Methods for the expression of antibody-multimeric fusions

Abstract

The present invention provides a method for producing an antibody-multimeric fusion polypeptide, comprising: (a) an antibody heavy chain and an antibody light chain; and (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; and a second fusion polypeptide comprising, from N-terminus to C-terminus, a second portion of the non-antibody multimeric polypeptide and an antibody light chain constant domain if the first polypeptide comprises the antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises the antibody light chain constant domain. and a fusion polypeptide of (a) or (b), wherein (i) the antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond, and wherein the antibody-multimeric fusion is expressed by a recombinant mammalian cell obtained by transfecting the mammalian cell with expression cassettes for the antibody heavy chain, the antibody light chain, the first fusion polypeptide, and the second fusion polypeptide in a 1:1:2:1 stoichiometric ratio.

Description

[Technical Field]

[0001] The present invention relates to the field of cell line engineering and polypeptide production. More precisely, the present invention reports the generation of recombinant mammalian producer cells by transfecting non-producing mammalian cells with expression cassettes for the individual polypeptides of an antibody-multimer fusion in a defined ratio of n. Such cells can be used in methods for the production of antibody-multimer fusions. [Background technology]

[0002] Background of the Invention Secreted glycosylated polypeptides, such as antibodies, are usually produced by recombinant expression in eukaryotic cells, either as stable or transient expression.

[0003] The higher the complexity of the polypeptide to be produced, i.e., the greater the number of different polypeptides or polypeptide chains required to form the polypeptide of interest within the cell, the more important it becomes to control the expression ratio of the different polypeptides or polypeptide chains relative to each other. Control of the expression ratio is necessary to allow efficient expression, correct assembly, and successful secretion of the polypeptide of interest with high expression yields.

[0004] One strategy for generating recombinant cells that express an exogenous polypeptide of interest involves random integration of a nucleotide sequence encoding the polypeptide of interest, followed by secretion and isolation steps.

[0005] Targeted integration by recombinase-mediated cassette exchange (RMCE) is a method for specifically and efficiently directing foreign DNA into predetermined sites in the eukaryotic host genome (Turan et al., J. Mol. Biol. 407 (2011) 193-221).

[0006] Crawford et al. reported that they used a combination of φC31 integrase and CRE-Lox technologies to rapidly identify reliable hosts for targeted cell line development through limited genomic screening (Biotechnol. Prog. 29 (2013) 1307-1315).

[0007] Rajendra et al. reported that a single quad vector is a simple yet effective alternative approach for generating stable CHO cell lines, which may accelerate the development of cell lines for clinical heterologous mAb therapeutics (Biotechnol. Prog. 33 (2017) 469-477).

[0008] Bahr et al. reported the development of a platform expression system using targeted integration in Chinese hamster ovary cells (Proceedings of Cell Culture Engineering XVI, 2018).

[0009] Carver et al. reported that optimizing the dosage and location of subunit genes maximizes antibody production in targeted integration hosts (Biotechnol. Prog. (2020) e2967). Summary of the Invention

[0010] Summary of the Invention The invention is defined by the following independent aspects and dependent embodiments.

[0011] 1. A first aspect of the present invention is a method for producing an antibody-multimeric fusion polypeptide, comprising: (a) an antibody heavy chain and an antibody light chain; (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; and a second fusion polypeptide comprising, from N-terminus to C-terminus, a second portion of the non-antibody multimeric polypeptide, and an antibody light chain constant domain if the first polypeptide comprises the antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises the antibody light chain constant domain. Including, (i) the antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond; the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to an antigen; wherein the antibody-multimeric fusion polypeptide is expressed by a recombinant mammalian cell obtained by transfecting a (parent) mammalian cell with expression cassettes for the antibody heavy chain, the antibody light chain, the first fusion polypeptide and the second fusion polypeptide in a 1:1:2:1 stoichiometric ratio.

[0012] 2. The method of aspect 1, wherein said antibody-multimeric fusion polypeptide is transiently or stably expressed.

[0013] 3. The method of aspect 1 or embodiment 2, wherein said mammalian cells are CHO cells, preferably CHO-K1 cells or HEK cells.

[0014] 4. The method of any one of aspect 1 and embodiment 2 or 3, wherein said transfecting is of four vectors, each vector comprising exactly one said expression cassette.

[0015] 5. The method of any one of aspect 1 and embodiment 2 or 3, wherein said transfecting is of three vectors, two vectors containing exactly two of said expression cassettes, and one vector containing exactly one of said expression cassettes.

[0016] 6. The method of embodiment 4, wherein the transfecting is of three vectors, a first vector comprising the expression cassettes for an antibody heavy chain and an antibody light chain, a second expression vector comprising the expression cassettes for the first fusion polypeptide and the second fusion polypeptide, and a third vector comprising one expression cassette for the first fusion polypeptide.

[0017] 7. The method of any one of aspect 1 and embodiments 2-6, wherein the first fusion polypeptide comprises, as a first portion of the non-antibody multimeric polypeptide, two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a peptide linker, and the second fusion polypeptide comprises, as a second portion of the non-antibody multimeric polypeptide, only one ectodomain or fragment thereof of the TNF ligand family member, or vice versa.

[0018] 8. (a) the first fusion polypeptide comprises, as a first portion of the non-antibody multimeric polypeptide, a first ectodomain of a TNF ligand family member or a fragment thereof, a spacer domain, and a second ectodomain of the TNF ligand family member or a fragment thereof; - the spacer domain is a polypeptide and comprises at least 25 amino acid residues; - the first ectodomain of a TNF ligand family member or a fragment thereof is fused to the N-terminus of the spacer domain either directly or via a first peptide linker; - the second ectodomain of the TNF ligand family member or a fragment thereof is fused to the C-terminus of the spacer domain either directly or via a second peptide linker; (b) the second fusion polypeptide comprises, as a second portion of the non-antibody multimeric polypeptide, a third ectodomain of the TNF ligand family member, or a fragment thereof, either directly or via a third peptide linker; - either to the C-terminus of the second ectodomain of the TNF ligand family member in the first fusion polypeptide or to the C-terminus of a spacer domain in the second fusion polypeptide; or 8. The method of embodiment 7, wherein the second portion of the antigen-binding domain is fused to the C-terminus of the second ectodomain of the TNF ligand family member in the first fusion polypeptide when the second portion of the antigen-binding domain is fused to the C-terminus of the spacer domain of the second fusion protein.

[0019] 9. The first fusion polypeptide comprises, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; 9. The method of any one of Aspect 1 and Embodiments 2 to 8, wherein the second fusion polypeptide comprises, from N-terminus to C-terminus, the second portion of the non-antibody multimeric polypeptide and an antibody heavy chain CH1 domain.

[0020] 10. The first fusion polypeptide comprises a knob mutation; The method of any one of aspects 1 and embodiments 2-9, wherein the antibody heavy chain comprises a hole mutation.

[0021] 11. The method of aspect 1 and any one of embodiments 2 to 10, wherein the antibody heavy chain of (a) and the first fusion polypeptide of (b) form an Fc region.

[0022] 12. The method of aspect 1 and any one of embodiments 2 to 11, wherein the antibody heavy chain of (a) and the first fusion polypeptide of (b) form an IgG1 Fc region or an IgG4 Fc region.

[0023] 13. The method of aspect 1 and any one of embodiments 2 to 12, wherein the Fc region is an IgG1 Fc region further comprising amino acid substitutions at positions 234 and 235 and / or 329 (Kabat EU numbering).

[0024] 14. The method of aspect 1 and any one of embodiments 2 to 13, wherein the Fc region is an IgG1 Fc region further comprising amino acid substitutions at positions L234A, L235A and / or P329G (Kabat EU numbering).

[0025] 15. The method of any one of embodiments 7 to 14, wherein in the first fusion polypeptide, the two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a first peptide linker are fused at their C-terminus to a CH1 domain by a second peptide linker, and in the second fusion polypeptide, the one ectodomain or fragment thereof of a TNF ligand family member is fused at its C-terminus to the antibody light chain constant domain by a third peptide linker.

[0026] 16. The method of any one of embodiments 7 to 14, wherein in the first fusion polypeptide, the two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a first peptide linker are fused at their C-terminus to a light chain constant domain by a second peptide linker, and in the second fusion polypeptide, the one ectodomain or fragment thereof of the TNF ligand family member is fused at its C-terminus to the heavy chain CH1 domain by a third peptide linker.

[0027] 17. The method of any one of aspect 1 and embodiments 2 to 16, wherein in the CL domain adjacent to said portion of the non-antibody multimeric polypeptide, the amino acid at position 123 (Kabat EU numbering) is replaced by arginine (R) and the amino acid at position 124 (Kabat EU numbering) is replaced by lysine (K), and in the CH1 domain adjacent to said portion of the non-antibody multimeric polypeptide, the amino acids at positions 147 (Kabat EU numbering) and 213 (Kabat EU numbering) are replaced by glutamic acid (E).

[0028] 18. The method of aspect 1 and any one of embodiments 2 to 17, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to a cell surface antigen.

[0029] 19. The method of any one of aspects 1 and embodiments 2-18, wherein the variable domains of the antibody heavy chain and the antibody light chain form binding sites that specifically bind to a cell surface antigen selected from the group consisting of fibroblast activation protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and CD33.

[0030] 20. The method of any one of embodiments 7 to 19, wherein said TNF ligand family member costimulates human T cell activation.

[0031] 21. The method of any one of embodiments 7 to 20, wherein said TNF ligand family member is selected from 4-1-BBL and OX40L.

[0032] 22. The method of any one of embodiments 7 to 21, wherein said TNF ligand family member is 4-1-BBL.

[0033] 23. The method of any one of embodiments 7 to 22, wherein the ectodomain of the TNF ligand family member comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 01, SEQ ID NO: 02, SEQ ID NO: 03, SEQ ID NO: 04, SEQ ID NO: 56, SEQ ID NO: 100, SEQ ID NO: 101 and SEQ ID NO: 102.

[0034] 24. The method of any one of embodiments 7 to 23, wherein the ectodomain of the TNF ligand family member comprises the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO: 56.

[0035] 25. (a) the antibody heavy chain and the antibody light chain form a binding site capable of specifically binding to a target cell antigen; (b) The method of any one of aspects 1 and embodiments 2 to 24, wherein the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 05, SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, and the second fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 01, SEQ ID NO: 56, SEQ ID NO: 03, and SEQ ID NO: 04.

[0036] 26. The method of any one of embodiments 7 to 21, wherein said TNF ligand family member is OX40L.

[0037] 27. The method according to any one of embodiments 7 to 21 and 26, wherein the ectodomain of the TNF ligand family member comprises the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44, in particular the amino acid sequence of SEQ ID NO: 43.

[0038] 28. The antibody-multimer fusion comprises: (a) at least one moiety capable of specifically binding to a target cell antigen, and (b) The method according to any one of embodiments 7 to 21 and 26 to 27, wherein the antigen-binding molecule comprises the first fusion polypeptide and the second fusion polypeptide linked to each other by a disulfide bond, wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 99 or SEQ ID NO: 100, and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44.

[0039] 29. The method of aspect 1 and any one of embodiments 2-28, wherein the antigen is fibroblast activation protein (FAP).

[0040] 30. The method of any one of aspects 1 and embodiments 2-29, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to a FAP, and comprise a VH domain that comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 06 or SEQ ID NO: 60, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 07 or SEQ ID NO: 61, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 08 or SEQ ID NO: 62, and a VL domain that comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 09 or SEQ ID NO: 63, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 64, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 65.

[0041] 31. The method of any one of aspects 1 and embodiments 2-30, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to a FAP, and comprise a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 15 and a variable light domain comprising the amino acid sequence of SEQ ID NO: 16, or a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 66 and a variable light domain comprising the amino acid sequence of SEQ ID NO: 67, or wherein the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 97 and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 98.

[0042] 32. The method of any one of aspect 1 and embodiments 2-31, wherein (i) the antibody heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and the antibody light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or wherein the antibody heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and the antibody light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 67; and (ii) the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73, and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 74.

[0043] 33. The method of any one of aspects 1 and embodiments 2-32, wherein (i) the antibody heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 15 and the antibody light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 16, or wherein the antibody heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 66 and the antibody light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 67; and (ii) the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 82, and the second fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 83.

[0044] 34. The method of any one of claims 1 to 28 and 42 to 47, wherein the antibody heavy chain and the antibody light chain form a binding site that specifically binds to (human) FAP, the antibody heavy chain having the amino acid sequence of SEQ ID NO: 141, the light chain having the amino acid sequence of SEQ ID NO: 142, the first fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 79, and the second fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 80.

[0045] 35. The method of aspect 1 or any one of embodiments 2-28, wherein the antigen is CEA.

[0046] 36. The method of aspect 1 and any one of embodiments 2 to 28 and 35, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to CEA, and comprise a VH domain that comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 84, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 85, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 86, and a VL domain that comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 87, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 89.

[0047] 37. The method of aspect 1 and any one of embodiments 2 to 28 and 35 or 36, wherein the variable domains of the antibody heavy chain and the antibody light chain form binding sites that specifically bind to CEA, and comprise a variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 90 and a variable light chain domain comprising the amino acid sequence of SEQ ID NO: 91.

[0048] 38. The antibody-multimer fusion comprises: (i) a heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 91; (ii) a first fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73; (iii) a second fusion polypeptide comprising the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72, and SEQ ID NO: 74; The method of aspect 1 and any one of embodiments 2-28 and 35-37, comprising:

[0049] 39. The antibody-multimer fusion comprises: (i) a heavy chain comprising a VH domain comprising the amino acid sequence of SEQ ID NO: 90 and a light chain comprising a VL domain comprising the amino acid sequence of SEQ ID NO: 91; (ii) a first fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, and SEQ ID NO: 82; (iii) a second fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, and SEQ ID NO: 83; The method of aspect 1 and any one of embodiments 2-28 and 35-38, comprising:

[0050] 40. The antibody-multimer fusion comprises: (i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 93, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 94, and two light chains comprising the amino acid sequence of SEQ ID NO: 92, or (ii) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 95, a second heavy chain comprising the amino acid sequence of SEQ ID NO: 96, and two light chains comprising the amino acid sequence of SEQ ID NO: 92; 40. The method of aspect 1 and any one of embodiments 2-28 and 35-39, comprising:

[0051] 41. The method of aspect 1 or any one of embodiments 2-28 and 35-40, wherein the antibody heavy chain and the antibody light chain form a binding site that specifically binds to (human) CEA, wherein the antibody heavy chain has the amino acid sequence of SEQ ID NO: 143, the light chain has the amino acid sequence of SEQ ID NO: 92, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 79, and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 80.

[0052] 42. The method of aspect 1 or any one of embodiments 2-28, wherein the antigen is CD19.

[0053] 43. The method of aspect 1 or any one of embodiments 2 to 28 and 42, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to CD19, and comprise a VH domain that comprises (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 104 or SEQ ID NO: 105, (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 106 or SEQ ID NO: 107, and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 108 or SEQ ID NO: 109, and a VL domain that comprises (iv) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 110 or SEQ ID NO: 111, (v) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 112 or SEQ ID NO: 113, and (vi) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 115.

[0054] 44. The method of aspect 1 or any one of embodiments 2 to 28 and 42 or 43, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to CD19, and comprise a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 116 and a variable light domain comprising the amino acid sequence of SEQ ID NO: 117, or comprise a variable heavy domain comprising the amino acid sequence of SEQ ID NO: 118 and a variable light domain comprising the amino acid sequence of SEQ ID NO: 119.

[0055] 45. (i) the heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 116 and the light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 117, or the heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and the light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 119; (ii) the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 68, SEQ ID NO: 71, and SEQ ID NO: 73; (iii) The method of any one of aspects 1 or embodiments 2 to 28 and 42 to 44, wherein the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 14, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74.

[0056] 46. ​​(i) the heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 116 and the light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 117, or the heavy chain comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 118 and the light chain comprises a VL domain comprising the amino acid sequence of SEQ ID NO: 119; (ii) the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, and SEQ ID NO:82; (iii) The method of aspect 1 or any one of embodiments 2 to 28 and 42 to 45, wherein the second fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80 and SEQ ID NO: 83.

[0057] 47. (i) the heavy chain comprises the amino acid sequence of SEQ ID NO: 120, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 121, and the light chain comprises the amino acid sequence of SEQ ID NO: 122; or (ii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 123, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 124, and the light chain comprises the amino acid sequence of SEQ ID NO: 122; or (iii) the heavy chain comprises the amino acid sequence of SEQ ID NO: 125, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 126, and the light chain comprises the amino acid sequence of SEQ ID NO: 127; or (iv) The method of aspect 1 or any one of embodiments 2 to 28 and 42 to 45, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 128, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 129, and the light chain comprises the amino acid sequence of SEQ ID NO: 127.

[0058] 48. The method of aspect 1 or any one of embodiments 2-28 and 42-47, wherein the antibody heavy chain and the antibody light chain form a binding site that specifically binds to (human) CD19, wherein the antibody heavy chain has the amino acid sequence of SEQ ID NO: 144, the light chain has the amino acid sequence of SEQ ID NO: 127, the first fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 79, and the second fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 80.

[0059] 49. The method of aspect 1 or any one of embodiments 2 to 48, wherein transfecting said (parent) mammalian cells is targeted integration transfection.

[0060] 50. The method of embodiment 49, wherein the targeted integration transfection is a double recombinase-mediated cassette exchange.

[0061] 51. The method of embodiment 49 or 50, wherein the (parent) mammalian cell is a CHO cell with the landing site integrated at a single site within a locus in its genome.

[0062] 52. The method of embodiment 51, wherein the landing site comprises a first selection marker and a second selection marker flanked by two RRSs, and the first selection marker is different from the second selection marker.

[0063] 53. The method of embodiment 52, wherein the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein.

[0064] 54. The method of embodiment 52, wherein the integrated landing site comprises a thymidine kinase selectable marker and a HYG selectable marker.

[0065] 55. The method of any one of embodiments 52 to 54, wherein the two RRSs flanking both selectable markers are different.

[0066] 56. The method of any one of embodiments 51 to 55, wherein the landing site comprises three heterospecific loxP sites for Cre recombinase-mediated DNA recombination.

[0067] 57. The method of embodiment 56, wherein the heterospecific loxP sites are L3, LoxFas, and 2L.

[0068] 58. The method of embodiment 57, wherein the L3 and 2L are adjacent to the landing site at the 5' and 3' ends, respectively, and LoxFas is located between the L3 and 2L sites.

[0069] 59. The method of any one of embodiments 51 to 58, wherein the landing site further comprises a bicistronic unit linking expression of an IRES-mediated selection marker to expression of a fluorescent GFP protein.

[0070] 60. The antibody-multimeric fusion polypeptide comprises, from 5' to 3': - a first expression cassette encoding said first fusion polypeptide; - a second expression cassette encoding said first fusion polypeptide; - a third expression cassette encoding said second fusion polypeptide; - a fourth expression cassette encoding said antibody heavy chain; - a fifth expression cassette encoding the antibody light chain; 60. The method of aspect 1 or any one of embodiments 2-59, wherein the nucleic acid sequence is expressed from a deoxyribonucleic acid integrated into the genome of said cell, comprising:

[0071] 61. The method of aspect 1 or any one of embodiments 2 to 60, wherein the deoxyribonucleic acid encoding the antibody-multimeric fusion polypeptide is stably integrated into the genome of the mammalian cell at a single site or locus.

[0072] 62. The deoxyribonucleic acid encoding the antibody-multimer fusion polypeptide comprises: - a first recombination recognition sequence located 5' to the first (5'-most) expression cassette; - a second recombination recognition sequence located 3' to the fifth (3'-most) expression cassette; - a third recombination recognition sequence, - between the first recombination recognition sequence and the second recombination recognition sequence, and - a third recombination recognition sequence located between the third expression cassette and the fourth expression cassette; Further comprising: 62. The method of embodiment 60 or 61, wherein all recombination recognition sequences are different.

[0073] 63. The method of any one of embodiments 60 to 62, wherein the deoxyribonucleic acid encoding the antibody-multimeric fusion polypeptide further comprises an additional expression cassette encoding a selectable marker.

[0074] 64. The expression cassette encoding a selectable marker is linked to the third recombination recognition sequence: i) 5', or ii) 3', or iii) Partially 5' and partially 3' 64. The method of embodiment 63, wherein the .alpha.-.alpha.

[0075] 65. The method of embodiment 63 or 64, wherein the expression cassette encoding a selectable marker is located partially 5' and partially 3' to the third recombination recognition sequence, the 5'-located portion of the expression cassette comprises a promoter and a start codon, and the 3'-located portion of the expression cassette comprises a coding sequence without a start codon and a polyA signal.

[0076] 66. The method of any one of embodiments 60 to 65, wherein the deoxyribonucleic acid encoding the antibody-multimer fusion polypeptide comprises a further expression cassette encoding a selectable marker, wherein the expression cassette encoding the selectable marker is located partially 5' and partially 3' to the third recombination recognition sequence, wherein the 5'-located portion of the expression cassette comprises a promoter and a start codon, and the 3'-located portion of the expression cassette comprises a coding sequence without a start codon and a polyA signal, and wherein the start codon is operably linked to the coding sequence.

[0077] 67. The method of embodiment 65 or 66, wherein the start codon is ATG. DETAILED DESCRIPTION OF THE INVENTION

[0078] Detailed Description of Embodiments of the Invention The present invention is based, at least in part, on the discovery that for expression of antibody-multimer fusions, which are complex molecules comprising different polypeptides, i.e., heteromultimers, the use of defined expression cassette ratios results in efficient expression and production of antibody-multimer fusions in mammalian cells, such as HEK or CHO cells.

[0079] The present invention is based, at least in part, on the discovery that for transient and stable expression of antibody-multimer fusions that are complex molecules comprising different polypeptides, i.e., heteromultimers, the use of the same defined expression cassette ratios results in the highest expression yields and product quality.

[0080] I. Definition As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as set forth in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat." Specifically, the Kabat numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which is further clarified in this case by saying "numbering is according to the Kabat EU index").

[0081] Knob-into-hole dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway JBB; Presta LG: Immunotechnology 2 (1996) 73-73(1).

[0082] General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0083] Useful methods and techniques for practicing the present invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, ND, and Hames, BD, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture—a practical approach, IRL Press Limited (1986); Watson, JD, et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, EL, From Genes to Clones, NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., NY (1987).

[0084] The use of recombinant DNA technology makes it possible to produce derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion, or insertion, at individual or several nucleotide positions. Modification or derivatization can be carried out, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization—a practical approach (1985) IRL Press, Oxford, England).

[0085] It should be noted that as used in this specification and the accompanying embodiments, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" can be used interchangeably.

[0086] The term "about" refers to a range of + / - 20% of the preceding numerical value. In one embodiment, the term about refers to a range of + / - 10% of the preceding numerical value. In one embodiment, the term about refers to a range of + / - 5% of the preceding numerical value.

[0087] The term "comprising" also includes the term "consisting of."

[0088] As used herein, the term "recombinant cell" refers to a cell after final genetic modification, e.g., a cell that expresses a polypeptide of interest and can be used for the production of said polypeptide of interest on any scale. For example, a "mammalian cell comprising an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), thereby introducing the coding sequence for the polypeptide of interest into the genome of the host cell, is a "recombinant cell." The cell is still capable of performing further RMCE reactions, but it is not intended to do so.

[0089] As used herein, the term "recombinant mammalian cell" refers to a mammalian cell containing an exogenous nucleotide sequence capable of expressing a polypeptide. Such a recombinant mammalian cell is a cell into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such a cell. Thus, the term "mammalian cell containing a nucleic acid encoding a heterologous polypeptide" refers to a cell containing an exogenous nucleotide sequence that has been integrated into the genome of the mammalian cell and is capable of expressing a heterologous polypeptide. In one embodiment, a mammalian cell containing an exogenous nucleotide sequence is a cell containing an exogenous nucleotide sequence integrated into a single site within a locus in the genome of the host cell, wherein the exogenous nucleotide sequence comprises first and second recombination recognition sequences flanking at least one first selectable marker, and a third recombination recognition sequence located between the first and second recombination recognition sequences, and wherein the recombination recognition sequences are all different.

[0090] Both "mammalian cells containing an exogenous nucleotide sequence" and "recombinant cells" are "transformed cells." This term includes the primary transformed cell and progeny derived therefrom, regardless of the number of transfers. The progeny may not be completely identical to the parent cell in nucleic acid content, for example, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0091] The term "integration site" refers to a nucleic acid sequence within a cell's genome into which an exogenous nucleotide sequence is inserted. In certain embodiments, the integration site is between two adjacent nucleotides in the cell's genome. In certain embodiments, the integration site comprises a stretch of nucleotide sequence. In certain embodiments, the integration site is located within a specific locus in the genome of a mammalian cell. In certain embodiments, the integration site is within an endogenous gene of a mammalian cell.

[0092] The terms "vector" or "plasmid" can be used interchangeably and, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term encompasses vectors as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of a nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0093] The term "binding" refers to the binding of a binding site to its target. For example, the binding of an antibody binding site comprising an antibody heavy chain variable domain and an antibody light chain variable domain to its respective antigen. This binding can be measured, for example, using a BIAcore® assay (GE Healthcare, Uppsala, Sweden). That is, the term "binding (to an antigen)" refers to the binding of an antibody to its antigen(s) in an in vitro assay. In one embodiment, binding is determined in a binding assay in which the antibody is bound to a surface and the binding of the antigen to the antibody is measured by surface plasmon resonance (SPR). Binding refers to, for example, binding of an antibody to its antigen(s). -8 M or less, in some embodiments 10 -13 ~10 -8 M, in some embodiments 10 -13 ~10 -9 Binding affinity (K D The term "bind" also includes the term "specifically bind."

[0094] For example, in one possible embodiment of a BIAcore® assay, an antigen is bound to a surface and the binding of the antibody, i.e., its binding site(s), is measured by surface plasmon resonance (SPR). The affinity of binding is measured by the term k a (association constant: rate constant for association to form a complex), k d (dissociation constant, rate constant for dissociation of the complex), and K D (k d / k a Alternatively, the binding signal of an SPR sensorgram can be directly compared with the response signal of a reference in terms of resonance signal height and dissociation behavior.

[0095] The term "binding site" refers to any proteinaceous entity that exhibits binding specificity to a target. It may be, for example, a receptor, a receptor ligand, anticalin, an affibody, an antibody, etc. Thus, the term "binding site" as used herein refers to a polypeptide that is capable of specifically binding to or being specifically bound by a second polypeptide.

[0096] As used herein, the term "exogenous" refers to a nucleotide sequence that is not native to a particular cell but is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. Thus, an exogenous nucleotide sequence is an artificial sequence, and artifacts can result, for example, from the combination of subsequences of different origins (e.g., the combination of a recombinase recognition sequence with an SV40 promoter and a coding sequence for green fluorescent protein is an artificial nucleic acid), or from partial deletion or nucleic acid base mutation of a sequence (e.g., a sequence or cDNA encoding only the extracellular domain of a membrane-bound receptor). The term "endogenous" refers to a nucleotide sequence that originates from a cell. An "exogenous" nucleotide sequence may have an "endogenous" counterpart with identical base composition, but an "exogenous" sequence has been introduced into a cell, for example, by recombinant DNA technology.

[0097] As used herein, the term "selection marker" refers to a gene that allows cells carrying a gene to be specifically selected or specifically eliminated in the presence of a corresponding selection agent. For example, but not limited to, a selection marker can allow host cells transformed with the selection marker gene to be positively selected in the presence of the respective selection agent (selective culture conditions), while untransformed host cells cannot grow or survive under selective culture conditions. A selection marker can be positive, negative, or bifunctional. A positive selection marker can allow selection of cells carrying the marker, while a negative selection marker can allow selective elimination of cells carrying the marker. A selection marker can confer resistance to a drug in a host cell or complement a metabolic or catabolic defect. In prokaryotic cells, genes that confer resistance to ampicillin, tetracycline, kanamycin, or chloramphenicol, among others, can be used. Resistance genes useful as selectable markers in eukaryotic cells include, but are not limited to, genes for aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D)), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Additional marker genes are described in WO 92 / 08796 and WO 94 / 28143.

[0098] In addition to facilitating selection in the presence of a corresponding selection agent, a selection marker may alternatively be a molecule not normally present in cells, such as green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire. Cells expressing such molecules can be distinguished from cells that do not harbor the gene based, for example, on the detection or absence, respectively, of fluorescence emitted by the encoded polypeptide.

[0099] The term "fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any native FAP derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed FAPs and any form of FAP resulting from processing in cells. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 17) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid sequence and nucleotide sequence of His-tagged human FAP ECD are shown in SEQ ID NO: 14 and SEQ ID NO: 15, respectively. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 18) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NO: 19 and SEQ ID NO: 20 show the amino acid sequence and nucleotide sequence, respectively, of His-tagged mouse FAP ECD. SEQ ID NO: 21 and SEQ ID NO: 22 show the amino acid sequence and nucleotide sequence, respectively, of His-tagged cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecules of the present invention bind to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in WO 2012 / 020006.

[0100] The term "TNF ligand family member" or "TNF family ligand" refers to a pro-inflammatory cytokine. Cytokines in general, and members of the TNF ligand family in particular, play important roles in stimulating and regulating the immune system. Currently, 19 cytokines have been identified as members of the TNF (tumor necrosis factor) ligand superfamily based on sequence, functional, and structural similarities. All of these ligands are type II transmembrane proteins with a C-terminal extracellular domain (ectodomain), an N-terminal intracellular domain, and a single transmembrane domain. The C-terminal extracellular domain, known as the TNF homology domain (THD), shares 20-30% amino acid identity among superfamily members and is responsible for receptor binding. The TNF ectodomain also carries the TNF ligands, forming trimeric complexes that are recognized by their specific receptors.

[0101] Members of the TNF ligand family include lymphotoxin alpha (also known as LTA or TNFSF1), TNF (also known as TNFSF2), LTβ (also known as TNFSF3), OX40L (also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL (also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70 or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1-BBL (also known as TNFSF9), TRAIL (APO2L, CD253 or TNFSF1), and OX40L (also known as TNFSF1). 10), RANKL (also known as CD254 or TNFSF11), TWEAK (also known as TNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also known as CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A (also known as VEGI or TNFSF15), GITRL (also known as TNFSF18), EDA-A1 (also known as ectozyplasin A1), and EDA-A2 (also known as ectozyplasin A2). Unless otherwise specified, the term refers to a natural TNF family ligand from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). In specific embodiments of the invention, the TNF ligand family member is selected from the group consisting of OX40L, FasL, CD27L, TRAIL, 4-1-BBL, CD40L and GITRL. In certain embodiments, the TNF ligand family member is selected from 4-1-BBL and OX40L.

[0102] Further information on TNF ligand family members, particularly sequences, can be obtained from publicly accessible databases such as UniProt (www.uniprot.org). For example, human TNF ligands have the following amino acid sequences: human lymphotoxin alpha (UniProt Accession No. P01374, SEQ ID NO: 24), human TNF (UniProt Accession No. P01375, SEQ ID NO: 25), human lymphotoxin alpha (UniProt Accession No. Q06643, SEQ ID NO: 26), human OX40L (UniProt Accession No. P23510, SEQ ID NO: 27), human CD40L (UniProt Accession No. P29965, SEQ ID NO: 28), human FasL (UniProt Accession No. P48023, SEQ ID NO: 29), human CD27L (UniProt Accession No. P32970, SEQ ID NO: 30), human CD30L (UniProt Accession No. P32971, SEQ ID NO: 31), 4-1-BBL (UniProt Accession No. P32972, SEQ ID NO: 32), and 4-1-BBL (UniProt Accession No. P32973, SEQ ID NO: 33). accession number P41273, SEQ ID NO: 32), TRAIL (UniProt accession number P50591, SEQ ID NO: 33), RANKL (UniProt accession number O14788, SEQ ID NO: 34), TWEAK (UniProt accession number O43508, SEQ ID NO: 35), APRIL (UniProt accession number O75888, SEQ ID NO: 36), BAFF (UniProt accession number Q9Y275, SEQ ID NO: 37), LIGHT (UniProt accession number O43557, SEQ ID NO: 38), TL1A (UniProt accession number O95150, SEQ ID NO: 39), GITRL (UniProt accession number Q9UNG2, SEQ ID NO: 40), and ectodiplaslin A (UniProt accession number Q92838, SEQ ID NO: 41).

[0103] An "ectodomain" is a domain of a membrane protein that extends into the extracellular space (i.e., the space outside a target cell). The ectodomain is usually the portion of a protein that initiates contact with a surface, resulting in signal transduction. Thus, the ectodomain of a TNF ligand family member as defined herein refers to the portion of the TNF ligand protein that extends into the extracellular space (the extracellular domain), but also includes shorter portions or fragments thereof that are responsible for trimerization and binding to the corresponding TNF receptor. Thus, the term "ectodomain of a TNF ligand family member or fragment thereof" refers to the extracellular domain of a TNF ligand family member that forms the extracellular domain or the portion thereof that is still capable of binding to the receptor (receptor-binding domain).

[0104] The term "costimulatory TNF ligand family member" or "costimulatory TNF family ligand" refers to a subgroup of TNF ligand family members that can costimulate T cell proliferation and cytokine production. These TNF family ligands can costimulate TCR signaling upon interaction with their corresponding TNF receptors, and interaction with these receptors results in the recruitment of TNFR-associated factors (TRAFs) that initiate a signaling cascade leading to T cell activation. The costimulatory TNF family ligand is selected from the group consisting of 4-1-BBL, OX40L, GITRL, CD70, CD30L, and LIGHT; more particularly, the costimulatory TNF ligand family member is selected from 4-1-BBL and OX40L.

[0105] As previously described herein, 4-1-BBL is a type II transmembrane protein and a member of the TNF ligand family. It has been described that intact or full-length 4-1-BBL, having the amino acid sequence of SEQ ID NO: 32, forms trimers on the surface of cells. Trimer formation is enabled by a specific motif in the ectodomain of 4-1-BBL. This motif is referred to herein as the "trimerization region." Amino acids 50 to 254 of the human 4-1-BBL sequence (SEQ ID NO: 42) form the ectodomain of 4-1-BBL, although even fragments thereof can form trimers. In specific embodiments of the present invention, the term "ectodomain of 4-1-BBL or a fragment thereof" refers not only to a polypeptide having an amino acid sequence selected from SEQ ID NO: 04 (amino acids 52 to 254 of human 4-1-BBL), SEQ ID NO: 01 (amino acids 71 ​​to 254 of human 4-1-BBL), SEQ ID NO: 03 (amino acids 80 to 254 of human 4-1-BBL), and SEQ ID NO: 02 (amino acids 85 to 254 of human 4-1-BBL), or to a polypeptide having an amino acid sequence selected from SEQ ID NO: 56 (amino acids 71 ​​to 248 of human 4-1-BBL), SEQ ID NO: 102 (amino acids 52 to 248 of human 4-1-BBL), SEQ ID NO: 101 (amino acids 80 to 248 of human 4-1-BBL), and SEQ ID NO: 100 (amino acids 85 to 248 of human 4-1-BBL), but also to other fragments of the ectodomain capable of trimerization.

[0106] As previously described herein, OX40L is another type II transmembrane protein and a further member of the TNF ligand family. Full-length or full-length human OX40L has the amino acid sequence of SEQ ID NO:27. Amino acids 51-183 of the OX40L sequence (SEQ ID NO:43) form the extracellular domain of OX40L, although even fragments thereof can form trimers. In specific embodiments of the invention, the term "ectodomain of OX40L or a fragment thereof" refers to a polypeptide having an amino acid sequence selected from SEQ ID NO:43 (amino acids 51-183 of human OX40L) or SEQ ID NO:44 (amino acids 52-183 of human OX40L), although other fragments of the ectodomain capable of trimerization are also encompassed herein.

[0107] The term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides are, for example, (G4S)n, (SG4)n, or G4(SG4)n peptide linkers, where "n" is generally a number between 1 and 10, typically between 1 and 4, and particularly 2, i.e., selected from the group consisting of GGGGS (SEQ ID NO:81), GGGGSGGGGS (SEQ ID NO:12), SGGGGSGGGG (SEQ ID NO:45), and GGGGSGGGGSGGGG (SEQ ID NO:46), but also including GSPGSSSSGS (SEQ ID NO:47), GSGSGSGS (SEQ ID NO:48), GSGSGNGS (SEQ ID NO:49), GGSGSGSG (SEQ ID NO:50), GGSGSG (SEQ ID NO:51), GGSG (SEQ ID NO:52), GGSGNGSG (SEQ ID NO:53), GGNGSGSG (SEQ ID NO:54), and GGNGSG (SEQ ID NO:55). Particularly interesting peptide linkers are (G4S)1 or GGGGS (SEQ ID NO: 81), (G4S)2 or GGGGSGGGGS (SEQ ID NO: 12) and GSPGSSSSGS (SEQ ID NO: 47), more particularly (G4S)2 or GGGGSGGGGS (SEQ ID NO: 12) and GSPGSSSSGS (SEQ ID NO: 47).

[0108] By "fused" or "connected" is meant that the components (e.g., the polypeptide and ectodomain of the TNF ligand family member) are linked by a peptide bond directly or via one or more peptide linkers.

[0109] General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0110] The term "heavy chain" is used herein in its original sense, i.e., to refer to the two larger of the four polypeptide chains that form an antibody (see, e.g., Edelman, GM and Gally JA, J. Exp. Med. 116 (1962) 207-227). The term "larger" in this context can refer to either molecular weight, length, or number of amino acids. The term "heavy chain" is independent of the sequence and number of individual antibody domains present therein. It is assigned solely on the basis of the molecular weight of the respective polypeptide.

[0111] The term "light chain" is used herein in its original sense, i.e., to refer to the smaller of the four polypeptide chains that form an antibody (see, e.g., Edelman, GM and Gally JA, J. Exp. Med. 116 (1962) 207-227). The term "smaller" in this context can refer to either molecular weight, length, or number of amino acids. The term "light chain" is independent of the sequence and number of individual antibody domains present therein. It is assigned solely on the basis of the molecular weight of the respective polypeptide.

[0112] As used herein, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbering system as set forth in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), and are referred to herein as "numbering according to Kabat." Specifically, the Kabat numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 647-660) is used for the light chain constant domains CL of kappa and lambda isotypes, and the Kabat EU index numbering system of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see pages 661-723) is used for the heavy chain constant domains (CH1, hinge, CH2, and CH3, which is further clarified in this specification by saying that the numbering is according to the Kabat EU index).

[0113] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, full-length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment fusions, and combinations thereof.

[0114] The term "antibody binding site" refers to a pair of heavy-chain variable domain and light-chain variable domain. To ensure proper binding to an antigen, these variable domains are cognate, i.e., belong together. An antibody binding site comprises at least three HVRs (e.g., in the case of a VHH) or three to six HVRs (e.g., in the case of a naturally occurring, i.e., conventional antibody with a VH / VL pair). Generally, the amino acid residues of an antibody involved in antigen binding form the binding site. These residues are usually contained in a pair of an antibody heavy-chain variable domain and a corresponding antibody light-chain variable domain. An antibody antigen-binding site comprises amino acid residues from "hypervariable regions" or "HVRs." "Framework" or "FR" regions are variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from the N-terminus to the C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy-chain variable domain is the region that contributes most to antigen binding and defines the binding specificity of the antibody. A "functional binding site" is capable of specifically binding to its target. The term "specifically binds" refers to the binding of a binding site to its target in an in vitro assay, in one embodiment, in a binding assay. Such a binding assay can be any assay as long as a binding event can be detected. For example, a binding assay in which an antibody is bound to a surface and the binding of antigen(s) to the antibody is measured by surface plasmon resonance (SPR). Alternatively, a bridging ELISA can be used.

[0115] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain comprising stretches of amino acid residues which are hypervariable in sequence ("complementarity determining regions" or "CDRs") and / or regions of an antibody variable domain which form structurally defined loops ("hypervariable loops") and / or regions of an antibody variable domain containing residues which contact the antigen ("antigen contacts"). Typically, antibodies comprise six HVRs: three in the heavy chain variable domain, VH (H1, H2, H3) and three in the light chain variable domain, VL (L1, L2, L3).

[0116] HVR includes: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c) comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0117] Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0118] The "class" of an antibody refers to the type of constant domain or constant region, preferably the Fc region, possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0119] The term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain that contains the constant domains, i.e., the CH1 domain, hinge region, CH2 domain, and CH3 domain in the case of a native immunoglobulin, or the first constant domain, hinge region, second constant domain, and third constant domain in the case of a full-length immunoglobulin. In one embodiment, a human IgG heavy chain constant region extends from Ala118 to the carboxyl terminus of the heavy chain (numbering according to the Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to the Kabat EU index). The term "constant region" refers to a dimer comprising two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0120] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the hinge region (middle and lower hinge regions), the second constant domain (e.g., CH2 domain), and the third constant domain (e.g., CH3 domain). In one embodiment, a human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (numbering according to the Kabat EU index). Thus, the Fc region is smaller than the constant region but at the same C-terminal end. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (numbering according to the Kabat EU index). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0121] The constant region of an antibody, more precisely the Fc region (and similarly the constant region), is directly involved in complement activation, C1q binding, C3 activation and Fc receptor binding. The effect of an antibody on the complement system depends on the specific conditions, but binding to C1q is caused by a defined binding site in the Fc region. Such binding sites are known in the prior art and are described, for example, in Lukas, TJ, et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, JJ, Mol. Immunol. 16 (1979) 907-917; Burton, DR, et al., Nature 288 (1980) 338-344; Thommesen, JE, et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE, 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 European Patent No. 0307434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat). Antibodies of the subclasses IgG1, IgG2, and IgG3 typically exhibit complement activation, C1q binding, and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q, and does not activate C3. The term "Fc region of an antibody" is well known to those skilled in the art and is defined based on papain cleavage of an antibody.

[0122] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the exception of possible variant antibodies that contain, for example, naturally occurring mutations or that arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci.

[0123] The term "valency," as used herein, refers to the presence of a particular number of binding sites in an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antibody.

[0124] A "monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2), or combinations thereof (e.g., full-length antibodies with additional scFv or Fab fragments). Monospecific antibodies need not be monovalent; i.e., they may contain two or more binding sites that specifically bind to one antigen. For example, naturally occurring antibodies are monospecific but bivalent.

[0125] The "knob-into-hole" dimerization module and its use in antibody engineering is described in Carter P.; Ridgway JBB; Presta LG: Immunotechnology 2 (1996) 73-73(1).

[0126] The CH3 domains of antibody heavy chains can be modified using the "knob-into-hole" technique. This technique is described in detail, with some examples, in, for example, WO 96 / 027011; Ridgway, JB, et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM, et al., Nat. Biotechnol. 16 (1998) 677-681. This method involves modifying the interaction surfaces of two CH3 domains to increase heterodimerization of these two CH3 domains, thereby increasing heterodimerization of polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) can be a "knob," and the other a "hole." The introduction of disulfide bridges further stabilizes the heterodimer (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681; Atwell, S., et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0127] The mutation T366W in the CH3 domain (of an antibody heavy chain) is designated as a "knob mutation" or "mutated knob," and the mutations T366S, L368A, and Y407V in the CH3 domain (of an antibody heavy chain) are designated as "hole mutations" or "mutated hole" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, AM, et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a S354C mutation in the CH3 domain of a heavy chain with a "knob mutation" (designated as a "knob-cys-mutation" or "mutated knob-cys") and a Y349C mutation in the CH3 domain of a heavy chain with a "hole mutation" (designated as a "hole-cys-mutation" or "mutated hole-cys") (numbering according to the EU index of Kabat).

[0128] The term "domain crossover", as used herein, means that in a pair of antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., antibody Fab (fragment antigen binding), the domain sequence deviates from that of the native antibody in that at least one heavy chain domain is replaced by the corresponding light chain domain, or vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and the domain crossover in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain having a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and the domain crossover in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of a complete light chain (VL-CL) and a complete VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and the domain crossover results in a heavy chain fragment with a VL-CL domain sequence (all domain sequences listed above are in the N-terminal to C-terminal direction).

[0129] As used herein, the term "replaced by one another" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when the CH1 and CL domains are "replaced by one another," the term refers to the domain crossover described under item (i) and the resulting heavy and light chain domain sequences. Thus, when the VH and VL are "replaced by one another," the term refers to the domain crossover described under item (ii), and when the CH1 and CL domains are "replaced by one another" and the VH and VL domains are "replaced by one another," the term refers to the domain crossover described under item (iii). Bispecific antibodies containing domain crossovers have been reported, for example, in International Publication Nos. 2009 / 080251, 2009 / 080252, 2009 / 080253, and 2009 / 080254, and Schaefer, W., et al., Proc. Natl. Acad. Sci USA 108 (2011) 11187-11192. Such antibodies are generally referred to as CrossMabs.

[0130] In one embodiment, the multispecific antibody also comprises at least one Fab fragment comprising either the domain crossover of the CH1 and CL domains described in the above item (i), or the domain crossover of the VH and VL domains described in the above item (ii), or the domain crossover of the VH-CH1 and VL-VL domains described in the above item (iii). In the case of a multispecific antibody with domain crossover, Fabs that specifically bind to the same antigen(s) are constructed to have the same domain sequence. Therefore, when more than one Fab with domain crossover is included in a multispecific antibody, the Fab(s) specifically bind to the same antigen.

[0131] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0132] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric, humanized, and human) that are prepared, expressed, produced, or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, and CHO cells.

[0133] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabody, linear antibody, and single-chain antibody molecules (e.g., scFv or scFab).

[0134] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabody, linear antibody, and single-chain antibody molecules (e.g., scFv or scFab).

[0135] "Target cell antigen," as used herein, refers to an antigenic determinant displayed on the surface of a target cell, e.g., a cell within a tumor, such as a tumor cell or a cell of the tumor stroma. In certain embodiments, the target cell antigen is an antigen on the surface of a tumor cell. In one embodiment, the target cell antigen is selected from the group consisting of fibroblast activation protein (FAP), carcinoembryonic antigen (CEA), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), CD19, CD20, and CD33. Specifically, the target cell antigen is fibroblast activation protein (FAP).

[0136] The term "CD19" refers to the B lymphocyte antigen CD19, also known as B lymphocyte surface antigen B4 or T cell surface antigen Leu-12, and includes any native CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CD19 is set forth in UniProt Accession No. P15391 (Version 160, SEQ ID NO: 103). This term encompasses "full-length," unprocessed human CD19, as well as any form of human CD19 resulting from processing in cells, so long as the antibodies described herein bind to it. CD19 is a structurally distinct cell surface receptor expressed on the surface of human B cells, including, but not limited to, pre-B cells, early developmental B cells (i.e., immature B cells), mature B cells through terminal differentiation to plasma cells, and malignant B cells. CD19 is expressed by most pre-B acute lymphoblastic leukemias (ALL), non-Hodgkin's lymphomas, B-cell chronic lymphocytic leukemia (CLL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphocytic leukemia, and some null acute lymphoblastic leukemias. Expression of CD19 on plasma cells further suggests that it may be expressed on differentiated B-cell tumors such as multiple myeloma. Therefore, the CD19 antigen is a target for immunotherapy in the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and / or acute lymphoblastic leukemia.

[0137] The term "fibroblast activation protein (FAP)," also known as prolyl endopeptidase FAP or seprase (EC 3.4.21), refers to any native FAP derived from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses "full-length," unprocessed FAPs and any form of FAP resulting from processing in cells. The term also encompasses naturally occurring variants of FAPs, such as splice variants or allelic variants. In one embodiment, the antigen-binding molecule of the present invention is capable of specifically binding to human, mouse, and / or cynomolgus monkey FAP. The amino acid sequence of human FAP is shown in UniProt (www.uniprot.org) accession number Q12884 (version 149, SEQ ID NO: 17) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_004451.2. The extracellular domain (ECD) of human FAP extends from amino acid position 26 to 760. The amino acid and nucleotide sequences of His-tagged human FAP ECD are shown in SEQ ID NOs: 14 and 15, respectively. The amino acid sequence of mouse FAP is shown in UniProt accession number P97321 (version 126, SEQ ID NO: 18) or NCBI RefSeq NP_032012.1. The extracellular domain (ECD) of mouse FAP extends from amino acid position 26 to 761. SEQ ID NOs: 19 and 20 show the amino acid and nucleotide sequences, respectively, of His-tagged mouse FAP ECD. SEQ ID NOs: 21 and 22 show the amino acid sequence and nucleotide sequence, respectively, of His-tagged cynomolgus monkey FAP ECD. Preferably, the anti-FAP binding molecules of the present invention bind to the extracellular domain of FAP. Exemplary anti-FAP binding molecules are described in WO 2012 / 020006.

[0138] The term "carcinoembryonic antigen (CEA)," also known as carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), refers to any naturally occurring CEA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The amino acid sequence of human CEA is set forth in UniProt accession number P06731 (version 151, SEQ ID NO: 23). CEA has long been identified as a tumor-associated antigen (Gold and Freedman, J Exp. Med., 121:439-462, 1965; Berinstein NL, J Clin. Oncol., 20:2197-2207, 2002). Originally classified as a protein expressed exclusively in fetal tissues, CEA has now been identified in several healthy adult tissues. These tissues are primarily epithelial in origin, including cells of the gastrointestinal, respiratory, and urinary tracts, as well as cells of the colon, cervix, sweat glands, and prostate (Nap et al., Tumor Biol., 9 (1988) 145-153; Nap et al., Cancer Res., 52 (1992) 2329-2339). Epithelial-derived tumors and their metastases contain CEA as a tumor-associated antigen. The presence of CEA itself does not imply transformation into cancerous cells, but it does indicate that CEA is distributed. In healthy tissues, CEA is generally expressed on the apical surface of cells (Hammarstroem S., Semin. Cancer Biol. 9 (1999) 67-81), making it inaccessible to antibodies in the bloodstream. In contrast to healthy tissues, CEA is expressed over the entire surface of cancerous cells (Hammarstroem S., Semin. Cancer Biol. 9 (1999) 67-81). This change in expression pattern makes CEA more susceptible to antibody binding within cancerous cells. Furthermore, CEA expression increases in cancerous cells. Furthermore, increased CEA expression can increase cell-cell adhesion, leading to metastasis (Marshall J., Semin Oncol., 30 (2003) (a Suppl. 8) 30-36). The prevalence of CEA expression in various tumor entities is generally very high.In agreement with published data, our own analyses performed on tissue samples confirmed its high prevalence, approximately 95% in colon carcinoma (CRC), 90% in pancreatic cancer, 80% in gastric cancer, 60% in non-small cell lung cancer (NSCLC co-expressing HER3), and 40% in breast cancer, with lower expression in small cell lung cancer and glioblastoma.

[0139] CEA is rapidly cleaved from the cell surface and enters the bloodstream either directly from tumors or via the lymphatic system. Because of this property, serum CEA levels have been used as a clinical marker for cancer diagnosis and screening for cancer recurrence, particularly colorectal cancer (Goldenberg, D. M., Int. J. Biol. Mark. 7 (1992) 183-188; Chau I., et al., J. Clin. Oncol. 22 (2004) 420-1429; Flamini, et al., Clin. Cancer Res. 12 (2006) 6985-6988).

[0140] As used herein, the term "heterologous" indicates that a polypeptide is not native to a particular cell, but rather that the respective encoding nucleic acid is introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. A heterologous polypeptide is therefore an artificial polypeptide relative to the cell that expresses it, and therefore does not depend on whether the polypeptide is a naturally occurring polypeptide from a different cell / organism or an artificial polypeptide.

[0141] As used herein, the term "operably linked" refers to the juxtaposition of two or more components in a relationship permitting them to function in a desired manner. For example, a promoter and / or enhancer is operably linked to a coding sequence if it functions to regulate the transcription of the coding sequence. In certain embodiments, DNA sequences that are "operably linked" are contiguous and adjacent on a single chromosome. In certain embodiments, when it is necessary to join two protein coding regions, e.g., a secretory leader and a polypeptide, these sequences are contiguous, adjacent, and in the same reading frame. In certain embodiments, an operably linked promoter can be located upstream of and adjacent to the coding sequence. In certain embodiments, for example, with an enhancer sequence regulating expression of a coding sequence, two components can be operably linked even though they are not adjacent. An enhancer is operably linked to a coding sequence if it increases the transcription of the coding sequence. An operably linked enhancer can be located upstream, within, or downstream of a coding sequence, and can be located a substantial distance from the promoter of the coding sequence. Operable linkage can be achieved by recombinant methods known in the art, for example, using PCR methods and / or by ligation at convenient restriction sites. If convenient restriction sites are not present, synthetic oligonucleotide adapters or linkers can be used in accordance with conventional procedures. An internal ribosome entry site (IRES) is operably linked to an open reading frame (ORF) if the IRES is capable of initiating translation of the ORF at an internal location in a 5'-end-independent manner.

[0142] II. Compositions and Methods Typically, recombinant large-scale production of a polypeptide of interest, such as a therapeutic polypeptide, requires cells that stably express and secrete the polypeptide. These cells are called "recombinant cells" or "recombinant production cells," and the methods used to generate such cells are called "cell line development." In the first step of the cell line development process, suitable host cells, such as CHO cells, are transfected with a nucleic acid sequence suitable for expression of the polypeptide of interest. In the second step, cells that stably express the polypeptide of interest are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the polypeptide of interest.

[0143] A nucleic acid that encodes a polypeptide, i.e., a coding sequence, is called a structural gene. Such structural genes are simple information and require additional control elements for their expression. Therefore, structural genes are usually incorporated into expression cassettes. The minimum control elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream, i.e., 5', of the structural gene, and a polyadenylation signal sequence functional in mammalian cells located downstream, i.e., 3', of the structural gene. The promoter sequence, structural gene sequence, and polyadenylation signal sequence are arranged in an operably linked form.

[0144] If the polypeptide of interest is a heteromultimeric polypeptide composed of different (monomeric) polypeptides, not only one expression cassette but multiple expression cassettes containing different structural genes are required, i.e., at least one expression cassette for each of the different (monomeric) polypeptides of the heteromultimeric polypeptide. For example, an antibody-multimeric fusion polypeptide is a heteromultimeric polypeptide containing one light chain, one heavy chain, one heavy chain constant domain fusion polypeptide, and one light chain constant domain fusion polypeptide. Thus, an antibody-multimeric fusion polypeptide is composed of four different polypeptides. Therefore, expression of an antibody-multimeric fusion polypeptide requires four expression cassettes: one for the light chain, one for the heavy chain, one for the heavy chain constant region fusion polypeptide, and one for the light chain constant region fusion polypeptide.

[0145] The expression cassette(s) for the polypeptide of interest are in turn incorporated into so-called "expression vectors." An "expression vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene(s) in mammalian cells. Typically, an expression vector comprises a prokaryotic plasmid propagation unit, which in the case of, for example, E. coli, contains an origin of replication and a prokaryotic as well as a eukaryotic selection marker, as well as the expression cassette required for expression of the structural gene(s) of interest. An "expression vector" is a delivery vehicle for introducing an expression cassette into mammalian cells.

[0146] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the greater the number of different expression cassettes required. Essentially, the size of the nucleic acid to be integrated into the genome of the host cell increases with the number of expression cassettes. Concurrently, the size of the expression vector also increases. However, the practical upper limit of vector size is in the range of about 15 kbp, above which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors, whereby the expression cassette can be shared between different expression vectors, each containing only a portion of the expression cassette.

[0147] Generally, cell line development (CLD) relies on random integration (RI) or targeted integration (TI) of an expression cassette for a polypeptide of interest.

[0148] II.a Methods according to the invention The present invention is based, at least in part, on the discovery that for expression of antibody-multimer fusions, which are complex molecules comprising different polypeptides, i.e., heteromultimers, the use of defined expression cassette ratios results in efficient expression and production of antibody-multimer fusions in mammalian cells, such as CHO or HEK cells.

[0149] The present invention is based, at least in part, on the discovery that for transient and stable expression of antibody-multimer fusions that are complex molecules comprising different polypeptides, i.e., heteromultimers, the use of the same defined expression cassette ratios results in the highest expression yields and product quality.

[0150] The present invention is based, at least in part, on the discovery that for the expression of antibody-multimer fusions by recombinant mammalian cells, it is advantageous to use a ratio of expression cassettes for the antibody heavy chain, antibody light chain, first fusion polypeptide, and second fusion polypeptide that must be in a stoichiometric ratio of 1:1:2:1. By using this ratio, improved antibody-multimer fusion expression can be obtained with respect to yield and by-product formation. Furthermore, it has been found that this ratio is independent of the expression method, i.e., transient and stable cell lines can be obtained with the same ratio, as well as using vector configurations, i.e., single expression cassettes or multiple expression cassette vectors.

[0151] In the following, the present invention is exemplified by a heteromultimeric antibody-multimer fusion comprising a pair of antibody heavy and light chains that form a binding site for human FAP, and a first fusion polypeptide and a second fusion polypeptide, the multimer being trimeric human 4-1-BBL. These experiments are presented merely to illustrate the concept of the present invention and should not be construed as limiting the present invention. Any pair of antibody chains and any multimeric polypeptide can be used as well.

[0152] Random integration, transient transfection, single expression cassette vectors In the first series of experiments, transient production of anti-FAP antibody-4-1-BBL multimeric fusions was performed. A set of vectors, each containing only a single expression cassette for a single polypeptide of the antibody-multimeric fusion, was used at different defined stoichiometric ratios. The results are shown in the table below: HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the expression cassette ratio of 1:1:2:2). JPEG0007727711000001.jpg39170

[0153] It can be seen that an expression cassette ratio of 1:1:2:1 gives the best results, resulting in an overall 20% higher effective titer and 35% higher effective titer than the 1:1:2:2 expression cassette ratio.

[0154] Random integration, transient transfection, and multiple expression cassette vectors In a second series of experiments, transient production of anti-FAP antibody-4-1-BBL multimeric fusions was performed. A set of vectors containing one or two expression cassettes for each single polypeptide of the antibody-multimeric fusion was used at different defined stoichiometric ratios. The results are shown in the table below: HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the expression cassette ratio of 1:1:2:2). JPEG0007727711000002.jpg44170 Vector 1 = dual expression cassette vector with expression cassettes for HC and LC; Vector 2 = dual expression cassette vector carrying expression cassettes for FH and FL; Vector 3 = Single expression cassette vector carrying an expression cassette for FH

[0155] It can be seen that an expression cassette ratio of 1:1:2:1 gave the best results, resulting in an overall 40% higher effective titer and 67% higher effective titer than the 1:1:2:2 expression cassette ratio.

[0156] Stable random integration, multiple expression cassette vectors In a third series of experiments, stable production of anti-FAP antibody-4-1-BBL multimeric fusions was performed. A set of vectors containing one or two expression cassettes for each single polypeptide of the antibody-multimeric fusion was used at different defined stoichiometric ratios. The results are shown in the table below: HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the expression cassette ratio of 1:1:2:2). JPEG0007727711000003.jpg23170 Vector 1 = dual expression cassette vector with expression cassettes for HC and LC; Vector 2 = dual expression cassette vector carrying expression cassettes for FH and FL; Vector 3 = Single expression cassette vector carrying an expression cassette for FH

[0157] Sixty plates containing single cell clones were cultured for each of the ratios in Experiment 1 and Experiment 2. The plate wells were analyzed for recovery, titer and product quality, as well as batch culture product quality (% main peak in CE-SDS). The results are presented in the table below. JPEG0007727711000004.jpg29170JPEG0007727711000005.jpg36170

[0158] It can be seen that a 1:1:2:1 expression cassette ratio (1:1 + 1 vector ratio) results in better growth (recovery), twice as many wells with greater than 20% confluence, and twice as many titer-positive clones compared to a 1:1:2:2 expression cassette ratio (1:2 vector ratio).

[0159] More specifically, selection was based on confluence and titer (>5% confluence and IgG positive). All titer-positive clones were processed for the next selection step. A double number of clones was obtained from plates containing cells transfected with an expression cassette ratio of 1:1:2:1 (1:1 + 1 vector ratio).

[0160] A total of 1056 clones were selected for further ELISA retest analysis, of which one-third were clones obtained with an expression cassette ratio of 1:1:2:2 (vector ratio 1:2; 352 clones) and two-thirds were clones obtained with an expression cassette ratio of 1:1:2:1 (vector ratio 1:1 + 1,704 clones).

[0161] The second selection was based on ELISA binding and cross-linking assays.

[0162] Of a total of 1056 clones, 220 clones were found to express both parts of the antibody-multimer fusion (major product). Over four times as many clones were derived from an expression cassette ratio of 1:1:2:1 (vector ratio 1:1 + 1) (20% (45 clones) were derived from an expression cassette ratio of 1:1:2:2 (vector ratio 1:2) and 80% (175 clones) were derived from an expression cassette ratio of 1:1:2:1 (plasmid ratio 1:1:1 (1:1 + 1))).

[0163] In summary, (45 / 352)*100%=12.78% of clones obtained with an expression cassette ratio of 1:1:2:2 (plasmid ratio 1:2) showed good product quality in ELISA retests, whereas (175 / 704)*100%=24.86% of clones obtained with an expression cassette ratio of 1:1:2:1 (vector ratio of 1:1:1) show good product quality in ELISA retests.

[0164] Thus, stable transfection confirms the results obtained with transient transfection.

[0165] Random integration, transient transfection, single expression cassette vectors In the fourth set of experiments, transient production of anti-CEA antibody-4-1-BBL multimeric fusions was performed. A set of vectors, each containing a single expression cassette, was used at different defined stoichiometric ratios. The results are shown in the table below: HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the expression cassette ratio of 1:1:2:2). JPEG0007727711000006.jpg51170

[0166] It can be seen that an expression cassette ratio of 1:1:2:1 gave the best results, resulting in an overall 15% higher effective titer and 24% higher effective titer than the 1:1:2:2 expression cassette ratio.

[0167] Targeted integration, stable transfection, and dual RMCE In the fifth set of experiments, stable production of anti-FAP antibody-4-1-BBL multimeric fusions was performed using targeted integration. A set of vectors, namely, front and back vectors, was used. Targeted integration was performed using a dual recombinase-mediated cassette exchange reaction with Cre recombinase. The front and back vectors contained different expression cassettes as outlined in the table below, where HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, and FL = second fusion polypeptide. JPEG0007727711000007.jpg35170

[0168] Based on the different numbers of expression cassettes contained in the front and back vectors, different defined stoichiometric ratios were used. The results obtained from stable transfected cell pools are shown in the table below (n=2). HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the expression cassette ratio of 1:1:2:2). JPEG0007727711000008.jpg40170

[0169] It can be seen that an expression cassette ratio of 1:1:2:1 gives the best results, resulting in an overall 45% higher effective titer as well as a 45% higher effective titer than the 1:1:2:2 expression cassette ratio.

[0170] Random integration, transient transfection, single expression cassette vectors In the sixth set of experiments, transient production of anti-CD19 antibody-4-1-BBL multimeric fusions was performed. A set of vectors, each containing only a single expression cassette for a single polypeptide of the antibody-multimeric fusion, was used at different, defined stoichiometric ratios. The results are shown in the table below: HC = antibody heavy chain, LC = antibody light chain, FH = first fusion polypeptide, FL = second fusion polypeptide, exp. = number of experiments, eff.titer = effective titer (titer and main peak yield), rel.eff.titer = relative effective titer (relative titer normalized to the 1:1:2:2 expression cassette ratio). JPEG0007727711000009.jpg39170

[0171] It can be seen that an expression cassette ratio of 1:1:2:1 gave the best results, resulting in an overall 9% higher effective titer and 23% higher effective titer than the 1:1:2:2 expression cassette ratio.

[0172] overview: Therefore, regardless of the method used to generate the cell line, an expression cassette ratio of 1:1:2:1 (HC:LC:FH:FL) results in improved product quality, i.e., effective titer. Furthermore, an increased number of suitable stable clones for expressing antibody-multimer fusions can be obtained.

[0173] II.b Ligands that interact with TNF molecules Ligands that interact with molecules in the TNF (tumor necrosis factor) receptor superfamily play crucial roles in the organization and function of the immune system. While regulating normal functions such as immune responses, hematopoiesis, and morphogenesis, TNF family ligands (also called cytokines) play a role in tumorigenesis, transplant rejection, septic shock, viral replication, bone resorption, rheumatoid arthritis, and diabetes (Aggarwal, 2003). The TNF ligand family contains 18 genes encoding 19 type II (i.e., intracellular N-terminus and extracellular C-terminus) transmembrane proteins characterized by the presence of a conserved C-terminal domain, designated the "TNF homology domain" (THD). This domain is involved in receptor binding and is therefore critical for the biological activity of TNF ligand family members. Sequence identity among family members is approximately 20–30% (Bodmer, 2002). TNF ligand family members exert their biological functions as self-assembling, noncovalently linked trimers (Banner et al., Cell 73 (1993) 431–445). Thus, TNF family ligands form trimers that are able to bind to and activate corresponding receptors of the TNFR superfamily.

[0174] 4-1-BB (CD137), a member of the TNF receptor superfamily, was first identified as a molecule whose expression is induced by T cell activation (Kwon and Weissman, 1989). Subsequent studies demonstrated its expression in T and B lymphocytes (Snell et al., 2011; Zhang et al., 2010), NK cells (Lin et al., 2008), NKT cells (Kim et al., 2008), monocytes (Kienzle and von Kempis, 2000; Schwarz et al., 1995), neutrophils (Heinisch et al., 2000), obese individuals (Nishimoto et al., 2005), and dendritic cells, as well as cells of nonhematopoietic origin, such as endothelial cells and smooth muscle cells (Broll et al., 2001; Olofsson et al., 2008). Expression of 4-1-BB in various cell types is largely inducible and driven by various stimulatory signals, such as triggering of the T cell receptor (TCR) or B cell receptor, as well as signaling elicited through costimulatory molecules or receptors for proinflammatory cytokines (Diehl et al., 2002; von Kempis et al., 1997; Zhang et al., 2010).

[0175] Expression of 4-1-BB ligand (4-1-BBL or CD137L) is more restricted and is observed on professional antigen-presenting cells (APCs) such as B cells, dendritic cells (DCs), and macrophages. Inducible expression of 4-1-BBL is characteristic of T cells, including both αβ and γδ T cell subsets, as well as endothelial cells (reviewed in Shao and Schwarz, 2011).

[0176] CD137 signaling is known to stimulate IFNγ secretion and proliferation of NK cells (Buechele et al., 2012; Lin et al., 2008; Melero et al., 1998), as well as promote DC activation, as indicated by their increased survival and increased capacity to secrete cytokines, and upregulate costimulatory molecules (Choi et al., 2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137 is best characterized as a costimulatory molecule that regulates TCR-induced activation of both CD4+ and CD8+ subsets of T cells. In combination with TCR triggering, agonistic 4-1-BB-specific antibodies enhance T cell proliferation, stimulate lymphokine secretion, and reduce the susceptibility of T lymphocytes to activation-induced cell death (reviewed in Snell et al., 2011).

[0177] Together with these costimulatory effects of 4-1-BB antibodies on T cells in vitro, their administration to tumor-bearing mice has led to potent antitumor effects in many experimental tumor models (Melero et al., 1997; Narazaki et al., 2010). However, 4-1-BB typically only exhibits efficacy as an antitumor agent when administered in combination with other immunomodulatory compounds (Curran et al., 2011; Guo et al., 2013; Morales-Kastresana et al., 2013; Teng et al., 2009; Wei et al., 2013), chemotherapeutic agents (Ju et al., 2008; Kim et al., 2009), tumor-specific vaccination (Cuadros et al., 2005; Lee et al., 2011), or radiation therapy (Shi and Siemann, 2006). In vivo depletion experiments have shown that CD8+ T cells play the most important role in the antitumor effects of 4-1-BB-specific antibodies. However, depending on the tumor model or combination therapy involving anti-4-1-BB, the contribution of other cell types, such as DCs, NK cells, or CD4+ T cells, has been reported (Melero et al., 1997; Murillo et al., 2009; Narazaki et al., 2010; Stagg et al., 2011).

[0178] In addition to their direct effects on different lymphocyte subsets, 4-1-BB agonists can also induce the infiltration and retention of activated T cells within tumors via 4-1-BB-mediated upregulation of intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) on tumor vascular endothelium (Palazon et al., 2011).

[0179] Induction of 4-1-BB can also reverse states of T cell anergy induced by exposure to soluble antigens in the tumor microenvironment or during chronic infection (Wilcox et al., 2004), which may contribute to the breakdown of immune tolerance.

[0180] The immunomodulatory properties of 4-1-BB agonist antibodies in vivo require the presence of a wild-type Fc portion on the antibody molecule, thereby implicating Fc receptor binding as a key event required for the pharmacological activity of these agents, as described for agonist antibodies specific for other proapoptotic or immunomodulatory members of the TNFR superfamily (Li and Ravetch, 2011; Teng et al., 2009). However, systemic administration of 4-1-BB-specific agonist antibodies with a functionally active Fc domain also induces CD8+ T cell expansion in mice, which is associated with hepatotoxicity that is reduced or significantly ameliorated in the absence of functional Fc receptors (Dubrot et al., 2010). In a human clinical trial (ClinicalTrials.gov, NCT00309023), an Fc-competent 4-1-BB agonist antibody (BMS-663513) administered every 3 weeks for 12 weeks induced disease stabilization in patients with melanoma, ovarian, or renal cell carcinoma. However, the same antibody administered in a separate trial (NCT00612664) caused grade 4 hepatitis, leading to study termination (Simeone and Ascierto, 2012).

[0181] Collectively, available preclinical and clinical data clearly demonstrate a high clinical need for effective 4-1-BB agonists. However, new drug candidates must not only effectively engage 4-1-BB on the surface of hematopoietic and endothelial cells, but also achieve this via mechanisms other than Fc receptor binding to avoid uncontrollable side effects. The latter can be achieved by preferential binding and oligomerization to tumor-specific or tumor-associated moieties.

[0182] Fusion proteins have been created that consist of one extracellular domain of a 4-1-BB ligand and a single-chain antibody fragment (Mueller et al., 2008; Hornig et al., 2012) or a single 4-1-BB ligand fused to the C-terminus of the heavy chain (Zhang et al., 2007). WO 2010 / 010051 discloses the creation of a fusion protein consisting of three TNF ligand ectodomains linked together and fused to an antibody moiety.

[0183] However, there remains a need for new antigen-binding molecules that combine a moiety capable of preferentially binding to a tumor-specific or tumor-associated target with a moiety capable of forming a costimulatory TNF ligand trimer, and that are stable enough to be pharmaceutically useful. The antigen-binding molecules of the present invention contain both, and surprisingly, they provide a trimeric, and therefore biologically active, TNF ligand, but one of the trimerized TNF ligand ectodomains is located on a separate polypeptide from the other two TNF ligand ectodomains of the molecule.

[0184] II.c Recombinant Methods and Compositions Antibodies can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acid(s) encoding the antibody are provided.

[0185] In one aspect, there is provided a method of making an antibody-multimeric fusion polypeptide, the method comprising culturing a host cell comprising nucleic acid(s) encoding the antibody-multimeric fusion polypeptide obtained by a method according to the invention under conditions suitable for expression of the antibody-multimeric fusion polypeptide, and optionally recovering the antibody-multimeric fusion polypeptide from the host cell (or host cell culture medium).

[0186] For recombinant production of antibody-multimeric fusion polypeptides, for example, nucleic acids encoding antibody-multimeric fusion polypeptides as described herein are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures, or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0187] Typically, recombinant large-scale production of a polypeptide of interest, such as a therapeutic antibody-multimeric fusion polypeptide, requires cells that stably express and secrete the polypeptide. These cells are called "recombinant cells" or "recombinant production cells," and the methods used to generate such cells are called "cell line development." In the first step of the cell line development method, suitable host cells, such as CHO cells, are transfected with a nucleic acid sequence suitable for expression of the polypeptide of interest. In the second step, cells stably expressing the polypeptide of interest are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the polypeptide of interest.

[0188] A nucleic acid that encodes a polypeptide, i.e., a coding sequence, is called a structural gene. Such a structural gene is a simple message, and its expression requires additional control elements. Therefore, a structural gene is usually incorporated into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in mammalian cells are a promoter functional in mammalian cells located upstream, i.e., 5', of the structural gene, and a polyadenylation signal sequence functional in mammalian cells located downstream, i.e., 3', of the structural gene. The promoter sequence, structural gene sequence, and polyadenylation signal sequence are arranged in an operably linked form.

[0189] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the greater the number of different expression cassettes required. Essentially, the size of the nucleic acid to be integrated into the genome of the host cell increases with the number of expression cassettes. Concurrently, the size of the expression vector also increases. However, the practical upper limit of vector size is in the range of about 15 kbp, above which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors. The expression cassette can then be divided between different expression vectors, each containing only a portion of the expression cassette, resulting in a reduction in size.

[0190] Cell line development (CLD) for generating recombinant cells expressing heterologous polypeptides, such as antibody-multimeric fusion polypeptides, uses either random integration (RI) or targeted integration (TI) of nucleic acid(s) containing the respective expression cassettes required for expression and production of the targeted integration(s) of interest.

[0191] Using RI, multiple vectors or fragments thereof are generally integrated into the genome of a cell at the same or different loci.

[0192] Using TI, typically a single copy of a transgene containing different expression cassettes is integrated into a defined "hot spot" in the host cell's genome.

[0193] Suitable host cells for the expression of (glycosylated) antibodies are generally derived from multicellular organisms, such as vertebrates.

[0194] II.d Host cells Any mammalian cell line adapted to grow in suspension can be used in the method according to the invention. Moreover, regardless of the integration method, i.e., in the case of RI and TI, any mammalian host cell can be used.

[0195] Examples of useful mammalian host cell lines include human amniotic cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) p. 133); monkey kidney CV1 cells transformed with SV40 (COS-7); human embryonic kidney cells (e.g., HEK293 cells or HEK293T cells as described in Graham, F. et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described by Mather, J.P. et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0196] In one embodiment, the mammalian host cell is, for example, a Chinese hamster ovary (CHO) cell (e.g., CHO K1, CHO DG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp20 cell), or a human amniotic cell (e.g., CAP-T, etc.). In a preferred embodiment, the mammalian host cell is a CHO cell.

[0197] Targeted integration allows an exogenous nucleotide sequence to be integrated into a predetermined site in the genome of a mammalian cell. In certain embodiments, targeted integration is mediated by a recombinase that recognizes one or more recombination recognition sequences (RRS) present in the genome and the exogenous nucleotide sequence to be integrated. In certain embodiments, targeted integration is mediated by homologous recombination.

[0198] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location in a nucleotide sequence where a recombination event is expected to occur.

[0199] In certain embodiments, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase. In certain embodiments, the RRS can be recognized by φC31 integrase.

[0200] In certain embodiments, if the RRS is a LoxP site, the cells require Cre recombinase to effect recombination. In certain embodiments, if the RRS is an FRT site, the cells require FLP recombinase to effect recombination. In certain embodiments, if the RRS is a Bxb1 attP site or a Bxb1 attB site, the cells require Bxb1 integrase to effect recombination. In certain embodiments, if the RRS is a φC31 attP site or a φC31 attB site, the cells require φC31 integrase to effect recombination. These recombinases can be introduced into cells using expression vectors containing coding sequences for these enzymes, or as proteins or mRNAs.

[0201] For TI, any known or future mammalian host cell suitable for TI that contains a landing site described herein integrated at a single site within a genomic locus can be used in the present invention. Such cells are referred to as mammalian TI host cells. In certain embodiments, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell that contains a landing site as described herein. In a preferred embodiment, the mammalian TI host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese hamster ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO K1 M cell that contains a landing site described herein integrated at a single site within a genomic locus.

[0202] In certain embodiments, the mammalian TI host cell comprises an integrated landing site, the landing site comprising one or more recombination recognition sequences (RRS). The RRS can be recognized by a recombinase, such as Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRSs can be independently selected from the group consisting of LoxP, LoxP L3, LoxP 2L, LoxFas, Lox511, Lox2272, Lox2372, Lox5171, Loxm2, Lox71, Lox66, FRT, Bxb1 attP, Bxb1 attB, φC31 attP, and φC31 attB. If multiple RRSs are to be present, the selection of each sequence is dependent on the other, provided that non-identical RRSs are selected.

[0203] In certain embodiments, the landing site comprises one or more recombination recognition sequences (RRSs), wherein the RRSs can be recognized by a recombinase. In certain embodiments, the integrated landing site comprises at least two RRSs. In certain embodiments, the integrated landing site comprises three RRSs, wherein the third RRS is located between the first and second RRSs. In certain preferred embodiments, all three RSSs are different. In certain embodiments, the landing site comprises a first RRS, a second RRS, and a third RRS, and at least one selectable marker located between the first and second RRSs, wherein the third RRS is different from the first RRS and / or the second RRS. In certain embodiments, the landing site further comprises a second selectable marker, wherein the first and second selectable markers are different. In certain embodiments, the landing site further comprises a third selectable marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selectable marker. The third selection marker can be different from the first or second selection marker.

[0204] While the present invention is exemplified herein using HEK and CHO cells, this is presented solely for the purpose of illustrating the invention and should not be construed as limiting in any way, with the true scope of the invention being set forth in the following claims.

[0205] An exemplary mammalian TI host cell suitable for use in the methods according to the invention is a CHO cell that has a landing site integrated at a single site within its genomic locus, the landing site containing three heterospecific loxP sites for Cre recombinase-mediated DNA recombination.

[0206] In this example, the heterospecific loxP sites are L3, LoxFas, and 2L (see, e.g., Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a bicistronic unit that couples expression of an IRES-mediated selectable marker to expression of a fluorescent GFP protein, allowing for positive selection to stabilize the landing site and for selection of absent sites (negative selection) after transfection and Cre recombination. Green fluorescent protein (GFP) is useful for monitoring the RMCE reaction.

[0207] This organization of the landing sites, as outlined in the previous paragraph, allows the simultaneous integration of two vectors, e.g., a so-called front vector containing an L3 site and a LoxFas site, and a back vector containing an internal LoxFas site and a 2L site. Functional elements of the selectable marker gene, distinct from those present in the landing sites, can be distributed between both vectors: the promoter and start codon can be located on the front vector, whereas the coding region and polyA signal are located on the back vector. Only correct recombinase-mediated integration of the nucleic acids from both vectors induces resistance to the respective selection agents.

[0208] Typically, a mammalian TI host cell is a mammalian cell that contains a landing site for integration at a site within a locus in the genome of the mammalian cell, the landing site comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0209] The selectable marker(s) may be selected from the group consisting of aminoglycoside phosphotransferases (APHs) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. The selectable marker(s) may also be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0210] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell, but can be introduced into that cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, the mammalian TI host cell comprises at least one landing site that is integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the landing site is integrated into one or more integration sites within a specific locus in the genome of the mammalian cell.

[0211] In certain embodiments, the integrated landing site comprises at least one selectable marker. In certain embodiments, the integrated landing site comprises a first, second, and third RRS, and at least one selectable marker. In certain embodiments, the selectable marker is located between the first and second RRS. In certain embodiments, the two RRSs are adjacent to the at least one selectable marker. That is, the first RRS is located 5' (upstream) of the selectable marker, and the second RRS is located 3' (downstream) of the selectable marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selectable marker, and the second RRS is adjacent to the 3' end of the selectable marker. In certain embodiments, the landing site comprises a first, second, and third RRS, and at least one selectable marker located between the first and third RRS.

[0212] In certain embodiments, the selectable marker is located between a first RRS and a second RRS, and the two flanking RRSs are different. In certain preferred embodiments, the first flanking RRS is a LoxP L3 sequence, and the second flanking RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 5' of the selectable marker, and the LoxP 2L sequence is located 3' of the selectable marker. In certain embodiments, the first flanking RRS is a wild-type FRT sequence, and the second flanking RRS is a mutant FRT sequence. In certain embodiments, the first flanking RRS is a Bxb1 attP sequence, and the second flanking RRS is a Bxb1 attB sequence. In certain embodiments, the first flanking RRS is a φC31 attP sequence, and the second flanking RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are positioned in the same orientation. In certain embodiments, the two RRSs are both oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite directions.

[0213] In certain embodiments, the integrated landing site comprises a first selection marker and a second selection marker flanked by two RSSs, wherein the first selection marker is different from the second selection marker. In certain embodiments, both of the two selection markers are, independently of each other, selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418). The first selectable marker is selected from the group consisting of genes encoding resistance to GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent protein. In certain embodiments, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein, hi certain embodiments, the two RRSs flanking both selection markers are different.

[0214] In certain embodiments, the selectable marker is operably linked to a promoter sequence. In certain embodiments, the selectable marker is operably linked to an SV40 promoter. In certain embodiments, the selectable marker is operably linked to a human cytomegalovirus (CMV) promoter.

[0215] II.e Targeted Integration One method for producing recombinant mammalian cells according to the present invention is targeted integration (TI).

[0216] Targeted integration uses site-specific recombination to introduce exogenous nucleic acid into a specific locus in the genome of a mammalian TI host cell. This is an enzymatic process in which the sequence at the integration site in the genome is exchanged with the exogenous nucleic acid. One system used to perform this nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The sequence to be exchanged is defined by the location of two lox(P) sites in the genome and in the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. No further steps are required; no ATP is required. The Cre-lox system was originally discovered in bacteriophage P1.

[0217] The Cre-lox system functions in a variety of cell types, including mammalian, plant, bacterial, and yeast cells.

[0218] In one embodiment, exogenous nucleic acids encoding antibody-multimeric fusion polypeptides are integrated into mammalian TI host cells by single or double recombinase-mediated cassette exchange (RMCE), resulting in recombinant mammalian cells, such as recombinant CHO cells, in which a defined, specific expression cassette sequence is integrated into the genome at a single locus, which in turn results in efficient expression and production of the antibody-multimeric fusion polypeptides.

[0219] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intramolecular and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp nonpalindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeats, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. When two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences into a covalently closed loop. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre recombinase-mediated recombination will reverse the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are located on two different DNA molecules and one DNA molecule is circular, Cre recombinase-mediated recombination will result in the integration of the circular DNA sequence.

[0220] The term "matched RRS" indicates that recombination occurs between two RRSs. In certain embodiments, the two matched RRSs are the same. In certain embodiments, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matched RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matched RRS is a Bxb1 attP sequence and the second matched RRS is a Bxb1 attB sequence. In certain embodiments, the first matched RRS is a φC31 attB sequence and the second matched RRS is a φC31 attB sequence.

[0221] In certain embodiments of the present invention, a "two-plasmid RMCE" strategy or "double RMCE" is used using a combination of two vectors. For example, but not limited to, the integrated landing site can include three RRSs, e.g., an arrangement in which a third RRS ("RRS3") is located between a first RRS ("RRS1") and a second RRS ("RRS2"), where the first vector includes two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector includes two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0222] The two-plasmid RMCE strategy involves simultaneously performing two independent RMCEs using three RRS sites. Therefore, the landing site for mammalian TI host cells using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross-reactivity with either the first RRS site (RRS1) or the second RRS site (RRS2). The two targeted plasmids require identical flanking RRS sites for efficient targeting: one plasmid (front) is flanked by RRS1 and RRS3, and the other (back) is flanked by RRS3 and RRS2. Furthermore, two selectable markers are also required in two-plasmid RMCE. One selectable marker expression cassette is split into two parts: the front plasmid contains a promoter followed by an initiation codon and the RRS3 sequence. The back plasmid lacks an initiation codon (ATG) and has the RRS3 sequence fused to the N-terminus of the selectable marker coding region. To ensure in-frame translation of the fusion protein, i.e., operable linkage, additional nucleotides may need to be inserted between the RRS3 site and the selectable marker sequence. Only when both plasmids are correctly inserted will the complete expression cassette for the selectable marker be assembled, thus conferring resistance to the respective selection agent to the cells.

[0223] Two-plasmid RMCE involves a recombinase-catalyzed double recombination crossover event between two heterospecific RRSs within a target genomic locus and a donor DNA molecule. Two-plasmid RMCE is designed to introduce copies of the combined DNA sequences from the front and back vectors into a predetermined locus in the mammalian TI host cell genome. RMCE can be performed such that no sequences from the prokaryotic vector are introduced into the mammalian TI host cell genome, thus reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0224] In certain embodiments, targeted integration is achieved by two rounds of RMCE, in which two different DNA sequences are both integrated into a predetermined site in the genome of a matching RRS of the mammalian TI host cell, with each DNA sequence comprising at least one expression cassette encoding a portion of an antibody-multimer fusion polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, targeted integration is achieved by multiple rounds of RMCE, in which DNA sequences from multiple vectors are all integrated into a predetermined site in the genome of the mammalian TI host cell, with each DNA sequence comprising at least one expression cassette encoding a portion of an antibody-multimer fusion polypeptide and / or at least one selectable marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selectable marker may be partially encoded in a first vector and partially encoded in a second vector, such that expression of the selectable marker is only possible if both are correctly integrated by double RMCE.

[0225] In certain embodiments, targeted integration by recombinase-mediated recombination integrates various expression cassettes for selectable markers and / or antibody-multimer fusion polypeptides into one or more predetermined integration sites of the host cell genome, without including sequences derived from the prokaryotic vector. SEQ ID NO: 130: Exemplary sequence of an L3 recombinase recognition sequence SEQ ID NO: 131: Exemplary sequence of 2L recombinase recognition sequence SEQ ID NO: 132: Exemplary sequence of a LoxFas recombinase recognition sequence SEQ ID NOs: 133-5: Exemplary variants of the human CMV promoter SEQ ID NO: 136: Exemplary SV40 polyadenylation signal sequence SEQ ID NO: 137: Exemplary bGH polyadenylation signal sequence SEQ ID NO: 138: Exemplary hGT terminator sequence SEQ ID NO: 139: Exemplary SV40 promoter sequence SEQ ID NO: 140: Exemplary GFP nucleic acid sequence

[0226] In addition to the various embodiments shown and embodied, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed and embodied herein. Thus, specific features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of features disclosed herein. The foregoing descriptions of specific embodiments of the disclosed subject matter have been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the disclosed subject matter to those disclosed embodiments.

[0227] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the presently disclosed subject matter without departing from the spirit or scope of the presently disclosed subject matter. Accordingly, it is intended that the presently disclosed subject matter include modifications and variations within the scope of the embodiments and their equivalents.

[0228] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated herein by reference in their entireties.

[0229] The following examples and sequences are provided to aid the understanding of the present invention, the true scope of which is set forth in the accompanying embodiments.

[0230] quotation Ascierto, PA, et al. Semin Oncol 37:508-516. Aggarwal BB, Nat. Rev. Immunol. 3 (2003) 745-56. Banner D. et al., Cell 73 (1993) 431-445. Bodmer J.,et al.,Trends Biochem.Sci.27(1),19-26. Broll,K.,et al.,Am.J.Clin.Pathol.115,543-549. Buechele,C.,et al.,Eur.J.Immunol.42,737-748. Choi,BK,et al.,J.Immunol.182,4107-4115. Cuadros,C.,et al.,Int.J.Cancer 116,934-943. Curran,MA,et al.,PLoS One 6,e19499. Diehl,L.,et al.,J.Immunol.168,3755-3762. Dubrot,J.,et al.,Cancer Immunol.Immunother.59,1223-1233. Futagawa,T.,et al.,Int.Immunol.14,275-286. Guo,Z.,et al.,J Transl.Med.11,215. Heinisch,IV,et al.,Eur.J.Immunol.30,3441-3446. Hornig,N.,et al.,J.Immunother.35,418-429. Ju,SA,et al.,Int.J.Cancer 122,2784-2790. Kienzle,G.,and von Kempis,J.Int.Immunol.12,73-82. Kim,DH,et al.,J.Immunol.180,2062-2068. Kim,YH,et al.,Mol.Cancer Ther.8,469-478. Kwon,BS,and Weissman,SMProc.Natl.Acad.Sci USA 86,1963-1967. Lee,H.,et al.,J.Surg.Res.169,e43-50. Levitsky,V.,et al.,J.Immunol.161,594-601. Li,F.,and Ravetch,JV,Science 333,1030-1034. Lin, W., et al., Blood 112,699-707. Melero,I.,et al.,Cell Immunol.190,167-172. Melero,I.,et al.,Nat.Med.3,682-685. Merchant, AM, et al., Nat.Biotechnol.16, 677-681. Morales-Kastresana,A.,et al.,Clin.Cancer Res.19,6151-6162. Mueller,D.,et al.,J.Immunother.31,714-722. Murillo,O.,et al.,Eur.J.Immunol.39,2424-2436. Narazaki, H., et al., Blood 115, 1941-1948. Nishimoto,H.,et al.,Blood 106,4241-4248. Olofsson, PS, et al., Circulation 117, 1292-1301. Palazon,A.,et al.,Cancer Res.71,801-811. Schwarz, H., et al., Blood 85, 1043-1052. Shao,Z.,and Schwarz,HJLeukoc.Biol.89,21-29. Shi,W.,and Siemann,DWAnticancer Res.26,3445-3453. Simeone,E.,and Ascierto,PAJ Immunotoxicol.9,241-247. Snell, LM, et al., Immunol. Rev. 244, 197-217. Stagg, J., et al., Proc. Natl. Acad. Sci USA 108, 7142-7147. Teng, MW, et al., J. Immunol. 183, 1911-1920. von Kempis, J., et al., Osteoarthritis Cartilage 5, 394-406. Wei, H.,et al.,PLoS One 8,e84927. Wilcox, RA, et al., J. Immunol. 168, 4262-4267. Wilcox, RA, et al., Blood 103, 177-184. Zhang, N., et al., Clin. Cancer Res. 13, 2758-2767. Zhang, X., et al., J. Immunol. 184, 787-795. [Example]

[0231] Example 1 general technology recombinant DNA technology DNA was manipulated using standard methods as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1989). Molecular biological reagents were used according to the manufacturer's instructions.

[0232] Gene synthesis The desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into E. coli plasmids for propagation / amplification. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Alternatively, short synthetic DNA fragments were constructed by annealing chemically synthesized oligonucleotides or via PCR. The respective oligonucleotides were prepared by metabion GmbH (Planeg-Martinsried, Germany).

[0233] DNA sequencing DNA sequences were determined by double-strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

[0234] DNA and protein sequence analysis and sequence data management The EMBOSS (European Molecular Biology Open Software Suite) software package and Invitrogen's Vector NTI version 11.5 or Geneious prime were used for sequence generation, mapping, analysis, annotation, and illustration.

[0235] reagent Unless otherwise stated, all commercially available chemicals, antibodies, and kits were used as provided according to the manufacturer's protocols.

[0236] Protein quantification The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.

[0237] Determination of antibody concentration in the supernatant 1) Protein A beads The antibody concentration in the cell culture supernatant was estimated by immunoprecipitation with protein A agarose beads (Roche Diagnostics GmbH, Mannheim, Germany). Therefore, 60 μL of protein A agarose beads were washed three times with TBS-NP40 (150 mM Tris buffer, pH 7.5, supplemented with 50 mM NaCl and 1% Nonidet-P40). Then, 1–15 mL of cell culture supernatant was applied to the protein A agarose beads pre-equilibrated in TBS-NP40. After 1 h of incubation at room temperature, the beads were loaded onto an Ultrafree-MC-filter column (Amicon) and washed once with 0.5 mL of TBS-NP40, twice with 0.5 mL of 2x phosphate-buffered saline (2x PBS, Roche Diagnostics GmbH, Mannheim, Germany), and briefly washed four times with 0.5 mL of 100 mM Na-citrate buffer (pH 5.0). Bound antibody was eluted by adding 35 μL of NuPAGE® LDS sample buffer (Invitrogen). Half of the sample was combined with or without NuPAGE® sample reducing agent and heated at 70°C for 10 min. Five to 30 μL of the resulting solution was applied to a 4-12% NuPAGE® Bis-Tris SDS-PAGE gel (Invitrogen) (containing MOPS buffer for non-reducing SDS-PAGE and MES buffer with NuPAGE® antioxidant running buffer additive (Invitrogen) for reducing SDS-PAGE) and stained with Coomassie blue.

[0238] 2) Affinity HPLC The antibody concentration in the cell culture supernatant was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatant containing antibody that binds to Protein A was applied to an Applied Biosystems Poros A / 20 column in 200 mM KH2PO4, 100 mM sodium citrate (pH 7.4) and eluted with 200 mM NaCl, 100 mM citric acid (pH 2.5) on an Agilent HPLC 1100 system. The eluted antibody was quantified by UV absorbance and peak area integration. A purified standard IgG1 antibody was used as a standard.

[0239] 3) Sandwich ELISA The concentrations of antibodies and derivatives in cell culture supernatants were measured by sandwich IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A 96-well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) were filled with 100 μL / well of biotinylated anti-human IgG capture molecule F(ab')2.<h-Fcγ> Plates were coated with BI (Dianova) at 0.1 μg / mL for 1 hour at room temperature or overnight at 4°C, followed by washing three times with 200 μL / well of PBS, 0.05% Tween (PBST, Sigma). 100 μL / well of a dilution series of each antibody-containing cell culture supernatant in PBS (Sigma) was then added to the wells and incubated for 1-2 hours on a shaker at room temperature. The wells were washed three times with 200 μL / well of PBST, and the bound antibody was removed by adding 100 μL of 0.1 μg / mL F(ab')2 as a detection antibody by incubating for 1-2 hours on a shaker at room temperature.<hFcγ> Detection was performed using POD (Dianova). Unbound detection antibody was removed by washing three times with 200 μL / well of PBST. Bound detection antibody was detected by adding 100 μL ABTS / well followed by incubation. Absorbance was determined on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

[0240] CHO host cell line culture CHO host cells were cultured at 37°C in a humidified incubator with 85% humidity and 5% CO2. They were cultured in a proprietary DMEM / F12-based medium containing 300 μg / mL hygromycin B and 4 μg / mL of a second selection marker. Cells were split every 3 or 4 days at a concentration of 0.3 x 10E6 cells / mL into a total volume of 30 mL. For culture, 125 mL unbaffled Erlenmeyer shake flasks were used. Cells were shaken at 150 rpm with a shaking amplitude of 5 cm. Cell number was determined using a Cedex HiRes Cell Counter (Roche Diagnostics GmbH, Mannheim, Germany). Cells were continued to be cultured until they reached 60 days of age.

[0241] Transform 10 beta-competent E. coli cells For transformation, 10 beta-competent E. coli cells were thawed on ice. 2 μL of plasmid DNA was then pipetted directly into the cell suspension. The tube was flicked and placed on ice for 30 minutes. The cells were then placed in a 42°C warm thermal block for a heat shock of exactly 30 seconds. Immediately after, the cells were cooled on ice for 2 minutes. 950 μL of NEB10 beta growth medium was added to the cell suspension. The cells were incubated at 37°C for 1 hour with shaking. Next, 50–100 μL was pipetted onto prewarmed (37°C) LB-Amp agar plates and spread with a disposable spatula. The plates were incubated overnight at 37°C. Only bacteria that had successfully integrated the plasmid and carried the ampicillin resistance gene could grow on these plates. The next day, single colonies were picked and cultured in LB-Amp medium for subsequent plasmid preparation.

[0242] bacterial culture E. coli was cultured in Luria Bertani LB medium, supplemented with 1 mL / L of 100 mg / mL ampicillin to achieve an ampicillin concentration of 0.1 mg / mL. For the different plasmid preparations, the following amounts were inoculated onto single bacterial colonies: JPEG0007727711000010.jpg36170

[0243] For minipreps, 96-well 2 mL deep-well plates were filled with 1.5 mL of LB-Amp medium per well. Colonies were picked by pressing a toothpick into the medium. Once all colonies were picked, the plate was sealed with an adhesive air-porous membrane. The plate was incubated at 37°C in an incubator with a shaking speed of 200 rpm for 23 hours.

[0244] For minipreps, 15 mL tubes (with ventilated lids) were filled with 3.6 mL of LB-Amp medium and inoculated evenly with bacterial colonies. The toothpicks were left in the tubes during incubation. Similar to the 96-well plates, the tubes were incubated at 37°C and 200 rpm for 23 hours.

[0245] For maxipreps, 200 mL of LB-Amp medium was filled into an autoclaved 1 L glass Erlenmeyer flask and inoculated with 1 mL of a 5-hour-old bacterial day culture. The Erlenmeyer flask was closed with a paper stopper and incubated at 37°C and 200 rpm for 16 hours.

[0246] Plasmid preparation For minipreps, 50 μL of bacterial suspension was transferred to a 1 mL deep-well plate. The bacterial cells were then centrifuged in the plate at 3000 rpm at 4°C for 5 minutes. The supernatant was removed, and the plate containing the bacterial pellet was placed in an EpMotion. After approximately 90 minutes, analysis was performed, and the eluted plasmid DNA could be removed from the EpMotion for further use.

[0247] For minipreps, the 15 mL tubes were removed from the incubator and 3.6 mL of bacterial culture was divided into two 2 mL Eppendorf tubes. The tubes were centrifuged at 6,800 × g for 3 minutes at room temperature in a tabletop microcentrifuge. Minipreps were then performed using the Qiagen QIAprep Spin Miniprep Kit according to the manufacturer's instructions. Plasmid DNA concentrations were measured using a Nanodrop™.

[0248] Maxi-Prep was performed using the Macherey-Nagel NucleoBond® Xtra Maxi EF kit according to the manufacturer's instructions. DNA concentration was measured with a Nanodrop.

[0249] Ethanol precipitation The DNA solution volume was mixed with 2.5 volumes of 100% ethanol. The mixture was incubated at -20°C for 10 minutes. The DNA was then centrifuged at 14,000 rpm at 4°C for 30 minutes. The supernatant was carefully removed, and the pellet was washed with 70% (v / v) ethanol. Again, the tube was centrifuged at 14,000 rpm at 4°C for 5 minutes. The supernatant was carefully removed by pipetting, and the pellet was allowed to dry. Once the ethanol had evaporated, an appropriate amount of endotoxin-free water was added. The DNA was allowed to redissolve in water overnight at 4°C. A small aliquot was taken, and the DNA concentration was measured using a Nanodrop device.

[0250] Preparative antibody purification Antibodies were purified from filtered cell culture supernatants according to standard protocols. Briefly, the antibodies were applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS. Antibody elution was achieved at pH 2.8 and immediately neutralized. Aggregated proteins were separated from monomeric antibodies by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS or 20 mM histidine buffer (pH 6.0) containing 150 mM NaCl. Monomeric antibody fractions were pooled and concentrated (if necessary) using, for example, a MILLIPORE Amicon Ultra (30 MWCO) centrifugal concentrator, and stored frozen at -20°C or -80°C. Portions of these samples were submitted for subsequent protein analysis and analytical characterization, for example, by SDS-PAGE, size-exclusion chromatography (SEC), or mass spectrometry.

[0251] SDS-PAGE The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions, specifically, 10% or 4-12% NuPAGE® Novex® Bis-TRIS Pre-Cast gels (pH 6.4) and NuPAGE® MES (reducing gels, supplemented with NuPAGE® antioxidant running buffer) or MOPS (non-reducing gels) running buffer.

[0252] CE-SDS Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Five microliters of antibody solution for CE-SDS analysis was prepared using the HT Protein Express Reagent Kit according to the manufacturer's instructions and analyzed on a Labchip GXII system using the HT Protein Express chip. Data were analyzed using Labchip GX Software.

[0253] Analytical Size Exclusion Chromatography Size exclusion chromatography (SEC) to determine the aggregation and oligomeric state of the antibody was performed by HPLC chromatography. Briefly, Protein A-purified antibody was applied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mM KH2PO4 / K2HPO4 (pH 7.5) on a Dionex Ultimate® system or to a Superdex 200 column (GE Healthcare) in 2x PBS on a Dionex HPLC-System. Eluted antibody was quantified by UV absorbance and peak area integration. BioRad Gel Filtration Standard 151-1901 was used as the standard.

[0254] mass spectrometry In this chapter, we describe the characterization of antibody / fusion proteins with emphasis on their correct assembly. The predicted primary structures were analyzed by electrospray ionization mass spectrometry (ESI-MS) of deglycosylated intact antibodies and, in special cases, deglycosylated / limited LysC-digested antibodies.

[0255] Antibodies / fusion proteins were deglycosylated with N-glycosidase F at a protein concentration of 1 mg / mL in phosphate buffer or Tris buffer at 37°C for up to 17 hours. Restrictive LysC (Roche Diagnostics GmbH, Mannheim, Germany) digestion was performed with 100 μg of deglycosylated antibody in Tris buffer (pH 8) at room temperature for 120 hours or at 37°C for 40 minutes, respectively. Prior to mass spectrometry analysis, samples were desalted by HPLC on a Sephadex G25 column (GE Healthcare). Total mass was determined by ESI-MS on a maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with a TriVersa NanoMate source (Advion).

[0256] Example 2 Plasmid preparation for random integration For the expression of antibodies / fusion proteins, expression vectors for transient expression (e.g. in HEK293 cells) based either on cDNA constructs with or without the CMV-intron A promoter, or on genomic constructs with the CMV promoter can be applied.

[0257] Expression cassette composition For expression of antibody chains, transcription units containing the following functional elements were used: - the immediate early enhancer and promoter from human cytomegalovirus, including intron A; - human heavy chain immunoglobulin 5' untranslated region (5'UTR), - mouse immunoglobulin heavy chain signal sequence, - nucleic acids encoding each antibody chain, - bovine growth hormone polyadenylation sequence (BGH pA), and - optionally, a human gastrin terminator (hGT).

[0258] Besides the expression unit / cassette containing the desired gene to be expressed, a basic / standard mammalian expression plasmid contains: - the origin of replication from the vector pUC18, allowing replication of this plasmid in E. coli, and - Contains the beta-lactamase gene that confers ampicillin resistance to E. coli.

[0259] Fusion genes encoding antibody chains were generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques by joining the nucleic acid segments together using unique restriction sites in each vector. The subcloned nucleic acid sequences were confirmed by DNA sequencing. For transient transfections, larger quantities of vector were prepared by vector preparation from transformed E. coli cultures (NucleoBond AX, Macherey-Nagel).

[0260] For all constructs, the knob-into-hole heterodimerization technique was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A, and Y407V) in the second CH3 domain (and two additional introduced cysteine ​​residues S354C / Y349C) (contained in each corresponding heavy chain (HC) sequence described above).

[0261] Example 3 Transient expression HEK 293 cells Transient expression was performed in suspension-adapted HEK293F (FreeStyle 293-F cells; Invitrogen) cells without transfection reagent 293 (Novagen).

[0262] After thawing in 125 ml shake flasks (incubated / shaken at 37°C, 7% CO2, 85% humidity, 135 rpm), cells were passaged by dilution at least four times (30 mL volume).

[0263] Cells were grown to 3 x 10E5 cells / mL in a 250 mL volume. After 3 days, cells were split and cultured in 1 liter shake flasks at 7 x 10E5 cells / mL in a 250 mL volume. 5 Transfections were performed at a density of approximately 1.4–2.0 × 10 cells / mL. 6 After 24 h at a cell density of 100 cells / mL.

[0264] Prior to transfection, 250 μg of plasmid DNA was diluted to a final volume of 10 ml with preheated (water bath, 37°C) Opti-MEM (Gibco). The solution was gently mixed and incubated at room temperature for no more than 5 minutes. 333.3 μL of 293-free transfection reagent was then added to the DNA-OptiMEM solution. The solution was then gently mixed and incubated at room temperature for 15–20 minutes. The entire volume of the mixture was added to a 1 L shake flask. The cells were incubated at 37°C, 7% CO2, 85% humidity, and 135 rpm for 6 or 7 days.

[0265] The supernatant was collected by a first centrifugation step at 2,000 rpm for 10 min at 4° C. The supernatant was then transferred to a new centrifuge flask and subjected to a second centrifugation step at 4,000 rpm for 20 min at 4° C. The cell-free supernatant was then filtered through a 0.22 μm bottle-top filter and stored in a freezer (−20° C.).

[0266] CHO-K1 cells Transient expression was performed in suspension-adapted CHO-K1 cells using the transfection reagent Nucleofector solution V (Lonza) and an Amaxa nucleoporator for electroporation.

[0267] After thawing in 125 ml shake flasks (incubation at 37°C, 7% CO , 85% humidity, 140 rpm), cells were passaged by dilution at least four times (volume 30 mL).

[0268] Cells were cultured at 3 x 10 in a 60 mL volume in a 250 mL shake flask. 5 The cells were grown to approximately 1.2-2.0 x 10 cells / mL. 6 The cells are ready for transfection at a cell density of 1 x 10 cells / mL. A total volume of 1 x 10 cells is transfected by centrifugation at 1000 rpm for 10 minutes at room temperature. 7 Cells were harvested. The cell pellet was dissolved in 100 μL of pre-warmed (room temperature) Nucleofector solution (Lonza). A total of 1.2 pmol or up to 10 μg of plasmid DNA (diluted in water) was mixed in a final volume of 10 μL, followed by 100 μL of transfection reagent and 1 × 10 7 The plasmid DNA, transfection reagent, and cells were then mixed into an electroporation cuvette. The mixture was then transferred to the Nucleofector system directly without any incubation period. After electroporation using program "U-24," 500 μL of prewarmed (37°C) medium was added to the cuvette. The entire mixture was transferred to a 125 mL shake flask containing 30 mL of prewarmed (37°C) chemically defined medium (Invitrogen).

[0269] CHO-K1 TI cells Transient expression was performed in suspension adapted CHO-K1 TI host cells using transfection reagent PE buffer and MaxCyte (OC-400 processing assembly) for electroporation.

[0270] Cells were passaged at least four times after thawing in 125 mL shake flasks (incubation at 37°C, 5% CO2, 85% humidity, 150 rpm).

[0271] Cells were cultured at 4 x 10 on day 1 in shake flasks. 5 On day 3, the cells were grown to approximately 1-2 x 10 cells / mL. 6 Ready for transfection at a cell density of 1000 cells / mL.

[0272] Centrifuge at 1000 rpm for 10 min at room temperature to remove a total of 3 x 10 6 Cells were harvested. The cell pellet was dissolved in 300 μL of PE buffer (MaxCyte), followed by the addition of up to 25 μg of plasmid DNA. The mixture of plasmid DNA, transfection reagent, and cells was then transferred to an electroporation cuvette, followed by electroporation using the "CHO-2" and process assembly protocols. After electroporation, the entire mixture was transferred to a shake flask without agitation at 37°C for 30 minutes. After 30 minutes, 30 mL of recovery medium was added.

[0273] The transfected cells were incubated at 37°C, 5% CO2, 85% humidity with shaking at 100 rpm for 7 days.

[0274] ExpiCHO cells Transient expression was performed using the ExpiCHO-S expression system (A29133; Gibco).

[0275] Thawing, passaging and transfection were performed according to the manufacturer's instructions (see ExpiCHO Expression System (A 29133) User Guide). For transfection, the "Standard Protocol" (see page 12, A29133, manual) was used.

[0276] The transfected cells were incubated at 37°C, 7% CO2, 85% humidity, with shaking at 140 rpm for 7 days.

[0277] The supernatant was collected by a first centrifugation step at 1,000 rpm for 10 min at 4° C. The supernatant was transferred to a new centrifuge flask and subjected to a second centrifugation step at 4,000 rpm for 20 min at 4° C. The cell-free supernatant was then filtered through a 0.22 μm bottle-top filter and stored in a freezer (−20° C.) until further use.

[0278] Example 4 Stable expression and purification Generation of stable cell lines The two double plasmids and one single plasmid were used to cotransfect the host cell line CHO K1-M. The double plasmid contained DNA fragments encoding the VL and VH domains of FAP, the monomer and dimer of the 4-1-BBL fusion protein, and the CH and CL domains of FAP and 4-1-BBL, while the single plasmid contained only the 4-1-BBL dimer. All sequences were chemically synthesized and combined with heterologous DNA elements including: a) a 5'-UTR containing a 5'-Kozak sequence, b) a DNA segment encoding a leader sequence (LL), and c) restriction endonuclease sites suitable for cloning added to the 5'- and 3'-ends of the synthesized DNA segments.

[0279] The host cell line is derived from the proline auxotrophic strain K1, established from the CHO cell line introduced by Puck et al. (Kao and Puck, 1967). CHO K1 cells were obtained as a frozen stock (accession number CCL-61) from the American Type Culture Collection (ATCC) and subsequently adapted to growth in chemically defined medium and suspension at Roche Pharma, Penzberg, and then designated "CHO ​​K1-M."

[0280] One ampoule of CHO K1-M WCB was used for transfection. Transfection was performed using linearized DNA in chemically defined medium. Clones that stably integrated the recombinant DNA were selected based on the DHFR / MTX expression system. For selection of stable transfectants, cells were transferred to 500-well plates at a density of 384 cells / well and cultured in chemically defined medium containing 400 nM MTX. These conditions ensured that only cells that overexpressed the DHFR gene from the double and single plasmids survived.

[0281] Three weeks after transfection, supernatants were screened for the presence of human IgG by anti-human Fc ELISA. Clones positive for antibody titer were expanded to a 96-well format, and the MTX concentration was then reduced to 250 nmol / L MTX. One week later, clones were tested for binding to the FAP antigen and 4-1-BB receptor by ELISA to ensure the functionality of the produced FAP antibody-4-1-BBL fusion protein molecule. Positive clones were expanded and banked.

[0282] Antibody purification The antibody-containing culture supernatant was filtered and purified by two chromatography steps. The antibody was captured by affinity chromatography using a HiTrap MabSelectSuRe (GE Healthcare) column equilibrated with PBS (1 mM KH2PO4, 10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl), pH 7.4. Unbound proteins were removed by washing with the equilibration buffer, and the antibody was recovered with 50 mM citrate buffer (pH 2.8) and neutralized to pH 6.0 with 1 M Tris base (pH 9.0) immediately after elution. Size-exclusion chromatography on a Superdex 200™ (GE Healthcare) was used as the second purification step. Size-exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The antibody-containing solution was concentrated with an Ultrafree-CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, MA) and stored at −80°C.

[0283] Example 5 Plasmid preparation for targeted integration To construct the two-plasmid antibody constructs, each structural gene was cloned into a front vector backbone containing L3 and LoxFas sequences, and a back vector containing LoxFas and 2L sequences and the pac selection marker. Cre recombinase plasmids (see Wong, E.T., et al., Nucl. Acids Res. 33 (2005) e147; O'Gorman, S., et al., Proc. Natl. Acad. Sci. USA 94 (1997) 14602-14607) were used for all RMCE processes. See also WO 2019 / 126634, which is incorporated herein by reference in its entirety.

[0284] cDNA encoding each polypeptide was generated by gene synthesis (Geneart, Life Technologies Inc.). The gene synthesis and backbone vectors were digested with HindIII-HF and EcoRI-HF (NEB) at 37°C for 1 hour and separated by agarose gel electrophoresis. The insert and backbone DNA fragments were excised from the agarose gel and extracted using a QIAquick Gel Extraction Kit (Qiagen). The purified insert and backbone fragments were ligated at a 3:1 insert / backbone ratio using a rapid ligation kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. The ligation approach was then transformed into competent E. coli DH5α by heat shock at 42°C for 30 seconds, incubated at 37°C for 1 hour, and plated on agar plates containing selective ampicillin. The plates were incubated at 37°C overnight.

[0285] The next day, clones were picked and incubated overnight at 37°C with shaking for Mini or Maxi preparation, which was performed using an EpMotion® 5075 (Eppendorf) or a QIAprep Spin Miniprep Kit (Qiagen) / NucleoBond Xtra Maxi EF Kit (Macherey & Nagel), respectively. All constructs were sequenced to ensure the absence of unwanted mutations.

[0286] In the second cloning step, the previously cloned vector was digested with KpnI-HF / SalI-HF and SalI-HF / MfeI-HF using the same conditions as in the first cloning. The TI backbone vector was digested with KpnI-HF and MfeI-HF. Isolation and extraction were performed as described above. Ligation of the purified insert and backbone was performed overnight at 4°C using T4 DNA ligase (NEB) according to the manufacturer's protocol at an insert / insert / backbone ratio of 1:1:1, followed by inactivation at 65°C for 10 minutes. The following cloning steps were performed as described above.

[0287] Example 6 Generation of stable cell lines by targeted integration CHO-K1 TI host cells containing a GFP expression cassette in the TI landing site were grown in a proprietary DMEM / F12-based medium under standard humidified conditions (95% rH2O, 37°C, and 5% CO2) at a constant agitation rate of 150 rpm in disposable 125 ml vented shake flasks. Every 3–4 days, cells were seeded at a density of 3 x 10E5 cells / mL in chemically defined medium containing effective concentrations of selectable marker 1 and selectable marker 2. Culture density and viability were measured using a Cedex HiRes cell counter (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

[0288] For stable transfection, equimolar amounts of the front and back vectors were mixed, and 1 μg of Cre expression plasmid was added per 5 μg of the mixture (i.e., 5 μg of Cre expression plasmid or Cre mRNA was added to 25 μg of the front and back vector mixture).

[0289] Two days before transfection, TI host cells were seeded in fresh medium at a density of 4 x 10E5 cells / mL. Transfection was performed in a Nucleofector device using the Nucleofector Kit V (Lonza, Switzerland) according to the manufacturer's protocol. 3 x 10E7 cells were transfected with a total of 30 μg of nucleic acid, i.e., 30 μg of plasmid (5 μg of Cre plasmid and 25 μg of front and back vector mixture). After transfection, cells were seeded in 30 mL of medium without selection agent.

[0290] Five days after seeding, the cells were centrifuged and transferred to 80 mL of chemically defined medium containing puromycin (selection agent 1) and 1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU; selection agent 2) at an effective concentration of 6 x 10E5 cells / mL for selection of recombinant cells. The cells were cultured without splitting from this day on at 37°C, 150 rpm, 5% CO2, and 85% humidity. The cell density and viability of the cultures were monitored regularly. When the viability of the cultures began to increase again, the concentrations of selection agents 1 and 2 were reduced to approximately half of the amounts previously used. More specifically, to facilitate cell recovery, the selection pressure was reduced when the viability was greater than 40% and the viable cell density (VCD) was greater than 0.5 x 10E6 cells / mL. Therefore, 4 x 10E5 cells / mL were centrifuged and resuspended in 40 ml of Selection Medium II (chemically defined medium, 1 / 2 selection markers 1 and 2). These cells were incubated under the same conditions as before, and again without splitting.

[0291] Ten days after the start of selection, the success of Cre-mediated cassette exchange was confirmed by flow cytometry, measuring the expression of intracellular GFP and the cell surface-bound extracellular heterologous fusion polypeptide. APC antibody (allophycocyanin-conjugated F(ab')2 fragment goat anti-human IgG) against the light and heavy chains of human antibodies was used for FACS staining. Flow cytometry was performed using a BD FACS Canto II flow cytometer (BD, Heidelberg, Germany). Ten thousand events were measured per sample. Live cells were gated on a plot of forward scatter (FSC) versus side scatter (SSC). A live cell gate was defined on untransfected TI host cells and applied to all samples using FlowJo 7.6.5 EN software (TreeStar, Olten, Switzerland). GFP fluorescence was quantified in the FITC channel (excitation at 488 nm, detection at 530 nm). Heterologous fusion polypeptide was measured in the APC channel (excitation at 645 nm, detection at 660 nm). Parental CHO cells, i.e., the cells used to generate the TI host cells, were used as a negative control for GFP and fusion polypeptide expression. After 14 days of selection, the viability was greater than 90% and the selection was considered complete.

[0292] Example 7 FACS screening FACS analysis was performed to examine the transfection efficiency and RMCE efficiency of the transfection. 4 × 10E5 cells from the transfected approach were centrifuged (1200 rpm, 4 min) and washed twice with 1 mL of PBS. After the washing step with PBS, the pellet was resuspended in 400 μL of PBS and transferred to a FACS tube (Falcon® round-bottom tube with cell strainer cap, Corning). Measurements were performed using a FACS Canto II, and data were analyzed using the software FlowJo.

[0293] Example 8 Fed-batch culture Fed-batch production cultures were performed in shake flasks or Ambr15 vessels (Sartorius Stedim) containing proprietary chemically defined medium. Cells were seeded at 1 × 10 cells / mL on day 0. Cultures received proprietary feed medium on days 3, 7, and 10. Viable cell counts (VCC) and percent viability of cells in the cultures were measured on days 0, 3, 7, 10, and 14 using a Cedex HiRes instrument (Roche Diagnostics GmbH, Mannheim, Germany). Glucose, lactate, and product titer concentrations were measured on days 3, 5, 7, 10, 12, and 14 using a Cobas analyzer (Roche Diagnostics GmbH, Mannheim, Germany). On day 14 after the start of the fed-batch culture, supernatants were harvested by centrifugation (10 min, 1000 rpm and 10 min, 4000 rpm) and clarified by filtration (0.22 μm). Protein A affinity chromatography with UV detection was used to determine titers on day 14. Product quality was determined by Caliper Labchip (Caliper Life Sciences).

[0294] Example 9 Quantification of fusion polypeptide The titer in the culture medium was measured by anti-human IgG sandwich ELISA. Briefly, the fusion polypeptide was captured from the cell culture medium with an anti-human Fc antibody bound to a MaxiSorp microtiter plate (Nunc™, Sigma-Aldrich) and detected with an anti-human Fc POD conjugate that binds to a different epitope than the capture antibody. The secondary antibody was quantified by chemiluminescence using BM chemiluminescent ELISA substrate (POD) (Sigma-Aldrich).

Claims

1. 1. A method for producing an antibody-multimeric fusion polypeptide, comprising: (a) an antibody heavy chain and an antibody light chain; (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain; and a second fusion polypeptide comprising, from N-terminus to C-terminus, a second portion of the non-antibody multimeric polypeptide, and an antibody light chain constant domain if the first polypeptide comprises an antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises an antibody light chain constant domain. Including, (i) the antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond; the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to an antigen; 10. A method according to claim 1, wherein said antibody-multimeric fusion polypeptide is expressed by a recombinant mammalian cell obtained by transfecting a (parent) mammalian cell with expression cassettes for said antibody heavy chain, said antibody light chain, said first fusion polypeptide and said second fusion polypeptide in a stoichiometric ratio of 1:1:2:

1.

2. 10. The method of claim 1, wherein the antibody-multimeric fusion polypeptide is transiently or stably expressed.

3. 3. The method of claim 1 or claim 2, wherein the mammalian cells are CHO cells.

4. 4. The method of any one of claims 1 to 3, wherein said transfecting is of four vectors, each vector containing exactly one said expression cassette.

5. 4. The method of any one of claims 1 to 3, wherein said transfecting is of three vectors, two vectors containing exactly two of said expression cassettes and one vector containing exactly one of said expression cassettes.

6. 6. The method of claim 5, wherein the transfecting is of three vectors, a first vector containing the expression cassettes for an antibody heavy chain and an antibody light chain, a second expression vector containing the expression cassettes for the first fusion polypeptide and the second fusion polypeptide, and a third vector containing one expression cassette for the first fusion polypeptide.

7. 7. The method of any one of claims 1 to 6, wherein the first fusion polypeptide comprises, as a first portion of the non-antibody multimeric polypeptide, two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a peptide linker, and the second fusion polypeptide comprises, as a second portion of the non-antibody multimeric polypeptide, only one ectodomain or fragment thereof of the TNF ligand family member, or vice versa.

8. 8. The method of claim 7, wherein in the first fusion polypeptide, the two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a first peptide linker are fused at their C-terminus to a CH1 domain by a second peptide linker, and in the second fusion polypeptide, the one ectodomain or fragment thereof of the TNF ligand family member is fused at its C-terminus to the antibody light chain constant domain by a third peptide linker.

9. 8. The method of claim 7, wherein in the first fusion polypeptide, the two ectodomains or fragments thereof of a TNF ligand family member connected to each other by a first peptide linker are fused at their C-terminus to a light chain constant domain by a second peptide linker, and in the second fusion polypeptide, the one ectodomain or fragment thereof of a TNF ligand family member is fused at its C-terminus to a heavy chain CH1 domain by a third peptide linker.

10. 10. The method of any one of claims 1 to 9, wherein the variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to a cell surface antigen selected from the group consisting of fibroblast activation protein (FAP), melanoma-associated chondroitin sulfate proteoglycan (MCSP), epidermal growth factor receptor (EGFR), carcinoembryonic antigen (CEA), CD19, CD20, and CD33.

11. The method according to any one of claims 7 to 10, wherein the TNF ligand family member is 4-1-BBL.

12. The method of any one of claims 7 to 11, wherein the ectodomain of a TNF ligand family member comprises the amino acid sequence of SEQ ID NO: 01 or SEQ ID NO:

56.

13. (a) the antibody heavy chain and the antibody light chain form a binding site capable of specifically binding to a target cell antigen; (b) the first fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:05, SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59, and the second fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:01, SEQ ID NO:56, SEQ ID NO:03, and SEQ ID NO:

04.

14. The method of any one of claims 1 to 13, wherein the antigen is fibroblast activation protein (FAP).

15. The method of any one of claims 1 to 13, wherein the antigen is CEA.

16. The method of any one of claims 1 to 13, wherein the antigen is CD19.

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