The present invention relates to a therapy which comprises a combination of neutrophil-like cell depletion and administration of a T cell engager.

BACKGROUND OF THE INVENTION

T cell immunotherapy is a promising approach for the treatment of disorders including cancer and autoimmune disorders. The present invention relates to combination therapy that provides improvements in T cell immunotherapy.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that the depletion or inactivation of neutrophil-like cells enhances the therapeutic effect of a T cell engager, thereby increasing the efficacy of a T-cell in killing a target cell.

In one aspect of the invention, there is provided a method for increasing the activity of a T cell engager in a subject, the method comprising depleting or inactivating neutrophil-like cells from the subject prior to, during and/or following administration of the T cell engager, wherein the T cell engager activates a T-cell to target a cell, thereby leading to the death of the cell.

In a further aspect of the invention, there is provided a method for treating cancer in a subject, the method comprising depleting or inactivating neutrophil-like cells from the subject prior to, during and/or following administration of a T cell engager, wherein the T cell engager activates the T -cell to target a cancer cell, thereby leading to the death of the cancer cell, and wherein the T cell engager binds to a cancer antigen expressed on the cell.

In a further aspect of the invention, there is provided a method for treating a disorder associated with BCMA expression in a subject, the method comprising depleting or inactivating neutrophil-like cells from the subject prior to, during and/or following administration of a T cell engager, wherein the T cell engager binds to BCMA and activates the T -cell to target a cell expressing BCMA, thereby leading to the death of the cell.

In some embodiments, the neutrophil-like cells are depleted or inactivated with a neutrophil-like cell depleting agent. Aspects and embodiments of the invention are set out in the appended claims. These and other aspects and embodiments of the invention are also described herein.

BRIEF DESCRIPTION OF FIGURES

The present invention will now be described in more detail with reference to the attached Figures, in which:

Figure 1 illustrates different formats of bispecific bivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (CD3 is illustrated) and a cancer antigen (BCMA is illustrated) in the format Fab BCMA- Fc - Fab CD3. The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions“RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab are linked to each other with flexible linkers.

Figure 2 illustrates different formats of bispecific trivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (the T cell antigen CD3 is illustrated) and a cancer antigen (BCMA is illustrated) in the following formats: Fab BCMA - Fc - Fab CD3 - Fab BCMA (A,B); Fab BCMA - Fc - Fab BCMA - Fab CD3 (C,D). The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions“RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab are linked to each other with flexible linkers.

Figure 3 illustrates further formats of bispecific bivalent antibodies for use in the present invention, which comprise Fab fragments binding to a T cell antigen (the T cell antigen CD3 is illustrated) and a cancer antigen (BCMA is illustrated) in the following formats: Fc - Fab CD3 - Fab BCMA (A,B); Fc - Fab BCMA - Fab CD3 (C,D). The CD3 Fab may include a VH-VL crossover to reduce light chain mispairing and side-products. Amino acid substitutions“RK/EE” may be introduced in CL-CH1 to reduce light chain mispairing/side products in production. The CD3 Fab and BCMA Fab are linked to each other with flexible linkers

Figure 4A illustrates that T lymphocyte proliferation decreases when the most mature neutrophil subpopulation is in contact with lymphocytes (0.5-fold; p=.03). Figure 4B illustrates that multiple myeloma cell death significantly increases on depletion of the tumor-infiltrating bone marrow neutrophil subsets CD1 lb+CDl31o/-CDl6- or CD1 lb+CDl3+CDl6+ (3-fold and 4-fold respectively; /?< 04) DETAILED DESCRIPTION

As used herein, the articles "a" and“an” may refer to one or to more than one ( e.g . to at least one) of the grammatical object of the article.

“About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values.

Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of’ and/or consisting essentially of such features.

The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

Depleting or inactivating neutrophil-like cells

The present invention involves the blocking, inhibition, depletion and/or inactivation of neutrophil-like cells in the subject. As used herein, depletion means that the number of neutrophil-like cells in a subject is reduced as compared to a subject who has not been treated.

In some embodiments of the invention, the neutrophil-like cells are depleted or inactivated prior to, during and/or following administration of the T cell engager. In preferred embodiments, the neutrophil- like cells are depleted or inactivated prior to administration of the T cell engager.

In some embodiments, the neutrophil-like cells are neutrophils. In other, more preferred embodiments, the neutrophil-like cells are myeloid-derived suppressor cells (MDSCs), e.g. granulocytic myeloid- derived suppressor cells. MDSCs are a heterogeneous population of cells which inhibit innate and adaptive immunity. In some embodiments, a subset of neutrophil-like cells ( e.g . MDSCs) is blocked, inhibited, depleted or inactivated. The subset may be selected from the group consisting of mature neutrophil-like cells (e.g. mature MDSCs), mature and intermediate neutrophil-like cells (e.g. mature and intermediate MDSCs), and intermediate neutrophil-like cells (e.g. intermediate MDSCs). In preferred embodiments, the subset consists essentially of or consists of mature neutrophil-like cells (e.g. mature MDSCs).

As used herein, the term“mature neutrophil-like cells” refers to cells (e.g. MDSCs) displaying the markers CD1 lb+ CD13+ CD16+. The cells may also display one or more of the markers selected from CD 14-, CD15+, CD33+ and HLADR-.

As used herein, the term“intermediate neutrophil-like cells” refers to cells (e.g. MDSCs) displaying the markers CD1 lb+ CDl31o/- CD16-. The cells may also display one or more of the markers selected from CD 14-, CD15+, CD33+ and HLADR-.

In some embodiments, the subset of neutrophil-like cells (e.g. MDSCs) that is blocked, inhibited, depleted or inactivated has one or more of the markers selected from the group consisting of: CDl lb+; CD 131o, CD13- or CD13+; and CD16+ or CD16-. In preferred embodiments, the subset of cells has the markers CD1 lb+, CDl31o and CD16- or CD1 lb+, CD13- and CD16-. In other preferred embodiments, the subset of cells has the markers CD1 lb+, CD13+ and CD16+.

The term“neutrophil-like cell depleting agent” is used herein to refer to an agent (e.g. a therapeutic agent) that blocks, inhibits, depletes or inactivates neutrophil-like cells. In some embodiments, the agent (e.g. therapeutic agent) blocks, inhibits, depletes or inactivates neutrophils. In other, more preferred embodiments, the agent (e.g. therapeutic agent) blocks, inhibits, depletes or inactivates myeloid-derived suppressor cells (MDSCs) e.g. granulocytic myeloid-derived suppressor cells.

The neutrophil-like cell depleting agent may be a small molecule, antibody or nucleic acid inhibitor.

Methods to deplete or inactivate MDSCs are known in the art (e.g. Wang et al., Oncoimmunology. 2017; 6(7): el33l 807 and Albeituni al. Cancer J. 2014; 19(6): 490-501, both of which are incorporated by reference). By way of example, neutrophil-like cell depleting agents (e.g. MDSC depleting agents) include agents that block the suppressive effects of MDSC, such as tyrosine kinase inhibitors (e.g., sunitinib), MDSC differentiating agents (e.g., all-trans retinoic acid), reactive nitrogen inhibitors (e.g., aminoguanidine or similar drugs), arginase enzyme inhibitors, indoleamine deoxygenase enzyme inhibitors, reactive oxygen species inhibitors, TGF-b inhibitors, IL-10 inhibitors, VEGF inhibitors, and PGE2 synthesis inhibitors. In some embodiments, the neutrophil-like cell depleting agents ( e.g . MDSC depleting agents) may comprise a bisphosphonate drug, such as clodronate, zoledronate, pamidronate, etidronate, or any other type of drug that is capable of depleting or inhibiting neutrophil-like cells. In some embodiments, the bisphosphonate drug is a liposomal conjugated agent, such as liposomal clodronate.

Additional examples of neutrophil-like cell depleting agents (e.g. MDSC depleting agents) of the invention include liposome-encapsulated bisphosphonate drugs, liposomes encapsulating other apoptosis inducing agents, or liposomes encapsulating siRNA or other RNA targeting molecules that induce MDSC apoptosis. By way of further example, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) may comprise virtually any agent demonstrated to deplete and/or inhibit the migration, accumulation or activity of MDSC, thus providing for an inhibition of the immunosuppressive activity of the MDSCs.

In some embodiments, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) may comprise antibodies targeted to neutrophil-like cells (e.g. MDSCs), including antibody-drug conjugates. The antibodies may target a subset of neutrophil-like cells (e.g. MDSCs), including the mature neutrophil- like cells (e.g. MDSCs), mature and intermediate neutrophil-like cells (e.g. MDSCs), and intermediate neutrophil-like cells (e.g. MDSCs). In preferred embodiments, the antibodies may target a subset consisting essentially of or consisting of mature neutrophil-like cells (e.g. MDSCs).

Additional neutrophil-like cell depleting agents (e.g. MDSC depleting agents) include agents that block neutrophil-like cell release from bone marrow (CCL2 or CCR2 inhibitors, competitors or agonists, M- CSF inhibitors, GM-CSF inhibitors).

In other embodiments, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) consist of agents that inhibit the recruitment and/or migration of MDSC in the periphery to tumor sites, particularly to solid tumors and hematological tumors outside of the bone marrow. These drugs would consist most specifically of small molecule inhibitors of the receptor for CCL2 (e.g. MCP-l), which is known as CCR2. These CCR2 receptor inhibitors block the egress of neutrophil-like cells from the bone marrow into the bloodstream. Specific inhibitors in this family include RS 102895 (Sigma- Aldrich) and other similar molecules.

Other similar agents would include chemokine/cytokine inhibitors, such as inhibitors of CXCR2, CXCR4, CSFR, G-CSF, M-CSF, GM-CSF, tumor necrosis factor, IL-lp, IL-3, IL-6, IL-8, SCF, VEGF, and prostaglandins, or receptors for these cytokines and chemokines. Other candidates for inhibition would include the S100 family of proteins, including especially S100A8/A9. These agents may be small molecules or antibodies.

In other embodiments, the neutrophil-like cell depleting agents ( e.g . MDSC depleting agents) consist of agents that inhibit the recruitment and/or migration of MDSC or both MDSC and regulatory T cells to tumor sites. These agents could consist of chemokine/chemokine receptor blockades targeting CCR2, CCR4, and CCR5 pathways. Other examples include CSFR inhibitors, CCL2 inhibitors, CXCR2 antagonist, CXCR4 antagonist, G-CSF inhibitors, CSF1R inhibitors and B-Raf inhibitors (e.g. vemurafenib).

In other embodiments, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) comprise agents that inhibit neutrophil-like cells (e.g. MDSC) suppressive function. These agents could consist of amiloride, an inhibitor of reactive nitrogen species (e.g. AT38), COX-2 and PGE2 inhibitors (e.g. indomethacin, celecoxib, meloxicam and acetylsalicylic acid (ASA)), nitroaspirin (NCX4060, NCX4016), PDE-5 inhibitor (e.g. sildenafil), triterpenoids (e.g. CDDO-Me) and VSSP (very small size proteoliposomes).

In other embodiments, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) comprise agents that inhibit differentiation of MDSC. These agents could consist of ATRA, CpG, curcumin, vitamin D3, sunitinib, taxanes (e.g. docetaxel and paclitaxel) and whole-glucan particles (WGP).

In other embodiments, the neutrophil-like cell depleting agents (e.g. MDSC depleting agents) comprise agents that inhibit expansion of MDSC. These agents could consist of sunitinib, amino-bisphophonate (e.g. clodronate, zoledronate, pamidronate, etidronate), COX-2 or PGE2 inhibitors (e.g. indomethacin, celecoxib , meloxicam and acetylsalicylic acid (ASA)), gemcitabine, prokineticin 2-specific antibody, SCF inhibitors (e.g. siRNA or anti-ckit antibody), chemotherapeutic agents (e.g. doxorubicin and cyclophosphamide) and 5-FU.

In some embodiments, the neutrophil-like cells are depleted or inactivated ex vivo. In some embodiments, the neutrophil-like cells are depleted using flow cytometry. In some embodiments, the neutrophil-like cells are depleted or inactivated using a combination of flow cytometry and a neutrophil-like cell depleting agent. T cell engager

The term“T cell engager” is used herein to refer to an agent that mediates activation of a T cell so as activate the T-cell to target a cell, thereby leading to the death of the cell. Accordingly, a T-cell engager can harness a T cell to eradicate target cells, such as cancer cells or BCMA expressing cells.

In some embodiments, the T cell engager comprises an antibody molecule, a receptor molecule (e.g., a receptor, a receptor fragment or functional variant thereof) including a chimeric antigen receptor, or a ligand molecule (e.g., a ligand, a ligand fragment or functional variant thereof), or a combination thereof. In preferred embodiments, the T cell engager is an antibody or a chimeric antigen receptor. In particularly preferred embodiments, the T cell engager is an antibody, e.g. a bispecific antibody.

In some aspects of the invention, the T cell engager binds to a cell expressing BCMA. Thus, the T-cell engager binds to the cell expressing BCMA and causes the T-cell to kill the cell expressing BCMA.

In other aspects of the invention, the T cell engager binds to a cancer antigen on a cell. The cancer antigen may be present on a hematological cancer, a solid tumor, a metastatic cancer, soft tissue tumor, metastatic lesion, or a combination thereof. Thus, the T-cell engager binds to the cancer antigen and causes the T- cell to kill the cancer cell.

The terms“cancer antigen”,“cancer associated antigen”,“tumour associated antigen”, and“tumour antigen” are used interchangeably herein and refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC/peptide). In some embodiments, a cancer antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a cancer antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, l -fold over expression, 2-fold overexpression, 3 -fold overexpression or more in comparison to a normal cell. In some embodiments, a cancer antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a cancer antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell. In some embodiments, exemplary cancer antigens include: CD19; CD123; CD22; CD30; CD171 ; CS-l ; C-type lectin-like molecule-l, CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (Gal Aca-Ser/Thr)); prostate- specific membrane antigen (PSMA); Receptor tyrosine kinase- like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2; Mesothelin; Interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); Protease Serine 21 ; vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage- specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAEX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl- GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A vims cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la); Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen- 1, melanoma antigen recognized by T cells 1 ; Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML- IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P4501B1 (CYP1B1); CCCTC- Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1); TNF receptor family member; Fms-Like Tyrosine Kinase 3 (FL T3); CD10; CD19; CD20; CD21; CD22; CD23; CD24; CD25; CD37; CD38; CD53; CD72; CD73; CD74; CD75; CD77; CD79a; CD79b; CD80; CD81; CD82; CD83; CD84; CD85; ROR1; BCMA; CD86; CD179b; CDla; CDlb; CDlc; CDld; CD2; CD5; CD6; CD9; CDl la; CDl lb; CDl lc; CD17; CD18; CD26; CD27; CD29; CD30; CD31; CD32a; CD32b; CD35; CD38; CD39; CD40; CD44; CD45; CD45RA; CD45RB; CD45RC; CD45RO; CD46; CD47; CD48; CD49b; CD49c; CD49d; CD50; CD52; CD54; CD55; CD58; CD60a; CD62L; CD63; CD63; CD68 CD69; CD70; CD85E; CD85I; CD85J; CD92; CD95; CD97; CD98; CD99; CD100; CD102; CD108; CD119; CD120a; CD120b; CD121b; CD122; CD124; CD125; CD126; CD130; CD132; CD137; CD138; CD139; CD147; CD148; CD150; CD152; CD162; CD164; CD166; CD167a; CD170; CD175; CD175s; CD180; CD184; CD185; CD192; CD196; CD197; CD200; CD205; CD210a; CDw210b; CD212; CD213al; CD213a2; CD215; CD217; CD218a; CD218b; CD220; CD221; CD224; CD225; CD226; CD227; CD229; CD230; CD232; CD252; CD253; CD257; CD258; CD261 ; CD262; CD263; CD264; CD267; CD268; CD269; CD270; CD272; CD274; CD275; CD277; CD279; CD283; CD289; CD290; CD295; CD298; CD300a; CD300c; CD305; CD306; CD307a; CD307b; CD307c; CD307d; CD307e; CD314; CD315; CD316; CD317; CD319; CD321 ; CD327; CD328; CD329; CD338; CD351 ; CD352; CD353; CD354; CD355; CD357; CD358; CD360; CD361; CD362; CD363; and a peptide of any of these antigens presented on MHC.

Preferred cancer antigens include ROR1 and BCMA. In particularly preferred embodiments, the cancer antigen is BCMA.

The term“ROR1” as used herein relates to tyrosine-protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase, receptor-related 1 (NTRKR1) (UniProtKB Q01973), which is a tyrosine-protein kinase receptor that belongs to the receptor tyrosine kinase-like orphan receptor (ROR) family of cell surface receptors. The receptor is described in Masiakowski P., Carroll R.D., J. Biol. Chem. 267:26181 -26190(1992)“A novel family of cell surface receptors with tyrosine kinase-like domain”. The extracellular domain of ROR1 consists according to UniProt of amino acids 30-406.

The term“BCMA” as used herein relates to human B cell maturation antigen (BCMA), also known as TR17 HUMAN or TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of BCMA consists according to UniProt of amino acids 1 - 54 (or 5-51).

In preferred embodiments, the T cell engager binds to BCMA and comprises an anti-BCMA antibody. Exemplary anti-BCMA antibodies that can be used in the present invention are set forth in Tables 1A and IB and in WO 2017/021450, which is hereby incorporated by reference in its entirety.

In preferred embodiments, the T cell engager binds to BCMA and comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of : a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

g) CDR1H region of SEQ ID NO:15 and CDR2H region of SEQ ID NO:16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19.

In some embodiments, the T cell engager binds to BCMA and comprises a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12,

b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13, c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14,

d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12,

e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12,

f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO:12, or

g) a VH region of SEQ ID NO:9 and a VL region of SEQ ID NO: 11.

In particularly preferred embodiments, the T cell engager binds to BCMA and comprises a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from the group of: i) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28;

ii) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24; or

iii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26.

In some embodiments, the T cell engager binds to BCMA and comprises a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13 or a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

Antibody T cell engager

In some embodiments, the T cell engager comprises an antibody (e.g., an antibody that includes at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains), or an antigen-binding fragment. The antibody may bind to (a) an antigen that promotes activation of one or more T cells and (b) a target antigen (e.g. a cancer antigen). In some embodiments, the antigen that promotes activation of one or more T cells is selected from the group consisting of CD3, TCRa, TCRp, TCRy, TCR , ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226. In preferred embodiments, the antigen that promotes activation or targeting of one or more T cells is CD3.

The T cell engager may comprise a multispecific antibody, a bispecific antibody, a single chain variable fragment (scFv) such as a bispecific T cell engager, diabody, or tandem scFv, an antibody mimetic such as DARPin, a naked monospecific antibody, a single domain antibody, a camelid antibody or an antibody drug conjugate.

Generation of multispecific or bispecific antibodies in different formats with or without an Fc portion is known in the state of the art. Bispecific antibody formats are well known in the state of the art and e.g. are also described in Kontermann RE, mAbs 4:2 1-16 (2012); Holliger P., Hudson PJ, Nature Biotech.23 (2005) 1126- 1136 and Chan AC, Carter PJ Nature Reviews Immunology 10, 301 -316 (2010) and Cuesta AM et al„ Trends Biotech 28 (2011) 355-362.

In some embodiments, the T cell engager is a multispecific, e.g. trispecific or bispecific, antibody. Preferably the T cell engager is a bispecific antibody. In some embodiments, the T cell engager is a multivalent, e.g. bivalent or trivalent, antibody. Preferably the T cell engager is a trivalent antibody.

In some embodiments, the T cell engager is a multispecific, e.g. bispecific, antibody that binds to (a) an antigen that promotes activation of one or more T cells and (b) a target antigen (e.g. a cancer antigen). In some embodiments, the antigen that promotes activation of one or more T cells is selected from the group consisting of CD3, TCRa, TCRp, TCRy, TCR , ICOS, CD28, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, SLAM, CD2, or CD226.

In preferred embodiments, the antigen that promotes activation or targeting of one or more T cells is CD3. Thus, the antibody may specifically bind to human CD3, preferably CD35 and/or CD3s. Human CD35 is described under UniProt P04234 (CD3D HUMAN). Human CD3s is described under UniProt P07766 (CD3E_HUMAN).

Antibodies against CD3 are known in the art. Examples of anti-CD3 antibodies include OKT3, TR66, APA 1/1, SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BCG, H2C, HuM291 (visibzumab), Hu291 (PDL), ChAglyCD3 (Otelixizumab), 1iOKT3g 1 (Ala-Ala) (Teplizumab) and NI-0401 (Foralumab).

The first anti-CD3 antibody generated was OKT3 (muromonab-CD3), a murine antibody binding to the CD3s domain. Subsequent anti-CD3 antibodies include humanized or human antibodies, and engineered antibodies, for example antibodies comprising modified Fc regions.

Anti-CD3 antibodies may recognise an epitope on a single polypeptide chain, for example APA 1/1 or SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100), or a conformational epitope located on two or more subunits of CD3, for example WT31, 7D6, UCHT-1 (see W02000041474) and Leu-4. Clinical trials have been carried out using several anti-CD3 antibodies, including BC-3 (Anasetti et al., Transplantation 54: 844 (1992) and H2C (WO2008119567A2). Anti-CD3 antibodies in clinical development include HuM291 (visibzumab) (Norman et al., Transplantation. 2000 Dec 27;70(12):1707- 12.) Hu291 (PDL), ChAglyCD3 (Otelixizumab) (H Waldmann), 1iOKT3g 1 (Ala-Ala) (Teplizumab) (J Bluestone and Johnson and Johnson) and (NI-0401) Foralumab,

Any anti-CD3 antibody or antigen-binding fragment thereof may be suitable for use in the T cell engagers of the present invention. For example, the T cell engagers may comprise an anti-CD3 antibody selected from OKT3, TR66, APA 1/1 , SP34, CH2527, WT31, 7D6, UCHT-1, Leu-4, BC-3, H2C, HuM291 (visilizumab), Hu291 (PDL), ChAglyCD3 (Otelixizumab), hOKT3yl (Ala-Ala) (Teplizumab) and NI- 0401 (Foralumab). In some embodiments, the T cell engagers comprise a humanized SP34 antibody or antigen-binding fragment thereof.

In preferred embodiments, the T cell engager is a multispecific, e.g. bispecific, antibody that comprises an anti-CD3 antibody comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H, CDR2H and CDR3H and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1L, CDR2L and CDR3L. In some embodiments, the multispecific, e.g. bispecific, antibody comprises an anti-CD3 antibody comprising the variable domains of SEQ ID NO:7 (VH) and SEQ ID NO: 8 (VL).

In embodiments in which the bispecific antibodies are bivalent, they may comprise one anti-cancer antigen antibody and one anti-CD3 antibody (referred to herein as the“1+1” format).

In embodiments in which the bispecific antibodies are trivalent, they may comprise two anti-cancer antigen antibodies and one anti-CD3 antibody (referred to herein as the“2+1” format). The two anti cancer antibodies may be the same or different. Preferably, the two anti-cancer antibodies are the same, and target BCMA or ROR-1.

In preferred embodiments, the T cell engager is a bispecific trivalent antibody which comprises two Fab fragments of an anti-cancer antigen (e.g. BCMA) antibody, one Fab fragment of an anti-CD3 antibody, and one Fc portion, wherein the bispecific antibody is in the format cancer antigen Fab - Fc - CD3 Fab - cancer antigen Fab. Especially preferred are bispecific antibodies comprising only the Fab fragments and the Fc part as specified, with or without amino acid substitutions:

Fab BCMA-Fc-Fab CD3 (bispecific format fig. 1A or IB),

Fab BCMA-Fc-Fab CD3-Fab BCMA (bispecific format fig.2A or 2B),

Fab BCMA-Fc-Fab BCMA-Fab CD3 (bispecific format fig.2C or 2D),

Fc-Fab CD3-Fab BCMA (bispecific format fig.3A or 3B),

Fc-Fab BCMA-Fab CD3 (bispecific format fig. 3C or 3D).

As shown in Figures 1 to 3“Fab BCMA-Fc,“Fab BCMA-Fc-Fab CD3” and“Fab BCMA-Fc-Fab CD3” means that the Fab fragment(s) is (are) bound via its (their) C-terminus to the N-terminus of the Fc fragment.“Fab CD3- Fab BCMA” means that the Fab CD3fragment is bound with its N-terminus to the C-terminus of the Fab BCMA fragment.“Fab BCMA - Fab CD3” means that the Fab BCMA fragment is bound with its N-terminus to the C-terminus of the Fab CD3 fragment. In preferred embodiments, the T cell engager is a multispecific, e.g. bispecific, antibody that comprises the anti-BCMA antibody described herein.

In preferred embodiments, the multispecific, e.g. bispecific, antibody comprises an anti-BCMA antibody comprising a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

g) CDR1H region of SEQ ID NO:15 and CDR2H region of SEQ ID NO:16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19.

In some embodiments, the multispecific, e.g. bispecific, antibody comprises an anti-BCMA antibody comprising a VH and a VL selected from the group consisting of: a) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 12,

b) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13,

c) a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14,

d) a VH region of SEQ ID NO:38 and a VL region of SEQ ID NO: 12,

e) a VH region of SEQ ID NO:39 and a VL region of SEQ ID NO: 12,

f) a VH region of SEQ ID NO:40 and a VL region of SEQ ID NO:12, or

g) a VH region of SEQ ID NO:9 and a VL region of SEQ ID NO: 11.

In particularly preferred embodiments, the multispecific, e.g. bispecific, antibody comprises an anti- BCMA antibody comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from the group of: i) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28;

ii) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24; or

iii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26.

In some embodiments, the multispecific, e.g. bispecific, antibody comprises an anti-BCMA antibody comprising a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13 or a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

In embodiments in which the bispecific antibody specifically binds to the extracellular domain of human BCMA and to human CD3, the bispecific antibody may comprise an anti-BCMA antibody comprising a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO:20 and a CDR1H, CDR2H, CDR1L, and CDR2L region combination selected from the group of: a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24,

b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25, and CDR2L region of SEQ ID NO:26,

c) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27, and CDR2L region of SEQ ID NO:28,

d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32,

f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO:31, and CDR2L region of SEQ ID NO:32, and

g) CDR1H region of SEQ ID NO:15 and CDR2H region of SEQ ID NO:16, CDR1L region of SEQ ID NO: 18, and CDR2L region of SEQ ID NO: 19, and an anti-CD3 antibody comprising a CDR1H region of SEQ ID NO:l, a CDR2H region of SEQ ID NO:2, a CDR3H region of SEQ ID NO:3, a CDR1L region of SEQ ID NO:4, a CDR2L region of SEQ ID NO:5 and a CDR3L region of SEQ ID NO:6.

Minor variations in the amino acid sequences of antibodies are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 75%, more preferably at least 80%, at least 90%, at least 95%, and most preferably at least 99% sequence identity to the antibody or antigen-binding fragment thereof as defined anywhere herein.

Antibodies of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. In one embodiment, amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. In particular, conservative amino acid replacements are contemplated.

A“conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in an antibody of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.

“Non-conservative amino acid substitutions” include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, He or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

The term“antibody” as used herein refers to a monoclonal antibody. An antibody consists of two pairs of a“light chain” (LC) and a“heavy chain” (HC) (such light chain (LC) /heavy chain pairs are abbreviated herein as LC/HC). The light chains and heavy chains of such antibodies are polypeptides consisting of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises the heavy chain constant domains CHI , CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The“constant domains” of the heavy chain and of the light chain are not involved directly in binding of an antibody to a target, but exhibit various effector functions.

The term“antibody” as used herein refers also to the portion of an antibody which is needed at least for specific binding to the antigen. Therefore such an antibody (or antibody portion) can be in one embodiment a Fab fragment. The antibody according to the invention can also be a Fab’, F(ab’) ₂ , Fv, a scFv, a di-scFv, or a bi-specific T-cell engager (BiTE).

The term“antibody” includes e.g. mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies) as long as their characteristic properties are retained. Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies. Further embodiments are heterospecific antibodies (bispecific, trispecific etc.) and other conjugates, e.g. with cytotoxic small molecules.

The term“bispecific antibody” as used herein refers in one embodiment to an antibody in which one of the two pairs of heavy chain and light chain (HC/LC) specifically binds to a T cell antigen such as CD3 and the other one specifically binds to an antigen (e.g. a cancer antigen such as BCMA) . The term also refers to other formats of bispecific antibodies according to the state of the art, in one embodiment to bispecific single-chain antibodies.

The term“naked antibody” as used herein refers to an antibody which specifically binds to an antigen (e.g. a cancer antigen such as BCMA), comprising an Fc part and is not conjugated with a therapeutic agent e.g. with a cytotoxic agent or radiolabel. The term“conjugated antibody, drug conjugate” as used herein refers to an antibody which specifically binds to an antigen (e.g. a cancer antigen such as BCMA), and is conjugated with a therapeutic agent, e.g. with a cytotoxic agent or radiolabel.

The term“bispecific single-chain antibody” as used herein refers to a single polypeptide chain comprising in one embodiment two binding domains, one specifically binding to an antigen (e.g. a cancer antigen such as BCMA) and the other one in one embodiment specifically binding to a T cell antigen such as CD3. Each binding domain comprises one variable region from an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to the T cell antigen, and the VH region of the second binding domain specifically binds to the antigen (e.g. cancer antigen). The two binding domains are optionally linked to one another by a short polypeptide spacer. A non-limiting example for a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) and repeats thereof. Each binding domain may additionally comprise one variable region from an antibody light chain ("VL region"), the VH region and VL region within each of the first and second binding domains being linked to one another via a polypeptide linker, long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain to pair with one another such that, together, they are able to specifically bind to the respective first and second binding domains (see e.g. EP0623679).Bispecific single-chain antibodies are also mentioned e.g. in Choi BD et al, Expert Opin Biol Ther. 2011 Jul;l l(7):843-53 and Wolf E. et al., Drug Discov Today. 2005 Sep 15; 10(18): 1237-44.

The term“diabody” as used herein refers to a small bivalent and bispecific antibody fragment comprising a heavy (VH) chain variable domain connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) connected by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen binding sites. To construct bispecific diabodies for use in the invention, the V-domains of an anti-T cell antigen (e.g. CD3) antibody and an anti-cancer antigen (e.g. BCMA) antibody are fused to create the two chains VH(CD3)-VL(BCMA), VH(BCMA)-VL(CD3). Each chain by itself is not able to bind to the respective antigen, but recreates the functional antigen binding sites of anti-CD3 antibody and anti-BCMA antibody on pairing with the other chain. The two scFv molecules, with a linker between heavy chain variable domain and light chain variable domain that is too short for intramolecular dimerization, are co-expressed and self-assemble to form bi-specific molecules with the two binding sites at opposite ends. By way of example, the variable regions encoding the binding domains for BCMA and CD3, respectively, can be amplified by PCR from DNA constructs obtained as described, such that they can be cloned into a vector like pHOG, as described in Kipiriyanov et al., J. Immunol, Methods, 200, 69- 77 (1997a). The two scFv constructs are then combined in one expression vector in the desired orientation, whereby the VH- VL linker is shortened to prevent backfolding of the chains onto themselves. The DNA segments are separated by a STOP codon and a ribosome binding site (RBS). The RBS allows for the transcription of the mRNA as a bi-cistronic message, which is translated by ribosomes into two proteins which non-covalently interact to form the diabody molecule. Diabodies, like other antibody fragments, have the advantage that they can be expressed in bacteria (E. coli) and yeast (Pichia pastoris) in functional form and with high yields (up to Ig/1).

The term“tandem scFvs” as used herein refers to a single chain Fv molecule (i.e. a molecule formed by association of the immunoglobulin heavy and light chain variable domains, VH and VL, respectively) as described e.g., in WO 03/025018 and WO 03/048209. Such Fv molecules, which are known as TandAbs®, comprise four antibody variable domains, wherein (i) either the first two or the last two of the four variable domains bind intramolecularly to one another within the same chain by forming an antigen binding scFv in the orientation VH/VL or VL/VH (ii) the other two domains bind intermolecularly with the corresponding VH or VL domains of another chain to form antigen binding VH/VL pairs. In a preferred embodiment, as mentioned in WO 03/025018, the monomers of such Fv molecule comprise at least four variable domains of which two neighboring domains of one monomer form an antigen-binding VH- VL or VL- VH scFv unit.

The term“DARPins” as used herein refers to a bispecific ankyrin repeat molecule as described e.g. in US 2009082274. These molecules are derived from natural ankyrin proteins, which can be found in the human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for the initial design and another 2200 for subsequent refinement. The modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is composed of 4 to 6 modules. Because each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of binders is done by ribosome display, which is completely cell- free and is described in He M and Taussig ML, Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.

The term“T cell bispecific engager” refers to a fusion protein consisting of two single-chain variable fragments (scFvs) of different antibodies, or amino acid sequences from four different genes, on a single peptide chain of about 55 kilodaltons. In preferred embodiments, one of the scFvs binds to T cells via the CD3 receptor, and the other to a BCMA.

There are five types of mammalian antibody heavy chains denoted by the Greek letters: a, d, e, g, and m (Janeway CA, Jr et al (2001). Immunobiology. 5th ed., Garland Publishing). The type of heavy chain present defines the class of antibody; these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades RA, Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). Distinct heavy chains differ in size and composition; a and g contain approximately 450 amino acids, while m and e have approximately 550 amino acids.

Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotype. Heavy chains g, a and d have a constant region composed of three constant domains CHI, CH2, and CH3 (in a line), and a hinge region for added flexibility (Woof J, Burton D Nat Rev Immunol 4 (2004) 89-99); heavy chains m and e have a constant region composed of four constant domains CHI, CH2, CH3, and CH4 (Janeway CA, Jr et al (2001). Immunobiology. 5th ed., Garland Publishing). The variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single antibody domain.

In mammals there are only two types of light chain, which are called lambda (l) and kappa (K). A light chain has two successive domains: one constant domain CL and one variable domain VL. The approximate length of a light chain is 211 to 217 amino acids. In one embodiment the light chain is a kappa (K) light chain, and the constant domain CL is in one embodiment derived from a kappa (K) light chain (the constant domain CK).

The terms“monoclonal antibody” or“monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of a single amino acid composition.

The“antibodies” according to the invention can be of any class ( e.g . IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl), whereby both antibodies, from which the bivalent bispecific antibody according to the invention is derived, have an Fc part of the same subclass (e.g. IgGl, IgG4 and the like, preferably IgGl), preferably of the same allotype (e.g. Caucasian).

The term“chimeric antibody” refers to an antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of“chimeric antibodies” encompassed by the present invention are those in which the constant region has been modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also referred to as "class-switched antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques are well known in the art. See, e.g., Morrison, S.L., et al., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; US Patent Nos. 5,202,238 and 5,204,244.

The term“humanized antibody” refers to antibodies in which the framework or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In a preferred embodiment, a murine CDR is grafted into the framework region of a human antibody to prepare the“humanized antibody.” See, e.g., Riechmann, L., et al, Nature 332 (1988) 323-327; and Neuberger, M.S., et al, Nature 314 (1985) 268- 270. Other forms of“humanized antibodies” encompassed by the present invention are those in which the constant region has been additionally modified or changed from that of the original antibody to generate the properties according to the invention, especially in regard to Clq binding and/or Fc receptor (FcR) binding.

The term“human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well- known in the state of the art (van Dijk, M.A., and van de Winkel, J.G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551 -2555; Jakobovits, A., et al., Nature 362 ( 1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H.R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J.D., et al, J. Mol. Biol. 222 (1991) 581 -597). The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boemer, P., et al, J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention the term“human antibody” as used herein also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in regard to Clq binding and/or FcR binding, e.g. by“class switching” i.e. change or mutation of Fc parts (e.g. from IgGl to IgG4 and/or IgGl/IgG4 mutation.)

The term“recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell such as an NS0 or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged form. The recombinant human antibodies have been subjected to in vivo somatic hypermutation. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo. The“variable domain” (variable domain of a light chain (VL), variable region of a heavy chain (VH)) as used herein denotes each of the pair of light and heavy chains which is involved directly in binding the antibody. The domains of variable human light and heavy chains have the same general structure and each domain comprises four framework (FR) regions whose sequences are widely conserved, connected by three“hypervariable regions” (or complementarity determining regions, CDRs). The framework regions adopt a b-sheet conformation and the CDRs may form loops connecting the b-sheet structure. The CDRs in each chain are held in their three-dimensional structure by the framework regions and form together with the CDRs from the other chain the binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affmity of the antibodies and therefore provide a further object of the invention.

The terms“hypervariable region” or“target-binding portion of an antibody” when used herein refer to the amino acid residues of an antibody which are responsible for target-binding. The hypervariable region comprises amino acid residues from the “complementarity determining regions” or “CDRs”. “Framework” or“FR” regions are those variable domain regions other than the hypervariable region residues as herein defined. Therefore, the light and heavy chains of an antibody comprise from N- to C- terminus the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. CDRs on each chain are separated by such framework amino acids. Especially, CDR3 of the heavy chain is the region which contributes most to target binding. CDR and FR regions are determined according to the standard definition of Rabat et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The terms“CDR1H, CDR2H and CDR3H” as used herein refer to the respective CDRs of the heavy chain located in the variable domain VH. The terms“CDR1L, CDR2L and CDR3L” as used herein refer to the respective CDRs of the light chain located in the variable domain VL.

For heavy chain constant region amino acid positions discussed in the invention, numbering is according to the EU index first described in Edelman, G.M., et al, Proc. Natl. Acad. Sci. USA 63 (1969) 78-85). The EU numbering of Edelman is also set forth in Rabat et al. (1991) {supra.). Thus, the terms“EU index as set forth in Rabat”,“EU Index”.“EU index of Rabat” or“EU numbering” in the context of the heavy chain refers to the residue numbering system based on the human lgGl EU antibody of Edelman et al. as set forth in Rabat et al. (1991).

The numbering system used for the light chain constant region amino acid sequence is similarly set forth in Rabat et al. (supra.). Thus, as used herein,“numbered according to Rabat” refers to the Rabat set forth in Rabat et al. (supra.). The constant heavy chain domain CHI by which the heavy chain domain CH3 is replaced can be of any Ig class ( e.g . IgA, IgD, IgE, IgG, and IgM), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2). The constant light chain domain CL by which the heavy chain domain CH3 is replaced can be of the lambda (l) or kappa (K) type, preferably the kappa (K) type.

The term“epitope” includes any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, epitope determinant include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments, may have specific three dimensional structural characteristics, and or specific charge characteristics. An epitope is a region of a target that is bound by an antibody.

An“Fc part of an antibody” is a term well known to the skilled artisan and defined on the basis of papain cleavage of antibodies. In one embodiment of the invention the T cell engager contains an Fc part, in one embodiment a Fc part derived from human origin and preferably all other parts of the human constant regions. The Fc part of an antibody is directly involved in complement activation, Clq binding, C3 activation and Fc receptor binding. While the influence of an antibody on the complement system is dependent on certain conditions, binding to Clq is caused by defined binding sites in the Fc part. Such binding sites are known in the state of the art and described e.g. by Lukas, TJ., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., Mol. Immunol. 16 (1979) 907-917; Burton, D.R., et al, Nature 288 (1980) 338-344; Thommesen, J.E., et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M„ et al., J. Virol. 75 (2001) 12161 -12168; Morgan, A., et al, Immunology 86 (1995) 319-324; and EP 0 307 434.

Such binding sites are e.g. L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Rabat). Antibodies of subclass IgGl, IgG2 and IgG3 usually show complement activation, Clq binding and C3 activation, whereas IgG4 do not activate the complement system, do not bind Clq and do not activate C3. In one embodiment the Fc part is a human Fc part.

In one embodiment of the invention the T cell engager comprises an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising an amino acid substitution at position Pro329 and at least one further amino acid substitution, wherein the residues are numbered according to the EU index of Rabat, and wherein said antibody exhibits a reduced affinity to the human FcyRIIIA and/or FcyRIIA and /or FcyRI compared to an antibody comprising the wildtype IgG Fc region, and wherein the ADCC induced by said antibody is reduced to at least 20% of the ADCC induced by the antibody comprising a wild-type human IgG Fc region. In a specific embodiment Pro329 of a wild-type human Fc region in the bispecific antibody is substituted with glycine or arginine or an amino acid residue large enough to destroy the proline sandwich within the Fc/Fcy receptor interface, that is formed between the proline329 of the Fc and tryptophan residues Trp 87 and Tip 110 of FcyRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In a further aspect of the invention the at least one further amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D, or P331S and still in another embodiment said at least one further amino acid substitution is L234A and L235A of the human IgGl Fc region or S228P and L235E of the human IgG4 Fc region. Such Fc variants are described in detail in W02012130831.

By“effector function” as used herein is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include but are not limited to ADCC, ADCP, and CDC. By“effector cell” as used herein is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, and gd T cells, and may be from any organism including but not limited to humans, mice, rats, rabbits, and monkeys. By“library” as used herein is meant a set of Fc variants in any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions at variable positions, a physical library comprising nucleic acids that encode the library sequences, or a physical library comprising the Fc variant proteins, either in purified or unpurified form.

By“Fc gamma receptor” or“FcyR” as used herein is meant any member of the family of proteins that bind the IgG antibody Fc region and are substantially encoded by the FcyR genes. In humans this family includes but is not limited to FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRlla (including allotypes H131 and R131), FcyRllb (including FcyRllb-l and FcyRllb-2), and FcyRllc; and FcyRIII (CD16), including isoforms FcyRllla (including allotypes V158 and F158) and FcyRlllb (including allotypes FcyRlllb-NAl and FcyRlllb- NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65), as well as any undiscovered human FcyRs or FcyR isoforms or allotypes. An FcyR may be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcyRs include but are not limited to FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII- 2 (CD16-2), as well as any undiscovered mouse FcyRs or FcyR isoforms or allotypes.

“Fc variant with increased effector function” as used herein is meant an Fc sequence that differs from that of a parent Fc sequence by virtue of at least one amino acid modification or relates to other modifications like amendment of glycosylation at e.g. Asn279 that increase effector functions. Such modifications are e.g. mentioned in Duncan et al., 1988, Nature 332:563-564; Lund et al, 1991 , J Immunol 147:2657- 2662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al, 1995, Proc Natl Acad Sci U S A 92:11980-11984; Jefferis et al, 1995, //77muno/ Lett 44:111 -117; Lund et al., 1995, Faseb J 9: 115-119; Jefferis et al., 1996, Immunol Lett 54:101 -104; Lund et al., 1996, J Immunol 157:4963- 4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178- 4184; Reddy et al., 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al, 2001 , J Immunol 166:2571 -2575; Shields et al., 2001 , J Biol Chem 276:6591 -6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490; US5624821; US5885573; US6194551; W0200042072; W0199958572. Such Fc modifications also include according to the invention engineered glycoforms of the Fc part. By "engineered glycoform" as used herein is meant a carbohydrate composition that is covalently attached to an Fc polypeptide, wherein said carbohydrate composition differs chemically from that of a parent Fc polypeptide. Engineered glycoforms may be generated by any method, for example by using engineered or variant expression strains, by co-expression with one or more enzymes, for example Dl-4- N- acetylglucosaminyltransferase III (GnTUI), by expressing an Fc polypeptide in various organisms or cell lines from various organisms, or by modifying carbohydrate(s) after the Fc polypeptide has been expressed. Methods for generating engineered glycoforms are known in the art and mentioned in Umana et al, 1999, Nat Biotechnol 17:176-180; Davies et al, 2001 , Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) US6602684; W0200061739; WO200129246; W0200231140; W0200230954; Potelligent™ technology (Biowa, Inc., Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland)). Engineered glycoform typically refers to the different carbohydrate or oligosaccharide composition than the parent Fc polypeptide.

In one embodiment of the invention, the antibody comprises an Fc variant with increased effector function show high binding affinity to the Fc gamma receptor III (FcyRIII, CD 16a). High binding affinity to FcyRIII denotes that binding is enhanced for CD16a/F158 at least 10-fold in relation to the parent antibody (95 % fucosylation) as reference expressed in CHO host cells, such as CHO DG44 or CHO K1 cells, or/and binding is enhanced for CD16a/V158 at least 20-fold in relation to the parent antibody measured by Surface Plasmon Resonance (SPR) using immobilized CD 16a at an antibody concentration of 100 nM. FcyRIII binding can be increased by methods according to the state of the art, e.g. by modifying the amino acid sequence of the Fc part or the glycosylation of the Fc part of the antibody (see e.g. EP2235061). Mori, K et al, Cytotechnology 55 (2007)109 and Satoh M, et al, Expert Opin Biol Ther. 6 (2006) 1161 -1173 relate to a FUT8 (a-l,6-fucosyltransferase) gene knockout CHO line for the generation of afiicosylated antibodies. In preferred embodiments, the T cell engager is a multispecific antibody ( e.g . bispecific antibody). Such antibodies may be heteromultimeric and may comprise mutations in regions involved interactions between antibody chains to promote correct assembly of the antibodies.

For example, the multispecific, e.g. bispecific, antibodies may comprise one or more mutation(s) in the CH2 and CH3 domain to enforce Fc heterodimerization. Alternatively or in addition, the multispecific, e.g. bispecific, antibodies may comprise mutations in the CHI and CL region to promote preferential pairing between the heavy chain and light chain.

A number of strategies exist for promoting heterodimerization. These strategies may include the introduction of asymmetric complementary mutations into each of two antibody chains, such that both chains are compatible with each other and thus able to form a heterodimer, but each chain is not able to dimerize with itself. Such mutations may encompass insertions, deletions, conservative and non conservative substitutions and rearrangements.

A further strategy for promoting heterodimerization is to rearrange portions of the antibody chains such that each chain remains compatible only with a chain comprising corresponding rearrangements. For example, CrossMAb technology involves exchange of the CHI and CL or VH and VL portions of a Fab. Other approaches to promoting heterodimerization include the use of a strand exchange engineered domain (SEED) (Davis et al, 2010. Protein Eng Des Sel, 23 (4); 195- 202).

The above heterodimerization strategies are described in more detail below. A combination of these strategies may be used (e.g. knob-into-holes with CrossMAb, knob-into-holes with CHI and CL heterodimerization mutations, CrossMAb with CHI and CL heterodimerization mutations, or most preferably knob-into-holes with CrossMAb and CHI and CL heterodimerization mutations) in the multispecific, e.g. bispecific, antibodies of the invention.

The multispecific, e.g. bispecific, antibodies may comprise one or more “knob-into-holes” modification(s), which are described in detail with several examples in e.g. WO 96/027011, Ridgway, J.B., et al., Protein Eng. 9 (1996) 617-621, Merchant, A.M. et al., Nat. Biotechnol. 16 (1998) 677-68, and WO 98/050431.

In this method, the interaction surfaces of the two CH3 domains are altered to increase the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". Accordingly, the multispecific, e.g. bispecific, antibodies may comprise two CH3 domains in the heavy chains, wherein the first CH3 domain of the first heavy chain and the second CH3 domain of the second heavy chain each meet at an interface which comprises an original interface between the antibody CH3 domains, wherein said interface is altered to promote the formation of the antibody.

In some embodiments:

(i) the CH3 domain of one heavy chain is altered, so that within the original interface of the CH3 domain of one heavy chain that meets the original interface of the CH3 domain of the other heavy chain within the antibody according to the invention, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a protuberance within the interface of the CH3 domain of one heavy chain which is positionable in a cavity within the interface of the CH3 domain of the other heavy chain; and ii) the CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the antibody according to the invention an amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain within which a protuberance within the interface of the first CH3 domain is positionable.

Preferably, said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W). Preferably, the amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), valine (V).

In some embodiments, the multispecific, e.g. bispecific, antibodies comprise a first CH3 domain comprising a modification at position T366 (“knob mutation”), e.g. T366W (numbering according to EU Index).

In some embodiments, the multispecific, e.g. bispecific, antibodies comprise a second CH3 domain comprising one or more modification(s) at positions T366, L368 and/or Y407 (“hole mutation(s)”), e.g. T366S, L368A, and/or Y407V (numbering according to EU Index).

In some embodiments, in the multispecific, e.g. bispecific, antibodies both CH3 domains are further altered by the introduction of cysteine (C) as amino acid in the corresponding positions of each CH3 domain. In the multispecific, e.g. bispecific, antibodies, one or more of the immunoglobulin heavy chains and light chains may comprise one or more modification(s), e.g. amino acid modifications, that are capable of promoting preferential pairing of a specific heavy chain with a specific light chain when heavy chains and light chains are co-expressed or co-produced.

The amino acid modifications may be substitutions of charged amino acids with opposite charges (for example in the CHI/CL interface) which reduce light chain mispairing, e.g. Bence-Jones type side products.

In preferred embodiments, the one or more modification(s) assist light and heavy chain heterodimerization are amino acid modifications in the light and heavy chains outside of the CDRs.

The one or more modification(s) may be present in the anti-cancer antigen (e.g. BCMA) antibody. Alternatively, the one or more modification(s) may be present in the anti-T cell antigen (e.g. CD3) antibody. In preferred embodiments, the one or more modification(s) are present in the anti-cancer antigen (e.g. BCMA) antibody. In embodiments in which the multispecific, e.g. bispecific, antibody comprises a second anti-cancer antigen (e.g. BCMA) antibody which is the same as the first anti-cancer antigen antibody, the second anti-cancer antigen antibody may comprise the same amino acid modifications to promote preferential pairing of the heavy chain with the light chain.

Bispecific antibodies against BCMA and CD3 having charge variants are described in EP14179705, disclosed by reference (further called as“charge variants resp. charge variant exchange”).

In some embodiments, the one or more modification(s) are present a constant domain CL, wherein the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Rabat), and in the respective constant domain CHI the amino acid at position 147 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to EU index).

In one embodiment in addition to the amino acid replacement at position 124 in the constant domain CL of the first or second light chain the amino acid at position 123 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Rabat).

In one embodiment in the constant domain CL the amino acid at position 124 is substituted by lysine (K) (numbering according to Rabat), and in the constant domain CHI the amino acid at position 147 and the amino acid at position 213 are substituted by glutamic acid (E) (numbering according to EU index), optionally wherein in addition in the constant domain CL in the amino acid at position 123 is substituted by arginine (R) (numbering according to Kabat).

The multispecific, e.g. bispecific, antibodies may comprise CrossMAb technology. CrossMAb refers to when either the variable or the constant regions of the antibody light chain and the antibody heavy chain are exchanged. It is used to facilitate multispecific antibody formation. CrossMAb technology is known in the state of the art. Bispecific antibodies wherein the variable domains VL and VH or the constant domains CL and CHI are replaced by each other are described in W02009080251 and W02009080252.

In one or more of the antibodies within the multispecific, e.g. bispecific, antibodies, the variable domains VL and VH or the constant domains CL and CHI may be replaced by each other.

In some embodiments in which the T cell engager comprises a bispecific antibody comprising an anti- CD3 antibody and an anti-BCMA antibody, the variable domains VL and VH or the constant domains CL and CHI of the anti-CD3 antibody are replaced by each other. More preferably, the variable domains VL and VH of the anti-CD3 antibody are replaced by each other.

It is especially preferred for bispecific antibodies having the 2+1 format to comprise CrossMAb technology. Thus, in embodiments in which the bispecific antibodies have two anti-cancer antigen (e.g. BCMA) antibodies and one anti-T cell antigen (e.g. CD3) antibody, the variable domains VL and VH or the constant domains CL and CHI of the anti-CD3 antibody or antigen binding fragment thereof may be replaced by each other. In preferred embodiments, the variable domains VL and VH are replaced by each other.

In one embodiment of the invention the bispecific antibody comprises: a) the first light chain and the first heavy chain of a first antibody which specifically binds to BCMA; and

b) the second light chain and the second heavy chain of a second antibody which specifically binds to CD3, and wherein the variable domains VL and VH in the second light chain and second heavy chain of the second antibody are replaced by each other; and

c) wherein in the constant domain CL of the first light chain under a) the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein in the constant domain CHI of the first heavy chain under a) the amino acid at position 147 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to EU Index) (see e.g. Figures 1A, 2A, 2C, 3A, 3C). In one embodiment said bispecific antibody described in the last preceding paragraph is further characterized in that said bispecific antibody comprises in addition a Fab fragment of said first antibody (further named also as“BCMA-Fab”) and in the constant domain CL said BCMA-Fab the amino acid at position 124 is substituted independently by lysine (K), arginine (R) or histidine (H) (numbering according to Rabat), and wherein in the constant domain CHI of said BCMA-Fab the amino acid at positions 147 and the amino acid at position 213 is substituted independently by glutamic acid (E), or aspartic acid (D) (numbering according to EU index) (see e.g. Figures 2A, 2C).

In one embodiment, the T cell engager is a bispecific antibody consisting of one Fab fragment of an antibody specifically binding to CD3 (further named also as“CD3-Fab”), and one Fab fragment of an anti-BCMA antibody (further named also as“BCMA-Fab(s)”) and a Fc part, wherein the CD3-Fab and the BCMA-Fab are linked via their C-termini to the hinge region of said Fc part. Either the CD3-Fab or the BCMA-Fab comprises amino acid substitutions and the CD3-Fab comprises crossover (Figures 1A and IB).

In one embodiment, the T cell engager is a bispecific antibody consisting of one CD3-Fab, and one BCMA-Fab and a Fc part, wherein the CD3-Fab and the BCMA-Fab are linked via their C-termini to the hinge region of said Fc part, and a second BCMA-Fab, which is linked with its C-terminus to the N- terminus of the CD3-Fab. The CD3-Fab comprises CrossMAb and either the CD3-Fab or both BCMA- Fabs comprise amino acid substitutions (Figures 2A and 2B). Especially preferred is a bispecific antibody comprising BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs comprise amino acid substitutions and the CD3-Fab comprises VL/VH crossover (Figure 2A). Especially preferred is a bispecific antibody consisting of BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fabs comprise the amino acid substitutions Q124K, E123R, K147E and K213E and the CD3-Fab comprises VL/VH crossover. Especially preferred is that both BCMA-Fabs comprise as CDRs the CDRs of antibody 21, 22, or 42, or as VH/VL the VH/VL of antibody 21, 22, or 42 (for antibodies 21, 22 and 42 see Table 1A and IB later in the text).

In one embodiment, the T cell engager is a bispecific antibody consisting of two BCMA-Fabs, one CD3 Fab and an Fc part, wherein one BCMA-Fab and the CD3 Fab are linked via their C-termini to the hinge region of said Fc part and the second BCMA-Fab is linked with its C-terminus to the N-terminus of the CD3-Fab. The CD3-Fab comprises CrossMAb and either the CD3-Fab or both BCMA-Fabs comprise amino acid substitutions (Figures 2A and 2B).

In one embodiment, the T cell engager is a bispecific antibody consisting of two BCMA-Fabs, one CD3- Fab and an Fc part, wherein the BCMA-Fabs are linked via their C-termini to the hinge region of said Fc part and a CD3-Fab, which is linked with its C-terminus to the N-terminus of one BCMA-Fab. The CD3- Fab comprises CrossMAb and either the CD3-Fab or both BCMA-Fabs comprise amino acid substitutions (Figures 2C and 2D).

In one embodiment, the T cell engager is a bispecific antibody consisting of one CD3-Fab, which is linked via its C-terminus to the hinge region of the Fc part and a BCMA-Fab, which is linked with its C- terminus to the hinge region of the Fc part. The CD3-Fab comprises CrossMAb and either the CD3-Fab or the BCMA-Fab comprise amino acid substitutions (Figures 1 A and IB).

In one embodiment, the T cell engager is a bispecific antibody consisting of one CD3-Fab, which is linked via its C-terminus to the hinge region of said Fc part and a BCMA-Fab, which is linked with its C- terminus to the N-terminus of the CD3-Fab. The CD3-Fab comprises CrossMAb and either the CD3-Fab or the BCMA-Fab comprise amino acid substitutions (Figures 3A and 3B).

In one embodiment, the T cell engager is a bispecific antibody consisting of one BCMA-Fab, which is linked via its C-terminus to the hinge region of said Fc part and a CD3-Fab, which is linked with its C- terminus to the N-terminus of the BCMA-Fab. The CD3-Fab comprises CrossMAb and either the CD3- Fab or the BCMA-Fab comprise amino acid substitutions (Figures 3C and 3D).

Preferably, the Fab fragments are chemically linked together by the use of an appropriate linker according to the state of the art. In one embodiment a (Gly4-Serl)3 linker is used (Desplancq DK et ak, Protein Eng. 1994 Aug;7(8):1027-33 and Mack M. et ak, PNAS July 18, 1995 vol. 92 no. 15 7021-7025). Linkage between two Fab fragments is performed between the heavy chains. Therefore the C-terminus of CHI of a first Fab fragment is linked to the N-terminus of VH of the second Fab fragment (no crossover) or to VL (crossover). Linkage between a Fab fragment and the Fc part is performed as linkage between CHI and CH2.

The bispecific antibodies can comprise instead of the Fabs single chains consisting of the same domains.

Methods of making the multispecific antibodies disclosed herein are disclosed in WO 2017/021450, the contents of which are hereby incorporated by reference in their entirety.

In embodiments in which the T cell engager binds to BCMA and CD3 is in a 2+1 and contains an Fc, the T cell may comprise the following SEQ ID NOs (as mentioned in Tables 1A and 2B below):

83A10-TCBcv: 45, 46, 47 (x2), 48 (Figure 2A)

21 -TCBcv: 48, 49, 50, 51 (x2) (Figure 2A)

22-TCBcv: 48, 52, 53, 54 (x2) (Figure 2A) 42-TCBcv: 48, 55, 56, 57 (x2) (Figure 2A)

The term“83A10-TCBcv” as used herein refer to a bispecific antibody specifically binding to BCMA and CD3 as specified by its heavy and light chain combination of SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47 (2x), and SEQ ID NO:48, and as shown in Figure 2A and described in EP14179705.

The terms“21-TCBcv, 22-TCBcv, 42-TCBcv” as used herein refer to the respective bispecific antibodies of Mab21, as specified by its heavy and light chain combination of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51 (2x), Mab 22 as specified by its heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53, and SEQ ID NO:54 (2x), and Mab42 as specified by its heavy and light chain combination of SEQ ID NO:48, SEQ ID NO:55, SEQ ID NO:56, and SEQ ID NO:57-(2x), and as shown in Figure 2A and described in WO 2017/021450.

CAR T cell engager

In some embodiments, the T cell engager comprises a chimeric antigen receptor (CAR) or a T cell comprising a CAR.

Chimeric antigen receptors (CARs) are artificial receptors that are expressed on the surface of immune cells, for example, T cells, in order to direct the immune cell to a target cell expressing a specific antigen, e.g. a cancer antigen such as BCMA or ROR1, and induce T cell-mediated killing of the antigen expressing target cell.

In some embodiments of the present invention, the T cell engager comprises a CAR, wherein the CAR comprises an antigen recognition moiety directed against a cancer antigen, a transmembrane moiety and a T-cell activation moiety. In preferred embodiments, the antigen recognition moiety is directed against a cancer antigen, such as BCMA or ROR1. In particularly preferred embodiments, the antigen recognition moiety is directed against BCMA.

In some embodiments, the T cell engager is a CAR that comprises an anti-BCMA antibody described herein.

In particularly preferred embodiments, the CAR comprises an anti-BCMA antibody comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from the group of: i) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28; ii) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24; or

iii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26.

In some embodiments, the anti-BCMA antibody comprises a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13 or a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

Such a CAR is then usually transferred by using a vector, preferably a retroviral vector, comprising the sequence encoding said CAR, into an immune effector cell for which herein the term“a CAR T-cell” is used. Such CAR T-cells are also provided as an aspect of the invention.

The CAR or the respective CAR T-cell may comprise:

(i) a cancer antigen recognition moiety;

(ii) a spacer domain; and

(ii) a transmembrane domain; and

(iii) an intracellular T cell signaling domain.

The CAR may comprise a T -cell activation moiety that can be any suitable moiety derived or obtained from any suitable molecule. In one embodiment, for example, the T-cell activation moiety comprises a transmembrane domain. The transmembrane domain can be any transmembrane domain derived or obtained from any molecule known in the art. For example, the transmembrane domain can be obtained or derived from a CD8a molecule or a CD28 molecule. CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T-cell receptor (TCR), and is expressed primarily on the surface of cytotoxic T-cells. The most common form of CD8 exists as a dimer composed of a CD8 alpha and CD8 beta chain. CD28 is expressed on T-cells and provides co-stimulatory signals required for T-cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, the CD8 alpha and CD28 are human. In addition to the transmembrane domain, the T-cell activation moiety further comprises an intracellular (i.e., cytoplasmic) T-cell signaling domain. The intercellular T-cell signaling domain can be obtained or derived from a CD28 molecule, a CD3 zeta molecule or modified versions thereof, a human Fc receptor gamma (FcRy) chain, a CD27 molecule, an 0X40 molecule, a 4-IBB molecule, or other intracellular signaling molecules known in the art. As discussed above, CD28 is a T-cell marker important in T-cell co-stimulation. CD3 zeta, associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (FTAMs). 4-IBB, also known as CD137, transmits a potent costimulatory signal to T-cells, promoting differentiation and enhancing long-term survival of T lymphocytes. In one embodiment, the CD28, CD3 zeta, 4-IBB, 0X40, and CD27 are human. In some aspects, there is provided a T cell engager for use in the treatment of a disorder associated with BCMA expression, wherein the treatment comprises depleting or inactivating neutrophil-like cells and wherein the T cell engager is a CAR that comprises an anti-BCMA antibody described herein.

In particularly preferred embodiments, the CAR comprises an anti-BCMA antibody comprising a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO:20 and a CDR1L and CDR2L region combination selected from the group of: i) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28;

ii) CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24; or

iii) CDR1L region of SEQ ID NO:25 and CDR2L region of SEQ ID NO:26.

In some embodiments, the anti-BCMA antibody comprises a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 13 or a VH region of SEQ ID NO: 10 and a VL region of SEQ ID NO: 14.

Therapy

The present invention provides improvements in T cell immunotherapy. T cell immunotherapy is a promising approach for the treatment of disorders including cancer and autoimmune disorders.

Accordingly, the present invention can be used for the treatment of cancer. The cancer may be a haematological cancer or a solid cancer.

In embodiments in which the cancer is a haematological cancer, the cancer may be selected from the group consisting of: B-cell chronic lymphocytic leukemia (CLL)/hairy cell leukemia (HCL) acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), acute myeloid leukemia (AML), plasmacytoma, plasma cell leukemia, Waldenstrom's macroglobulinemia, B cell lymphoma and lymphoplasmactyic lymphoma. In preferred embodiments, the haematological cancer is multiple myeloma or CLL. In particularly preferred embodiments, the haematological cancer is multiple myeloma.

In embodiments in which the cancer is solid cancer, the cancer may be selected from the group consisting of: ovarian cancer, breast cancer, including triple negative breast cancer, lung cancer, including lung adenocarcinoma, colon cancer and pancreatic cancers, testicular cancer, bladder cancers, uterus cancers, prostate cancers, and skin cancer including melanoma. In some aspects and embodiments of the invention, the T cell engager binds to BCMA. Accordingly, the present invention can be used for the treatment of a disorder associated with BCMA expression. Such disorders include multiple myeloma and other plasma cell disorders expressing BCMA.

Multiple myeloma is a plasma cell malignancy characterized by a monoclonal expansion and accumulation of abnormal plasma cells in the bone marrow compartment. Multiple myeloma also involves circulating clonal plasma cells with same IgG gene rearrangement and somatic hypermutation. Multiple myeloma arises from an asymptomatic, premalignant condition called monoclonal gammopathy of unknown significance (MGUS), characterized by low levels of bone marrow plasma cells and a monoclonal protein. Multiple myeloma cells proliferate at low rate. Multiple myeloma results from a progressive occurrence of multiple structural chromosomal changes ( e.g . unbalanced translocations). Multiple myeloma involves the mutual interaction of malignant plasma cells and bone marrow microenvironment (e.g. normal bone marrow stromal cells). Clinical signs of active multiple myeloma include monoclonal antibody spike, plasma cells overcrowding the bone marrow, lytic bone lesions and bone destruction resulting from overstimulation of osteoclasts (Dimopulos & Terpos, Ann Oncol 2010; 21 suppl 7: viil43-150).

Another plasma cell disorder involving plasma cells i.e. expressing BCMA is systemic lupus erythematosus (SLE), also known as lupus. SLE is a systemic, autoimmune disease that can affect any part of the body and is represented with the immune system attacking the body’s own cells and tissue, resulting in chronic inflammation and tissue damage. It is a Type III hypersensitivity reaction in which antibody-immune complexes precipitate and cause a further immune response (Inaki & Lee, Nat Rev Rheumatol 2010; 6: 326-337).

Further plasma cell disorders are plasma cell leukemia and AL-Amyloidosis. In all of these plasma cell disorders depletion of plasma cells/malignant plasma cells by T cell engagers according to the present invention is expected to be beneficial for the patients suffering from such a disease.

In some embodiments, the disorder associated with BCMA expression is a plasma cell disorder, preferably wherein the plasma cell disorder is multiple myeloma.

In some embodiments, the disorder associated with BCMA expression is AL-amyloidosis.

In some embodiments, the disorder associated with BCMA expression is an autoimmune disorder. The autoimmune disorder may be selected from the group consisting of arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis/dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus cerebritis, juvenile onset diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T- lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis and Churg- Strauss disease, agranulocytosis, vasculitis (including hypersensitivity vasculitis/angiitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AGHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti- phospholipid antibody syndrome, allergic neuritis, Behcet disease, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens- Johnson syndrome, solid organ transplant rejection, graft versus host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathies, seronegative spondyloarthropathies, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), Henoch- Schonlein purpura, autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant), non-specific interstitial pneumonia (NSIP), Guillain- BarreSyndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu's) arteritis), medium vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa), polyarteritis nodosa (PAN) ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, Celiac sprue (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans.

In some embodiments, the disorder associated with BCMA expression is antibody-mediated allograft rejection.

In some embodiments of the invention, the T cell engager binds to ROR1. Accordingly, the present invention can be used for the treatment of a disorder associated with ROR1 expression, e.g. as set forth below.

ROR1 is overexpressed in haematological malignancies, including B-cell chronic lymphocytic leukemia (CLL)/hairy cell leukemia (HCL) (Baskar et al. (2008), Clinical Cancer Research, 14 (2): 396M04; Daneshmanesh et al. (2008), International Journal of Cancer, 123 (5): 1190-5), acute lymphoblastic leukemia (ALL) (Shabani M, et al. (2007), Tumour Biology, 28 (6): 318-26; Shabani M et al. (2008), Leukemia & Lymphoma, 49 (7): 1360-7), chronic myeloid leukemia (CML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL) and acute myeloid leukemia (AML) (Daneshmanesh et al. (2013), Leukemia & Lymphoma 54 (4)).

ROR1 is also known to be expressed in solid tumors, including ovarian cancers (Zhang et al. (2014), Scientific Reports 4: 5811), breast cancers including triple negative breast cancer (Zhang et al. (2012), PLoS One 7(3): e31127), lung cancers including lung adenocarcinomas (Yamaguchi et al. (2012), Cancer Cell 21(3):348), colon cancers (Zhou et al, 2017, Oncotarget. 8(20): 32864-32872) pancreatic cancers (Gohil et al., 2017, Oncolmmunology, 6:7), testicular cancers, bladder cancers, uterine cancers, prostate cancers (Zhang et al. 2012, Am. J. of Pathol. 181(6): 1903-1910) and skin cancers including melanoma (Fernandez, et al. (2016), Mol. Carcinog. , 55: 1772-1785).

Pharmaceutical composition

Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of the T cell engager and a neutrophil-like cell depleting agent, and a pharmaceutically acceptable excipient.

In one aspect, the pharmaceutical composition comprises a therapeutically effective amount of a T cell engager and a neutrophil-like cell depleting agent, and a pharmaceutically acceptable excipient, wherein the T cell engager activates a T-cell to target a cancer cell, thereby leading to the death of the cancer cell.

In another aspect, the pharmaceutical composition comprises a therapeutically effective amount of a T cell engager and a neutrophil-like cell depleting agent, and a pharmaceutically acceptable excipient, wherein the T cell engager activates the T-cell to target a cell expressing BCMA, thereby leading to the death of the cell

Examples of suitable excipients include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as any combination thereof. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, or sodium chloride in the composition. In particular, relevant examples of suitable excipients include: (1) Dulbecco's phosphate buffered saline, pH.about.7.4, containing or not containing about 1 mg/mL to 25 mg/mL human serum albumin, (2) 0.9% saline (0.9% w/v sodium chloride (NaCl)), and (3) 5% (w/v) dextrose; and may also contain an antioxidant such as tryptamine and a stabilizing agent such as Tween 20 ^® .

A person skilled in the art would understand that the appropriate choice of excipient or excipients for use with the T cell engager would depend on the desired properties of the pharmaceutical composition.

The pharmaceutical compositions of the invention can be administered to a patient by any appropriate systemic or local route of administration. For example, administration may be oral, buccal, sublingual, ophthalmic, intranasal, intratracheal, pulmonary, topical, transdermal, urogenital, rectal, subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intracranial, intrathecal, epidural, intraventricular or intratumoral. In preferred embodiments, the pharmaceutical compositions are administered subcutaneously.

Pharmaceutical compositions of the invention can be formulated for administration by any appropriate means, for example by epidermal or transdermal patches, ointments, lotions, creams, or gels; by nebulizers, vaporisers, or inhalers; by injection or infusion; or in the form of capsules, tablets, liquid solutions or suspensions in water or non-aqueous media, drops, suppositories, enemas, sprays, or powders. The most suitable route for administration in any given case will depend on the physical and mental condition of the subject, the nature and severity of the disease, and the desired properties of the formulation. Preferably, the pharmaceutical composition will be administered subcutaneously. When administering therapeutic antibodies by injection, the administration may be by continuous infusion or by single or multiple boluses.

The depletion or inactivation of neutrophil-like cells in the subject results in an improved cytotoxicity of the T cell engagers. Due to this superior cytotoxicity, they can be administered to the subject at a lower magnitude of clinical dose range as compared to without depletion or inactivation of neutrophil-like cells.

It is envisaged that subcutaneous administration is preferred in the clinical settings ( e.g . in the dose range of 0.1 - 10 mg/m ² once or twice a week). In embodiments in which the T cell engager is a multispecific, e.g. bispecific, antibody, the T cell engager may eliminated with a half-life of about several days which allows at least once or twice/week administration. Accordingly, in preferred embodiments the T cell engager is administered once or twice a week, preferably via subcutaneous administration (e.g. preferably in the dose range of 0.1 to 10 mg/m ² once or twice a week). The exact dose can be determined by a person of relevant skill, such as a clinician, according to principles known in the art, for example the patient's age, weight, height, sex, general medical condition and previous medical history.

The following example, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. Table 1A: Antibody sequences

Remark: SEQ ID NO:20 and SEQ ID NO:33 are identical

Table IB: Antibody sequences (short list)

Table 2A: Additional constructs

Table 2B: Additional constructs

Example

Detailed Phenotypic, Molecular and Functional Profiling of Myeloid Derived Suppressor Cells (MDSCs) in the Tumor Immune MicroEnvironment (TIME) of Multiple Myeloma (MM) Background: Deep understanding of the complexity and diversity of the tumor immune microenvironment (TIME) and its influence on response to therapy is needed to improve the ability to predict, monitor and guide immunotherapeutic responsiveness. Among different cell types in the MM- TIME, granulocytic MDSCs (G-MDSCs) have a prominent role in promoting tumor growth and inducing immune suppression; however, their identification and monitoring is imprecise because the phenotypic profile of MDSCs in the MM-TGME is not well-established.

Aim: To provide the detailed phenotypic profile of G-MDSCs based on the immune suppressive potential, gene regulatory network and clinical correlates of distinct granulocytic subsets in the MM- TIME.

Methods: We used multidimensional flow cytometry (MFC) to characterize the phenotype of granulocytes in bone marrow (BM) samples from controls (n=4) and MM patients (n=5). We used principal component analysis (PCA) to unbiasedly identify different granulocytic subsets in the MM- TIME, and FACSorted them for in vitro experiments to determine their immune suppressive potential (n=9) and for RNAseq to analyze the molecular profile of G-MDSCs in MM (n=5) vs controls (n=5). The clinical significance of the different granulocytic subsets was investigated by comparing their numbers at diagnosis, in MM patients (n=124) achieving MRD-negativity vs MRD-positivity after treatment with VRD induction (x6) followed by autologous transplant and VRD consolidation (x2) (GEM2012MENOS65 clinical trial). Results: In humans, G-MDSCs have been characterized by a unique cluster displaying a CD1 lb-, CD14-, CD15+, CD33+ and HLADR- phenotype, comprising 1% of total BM nucleated cells in healthy individuals and approximately 25% in MM patients. Surprisingly, we found that the percentage of cells with a CD1 lb-CD14-CD15+CD33+HLADR- phenotype was similar in the BM of controls and MM patients (median of 8% in both, p>.99). Since these cells were not expanded in MM and represented only 24% of total neutrophils, we next used MFC and PCA to unbiasedly identify other cell clusters within neutrophils. Accordingly, 3 major subsets were identified in neutrophils from controls and MM patients, based on homogeneous CD14-CD15+CD33+HLADR- expression but differential reactivity against CDl lb, CD13 and CD16: CDl lb-CD13lo/-CD16- (19% and 24%), CDl lb+CD13lo/-CD16- (46% and 47%) and CD1 lb+CD13+CD16+ (35% and 29%). Afterwards, we used FACSorting to deplete or isolate individually, each of the 3 neutrophil subsets from the BM MM-TGME and determine its immune suppressive potential in 2 functional assays: 1) the proliferation rate of autologous T cells in presence of CD3/CD28 stimulatory beads and, 2) the cytotoxic potential of autologous T -cells against MM cells using a BCMAxCD3 bispecific antibody. Interestingly, we noted a significant decrease in T cell proliferation when these were stimulated in the presence of CDl lb+CD13+CD16+ neutrophils (0.5-fold, /;=.()3) but not the CDl lb-CD13lo/-CD16- and CDl lb+CD13lo/-CD16- subsets (see Figure 4A). In addition, we noted that the cytotoxic potential of T cells engaged by the BCMAxCD3 bispecific antibody significantly increased with the depletion of CD1 lb+CD13lo/-CD16- and CDl lb+CD13+CD16+ subsets (3-fold and 4-fold, respectively; p£.04) but not CDl lb-CD13lo/-CD16- neutrophils (see Figure 4B). Furthermore, RNAseq of the 3 subsets in controls and MM patients revealed that genes related with the IL-4, IL-10 and IL-13 immunosuppressive pathways were specifically upregulated in the CDl lb+CD13+CD16+ subset. Finally, based on the surrogacy between the achievement of MRD-negativity and prolonged survival, we compared the distribution of the 3 granulocytic subsets in the BM-TGME at diagnosis and observed that patients reaching MRD-negativity (n=56) displayed significantly lower percentages of total neutrophils (46% vs 52%, p=.007), particularly of the CDl lb+CD13lo/-CD16- (11% vs 15%, /?=.008) or CDl lb+CD13+CD16+ (31% vs 35%, /;=.()3) subsets vs MRD-positive cases (n=68).

Conclusions: In this comprehensive analysis using MFC, RNAseq, functional and clinical studies, we have determined the exact correlation between the phenotypic, molecular and immunosuppressive potential of well-defined granulocytic subsets, thereby providing for the first time, a set of optimal markers (i.e. CD1 lb, CD13, CD16) for monitoring G-MDCSs in patients with MM.