1 binding was detected, but no difference was seen between the Ck- and CH ₃ -variant (Fig. 2B).

[132] In addition, PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared also showing no obvious difference to each other (Fig. 2C). [133] These data clearly demonstrate that PD-L1 binding and PD-L1/PD-1 blocking is unaffected by the location of the anti-PD-L1 scF _v , whether it is fused to the constant domain of the light chain or the CH ₃ domain of the heavy chain.

[134] Different ELISAs were developed to analyze PD-L1 binding and PD-L1/PD-1 blocking capacity of antibodies capable of binding to PD-L1 . In the PD-L1 antigen ELISA, the commercially available antigen human PD-L1 (Sino) is immobilized on Maxisorp 96 well plates. Coated wells are blocked with 2% BSA in PBS to prevent unspecific binding of antibodies. Serial dilutions of test antibodies in 1 % BSA PBS are incubated to the immobilized antigen for binding and detected by an enzyme-labelled secondary goat-anti human IgG F _c antibody (Jackson Immuno Research). The enzyme POD converts the substrate TMB into a dye, which is quantified photometrically after acidification with diluted sulphuric acid at 450 nm.

The PD-L1/PD-1 blocking ELISA is considered as the most relevant ELISA for antibodies capable of binding to PD-L1 by depicting the blocking situation between PD-1 and PD-L1. F _c - tagged human PD-L1 (tebu-bio/BPS bioscience) was coated on Maxisorp 96 well plates. After washing and blocking, a fixed concentration of biotinylated human PD-1 (tebu-bio/BPS bioscience) in presence of serial dilutions of test antibodies were added thereby competing for the binding to PD-1. After washing, binding of PD-1 was detected by Streptavidin-POD and TMB. As result, the higher the inhibition of the interaction between PD-1 and PD-L1 by antibodies capable of binding to PD-L1 , the lower is the resulting OD at 450 nm.

[135] Example 3: Ck- and CH ₃ -variants of PM-PDL-GEX show comparable binding to FcYRIIIa.

[136] The relative potency of fucose-reduced PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc- /CH ₃ was higher compared to normal-fucosylated variants PM-PDL-GEX H9D8/Ck and PM- PDL-GEX H9D8/CH ₃ (Fig. 3). This was expected since it is known that reduced fucosylation of hlgG1 leads to better FcYRIIIa-binding. No obvious difference between the Ck- and the CH ₃ - varaints of the fucose-reduced and the normal-fucosylated, respectively, was detected.

[137] In order to characterize binding of the antibody F _c part to FcYRIIIa on a molecular level, a new assay using a bead-based technology of Perkin Elmer (AlphaScreen®) was developed. The extracellular domain of recombinant human FcYRIIIa (produced recombinantly by Glycotope in the GEX-H9D8 cell line) was used in this assay. His-tagged FcYRIIIa was captured by Ni-chelate donor beads. The test antibodies and rabbit-anti-mouse coupled acceptor beads compete for binding to FcYRIIIa. In case of interaction of FcYRIIIa with rabbit-anti-mouse acceptor beads only, donor and acceptor beads come into close proximity, which leads upon laser excitation to light emission by chemiluminescence. A maximum signal is achieved. In case of competition of the test antibody binding to FcyRllla with the acceptor beads the maximum signal is reduced in a concentration dependent manner. The chemiluminescence was quantified by measurement at 520-620 nm. As a result, a concentration dependent sigmoidal dose- response curve was received, which is defined by top-plateau, bottom-plateau, slope and EC50. The EC50 equals the effective antibody concentration needed for 50% of maximum binding to FcyRllla.

[138] Example 4: Ck- and CH ₃ -variants of PM-PDL-GEX show comparable cell binding.

[139] Whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or whether it is coupled to the CH ₃ domain of the heavy chain has no influence on binding to cell lines expressing TA-MUC1 and/or PD-L1 .

[140] The binding properties of the different variants of PM-PDL-GEX were analyzed using the breast cancer cell line ZR-75-1 , which strongly expressed TA-MUC1 and the prostate carcinoma cell line DU-145, whose PD-L1 expression was higher compared to TA-MUC1 (Fig. 4A). The cell lines were characterized for their expression of TA-MUC1 and PD-L1 using 100μg/ml TA- MUC1 -specific antibody and 100μg/ml mono-specific anti-PD-L1 antibody PDL-GEX Fuc-. Binding was detected using a fluochrome-labeled secondary antibody and flow cytometric analysis.

[141] Further, to test binding of the different PM-PDL-GEX variants, target cells were incubated with indicated antibodies in serial dilutions. Again, binding was detected using a fluochrome-labeled secondary antibody. Afterwards, cells were analyzed via flow cytometry. Comparable binding of the tested antibodies (PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ ) is most likely due to specificity towards TA-MUC1 since ZR-75-1 shows only weak expression of PD-L1 compared to TA-MUC1 (Fig. 4B).

[142] Comparable binding to Du-145 was also demonstrated by using PM-PDL-GEX Fuc-/Ck, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ and in particular by using the Ck- and CH ₃ -variant of PM-PDL-GEX Fuc- being directly compared to each other (Fig. 4C and D).

[143] Therefore it can be concluded that whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or to the CH ₃ domain of the heavy chain has no influence on binding to cell lines expressing TA-MUC1 and/or PD-L1 .

[144] Example 5: CH ₃ -variants of PM-PDL-GEX show lower ADCC activity against certain target cells lines compared to Ck-variants.

[145] Whether the anti-PD-L1 scF _v is fused to the constant domain of the light chain or whether it is coupled to the CH ₃ domain of the heavy chain has no influence on antigen binding to PD-L1 (see Example 2) and TA-MUC1 (see Example 4), the blocking of PD-L1/PD-1 interaction (see Example 2), or the binding to FcyRllla (see Example 3), but the ADCC against particular cancer cell lines is affected. The hypothesis is that the activation of FcyRllla (or other FcyRs) is surprisingly impaired when the anti-PDL1 scF _v is fused to the CH ₃ domain of the heavy chain, despite the fact, that binding to FcYRIIIa has been shown to be unaffected.

[146] The capacity to mediate ADCC against tumor cells was analyzed using a europium release assay. Briefly, target cells were loaded with europium (Eu2 ⁺ ) by electroporation and incubated with an FcyRllla-transfected NK cell line in the presence of test antibodies for 5 hours. Four different cancer cell lines were used for the ADCC assays: I) the breast cancer cell line ZR-75-1 , II) the breast cancer cell line T47-D, III) the prostate carcinoma cell line DU-145 and IV) the head and neck squamous cell carcinoma cell line HSC-4. The E:T-ratio for ZR-75-1 , T-47D and Du-145 as target cells was 30:1 , for HSC-4 as target cells 10:1 . Europium release to the supernatant (indicating antibody mediated cell death) was quantified using a fluorescence plate reader. Maximal release was achieved by incubation of target cells with triton-X-100 and spontaneous release was measured in samples containing only target cells but no antibody and no effector cells. Specific cytotoxicity was calculated as:

% specific lysis = (experimental release - spontaneous release) / (maximal release - spontaneous release) x100.

[147] The cell lines T-47D and HSC-4 cell lines were characterized for their expression of TA- MUCI and PD-L1 as described in Example 4 for ZR-75-1 and Du-145. Similar to ZR-75-1 (see Example 4), T47-D showed high expression of TA-MUC1 and in comparison the PD-L1 expression was lower. In comparison to T-47D, the TA-MUC1 expression of HSC-4 was on a very low level. The TA-MUC1 expression on HSC-4 was slightly higher compared to PD-L1 , but it is to mention that the difference between TA-MUC1 and PD-L1 expression was not as drastic as observed for ZR-75-1 and T47-D (Fig. 5A).

[148] Next, the capacity to mediate ADCC of the different PM-PDL-GEX variants was analyzed against ZR-75-1 . PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ showed strongly enhanced ADCC activity compared to PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ . This was expected since PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ are fucose- reduced (see Example 1 ) and therefore have higher affinity to FcYRIIIa (see Example 3). No obvious difference between the Ck- and CH ₃ -variants was seen. The ADCC mediated by PM- PDL-GEX Fuc-/Ck, PM-PDL-GEX Fuc-/CH ₃ , PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ against ZR-75-1 was most likely primarily due to TA-MUC1 specificity since a fucose-reduced mono-specific anti-PD-L1 hlgG1 (PDL-GEX Fuc-) induced no ADCC effect (Fig. 5B).

[149] Then, PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ were tested in an ADCC against T-47D. Both antibodies mediated comparable ADCC activity, hence no difference between Ck- and CH ₃ -variants were seen. As mentioned for ZR-75-1 , the ADCC due to PM- PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ was probably mediated via the TA-MUC1 specificity because no ADCC effect was detected using a fucose-reduced mono-specific anti- PD-L1 hlgG1 (PDL-GEX Fuc-) (Fig. 5C).

[150] Further, PM-PDL-GEX H9D8/Ck, PM-PDL-GEX H9D8/CH ₃ , PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were tested in an ADCC assay against Du-145. As expected, the fucose-reduced PM-PDL-GEX variants showed strongly enhanced ADCC compared to the normal-fucosylated variants. However, using Du-145 as a target cell line resulted in different ADCC activity of the Ck- and CH ₃ -varaints. In presence of PM-PDL-GEX Fuc-/Ck the ADCC effect was increased compared to PM-PDL-GEX Fuc-/CH ₃ . In line with that, PM-PDL-GEX H9D8/Ck showed ADCC activity, whereas no ADCC activity was observed using PM-PDL-GEX H9D8/CH ₃ . In contrast to ZR-75-1 and T-47D, the ADCC of PM-PDL-GEX variants against Du- 145 was primarily mediated due to the PD-L1 specificity since an anti-TA-MUC1 hlgG1 only mediated minimal ADCC activity (Fig. 5D).

[151] At last, the different PM-PDL-GEX variants were tested against HSC-4 for their capacity to mediate ADCC. Similar to the results with Du-145 as target cells, drastically differences between the Ck- and CH ₃ -variants were seen. PM-PDL-GEX Fuc-/Ck mediated increased ADCC compared to PM-PDL-GEX Fuc-/CH ₃ and PM-PDL-GEX H9D8/Ck mediated ADCC was stronger compared to PM-PDL-GEX H9D8/CH ₃ . A mono-specific anti-TA-MUC1 hlgG1 showed only minimal ADCC activity, therefore it can be concluded that ADCC mediated by the PM-PDL- GEX antibody is mainly due to PD-L1 specificity (Fig. 5E).

[152] Example 6: CH ₃ -variants of PM-PDL-GEX show less T cell activation compared to Ck-variants.

[153] The variants of PM-PDL-GEX with the anti-PD-L1 scF _v coupled to the constant domain of the light chain have higher activity in relevant mode of actions such as ADCC and T cell activation compared to the counterparts with the scF _v coupled to the CH ₃ domain of the heavy chain.

[154] This was analyzed with a mixed lymphocyte reaction (MLR). The MLR is a functional assay which was established to analyze the effect of PD-L1 blocking antibodies on the suppression of PD-1 expressing T cells by PD-L1 expressing antigen presenting cells. The assay measures the response of T cells from one donor as responders to monocyte-derived dendritic cells (moDCs) from another donor as stimulators (= allogenic MLR). Briefly, monocytes were isolated from buffy coat via negative selection using magnetic-activated cell sorting and then differentiated to moDCs with IL-4 and GM-CSF for 7 days. After differentiation, moDCs were cultivated with isolated T cells with a stimulator/responder-ratio of 1 :10. After 5 days, T cell activation was measured using appropriate fluochrome-labeled antibodies and flow cytometric analysis. [155] In a first allogeneic MLR, I pg/ml PM-PDL-GEX H9D8/Ck and PM-PDL-GEX H9D8/CH ₃ were compared to each other. Both antibodies showed activation of CD8 T cells (CD3 ⁺ CD8 ⁺ cells) measured via the activation marker CD25 (Fig. 6A) and the co-stimulatory molecule CD137 (Fig. 6B) compared to the medium control. However, the Ck-variant showed increased activation compared to the CH ₃ -variant.

[156] In a second allogeneic MLR, serial dilutions of PM-PDL-GEX Fuc-/Ck and PM-PDL-GEX Fuc-/CH ₃ were compared. PM-PDL-GEX Fuc-/Ck showed a strong concentration-dependent activation of T cells measured by CD25 (Fig. 6C) and CD137 (Fig. 6D) expression, whereas incubation with PM-PDL-GEX Fuc-/CH ₃ resulted only in a slightly increased activation compared to the medium control.

[157] Example 7: CH ₃ -varaints of a bispecific anti-TA-MUC1/CD3 antibody and of a bispecific anti-HER2/CD3 antibody show lower NK cell-mediated ADCC activity compared to the respective Ck-variants

[158] A bispecific anti-TA-MUC1/CD3 (PM-CD3-GEX) antibody and a bispecific anti- HER2/CD3 (TM-CD3-GEX) antibody were tested for their capacity to mediate ADCC in an europium release assay as described in Example 5. CD3-expressing Jurkat cells were used as target cells and a FcyRllla-transfected NK cell line as effector cell in a effector to target ratio of 10:1 . Cells were incubated for 4h. Both antibodies were added in two different variants: I) the anti-CD3 scFvs coupled to the constant domain of the light chain (PM-CD3-GEX Ck and TM- CD3-GEX Ck) and II) the anti-CD3 scF _v s coupled to the CH ₃ -domain of the heavy chain (PM- CD3-GEX CH ₃ and TM-CD3-GEX CH ₃ ).

[159] The Ck-variants of both, the PM-CD3-GEX (Fig. 7A) and the TM-CD3-GEX showed increased ADCC-activity compared to the respective CH ₃ -variants (Fig. 7B).

[160] Example 8: PM-PDL-GEX CDR mutants show comparable binding and blocking capacity compared to the non-mutated counterpart.

[161] Different CDR mutants of PM-PDL-GEX Fuc- /Ck were generated:

PM-PDL-GEX Fuc- CDRmut a (SEQ ID No. 75)

PM-PDL-GEX Fuc- CDRmut b (SEQ ID NO. 77 + SEQ ID NO. 83),

and tested in various assays:

I) For their PD-L1 binding capacity using PD-L1 antigen ELISA. Therefore, human PD-L1 was coated on Maxisorp 96 well plates. After washing and blocking, serial dilutions of test antibodies were added. After washing, binding of test antibody was determined using POD- coupled secondary antibody and TMB (Fig. 8A).

II) For their blocking capacity in an PD-L1/PD-1 blocking ELISA as descripted in Example 2 (Fig. 8B). III) For their TA-MUC1 binding capacity using TA-MUC1 expressing T-47D and flow cytometric analysis (Fig. 8C).

Mutation of the CDR part had no obvious effect on PM-PDL-GEX/Ck binding to PD-L1 , blocking of PD-L1/PD1 interaction and TA-MUC1 binding.

[155] Example 9: PM-PDL-GEX CDR mutants show comparable enhanced activation of CD8 T cells to the non-mutated counterparts

[156] Different CDR mutants of PM-PDL-GEX H9D8/Ck and PM-PDL-GEX Fuc- /Ck were generated:

PM-PDL-GEX H9D8 CDRmut a (SEQ ID No. 75)

PM-PDL-GEX H9D8 CDRmut b (SEQ ID NO. 77 + SEQ ID NO. 83)

PM-PDL-GEX Fuc- CDRmut a (SEQ ID No. 75)

PM-PDL-GEX Fuc- CDRmut b (SEQ ID NO. 77 + SEQ ID NO. 83),

and tested for their capacity to activate T cells in an allogeneic MLR as described in Example 6. The CDR mutated PM-PDL-GEX Fuc-/Ck variants activated CD8 T cells (CD25+ cells of CD8 T cells) comparable to non-mutated PM-PDL-GEX Fuc-/Ck. The CDR mutated PM-PDL-GEX H9D8/Ck variants activated CD8 T cells comparable to non-mutated PM-PDL-GEX H9D8/Ck (Fig. 9).